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Polímeros VOLUME XXIX - Issue IV - Oct./Dec., 2019

October 27-31, 2019 Dall´Onder Grande Hotel Bento Gonçalves, RS, Brazil


UBE lança ETERNATHANE®, pré-polímeros de poliuretano à base de policarbonato-diol para elastômeros de alto desempenho e durabilidade A UBE é uma indústria multinacional Japonesa que atua nos setores de químicos, máquinas, fármacos, energia e construção. Com escritórios ao redor do mundo e fábricas no Japão, Tailândia e Espanha, há um destaque na produção de caprolactama, poliamidas, fertilizantes e produtos químicos nos. O poliuretano para elastômeros tornou-se cada vez mais soosticado para atender às exigências do mercado atual. Neste contexto, a UBE desenvolveu o ETERNACOLL® e o ETERNATHANE®, uma grande plataforma de soluções que oferecem possibilidades personalizáveis aos materiais de poliuretano, bem como retenção de desempenho superior e a longo prazo, como estabilidade térmica, resistência a óleo, estabilidade hidrolítica, resistência à intempéries e resistência química.

retenção das propriedades mecânicas após exposição a altas temperaturas

redução da absorção de água

retenção das propriedades originais após severa agressão hidrolííca e química

redução da perda de volume quando exposto à abrasão extrema

Os pré-polímeros de poliuretano ETERNATHANE®, à base de policarbonato-dióis ETERNACOLL® e terminados em isocianatos, são aplicados em elastômeros de alto desempenho. Através do aprimoramento das propriedades de resistência mecânica, química e térmica dos poliuretanos tradicionais, os novos elastômeros obtidos podem ser aplicados a novos usos e funções não disponíveis até o momento para novos mercados e clientes, tais como: petróleo e mineração, revestimento de rolos, membranas elastoméricas, pisos, elastômeros fundidos, TPU, rodas e pneus, compostos de poliu poliuretano, selantes, eletrônicos e encapsulamento, entre outros.

https://www.ube.com/contents/pcd/index.html

Rua Iguatemi, 192, cj. 134 Itaim Bibi - São Paulo +55 11 30785424


ISSN 0104-1428 (printed) ISSN 1678-5169 (online)

P o l í m e r o s - I ss u e I V - V o l u m e X X I X - 2 0 1 9 I n d e x e d i n : “ C h e m ic a l A b s t r a c t s ” — “ RA P RA A b s t r a c t s ” — “A l l - R u s s i a n I n s t i t u t e o f S ci e n c e ­T e c h n ic a l I n f o r m a t i o n ” — “ R e d d e R e v i s t a s C i e n t i f ic a s d e A m e r ic a L a t i n a y e l C a r i b e ” — “ L a t i n d e x ” — “ W e b o f S ci e n c e ”

and

Polímeros E d i t o r i a l C o u nci l Antonio Aprigio S. Curvelo (USP/IQSC) - President

Editorial Committee Sebastião V. Canevarolo Jr. – Editor-in-Chief

Members Adhemar C. Ruvolo Filho (UFSCar/DQ) Ailton S. Gomes (UFRJ/IMA) Alain Dufresne (Grenoble INP/Pagora) Antonio Aprigio S. Curvelo (USP/IQSC) Bluma G. Soares (UFRJ/IMA) César Liberato Petzhold (UFRGS/IQ) Cristina T. Andrade (UFRJ/IMA) Edson R. Simielli (Simielli - Soluções em Polímeros) Edvani Curti Muniz (UEM/DQI) Elias Hage Jr. (UFSCar/DEMa) Eloisa B. Mano (UFRJ/IMA) João B. P. Soares (UAlberta/DCME) José Alexandrino de Sousa (UFSCar/DEMa) José António C. Gomes Covas (UMinho/IPC) José Carlos C. S. Pinto (UFRJ/COPPE) Júlio Harada (Harada Hajime Machado Consutoria Ltda) Laura H. de Carvalho (UFCG/DEMa) Luiz Antonio Pessan (UFSCar/DEMa) Luiz Henrique C. Mattoso (EMBRAPA) Marco-Aurelio De Paoli (UNICAMP/IQ) Osvaldo N. Oliveira Jr. (USP/IFSC) Paula Moldenaers (KU Leuven/CIT) Raquel S. Mauler (UFRGS/IQ) Regina Célia R. Nunes (UFRJ/IMA) Richard G. Weiss (GU/DeptChemistry) Rodrigo Lambert Oréfice (UFMG/DEMET) Sadhan C. Jana (UAKRON/DPE) Sebastião V. Canevarolo Jr. (UFSCar/DEMa) Silvio Manrich (UFSCar/DEMa)

A ss o ci at e E d i t o r s Adhemar C. Ruvolo Filho Alain Dufresne Bluma G. Soares César Liberato Petzhold José António C. Gomes Covas José Carlos C. S. Pinto Paula Moldenaers Richard G. Weiss Rodrigo Lambert Oréfice

Sadhan C. Jana

D e s k t o p P u b l is h in g

www.editoracubo.com.br

“Polímeros” is a publication of the Associação Brasileira de Polímeros São Paulo 994 St. São Carlos, SP, Brazil, 13560-340 Phone: +55 16 3374-3949 emails: abpol@abpol.org.br / revista@abpol.org.br http://www.abpol.org.br Date of publication: December 2019

Financial support:

Available online at: www.scielo.br

Polímeros / Associação Brasileira de Polímeros. vol. 1, nº 1 (1991) -.- São Carlos: ABPol, 1991Quarterly v. 29, nº 4 (Oct./Dec. 2019) ISSN 0104-1428 ISSN 1678-5169 (electronic version)

Website of the “Polímeros”: www.revistapolimeros.org.br

1. Polímeros. l. Associação Brasileira de Polímeros. Polímeros, 29(4), 2019

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Editorial Section News....................................................................................................................................................................................................E3 Agenda.................................................................................................................................................................................................E4 Funding Institutions.............................................................................................................................................................................E5 15º CBPol.............................................................................................................................................................................................E7

O r i g in a l A r t ic l e Positron annihilation spectroscopy of chain‑end‑functionalized polystyrenes with definite numbers of benzyl alcohol and perfluorooctyl groups Kamal Reyad Mahmoud , Ashraf El-Shehawy and Hoda Atta........................................................................................................................ 1-10

Evaluation of commercial arrowroot starch/CMC film for buccal drug delivery of glipizide Dhanasekaran Gayathri and Lakshmanan Saraswathy Jayakumari ............................................................................................................... 1-9

Effects of polyether siloxane surfactant on the hydrophilic capacity of polypropylene films Lucas Fiamenghi Antunes , Douglas Alexandre Simon, Rudinei Fiorio and Edson Francisquetti................................................................... 1-5

Investigation of TPP-Chitosomes particles structure and stability as encapsulating agent of cholecalciferol Aline Sayuri Lima Iida, Karina Novais Luz, Taís Téo Barros-Alexandrino, Carmen Sílvia Fávaro-Trindade, Samantha Cristina de Pinho, Odílio Benedito Garrido Assis and Milena Martelli-Tosi ............................................................................................................................... 1-8

Application of metric entropy to determine properties of structural materials Grzegorz Garbacz and Lesław Kyzioł............................................................................................................................................................... 1-9

Stabilization of gelatin and carboxymethylcellulose water-in-water emulsion by addition of whey protein Mayara Rocha Laranjo, Bernardo de Sá Costa and Edwin Elard Garcia-Rojas ............................................................................................ 1-8

Evaluation of antimicrobial action of silver composite microspheres based on styrene-divinylbenzene copolymer Maria Aparecida Larrubia Granado Moreira Rodrigues Mandu, Luciana da Cunha Costa, Rodrigo Bernardes Tiosso, Rômulo Pires Grasso and Mônica Regina da Costa Marques Calderari ...................................................................................................... 1-10

Synthesis and performance of AM/SSS/THDAB as clay hydration dispersion inhibitor Wei-Chao Du , Xiang-Yun Wang, Man Liu, Tai-Fei Bi, Shun-Xi Song, Jie Zhang and Gang Chen.................................................................. 1-7

Effect of nanoclay addition and chemical treatment on static and dynamic mechanical analysis of jute fibre composites Seetharaman Arulmurugan and Narayanan Venkateshwaran......................................................................................................................... 1-8

Kraft lignin and polyethylene terephthalate blends: effect on thermal and mechanical properties Lívia Lazzari, Eloilson Domingos, Letícia Silva, Alexei Kuznetsov, Wanderson Romão and Joyce Araujo .................................................... 1-9

Synthesis of immobilized biocatalysts for wastewater decontamination Thâmara Machado e Silva, Leonardo Luiz Borges, Eli Regina Barboza e Souza and Samantha Salomão Caramori ................................... 1-8

Design of a polymeric composite material femoral stem for hip joint implant Romeu Rony Cavalcante da Costa , Fellipe Roberto Biagi de Almeida, Amanda Albertin Xavier da Silva, Sandra Mara Domiciano and André Ferreira Costa Vieira.............................................................................................................................. 1-8

Mechanical characterization of HDPE reinforced with cellulose from rice husk biomass Mariane Weirich Bosenbecker, Gabriel Monteiro Cholant, Gabriela Escobar Hochmuller da Silva, Oscar Giordani Paniz, Neftali Lenin Villarreal Carreño, Juliano Marini and Amanda Dantas de Oliveira .............................................................................................. 1-7

Acoustic approach of weldability for nanocomposite (nanosilica/PA6) welded by ultrasonic welding Anderson Ribeiro , Jaime Casanova, Sérgio Duarte Brandi and Diego de Moura Pinheiro........................................................................... 1-7

Design of chitosan-alginate core-shell nanoparticules loaded with anacardic acid and cardol for drug delivery João Campos Paiva Filho, Selene Maia de Morais, Antonio Carlos Nogueira Sobrinho, Gessica Soares Cavalcante, Nilvan Alves da Silva and Flávia Oliveira Monteiro da Silva Abreu ............................................................................................................ 1-10

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Molecular Fusion Technology Permanently Embeds Antimicrobials in Polymer Substrates

Borealis launches innovative, ADCA-free compound for telecommunications cables

A new proprietary line of polymer fusion products with antimicrobial blocking agents has been introduced by Polyfuze Graphics Corp. The products are designed to exceed the rigid labeling standards of the medical, food service, reusable packaging, and health services industries. The labeling technology for olefins developed by the Clarkdale, AZ–based company relies on pigmented polymers to create permanently fused labels for products in the medical, food packaging, outdoor sports and automotive sectors. Polyfuze fusion technology has unique applicability across all olefin thermoplastic polymer materials where safety, health, and warning labels are required, said the company. Its unique molecular fusion technology permanently unites antimicrobial additives with polymer substrates in critical products such as sharps and biohazardous waste containers, medical beds, medical carts, and hospital laundry carts. It eliminates the sanitary and quality issues of other labeling methods that use surface applications where label edges, layers and adhesives create shelters where microorganisms can thrive and grow despite cleaning with sprays and cleaners. By integrating proprietary olefinic polymer antimicrobial agents in its molecular fusion technology, Polyfuze Graphics helps manufacturers create “label-free” products that are easy to clean and disinfect. Matthew Stevenson, Polyfuze President and CEO remembers that the cost of healthcare-associated infections ranges between $28 billion to $45 billion every year. By preventing just one with the combination of these technologies, Polyfuze can contribute to the protection of human life and help reduce the costs associated with these infections.” Source: Plastics Today - www.plasticstoday.com

World’s first chemically-foamed, high density polyethylene grade free of azodicarbonamide (ADCA), intended for the production of telecommunications cables is been announced as a global commercial launch by Borealis. This launch builds on the long Borealis track record of innovation in the most demanding Wire & Cable segments. Borealis is using its innovation expertise to enable cable manufacturers to future-proof their production operations: its use allows them to bypass altogether any issues arising from the planned inclusion of ADCA on the EU’s Annex XIV of REACH (“Authorisation List”). Thus, cable manufacturers can rely on being able to maintain seamless production, irrespective of any potential disruption caused by legislative changes. The ADCA compound is mainly used as a blowing agent in the production of foamed plastics. At present, it is classified as a Substance of Very High Concern (SVHC) by the European Chemicals Agency. However, it has recently been recommended for inclusion in the Annex XIV of REACH, the so-called “Authorisation List”. Inclusion on this list would require producers, converters, and other downstream users to seek special temporary permission for the use of this substance in production. Given the continued growth of the cable sector due to digitalisation, such an action may have adverse effects on the global supply chain. Anticipating the potentially disruptive effects to the cable industry, Borealis developed a solution for its global customers by creating this new HDPE compound. Free of ADCA and all other SVHCs, it matches the technical performance of other chemically foamed HDPE grades currently on the market. It combines the proven advantages of its predecessors: superb processability, excellent stabilization, and the requisite toughness for fast multi-pair assembly. Its optimal cell structure makes it highly suitable for applications such as foam or foam-skin insulation for telephone singles and data cables with a typical expansion of 35%-40%, as well as for dry core and petroleum jelly-filled cables. It was successfully tested in a series of customer trials before its global launch in 2019. Source: Borealis - www.borealisgroup.com

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A A A A A A A A A A A A A A A A A A A A A

February

June

Performance Polypropylene Europe Date: February 09-10, 2021 Location: Cologne, Germany Website: www.ami.international/events/event?Code=C1119 Polymers and Nanotechnology Date: February 21-24, 2021 Location: Napa Valley, United States Website: www.polyacs.net/21polynano

Gordon Research Conference — Polymers Date: June 5-6, 2021 Location: South Hadley, United States Website: www.grc.org/polymers-conference/2021 Biopolymer – Processing & Moulding Date: June 15-16, 2021 Location: Halle (Saale), Germany Website: https://polykum.de/en/biopolymer-mkt-2021 Gordon Research Conference — Polyamines Date: June 27 - July 2, 2021 Location: Waterville Valley, United States Website: www.grc.org/polyamines-conference/2021

April PVC Formulation Date: April 19-21, 2021 Location: Cologne, Germany Website: www.ami.international/events/event?Code=C1104 37th International Conference of the Polymer Processing Society Date: April 19-23, 2021 Location: Fukuoka, Japan Website: www.pps-37.org Plastic Pouches Date: April 26-28, 2021 Location: Barcelona, Spain Website: www.ami.international/events/event?Code=C1097

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September CIRM – Workshop — Directed Polymers and Folding Date: September 6-10, 2021 Location: Marseille, France Website: https://conferences.cirm-math.fr/2021-calendar.html

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ABPol Associates Sponsoring Partners

Collective Members Master Polymers Ltda. Nexo International Ltda. Nitriflex S/A Ind. e Com. Radici Plastics Ltda. Uniflon - Fluoromasters Polimeros Ind .Com. Imp. Export.Ltda

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15th Brazilian Polymer Conference - 15th CBPol Bento Gonçalves, RS, October 27 to 31, 2019

The Brazilian Polymer Conference (CBPol) is the main scientific forum in the field of polymer science and technology in Brazil. The CBPol has been held every two years since 1991 and attracts around 800 participants per event. Its 15th edition was organized by a group of faculties from the University of Caxias do Sul (UCS), Federal University of Rio Grande do Sul (UFRGS), Federal University of Pelotas (UFPel) and Federal University of Sao Carlos (UFSCar) in partnership with the Brazilian Polymer Association (ABPol). The conference was held in the DallOnder Convention Center in Bento Gonçalves, from October 27 to 31, 2019. Bento Gonçalves, known as the Brazilian Capital of Wine, is a pleasant town in the “Serra Gaúcha”, a tourist region located in the northeast of Rio Grande do Sul State in southern Brazil. The 15th CBPol was sponsored by the Brazilian Funding Agencies CAPES, CNPq and FAPESP (for the participants from Sao Paulo State) and the companies Anton Paar, TA Instruments, UBE, Waters and Zwick Roell. The opening session of the 15th CBPol took place on the evening of October 27th 2019 (Sunday). The participants in charge of the session were Dr. José Donato Ambrósio (ABPol President), Prof. Otávio Bianchi (Conference Chair), Prof. André Ricardo Fajardo (Conference Vice-Chair), Prof. Leonardo Bresciani Canto (Coordinator of the Scientific Committee), Prof. Antonio José Felix de Carvalho (ABPol representative), Prof. Cesar Liberato Petzhold (ABPol representative), Prof. Rodrigo Lambert Oréfice (ABPol representative), Marco Antonio Cione (ABPol representative) and Prof. Odacir D. Graciolli (Vice-Rector / UCS) (Figure 1). The ABPol president Dr. José Donato Ambrósio made a speech emphasizing the importance of ABPol and CBPol to the continued advance of the science and technology of polymers in Brazil. In his speech, the Conference Chair Otávio Bianchi highlighted the high number of student participants at the conference and their importance to the future of polymer science and technology in Brazil. Following the speeches, the ABPol representative Prof. Cesar Liberato Petzhold paid tribute (in memoriam) to the Brazilian scientist Profa. Eloisa B. Mano, who was a pioneer in the field of polymers. Awards were then given to remarkable Brazilian researches: Prof. Luiz Antonio Pessan (UFSCar) received the ABPol “Profa Eloisa Mano” Polymer Science prize, Prof. André Ricardo Fajardo (UFPel) received the “ABPol Young Researcher” prize and the ABPol “Polymer Technology” prize was awarded to Eng. Luis Fernando Cassineli (FAPESP) (Figure 2). The ceremony was closed with a concert performed by the University of Caxias do Sul (UCS) Orchestra, followed by a welcome cocktail for all participants (Figure 3).

Figure 1: Images of the opening session of the 15th CBPol. First image (from left to right): Antonio José Felix de Carvalho (ABPol representative), Marco Antonio Cione (ABPol representative), Prof. André Ricardo Fajardo (Conference Vice-Chair), Prof. Otávio Bianchi (Conference Chair), Dr. José Donato Ambrósio (ABPol President), Prof. Leonardo Bresciani Canto (Coordinator of the Scientific Committee), Prof. Cesar Liberato Petzhold (ABPol representative), Prof. Rodrigo Lambert Oréfice (ABPol representative) and Prof. Odacir D. Graciolli (Vice-Rector / UCS).

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Figure 2: Images of the ABPol awards. From top to bottom, left to right: Prof. Luiz Antonio Pessan (ABPol “Profa Eloisa Mano” prize winner) and Prof. Cesar Liberato Petzhold (ABPol representative); Prof. Rodrigo Lambert Oréfice (ABPol representative) and Prof. André Ricardo Fajardo (ABPol “Young Researcher” prize winner and Vice-Chair); Luis Fernando Cassineli (ABPol “Polymer Technology” prize winner) and Marco Antonio Cione (ABPol representative).

Figure 3: Images of the concert performed by the University of Caxias do Sul Orchestra (left) and welcome cocktail (right).

On Sunday afternoon, the ABPol administrative assistants Mr. Marcelo Perez Gomes and Mr. Charles Fernandes de Souza delivered the conference documents to the participants (Figure 4). In parallel, two short-courses (40 places each) were offered to the participants, namely: “Introduction to Composite Materials and Micromechanics” given by Prof. Sandro Campos Amico (Federal University of Rio Grande do Sul – UFRGS / Brazil) and “Physics of Polymer Networks” given by Prof. Bradley D. Olsen (Massachusetts Institute of Technology – MIT / USA).

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Figure 4. Image of the conference reception desk. ABPol administrative assistants Mr. Marcelo Perez Gomes and Mr. Charles Fernandes de Souza.

In the following four days (Monday to Thursday), the activities of each period of the day (morning and afternoon) started with a plenary lecture followed by keynote lectures and oral presentations in 5 parallel sessions. At the same time, there were stands for the exhibition of products and equipment associated with polymers research and related branches of science hosted by the manufacturing companies: Altmann, Anton Paar, Brucker, dpUNION, KCEN, Netzsch, Perkin Elmer, PolyAnalytik, ReoTerm, Shimadzu, TA Instruments, UBE, Waters and Zwick Roell (Figure 5). On Monday to Wednesday, at the end of each day (late afternoon), poster sessions took place (Figure 5). A coffee-break was offered in each period of the day. In addition, a banquet dinner (on Tuesday evening) (Figure 6) and a conference party (on Wednesday evening) were offered to the participants. These social activities provided an extra opportunity for networking among the participants.

Figure 5: Images of the exhibition stands (left) and poster session (right).

Figure 6. Image of the banquet dinner.

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The 15th CBPOL had 880 registered participants from the following countries: South America - Brazil (857), Argentina (1), Chile (9) and Peru (1); North America - USA (1) and Canada (2); and Europe - Belgium (1), France (1), Great Britain (1), Portugal (5) and Spain (1) (Figure 6). Of the total, 60% were students and 40% professors or researchers (55% women and 45% men). The number of registered participants from Brazil per state (federative regions) is shown in Figure 7. The most significant number of researchers was from the state of SĂŁo Paulo, followed by Rio Grande do Sul, Rio de Janeiro and Minas Gerais, which is similar to previous events.

Figure 7. Number of registered participants per state (federative region) of Brazil.

A total of 1,075 papers from 2725 authors were presented at the conference. All of the papers were previously subjected to a peer review. The review of the papers was coordinated by Prof. Leonardo Bresciani Canto (UFSCar) with the collaboration of 21 symposia coordinators and 185 ad hoc referees. The 1,075 papers accepted were divided as follows: 7 plenary lectures (60 min each), 60 keynote lectures (30 min each), 84 oral presentations (20 min each) and 924 posters (Figure 8).

Figure 8. Number of presentations at the 15th CBPol

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The papers were classified into 11 thematic symposia according to their subjects: 1) Biopolymers, eco-friendly polymers and biodegradable polymers; 2) Functional Polymers for biomedical, energy, optical, acoustic, sensors, membranes & barriers or smart packaging applications, and biomimetics; 3) Polymer nanocomposites and nanostructured polymers; 4) Polymer composites and polymers for structural applications; 5) Polymer blends and plasticized polymers; 6) Rubber, elastomers and thermosets; 7) Synthesis and chemical modification of polymers; 8) Polymer degradation and stabilization; 9) Processing, modeling and simulation and recycling of polymers; 10) Polymer characterization techniques; and 11) Technical papers. The biopolymers symposium was the one that received the most significant number of papers, followed by functional polymers, nanocomposites, and composites symposia (Figure 9). This reflects the global scenario of research in polymers.

Figure 9: Number of papers in each symposium presented at the 15th CBPol. The category numbers relate to the classification of the thematic symposia given above.

The plenary lectures were given by renowned Brazilian and foreign researchers (Table 1 and Figure 10), which encompassed emerging areas of polymer research around the world.

Table 1: Plenary lectures of the 15th CBPol Title

Lecturer

Responsive polymers and supramolecular chemistry as basis for Richard Hoogenboom (Ghent University, Belgium) sensors Biofunctionality and bioengineered poly(lactic-co-glycolic) Bruno Sarmento (University of Porto / Portugal) acid in biomedicine Hybrid polymer blend nanocomposites for electrical, charge Uttandaraman Sundararaj (University of Calgary/Canada) storage, ESD and EMI shielding applications New Theory, New Experiments, and Big Data to Understand Bradley D. Olsen (Massachusetts Institute of Technology / USA) Polymer Networks Multifunctional Polymeric Materials for Gas Barrier Packaging, Luiz Antonio Pessan (Federal University of Sao Carlos - UFSCar / Brazil) Hydrogen Energy Storage and Additive Manufacturing Use of Renewable Monomers for Manufacture of Polymer José Carlos Pinto (Federal University of Rio de Janeiro - UFRJ /Brazil) Particles: Some Challenges and Solutions Modeling and Experiments in FDM/FFF 3D printing

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José António Covas (University of Minho / Portugal)

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Figure 10. Images of the plenary lecturers of the 15th CBPol. From top to bottom, left to right: Prof. Richard Hoogenboom, Prof. Bruno Sarmento, Prof. Uttandaraman Sundararaj, Prof. Bradley D. Olsen, Prof. Luiz Antonio Pessan, Prof. José Carlos Pinto, Prof. José António Covas.

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On Tuesday evening, immediately after the poster session, there was a meeting of the editorial board of the Journal “Polimeros Ciência e Tecnologia” (Figure 11).

Figure 11. Image of the editorial board meeting of the Journal “Polimeros Ciência e Tecnologia”. At the bottom: (seated) Sebastião V. Canevarolo (Editor-in-chief) and (standing) Prof. Antonio Aprigio S. Curvelo (President of the Editorial Council).

The poster prizes were awarded at the closing ceremony on October 31th (Thursday) at midday, which was overseen by Prof. Dr. André R. Fajardo (Vice-Chair). The top three students of each category (doctorate, master’s and undergraduate) were awarded. The first places of each category received a voucher for EUR 150 to purchase books from the publisher Wiley. The second place in each category was offered a voucher to purchase the book “Polymer Science: A Textbook for Engineers and Technologists” (Hanser Publications) authored by Prof. Sebastião S. Canevarolo. The third place in each category received a voucher for EUR 65 to purchase books from the ABPol collection. The award-winning students are listed below by category:

Doctorate students 1st place: Stéphanie Cardoso de Sá (Federal University of Rio Grande do Sul - UFRGS) for the study entitled “Evaluation of the use of calcinated rice husk as a silicon source in intumescent coatings for the protection of steel against fire”. Coauthors: Andreza P. Cardoso (UFRGS) and Carlos A. Ferreira (UFRGS). 2nd place: André Moreira Castro (Federal University of Rio de Janeiro - UFRJ) for the study entitled “Rheology of melt polyethylene using multipass rheometer and computional simulation in OpenFOAM”. Co-authors: Juliana O. Pereira (UFRJ), Veronica M. A. Calado (UFRJ) and Argimiro Resende Secchi (UFRJ). 3rd place: Lilian Ribeiro Batista (Federal University of Goias - UFG) ) for the study entitled “Production of lubricating grease developed from chemically modified soybean oil”. Co-authors: Aline Silva Muniz (UFG, UFPR), Lucas O. Gomes (UFG), Maríthiza G. Vieira (UFG) and Nelson R. Antoniosi Filho (UFG).

Master’s students 1st place: Rodrigo Duarte Ribeiro (Federal University of Rio de Janeiro - UFRJ) for the study entitled “Evaluation of Composite Hull for Subsea Separator”. Co-authors: Marysilvia F. Costa (UFRJ) and Ilson P. Pasqualino (UFRJ). 2nd place: Barbara Fornaciari (University of Sao Paulo - USP) for the study entitled “Chitosan-Tripolyphosphate‑Pol y(ethyleneglycol) nanoparticles for encapsulation of the diruthenium(II,III) anticancer metallodrug”. Co-author: Denise Oliveira Silva (USP). 3rd place: Matheus Colovati Saccardo (Federal University of Sao Carlos - UFSCar) for the study entitled “Counter-ion and relative humidity influence in the water absorption kinects of Nafion and Ionomeric Polymer Metal Composites”. Coauthors: Ariel G. Zuquello (UFSCar), Roger Gonçalves (UFSCar), Thiago P. Dardis (UFSCar), Laos A. Hirano (UniFal) and Carlos H. Scuracchio (UFSCar).

Undergraduate students 1st place: Herik Grillo Brogliato (Federal Institute for Education, Science, and Technology of Rio Grande do Sul IFRS) for the study entitled “Energy absorption by variable metamaterials geometry structures in 3D printing”. Co‑authors: Luciano A. Ferri (UFRS), Douglas A. Simon (UFRS).

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2nd place: Amanda Rinaldi Sorigotti (University of Sao Paulo - USP) for the study entitled “Silk fibroin nanofibers hybridized with organosilanes for biomedical applications”. Co-authors: Rafaella T. Paschoalin (USP), Robson R. Silva (Chalmers University of Technology / Sweden), Caio G. Otoni, Hernane S. Barud (UNIARA), Sidney J. L. Ribeiro (UNESP), Osvaldo N. Oliveira Jr (USP) and Luiz H. C. Mattoso (EMBRAPA). 3rd place: Leonardo Patrick Pereira Gonçalves (Federal University of Minas Gerais - UFMG) for the study entitled “Colloidal stability and external stimuli multi responsiveness of polyetheramine-cellulose nanocrystals aqueous nanofluids for applications in Ht-Hp conditions and high salinity”. Co-authors: Éder José Siqueira (Université Grenoble Alpes / France), Bruno Jean (CERMAV / France).

Figure 12: Image of the poster awards ceremony, from left to right: Prof. André R. Fajardo (Vice-Chair); Prof. José Carlos Pinto (Wiley representative); Prof. Sebastião V. Canevarolo (Hanser Publications representative and book author); Marcia C. Branciforti (Awards Committee Chair); Awarded students: Lilian Ribeiro Batista (UFG), André Moreira Castro (UFRJ), Leonardo Patrick Pereira Gonçalves (UFMG), Herik Grillo Brogliato (IFRS) and Amanda Rinaldi Sorigotti (USP).

In the closing ceremony (Figure 12) at midday on October 31th (Thursday) the ABPol president Dr. José Donato Ambrósio presented an overview of the conference and announced the new ABPol board for the biennium 2020-2021, which will be managed by the elected-president Prof. Marco Aurélio De Paoli. The Conference Chair Prof. Dr. Otávio Bianchi gave a presentation on the scientific highlights of the conference. Finally, three proposals for the organization of the next conference were delivered.

Figure 12: Images of the closing session of the 15th CBPol. From top to bottom, left to right: Dr. José Donato Ambrósio (ABPol president) and the audience; Otávio Bianchi (Conference Chair) and Prof. Marco Aurélio De Paoli (ABPol elected-president).

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Some aspects of the 15th CBPol are particularly noteworthy. Firstly, there was a notably high number of registered participants (880) and papers presented (1,075), based on the historical average of the event. Secondly, we should highlight the impressively high scientific and technological quality of the papers presented at the event. Lastly, the scientific and social activities were a great success, providing a favorable environment for discussion and networking among the participants. We would like to thank the organizing and scientific committee as well as the symposia coordinators and the ad hoc referees, who played a key role in achieving the excellent scientific quality of the conference. We also thank the funding agencies that supported the event, FAPESP, CAPES and CNPq, as well as the sponsors and exhibitors. We also gratefully acknowledge ABPol, its board and employees (Mr. Marcelo P. Gomes and Mr. Charles F. de Souza), for all of their support in the organization of the event. Finally, we thank all of the participants of the 15th CBPol who contributed significantly to the success of the conference. We look forward to seeing you at the 16th CBPol!

Otávio Bianchi – UCS/UFRGS (Chair) André Ricardo Fajardo – UFPel (Vice-Chair) Leonardo Bresciani Canto – UFSCar (Scientific Coordinator)

Polímeros, 29(4), 2019

E15


ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.04619

Positron annihilation spectroscopy of chain‑end‑functionalized polystyrenes with definite numbers of benzyl alcohol and perfluorooctyl groups Kamal Reyad Mahmoud1* , Ashraf El-Shehawy2 and Hoda Atta1 Physics Department, Faculty of Science, Kafrelsheikh University, El Gaish Street, Kafr El-Sheikh, Egypt Chemistry Department, Faculty of Science, Kafrelsheikh University, El Gaish Street, Kafr El-Sheikh, Egypt 1

2

*kamalreyad@sci.kfs.edu.eg

Abstract A series of well-defined chain-end-functionalized polystyrenes with a definite number of benzyl alcohol and perfluorooctyl groups [PS(BnOH)n & PS(BnORf)n, respectively] linearly aligned in a double line at the chain-ends were prepared and investigated using XRD, SEM, PALS and DBAR spectroscopy. XRD studies showed that PS(BnOH)n are crystalline and the degree of crystallinity increases with increasing the number of benzyl alcohol functionalities, while XRD pattern of PS(BnORf)n revealed that incorporating perfluorooctyl groups resulting in some fractions of polystyrene chains that were intercalated or broken between the interlayer spacing. PALS measurements yielded three lifetime components and the formation probabilities as well as lifetime of ortho-positronium in polymer series were found to be dependent on the chain-end polymer structure. DBAR measurements suggested that only one type of defect is present in the polymer samples. Keywords: benzyl alcohol and perfluorooctyl groups, hyperbranched polymers, chain-end-functionalized polystyrene, doppler broadening, free volume, positron annihilation. How to cite: Mahmoud, K. R., El-Shehawy, A., & Atta, H. (2019). Positron annihilation spectroscopy of chain-end-functionalized polystyrenes with definite numbers of benzyl alcohol and perfluorooctyl groups. Polímeros: Ciência e Tecnologia, 29(4), e2019046. https://doi.org/10.1590/0104-1428.04619

1. Introduction Dendrimers and hyperbranched polymers are highly branched structures of three dimensional globular macromolecules. The globular and dendritic architectures of these polymers endow them with unique structures and properties such as abundant functional groups, intramolecular cavities, low viscosity, and high solubility. The synthesis of the hyperbranched polymers and the development in their structure are of great interest due to their unique properties in a wide variety of applications. These applications not only restricted for using in medicine and pharmacy as drug delivery systems but also in solving some ecological and biological problems, as well as in modern and nano technologies[1-5]. Moreover, hyperbranched polymers have been widely applied in various fields such as light emitting materials, hybrid materials and composites, supramolecular chemistry, biomaterials, nanoscience and technology, coatings, adhesives, and modifiers. These materials have different physical, mechanical and chemical properties, which can be determined by structures of their macromolecular chains and condensed states[2, 6-8]. Positron annihilation lifetime spectroscopy (PALS) is a non destructive technique that has been used as a sensitive microprobe to study the nano scale microstructure of molecular solids. This technique is based on positron (e+) implantation from radioactive source into the molecular

Polímeros, 29(4), e2019046, 2019

solids. The positron is either annihilate with an electron of the atoms of materials or compound with an electron to form positronium (Ps) atom. Two positronium states can be formed: a single state (para-Ps, p-Ps) and a triplet state (ortho-Ps, o-Ps). Their formation probability (Ii) and lifetimes (τi) can be measured, which provide information about the physical and chemical properties of such solids[9]. The Doppler broadening (DB) of the annihilation γ-rays represents another technique related to the positron annihilation process. The DB experiments provide useful information on the contribution of the inner electronic shells and gives valuable data on chemical surrounding of the annihilation site. Two important parameters can be identified from the DB measurements are the S and W-parameters[10]. The S-parameter is defined as ratio of area under the central part of the 511 keV line to the area under the whole annihilation line. The S-parameter characterizes the contribution of positron annihilations with the low momentum electrons, which may be present in open volume defects. Therefore, this parameter is sensitive to the average density of the open volume defects in the medium. In addition, the W-parameter can be defined as the ratio of the area under the fixed wing region of the annihilation line to the area under the whole annihilation line. This parameter is related to annihilation of positrons with deeply bound core electrons, which provides

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O O O O O O O O O O O O O O O O


Mahmoud, K. R., El-Shehawy, A. & Atta, H. information about chemical environment of the defect. Several studies on polymers including hyperbranched and grafted polymers have been advantageously performed by means of the PAL technique[11-14]. The PAL technique was used to study the experimental evidence of the free volume change during the investigated processes[15]. The high sensitivity of PAS in probing free volume properties arises from the fact that the positronium atom (a positron-electron bound state) is preferentially trapped (localized) in atomic scale free volume holes located between the grain surface and the surrounding innermost polymer layer. The lifetime of positrons trapped at the grain surface defects and the grain polymer interface decreased with increasing grain size[16]. As part of our research[17-21] with a goal to study the nano scale microstructure of molecular solids, this study used PALS to evaluate how the number of end-functional groups affected the size and distribution of molecular level free volume in the chain-end-functionalized polystyrenes having a definite number of benzyl alcohol through systematic comparison with their corresponding polymers having the same definite number of C8F17 groups. These chain-end-functionalized polystyrenes are distributed and linearly aligned in a double line at their chain-ends.

2. Materials and Methods 2.1 Materials All chemicals (> 98% purities) were purchased from Tokyo Kasei Co. Ltd., Japan and used as received unless otherwise noted. Tetrahydrofuran (THF) was refluxed over sodium wire for 5 h and then distilled over LiAlH4 under nitrogen. It was finally distilled from its sodium naphthalenide solution on the vacuum line. N,N-Dimethylformamide (DMF), dichloromethane, and pyridine, were distilled over CaH2 under nitrogen. 3-Perfluorooctylpropyl bromide [C8F17(CH2)3Br] was synthesized by the reaction of C8F17(CH2)3OH with carbon tetrachloride/triphenylphosphine in THF/dichloromethane according to the modified procedure previously reported[22].

2.2 Measurements 1 H NMR spectra were recorded on a Bruker DPX300 (300 MHz) in CDCl3 for all polymers. Chemical shifts were recorded in ppm relative to tetramethylsilane (δ 0). Size-exclusion chromatography (SEC) was performed on a TOSOH HLC-8020 instrument with UV (254 nm) and refractive index detection. THF was used as a carrier solvent at a flow rate of 1.0 mL/min at 40 °C. Three polystyrene gel columns (pore size (bead size)): 650 Å (9 μm), 200 Å (5 μm), and 75 Å (5 μm)) were used. Measurable molecular weight ranges are 103 ~ 4×105 (g/mol). Calibration curve was made with standard polystyrene samples for determining both Mn and Mw/Mn values. Positron annihilation lifetime spectrometer used in this work is a fast-fast coincidence spectrometer[23] with a resolution of ~350ps measured using a 60Co source at room temperature for the positron lifetime measurements. About 15μCi of 22Na activity was deposited and dried on a thin Kapton foils (7.6μm thick), covered with an identical foil and were afterward glued with epoxy glue. This assembly was used as the positron source sandwiched between two

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identical samples. Each sample was measured at least 2-3 times differed by a total number of elementary annihilation events in the range of 1-2 million counts. The obtained spectra were analyzed using the LT computer program of Kansy[24], with a suitable correction for the positrons annihilated in the Kapton. Three lifetime components (τ1, τ2 and τ3) were obtained from the analysis of the measured spectra. The first lifetime component τ1 is attributed to the para-positronium (p-Ps) atom, which fixed to the value 0.125 ns (below the time resolution limit of the equipment). The intermediate lifetime component τ2 is due to the annihilation of positrons with free electrons, while the third lifetime component τ3 which is the longest lifetime component is related to annihilation of ortho-positronium by “pick off” mechanism in the free volume sites present in the amorphous regions. All these components were determined by the fit’s variance ranged from 1.005 to 1.180. The determination of the o-Ps components, τ3, provides valuable information about the mean size of free volume cavities probed by o-Ps. According to the free‑volume model[25], the lifetime of the o-Ps localized inside a rigid and spherical potential well of radius R0, and free volume of radius R, below which no electrons are found, is given by the following expression[26, 27]:  R   2πR  −1 τo − Ps = 0.5[1 −   + (1 / 2π ) Sin  ] R  o  Ro 

(1)

Where δR= R0- R = 1.656Å is the fitted empirical electron layer thickness. With this value of δR, the free volume radius R was calculated from Equation 1 and the average size of the free volume holes Vf was evaluated as Vf= ( 4 / 3) πR 3 (in Å3). Doppler broadening measurements: A p-type high‑purity germanium detector (Ortec, GEM series) with an energy resolution (FWHM) of 1.6 keV for 1.33 MeV gamma line of 60Co and relative efficiency of 25% was used to determine the Doppler broadening line-shape parameters (S and W). The amplified signals from an Ortec 570 amplifier were acquired with an Ortec 919 multichannel analyzer (MCA). A 3µCi 22Na source was prepared using a droplet of 22NaCl solution dried onto two identical Kapton foils (7.5 µm thick), which were afterward glued by epoxy glue. Two disk samples were arranged with the 22Na source in a 4π configuration. The energy calibration (~68 eV/channel) was achieved using the 133Ba source. The Doppler broadening spectra were measured in air at room temperature. More than one million counts in the annihilation line were accumulated for each spectrum. The analysis of the obtained Doppler broadening spectra was done using SP ver. 1.0 program[28]. The centroid channel with maximum counts of the 511 keV peak was carefully defined as it is a base for calculations of S- and W-parameters. The input data for this program are fixed for all spectra of the studied samples.

2.3 Sample preparation The synthesized chain-end-functionalized polymers having benzyl alcohol and perfluorooctyl groups [PS(BnOH)n and PS(BnORf)n, respectively] were ground and the powder was pressed into disc-shaped samples (1 cm diameter and ~ 2mm thickness) using stainless steel dies. The samples were pressed at 3×108 N/m2. To understand the crystalline Polímeros, 29(4), e2019046, 2019


Positron annihilation spectroscopy of chain-end-functionalized polystyrenes with definite numbers of benzyl alcohol and perfluorooctyl groups domains and intercalation of PS between the interlayer spacing of the materials, X-ray powder diffraction data were recorded using an X-ray powder diffractometer (Shimadzu X-ray Diffractometer, XRD-6000) with CuKα radiation (wavelength, 1·53 Ao) source operated at 40 kV and 30 mA. To visualize the phase morphology and intercalated structures in the chemical compound, high resolution Scanning Electron Microscopy (SEM) (Model JSM-IT100, JEOL) was employed. 2.3.1 Synthesis of polystyrenes end-functionalized with benzyl alcohol groups PS(BnOH)N A series of PS(BnOH)n were readily obtained by treatment the polystyrene end-functionalized with silyl protected alcohols PS[BnOSi(CH3)2C(CH3)3]n, prepared by our previously reported method[29] with a 5-fold excess of (C4H9)4NF in THF and the reaction mixture was stirred at 25 °C for 12 h. The reaction was quenched with a small amount of water and the reaction mixture was poured into 1N HCl to precipitate the polymer. The resulting polymers were purified by reprecipitation using THF/methanol twice and freeze-drying from their absolute benzene solutions for 24 h to afford the objective polymers PS(BnOH)n in quantitative yields. All of the benzyl alcohol-functionalized polymers showed sharp and symmetrical monomodal SEC distributions similar to those of their parent polymers. In each case, quantitative deprotection of the silyl-protecting group was confirmed by the complete disappearances of tert‑butyldimethylsilyl protons (0.1 and 0.9 ppm) in 1 H NMR[29].

at 0 °C and the reaction mixture was stirred at 25 °C for 18  h. Water was cautiously added to quench the excess NaH. The resulting mixture was poured into 1N HCl methanolic solution to precipitate the polymer. The polymer was purified by reprecipitation with THF/methanol twice affording PS(BnORf)8 (0.24 g) in 92% yield. All other chain-end-functionalized Rf polymers were synthesized under the same conditions. All polymers were purified by reprecipitation twice, followed by freeze-drying from their benzene solutions for 24 h. Yields of polymers isolated were usually around 90%. They were characterized by 1H NMR, and SEC[29]. The assignments of 1H NMR spectra of these polymer series were shown below: PS[BnOC3H8C8F17]2: δ 0.5-0.7 (br, 6H, CH3), 1.2-2.4 (br, 570H, CH2), 3.2-3.5 (br, 4H, ArCH2), 4.3-4.4 (br, 4H, ArCH2OCH2), 6.3-7.2 (br, 942H, Ar). PS[BnOC3H8C8F17]4: δ 0.3-0.8 (br, 12H, CH3), 1.2-2.3 (br, 597H, CH2), 3.2-3.5 (br, 8H, ArCH2), 4.3-4.4 (br, 8H, ArCH2OCH2), 6.3-7.2 (br, 980H, Ar). PS[BnOC3H8C8F17]6: δ 0.3-0.8 (br, 6H, CH3), 1.2-2.4 (br, 597H, CH2), 3.2-3.5 (br, 12H, ArCH2), 4.3-4.4 (br, 12H, ArCH2OCH2), 6.3-7.3 (br, 979H, Ar). PS[BnOC3H8C8F17]8: δ 0.2-0.8 (br, 12H, CH3), 1.2-2.4 (br, 570H, CH2), 3.2-3.5 (br, 16H, ArCH2), 4.3-4.4 (br, 16H, ArCH2OCH2), 6.3-7.2 (br, 988H, Ar). PS[BnOC3H8C8F17]10: δ 0.5-0.7 (br, 6H, CH3), 1.1-2.3 (br, 761H, CH2), 3.1-3.5 (br, 20H, ArCH2), 4.2-4.5 (br, 20H, ArCH2OCH2), 6.2-7.3 (br, 1230H, Ar).

2.3.2. Introduction of C3H8C8F17 (Rf) groups to benzyl alcohol functionalities of PS(BnOH)n by Williamson reaction

3. Results and Discussions

It can be readily synthesized by the Williamson reaction of the corresponding PS(BnOH)n with C8F17(CH2)3Br. Typical example is as follows: Under nitrogen, NaH (40.0 mg, 1.67 mmol) was added to PS(BnOH)8 (0.204 g, Mn = 19.8 x 103 g/mol, benzyl alcohol moiety = 0.082 mmol) dissolved in a mixture of THF (10 mL) and DMF (3 mL) at 0 °C and the resulting suspension was allowed to stir for 2 h at 25 °C. Then, C8F17(CH2)3Br (0.903 g, 1.67 mmol) in THF (3.00 mL) was added slowly to this suspension

In this work, well-defined chain-end-functionalized polystyrenes with a definite number of benzyl alcohol moieties [PS(BnOH)n] linearly aligned in a double line at their chain-ends were prepared via our previously reported synthetic methodology[29] and used as precursory polymers for introducing perfluorooctyl (C8F17) groups on their benzyl alcohol (BnOH) functionalities, see Figure 1. They may also referred as LB-n, where “n” indicates the number of either BnOH or C3H8C8F17 groups in each polymer series.

Figure 1. Structures of chain-end-functionalized polymers having benzyl alcohol and perfluorooctyl groups [PS(BnOH)n and PS(BnORf)n, respectively]. Polímeros, 29(4), e2019046, 2019

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Mahmoud, K. R., El-Shehawy, A. & Atta, H. In order to prepare chain-end functionalized polystyrenes having two, four, six, eight, and ten BnOH functionalities, we should first prepare the corresponding precursory polymers having similar numbers of silyl-protected benzyl alcohol functionalities PS[BnOSi(CH3)2C(CH3)3]n, followed by deprotecting the BnOH functionalities using a 5-fold excess of (C4H9)4NF in THF at 25 °C for 12 h to afford the desired polymers PS(BnOH)n[29]. Two, four, six, eight and ten C8F17 groups were then introduced at the polymer chain-ends by reacting PS(BnOH)n (n = 2, 4, 6, 8 or 10) with NaH, followed by treatment with C8F17(CH2)3Br, a typical example is shown in Scheme 1. Thus, benzyl ether linkage was used to connect C3H8C8F17 (Rf) groups to the main polystyrene chains in these polymers. They referred to as PS(BnORf)n (n = 2, 4, 6, 8 or 10). The “n” indicates the number of C8F17 group in each polymer series. They referred to as PS(BnORf)n and the “n” indicates the number of C8F17 group in each polymer series.

As can be seen, all of these polymers exhibited sharp, symmetrical monomodal SEC distributions, indicating that all reactions proceed cleanly without any side reactions leading to chain coupling and degradation. However, small high molecular weight shoulders (< 5%), which double the molecular weight of the parent polymers, were sometimes formed during the course of the Williamson reaction. In such cases, they were completely removed by fractional precipitation using a mixture of cyclohexane and hexane. The degrees of C8F17-end-functionalization measured by 1H NMR analysis were in good agreement with those expected in all cases within error limits. The molecular weights (Mn values) of the polymers were determined by 1 H NMR as follows: The molecular weight of polystyrene

Fortunately, the reaction proceeded efficiently to quantitatively introduce C8F17 groups. The objective C8F17-chain-end-functionalized polystyrenes as well as their precursory polymers synthesized in all iteration processes possess predictable molecular weights and narrow molecular weight distributions. All analytical data are summarized in Table 1. The Mn values observed by SEC and 1H NMR agreed quite well with those calculated in all cases. SEC profiles of the polymer samples exhibited symmetrical monomodal distributions and narrow molecular weight distributions were always attained. Neither shoulder nor tailing is observed in each polymer sample. In all polymer samples, the main polystyrene chains were also designed and adjusted to be around 20 kg/mol in Mn value. Figure 2 shows SEC profiles of chain-end-functionalized polystyrenes with two, four, six, eight, and ten perfluorooctyl groups (C3H8C8F17 groups) (a-e, respectively).

Figure 2. SEC curves for chain-end-functionalized polystyrenes with two-ten perfluorooctylpropyl ether functions (a-e, respectively).

Scheme 1. Typical reaction for deprotecting the hydroxyl groups & introducing the perfluorooctyl groups at the polymer chain-ends. Table 1. Analytical data for the chain-end-functionalized polystyrenes with definite numbers (two, four, six, eight, and ten) benzyl alcohol and perfluorooctyl groups [PS(BnOH)n and PS(BnORf)n, respectively]. C8F17-End-Functionalized Polystyrene Code

a

Mn(Kg/mol)

LB-2

Calcdb 20.5

Obsda 20.7

LB-4

21.4

22.6

LB-6

22.4

LB-8 LB-10

Mw/Mn

BnOH-End-Functionalized Polystyrene

Functionality

Mn(Kg/mol)

1.05

Calcdb 2

Obsda 1.96

Calcdb 19.6

Obsda 19.8

1.03

4

3.98

19.6

20.8

23.5

1.04

6

5.98

19.6

20.8

23.5

24.7

1.03

8

7.99

19.8

21.1

29.6

30.8

1.04

10

9.94

25.0

26.1

Mw/Mn

Functionality

1.03

Calcdb 2

Obsda 1.99

1.04

4

3.99

1.03

6

5.96

1.04

8

7.96

1.05

10

9.95

Determined by 1H NMR; b Determined by SEC.

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Positron annihilation spectroscopy of chain-end-functionalized polystyrenes with definite numbers of benzyl alcohol and perfluorooctyl groups part was first determined by comparing 1H NMR signal area ratio of aromatic protons of the main chain (6.1-7.4 ppm) with methyl protons of the initiator fragment (0.2-0.8 ppm) (as well as SEC relative to standard polystyrene). The degree of C8F17-functionalization was determined by 1H NMR signal area ratio of methyl protons of the initiator fragment (0.2-0.8 ppm) to methylene protons of OCH2CH2CH2C8F17 (3.8-4.5 ppm for ether linkage). Then, the total molecular weight of the polymer was obtained by adding the molecular weight of C8F17 part to that of the polystyrene part.

Figure 3a shows XRD pattern of the chain-end functionalized polystyrenes with a definite number of benzyl alcohol groups PS(BnOH)n with number of branches n = 2 and n = 10. These results provide beneficial information about the degree of crystallinity of the measured samples. The XRD pattern of benzyl alcohol group with n = 2 shows a halo broad peak at approximately 2θ = 18.4° which may be attributed to the (100) plane due to the presence of somewhat low crystallinity and small particle size[30]. Further peaks at 2θ values of 37.7◦, 43·93◦, 64.3 and 77·37◦ are assigned to (111), (200), (220) and (311) crystal planes, respectively. This means that both amorphous (halo broad peak) and

crystalline regions are found in this sample. On the other hand, as the number of branches increases to n = 10, the amorphous halo broad peak (100) plane disappeared as shown in Figure 3a indicating that the degree of crystallinity increases with increasing branches until the material become highly crystalline. The XRD patterns for the polystyrenes end‑functionalized with perfluorooctyl groups PS(BnORf)n, where n = 2 and 10, are shown in Figure 3b. It is observed that the XRD pattern of PS(BnORf)2 shows five peaks at 2θ with values of 31.08, 37.72, 43.96, 64.32 and 77.43◦ which correspond to 110, 111, 200, 220 and 311 crystal planes, respectively. On the other hand, the XRD pattern of PS(BnORf)10 shows only four peaks at 2θ values of 37.72, 43.96, 64.32 and 77.43 which correspond to 111, 200, 220 and 311 miller indices, respectively. It is worth mention that the peak at 2θ = 31.08◦ is absent in the XRD pattern of PS(BnORf)10 and this may be because some fractions of polystyrene chains were intercalated or broken between the interlayer spacing. Figure  4 shows the representative SEM images and the typical surface morphology of PS(BnOH)n: (a) n =2; (b) n = 10. Indeed, as revealed by SEM, a sizeable shows

Figure 3. X-ray diffraction pattern of the chain-end-functionalized polystyrenes samples with a definite number of: (a) benzyl alcohol groups PS(BnOH)n; (b) perfluorooctyl groups PS(BnORf)n with n =2 and n = 10.

Figure 4. SEM surface micrographs of the chain-end functionalized polystyrenes with a definite number of benzyl alcohol group PS(BnOH)n: (a) n =2; (b) n = 10. Polímeros, 29(4), e2019046, 2019

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Mahmoud, K. R., El-Shehawy, A. & Atta, H. that roughness of the surface exists. Figure 4 shows a number of cracks and voids leading to relatively porous surface, as well as outgrowths at the junctions of the grains in the samples are apparent. However, the polymer sample with n = 10 appears in more fine structure than polymer sample with n = 2. All polymer samples have been investigated using PALS to correlate the parameters of PAL measurements with chemical structure. All PALS spectra of the investigated polymer samples showed three lifetime components (τ1, τ2 and τ3) with their respective intensities (I1, I2 and I3). The only exception was found for the sample of functionalized polymer having 10 Rf groups which are resolved into four lifetime components (τ1, τ2, τ3 and τ4) with their respective intensities (I1, I2, I3 and I4). These components can be attributed to various states of positron annihilation in the polymer.

Figure 5. Variation of the lifetime (τ2) and its intensity I2 (%) for chain-end-functionalized polymers having benzyl alcohol PS(BnOH)n and perfluorooctyl groups PS(BnORf)n with the number of functionalities. The solid lines are only drawn to guide the eyes.

In all cases the short‑lived component, τ1, is attributed to para-positronium atom (p-Ps) which is especially difficult to determine precisely since its value is very sensitive to small changes in spectrometer time resolution function. In order to reduce the scatter of the other parameters extracted from the analysis, the lifetimes were analyzed with (τ1) fixed at the theoretical vacuum para-Ps lifetime, 0.125 ns. This constraint did not increase the “variance of the fit”[25]. The intermediate lifetime component τ2 is directly related to annihilation of the positrons without forming Ps, i.e., free positron annihilation, and I2 its intensity. Figure 5 represents the variation of the lifetime component τ2 and its intensity I2 with number of end-functional groups for polymer samples of PS(BnOH)n and PS(BnORf)n. The obtained results showed that τ2 ranged from 0.378 – 0.451 and 0.348 – 0.501 ns, whereas, the range of intensity I2 is 32 – 48% and 30.1 – 44.4% for the measured samples, respectively. The reduction in τ2 and increase of I2 can be explained by an enhancement in the electron density. A great caution must be taken when interpreting short lifetimes[31]. The longest-lived component τ3 is attributed to pick‑off annihilation of o-Ps localized in the open nano-spaces in the polymer structure, which are very sensitive to the microstructural changes. In this case, the o-Ps localized in a cavity or a free volume holes in polymer annihilates via the pick-off annihilation mechanism with an electron of antiparallel spin from molecules forming the cavity wall. According to the simple quantum-mechanical of Tao and Eldrop model[25,32], the o-Ps lifetime and its intensity extracted from the lifetime spectra provide valuable information on the mean size of free volume holes where the ortho-positronium was annihilated. Figure 6 shows the lifetime τ3 and its intensity I3 which describe the annihilation parameters of the o-Ps in the measured polymer samples as a function of number of benzyl alcohol groups in PS(BnOH)n and perfluorooctyl groups in PS(BnORf)n.

Figure 6. Variation of the lifetime (τ3) and its intensity I3 (%) with increasing the number of benzyl alcohol and perfluorooctyl groups. The solid lines are only drawn to guide the eyes. 6/10

Polímeros, 29(4), e2019046, 2019


Positron annihilation spectroscopy of chain-end-functionalized polystyrenes with definite numbers of benzyl alcohol and perfluorooctyl groups The obtained results showed that τ3 is almost constant within one error bar with increasing the number of BnOH groups with a decrease in I3 at n=8 and n=10. The enhanced reduction in I3 from 25% to 12% suggests that (o-Ps) quenching processes take place either through chemical reactions in which a complex molecule including the positron may be formed. Consequently, the positron is no longer correlated with a single electron of parallel spin resulting in reducing I3.

It has been also observed that the τ3 and Vf slightly increased with increasing the number of Rf groups at the chain-ends up to n = 6, after that a steep decrease in τ3 and Vf for n > 6. The increase in τ3 and Vf can be explained according to the following features: i) The high electronegative fluorine atoms increased by increasing the number of branches in the Rf groups from 2-6 lead to the decrease of the electron density at the polymer and as a result lead to the slightly increase in τ3. ii) Further increasing the number of Rf groups (more than 6) resulting a steric hindrance which keep the free volume inside the polymer and hence a decrease in τ3 has been observed (see Figure 6). On the other hand, the variation of the average value of the relative intensity I3 with increasing the number of both BnOH and Rf groups are shown in Figure 6. The data showed that I3 increased with increasing the number of BnOH groups up to n = 6, then decreased with n > 6. Increasing of I3 may be due to the presence of hydroxyl group which is a good cation scavenger that enhances Ps formation by hole scavenging when a small amount is added to the polymer[33]. The same behavior of I3 was shown for the Rf groups, where I3 slightly increased with increasing the number up to n=4, then steeply decreased afterwards. The decrease in I3 might be explained by the decrease in o-Ps formation probability due to decrease in the size of free volumes. The calculated values of the mean free volume, Vf (Å), as a function of the number of BnOH and Rf functionalities are shown in Figure 7. The results showed that the variation of Vf with number of functional groups has the same trend as the τ3 (see Figure 6) and the same explanation can be suggested. As noted from Figure 7 that in Ps(BnORf),V1 decreases from 122.5 Ao3 to 71 Ao3 as n varies from n=6 to n=10. Also, there is exist two values of the free volume (V1 and V2) for the functionalized polymer having 10 Rf groups. This is may be due to the presence of the two o-Ps lifetime values τ3 and τ4 for this polymer sample which may be attributed to the unique molecular

architecture of the hyperbranched polymer, consisting of an interior cavity spaces with different sizes formed by loosely linked core and chain ends with a number of branches and functional groups. These results are in good agreements with that reported for hyperbranched poly(ether ketone) and hyperbranched-b-linear-b-hyperbranched poly(ether ketone) polymers[14]. Although the results from positron annihilation lifetime measurements pointed to the determination of the longer lifetime component to o-Ps, it seemed interesting to confirm this conclusion using another independent technique such as Doppler Broadening of Annihilation Radiation (DBAR). The calculated values of S– and W–parameters as a function of the number of BnOH and Rf groups in [PS(BnOH)n] and [PS(BnORf)n] from the DBAR measurements are shown in Figures 8. From Figure 8A, it has been shown that the values of S-parameter decrease and W-parameter increase with increasing the number of BnOH groups. This can be attributed to the decrease of the free volumes size as confermed by the PALS measurements (see Figures 6 and 7). As for the polymers end-functionalized with Rf groups, the S– and W–parameters increase and decrease respectively upon increase of the number of Rf groups from n=2 to n=4)  Figure  8B. This may be due to a decrease in the valence electrons and defect size and its concentration for these compounds. In addition, the values of S- and W-parameters linearly decreased and increased respectively for polymer samples with Rf groups having n>4 which can be attributed to the increase of the fluorine atoms by increasing the number of Rf groups from n = 4 ~ 10 indicating that the annihilation with low momentum valence electrons decreased and increasing the annihilation with high momentum core electrons at the vacancies of these polymer samples. In order to reveal some information about the number of defect types, the S-parameter can be plotted as a function of the W-parameter (S–W plots) for all the tested polymers (Figure 9). Krause-Rehberg and Leipner[34] pointed out that for samples with only one type of defect, the plot of S versus W dependence should be linear. A linear behavior between the S and W- parameter is shown in Figure 9. From these plots in Figure 9, one can easily notice that, the W-parameter increases as S-parameter decreases for all tested polymer samples. In addition there is a good correlation with

Figure 7. The calculated values of the mean free volume, Vf (Å), as a function of the number of benzyl alcohol and perfluorooctyl groups. The solid lines are only drawn to guide the eyes. Polímeros, 29(4), e2019046, 2019

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Mahmoud, K. R., El-Shehawy, A. & Atta, H.

Figure 8. The variation of S– and W–parameters as a function of the number of: (A) BnOH and (B) Rf groups.

Figure 9. The S versus W plot for chain-end-functionalized polymers with different number of functional groups: (A): for BnOH groups; (B): for Rf groups.

r2 = 0.943 and 0.928 between S-parameter and W-parameter for polymers end-functionalized with BnOH and Rf groups, respectively. These observations suggest that only one type of defect is present in these samples.

4. Conclusions A series of chain-end-functionalized polystyrenes with a definite number of benzyl alcohol PS(BnOH)n and perfluorooctyl groups PS(BnORf)n were investigated using XRD, SEM, PALS and DBAR spectroscopy techniques. XRD studies showed that the polymers end-functionalized with a definite number of BnOH groups are crystalline and the degree of crystallinity increases by with increasing the number of BnOH functionalities until the polymeric material become highly crystalline by the effect of interlinking between polymer chain-ends due the possible hydrogen bonding. While, XRD pattern of polymers end-functionalized with Rf groups revealed that by adding branches incorporating Rf groups some fractions of polystyrene chains were intercalated or broken between the interlayer spacing. The surface morphology of PS(BnOH)n investigated by SEM showed that several cracks and voids leading to relatively porous 8/10

surface, together with outgrowths at the junctions of the grains in the samples are apparent and the roughness of the surface exists but looks more fine structure by adding branches. Positron annihilation lifetime measurements show that formation of free volume vacancies takes place in all the investigated samples. For instance, In Ps(BnORf),V1 decreases from 122.5 Ao3 to 71 Ao3 as n varies from n=6 to n=10. The results of Doppler broadening of annihilation radiation suggested that only one type of defect is present in these polymer samples.

5. Reference 1. Paleos, C. M., Tsiourvas, D., Sideratou, Z., & Tziveleka, L.-A. (2010). Drug delivery using multifunctional dendrimers and hyperbranched polymers. Expert Opinion on Drug Delivery, 7(12), 1387-1398. http://dx.doi.org/10.1517/17425247.2010 .534981. PMid:21080860. 2. Yates, C. R., & Hayes, W. (2004). Synthesis and applications of hyperbranched polymers. European Polymer Journal, 40(7), 1257-1281. http://dx.doi.org/10.1016/j.eurpolymj.2004.02.007. 3. Jin, H., Huang, W., Zhu, X., Zhou, Y., & Yan, D. (2012). Biocompatible or biodegradable hyperbranched polymers: from self-assembly to cytomimetic applications. Chemical Polímeros, 29(4), e2019046, 2019


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properties studies for epoxy matrices reinforced with different nanoparticles. Journal of Polymer Research, 23(9), 181-192. http://dx.doi.org/10.1007/s10965-016-1074-6. 18. Mahmoud, K. R., Khodair, A. I., & Shaban, S. Y. (2015). Positron annihilation lifetime studies of changes in free volume on some biorelevant nitrogen heterocyclic compounds and their S-glycosylation. Applied Radiation and Isotopes, 105, 303-307. http://dx.doi.org/10.1016/j.apradiso.2015.07.002. PMid:26272166. 19. Shaban, S. Y., Mahmoud, K. R., & Sharshar, T. (2013). Positron annihilation studies of bio-related N2S2-tetradentate ligands and their zinc complexes. Radiation Physics and Chemistry, 82, 12-15. http://dx.doi.org/10.1016/j.radphyschem.2012.09.001. 20. Mahmoud, K. R., Refat, M. S., Sharshar, T., Adam, A. M. A., & Manaaa, E.-S. A. (2016). Synthesis of amino acid iodine charge transfer complexes in situ methanolic medium: chemical and physical investigations. Journal of Molecular Liquids, 222, 1061-1067. http://dx.doi.org/10.1016/j.molliq.2016.07.138. 21. Ismail, A. M., Mahmoud, K. R., & Abd-El Salam, M. H. (2015). Electrical conductivity and positron annihilation characteristics of ternary silicone rubber/carbon black/TiB nanocomposites. Polymer Testing, 48, 37-43. http://dx.doi. org/10.1016/j.polymertesting.2015.09.006. 22. Hooz, J., & Gilani, S. S. H. (1968). A rapid, mild procedure for the preparation of alkyl chlorides and bromides. Canadian Journal of Chemistry, 46(1), 86-87. http://dx.doi.org/10.1139/ v68-017. 23. Mahmoud, K. R., Al-Sigeny, S., Sharshar, T., & El-Hamshary, H. (2006). Positron annihilation study on free volume of amino acid modified, starch-grafted acrylamide copolymer. Radiation Physics and Chemistry, 75(5), 590-595. http://dx.doi. org/10.1016/j.radphyschem.2005.12.037. 24. Kansy, J. (1996). Microcomputer program for analysis of positron annihilation lifetime spectra. Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, 374(2), 235-244. http://dx.doi.org/10.1016/0168-9002(96)00075-7. 25. Eldrup, M., Lightbody, D., & Sherwood, J. N. (1981). The temperature dependence of positron lifetimes in solid pivalic acid. Chemical Physics, 63(1-2), 51-58. http://dx.doi. org/10.1016/0301-0104(81)80307-2. 26. McGonigle, E. A., Liggat, J. J., Pethrick, R. A., Jenkins, S. D., Daly, J. H., & Hayward, D. (2001). Permeability of N2, Ar, He, O2 and CO2 through biaxially oriented polyester films - Dependence on free volume. Polymer, 42(6), 2413-2426. http://dx.doi.org/10.1016/S0032-3861(00)00615-7. 27. Porto, A. O., Silva, G. G., & Magalha, W. F. (1999). Free volumesize dependence on temperature and average molecular-weight in poly(ethylene oxide) determined by positron annihilation lifetime spectroscopy. Journal of Polymer Science. Part B, Polymer Physics, 37, 219-226. http://dx.doi.org/10.1002/ (SICI)1099-0488(19990201)37:3<219::AID-POLB5>3.0.CO;2-I. 28. Jerzy Dryzek. (2019). Retrieved in 2019, October 18, from https://www.ifj.edu.pl/~mdryzek 29. El-Shehawy, A. A., Yokoyama, H., Sugiyama, K., & Hirao, A. (2005). Precise synthesis of novel chain-end-functionalized polystyrenes with a definite number of perfluorooctyl groups and their surface characterization. Macromolecules, 38(20), 8285-8299. http://dx.doi.org/10.1021/ma050457z. 30. Paul, P. K., Hussain, S. A., Bhattacharjee, D., & Pal, M. (2013). Preparation of polystyrene–clay nanocomposite by solution intercalation technique. Bulletin of Materials Science, 36(3), 361-366. http://dx.doi.org/10.1007/s12034-013-0498-4. 31. Wästlund, C., Eldrup, M., & Maurer, F. H. J. (1998). Interlaboratory comparison of positron and positronium lifetimes in polymers. Nuclear Instruments & Methods in Physics Research. Section 9/10


Mahmoud, K. R., El-Shehawy, A. & Atta, H. B, Beam Interactions with Materials and Atoms, 143(4), 575583. http://dx.doi.org/10.1016/S0168-583X(98)00400-5. 32. Tao, S. J. (1972). Positronium annihilation in molecular substances. The Journal of Chemical Physics, 56(11), 54995510. http://dx.doi.org/10.1063/1.1677067. 33. Ito, Y. (1988). Vacancy Spectroscopy of polymers using positronium. In D. M. Schrader & Y. C. Jean (Eds.), Positron and positronium chemistry (pp. 334-354). Elsevier Science: Amsterdam. https://doi.org/10.1021/bk-1998-0710.ch023.

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34. Krause-Rehberg, R., & Leipner, H. S. (1999). Positron annihilation in semiconductors: Defect Studies (Springer Series in Solid-State Sciences). Berlin: Springer-Verlag. http://dx.doi. org/10.1007/978-3-662-03893-2. Received: July 27, 2019 Revised: Oct. 18, 2019 Accepted: Oct. 22, 2019

PolĂ­meros, 29(4), e2019046, 2019


ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.06619

Evaluation of commercial arrowroot starch/CMC film for buccal drug delivery of glipizide Dhanasekaran Gayathri1 and Lakshmanan Saraswathy Jayakumari1*  1

Department of Rubber & Plastics Technology, Anna University, Madras Institute of Technology Campus, Chennai, Tamil Nadu, India *lsjayakumarimit@gmail.com

Abstract In the present work, commercial arrowroot starch (AR starch) has been successfully used as a base material with sodium salt of carboxy methyl cellulose (CMC) for buccal drug delivery system. Different ratio of CMC and AR starch has been prepared with constant ratio of drug. In our study, glipizide has been used as the drug for controlled drug delivery through buccal mucosa. Films were cast by solution casting method with glycerol as plasticizer. All the films were characterized for thickness, swelling index, moisture content, relative pH, drug content uniformity, compatibility of polymer and drug, surface morphology of films, muco adhesive property and in-vitro drug release study. Formulation F3 shows a residence time of 140 minutes with good mucoadhesive property, compatibility within polymers, drug content uniformity of 100 ± 6.0% and a controlled drug release among all the ratios. Keywords: arrowroot starch, buccal drug delivery, CMC, film, glipizide. How to cite: Gayathri, D., & Jayakumari, L. S. (2019). Evaluation of commercial arrowroot starch/CMC film for buccal drug delivery of glipizide. Polímeros: Ciência e Tecnologia, 29(4), e2019047. https://doi.org/10.1590/0104-1428.06619

1. Indroduction The development of buccal drug delivery has been focused in recent years[1,2] due to the advantages like controlled drug delivery, compatibility, rapid termination, flexibility, comfort, ease in accessibility, most convenient, easy and painless administration[3,4,5]. First pass hepatic metabolism is avoided in buccal drug delivery system as it access directly into the systemic circulation through jugular vein which makes more bioavailability than gastrointestinal route of drug release[6]. Mucoadhesive polymers are widely used as the base material to carry the drug and adhere to the mucosal surface of the buccal cavity[7]. Mucoadhesion is a process in which mucous membrane and the polymer get adhered[8]. In buccal drug delivery system, drugs can be administered in many forms such as ointments, patches, tablets, gels and films, but films are widely used because of its long residence time, flexibility, thinness, comfort and patient’s compliance[9]. Selection of suitable material is essential for the development of different drug dosage forms[10]. Many polymers such as polycarbophil, carbopol, hydroxy propyl methyl cellulose, hydroxy ethyl cellulose, alginate, sodium carboxymethyl cellulose, polyvinyl pyrrolidone and hydroxy propyl cellulose are widely used due to their mucoadhesion property, compatibility with drug, flexibility and film forming capacity[11-16]. The mechanism behind the interaction of mucus and mucoadhesive polymers results in physical entanglement, hydrogen bonding or Van-der-Waals attraction. The adhesion property mainly depends on the chemical groups present at the surface of the polymers such as hydroxyls, amines, amides and carboxyls[17-19] Cellulose

Polímeros, 29(4), e2019047, 2019

derivatives are considered to be a promising material in terms of mucoadhesion property and hydrogen bonding ability with mucous membrane. It has been reported that carboxymethylcelullose has good mucoadhesive property than other cellulose derivatives. Since CMC is an anionic polymer it has the ability to form hydrogen bonding but other derivatives like hydroxypropylmethylcellulose, which is non-ionic neutral derivative has a moderate mucoadhesive property due to the absence of proton donating carboxyl groups which facilitates the formation of hydrogen bonds[20,21]. Starch is a natural biopolymer, which is widely available and low cost[22]. In drug delivery and tissue engineering starch have been widely used[23]. Starch films are usually brittle with more tackiness. Starch can be used as oral films when it is suitably formulated with mucoadhesive polymers like CMC, HPMC, chitosan[24,25]. In the case of starch, amylose and amylopectin content are crucial for its mechanical and thermal properties. Higher the amylose content, more strong the film will be. Amylopectin contributes to the flexibility of the film. Generally all kind of starches have about 25% of amylose and 75% of amylopectin content. Strength of the film, processability, ability to resist water and thermal stability depends upon the molecular weight. When starch and CMC is blended it leads to improved mechanical properties in terms of increased tensile strength and decreased elongation at break[26,27]. CMC addition in starch increases the film forming properties of starch[28] and blending of starch and CMC results in clearer film without hazy appearance[29]. CMC/starch forms a biodegradable

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O O O O O O O O O O O O O O O O


Gayathri, D., & Jayakumari, L. S. film suitable for many applications such as edible film and coating, packaging and in some medical applications[26]. Glipizide is a second generation sulphonyl urea based drug which stimulates the β cells of pancreas to release insulin[30]. Glipizide is considered to be an antidiabetic drug with a biological half-life of 3 - 4 h, such a rapid absorbing drug will also have fast elimination rate[31,32]. Although glipizide is a widely consumed antidiabetic drug, it leads to gastrointestinal disturbance on chronic usage[33]. Therefore it is necessary to formulate a material which entraps glipizide and facilitates controlled and sustained drug release without affecting gastrointestinal track. In this present study, commercially available Indian arrowroot starch, which is low cost, has been effectively used as a matrix material for buccal drug delivery system. Glipizide, an anti-diabetic drug is successfully incorporated in the film with carboxymethylcellulose and Indian arrowroot starch to evaluate the basic film property, drug delivery efficacy, stability of the film and mucoadhesion property for buccal film, in vitro residence time and in vitro drug release.

2. Experimental

10 minutes after adding 0.5g of glycerol as the plasticizer. The above mixture was cast by solution casting method in a petri dish dried at 40 °C overnight in hot air oven. Different formulation of polymer and drug is given in the Table 1.

2.4 Film thickness and weight measurement The films of blend and the plain CMC and starch were cut into 2cm × 2cm dimension and each formulation was weighed accurately with three specimens in each sample with the digital balance. The film thickness was measured using the (model Baker 10mm) dial thickness gauge K130/3, at minimum five different points[9].

2.5 Surface pH of films Surface pH of the film was determined by preparing a film specimen of a dimension 2cm × 2cm and kept in the petri dish by wetting the film with 0.5ml of water and allowed to swell for 2 hours at room temperature, surface pH of the film was determined using pH paper. Acid or alkaline pH of the film may cause irritation to the mucosa therefore it is necessary to maintain the pH of the film around 7[35].

2.6 Swelling index

2.1 Materials Sodium salt of carboxymethylcellulose with medium viscosity was purchased from Sigma Aldrich, Arrowroot starch (curcuma angustifolia) was purchased from local market in Chennai (harvested between March-April 2017) and glycerol was purchased from Fischer Scientific. Double distilled water was used throughout the process. All the chemicals were used as purchased.

2.2 Amylose and amylopectin content of starch Amylose and amylopectin content in the arrowroot starch sample was determined using standard iodine calorimetric method according to ISO 6647-2[34]. Percentage of amylose and amylopectin content was found to be 46.8% and 53.2% respectively.

2.3 Preparation of drug loaded CMC/AR starch film Starch and CMC blends were formed with different weight ratios such as 100:0, 10:90, 30:70, 50:50, 70:30, 90:10 and 0:100 wt%. Starch was gelatinized with 30g of DI water at 90 °C for 15 minutes using magnetic stirrer with 300 rpm. CMC was dissolved in 100g of water and made to stir for 2 hours to form a homogeneous solution. Gelatinized starch solution was cooled to room temperature and was mixed with CMC solution and stirred for another

The swelling properties of blended films were measured by percentage swelling index. The film was cut into 2cm × 2cm dimension and its weight was measured accurately and denoted as W1. Then the film was placed in the petri dish containing 5 ml of phosphate buffer solution with a pH of 6.8. The swollen film was removed from the petri dish carefully and was wiped with filter paper to remove the excess solvent from the film[1]. The weight of the film was weighed and denoted as W2 . Swelling index was calculated by using the following Equation 1: %Swelling =

W 2 −W 1 x100 W1

(1)

Where, W1 = initial weight of the film. W2 = weight of the swelled film.

2.7 Folding endurance Folding endurance of the film was measured quantitatively by repeatedly folding a small strip with a dimension of 4cm × 1cm at 180° plane at the same place until it break or folded 300 times without breaking. The number of times film was folded without breaking is calculated and considered to be the measure of folding endurance[5].

Table 1. Formulation of AR starch/CMC blend system. Ratio 100:0 90:10 70:30 50:50 30:70 10:90 0:100

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CMC 4.00g 3.60g 2.80g 2.00g 1.20g 0.40g 0.00g

Starch 0.00g 0.40g 1.20g 2.00g 2.80g 3.60g 4.00g

Plasticizer 0.5g 0.5g 0.5g 0.5g 0.5g 0.5g 0.5g

Drug 0.5g 0.5g 0.5g 0.5g 0.5g 0.5g 0.5g

Formulation PC F1 F2 F3 F4 F5 PS

Polímeros, 29(4), e2019047, 2019


Evaluation of commercial arrowroot starch/CMC film for buccal drug delivery of glipizide 2.8 Moisture content The amount of moisture present in the film samples can be determined by simple weight measurement. 2cm × 2cm dimension of each samples were cut and weighed as the initial weight (W1). Then the samples were heated to 100 °C for nearly 2 hours to attain constant weight and it is considered as the final weight (W2) of the sample. The percentage weight difference between W1 and W2 are the moisture content present in the film[36].

2.9 Drug content uniformity The drug content uniformity was measured by taking three specimens from each sample with a dimension of 2cm × 2cm and dissolved in 100mL of phosphate buffer solution with a pH of 6.8 and was continuously stirred for 6 – 8 hours. The solution was filtered and diluted suitably and the drug content was measured in Varian Cary 50 UV spectrophotometer at an absorbance of 276nm[37]. Drug content uniformity is measured by the following equationDrug Content uniformity = experimental drug loading/ theoretical drug loading × 100.

2.10 Drug – polymer compatibility study Drug and polymer interaction can be determined by using ATR-FTIR. The different formulation of blended film with drug content was analysed with the help of Attenuated total reflectance Fourier transform infrared spectroscopy of the model Bruker tensor 27 FTIR spectrometer (Bruker, Billerica, MA, USA) at a resolution of 4cm-1 over the wave number region of 4000 – 400cm-1 [15].

2.11 Mucoadhesive property Mucoadhesion of polymer is important in buccal drug delivery system because it increases the residence time of the drug in the buccal cavity[38]. The buccal mucosa of goat was taken in phosphate buffer solution of pH 6.8 and the polymer film was adhered on to the surface of mucosa, buccal mucosa and the film was taped in a glass substrate within the beaker containing phosphate buffer and allowed to stir at an RPM of 200 at 37 °C using magnetic stirrer. Time taken by the film to detach from the mucosa is considered as the residence time and how long it attached to the mucosa gives the mucoadhesion property of the film[36].

2.12 Surface morphology studies Surface morphology and the presence of drug in the polymer film were studied by scanning electron microscopy using the model Hitachi S3400-N (Tokyo, Japan). All the films were gold coated using sputter coater. Films were analysed at an accelerating voltage of 20.0 kV and at a resolution of 400μm.

2.13 In vitro drug release study In vitro drug release study was performed by adhering the polymer film in the side walls of the beaker which contains phosphate buffer solution with a pH of 6.8, at a temperature of 37 °C. Assume that the drug releases from the film from one side only. Beaker was stirred gently in a magnetic stirrer and the buffer solution was sampled periodically in Polímeros, 29(4), e2019047, 2019

Varian Cary 50 UV spectrophotometer at 276nm to find the concentration of drug present in the sample. Solution withdrawn from the beaker was then replaced with the exact volume at each sampling[39].

3. Results and Discussion 3.1 Film thickness and weight measurement Uniformity in film thickness is necessary because it is directly related to drug concentration and mucoadhesive property of the polymer. Plasticizer used in the formulation will affect the thickness of the film and sometimes even the polymer itself will increase the thickness of the film (polyvinylpyrrolidone). An ideal film for buccal drug delivery system should have an optimum thickness of around 0.05 to 1.0 mm[36,40]. Film thickness was measured at minimum five different points and mean standard deviation values has been calculated and is presented in Table.2. All films have a random thickness in the range of 0.22 – 0.32 mm irrespective of the ratio of polymers present in the film; it shows that polymer does not influence much in the thickness. PC, formulation measured a minimum thickness of 0.22mm and F1 shows a maximum thickness of 0.32mm. Weight measurements are generally done to ensure the uniform drug loading in the sample. Weight variations should not be larger as it renders inconsistency of drug loading[41]. Weight of the film is measured using digital balance and mentioned in the Table 2. Weight of the film also varies according to the moisture content present in the samples. Formulation F2 and F3 has a maximum film weight of 5.32 and 5.30g respectively, this may be due to the presence of more moisture content in the film such as 24.6 ± 1.25% and 23.1± 0.72% for F2 and F3 respectively.

3.2 Swelling ratio Swelling characteristic is one of the important factors which affect the mucoadhesive property of the polymers. The optimum amount of water uptake by the polymer film is essential in exhibiting mucoadhesive property[7]. Generally hydrophilic polymers will absorb more water and undergo dissolution by making pores, formation of such pores facilitates the diffusion of drug from the film[42]. Swelling is affected by structure of the polymer, temperature, swelling medium, ionic strength, pH of the environment and cross linking of network[43]. Polymers which have the ability to form hydrogen bonds can form a strong network structure, therefore penetration of water becomes difficult, and such hydrogen bond can be between polymer - polymer, polymer - plasticizer and polymer- drug[44,45]. In hydrophilic polymers like CMC swelling is mainly due to hydrogen bond formation between water and the polymer chain. Starch contains readily available hydroxyl groups which attributes to the interaction of water and polymer chain for substantial hydrophilicity. Glycerol will also interact with polymer and water molecules, giving rise to hydrogen bonds and increase in swelling percentage[46]. In the present study constant amount of plasticizer and drug were used in all systems so, the interaction between drug - water and plasticizer - water does not influence much in swelling. PC formulation contain CMC, plasticizer and 3/9


Gayathri, D., & Jayakumari, L. S. Table 2. Physical properties of AR starch/CMC blend system. Formulation PC F1 F2 F3 F4 F5 PS

Thickness

Swelling

Moisture

(mm)

(%)

(g)

0.22± 0.01 0.32± 0.02 0.23± 0.01 0.24± 0.01 0.28± 0.05 0.27± 0.03 0.25± 0.51

dissolved 487 ± 1.7 455 ± 1.7 433 ± 1.0 418 ± 2.3 408 ± 2.4 355 ± 2.8

2.90± 0.05 3.90± 0.05 24.6± 1.25 23.1± 0.72 15.0± 0.58 12.1± 0.44 11.1± 0.72

drug, it swells faster and dissolve completely in the buffer solution of pH 6.8 because the polymer interacts more with water than drug and plasticizer. In PS formulation (355 ± 2.8), which has AR starch, drug and plasticizer, has a considerable swelling percentage due to the interaction of AR starch with plasticizer and drug, therefore starch is not able to interact more with water to form hydrogen bond and does not dissolve so it swells in the presence of water. On increasing the percentage of AR starch in CMC, there is a gradual increase in swelling percentage in the formulations F1(487 ± 1.7), F2 (455 ± 1.7) and F3 (433 ± 1.0), this may be due to the synergistic interaction of CMC and AR starch with water in the film. In F4 (418 ± 2.3) and F5 (408 ± 2.4) swelling percentage tend to decrease as the less interacting AR starch has increased more (Table 2).

3.3 pH of the film pH of the film is crucial in drug delivery system because the film should be compatible with the pH of the mucus surface which involved in absorbing the drug. pH is one of the factor which affects the mucoadhesion property of the polymer and therefore, optimum pH is necessary for required mucoadhesion[11,18]. pH of all the films are found to be in the range of saliva pH (6.5 - 6.8) and therefore it does not cause irritation at the place of administration[47]. pH of the film samples are given in the table.2.

3.4 Drug content uniformity Drug content uniformity is important because it ensures that the drug is uniformly distributed throughout the film. The limit of drug content uniformity is likely to be between 85 – 115%[48]. Drug content for all the samples was found to be between 100 to 105(table.2).

3.5 Moisture content Moisture content is essentially important as it helps in initial adhesion to the mucosa membrane. Optimum amount of moisture is needed to adhere the film to the mucosa membrane and excess moisture leads to tackiness in the film which leads to poor aesthetic appearance and difficulty in handling. CMC has more crystalline region than amorphous region in its molecular alignment. Therefore, it absorbs less moisture from the environment which appears in Formulation PC (CMC with drug) with least moisture content of 2.9 ± 0.05[16]. AR starch after gelatinization becomes more amorphous and has porous 4/9

Surface pH 6.9 ± 0.05 6.4 ± 0.05 6.6 ± 0.08 6.7 ± 0.03 6.8 ± 0.10 6.6 ± 0.05 6.9 ± 0.05

Film weight (g) 4.86± 0.05 4.93± 0.06 5.31± 0.05 5.29± 0.03 5.09± 0.03 5.07± 0.05 4.97± 0.05

Drug content

Folding

Uniformity

Endurance

(%) 102±1.5% 104± 2.6% 102± 5.5% 100± 6.0% 102± 5.2% 100±10.2% 104± 7.3%

(No) 289 ± 1.4 302 ± 1.4 315 ± 2.3 294 ± 2.9 274 ± 3.0 267 ± 1.4 252 ± 1.4

structure and absorbs more moisture compared to CMC which is exhibited in the formulation PS (AR starch with drug) with 11.1 ± 0.72 of moisture content[49]. Formulation F1 (3.9± 0.05), F2 (24.6 ± 1.25) and F3 (23.1± 0.72) shows a synergistic effect with the increased percentage of moisture with the gradual increment in the percentage of AR starch. Percentage of moisture present in the film samples was given in the Table 2.

3.6 Folding endurance Folding endurance value is expressed in terms of number of times the film is folded without breaking. Folding endurance value indirectly gives the mechanical property of the film, higher the folding endurance value, higher is the flexibility of the film[50]. The synergistic effect of CMC and AR starch shows an increase in the flexibility of the film in the Formulation F1 and F2 with an increased folding endurance value of 302 and 315 respectively.

3.7 Drug – polymer compatibility study Compatibility between polymer and the drug is studied through FTIR. The FTIR spectrum of pure glipizide drug is shown in the Figure 1A. A vibrational stretch at 3340cm-1 is attributed to the N-H bond present in the drug; similarly 2918cm-1, 1688cm-1, 1651cm-1 and 1529 cm-1 are corresponding to C-H stretching vibration, C=O, C=N and N-H bending vibrations respectively[51] are observed. In Figure 1B, spectra of AR starch, CMC, and AR starch/CMC with drug are shown. AR starch film shows a stretching vibration at 3283cm-1 due to the presence of hydroxyl group in AR starch as well as plasticizer (glycerol), 2928cm-1 attributes to the presence of C-H in the system. Peaks at 1015cm-1 and 854cm-1 are corresponding to the C-O-C stretching and bending vibrations present in AR starch respectively[52]. CMC film shows a stretching vibration of hydroxyl group at 3276cm-1 and presence of C-H bond shows a characteristic peak at 2923cm-1. Vibrational stretching present at 1589cm-1 attributes to the COO- (carboxyl) group. A vibrational stretch at 1029cm-1 attributes to the presence of C-O-C group in the film[53]. The blend film (starch-CMC with drug) shows no new characteristic peak in the spectrum, which means that CMC, AR starch and glipizide drug are compatible without forming bonds between them. Blend film shows a broad peak between 3800 and 3000cm-1 due to the presence of hydroxyl group in AR starch, CMC, plasticizer and the stretching vibration of Polímeros, 29(4), e2019047, 2019


Evaluation of commercial arrowroot starch/CMC film for buccal drug delivery of glipizide

Figure 1. FTIR spectrum of glipizide (A), FTIR of starch, CMC and Starch-CMC and drug (B).

Figure 2. SEM image of different formulations F1(A), F2(B), F3(C), F4(D), F5(E).

N-H group present in glipizide drug also overlaps to obtain a broad peak. Peak at 2921cm-1 attributes to the C-H present in AR starch, CMC and the drug. Vibrational stretching at 1621cm-1 is corresponding to the overlap of C=N present in the drug and COO- group present in CMC. C-O-C stretching vibration in the spectrum is shown at 1015cm-1 due to the presence of AR starch and CMC. All peaks appear in the blend of AR starch – CMC with glipizide drug with minor shift in the corresponding peak values.

3.8 Scanning electron microscopy Scanning electron microscopy is a useful tool to analyse the surface morphology and compatibility of the blend system. It is also useful in studying the dispersion of drug present Polímeros, 29(4), e2019047, 2019

in the blend composition. Formulation of F1(Figure 2A) and F5(Figure  2E) shows a brittle morphology in the image attributing to the lack of plasticizer and insufficient amount of AR and CMC in F1 and F5 respectively, but the presence of drug was found to be homogenous in both the images. Figure 2B belongs to formulation F2, showing some agglomeration of drug in the surface but without any appearance of crack which may be due to the optimum percentage of plasticizer and adequate proportion of CMC and AR starch in the blend. Figure 2D also shows some agglomeration in the surface which belongs to F4 formulation. Figure 2C has optimum ratio of plasticizer, blend proportion and dispersion of drug is also homogenous which can be shown as smooth surface 5/9


Gayathri, D., & Jayakumari, L. S.

Figure 3. SEM image of Plain CMC with drug (A) and Plain AR starch with drug (B).

respectively because F4 and F5 has less proportion of CMC so the mucoadhesion property was low in those formulations. PS (AR starch with drug) has a residence time of less than 15 minutes, due to least mucoadhesive property of AR starch. F3 was found to have good mucoadhesive property and its residence time was 140 ± 3 minutes without dissolving in the buffer medium which attributes that F3 has an optimum composition of CMC and AR starch (50:50) as a mucoadhesive film.

3.10 In vitro drug release study

Figure 4. In-vitro drug release profile.

in the image, therefore F3 is considered as the optimum composition of polymer as well as the drug. Formulation PC and PS also shows some agglomeration of drug in the system which is shown in the Figure 3A and B.

3.9 Mucoadhesive property Residence time of a film and mucoadhesive property is directly related to each other. In-vitro residence time of film in buccal mucosa of goat was examined and determined experimentally in minutes. F3 was found to have longest residence time of 140 ± 3 minutes and F5 has a least residence time of 30 ± 2 minutes. All the blend systems found to have mucoadhesive property with the mucosa membrane but the residence time differs between them according to the wt% of polymer present in the system. CMC was reported to be a mucoadhesive polymer by many research groups[12,54,55] therefore blend formulation with high proportion of CMC results in good mucoadhesive property. F1 and F2 initially swell faster when adhered to the surface of mucosa and dissolved within 80 ± 3 and 90 ± 3 minutes respectively. This may be due to the increased wt% of hydrophilic CMC present in the system. At the same time F4 and F5 loosen the bond strength from the mucosa membrane within 40 ± 2 and 30 ± 2 minutes 6/9

In-vitro drug release study was performed for 4 hours. Figure 4 demonstrates the drug release profile of different formulations. CMC has more affinity towards water than drug therefore it dissolves soon and releases the drug rapidly. This is also attributed in the swelling percentage of PC(dissolve). Formulation F1 (90:10) and F2 (70:30) shows a random release of drug which may be due to the presence of higher CMC ratio and non-uniform distribution of drug in the film and it is also confirmed by SEM image showing agglomeration and non-uniformity in the surface. PS has good interaction with CMC, drug, plasticizer, which helps in entrapping drug for sustained release. But, drug release percentage was unable to study for PS, as it adhered to the side walls of the beaker due to least mucoadhesive property. All blend system shows mucoadhesive property and controlled drug release due to the synergistic effect of PC and PS. From the graph it implies that F3 (50:50) shows a sustained drug release at each hour compared to that of all formulations. So, F3 can be considered as an optimum formulation of the blend system with respect to drug release profile and SEM image also supports by homogenous, uniform and smooth surface (Figure 2C). F4 and F5 exhibit a random release of drug similar to F1 and F2 showing that the drug is non-uniformly distributed in the film.

4. Conclusion In the present work, drug release through gastrointestinal route has been avoided and buccal route has been suggested because glipizide leads to gastrointestinal disturbance. In order to increase the residence time without losing the Polímeros, 29(4), e2019047, 2019


Evaluation of commercial arrowroot starch/CMC film for buccal drug delivery of glipizide mucoadhesive property of CMC, AR starch is successfully blended with the constant ratio of antidiabetic drug, glipizide. Drug content uniformity of the films was found to be between 100 to 105%. FTIR shows the compatibility between drug and the polymer materials. The smooth surface of the films by SEM also supports the compatibility of polymer and drug. Formulation F3 (50:50) was found to have suitable swelling ratio, smooth surface morphology, good muco- adhesive property and sustained in-vitro drug release profile. Therefore, the blend of commercially available, low cost Indian arrowroot starch and CMC can be used as buccal film for drug delivery system with suitable drug.

5. Acknowledgements This Research work has been fully supported by (Anna Centenary Research fellowship) Anna University, Chennai. Authors also wish to thank the DST- FIST for the fund provided for the improvement of infrastructure in the Department.

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tramadol for effective pain management. Revista Brasileira de Anestesiologia, 67(3), 231-237. http://dx.doi.org/10.1016/j. bjan.2016.10.006. PMid:27899200. 38. Davidovich-Pinhas, M., & Bianco-Peled, H. (2010). Mucoadhesion: a review of characterization techniques. Expert Opinion on Drug Delivery, 7(2), 259-271. http://dx.doi. org/10.1517/17425240903473134. PMid:20095946. 39. Hunt, J. A., Joshi, H. N., Stella, V. J., & Topp, E. M. (1990). Diffusion and drug release in polymer films prepared from ester derivatives of hyaluronic acid. Journal of Controlled Release, 12(2), 159-169. http://dx.doi.org/10.1016/0168-3659(90)90092-8. 40. Cao, N., Yang, X., & Fu, Y. (2009). Effects of various plasticizers on mechanical and water vapor barrier properties of gelatin films. Food Hydrocolloids, 23(3), 729-735. http://dx.doi. org/10.1016/j.foodhyd.2008.07.017. 41. Karki S., Kim H., Na S.J., Shin D., Jo K., Lee J. (2016). Thin films as an emerging platform for drug delivery. Asian Journal of Pharmaceutical Sciences, 11(5), 559-574. https:// doi.org/10.1016/j.ajps.2016.05.004. 42. Shidhaye, S. S., Saindane, N. S., Sutar, S., & Kadam, V. (2008). Mucoadhesive bilayered patches for administration of sumatriptan succinate. AAPS PharmSciTech, 9(3), 909-916. http://dx.doi.org/10.1208/s12249-008-9125-x. PMid:18679806. 43. Peppas, N. A., Bures, P., Leobandung, W., & Ichikawa, H. (2000). Hydrogels in pharmaceutical formulations. European Journal of Pharmaceutics and Biopharmaceutics, 50(1), 27-46. http://dx.doi. org/10.1016/S0939-6411(00)00090-4. PMid:10840191. 44. Baranowski, P., Karolewicz, B., Gajda, M., & Pluta, J. (2014). Ophthalmic drug dosage forms: characterisation and research methods. TheScientificWorldJournal, 2014, 1-14. http://dx.doi. org/10.1155/2014/861904. PMid:24772038. 45. Panomsuk, S. P., Hatanaka, T., Aiba, T., Katayama, K., & Koizumi, T. (1996). A study of the hydrophilic cellulose matrix: effect of drugs on swelling properties. Chemical & Pharmaceutical Bulletin, 44(5), 1039-1042. http://dx.doi. org/10.1248/cpb.44.1039. 46. Mali, S., Sakanaka, L. S., Yamashita, F., & Grossmann, M. V. (2005). Water sorption and mechanical properties of cassava starch films and their relation to plasticizing effect. Carbohydrate Polymers, 60(3), 283-289. http://dx.doi. org/10.1016/j.carbpol.2005.01.003. 47. Reddy, J. R., Muzib, Y. I., & Chowdary, K. P. (2013). Development and in-vivo characterization of novel trans buccal formulations of Amiloride hydrochloride. Journal of Pharmacy Research, 6(6), 647-652. http://dx.doi.org/10.1016/j.jopr.2013.04.051. 48. Bhyan, B., Jangra, S., Kaur, M., & Singh, H. (2011). Orally fast dissolving films: innovations in formulation and technology. International Journal of Pharmaceutical Sciences Review and Research, 9(2), 50-57. 49. Duan, X., Han, Y., Li, Y., & Chen, Y. (2014). Improved capacity retention of low cost sulfur cathodes enabled by a novel starch binder derived from food. RSC Advances, 4(105), 60995-61000. http://dx.doi.org/10.1039/C4RA10953H. 50. Irfan, M., Rabel, S., Bukhtar, Q., Qadir, M. I., Jabeen, F., & Khan, A. (2016). Orally disintegrating films: a modern expansion in drug delivery system. Saudi Pharmaceutical Journal, 24(5), 537-546. http://dx.doi.org/10.1016/j.jsps.2015.02.024. PMid:27752225. 51. Joshi A. S., Patil C. C., Shiralashetti S. S., Kalyane N. V. (2013). Design, characterization and evaluation of Eudragit microspheres containing glipizide. Drug Invention Today, 5(3), 229-234. https://doi.org/10.1016/j.dit.2013.06.009. 52. Sandoval Gordillo, C. A., Valencia, G. A., Vargas Zapata, R. A., & Agudelo Henao, A. C. (2014). Physicochemical characterization of arrowroot starch (Maranta arundinacea linn) and glycerol/arrowroot Polímeros, 29(4), e2019047, 2019


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55. Jimenez-Castellanos, M. R., Zia, H., & Rhodes, C. T. (1993). Assessment of an in vitro method for measuring the bioadhesiveness of tablets. International Journal of Pharmaceutics, 89(3), 223-228. http://dx.doi.org/10.1016/03785173(93)90247-D.

Received: Sept. 20, 2019 Revised: Oct. 30, 2019 Accepted: Nov. 25, 2019

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ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.06518

Effects of polyether siloxane surfactant on the hydrophilic capacity of polypropylene films Lucas Fiamenghi Antunes1* , Douglas Alexandre Simon1, Rudinei Fiorio1 and Edson Francisquetti1 Programa de Pós-graduação em Tecnologia e Engenharia de Materiais – PPGTEM, Instituto Federal do Rio Grande do Sul – IFRS, Farroupilha, RS, Brasil

1

*lucarque@yahoo.com.br

Abstract To evaluate the hydrophilic capacity, polypropylene and surfactant (polyether siloxane) samples were extruded in the proportions of 0.0, 0.5, 1.0 and 3.0 (wt%) and films were obtained in a heated press. The samples were submitted to measurements of contact angle, surface tension, melt flow index and surface roughness. The results indicated that increasing surfactant content promoted better wettability and consequently higher hydrophilicity. Using water, the increase in the surfactant content reduced the contact angle (92.58° to 68.10°) and increased the surface tension (26.7 to 56.9 mN.m-1). However, with ethylene glycol, increasing the surfactant content promoted a small variation on the contact angle (59.14° to 65.10°) and on the surface tension (5.5 to 5.0 mN.m-1). The surfactant promoted a slight change in the melt flow index but not affected the roughness of the samples. Keywords: hydrophilicity, polyether siloxane, polypropylene, wettability. How to cite: Antunes, L. F., Simon, D. A., Fiorio, R., & Francisquetti, E. (2019). Effects of polyether siloxane surfactant on the hydrophilic capacity of polypropylene films. Polímeros: Ciência e Tecnologia, 29(4), e2019048. https://doi. org/10.1590/0104-1428.06518

1. Introduction Polypropylene (PP) is currently one of the most studied polymers, mainly because of its interesting properties as moderate rigidity above glass transition temperature, relatively high melting point (allowing its use at temperatures higher than 100°C), low density, hydrophobic characteristics, diversity in mechanical properties, low cost and high availability[1,2]. PP can also be used for the manufacture of non-woven products, which are widely used in filtration processes, agriculture, hygiene products, protective clothing and also for the production of carpets[3]. The manufacture of non-woven products usually occurs through melt-blown or spunbond processes. The most common process is melt blown, which is an extrusion process in which the molten polymer passes through air jets after exiting the extrusion die, causing the diameter of the fiber to decrease when in contact with ambient air, forming a web[4]. Due to the wide range of non-woven applications, sometimes it is necessary to modify the characteristics of the resin from hydrophobic to hydrophilic in order to provide adsorption ability for certain fluids[2]. Several studies have been carried out to investigate this modification; the contact angle measurement is usually an indicative of wettability (hydrophilicity). The tendency of a liquid to spread or wet the surface of a solid increases as the contact angle decreases. Thus, the contact angle represents an inverse measurement of wettability[5]. When the contact angle (θ) is lower than 90°, the surface can be characterized as hydrophilic (considering water as

Polímeros, 29(4), e2019048, 2019

the liquid). When the angle is between 90° and 180°, it can be said that the surface is hydrophobic, being called predominantly non-wetting[6]. Andersen and Taboryski[7], after several analyses of the drops shapes, traced a drops profile as shown in Figure 1. The modification of the hydrophobic to hydrophilic capacity of the non-woven PP is commercially important, considering the applications for automotive components, such as: benches, internal part of doors, for the protection of the instruments of dashboards, aiming the replacement of the natural fibers, which degrade in a short period of time[8]. However, for the utilization of non-woven PP in substitution of natural fibers, the PP needs an industrially and economically viable modification. Wang  et  al.[9] investigated the improvement of the hydrophilic properties of the non-woven surface of polypropylene using a plasma atmosphere by dielectric barrier discharge in nitrogen at atmospheric pressure. The authors characterized the samples by contact angle measurements, scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), before and after the plasma treatment. Their results showed that the surface of the samples presented hydrophilic properties after the plasma treatment, evidenced by the reduction of the contact angle; the plasma treatment also promoted an increase in the surface roughness and the introduction of oxigen- and nitrogen-containing polar groups on the surface of PP.

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O O O O O O O O O O O O O O O O


Antunes, L. F., Simon, D. A., Fiorio, R., & Francisquetti, E.

Figure 1. Drops profile (1), (2), (5): θ < 90° = hydrophilic; (3), (4), (6), (7): θ > 90° = hydrophobic.

In order to modify the wettability of PP, Wanke et al.[10] compared the plasma surface treatment with the ultraviolet vacuum technique (VUV) aiming to introduce functional groups containing oxygen in the material. The samples were analyzed by FTIR, atomic force microscopy (AFM) and contact angle with the objective of evaluating the chemical and physical changes of the PP surface. The authors observed that plasma treated samples presented better efficiency when compared to those treated by VUV; the plasma treated samples required a shorter exposure time to obtain the same contact angle. The chemical modification that occurred in both processes was observed after a 14-day exposure to plasma and ultraviolet light, which was measured by FTIR. The AFM analysis did not show change in the roughness of the samples after the exposure period. Zhang, Sun and Xiao[11] studied the effects of the wettability of 16 additives introduced into the non-woven fibers produced by the melt-blown process. The effect of the additives on the surface tension of the polymers was determined by the measurement of the contact angle, and also the interference of the additive in the melt viscosity. The additives were divided into five groups according to their chemical functionality; the additive that presented the best performance reducing the surface tension was a surfactant of the family of the polyethers modified with trisiloxane. The aim of this study was to investigate and evaluate the effect of the addition of polyether siloxane in a polypropylene film in order to increase the hydrophilic characteristics, allowing the sorption of polar materials, such as water.

2. Materials and Methods 2.1 Materials The polypropylene (PP, H107, Braskem) used was a homopolymer, whose density is 905 kg/m3 and the melt flow index (MFI) is 80 g/10min (230°C/2.16kg). The surfactant used corresponds to a polyether siloxane (Tegopren 5840, Evonik), nonionic, liquid material.

2.2 Sample Preparation The compounds studied contained 0, 0.5, 1.0 and 3.0 wt% of surfactant, according to Table 1. Before the extrusion process, PP granules and the surfactant used (a liquid at room temperature) were manually mixed for 5 minutes. After the manual mixing, the samples remained at rest for seven days. After this period the samples were extruded in 2/5

Table 1. PP Samples + Surfactant. Product Quantity surfactant (%) Surfactant mass (g) PP mass (g)

Sample 1 2 0.0 0.50 0 10 2000 1990

3 1.0 20 1980

4 3.0 60 1940

a twin-screw co-rotating extruder (MScientific, Lab Tech Engineering Company), with mass temperature of 195°C and screw rotation of 220 rpm, obtaining pellets from which four films were produced in a heated press (210°C, 29.4 MPa, 4 minutes). The test specimens were obtained from these films.

2.3 Characterization 2.3.1 Contact angle measurements To measure the contact angles, deionized water, ethylene glycol, a high definition photographic camera and the software Surftens 4.7 were used. The method used to perform the contact angle measurements was the “sessile drop”. This method consists of depositing a drop of a liquid on a solid surface; the droplet is magnified with a lens, and the contact angle is measured by the principle of the goniometer. The contact angle was defined as the angle between the tangent of liquid interface - air and the tangent between the solid interface - air, as shown in Figure 2. For each sample, 6 measurements of the contact angle were made and the average of these measurements was used to express the result. 2.3.2 Surface tension The surface tension was obtained by using Young’s method, considering that the drop is in equilibrium, applying Young’s Equation 1[12]. γ= SV γ SL + γ LV .cosθ

(1)

• θ: drop contact angle; • γSV: surface tension of the solid vapor; • γLV: surface tension liquid vapor; • γSL: surface tension of solid liquid. Polímeros, 29(4), e2019048, 2019


Effects of polyether siloxane surfactant on the hydrophilic capacity of polypropylene films Table 2 shows the values of γLV and γSV under the normal conditions of temperature and pressure. The γSL value was obtained experimentally through the measurement of the contact angle.

2.3.3 Roughness

Figure 2. Contact angle setting.

2.3.4 Melt Flow Index (MFI)

Table 2. Tabulated Values γLV e γSV. Sample γLV (mN.m-1) γSV (mN.m-1) Deionized Water 72.8 -------Ethylene Glycol 47.7 -------Pure PP ------30.7

Reference [13] [14] [15]

The surface roughness was determined by the standard NBR ISO 4287[16]. The values were obtained from the arithmetic average of 6 measurements for each sample, using a TR110 surface roughness tester. The method used was the roughness average (Ra) that is obtained from the arithmetic average of the absolute values of the ordinates of the spacings of the roughness profile points, in relation to the medium line, within the measurement path, which in this case was 2 cm2. The melt flow index (MFI) was measured at 230 °C and 2.16 kg on an Instron plastomer according to the standard ASTM D1238[17]. The average results were obtained from 10 measurements with the objective of evaluating the influence of the surfactant on the fluidity of the compound and consequently associated with possible effects in the processing.

3.Results and Discussions 3.1 Contact angle

Figure 3. Contact angle in PP films with different surfactant concentrations.

The contact angle measurement was used to determine the hydrophobic/hydrophilic character of the polypropylene using deionized water and ethylene glycol. In this study an average contact angle of 92.58° was obtained for the unmodified PP in water. A similar result was found by Brow and Bhushan[13] who obtained a measure of 96° using water and PP. Figure 3 shows the average contact angle values obtained for each sample. For the deionized water, it was observed that the addition of the surfactant reduces the contact angle value. However, for the ethylene glycol, an increasing trend for the contact angle was observed with the increase in the surfactant content. This difference is related to the higher hydrophilic character of the water compared to the ethylene glycol. For the water, the lower contact angle result was obtained with the addition of 3.0 wt% of surfactant. A similar behavior was also observed by Zhang, Sun and Xiao[11] with deionized water. The contact angle change in ethylene glycol shows changes in the apolar component influenced by the surfactant characteristics (polyether siloxane). Wang, Bratko and Luzar[18] studied the contact angle variation in a natural (protein-based) and a synthetic (graphene layers functionalized with polar and apolar groups) surface. These authors observed that, along the surface, the angle varied according to the influence of neighboring groups. The influence of the polar (hydroxyl) and apolar (ethylene) segments of the ethylene glycol, as well as the chemical groups present in the surfactant (ether, methyl, and siloxane), can have the same effect.

3.2 Surface tension

Figure 4. Surface Tension in PP films with different surfactant concentrations. Polímeros, 29(4), e2019048, 2019

Figure 4 shows the averages obtained from surface tension (solid/liquid) for water and ethylene glycol. It is observed that a variation of the surface tension occurs as the concentrations of the surfactant are increased, possibly due to the fact that the surfactant is migrating to the surface of the samples, increasing the concentration of siloxane 3/5


Antunes, L. F., Simon, D. A., Fiorio, R., & Francisquetti, E. samples. Therefore, the contact angle variation observed was mainly related to the surfactant content. Grundke et al.[20] highlighted that the study of the relation between contact angle and roughness has increased greatly in the last decades, especially studying wettability as a factor for self-cleaning products. They proved that increasing the surface roughness causes variations in the contact angle measurement in the range of 40°. The relation between contact angle and roughness was studied by Morrow[21] which indicates that the preparation of a smooth solid surface is recognized as an important factor in obtaining reproducible results. In this work, it was not possible to observe a significant variation for the roughness, indicating that the surfactant promoted the modification of the contact angle values. Figure 5. Roughness in PP films with different surfactant concentrations.

3.4 Melt Flow Index (MFI) Figure 6 shows the melt flow index results for the studied samples. From Figure 6, it is observed that the addition of surfactant in concentrations of 1.0 and 3.0 wt% induced to a slight increase in the MFI. This was expected due to a lubricant effect of the surfactant.

4. Conclusions

Figure 6. MFI in PP films with different surfactant concentrations.

molecules on the PP surface. This migration can increase the hydrophilicity and the surface tension. Farris et al.[19] studied some features to improve the wettability and adhesion properties of polyolefin surfaces, proven through the reduction of the contact angle and consequently for the increase of the surface tension, which changed the shape of the drop from spherical to laminate. Similar behaviours are verified in Figures 3 and 4 for water where the decrease of the contact angle and the incresase of surface tension occurs. However, in ethylene glycol, the increase trend for the surface tension is possibly associated with an interaction with neighboring groups (methyl and ethers), as described by Wang, Bratko and Luzar[18]. Zhang,  et  al.[11] observed a variation of the surface tension when adding a polyether to the PP, and associated this change to a possible migration of the polyether from the solid interior to the surface of PP, thus increasing the PP hydrophilicity.

3.3 Roughness Figure 5 shows the average of the surface roughness obtained for the samples. It was observed that there was no significant variation among the roughness of the studied 4/5

The addition of the polyether siloxane provided a variation in the hydrophilic capacity of the polypropylene, where an addition of 3.0 wt% of surfactant promoted the highest increase in the hydrophilicity, substantially reducing the contact angle when deionized water was used. When ethylene glycol was used, no significant variation of the contact angle was observed. The surface tension variation suggests that the surfactant can migrate to the surface of the polypropylene film, increasing the wettability and consequently promoting a reduction in the contact angle value. The modification of the contact angle was not influenced by the roughness, since all the samples presented similar roughness. Increasing the surfactant content did not cause a large modification on the MFI values.

References 1. Spadetti, C., Alves da Silva Filho, E., Lopes de Sena, G., & Vital Paixão de Melo, C. (2017). Propriedades térmicas e mecânicas dos compósitos de Polipropileno pós-consumo reforçados com fibras de celulose. Polímeros: Ciência e Tecnologia, 27(número especial), 84-90. https://orcid.org/10.1590/0104-1428.2320. 2. Hutten, I. M. (2016). Handbook of nonwoven filter media. Delray Beach: Elsevier Ltd. 3. Soltani, I., & Macosko, C. W. (2018). Influence of rheology and surface properties on morphology of nanofibers derived from islands – in-the-sea meltblown nonwovens. Polymer, 145, 21-30. http://dx.doi.org/10.1016/j.polymer.2018.04.051. 4. Zhang, H., Liu, J., Zhang, X., Huang, C., & Jin, X. Online prediction of the filtration performance of polypropylene melt blown nonwovens by blue-colored glow. Journal of Applied Polymer Science [Internet]. 2017[cited 2019, October 26];135(10):1307. Available from: https://onlinelibrary.wiley. com/doi/epdf/10.1002/app.45948 Polímeros, 29(4), e2019048, 2019


Effects of polyether siloxane surfactant on the hydrophilic capacity of polypropylene films 5. Chan, C. M. (1994). Polymer surface modification and characterization. Munich: Hanser/Gardner Publications. 6. Lamour, G., Hamraoui, A., Buvailo, A., Xing, Y., Keuleyan, S., Prakash, V., Bafrooei, A. E., & Borguet, E. (2010). Contact angle measurements using a simplified experimental setup. Journal of Chemical Education, 87(12), 1403-07. http://dx.doi. org/10.1021/ed100468u. 7. Andersen, N. K., & Taboryski, R. (2017). Drop shape analysis for determination of dynamic contact angles by double sided elliptical fitting method. Measurement Science & Technology, 28(4), 047003. http://dx.doi.org/10.1088/1361-6501/aa5dcf. 8. Sullins, T., Pillay, S., Komus, A., & Ning, H. (2017). Hemp fiber reinforced polypropylene composites: the effects of material treatments. Composites. Part B, Engineering, 114, 15-22. http://dx.doi.org/10.1016/j.compositesb.2017.02.001. 9. Wang, K., Wang, W., Yang, D., Huo, Y., & Wang, D. (2010). Surface modification of polypropylene non-woven fabric using atmospheric nitrogen dielectric barrier discharge plasma. Applied Surface Science, 256(22), 6859-64. http://dx.doi. org/10.1016/j.apsusc.2010.04.101. 10. Wanke, C. H., Barbosa, L. G., Hübner, J. V. M., Horowitz, F., Mauler, R. S., & Oliveira, R. V. B. D. (2012). Recuperação hidrofóbica de polipropileno tratado por VUV ou plasma. Polímeros, 22(2), 158-63. http://dx.doi.org/10.1590/S010414282012005000027. 11. Zhang, D., Sun, C., & Xiao, J. (2006). Effect of selected additives on surface energy of fibers and meltblown nonwovens. Textile Research Journal, 76(3), 261-65. http:// dx.doi.org/10.1177/0040517506053905. 12. Kan, M., Kawsaki, H., & Suzumura, F. (2017). A wettability evaluation on super-hydrophobic and hydrophobic surface. In The 4th International Conference on Design Engineering and Science. Aachen, Germany: ICDES. Retrieved from http:// www.jsde.or.jp/icdes/proceedings/4th-2017/PDF/162.pdf 13. Brown, P. S., & Bhushan, B. (2017). Liquid-impregnated porous polypropylene surfaces for liquid repellency. Journal of Colloid and Interface Science, 487, 437-443. http://dx.doi. org/10.1016/j.jcis.2016.10.079. PMid:27814555.

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14. Dean, J. A. (1999). Lange’s handbook of chemistry. Knoxville: McGraw-Hill, Inc. 15. Ryntz, R. A., & Yaneff, P. V. (2003). Coating of polymers and plastics. New York: Marcel Dekker. http://dx.doi. org/10.1201/9780203912379. 16. Associação Brasileira de Normas Técnicas – ABNT. (2002). ABNT NBR ISO 4287: Especificações geométricas do produto (GPS). Rugosidade: método do perfil: termos, definições e parâmetros da rugosidade. Rio de Janeiro: ABNT. 17. American Society for Testing and Materials – ASTM. (2004). ASTM D1238-04: Standard test method for melt flow rates of thermoplastics by extrusion plastometer. West Conshohocken: ASTM. 18. Wang, J., Bratko, D., & Luzar, A. (2011). Probing surface tension additivity on chemically heterogeneous surfaces by a molecular approach. Proceedings of the National Academy of Sciences of the United States of America, 108(16), 6374-6379. http://dx.doi.org/10.1073/pnas.1014970108. PMid:21460249. 19. Farris, S., Pozzoli, S., Biagioni, P., Duó, L., Mancinelli, S., & Piergiovanni, L. (2010). The fundamentals of flame treatment for the surface activation of polyolefin polymers – A review. Polymer, 51(16), 3591-3605. http://dx.doi.org/10.1016/j. polymer.2010.05.036. 20. Grundke, K., Pöschel, K., Synytska, A., Frenzel, R., Drechsler, A., Nitschke, M., Cordeiro, A. L., Uhlmann, P., & Welzel, P. B. (2015). Experimental studies of contact angle hysteresis phenomena on polymer surfaces: toward the understanding and control of wettability for different applications. Advances in Colloid and Interface Science, 222, 350-376. http://dx.doi. org/10.1016/j.cis.2014.10.012. PMid:25488284. 21. Morrow, N. R. (1975). The effects of surface roughness on contact: Angle with special reference to petroleum recovery. Journal of Canadian Petroleum Technology, 14(04), 42-53. http://dx.doi.org/10.2118/75-04-04. Received: Sept. 21, 2018 Revised: Oct. 26, 2019 Accepted: Nov. 27, 2019

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ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.04119

Investigation of TPP-Chitosomes particles structure and stability as encapsulating agent of cholecalciferol Aline Sayuri Lima Iida1, Karina Novais Luz1, Taís Téo Barros-Alexandrino2,3, Carmen Sílvia Fávaro-Trindade1, Samantha Cristina de Pinho1, Odílio Benedito Garrido Assis2 and Milena Martelli-Tosi1*  1

Departamento de Engenharia de Alimentos, Faculdade de Zootecnia e Engenharia de Alimentos – FZEA, Universidade de São Paulo – USP, Pirassununga, SP, Brasil 2 Laboratório Nacional de Nanotecnologia para o Agronegócio, Embrapa Instrumentação, São Carlos, SP, Brasil 3 Programa de Pós-Graduação em Biotecnologia (PPG-Biotec) da Universidade Federal de São Cartos, UFSCar, São Carlos, SP, Brasil. *mmartelli@usp.br

Abstract Tripolyphosphate (TPP)-chitosomes were produced aiming at the encapsulation and conservation of vitamin D3. This hybrid system is made of liposomes, vesicles consisting of phospholipid bilayers, surrounded by chitosan wall ionic-crosslinked with TPP. Chitosan concentrations (2 and 4 mg mL-1) were tested and the vitamin stability in aqueous dispersions monitored for 49 days. The results confim that D3 remained stable throughout the analyzed period (49 days), whereas the non-encapsulated vitamin totally degrades after the second week of storage. The particle diameters ranged from 0.1 to 5 μm with good colloidal stability (+22 to +48 mV), and encapsulation efficiency of 97%. Thermal stability was also improved when using the TPP-chitosomes. The protection performed was attributed to the stable interactions conferred by the phospholipids crosslinking with the chitosan amino groups and a formation of a net of hydrogen bonds established amongst the hydroxyl groups of the interacting compounds as revealed by infrared spectroscopy. Keywords: liposomes, cholecalciferol, lecithin, encapsulation, vitamin stability. How to cite: Iida, A. S. L., Luz, K. N., Barros-Alexandrino, T. T., Fávaro-Trindade, C. S., Pinho, S. C., Assis, O. B. G., & Martelli-Tosi, M. (2019). Investigation of TPP-Chitosomes particles structure and stability as encapsulating agent of cholecalciferol. Polímeros: Ciência e Tenologia, 29(4), e2019049. https://doi.org/10.1590/0104-1428.04119

1. Introduction Vitamins are vital substances for the maintenance of human health and metabolic functions. Vitamin D, in particular, has the fundamental physiological property of stabilizing the calcium homeostasis and is also involved in several cellular processes, such as immunology and regulation of gene transcription[1]. The American Institute of Medicine, Food and Nutrition Board recommends a daily intake of 10 to 15 μg of vitamin D to achieve acceptable levels of serum 25-hydroxyvitamin D [25(OH)D][2]. This recommendation applies to both forms of vitamin-D, namely ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3), which are from vegetal and animal sources, respectively. However, some studies have shown that the administration of vitamin D3 is more effective in increasing serum 25(OH)D levels than vitamin D2[3], either from cutaneous synthesis or through dietary ingestion. The vitamin D insufficiency in general population is much common than currently reported[4], and the enrichment of food products can be an alternative to ensure the consumption of adequate levels of nutrients[5]. Vitamins, however, are sensitive compounds. Heat, light, variation of

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pH and exposure to atmospheric oxygen can accelerate their degradation[6,7], thus requiring appropriate means to increase the stability and preserve properties before consumption. Encapsulation is one of the strategies to overcome this problem and different materials are described to form protective matrices for vitamins. Gelatin[8], whey protein[9], maize prolamine protein[10], multilamenar liposomes[11], pickering emulsions stabilized by nanofibrillated cellulose[12], and nanostructured lipid carriers[13], have been tested to entrap vitamin D. Among these, the system based on liposomes encapsulation combined with chitosan has been suggested as a suitable approach to protect and achieve intestinal absorption[14]. Conventional liposomes have low resistance to gastric pH and are very susceptible to enzymatic lysis. The formation of a surrounding polymeric wall, such as chitosan, can improve significantly their stability[15]. Chitosan is a cationic polysaccharide consisting of glucosamine units of poly‑(β(1→4)-2-amino-2-deoxy-D-glucopyranose) largely evaluated as encapsulation matrix of active compounds[16]. Chitosan can be formulated into nanoparticles by ionic

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Iida, A. S. L., Luz, K. N., Barros-Alexandrino, T. T., Fávaro-Trindade, C. S., Pinho, S. C., Assis, O. B. G., & Martelli-Tosi, M. crosslinking, formed between the positively charged amino groups in the chitosan and the negatively charged species of an anionic polymer, such as the sodium tripolyphosphate (TPP). Such process is referred to as ionic gelation and has been successfully applied to encapsulation and delivery of vitamins B9, B12 and C[17,18]. By forming a stable three-dimensional network, the chitosan-TPP complex is also suitable to coat liposomes by binding to polar heads of lipid bilayers with no alteration in the phospholipids in the vesicles assembling[15,19]. In such hybrid system, the release occurred preferentially in an alkaline pH, what provides protection to acid stress. In the present study, the aim was to prepare and characterize the complexation of chitosan and TPP as liposome protective coating and evaluate the stability of this system in the retention of vitamin D3 in aqueous dispersion over time.

2. Material and Methods 2.1 Material Medium molecular weight chitosan (Product 448877, 190,000-310,000 Da with 75%-85% deacetylated units), sodium tripolyphosphate (TPP, Product 238503), and cholecalciferol (98% purity, Product C9756), were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Phospholipon 90G (Lipoid GmbH, Ludiwgshafen, Germany) and HPLC grade methanol (Merck KGaA, Darmstadt, Germany) were also used.

2.2 Preparation of Chitosan-TPP particles (Ch-Np) and vitamin loading The chitosan nanoparticles (Ch-Np) were synthesized following the ionic gelation method as described by Britto et al.[18]. In brief, the process consisted of solubilizing chitosan in 1.0 wt% acetic acid aqueous solution under moderate stirring for 16 h. Two chitosan concentrations (2 mg mL-1 and 4 mg mL-1) were prepared and identified as Ch2 and Ch4 respectively. Subsequently, a solution of TPP at a concentration of 0.6 mL min-1 was added, dropwise with stirring, to the chitosan solutions at a rate of 1.0 mL min-1.

The resultant nanoparticles were identified as Ch2‑Np and Ch4-Np. In parallel, 50 mg of vitamin D3 (V) was diluted in 2 mL of 96% ethanol and separately added to 48 mL of each chitosan solution, resulting in final vitamin D3 concentrations of 1 mg mL-1. The dispersion was mixed by an Ultraturrax T18 homogenizer (Ika, USA) for 2 min (at 13000 rpm) to ensure physical mixture. Then, the TPP solution (0.6 mg mL-1) was dropped to the mixture at 1.0 mL min-1, under magnetic stirring, generating the loaded nanoparticles, named Ch2-Np-V and Ch4-Np-V. All samples were prepared at room temperature (20-30oC) and away from light (flasks covered with aluminum foil).

2.3 Preparation of TPP-Chitosomes (TPP-Ch) Phospholipon (Lipoid 90G, 100 mg) was weighed, dispersed in 2 mL of 96% ethanol, added to 48 mL of deionized water and mechanically homogenized for 2 min at 13000 rpm (Ultraturrax T18, Ika, USA) to produce the liposomes. For TPP-chitosome preparation, the dispersion with Phospholipon was mixed with both chitosan solutions (2 and 4 mg mL-1) and the TPP added in analogous manner as previous outlined. For vitamin loading, 50 mg of vitamin D3 was first diluted in 2 mL of 96% ethanol and added to 46 mL of each chitosan solution. Then, 2 mL of the phospholipid dispersion was added and mechanically homogenized in an Ultraturrax for 2 min. TPP was then dropped to promote crosslinking and vitamin retention into the chitosomes particles (TPP‑Ch2-V and TPP-Ch4-V). The final compositions and all samples identification are summarized in Table 1. All samples were prepared at room temperature (20-30 °C) and protected from light.

2.4 Characterization of particles Suspensions of particles (with and without vitamin) were sonicated for 15 min in the ultrasonic bath (Ultra Cleaner 1400A, Unique, Brazil), in deionized water (1:10 v/v), cast on a silicon slides and allowed to dry for 48 h at room temperature. SEM‑FEG images were recorded by a JSM-6701F field emission microscope (JEOL, Japan) with

Table 1. Samples identification according to composition. Sample Ch2 Ch4 Ch2-Np Ch4-Np TPP-Ch2 TPP-Ch4 Liposomes Ch2-V Ch4-V Ch2-Np-V Ch4-Np-V TPP-Ch2-V TPP-Ch4-V Liposomes-V

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Chitosan (mg.mL-1) 2 4 2 4 2 4 2 4 2 4 2 2 0

TPP (mg.mL-1) 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 -

Vitamin D3 (mg.mL-1) 1 1 1 1 1 1 1

Phospholipon (mg.mL-1) 2 2 2 2 2 2

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Investigation of TPP-Chitosomes particles structure and stability as encapsulating agent of cholecalciferol an accelerating voltage of 2kV. Scannings were performed on non-coated surfaces. The particle size distribution and zeta potential were measured by dynamic light scattering (DLS) in a Zetasizer ZS 3600 (Malvern Instruments, USA). All determinations were carried out in triplicate at 25 °C. The final size distributions were considered as the average of three distinct measurements for each synthesis/group. The thermal stability of the nanoparticles was studied in a thermogravimetric analyser TGA-Q500 (TA Instruments, USA). The dispersions were freeze-dried and samples of about 10 mg sealed in platinum pans and heated from 20 °C to 600 °C (heating rate of 10 °C min-1), under nitrogen atmosphere (gas flow of 60 cm3 min-1). Mass changes were continuously recorded as a function of temperature. Attenuated total reflection Fourier-Transform Infrared spectra (ATR-FTIR) were recorded on IR Prestige-21 (Shimadzu, Kyoto, Japan), in absorbance mode (resolution of 2 cm-1, spectral region of 4000-400 cm-1), over 40 consecutive scans. Three spectra were acquired from three freeze-dried sample of each formulation. The average spectra were constructed using the OMNIC™ Software (Thermo Fisher Scientific, USA).

substrate, in irregular dark stains around the liposomes or as isolated circular patterns in the presence of chitosan. This aggregation occurs due to the lipophilic nature of the cholecalciferol molecules (not soluble in water), arranging them as a circular phase when in aqueous suspension, which stands out as contrasted spots on the dried surface. Dark spots observed in the center of loaded nanoparticles (Ch2-V and Ch4-V) are problably due to thermal degradation of unstable organic compounds during the electron beam exposure. For non-load chitosan nanoparticles predominates the circular shape in varying dimensions. In the presence of phospholipids, the interaction with TPP-Ch suspension generates smaller particles in irregular shapes and distribution. When the vitamin is added, the surface tension of the particles changes, affecting the colloidal equilibrium, resulting in a reduction of the interaction between the compounds. The resulted particles are not very well defined with evident aggregation of the smaller ones in large granules, which is clearly observed to TPP-Ch-V systems (Figure 1). These characteristics are confirmed by the size distribution

2.5 Vitamin quantification and encapsulation efficiency The amount of the vitamin D3 retained in the suspensions was quantified according to Staffas and Nyman[19]. An aliquot of 0.5 g of each sample was mixed with 10 mL of HPLC grade methanol, vortexed and placed in an ultrasonic bath for 5 min, followed by centrifugation for 5 min at 5000 rpm (Eppendorf AG, Germany) at 10 °C. After centrifugation, the supernatants were filtered through 0.45 μm membrane filter (Millipore Corp., USA) and injected into an HPLC system (Prominence Modular, Shimadzu, Japan) equipped with a C18 column (Merck, 4.6 mm × 250 mm) and an inline degasser (DGU-20A5, Shimadzu, Japan). Methanol‑acetonitrile (90:10 v/v) was used as the mobile phase at a flow rate of 1 mL min-1, and the column heated to 35 °C. The analytical curve was generated by injection of vitamin D3 at different concentrations and the vitamin stability measured every 7 days over a 49 days period. The  suspensions were stored in 50 mL Falcon conical tubes in the refrigerator at 4 °C and protected from light. Encapsulation efficiency (EE) was calculated as the difference between the amount of added vitamin D3 in the particle suspension [VitD3]ADDED and the amount of vitamin D3 quantified in the supernatant [VitD3]SUPERNATANT after centrifugation (5 mL for 10 min at 13000 rpm), according to the Equation 1. EE ( % ) =

[VitD3 ]added − [VitD3 ]supernatant * 100 [VitD3 ]added

(1)

3. Results and Discussions 3.1 Nanoparticles characterization Figure 1 provides the SEM-FEG micrographs of the empty systems and vitamin D3 loaded particles. It is generally possible to visualize agglomeration of vitamin D3 on the Polímeros, 29(4), e2019049, 2019

Figure 1. Photomicrographs of empty and vitamin D3 load systems: liposomes, according to adopted identification as displayed in Table 1. The inserted bar correspond to 1 μm for all images. 3/8


Iida, A. S. L., Luz, K. N., Barros-Alexandrino, T. T., Fávaro-Trindade, C. S., Pinho, S. C., Assis, O. B. G., & Martelli-Tosi, M. profiles as obtained in the zetasizer equipment (Figure 2). The processed nanoparticles without vitamin have all monodispersed distribution (Figure 2A). Liposome formation has the smallest average size, 32.7 nm, with narrowest size‑distribution associated to a slight negative zeta potential of -0.8 mV. The quasi-neutrality of the liposomes indicates

a stability of the three dimensional structure. The liposomes are composed of neutral lipids having one phosphatidyl group and one choline group in their molecules. According to the model presented by Makino et al.[20], the charge neutrality in the formed three dimensional structure resulted from the lipid molecules arranged in such a way that the neutral hydrophilic groups are on the particle surface. When loaded, the zeta potential is enhanced to –3 mV, indicating changes in the liposome dipole configuration due to the vitamin interaction with the head group region (internal cavity). The different concentrations of chitosan introduce slight changes in the particle sizes. Average dimensions of 68 nm (Ch2-Np) and 53 nm (TPP-Ch2) were measured for the concentration of 2 mg mL-1. 78 nm was the average size recorded for both Ch4-Np and TPP-Ch4 when reacting 4 mg mL-1 of chitosan. Zeta potential values were also preserved (+40 mV for Ch2-Np and +48 mV for Ch4-Np). When vitamin is added (Ch-Np-V and TPP-Ch-V series), the ionic formation of particles is disturbed in some way that the distribution assumes a bimodal profile with large aggregates as confirmed by microscopic analyses. Averages sizes as low as 28 nm and as large as 5560 nm were found for TPP-Ch2-V samples. Distributions centered at 91 nm and 459 nm are read for TPP-Ch4-V. In these, Zeta potential ranges from +23 mV to +38 mV suggesting reasonable to good stability of the loaded carriers[21]. Numerical data are summarized in Table 2.

3.2 Stability of the capsules

Figure 2. Particle size distributions of (A) empty and (B) vitamin D3 loaded-systems, as acquired from the Zetasizer (each curve represents the average of three measurements).

The instability index describes the portion of the samples that were separated by centrifugal forces and expressed by a normalized position of the flocculated/supernatant boundary. The higher the index value, the lower is the colloidal dispersion stability. According to measurements as obtained by the LUMiSizer (Table 2), the instability of empty particles increases as the chitosan concentration increases (Ch2-Np = 0.22 and Ch4-Np = 0.49). However, no significant difference was observed when these capsules are vitamin loaded (Ch2-V and Ch4-V). Both indexes were relatively small (0.02 and 0.01 respectively), which would mean high stability of this system. It is noteworthy that these samples were translucent, thus the response generated by the LUMiSizer may have underestimated the results.

Table 2. Zeta potential, particle size, instability index and encapsulation efficiency (EE) of the suspensions containing chitosan-TPP micro/nanoparticles at two chitosan concentrations: 2 and 4 mg mL-1 (Ch2 and Ch4). Sample Liposomes Liposomes-V Ch2-V Ch2-Np Ch2-Np-V TPP-Ch2 TPP-Ch2-V Ch4-V Ch4-Np Ch4-Np-V TPP-Ch4 TPP-Ch4-V

Zeta potential (mV) -0.80 ± 0.03a -3.02 ± 0.05b +44 ± 1c +40 ± 3c,e +28 ± 3f +26 ± 4f +23 ± 4f +46 ± 1e +48 ± 3e +38 ± 2c +22 ± 2f +35 ± 1g

Peak 1 32.7 ± 11.2 44 ± 16.6 106 ± 34.5 68.1 ± 34.8 220 ± 93.5 53.8 ± 40.7 28.2 ± 5.64 225 ± 83.6 78.8 ± 38.7 91.3 ± 31.8 78.8 ± 34.8 91.3 ± 36.8

Particle size (nm) Peak 2 825 ± 349 5560 ± 1358 1720 ± 1081 459 ± 119

Instability index * * 0.02 ± 0.00 0.22 ± 0.02 0.63 ± 0.06 0.83 ± 0.01 0.57 ± 0.05 0.01 ± 0.00 0.49 ± 0.24 0.11 ± 0.01 0.84 ± 0.01 0.29 ± 0.10

EE (%) 5 ± 1a 95 ± 2c 97 ± 1c 75 ± 10b 97 ± 1c

*Samples were very translucent. Means with different superscript letters in the same column are statistically different at p < 0.05.

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Investigation of TPP-Chitosomes particles structure and stability as encapsulating agent of cholecalciferol In general, the results evidenced that the vitamins encapsulated with 4 mg mL-1 of chitosan were more stable than those with 2 mg mL-1, namely by electrostatic repulsion between the particles. A higher concentration of chitosan favours the presence of residual charges on particles surface (as confirmed by zeta potential measurements). When submitted to centrifugation, the repulsive forces made the particles more difficult to agglomerate, leading to a slower flocculation. The highest instability was verified to suspensions containing phospholipids (TPP-Ch2 and TPP-Ch4), with indexes of 0.83 and 0.84, respectively. Both formulations resulted in the smallest particles in size with low zeta potential (little repulsion forces). The particles could eventually aggregate contributing to a condition of continuity of the matrix, which is interpreted as low stability of this system.

3.3 Vitamin D3 encapsulation efficiency in Ch-Np and TPP-chitosomes Values of vitamin encapsulation efficiency (EE), as estimated by centrifuging the dispersion and quantified as the percentage of vitamin in the sediment, are also listed in Table 2. Chitosan-TPP nanoparticles are able to retain 95% of free vitamin in the Ch2-Np-V system and 75% when the concentration of chitosan is doubled (Ch4-Np-V). In the conjugated liposome system, the efficiency is even greater: 97% are entrapped in both formulations (TPP‑Ch2-V and TPP-Ch4-V). These results were higher than those encountered by Chaves  et  al.[11], which encountered the EE of D3 in liposomes, produced by proliposomes hydration, of about 91% during the 42 days of storage. In other systems, the EE was 52% when D3 was encapsulated using zein nanoparticles, and was greatly improved to 88% when these particles were coated with carboxymethyl chitosan[10]. Therefore, the TPP-Ch system was effective on the entrapement of D3. Concerning the vitamin degradation in aqueous medium, the Figure 3 shows the temporal evolution of concentrations during storage at room temperature (1 to 49 days), in encapsulated conditions. As reference, dispersion of free-vitamin (non-encapsulated), identified as VitD3 was also essayed. The low stability of VitD3 in suspension is evident, with pronounced variation of concentration over time, reaching almost zero at the end of the first week in a nonlinear fashion. Vitamin physically trapped by chitosan (Chx-V) also follows an exponential decay but not with such a marked loss. The decrease in concentrations in these samples is more accentuated during the first 28 days, stabilizing onwards. In this period, the average vitamin concentration dropped to 22% in Ch2-V nanoparticles and to approximately 18% for Ch4-V. Similar behaviour is shown by the loaded nanoparticles when synthetized by chitosan-TPP crosslinking (Ch2-Np-V and Ch4-Np-V). In these samples however, the vitamin degradation is less pronounced over time, indicating some protection conferred when the encapsulation matrix is assembled by ionic interaction, independent of the chitosan concentration. The vitamin D3 deterioration in such exponential decay model fits a pseudo first-order kinetics since as the time increases, the rate of loss also increases[22]. The better description for this behaviour can be mathematically expressed by the Weibull model as Ct/Co = exp(–ktn), where Ct and Co are the momentary and initial concentration of the vitamin Polímeros, 29(4), e2019049, 2019

and (–ktn) express, in module, the rate of reaction, i.e., how fast the concentration curve drops with time. Conversely, for the TPP-chitosomes particles (TPP‑Ch2-V and TPP-Ch4-V), there were no significant vitamin losses during storage. In these systems the temporal variation of concentration can be adequately adjusted to a linear fit (tn = 1), indicating good stability with minimal reduction in concentration over time. Such indicates that liposomes grafted with TPP-chitosan nanoparticles can lead to great encapsulation efficiency, reducing potential degradations.

3.4 Thermal decomposition The thermal stability of the neat components is compared in the thermograms of Figure 4, according to TG analyses. The initial loss of mass of Phospholipon is recorded at 120 °C due to removal of the few absorbed moisture in its structure (approximately 3%). The second event was observed between 236 °C and 307 °C and assigned to the decomposition of the residual proteins present in the sample, in which around 17% of the total mass was lost (residual mass of 80%). The most severe degradation occurs in the sequence, resulting in an accentuated reduction to 12% of original mass at the end of the event at 358 °C. The decomposition at this temperature may be attributed to de degradation of the phospholipids[23].

Figure 3. Evolutions of vitamin D3 over 49 days of storage. VitD3 stands for free-vitam-in dispersion (non-encapsulated). For comparison, results were disposed as (A) and (B) according to the chitosan concentration: 2 mg mL-1 and 4 mg mL-1, respectively. Ct and Co are the concentration at certain time t and initially, respectively. Samples identifications according to composition displayed in Table 1. 5/8


Iida, A. S. L., Luz, K. N., Barros-Alexandrino, T. T., Fávaro-Trindade, C. S., Pinho, S. C., Assis, O. B. G., & Martelli-Tosi, M. From this temperature on the long tail reflects the formation of carbon dioxide and other volatile substances. Chitosan shows a three-phase decomposition. The first at 87 °C corresponds to the removal of physically and chemically bonded water (12%). The second and more intense, starting at around 250 °C, is related to oxidative backbone degradation mainly to the destruction of amine groups. A third event, from 310 °C, leads to a continuous processes of formation of water, CO, and CH4 followed by depolymerisation and decarbonylation until complete pyrolitic decomposition[24]. The vitamin D3, in turn, is the one with the lowest thermal stability. The vitamin is thermally stable until 200 °C and has a unique event of total mass loss (100% degradation). No water desorption is recorded due to the hydrophobic nature of this compound. For the suspensions prepared in the present study, the thermal degradation profiles are shown in Figure 5. In all samples predominates the typical appearance of chitosan degradation (Figure 4) with similar three distinct main events. The initial one (between 40 °C to 100 °C) corresponds to water evaporation with loss of mass varying from 5 to 12% according to the encapsulation formulation. The second event, observed to start above 200 °C, is probably related to the decomposition of the acetylated and deacetylated units of

the chitosan structure. The third event ranging from 300 °C to 400 °C, is related to polymeric structure degradation e vitamin decomposition. From the curves of Figure 5 is obvious the enhancing of stability introduced by the tripolyphosphate crosslinking. Two distinct groups of samples may be identified: one with an intense decomposition at temperatures above 300 °C, corresponding to vitamin physical entrapped by chitosan (non-cross-linked Chx-V samples), and that with a proportional higher stability, corresponding to TPP cross‑linked systems (Chx-Np-V and TPP-Chx-V particles). By 400 °C, the residual mass is approximately 43% for cross-linked samples while it was found to be as low as 18% for vitamin samples entrapped only by chitosan. Above this temperature, all samples undergo decarbonylation and pyrolitic decomposition in a more marked way non-crosslinked samples.

3.5 ATR-FTIR Valuable qualitative information about the particle chemical structures can be obtained by Infrared spectroscopy. The FTIR spectra for the neat components (chitosan, Phospholipon

Figure 4. Mass loss as a function of the temperature of the pure components: Phospholipon, vitamin D3 and chitosan.

Figure 5. Mass loss as a function of the temperature of vitamin D3-loaded systems: Ch2-Np-V, TPP-Ch2-V, Ch4-Np-V and TPP‑Ch4-V, as composition described in Table 1. 6/8

Figure 6. ATR-FTIR of (A) the pure components: Phospholipon, chitosan, vitamin D3 and (B) vitamin D3-loaded systems: Ch2-V, Ch2-Np-V, TPP-Ch2-V, Ch4-V, Ch4-Np-V andTPP-Ch4-V, as described in Table 1. Polímeros, 29(4), e2019049, 2019


Investigation of TPP-Chitosomes particles structure and stability as encapsulating agent of cholecalciferol and vitamin D3) are presented in Figure 6A and for the formed particles, displayed in Figure 6B. All samples present two main spectral ranges: a broad band between 3600 and 2500 cm-1 and a strong absorption region between 1800 and 400 cm-1, both characteristic of amine groups (proteins) and polysaccharide structures. The  broad band between 3600-3000 cm-1 is assigned to O-H and N-H groups stretching. The bands centred at 3000 and 2700 cm-1 may be attributed to the symmetric and asymmetric –CH group stretching in the glucosamine ring, which is a typical polysaccharide vibration. The characteristic spectrum of the phospholipids is typical of phospholipid materials[25]. The absorbance bands at 2923 and 2853 cm-1 are related to the alkane groups stretching. The band at 1736 cm-1, has been assigned to the carbonyl stretching vibration band C=O of saturated aliphatic ester, which is located between the hydrophilic tails and hydrophobic head groups in the phospholipid molecule[26]. The peaks around 1637 cm-1 are largely due to a combination of stretching and scissoring vibrations from C=O, C=C and N=C. Asymmetric and symmetric stretching of PO2 is identified as bands at 1241 and 1092 cm-1, respectively[25,27]. Neat chitosan presented typical polysaccharide spectrum with a broad O-H groups stretching band near 3250 cm-1. A low intensity band centred at 2870 cm-1 arose from the axial deformation of C-H from CH2 and CH3 groups from the glucosamine unit in the chitosan structure. A clear doublet at 1648 cm-1 was attributed to the C2 position in the NH2 group of amino. The peak signals at 1585 cm-1 correspond to the NH bending of the amide II in vibrational mode. A medium intensity vibration at 1317 cm-1 is characteristic of -OH, -NH2 or -CO groups. The more intense vibrations at 1026 cm-1 are associated to -C-O-C- stretching in the glycosidic linkage. Bands observed in the spectrum of vitamin D3 can be identified as the stretching of hydrogen bond O–H at 3290 cm−1 and the alkyl C–H vibration in the doublet at 2942 cm−1 and 2870 cm−1. Absorbance bands from C=O stretching at 1635 and 1440 cm−1, C-O group at 1054 cm−1 and CH2 group at 895 cm−1 are assigned. Other bands are related to several C-H bendings in the cholecalciferol structure[28]. In the particle spectra (Figure 6B), the main absorbance bands came from the chitosan structural absorbances. For  chitosan-vitamin D entrapment (Chx-V system), the broad band, related to O-H groups, has a shift to maximum at 3269 cm-1, differently from chitosan and vitamin D3 (peaks at 3250 and at 3290 cm-1 respectively), indicating hydrogenic bonds between the hydroxyl groups in chitosan with vitamin D3[29]. Peaks at 2928 and 2866 cm-1, despite low intensities, are visible and associated to non-bonded alkyl C–H vibration from vitamin D3. The vibrations peaks in the amide region (between 1648 and 1026 cm-1) had obvious shifts in all formulations suggesting the participation of –NH2 groups of chitosan in electrostatic interactions. The electrostatic interaction between the positively charged amino groups of chitosan and the negatively charged counter-ions of sodium tripolysophosphate (TPP) is the main mechanism of particle formation. In these cross-linked particles (Chx-Np-V and TPP-Chx-V), the main differences in the FTIR spectra, according to Martins et al.[30] refers to weak band at 1218 cm-1 assigned to PO stretching and a band at 892 cm−1 attributed to P-O-P asymmetric stretching. Polímeros, 29(4), e2019049, 2019

A shoulder around 1460 cm-1 is also considered as arising from the cross-linked amine vibrations[31]. In the samples containing phospholipids (TPP-Chx-V), some peaks can be easy identified, as those related to the alkane groups (2923 and 2853 cm-1); 1736 (C=O stretching from aliphatic ester), 1241 cm-1 (PO2 stretching) and several other CH vibrations of saturated and unsaturated carbon atoms at around these peaks. The PO vibration (at 1218 cm-1) characteristic of the TPP crosslinking is overlapped in this system by a more intense absorption at 1241 cm-1 for the phospholipids. The intense peak at 1154 cm-1 corresponds to entrapped vitamin D3 vibrations. In all crosslinked samples the peak at 892 cm-1 increases in intensity indicating the formation of a strong net of hydrogen bonds between the hydroxyl groups amongst the interacting compounds.

4. Conclusions Chitosan-based micro/nanoparticles are successfully produced by ionic gelation and can be adequately applied to the encapsulation of vitamin D3. The formed structures have zeta potential between + 22 to + 48 mV, with variable diameters but not exceeding 225 nm for load particles. Significant reduction of vitamin degradation in aqueous suspension and under thermal conditions can be attained by entrapping vitamin into liposomes and underwent subsequent formation of a coating with TPP-chitosan complex. Therefore, these systems were were more effective to protect vitamin D3 from time storage than the chitosan‑TPP nanoparticles. The infrared spectral analyses suggest good interaction between compounds by establishing an extensive intermolecular interaction. Electrostatic interaction occurs in the majority of amine groups present in chitosan with a formation of a three-dimensional net of hydrogen bonds amongst the hydroxyl groups of the others interacting compounds.. In conclusion, the present study sheds some lights in understanding the formation of liposomes/TPP‑chitosan encapsulation system and testifies its efficiency in preserving entrapped lipophilic compounds, such as vitamin D3.

5. Acknowledgements This study was supported by the São Paulo Research Foundation – FAPESP (grant number 2016/18788‑1), SiSNano (MCT) and Rede AgroNano (Embrapa). The authors gratefully acknowledge the University of São Paulo for the fellowships granted to Aline S. L. Iida and Karina N. Luz, and the Coordination for the Improvement of Higher Education Personnel (CAPES) for the doctoral fellowships of Taís T. Barros-Alexandrino.

6. References 1. Bouillon, R., Okamura, W. H., & Norman, A. W. (1995). Structure-function-relationships in the vitamin-D endocrine system. Endocrine Reviews, 16(2), 200-257. http://dx.doi. org/10.1210/edrv-16-2-200. PMid:7781594. 2. Ross, A. C., Taylor, C. L., Yaktine, A. L., & Del Valle, H. B. (2010). Dietary reference intakes for Calcium and Vitamin D. Washington: National Academy Press. Retrieved in 2019, August 9, from https://www.ncbi.nlm.nih.gov/books/NBK56070/ 3. Trang, H. M., Cole, D. E. C., Rubin, L. A., Pierratos, A., Siu, S., & Vieth, R. (1998). Evidence that vitamin D-3 increases serum 25-hydroxyvitamin D more efficiently than does vitamin D-2. The American Journal of Clinical Nutrition, 68(4), 854858. http://dx.doi.org/10.1093/ajcn/68.4.854. PMid:9771862. 7/8


Iida, A. S. L., Luz, K. N., Barros-Alexandrino, T. T., Fávaro-Trindade, C. S., Pinho, S. C., Assis, O. B. G., & Martelli-Tosi, M. 4. Holick, M. F., & Chen, T. C. (2008). Vitamin D deficiency: a worldwide problem with health consequences. The American Journal of Clinical Nutrition, 87(4), 1080S-1086S. http:// dx.doi.org/10.1093/ajcn/87.4.1080S. PMid:18400738. 5. Calvo, M. S., & Whiting, S. J. (2013). Survey of current vitamin D food fortification practices in the United States and Canada. The Journal of Steroid Biochemistry and Molecular Biology, 136, 211-213. http://dx.doi.org/10.1016/j.jsbmb.2012.09.034. PMid:23104118. 6. deMan, J. M. (1999). Principles of food chemistry (3rd ed.). Gaithersburg: Aspen Publishers. http://dx.doi.org/10.1007/9781-4614-6390-0. 7. Paucar, O. C., Tulini, F. L., Thomazini, M., Balieiro, J. C. C., Pallone, E., & Favaro-Trindade, C. S. (2016). Production by spray chilling and characterization of solid lipid microparticles loaded with vitamin D-3. Food and Bioproducts Processing, 100, 344-350. http://dx.doi.org/10.1016/j.fbp.2016.08.006. 8. Jannasari, N., Fathi, M., Moshtaghian, S. J., & Abbaspourrad, A. (2019). Microencapsulation of vitamin D using gelatin and cress seed mucilage: Production, characterization and in vivo study. International Journal of Biological Macromolecules, 129, 972-979. http://dx.doi.org/10.1016/j.ijbiomac.2019.02.096. PMid:30779987. 9. Abbasi, A., Emam-Djomeh, Z., Mousavi, M. A. E., & Davoodi, D. (2014). Stability of vitamin D3 encapsulated in nanoparticles of whey protein isolate. Food Chemistry, 143, 379-383. http:// dx.doi.org/10.1016/j.foodchem.2013.08.018. PMid:24054255. 10. Luo, Y., Zeng, T., & Wang, Q. (2012). Development of zein nanoparticles coated with carboxymethyl chitosan for encapsulation and controlled release of vitamin D3. Journal of Agricultural and Food Chemistry, 60(3), 836-843. http:// dx.doi.org/10.1021/jf204194z. PMid:22224939. 11. Chaves, M. A., Oseliero, P. L., Fo., Jange, C. G., SinigagliaCoimbra, R., Oliveira, C. L. P., & Pinho, S. C. (2018). Structural characterization of multilamellar liposomes coencapsulating curcumin and vitamin D3. Colloids and Surfaces. A, Physicochemical and Engineering Aspects, 549, 112-121. http://dx.doi.org/10.1016/j.colsurfa.2018.04.018. 12. Winuprasith, T., Khomein, P., Mitbumrung, W., Suphantharika, M., Nitithamyong, A., & McClements, D. J. (2018). Encapsulation of vitamin D3 in pickering emulsions stabilized by nanofibrillated mangosteen cellulose: impact on in vitro digestion and bioaccessibility. Food Hydrocolloids, 83, 153164. http://dx.doi.org/10.1016/j.foodhyd.2018.04.047. 13. Park, S. J., Garcia, C. V., Shin, G. H., & Kim, J. T. (2017). Development of nanostructured lipid carriers for the encapsulation and controlled release of vitamin D3. Food Chemistry, 225, 213-219. http://dx.doi.org/10.1016/j.foodchem.2017.01.015. PMid:28193417. 14. Alavi, S., Haeri, A., & Dadashzadeh, S. (2017). Utilization of chitosan-caged liposomes to push the boundaries of therapeutic delivery. Carbohydrate Polymers, 157, 991-1012. http://dx.doi. org/10.1016/j.carbpol.2016.10.063. PMid:27988018. 15. Caddeo, C., Diez-Sales, O., Pons, R., Carbone, C., Ennas, G., Puglisi, G., Fadda, A. M., & Manconi, M. (2016). Cross-linked chitosan/liposome hybrid system for the intestinal delivery of quercetin. Journal of Colloid and Interface Science, 461, 69-78. http://dx.doi.org/10.1016/j.jcis.2015.09.013. PMid:26397912. 16. Du, H., Yang, X., & Zhai, G. (2014). Design of chitosan-based nanoformulations for efficient intracellular release of active compounds. Nanomedicine, 9(5), 723-740. http://dx.doi. org/10.2217/nnm.14.8. PMid:24827846. 17. Alishahi, A., Mirvaghefi, A., Tehrani, M. R., Farahmand, H., Shojaosadati, S. A., Dorkoosh, F. A., & Elsabee, M. Z. (2011). Shelf life and delivery enhancement of vitamin C using chitosan nanoparticles. Food Chemistry, 126(3), 935-940. http://dx.doi. org/10.1016/j.foodchem.2010.11.086. 18. Britto, D., Moura, M. R., Aouada, F. A., Pinola, F. G., Lundstedt, L. M., Assis, O. B. G., & Mattoso, L. H. C. (2014). Entrapment characteristics of hydrosoluble vitamins loaded into chitosan and N,N,N-trimethyl chitosan nanoparticles. Macromolecular

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Research, 22(12), 1261-1267. http://dx.doi.org/10.1007/ s13233-014-2176-9. 19. Zhou, F., Xu, T., Zhao, Y. J., Song, H. X., Zhang, L. Q., Wu, X. D., & Lu, B. Y. (2018). Chitosan-coated liposomes as delivery systems for improving the stability and oral bioavailability of acteoside. Food Hydrocolloids, 83, 17-24. http://dx.doi. org/10.1016/j.foodhyd.2018.04.040. 20. Makino, K., Yamada, T., Kimura, M., Oka, T., Ohshima, H., & Kondo, T. (1991). Temperature- and ionic strength-induced conformational changes in the lipid head group region of liposomes as suggested by zeta potential data. Biophysical Chemistry, 41(2), 175-183. http://dx.doi.org/10.1016/03014622(91)80017-L. PMid:1773010. 21. Moore, J., & Cerasoli, E. (2017). Particle Light Scattering Methods and Applications A2 - Lindon, John C. In G. E. Tranter & D. W. Koppenaal (Eds.), Encyclopedia of spectroscopy and spectrometry (3rd ed., pp. 543-553). Oxford: Academic Press. http://dx.doi.org/10.1016/B978-0-12-803224-4.00040-6. 22. Gong, G., & Bell, L. N. (2013). Degradation kinetics of rebaudioside A in various buffer solutions. International Journal of Food Science & Technology, 48(2), 2500-2502. http://dx.doi.org/10.1111/ijfs.12241. 23. Scholfield, C. R. (1981). Composition of soybean lecithin. Journal of the American Oil Chemists’ Society, 58(10), 889892. http://dx.doi.org/10.1007/BF02659652. 24. Martins, C. S., Morgado, D. L., & Assis, O. B. G. (2016). Cashew gum-chitosan blended films: Spectral, mechanical and surface wetting evaluations. Macromolecular Research, 24(8), 691-697. http://dx.doi.org/10.1007/s13233-016-4103-8. 25. Nzai, J. M., & Proctor, A. (1999). Soy lecithin phospholipid determination by fourier transform infrared spectroscopy and the acid digest/arseno-molybdate method: a comparative study. Journal of the American Oil Chemists’ Society, 76(1), 61-66. http://dx.doi.org/10.1007/s11746-999-0048-9. 26. Tantipolphan, R., Rades, T., McQuillan, A. J., & Medlicott, N. J. (2007). Adsorption of bovine serum albumin (BSA) onto lecithin studied by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. International Journal of Pharmaceutics, 337(1), 40-47. http://dx.doi.org/10.1016/j. ijpharm.2006.12.021. PMid:17240095. 27. Ding, G.-J., Zhu, Y.-J., Qi, C., Lu, B.-Q., Chen, F., & Wu, J. (2015). Porous hollow microspheres of amorphous calcium phosphate: soybean lecithin templated microwave-assisted hydrothermal synthesis and application in drug delivery. Journal of Materials Chemistry B: Materials for Biology and Medicine, 3(9), 1823-1830. http://dx.doi.org/10.1039/C4TB01862A. 28. Kiani, A., Fathi, M., & Ghasemi, S. M. (2017). Production of novel vitamin D3 loaded lipid nanocapsules for milk fortification. International Journal of Food Properties, 20(11), 2465-2674. http://dx.doi.org/10.1080/10942912.2016.1240690. 29. Othayoth, R., Mathi, P., Bheemanapally, K., Kakarla, L., & Botlagunta, M. (2015). Characterization of vitamin-cisplatinloaded chitosan nano-particles for chemoprevention and cancer fatigue. Journal of Microencapsulation, 32(6), 578-588. http:// dx.doi.org/10.3109/02652048.2015.1065921. PMid:26218628. 30. Martins, A. F., Oliveira, D. M., Pereira, A. G. B., Rubira, A. F., & Muniz, E. C. (2012). Chitosan/TPP microparticles obtained by microemulsion method applied in controlled release of heparin. International Journal of Biological Macromolecules, 51(5), 1127-1133. http://dx.doi.org/10.1016/j.ijbiomac.2012.08.032. PMid:22975304. 31. Lawrie, G., Keen, I., Drew, B., Chandler-Temple, A., Rintoul, L., Fredericks, P., & Grøndahl, L. (2007). Interactions between alginate and chitosan biopolymers characterized using FTIR and XPS. Biomacromolecules, 8(8), 2533-2541. http://dx.doi. org/10.1021/bm070014y. PMid:17591747. Received: June 23, 2019 Revised: Aug. 09, 2019 Accepted: Oct. 01, 2019

Polímeros, 29(4), e2019049, 2019


ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.03119

Application of metric entropy to determine properties of structural materials Grzegorz Garbacz1* and Lesław Kyzioł2  Faculty of Production Engineering and Logistics, University of Technology, Opole, Poland 2 Faculty of Mechanical Engineering, Gdynia Maritime University, Gdynia, Poland

1

*g.garbacz@po.opole.pl

Abstract Composite materials have nowadays become a group of construction materials whose application in mechanical structure designing has been constantly increasing. There is therefore a real demand for an objective opinion on the mechanical properties of composites. According to the authors, determining those properties based on the recommended methods included in current standards requires objectivization. The difficulty is that those methods are usually based on the geometrical shape of stress curves in the strain function. The study proposes an new method of testing the mechanical properties of composites through analyzing the internal dynamics of measurement data based on uniaxial stretching tests. A tool in such an analysis is determining the Kolmogorov-Sinai metric entropy values of measurement data. Nine samples of composite materials having various compositions have been tested. In selecting these materials, the focus has been on the possibility of introducing composite recyclates as their structural components. Keywords: designing materials, Kolmogorov-Sinai metric entropy, mechanical properties, recycling, stretching curve. How to cite: Garbacz, G., & Kyzioł, L. (2019). Application of metric entropy to determine properties of structural materials. Polímeros: Ciência e Tecnologia, 29(4), e2019050. https://doi.org/10.1590/0104-1428.03119

1. Introduction Composite materials have been widely used in engineering applications as structural components. Property analysis of composite materials in terms of their engineering applications has an extensive bibliography. Referring to examples, the experimental methods of composite mechanics have been presented in the report[1], the mechanical properties of composites and the methods of damage assessment have been presented included in the report[2]. Computational analysis of the relationships between the structure and mechanical properties of the composite are discussed in[3]. The methods which allow to improve the desired properties of the composite are described in[4]. An experimental investigation on mechanical properties of the hybrid polymer nanocomposite has been recently reported in[5]. The analysis of fatigue damage has been presented in the report[6]. The  difficulties related to recycling as well as polymer and composite utilization have been presented in the report[7]. However, determining material constants of composites involves certain difficulties stemming from their structural specifics. Moreover, the composite stretching curve usually does not include characteristic points reflecting internal structural strain. A clear damage to the composite sample usually occurs instantly, just as in case of brittle materials. Engineering calculations, however, require a determination of the stage in the composite material deformation process, right before its destruction, at which essential changes from the point of view of its durability and strength occur. The key is to calculate the metric entropy of data measurement subsets based on the formula proposed by

Polímeros, 29(4), e2019050, 2019

Kolmogorov-Sinai (K-S). The essence of metric entropy is that its nature is dynamic, as it describes system movement typical for chaotic processes. Metric entropy measures the dynamic instability of a system, i.e. expresses a method of describing chaos in a quantitative way[8]. The report[9] presents the results of mechanical testing of structural materials and the prepared mathematical model supported by dedicated computer software, one that allows for calculating the Kolmogorov-Sinai entropy for the results of those tests. The calculations included data sets consisting of several hundred to over a dozen thousand measurement points. Based on the K-S entropy calculations, the report[10] includes determining material constants for metals and the report[11] for composites. Both analyses involved stretching samples, controlled by extensometer signal. Additionally, the report[11] analyzes phase images of metric entropy. The purpose of this article is to present a new method consisting in converting measurement data based on their internal strain dynamics for newly-invented composites[12].

2. Materials and Methods 2.1 Own research New composite materials[12] subjected to testing have been designed and produced in the laboratory of the Faculty of Mechanics, Technology Basics Institute at the Maritime Academy in Gdynia. Mechanical tests were carried out on

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O O O O O O O O O O O O O O O O


Garbacz, G. & Kyzioł, L. three composite materials whose structures were filled with different recyclate types. The composites consisted of the following components: Polimal 109A resin serving as the matrix, a glass mat serving as reinforcement and recyclate serving as the filler. The recyclate has been obtained through grinding industrial waste in the form of polyester-glass laminates and screening using a size 1.2 x 1.2 mm square mesh sieve.

also to determine the influence of the filler (recyclate) on the overall strength of the composite. Selected, typical parameters of Polimal 109 have been included in Table 2. This data originates from the product sheets, which describes only their indicative nature. The shape and dimensions of samples, measured prior to the test, have been presented in Figure 1. Samples were prepared by waterjet cutting with the greatest possible care. Sample cross-section dimensions, recognized as significant considering the calculation of nominal stress, have been measured with the accuracy of 0.01 mm and have been included in Table 3. The aim of this analysis is to present a test method, not to determine material constants intended for presentation in construction material tables, as it would also require conducting tests on a much larger number of samples.

The type A Polimal 109 composite component is an all-purpose, non-saturated, polyester structural resin. This resin is recommended for the manual manufacturing of large format, glass-fiber laminates. The percentage by weight contents of glass mat and other components has been included in Table 1. The materials obtained have been designated a K10, K30 and K0. The purpose of the tests is

Figure 1. Sample geometry. Table 1. Sample composition. Composite

Sample no.

Resin (%)

Glass mat (%)

K10 K30 K0

1, 2, 3 4, 5, 6 7, 8, 9

61.5 61 62

27 8 37

Glass mat layer number 10 2 12

Fiber (%) 10.5 30 0

Hardner and accelerant(%) 1 1 1

Table 2. Selected parameters of Polimal 109 resin. Parameter tensile strength tensile elongation Toughness Barcol hardness

Norm ISO 527 ISO 527 ISO 179 ASTM D 2583

Unit MPa % kJ / m2 o B

Value 70 1,8 10 42

Table 3. Sample dimensions. Sample number 1 2 3 4 5 6 7 8 9

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a 70 70 70 70 70 70 70 70 70

b 7.24 6.33 7.75 6.83 8.42 7.85 7.55 7.40 7.04

Dimensions (mm) c d 240 20 240 20 240 20 240 20 240 20 240 20 240 20 240 20 240 20

h 9.44 9.80 9.88 9.99 9.98 9.97 10.05 10.03 9.55

R 50 50 50 50 50 50 50 50 50

Polímeros, 29(4), e2019050, 2019


Application of metric entropy to determine properties of structural materials In accordance with the data included in Table 1, 3 and 4, nine samples underwent uniaxial stretching tests. For the purposes of calculations and the presentation of results included in this article, 18 registered records of data have been used, meaning 2 data records of force and strain have been made for each sample. The number of sets corresponds to the number of measurement points, and it is the same for a single test conducted until material destruction. Strain

was recorded using an extensometer with a 50 mm base. The average strain rate and the sample total time of stretching are presented in Table 4. The static stretching test results for each sample were used as the bases for mathematical calculations aiming at determining the changes within Kolmogorov-Sinai metric entropy. The stretching tests have been conducted by controlling the applied force of the Zwick/Roell MPMD P10 B type HB 100 universal testing machine. The standard recommendations (DIN EN ISO 527-1) for testing have been observed. The tests have been carried out at the Faculty of Marine Engineering at Gdynia Maritime University. Composite material samples have been subjected to uniaxial stretching. The figures (Figure  2) present sample scraps after rupture. Clear differences between samples containing recyclate and samples without recyclate, considering their external surfaces, are observable. The external surfaces of samples without recyclate (K0) are bright, and the surfaces of samples containing recyclate (K10 and K30) include bright grains of grinded polyester-glass dust.

2.2 Kolmogorov-Sinai metric entropy notion The Kolmogorov-Sinai metric entropy[13,14] which, for the discrete probability distribution, is expressed by the following Equation 1: N

S = − ∑ pi lnpi i =1

Figure 2. Image of sample scraps after rupture, in accordance with the numbering provided in Table 1 and Table 3. Table 4. Average strain rate and total time of stretching test. Sample number 1 2 3 4 5 6 7 8 9

Total time of stretching test (s) 124 121 137 55 62 75 130 145 136

Polímeros, 29(4), e2019050, 2019

Strain rate (s-1) 1.8·10-4 1.5·10-4 1.7·10-4 2.2·10-4 1.5·10-4 1.2·10-4 1.4·10-4 1.5·10-4 1.1·10-4

(1)

where: N - number of subintervals, into which the result measurement data set has been divided. The Kolmogorov‑Sinai measurement data set cardinality is indirect. At first, the cardinality is used to calculate the pi probability. Measurement data sets in reports are designated by the letter k. In fact, those are subsets distinguished from an entire record of test data and designated by the letter n. pi is probability of results in an ith interval (where the definition assumes p ln p ≡ 0, if p = 0). If the intervals are equally likely, meaning pi = 1/N for all instances of i, the entropy is expressed by formula S = ln N and assumes a maximum value. However, if the results are included in specific intervals, the entropy assumes a minimum value, S = 0 because pi = 1. K-S metric entropy is dynamic by nature; therefore, it is useful for describing and analyzing phenomena of that exact nature, e.g. construction material strain. Fluctuation of metric entropy involves changes within the dynamics of physical strain processes as well as includes dissipation of energy that accompanies those changes. Kolmogorov-Sinai entropy is calculated multiple times, each time for a certain number of subsets having a fixed, heuristically assumed cardinality of k, distinguished from the strain set n possible results registered during the stretching test. Internal dynamics of strains has been analyzed as the tests have been conducted by controlling the stretching force. In this case, the force diagram in the function of measurement points is linear. If the tests were conducted through controlling the stretching using the extensometer signal, it would be necessary to analyze the internal dynamic changes within the force or nominal stress. If an additional 3/9


Garbacz, G. & Kyzioł, L. transverse extensometer was used, it would be possible to analyze the actual stress. The analysis described in this article has been based on strain signal data records obtained through tests conducted using a single extensometer mounted longitudinally. In order to better illustrate the calculation process, k-element subsets are compared to a “caterpillar” crawling over an n element measurement data set. The “caterpillar” makes n minus k steps. The distinguished k element subsets are each time divided into N subintervals to perform the calculations of Kolmogorov-Sinai entropy. For each test, N has a heuristically assigned value and is fixed. Each position of

Figure 3. Demonstration of k subset “movement”.

a k set that determines its content allows for calculating the position of a single point on the entropy diagram. K-S metric entropy diagram in the function of subsequent measurement points consists of n minus k points. This situation has been schematically shown in Figure 3 and Figure 4.

2.3 Method of calculating a single entropy value corresponding to a single diagram point The cardinality of an n strain measurement data record and the cardinality of k-element subsets as well as the number of N subintervals to which the k-element subsets have been divided in relation to the strains of the examined sample 1 have been assumed in accordance with Table 5(first row). For a selected subset of k = 100 data, from measurement point 6301 to point 6400, a single value of K-S entropy has been calculated. The subset has been sorted in an ascending order from the minimum value to the maximum value. Based on the difference between max and min values, the limits for N =4 subintervals have been calculated, meaning the difference has been divided by 4. Point 6351 has been adopted as the central point of the subset and the K-S entropy for this point, and, based on the performed calculations as per Equation 2, equals 1.381. Subintervals 1, 2 and 3 are left-closed and right-open. The final subinterval is closed on both ends. The described actions have been presented in Table 6. Finally, the K-S entropy is: 4

S = − ∑ pi lnpi = −(− 0.353 − 0.328 − 0.353 − 0.347 ) ≈ 1.381 (2) i =1

According to Table 5, the n strain measurement data record for sample 1 equals 7077 points. The complete diagram of K-S entropy values presented in Figure  5 consists of 6977 points. The reason for this is that the k value has been deducted from the n value, meaning 100 points have been deducted from 7077 and the result of 6977 has been obtained.

3. Results and Discussions

Figure 4. Schematic presentation of the K-S entropy diagram development.

In the course of uniaxial stretching tests, records of force data and corresponding strain of the extensometer have been registered for each of the 9 composite samples presented in Figure 2. Stress has been calculated considering the data from cross-section areas of samples. Figures 6-8 present the results of tests in a coordinate system of strain - stress. The results from static stretching tests have proved that the highest mechanical properties were found in the

Table 5. n, k and N figures referring to the subsequent sample tests. Sample number 1 2 3 4 5 6 7 8 9

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Total number of measurement points n 7077 5189 6438 1782 2093 2158 7659 8168 7232

Number of points for subset k

Number of subset’s partitions N

100 100 100 80 80 80 100 100 100

4 4 4 4 4 4 4 4 4

Polímeros, 29(4), e2019050, 2019


Application of metric entropy to determine properties of structural materials K0 designated composite (recyclate 0%), a bit lower in K10 composite and significantly lower in K30 composite. In composite K30, a high amount of recyclate (30%) and a low amount of glass mat, which is basically responsible for bearing the loads, caused the structure of such composite to display low strength properties and very low plasticity. Strain, stress and K-S entropy in the function of subsequent measurement data diagrams have been prepared in order

to use the metric entropy calculations to detect changes within the structure of composites assessed based on the measurement data dynamics. Table 5 presents the records of n number of measurement points registered during each test as well as optional values k and N, adopted heuristically based on the observation of entropy diagram sharpness and expressiveness. The k values should be high enough to determine subintervals and to calculate probability, similarly

Table 6. Organized measurement data of strains and the calculation of probability for each subinterval. subinterval boundaries of subintervals measurement data of deformations

probability pi of finding the results of the measurement in the ith subinterval

pi ln pi

1 0.016638 0.016753 0.016638 0.016644 0.016650 0.016654 0.016658 0.016662 0.016666 0.016670 0.016674 0.016678 0.016681 0.016685 0.016688 0.016693 0.016697 0.016701 0.016705 0.016708 0.016712 0.016717 0.016722 0.016727 0.016734 0.016738 0.016742 0.016746 0.016750

2 0.016753 0.016868 0.016753 0.016757 0.016760 0.016764 0.016768 0.016772 0.016777 0.016781 0.016785 0.016790 0.016795 0.016799 0.016806 0.016816 0.016825 0.016831 0.016837 0.016841 0.016845 0.016853 0.016867

3 0.016868 0.016982 0.016873 0.016878 0.016882 0.016886 0.016890 0.016895 0.016901 0.016906 0.016912 0.016916 0.016920 0.016924 0.016928 0.016933 0.016937 0.016941 0.016944 0.016948 0.016952 0.016956 0.016959 0.016961 0.016964 0.016968 0.016971 0.016975 0.016978

4 0.016982 0.017097 0.016982 0.016985 0.016989 0.016992 0.016996 0.017000 0.017005 0.017010 0.017017 0.017025 0.017031 0.017036 0.017040 0.017045 0.017049 0.017055 0.017061 0.017067 0.017073 0.017077 0.017081 0.017085 0.017090 0.017093 0.017097

27/100

21/100

27/100

25/100

0.353

0.328

Figure 5. Diagram presenting the strain and K-S entropy for sample 1 with the marked set of k = 100 measurement points. Polímeros, 29(4), e2019050, 2019

0.353

amount of points

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

0.347

Figure 6. K10 composite samples stretching diagrams. 5/9


Garbacz, G. & Kyzioł, L. to developing a histogram. When excessive k values are adopted, the local dynamics is lost and the K-S entropy diagram does not include clear local minima. The adopted N value bears similar significance. Generally, higher k and N values are adopted for a large n record, meaning over a dozen thousand data. The method is not applicable to a small n number e.g. 100 measurement points. In data records of approx. 20,000 measurement points, it is usually difficult to distinguish between measurement noise and the response of the tested material. The purpose of the diagrams is to search for clear metric entropy drops, which serve as the basis for conclusions related to the material structure change.

Figure 7. K30 composite samples stretching diagrams.

Based on the shape of the K-S entropy diagram as well as Figure 9 and Figure10, a conclusion was drawn that the internal composite material damage begins at the 5,359 measurement point, which corresponds to the relative strain of 0.013 and a 79 MPa stress. Another significant entropy drop has been identified at point 5536, to which the relative strain of 0.14 and an 82 MPa stress correspond. A drastic drop in the entropy value can be observed right before sample rupture and applies to all tested samples. Based on the shape of the K-S entropy diagram as well as Figure 11 and Figure 12, a conclusion was drawn that the internal composite material damage begins at the test

Figure 10. Stress diagram in the function of subsequent measurement points for sample 1.

Figure 8. K0 composite samples stretching diagrams. Figure 11. K-S metric entropy diagram against relative strain for sample 2.

Figure 9. K-S metric entropy diagram against relative strain for sample 1. 6/9

Figure 12. Stress diagram in the function of subsequent measurement points for sample 2. Polímeros, 29(4), e2019050, 2019


Application of metric entropy to determine properties of structural materials starting point, in the area marked orange. However, the situation stabilizes around point 1700 and continues until measurement points 3854 and 4628 where major entropy drops are visible, which corresponds to relative strains of 0.013 and 0.014 as well as 63 and 75 MPa stresses. Figure 13 presents one major entropy drop corresponding to point 6276. The point corresponds to strain equal to 0.0205. Based on Figure 14 a stress equal to 87 MPa has been determined. The phenomenon occurred right before sample rupture. Figure 15 presents an entropy drop related to the final stretching stage, meaning sample rupture. This constitutes the 1406 measurement point as well as the corresponding relative strain of 0.0056, and according to Figure 16, stress equal to 21.3 MPa. Similarly to Figure 15 and Figure 16, Figure 17 and Figure 18 present an entropy drop related to the final stretching stage, meaning sample rupture. The 1905 measurement point as well as the corresponding relative strain of 0.0073, and according to Figure 18, stress equal to 23.54 MPa have been determined as demarcation points. The behaviour of the sample 6 during the tests followed that recorded for the sample 5 (respective diagrams are not shown). For the sample 6, it was found that the relative strain is 0.0084 and stress equals 27 MPa.

Figure 13. K-S metric entropy diagram against relative strain for sample 3.

Figure 14. Stress diagram in the function of subsequent measurement points for sample 3. Polímeros, 29(4), e2019050, 2019

The process of composite internal structure change within sample 7 starts with the beginning of stretching, which has been marked orange on Figure 19. The damage development and halt schemes are very similar to sample 2. Moreover, two significant measurement points have been distinguished – 5768, where the internal composite structure changes occur, and 6619 as the starting point for a definitive sample destruction. The stress and strain values for those points have been presented in Figure  19 and Figure  20. The results obtained for the sample 8 (not shown) are the same within a few percents as those which were determined for the sample 7. Figure  21 presents the 6029 measurement point that corresponds to the commencement of composite structure changes and point 7029 that directly precedes the sample destruction. Proper strain and stress values have been provided in Figure 21 and Figure 22. The described method is based on the entropy of macroscopic quantity, i.e., the mechanical stress of the sample. Since the sample break originates from its structural changes at the microscopic level, therefore, our method may provide an insight into the relationship between the mechanical properties of composites and their structure. However, further studies are required for this purposes, e.g., simultaneous recording of the stress of the sample and the changes of the sample structure by non-destructive testing methods.

Figure 15. K-S metric entropy diagram against relative strain for sample 4.

Figure 16. Stress diagram in the function of subsequent measurement points for sample 4. 7/9


Garbacz, G. & KyzioĹ&#x201A;, L.

Figure 17. K-S metric entropy diagram against relative strain for sample 5. Figure 21. K-S metric entropy diagram against relative strain for sample 9.

Figure 18. Stress diagram in the function of subsequent measurement points for sample 5.

Figure 22. Stress diagram in the function of subsequent measurement points for sample 9.

4. Conclusions 1. The presented method for determining the mechanical properties of composite materials can be used in the process of structural design.

Figure 19. K-S metric entropy diagram against relative strain for sample 7.

2. Due to the fact that the standards related to the strength of polymer composites basically do not consider the plasticity limit, this method becomes useful in the process of designing structures made of those materials. 3. The proposed method allows an objective assessment of the composite composition and the usefulness of material share from their recycling, considering designing materials having the required construction properties. 4. The proposed method of presenting the changes within the dynamics of data received based on strength tests conducted in laboratories or based on measuring actual objects may be used in design and modelling.

Data availability statement Figure 20. Stress diagram in the function of subsequent measurement points for sample 7. 8/9

The raw data required to reproduce these findings are available on request. The processed data required to reproduce these findings are available on request. PolĂ­meros, 29(4), e2019050, 2019


Application of metric entropy to determine properties of structural materials

5. References 1. Ochelski, S. (2004). Experimental methods of mechanics incomposite materials research Metody doświadczalne mechaniki kompozytów konstrukcyjnych, Warszawa: Wydawnictwo Naukowo-Techniczne. 2. Szymczak, T., & Kowalewski, Z. L. (2014). Mechanical properties of selected composites and methods of their fracture assessment. Transport Samochodowy, 4, 33-54. 3. Mishnaevsky, L. Jr, Mikkelsen, L. P., Gaduan, A. N., Lee, K.-Y., & Madsen, B. (2019). Nanocellulose reinforced polymer composites: computational analysis of structuremechanical properties relationships. Composite Structures, 224, 111024-111032. http://dx.doi.org/10.1016/j. compstruct.2019.111024. 4. Jong, L. (2019). Improved mechanical properties of silica reinforced rubber with natural polymer. Polymer Testing, 79, 106009. http://dx.doi.org/10.1016/j.polymertesting.2019.106009. 5. Kumar, G. N., Kumar, C. S., & Seshagiri Rao, G. V. R. (2019). An experimental investigation on mechanical properties of hybrid polymer nanocomposites. Materials Today: Proceedings, 19(2), 691-699. http://dx.doi.org/10.1016/j. matpr.2019.07.755. 6. Rutecka, A., Kowalewski, Z. L., Makowska, K., Pietrzak, K., & Dietrich, L. (2015). Fatigue damage of Al/SiC composites – Macroscopic and microscopic analysis. Archives of Metallurgy and Materials, 60(1), 101-105. http://dx.doi.org/10.1515/ amm-2015-0016. 7. Błędzki, A. K., Gorący, K., & Urbaniak, M. (2012). Possibilities of recycling and utilization of the polymeric materials and composite products. Polimery, 57(9), 620-626. http://dx.doi. org/10.14314/polimery.2012.620.

Polímeros, 29(4), e2019050, 2019

8. Dietrich, L., & Garbacz, G. (2008). Chaos taken into account in measurement of physical quantities. In: Proceedings of the 25th Danubia-Adria Symposium on Advances in Experimental Mechanics (p. 53-54). České Budějovice and Český Krumlov, Czech Republic: Czech Technical University in Prague Faculty of Mechanical Engineerin. 9. Garbacz, G. (2009). Processing of experimental data taking into account their chaotic nature (Doctoral thesis). Institute of Fundamental Technological Research Polish Academy of Sciences, Warsaw, Poland. 10. Garbacz, G., & Kyzioł, L. (2014). Determination of yield point of structural materials with using the metric entropy. Journal of KONES Powertrain and Transport, 21(3), 97-104. http:// dx.doi.org/10.5604/12314005.1133176. 11. Garbacz, G., & Kyzioł, L. (2017). Application of metric entropy for results interpretation of composite materials mechanical tests. Advances in Materials Science, 17(1), 70-81. http:// dx.doi.org/10.1515/adms-2017-0006. 12. Panasiuk, K., & Hajdukiewicz, G. (2017). Production of composites with added waste polyester-glass with their initial mechanical properties. Scientific Journals of the Maritime University of Szczecin, 57(124), 30-36. http://dx.doi.org/10.17402/242. 13. Kolmogorov, A. N. (1959). Entropy per unit time as a metric invariant of automorphism. Doklady of Russian Academy of Sciences, 124, 754-755. 14. Sinai, Y. G. (1959). On the notion of entropy of a dynamical system. Doklady of Russian Academy of Sciences, 124, 768771. Received: May 26, 2019 Revised: Sept. 21, 2019 Accepted: Oct. 14, 2019

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ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.03319

Stabilization of gelatin and carboxymethylcellulose water-in-water emulsion by addition of whey protein Mayara Rocha Laranjo1, Bernardo de Sá Costa2 and Edwin Elard Garcia-Rojas1,2*  Pós-graduação em Engenharia Mecânica – PGMEC, Escola de Engenharia, Universidade Federal Fluminense – UFF, Volta Redonda, RJ, Brasil 2 Departamento de Engenharia de Agronegócios – VEA, Universidade Federal Fluminense – UFF, Volta Redonda, RJ, Brasil

1

*edwinr@id.uff.br

Abstract Due to their aqueous nature and biocompatibility, water/water emulsions are particularly advantageous in the production of low calorie functional food and bioactive carrier microparticles. The aim of this study was to investigate the stability of water/water emulsions formed by gelatin and carboxymethycelullose through the Pickering effect, by addition of whey protein particles. The effect of phase composition and pH on emulsion stability over 3 days of storage was studied and the emulsion properties were characterized. Finally, the effect of the addition of different concentrations of whey protein particles on the emulsion stability was investigated. The added protein particles contributed to reduce the rate of phase separation and higher protein concentration showed this effect more clearly. The time of complete phase separation increased 12 h after addition of 15% (w/w) protein. Emulsions at pH 5.5 with protein particles, however, showed lower stability than those at pH 7.5 without particles. Keywords: biocompatibility, biopolymer, Pickering, phase separation. How to cite: Laranjo, M. R., Costa, B. S., & Garcia-Rojas, E. E. (2019). Stabilization of gelatin and carboxymethylcellulose water-in-water emulsion by addition of whey protein. Polímeros: Ciência e Tecnologia, 29(4), e2019051. https://doi. org/10.1590/0104-1428.03319

1. Introduction Kinetically stable water/water (W/W) emulsions have been focus of great interest due to their strategic potential application in cosmetics[1], foods[1-3] and biomedicine[4,5]. Originated from an aqueous binary polymeric system, W/W emulsions can be formed by completely biocompatible ingredients, such as proteins and polysaccharides[6], and are ideal for the formulation of fat free food or for the encapsulation of sensitive ingredients, such as cells and proteins[5-9]. Several studies on the properties and applications of W/W emulsions have been reported in the literature[1-9]. Because W/W emulsions are completely aqueous system, with extremely low interfacial tension and an ill- defined interface, they cannot be stabilized by conventional surfactants, as commonly occurs in water/oil (W/O) and oil/water (O/W) emulsions[1-14]. One of the notable mechanisms of stabilization is the Pickering method, in which a solid particle added to the system adsorbs at the droplet interface and provides a physical barrier against droplet coalescence[1-14]. Recent advances in the research of different substances to adsorb at interfaces between immiscible aqueous solutions have been reliably reported by Dickinson[14]. Protein microgels are an example that has already proved to be efficient in stabilizing W/W emulsions[10-12]. In this context, the present study aims to produce and characterize W/W emulsions from an aqueous two-phase

Polímeros, 29(4), e2019051, 2019

system composed of gelatin and carboxymethylcellulose and evaluate the effect of whey protein on kinetic stability.

2. Materials and Methods 2.1 Materials Gelatin type B and sodium carboxymethylcellulose (NaCMC) of medium viscosity (400-800 cps), purchased from Sigma Aldrich (Saint Louis, USA), and whey protein isolate (WPI) 90% (Glanbia Nutritionals, Springfield, United States) were used for the preparation of emulsions. Sodium chloride (NaCl), sodium hydroxide (NaOH), hydrochloric acid (HCl) and sodium azide were purchased from Vetec Química Fina (Duque de Caxias, Brazil). All solutions were prepared in ultrapure water with a conductivity of 0.05 ± 0.01µS.cm-1 (Gehaka-Master P&D, Brazil).

2.2 Molecular weight determination The average viscosity molecular weight of gelatin and NaCMC was determined by viscometric measurements[15]. Samples of gelatin (1-10 g.cm-3) and NaCMC (0.4 – 1.0 g.cm-3) were prepared using aqueous solutions of NaCl 0.1 mol.L-1as solvent. The relative viscosity was measured with a capillary (Schott, Cannon Fenske, Germany) in a thermostatic water bath at 25.0 ± 0.1°C (Schott, CT52, Germany). Intrinsic viscosity [ƞ] is defined as (Equation 1):

1/8

O O O O O O O O O O O O O O O O


Laranjo, M. R., Costa, B. S., & Garcia-Rojas, E. E. = [η ]

(η= sp / C )C →0

(ηred )C →0

(1)

The intrinsic viscosity was obtained by extrapolating the reduced viscosity, ƞRED ,vs the concentration (C) data to zero concentration. The intercept on the abscissa is the intrinsic viscosity. The average viscosity molecular weight was calculated based on the Mark-Houwink-Sakurada equation (Equation 2):

[η ] = kM v a

(2)

where the constants k=2.69x10-3 and a=0.88 are defined for gelatin[16] and k=1.23x10-2 and a=0.91 for NaCMC[17].

2.3.1. Phase diagram construction A phase diagram was constructed by visually observing the formation of two distinct layers and was used to choose the composition of the W/W emulsions[18]. A stock solution of gelatin (16% w/w) was prepared by diluting the protein in ultrapure water at 60.0 ± 1.0 °C under magnetic stirring for 1 h. A stock solution of NaCMC (2% w/w) was prepared by dilution in ultrapure water at room temperature under magnetic stirring for 12 h. Sodium azide (0.05% w/w) was added to prevent the microbial growth. The pH was adjusted to 6.0 by the addition of NaOH or HCl. The stock solutions were diluted to the appropriate ratio in a transparent glass tube and left in a thermostatic water bath (Huber, Germany) at 45.0 ± 0.1 °C, avoiding any gelation. The mixtures were stirred in a vortex (Phoenix, AP56, Brazil) for 1min and stored in a water bath for 48 h with temperature controlled at 45.0 ± 0.1 °C. 2.3.2. Emulsion preparation To induce the segregative phase separation (repulsive interactions between the polymers), which is necessary to form W/W emulsions[19], the pH of the stock solutions was adjusted to 5.5, 6.5 or 7.5. The solutions were placed in a glass tube (11 mm diameter and 150 mm height) and vortexed (Phoenix, AP56, Brazil) for 1 min. Thereafter, the emulsions were stored in a water bath (Huber, Germany) for 48 h with temperature controlled at 45.0 ± 0.1 °C. 2.3.3. Kinetic stability The kinetic stability of the emulsions was evaluated by visually observing the formation of two distinct layers over 3 days of storage[20]. The phase separation is expressed by the separation index (SI), which is calculated as the relation between the upper phase volume (V) and the original volume (Vo), as described by Equation 3[21,22]: V x100 V0

(3)

2.4 Emulsion characterization The emulsion properties were characterized only for those that remained stable for a period longer than 8 h. All measurements were performed immediately after emulsion preparation. 2/8

The superficial tension of the gelatin and NaCMC aqueous solutions was measured with the pendant drop method using a tensiometer with (Teclis Scientific, Easytrack, France) connected with a thermostatic water bath (Julabo, Corio-CD BC4, Germany) at 45.0 ± 0.1 °C. To create the air-water interface, a bubble was formed at the end of a needle connected with a syringe and immersed in a glass cuvette filled with a gelatin solution (6%, 8%, 10% and 12% w/w) or NaCMC solution (0.10%; 0.25%; 0.50% and 1.00% w/w). The superficial tension was determined by bubble shape analysis and measured during 1300 s from bubble formation. 2.4.2. Particle size and ζ potential analysis

2.3 Emulsion stability

SI =

2.4.1. Dynamic superficial tension

The size, size distribution and ζ potential were determined by laser diffraction using a Zetasizer (Malvern Instruments, Nano ZS90, England). Before the measurement, the emulsions were diluted in ultrapure water at a ratio 1:10 (v/v). The optical properties adopted were refraction index (1.335) and absorption (0.01). The results obtained correspond to the mean and corresponding standard deviation of three replicates. 2.4.3. Viscosity measurements The viscosity of the emulsions was measured by a rotational viscometer (Thermo Fisher Scientific, Haake Viscotester D, Germany) using the LCP spindle at different speeds. The viscosity and percentage of torque were manually recorded when the viscosity reading reached apparent equilibrium. The measurement temperature was controlled at 45.0 ± 0.5 °C with a circulating water bath (Quimis, 0214M2, Brazil). The measurements reported correspond to an average of three replicates.

2.5 Effect of the addition of whey protein isolate on emulsion stability The effect of the addition of WPI on emulsion stability was tested on samples that showed poor stability in previously performed tests. The preparation of WPI microgel particles was based on the method of Murray and Phisarnchananan[10]. A WPI solution (10% w/w) was prepared by the dispersing the protein in ultrapure water under magnetic stirring for 12 h. The solution was transferred to a glass bottle, heated in a thermostat water bath (Huber, Germany) at 90.0 ± 1.0 °C for 30 min and suddenly cooled under running water for 15 min. The gel formed was roughly broken with a spatula to obtain fine gel fragments which were diluted in water and homogenized by an Ultraturrax system (IKA, T25D, Germany) for 5 min at 10,000 rpm, and again by ultrasound (Hielscher, UP100H, Germany) for 2 min with an amplitude of 100%. The suspension obtained was centrifuged (Digicen 21R, Spain) with the RT504 rotor at 9,000 rpm until the microgel sedimented to leave a clear upper aqueous phase which was carefully removed via a pipette. To prepare the emulsions, different concentrations of the WPI microgel particles (5, 10 and 15% w/w) were added to the aqueous system of gelatin-NaCMC before homogenization in the vortex. The influence of the WPI on emulsion stability was statistically evaluated by application of the analysis of variance (ANOVA) using the software Statistical Analysis System version 9.2 (SAS Institute Inc, Cary, NC). Polímeros, 29(4), e2019051, 2019


Stabilization of gelatin and carboxymethylcellulose water-in-water emulsion by addition of whey protein immiscible aqueous phases caused by repulsive interactions between the polymers[19].

3. Results and Discussion 3.1 Molecular weight determination The intrinsic viscosity [ƞ] and the average viscosity molecular weight (Mv) of gelatin and NaCMC are presented in Table 1. In the literature, it is possible to find a wide variety of molecular weight values for gelatin, resulting from a polydisperse protein with broad molecular weight distribution in solution[23]. According to Ledward[24], the molecular weight of gelatin type B can vary between 100 and 500 kDa. Riihimaki[16] determined the molecular weight of gelatin type B from different origins using the viscometer method and found values between 45 and 170 kDa and Masuelli[25] found [ɳ]=48.65 cm3.g-1 in a 0.01 mol.L-1 NaCl solution and Mv=67.44 kDa, using the same method.

NaCMC, as well as many other derivative polysaccharides, has a heterogeneous molecular weight distribution and chemical composition, which explain the diversity of molecular weight values found in the literature. Vázquez et  al.[26] characterized the average molecular weight of NaCMC of medium viscosity using a capillary viscometer and found [ƞ]=535 mL.g-1 and Mv=124.94 kDa. Sharma  et  al.[27] determined [ɳ]=198 cm3.g-1 and Mv=90 kDa and Gomez‑Diáz and Navaza[28] found [ɳ]=643.9 cm3.g-1 and Mv=386 kDa. Rinaudo  et  al.[29] characterized CMC samples by size exclusion chromatography and found molecular weights between 55.83 and 578.58 kDa. CMC is highly heterogeneous polymer whose molecular weight depends on the internal structure, mainly the degree of polymerization and the degree of substitution[30].

3.2 Emulsion stability 3.2.1 Phase diagram construction Figure 1 shows the visual phase diagram constructed for gelatin and NaCMC solutions in water at pH 6.0 and 45.0 ± 0.1 °C. According to the phase diagram, two distinct regions could be visualized: a one-phase region (homogeneous system), corresponding to the area below the binodal line, and a two-phase region (non-homogeneous system), corresponding to the area above the bimodal line. At relatively low gelatin and NaCMC concentrations the systems formed a single phase. The minimum concentrations for phase separation are approximately 3.0% (w/w) gelatin and 0.1% (w/w) NaCMC. Furthermore, higher gelatin concentrations increased the minimum concentration of NaCMC necessary for macroscopic phase separation to occur. Soon after preparation, the solutions in the one‑phase region appeared clear, and those in the two-phase region initially appeared turbid followed by macroscopic phase separation after a few hours. This behavior suggests a segregative phase separation, with the formation of two

3.2.2 Kinetic stability In the test of kinetic stability, it was observed that both the phase composition and the pH of the solution influence on biopolymer interactions and, hence, on the kinetics of phase separation. Figure 2 shows the SI of the emulsions prepared at pH 5.5, 6.5 and 7.5 over 3 days of storage at 45.0 ± 0.1 °C. Because null values of SI for a long period of time are indicative of good stability of emulsions, it can be observed that, for all compositions tested, pH 5.5 is the condition of lowest stability while pH 7.5 is the condition of highest stability. At pH 7.5, macroscopic phase separation was not observed for 3 different compositions: 8% gelatin and 0.10% NaCMC, 10% gelatin and 0.50% NaCMC and 12% gelatin and 0.50% NaCMC. This result suggests that emulsions are more stable as pH moves from the isoelectric point of the protein due to an increase in repulsive interactions between the polymers. According to Dickinson[14], the pH of the solutions controls the molecular charge distribution and the higher the polymer charge is, the lower the tendency of phase separation. A similar result was verified by Perrechil and Cunha[18] who observed phase separation only at low pH values. In addition to pH, phase composition also influences the kinetics of phase separation. At pH 5.5, the emulsion with 6% gelatin and 0.25% NaCMC presented fastest phase separation, which was completed approximately 1 h after preparation, followed by emulsions with 8% gelatin and 0.1% NaCMC, 8% gelatin and 0.25% NaCMC, 10% gelatin and 0.50% NaCMC and, lastly, 12% gelatin and 0.50% NaCMC, which started phase separation approximately 8 h after preparation. The same sequence of phase separation was observed in emulsions prepared at pH 6.5 and 7.5.The increase in stability as a function of gelatin concentration can be explained by an increase in the viscosity of the continuous phase which limits the movement of the droplets and, therefore, their approximation and aggregation. Similar results were observed by Singh[19] and Perrechil and Cunha[18], where emulsions with higher polysaccharide concentrations were

Table 1. Intrinsic viscosity ([ƞ]) and average viscosity molecular weight (Mv) of gelatin and NaCMC. Polymer Gelatin NaCMC

[ƞ] (cm3g-1) 58.86 394.05

Polímeros, 29(4), e2019051, 2019

Mv (kDa) 85.48 89.38

Figure 1. Phase diagram of gelatin-NaCMC system at pH 6.0 and 45.0 ± 0.1 °C. The solid line represents the tendency of the binodal line. 3/8


Laranjo, M. R., Costa, B. S., & Garcia-Rojas, E. E.

Figure 2. Separation index of W/W emulsions at pH a) 5.5; b) 6.5; c) 7.5, over 3 days at 45.0 ± 0.1 °C (● 12.0% gelatin, 0.50% NaCMC; ○ 10.0% gelatin, 0.50% NaCMC, ★ 8.0% gelatin, 0.25% NaCMC; ■ 8.0% gelatin, 0.10% NaCMC; □ 6.0% gelatin, 0.25% NaCMC).

more viscous and stable. Furthermore, the small difference in density between the two phases contributes to a slow phase separation. According to Dickinson[14], a small difference in density between two aqueous phases implies a creaming rate up to 100 times lower than that of O/W droplets of the same size. Another important observation was that the final SI of the emulsions is connected with the concentration of the disperse phase, with lower concentrations inducing higher SI values. Emulsions with 0.50%, 0.25% and 0.10% NaCMC presented separation indexes of approximately 41, 52 and 62%, respectively. The picture of the emulsions at different moments during the analysis is presented in Figure 3. After phase separation, a translucent upper layer and a turbid bottom layer were observed, indicating that the NaCMC droplets sedimented because they were denser and more opaque than the gelatin solution.

3.3 Emulsion characterization 3.3.1 Dynamic superficial tension As shown in Figure 4, the gelatin-air and NaCMC‑air systems presented a reduction in superficial tension with time until they reached equilibrium, indicating the migration of one or more components in solution to the interface[31]. 4/8

Figure 3. Pictures of the W/W emulsions prepared at pH 5.5, 6.5 and 7.5, at different moments over 3 days of storage at 45.0 ± 0. 1°C. From left to right, sample compositions corresponds to: 6% gelatin and 0.25% NaCMC; 8% gelatin and 0.10% NaCMC, 8% gelatin and 0.25% NaCMC, 10% gelatin and 0.5% NaCMC and 12% gelatin, 0.10% NaCMC. Polímeros, 29(4), e2019051, 2019


Stabilization of gelatin and carboxymethylcellulose water-in-water emulsion by addition of whey protein This behavior can be explained by the partially hydrophobic nature of proteins and polysaccharides or by the presence of impurities in solution active in the interface. The NaCMC solutions presented an initial superficial tension of approximately 65 mN.m-1 followed by a fast reduction and a tendency for the rate of reduction to decrease until a steady state of approximately 50 mN.m-1 was reached. The gelatin samples presented lower initial superficial tension, approximately 47 mN.m-1, because of larger amount of solutes in solution. Compared to that of the NaCMC solutions, a low rate of reduction was observed, which was related to the poor interfacial adsorption. In addition, a low tension variation and short time to reach the steady value could be observed. The test of ANOVA showed that for NaCMC solutions, the concentration of the solutions does not have a significant influence (p<0.05) on the equilibrium superficial tension, which may be related to the small difference in density between the solutions. However, it was verified that the gelatin concentration significantly influences the equilibrium superficial tension (p<0.05). 3.3.2 Droplet size and ζ potential The mean diameter (dm) of the emulsion droplets prepared at pH 7.5, the polydispersity index (PDI) and the ζ potential, with the respective standard deviation are presented in Table 2. These data show the formation of nanoemulsions, provided by the low interfacial tension that requires low energy to promote droplet breaking. The highly varied droplet sizes and the high PDI values show the formation of emulsions with broad size distribution. This result can be related to the

Figure 4. Superficial tension (σ) with time (t) of the polymer‑air systems at 45.0 ± 0.1 °C (○ gelatin 6.0%, □ gelatin 8.0%, ∆ gelatin 10.0%, ∇ gelatin 12.0%, ■NaCMC 0.10%, ● NaCMC 0.25%,▲NaCMC 0.50% and ▼NaCMC 1.0%).

polydisperse characteristics of the polymers in addition to the occurrence of Ostwald Ripening, a common phenomenon in nanoemulsions, meaning that smaller particles submit themselves to the larger ones and start growing larger[32]. The magnitude of the ζ potential is indicative of the stability of the colloidal system. According to Freitas[33], a minimum ζ potential higher than |60 mV| is needed for excellent stability, and one higher than |30 mV| is needed for good physical stability. All the emulsions presented ζ< |20 mV|, indicating weak electrostatic repulsion between droplets. Thus, it may be assumed that any change in the physicochemical properties of the medium can cause instability in the system or that these emulsions would show phase separation if evaluated for longer periods. In addition, it can be considered that repulsive forces exceed attractive forces (van der Waals interactions), inhibiting the droplet approximation. The ζ potential value, however, is only one of many indications of emulsion stability and in some cases, this is not a relevant direct parameter to assess stability[34]. 3.3.3 Viscosity The measurement of emulsion viscosity showed that this property is highly dependent on phase composition. The mean apparent viscosity of emulsions with 6% gelatin and 0.25% NaCMC, 8% gelatin and 0.25% NaCMC and 8% gelatin and 0.10% NaCMC, as well as their respective standard deviation, are presented in Table 3. Emulsions with 10% gelatin and 0.50% NaCMC and 12% gelatin and 0.50% NaCMC presented different behaviors. In addition to the high viscosity, above 10 times the value of viscosity for the other compositions, it was observed that while shear is applied, the viscosity tends to increase and when shear is stopped, the emulsion reverts back to the original structure, a typical behavior of rheopectic fluids. By definition, rheopectic fluids show an increase in structure strength during the application of stress and consequent recovery of the structure and viscosity at the end of the stress period[35]. One of the main reasons for this behavior is that the shear increases both the frequency and the efficiency of collision between the droplets, which induces aggregation and, thus, increases the apparent viscosity[19]. Rheopexy in highly concentrated emulsions was discussed by Masalova et al.[36], according to them, the restoration of the initial viscosity can be explained by elastic deformations of the droplets in the disperse phase.

3.4 Effect of adding whey protein isolate on emulsion stability As shown in Figure 5, the WPI particles influenced the SI value and the rate of phase separation of emulsions prepared at pH 5.5. The extent of the effect was dependent

Table 2. Mean diameter (dm), polydispersity index (PDI) and ζ potential of the disperse phase of emulsions with different compositions at pH 7.5. Composition 12% gelatin, 0.50% NaCMC 10% gelatin, 0.50% NaCMC 8% gelatin, 0.25% NaCMC 8% gelatin, 0.10% NaCMC 6% gelatin, 0.25% NaCMC

Polímeros, 29(4), e2019051, 2019

dm (nm)

PDI

ζ (mV)

84.27 ± 5.52 110.90 ± 26.88 83.73 ± 9.71 144.00 ± 23.42 77.87 ± 22.91

0.95 ± 0.04 0.70 ± 0.12 1.00 ± 0.00 0.76 ± 0.33 0.84 ± 0.20

-17.00 ± 1.16 -18.00 ± 1.57 -15.94 ± 1.03 -10.96 ± 0.19 -16.44 ± 1.97

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Laranjo, M. R., Costa, B. S., & Garcia-Rojas, E. E.

Figure 5. SI of emulsions with a) 12% gelatin and 0.50% NaCMC; b) 10% gelatin and 0.50% NaCMC; c) 8% gelatin and 0.25% NaCMC; d) 8% gelatin and 0.10% NaCMC; e) 6% gelatin and 0.25% NaCMC over time with different concentration of WPI (● 0%; ○ 5%; ■ 10%; □ 15%) at pH 5.5. Table 3. Mean apparent viscosity (ƞ) of the emulsions with different phase composition measured at different rotation speed (v) at 45.0 ± 0.5 °C and pH 7.5. Composition 8% gelatin, 0.25% NaCMC 8% gelatin, 0.10% NaCMC 6% gelatin, 0.25% NaCMC

v (rpm) 30 60 50

ƞ (mPa.s) 15.44 ± 2.60 8.00 ± 0.41 9.94 ± 0.50

on the phase composition and the amount of protein added. The formation of a white and thick product in addition to high foaming was observed. Few minutes after the emulsion formation, it was possible to identify a thin clear upper layer, indicative of the beginning of the phase separation process, while emulsions without WPI remained homogeneous for approximately 1 h. However, whereas emulsions without 6/8

WPI showed complete phase separation in no more than 6 h after formation, those with WPI particles showed complete phase separation only 24 h after formation, demonstrating that the protein particles were able to slow the rate of phase separation. Another relevant effect was the reduction of the SI compared to the formations without WPI. The emulsion with 8% gelatin and 0.1% NaCMC presented this effect clearly: the SI, which was 62% before the addition of WPI, decreased to 36% after the addition of 15% WPI. It was also observed that the addition of WPI influenced the kinetics of phase separation of the emulsions at almost all compositions, with the only exception being those with 12% gelatin and 0.50% NaCMC. The addition of 15% WPI caused a lower rate of phase separation and higher SI reduction. The application of the ANOVA test ensured that Polímeros, 29(4), e2019051, 2019


Stabilization of gelatin and carboxymethylcellulose water-in-water emulsion by addition of whey protein the addition of WPI had a significant influence (p>0,05) on the SI value at any phase composition regardless of the amount of protein added. The use of WPI microgel particles as stabilizing agents in O/W emulsions is extensively well known[37-39] however, the use of these particles in stabilizing W/W emulsions is very recent and still question still remain about the best conditions for using them. This is because the heat treatment induces the aggregation of protein molecules in solution, and stable suspensions of protein-based soft hydrogels are obtained. These hydrogel particles could adsorb on the interface much more strongly than could native untreated protein. As a result, remarkable stable water-in-water emulsions could be obtained because of the Pickering mechanism[6,12]. Recent discoveries have revealed that the Pickering effect is efficient only when the particles undergo an aggregation process at the interface[10,12] and when the particle is preferably solvated by the continuous phase[14]. According to Dickinson[14], one significant disadvantage of WPI microgel as W/W emulsion stabilizers is the tendency of the particles to flocculate in the vicinity of the isoeletric point of the protein (pI~5) and thus, it is expected that the particles might be more efficient on stabilizing emulsions prepared at pH>5.5. Although the addition of WPI particles retarded the phase separation, the stability time of the emulsions prepared at pH 7.5 could not be exceeded without the addition of particles.

4. Conclusions Under specific conditions of pH and phase composition, it is possible to produce stable W/W emulsions for at least 3 days of storage without the addition of stabilizing agents. Emulsions prepared at the pH furthest from the isoelectric point of gelatin and with high protein concentration presented the best stability. WPI particles added to emulsions at pH 5.5 showed the ability to reduce the phase separation speed and 15% WPI showed this effect clearly. Emulsions at pH 5.5 with WPI remained less stable than those prepared at pH 7.5 and 12% gelatin without WPI. Reducing the rate of phase separation opens new possibilities for research using particles to stabilize emulsions with practical applications in the formulation of functional food and in the encapsulation of bioactive components.

5. Acknowledgements The authors thank to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Finance code 001) for financial support.

6. References 1. Zhang, J., Hwang, J., Antonietti, M., & Schmidt, B. V. (2018). Water-in-water Pickering emulsion stabilized by polydopamine particles and cross-linking. Biomacromolecules. http://dx.doi. org/10.1021/acs.biomac.8b01301. PMid:30395449. 2. Chen, J.-F., Guo, J., Zhang, T., Wan, Z.-L., Yang, J., & Yang, X.-Q. (2018). Slowing the starch digestion by structural modification through preparing zein/pectin particle stabilized Polímeros, 29(4), e2019051, 2019

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Laranjo, M. R., Costa, B. S., & Garcia-Rojas, E. E. handbook (Vol. 7). New York: John Wiley & Sons. http:// dx.doi.org/10.1002/0471532053.bra049. 18. Perrechil, F. A., & Cunha, R. L. (2012). Development of multiple emulsions based on the repulsive interaction between sodium caseinate and LBG. Food Hydrocolloids, 26(1), 126134. http://dx.doi.org/10.1016/j.foodhyd.2011.04.017. 19. Singh, P., Medronho, B., Miguel, M. G., & Esquena, J. (2018). On the encapsulation and viability of probiotic bacteria in edible carboxymethylcellulose-gelatin water-in-water emulsions. Food Hydrocolloids, 75, 41-50. http://dx.doi.org/10.1016/j. foodhyd.2017.09.014. 20. Mcclements, D. J. (2005). Food Emulsions: Principles, practices, and techniques. Florida: CRC Press. 21. Ushikubo, F. Y., & Cunha, R. L. (2014). Stability mechanisms of liquid water-in-oil emulsions. Food Hydrocolloids, 34, 145-153. http://dx.doi.org/10.1016/j.foodhyd.2012.11.016. 22. Gomes, A., Costa, A. L. R., Assis Perrechil, F., & Cunha, R. L. (2016). Role of the phases composition on the incorporation of gallic acid in O/W and W/O emulsions. Journal of Food Engineering, 168, 205-214. http://dx.doi.org/10.1016/j. jfoodeng.2015.07.041. 23. Masuelli, M. A., & Sansone, M. G. (2012). Hydrodynamic properties of gelatin-studies from intrinsic viscosity measurements. In: J. Verbeek. Products and pplications of biopolymers (pp. 85-116). Argentina: InTech. 24. Ledward, D. A. (2000). Gelatin. In G. O. Phillips & P. A. Williams, Handbook of hydrocolloids (pp. 67-86). United States of America: CRC Press. 25. Masuelli, M. A. (2014). Mark-Houwink parameters for aqueoussoluble polymers and biopolymers at various temperatures. Journal of Polymer and Biopolymer Physics Chemistry, 2(2), 37-43. http://dx.doi.org/10.12691/jpbpc-2-2-2. 26. Vázquez, M. J., Gómez-Amoza, J., Martínez-Pachelo, R., Souto, C., & Concheiro, A. (1995). Relationships between drug dissolution profile and gelling agent viscosity in tablets prepared with hydroxypropylmethylcellulose (HPMC) and sodium carboxymethylcellulose (NaCMC) mixtures. Drug Development and Industrial Pharmacy, 21(16), 1859-1874. http://dx.doi.org/10.3109/03639049509070862. 27. Sharma, R., Das, B., Nandi, P., & Das, C. (2010). Viscosity of sodium carboxymethylcellulose in ethylene glycol–water mixed solvent media: separation of the influences of polyion conformation and electrostatic interactions on the reduced viscosity. Journal of Polymer Science. Part B, Polymer Physics, 48(11), 1196-1202. http://dx.doi.org/10.1002/polb.22009. 28. Gómez-Díaz, D., & Navaza, J. M. (2003). Rheology of aqueous solutions of food additives: effect of concentration, temperature and blending. Journal of Food Engineering, 56(4), 387-392. http://dx.doi.org/10.1016/S0260-8774(02)00211-X. 29. Rinaudo, M., Danhelka, J., & Milas, M. (1993). A new approach to characterizing carboxymethylcelluloses by size exclusion

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chromatography. Carbohydrate Polymers, 21(1), 1-5. http:// dx.doi.org/10.1016/0144-8617(93)90109-H. 30. Caraschi, J., & Campana, S. P. (1999). Influência do grau de substituição e da distribuição de substituintes sobre as propriedades de equilíbrio de carboximetilcelulose em solução aquosa. Polímeros: Ciência e Tecnologia, 9(2), 70-77. http:// dx.doi.org/10.1590/S0104-14281999000200015. 31. Andreas, J. M., Hauser, E. A., & Tucker, W. B. (1938). Boundary tension by pendant drops. Journal of Physical Chemistry, 42(8), 1001-1019. http://dx.doi.org/10.1021/j100903a002. 32. Mason, T. G., Wilking, J. N., Meleson, K., Chang, C. B., & Graves, S. M. (2006). Nanoemulsions: Formation, structure, and physical properties. Journal of Physics Condensed Matter, 18(41), R635-R666. http://dx.doi.org/10.1088/0953-8984/18/41/ R01. 33. Freitas, C., & Müller, R. H. (1998). Effect of light and temperature on zeta potential and physical stability in solid lipid nanoparticle (SLN™) dispersions. International Journal of Pharmaceutics, 168(2), 221-229. http://dx.doi.org/10.1016/ S0378-5173(98)00092-1. 34. Roland, I., Piel, G., Delattre, L., & Evrard, B. (2003). Systematic characterization of oil-in-water emulsions for formulation design. International Journal of Pharmaceutics, 263(1-2), 85-94. http://dx.doi.org/10.1016/S0378-5173(03)00364-8. PMid:12954183. 35. Gabas, A. L., Menezes, R. S., & Telis-Romero, J. (2012). Reologia na Indústria de Biocombustíveis. Brazil: Indi. 36. Masalova, I., Taylor, M., Kharatiyan, E., & Malkin, A. Y. (2005). Rheopexy in highly concentrated emulsions. Journal of Rheology, 49(4), 839-849. http://dx.doi.org/10.1122/1.1940641. 37. Destribats, M., Rouvet, M., Gehin-Delval, C., Schmitt, C., & Binks, B. P. (2014). Emulsions stabilised by whey protein microgel particles: towards food-grade Pickering emulsions. Soft Matter, 10(36), 6941-6954. http://dx.doi.org/10.1039/ C4SM00179F. PMid:24675994. 38. Wu, J., Shi, M., Li, W., Zhao, L., Wang, Z., Yan, X., Norde, W., & Li, W. (2015). Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking. Colloids and Surfaces. B, Biointerfaces, 127, 96-104. http:// dx.doi.org/10.1016/j.colsurfb.2015.01.029. PMid:25660092. 39. Sarkar, A., Murray, B., Holmes, M., Ettelaie, R., Abdalla, A., & Yang, X. (2016). In vitro digestion of Pickering emulsions stabilized by soft whey protein microgel particles: Influence of thermal treatment. Soft Matter, 12(15), 3558-3569. http:// dx.doi.org/10.1039/C5SM02998H. PMid:26959339. Received: May 03, 2019 Revised: Sept. 04, 2019 Accepted: Oct. 19, 2019

Polímeros, 29(4), e2019051, 2019


ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.00219

Evaluation of antimicrobial action of silver composite microspheres based on styrene-divinylbenzene copolymer Maria Aparecida Larrubia Granado Moreira Rodrigues Mandu1, Luciana da Cunha Costa2, Rodrigo Bernardes Tiosso1, Rômulo Pires Grasso1 and Mônica Regina da Costa Marques Calderari1*  Programa de Pós-graduação em Química, Universidade do Estado do Rio de Janeiro – UERJ, Rio de Janeiro, RJ, Brasil 2 Programa de Pós-graduação em Ciência e Tecnologia Ambiental, Centro Universitário Estadual da Zona Oeste – UEZO, Rio de Janeiro, RJ, Brasil 1

*monicamarques@uerj.br

Abstract This article reports the evaluation of the antimicrobial activity of a silver composite based on sulfonic resin. The antimicrobial action of the composite was evaluated against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus through plate, batch and colunm experiments. In batch studies, the efficiency of the composite was evaluated as a function of composite mass, bacterial concentration and contact time. We also developed a method to evaluate the antimicrobial activity of this composite using column tests. The antimicrobial activity of the composite was similar against the three bacteria in halo inhibition and batch experiments. The antibacterial activity was 100% against all bacteria above 0.20 g of composite and for all concentrations of bacteria studied. Column studies showed that the composite (1 g) had 100% action against 48 cm3 of S. aureus and 55 cm3 of E. coli and P. aeruginosa suspensions (105 cells mL-1, 50 cm3 min-1). Keywords: biocidal polymers, styrene-divinylbenzene copolymers, sulfonic resins, silver composites. How to cite: Mandu, M. A. L. G. M. R., Costa, L. C., Tiosso, R. B., Grasso, R. P., & Calderari, M. R. C. M. (2019). Evaluation of antimicrobial action of silver composite microspheres based on styrene-divinylbenzene copolymer. Polímeros: Ciência e Tecnologia, 29(4), e2019052. https://doi.org/10.1590/0104-1428.00219

1. Introduction Among the various antimicrobial materials are nanomaterials, antimicrobial peptides and antimicrobial polymers[1]. Antimicrobial polymers have been extensively evaluated for disinfection of water[2-4]. They have many advantages over low molecular weight biocidal compounds, mainly greater stability with respect to volatilization, dissolution and diffusion to the environment[3,5]. The formation of biofilms on solid surfaces is a concern in several fields, such as the pharmaceutical industry. In these biofilms, the microorganisms are much more resistant to disinfectants[6]. Porous membranes used for water treatment in processes such as microfiltration, ultrafiltration and reverse osmosis are subject to membrane fouling and consequently biofilm formation[7]. Thus, a water pretreatment step with an antimicrobial polymer before traditional membrane treatment can reduce the formation of biofilms, consequently increasing the lifetimes of these membranes. Crosslinked copolymer microspheres such as copolymers based on divinylbenzene have been extensively used as supports for chemical catalysts and biocatalysts[8,9], ion imprinting polymers[10], and introduction of magnetic particles[11-14], adsorbents and absorbents[15,16] and antimicrobial groups[3,5,17-26]. The introduction of antimicrobial groups in crosslinked copolymers is commonly achieved by chemical modification

Polímeros, 29(4), e2019052, 2019

or impregnation of the antibacterial agent in the polymeric matrix[3]. The polymeric supports employed most often are crosslinked beads made of divinylbenzene (DVB), and the biocidal groups studied are mainly ammonium and phosphonium quaternary groups[3,17,19,22], charge transfer complexes involving iodine and quaternary ammonium groups[3,17,23], N-halamines[3,6], sulfo-derivatives[3,5,18] and metal particles[1-3,20,21,24-26]. According to Munoz-Bonilla and Fernández-Garcia[2], silver nanoparticles are the metal particles most commonly used as antimicrobial agents in polymeric nanocomposites. Also according to those authors, silver in its metallic state is ionized in the medium, releasing Ag+ ions, and among other effects, the Ag+ ions bind to thiol groups in essential proteins, provoking structural changes in the cell wall that can cause death. The introduction of silver particles in crosslinked Sty-DVB copolymers can be considered a good strategy to prepare antimicrobial polymers. Silver has strong antimicrobial activity against a broad spectrum of microorganisms[2,24-26] and Sty-DVB copolymers are inert supports with good mechanical stability and easy synthesis. Santa Maria et  al.[21] prepared composites through impregnation of silver nanoparticles in styrene-divinylbenzene copolymers containing sulfonic acid groups. The biocidal activity was evaluated against E. coli using the column

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O O O O O O O O O O O O O O O O


Mandu, M. A. L. G. M. R., Costa, L. C., Tiosso, R. B., Grasso, R. P., & Calderari, M. R. C. M. method. This activity of silver nanocomposites varied from 54 to 100%. However, the biocidal activity of the nanocomposite was not evaluated against other bacteria by applying the plate and batch methods. Column experiments were realized in syringes similar to those used in solid phase extraction (SPE), without control of flow rate. More recently, Yee et al.[24] prepared silver nanocomposites based on a commercial ion exchange resin (the sulfonic resin Dowex 50WX8-400) as support. This resin was impregnated with silver ions and reduced with sodium borohydride. The authors observed that the nanocomposite inhibited the attachment of Histioteuthis pacifica biofilms. The biocompatibility evaluations showed that this silver nanocomposite was not toxic to human cells. In this work, we investigated the antimicrobial activity of silver composite microspheres prepared by impregnation of silver particles in a sulfonic resin. The antimicrobial experiments were performed against three bacteria of medical importance: E. coli and P. aeruginosa (Gram-negative bacteria) and S. aureus (Gram-positive bacterium), by applying the plate, batch and column methods. A method was also developed to evaluate the antimicrobial activity of polymeric resins by using column experiments. Some data obtained from the column experiments, such as the breakthrough point, are important for practical application of this type of material on a macro scale.

w/v AgNO3 acidified with 0.1 cm3 of HNO3 in a sealed flask protected from light for 48 h. According to the manufacturer, the sulfonic resin VPOC1800 has a cation exchange capacity of 4.4 mEq g-1, so it was used with an excess of 10x of Ag+ to H+ ions. After this, the resin was washed with deionized water until negative test with 1% w/v NaCl. The reduction step was performed employing 2 mol dm-3 of hydroxylamine chloride, gelatin and 2-hydroxy-ethyl cellulose. The beads were washed repeatedly with deionized water until neutral pH, followed by ethanol (300 cm3), and dried at 60 °C for 48 h.

2. Materials and Methods

2.4 Antibacterial experiments

2.1 Chemicals

2.4.1 Preliminary experiments

The ion-exchange resin Lewatit VPOC1800 was donated by Lanxess - Bayer Chemicals. Nutrient broth, nutrient agar, cetrimide agar base, Levines EMB agar, mannitol salt agar base, tryptone soy agar and tryptone soy broth for use in microbiological culture media were purchased from HiMedia Laboratories PVT Limited (Mumbai, India). The Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 11229 and Pseudomonas aeruginosa ATCC 15442 strains were donated by the Microorganism Collection for Sanitary Vigilance Reference (INCQS-FIOCRUZ, Rio de Janeiro, RJ). Other reagents and solvents were purchased from Sigma-Aldrich Brazil Co. Ltda. (Rio de Janeiro, Brazil) and used as received.

Sterility of the culture medium was verified by incubation of 5% of the solutions at 37 °C for 48 h before the experiments. Lyophilized Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 11229 and Pseudomonas aeruginosa ATCC 15442 bacteria were hydrated and cultured on specific media according to Table 1. Bacterial glycerol stocks were prepared and kept frozen at -4 °C. Aliquots of 25 μL of these bacterial stocks were pipeted to fresh broth (3 mL) and incubated at 37 °C for 24 h. Then, 10 μL samples of these cultures were seeded on agar slants, and these cultures were used to prepare suspensions in saline solutions (3 mL, 0.9% m/v). These suspensions were adjusted to 3 x 108 cells/mL, where the concentration was determined by turbidimetry with a spectrophotometer (Hach DR 5000, 600 nm) employing the McFarland standard (chemical solution of barium chloride and sulfuric acid). These suspensions were diluted to attain the other concentrations. All antimicrobial experiments were accompanied by negative, positive and environmental controls. In the positive control experiments, bacterial suspensions were cultivated without sulfonic resin and silver composite. In the negative control experiments, bacterial suspensions were placed in

2.2 Preparation of the silver composite The silver composite was prepared by impregnation of silver particles in commercial Lewatit VPOC1800 sulfonic resin according to the method previously described[21]. Sulfonic resin (in sodium form) was pretreated with an aqueous solution of 2% v/v HCl for 24 h, followed by washing with deionized water until neutral pH, then acetone (300 cm3), and drying at 60 °C for 24 h. The impregnation of silver particles was performed with a solution of 7.7%

2.3 Characterization of the silver composite Surface area and pore volume of the sulfonic resin and composite derived from it were determined by nitrogen adsorption measurements, according to the BET and BJH methods (Micrometrics ASAP 2010 system). TG and DTG curves were obtained using a TA Q50 instrument in a temperature range of 30-700 °C at a constant heating rate of 20 °C min-1 under nitrogen atmosphere and a flow rate of 100 cm3 min-1. The shape and surface texture of the materials were monitored with an FEI Quanta 400 scanning electron microscope (SEM) operating at 20 keV, with magnification of 10 000 x. The presence of metal particles was confirmed by energy dispersive X-ray spectrometry using an EDX microprobe.

Table 1. Conditions for Hydration and Cultivation of Bacterial Strains. Bacterial strains Staphylococcus aureus ATCC 6538 Escherichia coli ATCC 11229 Pseudomonas aeruginosa ATCC 15442

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Hydration medium Tryptone soy broth Tryptone soy broth Nutrient broth

Cultivation medium Tryptone soy agar Tryptone soy broth Nutrient agar

Polímeros, 29(4), e2019052, 2019


Evaluation of antimicrobial action of silver composite microspheres based on styrene-divinylbenzene copolymer contact with Lewatit VPOC 1800 sulfonic resin. Environmental control experiments were performed by putting plates with culture medium under a laminar flow hood. 2.4.2 Plate method Bacterial cultures of S. aureus, E. coli and P. aeruginosa (3 x 108 cells mL-1) were spread onto plates with agar nutrient medium. The center of these plates was filled with silver composites. The plates were incubated at 37 °C for 48 h. After this, the inhibition zone was measured. Unmodified sulfonic resins were used as controls in the experiments.

was performed in a different column. For determination of the breakthrough curve, the elute was collected from the column effluent at intervals of 1-2 minutes. Aliquots of these suspensions were plated on LB nutrient medium and kept at 37 °C. After 48 h, the colonies formed (CFU) were counted. Unmodified sulfonic resins were used as controls in the experiments. The breakthrough curve provided data on the breakthrough point Vp (the point when the cells are first detected in the column effluent) and working biocidal activity Cr (Equation 2)[16]. Cr =

2.4.3 Batch experiments Bacterial suspensions of S. aureus, E. coli and P. aeruginosa were placed in glass flasks containing 2 mL of sterile 0.9% saline solution and a determined mass of composite. The  composite and bacteria were maintained in contact for varied periods of time. The experiments of variation of composite mass were conduced employing bacterial suspensions with concentration of 105 cells mL-1, contact time of 30 minutes and varied composite mass (0.05-0.8 g). The experiments involving variation of bacterial suspension concentrations were realized employing 0.2 g of composite, contact time of 30 minutes and varied concentrations of bacteria (103-107 cells mL-1). The experiments investigating variation of contact time were conduced employing a bacterial suspension of 105 cells mL-1, 0.2 g of silver composite and contact time of 30 to 180 seconds. Each experiment was conducted in a glass flask and all experiments were realized in triplicate. Aliquots of 1 mL of supernatant were plated on selective media and kept at 37 °C. After 48 h, the colonies formed (CFU) were counted. Unmodified sulfonic resins were used as controls in the experiments. The bactericidal activity of the polymers was estimated by calculating the decrease in the number of bacteria according to Equation 1[17,19]. Antimicrobial capacity = 100 × (CFUi – CFU f ) / CFUi (1)

where CFUi = colonies formed before contact between bacterial suspensions and polymers; CFUf = colonies formed after contact between bacterial suspensions and polymers. 2.4.4 Column experiments Column experiments were conducted in acrylic columns measuring 1.9 cm (inside diameter) and 30 cm (height) coupled to a peristaltic pump (Exatta Model A305). The column was packed with 30 g of resin (13 cm3) and the resin was submitted to swelling with sterile distilled water (60 cm3) for 24 h. After being eluted through the column, this water was analyzed by ion exchange chromatography (IEC) (DIONEX, Model ICS 300 DP). No silver ions were detected in this water. After the swelling period, suspensions of each bacterium (S. aureus, E. coli or P. aeruginosa) of 105 cells mL-1 were passed through the column at a flow rate of 50 mL min-1. Each experiment with a different bacterium

Vp • C o

Vj

(2)

where Vp =effluent volume at the breakpoint (cm3); Vj = volume of biocidal resin in the column (cm3); and Co = initial bacterial concentration (cells cm-3). 2.4.5 Statistical analysis All the tests results were compared by one-way analysis of variance (ANOVA) using the BioEstat software (version 5.0).

3. Results and Discussion 3.1 Characterization of silver composite The silver composite was prepared by treatment of Lewatit VPOC1800 sulfonic resin with AgNO3 followed by reduction reaction with hydroxylamine chloride. These reactions caused a drastic reduction in surface area and pore volume of the sulfonic resin (Table 2), indicating the agglomeration of silver particles in pores and channels of the resin[21]. The occurrence of impregnation was also evidenced by TGA (Table  2). The anchorage of silver particles increased the onset temperature and residual mass of the polymeric matrix, indicating that this anchorage increases the thermal resistance of the sulfonic resin. These results agree with other results of thermal decomposition of nanocomposites, demonstrating that the impregnation of metal particles improves the thermal resistance of the polymeric support[15]. The SEM micrographs of the Lewatit VPOC1800 resin impregnated with silver nanoparticles (Figure  1) demonstrated that the nanoparticles were distributed on the beads surface. The presence of elemental silver on the surface of the composite was also investigated by EDS spectra (Figure 2), confirming the silver impregnation of the sulfonic resin surface.

3.2 Plate method The silver nanocomposite and unmodified resin were evaluated for antibacterial activity by using the plate method (Figure 3). The inhibition zone was measured for three bacterial cultures: E. coli, S. aureus and P. aeruginosa.

Table 2. Morphological characteristics and TGA data for the resin Lewatit VPOC1800 before and after impregnation with silver. Materials VPOC1800 VPOC1800/Ag

S (m2 g-1)a 3.789 0.038

Vp (cm3 g-1)b 0.02 ndd

T onset (°C)c 344 463

Residue (%) 39.2 57.1

S: surface area (BET-ASAP); bVp: pore volume (BJH-ASAP); cthermogravimetric analysis (TGA); dnot detected.

a

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Mandu, M. A. L. G. M. R., Costa, L. C., Tiosso, R. B., Grasso, R. P., & Calderari, M. R. C. M.

Figure 1. Scanning electron micrographs of Lewatit VPOC1800 resin before (a, c) and after (b, d) impregnation with silver.

Figure 2. EDS spectra of silver composite.

As expected, the unmodified resin did not have antimicrobial action against the three bacterial species analyzed. Santa Maria et al.[21] also observed that the unmodified sulfonic resin (Lewatit VPOC1800) did not show antimicrobial action against E. coli. However, E. coli had a zone diameter of 1.4 ± 0.3 cm, P aeruginosa had a zone diameter of 1.8 ± 0.2 cm and S. aureus showed a zone diameter of 1.3 ± 0.2 cm for the silver nanocomposite. These data demonstrate the bacteriostatic effect of silver composite, i.e., the composites 4/10

ability to inhibit bacterial growth around polymeric particles. Kamrupi et  al.[27] also observed that nanocomposites of polystyrene containing silver nanoparticles had inhibitory effect against four bacterial species studied: Pseudomonas fluorescens BS3, Bacillus circulens BP2, Eschericia coli and Staphylococcus aureus. However, those authors did not measure the inhibition halo of the bacteria. Statistical analysis of the data of the inhibition halo observed in this study, performed by one-way analysis of Polímeros, 29(4), e2019052, 2019


Evaluation of antimicrobial action of silver composite microspheres based on styrene-divinylbenzene copolymer

Figure 3. Plate test for P. aeruginosa: (a) positive control, indicating bacteria growing on agar nutrient medium without nanocomposite (b) inhibition halo formed by composite in P. aeruginosa culture (c) negative control, indicating absence of inhibition halo for Lewatit VPOC1800 not modified with silver particles.

variance, demonstrated that the difference in zone diameter was not significant (p = 0.1361 and α = 0.01, i.e., p > α). Since Gram-negative bacteria (such as E. coli and P. aeruginosa) possess an additional cell wall that Gram-positive bacteria (such as S. aureus) do not have, we expected the biocidal action of the silver nanocomposite would be different for these two types of bacteria[27]. However, unlike expected, it was not possible to observe differences in the inhibition zones of the silver composite against the three bacteria species, i.e., no difference was observed in the halo test.

3.3 Batch experiments 3.3.1 Mass of composite In this study, bacterial suspensions were used with concentration of 105 cells mL-1, contact time of 30 minutes and composite masses of 0.05-0.80 g. As expected, an increase of composite mass provoked an increase of the biocidal activity. In general, the antibacterial activity was complete (100%) for all bacteria above 0.20 g of composite. The ANOVA results (α = 0.05) indicated that the variation of composite mass was highly significant (p < 0.0001), whereas the variation of bacteria was not significant (p > 0.5). The Tukey test indicated that the mass variation until 0.15 g was significant and increased the antimicrobial activity. Above 0.15 g, the differences in removal percentages were not significant (Table 3). Polímeros, 29(4), e2019052, 2019

The antimicrobial action of silver composite was similar to Gram-positive and Gram-negative bacteria, confirming the results of the plate experiments. The silver composite is a bactericide, i.e., this composite causes damage to bacteria that leads to their death, such as by metabolic inhibition, loss of membrane integrity or coagulation of intracellular material. This composite may have realeased the silver ions in the medium (a demand‑release disinfectant) or acted via contact between bacteria and silver particles trapped in polymeric matrix, causing damage to the bacteria (contact disinfectant), leading to death[28]. In both cases, the increasing mass of composite would cause rising biocidal activity. 3.3.2 Concentration of bacteria We employed bacterial suspensions of 103 to 107 cells mL-1, 0.2 g of silver composite and contact time of 30 minutes (Table 4). Valle et al.[17,19] demonstrated that the biocidal activity of vinyl pyridine copolymers containing quaternary ammonium groups and charge transfer complexes with iodine was influenced by concentration of bacterial suspension in contact with these polymers, and there was a significant reduction of the biocidal activity with high concentrations of bacterial suspensions (106 and 107 cells mL-1). Souza et al. [5] also observed that the biocidal activity of Sty-DVB copolymers with phosphoryl and sulfophosphoryl groups was influenced by concentration of bacterial suspension 5/10


Mandu, M. A. L. G. M. R., Costa, L. C., Tiosso, R. B., Grasso, R. P., & Calderari, M. R. C. M. Table 3. Antimicrobial activity (%) of silver composite with varying mass (g). Bacteria

Mass (g)

Pseudomona aueruginosa

0.05 0.10 0.15 0.20 0.40 0.80 0.05 0.10 0.15 0.20 0.40 0.80 0.05 0.10 0.15 0.20 0.40 0.80

Escherichia coli

Staphilococcus aureus

CFU mL-1 6.50x104 1.90x104 7.40x103 1.00 0.00 0.00 6.80x104 1.80 x104 8.20 x103 0.00 1.00 0.00 8.80x104 4.40x104 2.00x104 2.50x102 1.00 0.00

5.20x104 1.80x104 8.50x103 2.00 0.00 0.00 7.00x104 1.60 x104 9.15 x103 0.00 1.00 0.00 7.60x104 3.00x104 1.50x104 3.80x102 3.00 0.00

5.80x104 1.80x104 6.70x103 2.00 0.00 0.00 6.67 x104 1.19 x104 9.60 x103 2.00 2.00 0.00 6.70x104 3.00x104 1.60x104 4.90x102 2.00 0.00

6.40x104 1.90x104 9.80x103 4.00 0.00 0.00 5.94 x104 1.63 x104 1.11 x104 1.00 1.00 0.00 8.40x104 4.30x104 1.00x104 1.00 1.00 0.00

Antimicrobial capacity (%) 60 88 95 100 100 100 55 90 94 100 100 100 47 74 90 100 100 100

Analytic conditions: Initial concentration of bacteria, 105 cells mL-1; contact time of 30 minutes; and composite masses of 0.05-0.80 g. CFU: number of colonies formed after contact between bacterial suspensions and polymer.

Table 4. Animicrobial activity (%) of silver composite with varying bacterial concentrations (CFU mL-1). Bacterial Pseudomona aueruginosa

Escherichia coli

Staphilococcus aureus

Concentration (CFU mL-1) 1.50x 103 1.50x104 1.50x 105 1.50x 106 1.50x 107 1.50x 103 1.50x104 1.50x 105 1.50x 106 1.50x 107 1.50x 103 1.50x104 1.50x 105 1.50x 106 1.50x 107

CFU mL-1 0.00 0.00 1.00 0.00 50.00 0.00 0.00 1.00 20.00 100.00 0.00 0.00 1.00 2.00 200.00

1.00 0.00 0.00 0.00 67.00 1.00 0.00 0.00 15.00 88.00 0.00 0.00 1.00 3.00 167.00

1.00 0.00 0.00 1.00 30.00 1.00 0.00 0.00 17.00 97.00 0.00 0.00 1.00 1.00 230.00

0.00 0.00 1.00 2.00 77.00 0.00 0.00 1.00 12.00 77.00 0.00 0.00 1.00 2.00 177.00

Antimicrobial capacity (%) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

Analytic conditions: composite mass of of 0.2 g; contact time of 30 minutes; initial bacterial concentrations of 103 to 107 cells mL-1. CFU: number of colonies formed after contact between bacterial suspensions and polymer.

and noted a reduction of biocidal action of the polymers to bacterial suspensions with 106 and 107 cells mL-1. However, they observed that the variation in bacterial concentration did not cause a variation in biocidal activity of the silver composite. For the highest concentration of bacteria studied (1.5 x 107 cells mL-1), the biocidal activity was already 100%. 3.3.3 Contact time We used a bacterial suspension of 105 cells mL-1, 0.2 g of silver composite and contact times of 30 to 180 seconds (Table 5). A wide range of contact times between silver composite and bacterial suspensions was used in order to determine the minimum contact time between the suspensions and polymer needed to achieve maximum antimicrobial activity (100%). 6/10

The variation in contact time caused an increase of antimicrobial activity of the composite against all bacteria studied. The ANOVA results (α = 0.05) demonstrated that the variation in contact time was highly significant (p < 0.0001), whereas the variation of the type of bacterium was not significant (p > 0.5). The Tukey test indicated that the variation of contact time until 40 seconds was significant and contributed to increase biocidal activity, but above 40  seconds the differences in removal percentages were not significant. After short contact time between composite and suspensions, the maximum biocidal activity was already reached. Our first hypothesis for this result was the release of silver from the polymeric structure as a function of contact time. This hypothesis was partially confirmed by plate Polímeros, 29(4), e2019052, 2019


Evaluation of antimicrobial action of silver composite microspheres based on styrene-divinylbenzene copolymer Table 5. Antimicrobial activity (%) of the silver composite with varying contact times (sec). Bacteria

Time (sec)

Pseudomona aueruginosa

30 40 60 120 180 30 40 60 120 180 30 40 60 120 180

Escherichia coli

Staphilococcus aureus

CFU mL-1 2.00x104 2.50x102 2.00 1.00 0.00 1.00x104 2.5x102 50.00 0.00 0.00 2x104 2.5x102 1.00 0.00 0.00

1.00x104 1.20x102 0.00 0.00 0.00 1.5x104 3.8x102 2.00 0.00 0.00.00 1.5x104 3.8x102 5.00 0.00 0.00

1.20x104 95.00 10.00 0.00 0.00 1.6x104 4.9x102 4.00 0.00 0.00 1.6x104 2.9x102 2.00 0.00 0.00

1.00x104 80.00 0.00 0.00 0.00 1x104 1 1.00 0.00 0.00 1.00x104 1.00x102 1.00 0.00 0.00

Antimicrobial Capacity (%) 87 99 99 99 100 87 99 99 100 100 84 99 99 100 100

Analytic conditions: Initial bacterial concentration of 105 cells mL-1; composite mass of 0.2 g; contact times of:30-180 seconds. CFU: number of colonies formed after contact between bacterial suspensions and polymer.

studies, which demonstrated the composites ability to inhibit bacterial growth around polymeric particles. However, the subsequent column studies demonstrated the saturation point was reached only after passing a large volume of bacterial suspension through columns packed with composite, and it was not possible to observe a physical change of the composite, such as a discoloration, in these column studies. This indicates that the composite probably also acts as a contact biocide, where the electrostatic interation between silver ions and cell membrane causes damage to the bacteria leading to death[28, 29].

3.4 Column experiments The antimicrobial activity of the polymeric resins, composites and nanocomposites was evaluated by plate and batch methods by varying the concentration of bacteria, contact time and resin mass, using E. coli (a Gram-negative bacterium)[20,22]. The antimicrobial activity of these materials has not been sufficiently studied by column experiments, even though the use of these materials on a larger scale requires such studies. Jandrey et al.[23] developed a method for evaluation of biocidal activity of these polymers in column tests employing micro syringes (1.0 mL) filled with polymers (130-250 mg). This method has been employed by several researchers, such as Souza et al.[5], Valle et al.[17,19], Costa et al.[18] and Santa Maria[21]. The method to evaluate the biocidal activity of polymers through column experiments applied in this work as well as the determination of the breakthrough point, saturation point and working biocidal activities, can be considered novel because they are not described in the previous literature. Our column experiments were conducted in acrylic columns coupled to a peristaltic pump. Bacterial suspensions a (4000 cm3, 105 cells cm-3) were passed through the column packed with nanocomposite (30 g) at a flow rate of 50 cm3 min-1. Aliquots of these suspensions were plated on LB nutrient medium and kept at 37 °C, and after incubation Polímeros, 29(4), e2019052, 2019

the colonies formed (CFU) were counted. The breakthrough curve (Figure 4) and working biocidal activities of the silver composite were determined by employing a similar method to that used to determine the breakthrough curve and working activities of ion exchange resins[16]. The data obtained from these experiments are important for practical application of this type of material on a macro scale. The breakthrough point is the point when the efficiency of the resin becomes lower than 100%. This information is very important considering the need for water without pathogenic microorganisms. The breakthrough point occurred after passing 1650 cm3 of the suspensions of E. coli and P. aeruginosa and 1450 cm3 of the suspension of S aureus through the column. These results indicate that each gram of composite has complete biocidal action against 55 cm3 of E.  coli and P. aeruginosa suspensions and 48 cm3 of S.  aureus suspension (105 cells mL-1) at a flow rate of 50 cm3 min-1. The saturation point occurred after passing of 2250 cm3 of the suspension of S. aureus and 2450 cm3 of the suspensions of E. coli and P aeruginosa through the column. The working biocidal activities of the silver composite were 126 x 105 cells mL-1 against E. coli and P. aeruginosa and 112 x 105 cells mL-1 against S. aureus. According to Popa et al.[29], the reduction of CFU as a function of exposure between polymer and bacterial suspensions can be interpreted as “an adsorption-like phenomenon”, where the evolution is very similar to that of a monomolecular layer adsorption isotherm and culminates with the saturation process of the biocidal polymer with time. The results indicated that the antimicrobial activity of the silver composite against E. coli and P. aeruginosa (Gram-negative bacteria) was higher than against S. aureus (Gram-positive bacterium). Liang et al.[30] reported that antimicrobial polymers with polyquat structures, such as polymers with ammonium and phosphonium groups, have higher antimicrobial action against Gram-positive bacteria. This can be parcially explained considering that Gram-negative bacteria possess a cell wall 7/10


Mandu, M. A. L. G. M. R., Costa, L. C., Tiosso, R. B., Grasso, R. P., & Calderari, M. R. C. M.

Figure 4. Breakthrough curve for the silver composite in contact with suspensions of S. aureus, E. coli or P. aeruginosa.

of peptidoglycan and LPS, which impairs the action of these polyquats. However these researchers have also reported that other antimicrobial polymers such as N-halamine polymers are more efficient against Gram-negative than Gram-positive bacteria. The antimicrobial action this of type of polymer involves direct transfer of oxidative halogen to the cell and cell inactivation. It is well known that silver compounds are active against bacteria, viruses and fungi, but the bactericidal mechanism of these compounds is only partially understood. Probably the silver particles react with moisture and are ionized, releasing highly reactive Ag+ ions on the beads’ surface. The Ag+ ions have greater tendency to react with the sulfur sites present in proteins, causing structural changes in the cell wall and also in the nuclear membranes, which causes cell death. In addition Ag+ ions also form complexes with phosphorous sites present in DNA and RNA, inhibiting replication of microorganisms[31]. According to Thiel et al. [32], the bactericidal action of silver particles can be attributed to electronic effects resulting from changes in the electronic structure of the surfaces of the silver particles. Silver particles act on the bacterial membrane causing destabilization of the plasma-membrane potential and reduction of the levels of intracellular ATP, resulting on bacterial death. Our column data indicated that silver composite was more efficient against Gram-negative bacteria. Considering that silver particles provoke desestalization of the plasma‑membrane potential and depletion of ATP levels and Gram-negative bacteria have an additional cell wall, it is possible that this type of bacterium is more susceptible to antimicrobial action of silver particles. Additional studies involving evaluation of the antimicrobial action of silver particles against Gramnegative and Gram-positive bacteria are needed.

4. Conclusions Silver composite microspheres were prepared by impregnation of silver particles in the commercial sufonic resin Lewatit VPOC 1800. The impregnation reaction was achieved through reduction of Ag+ ions with hydroxylamine in the presence of gelatin and 2-hydroxi-ethyl-celullose 8/10

and characterized by thermogravimetry, SEM and EDS. The composite showed bactericidal activity against the three bacteria studied (E. coli, P. aeruginosa and S. aureus). The halo inhibition tests and batch experiments demonstrated that the antimcrobial activity of the composite was similar against the three bacterial species. However, the column studies indicated that the composite had stronger antimicrobial action against S. aureus than E. coli and P. aeruginosa. Each gram of the composite had complete antimicrobial action against 48 cm3 of S. aureus suspension and 55 cm3 of E. coli and P. aeruginosa suspensions (105 cells mL-1) at a flow rate of 50 cm3 min-1. The working antimicrobial activities of the silver composite were 126 x 105 cells mL-1 against E. coli and P. aeruginosa and 111.5 x 105 cells mL-1 against S. aureus. The method to evaluate the biocidal activity of polymers through column experiments reported here as well as the determination of the breakthrough point, saturation point and working biocidal activities can be considered novel and are important for practical application of this type of material on a macro scale. These results indicated that each gram of composite has complete biocidal action against 55 cm3 of E. coli and P. aeruginosa suspensions and 48 cm3 of S. aureus suspension (105 cells mL-1) at a flow rate of 50 cm3 min-1

5. Acknowledgements This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001. We also thank Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq for financial support and the companies Petroflex, Nitriflex and Metacril for donating the monomers.

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15. Simplício, S., Maria, L. C. S., Costa, M. A. S., Lucas, E. F., Queirós, Y. G. C., Marques, L. R. S., Costa, L. C., Hui, W. S., & Silva, M. R. (2013). Removal of phenol from aqueous solutions by polymeric composites containing Ni and Co particles. Polímeros: Ciência e Tecnologia, 23(5), 590-596. http://dx.doi.org/10.4322/polimeros.2013.092. 16. Evaristo, A. A. A., Santos, K. C. R., Costa, L. C., & Marques, M. R. C. (2013). Evaluation of ion exchange resins for recovery of metals from electroplating sludge. Polymer Bulletin, 70(8), 2239-2255. http://dx.doi.org/10.1007/s00289-013-0944-x. 17. Valle, A. S. S., Marques, M. R. C., Costa, L. C., Maria, L. C. S., Aguiar, A. P., & Merçon, F. (2013). Evaluation of bactericidal action of 2-vinylpiridine copolymers containing quaternary ammonium groups and their charge transfer complexes. Polímeros Ciência e Tecnologia, 23(2), 152-160. http://dx.doi. org/10.1590/S0104-14282013005000023. 18. Costa, L. C., Marques, M. R. C., Tiosso, R. B., Cantarim, J. P., & Merçon, F. (2012). Evaluation of the biocidal activity of hypercrosslinked resins containing dithiocarbamate groups. Macromolecular Symposia, 319(1), 121-128. http://dx.doi. org/10.1002/masy.201100175. 19. Valle, A. S. S., Costa, L. C., Marques, M. R. C., Silva, C. L. P., Maria, L. C. S., Merçon, F., & Aguiar, A. P. (2011). Preparação de copolímeros à base de 2-vinilpiridina com propriedades bactericidas. Quimica Nova, 34(4), 577-583. http://dx.doi. org/10.1590/S0100-40422011000400005. 20. Gangadharan, D., Harshvardan, K., Gnanasekar, G., Dixit, D., Popat, K. M., & Anand, P. S. (2010). Polymeric microspheres containing silver nanoparticles as a bactericidal agent for water disinfection. Water Research, 44(18), 5481-5487. http://dx.doi. org/10.1016/j.watres.2010.06.057. PMid:20673945. 21. Santa Maria, L. C., Oliveira, R. O., Mercon, F., Borges, M. E. R. S. P., Barud, H. S., Ribeiro, S. J. L., Messaddeq, Y., & Wang, S. H. (2010). Preparation and bactericidal effect of composites based on crosslinked copolymers containing silver nanoparticles. Polímeros: Ciência e Tecnologia, 20(3), 227230. http://dx.doi.org/10.1590/S0104-14282010005000028. 22. Ahmed, A. E. I., Hay, J. N., Bushell, M. E., Wardell, J. N., & Cavalli, G. (2008). Biocidal polymers (I): preparation and biological activity of some novel biocidal polymers based on uramil and its azo-dyes. Reactive & Functional Polymers, 68(1), 248-260. http://dx.doi.org/10.1016/j.reactfunctpolym.2007.09.004. 23. Jandrey, A. C., Aguiar, A. P., Aguiar, M. R. M. P., Santa Maria, L. C., Mazzei, J. L., & Felzenszwalb, I. (2007). Iodine-poly(2vinylpyridine-co-styrene-co-divinylbenzene) charge transfer complexes with antibacterial activity. European Polymer Journal, 43(11), 4712-4718. http://dx.doi.org/10.1016/j. eurpolymj.2007.07.042. 24. Yee, M. S.-L., Khiew, P. S., Tan, Y. F., Kok, Y.-Y., Cheong, K. W., Chiu, W. S., & Leong, C.-O. (2014). Potent antifouling silver-polymer nanocomposite microspheres using ionexchange resin as templating matrix. Colloids and Surfaces A, Physicochemical and Engineering Aspects, 457(1), 382-391. http://dx.doi.org/10.1016/j.colsurfa.2014.06.010. 25. Qu, X., Alvarez, P. J. J., & Li, Q. (2013). Applications of nanotechnology in water and wastewater treatment. Water Research, 47(12), 3931-3946. http://dx.doi.org/10.1016/j. watres.2012.09.058. PMid:23571110. 26. Mthombeni, N. H., Mpenyana-Monyatsi, L., Onyango, M. S., & Momba, M. N. B. (2012). Breakthrough analysis for water disinfection using silver nanoparticles coated resin beads in fixed-bed column. Journal of Hazardous Materials, 217-218(1), 133-140. http://dx.doi.org/10.1016/j.jhazmat.2012.03.004. PMid:22459979. 27. Kamrupi, I. R., Phukon, P., Konwer, B. K., & Dolui, S. K. (2011). Synthesis of silver-polystyrene nanocomposite 9/10


Mandu, M. A. L. G. M. R., Costa, L. C., Tiosso, R. B., Grasso, R. P., & Calderari, M. R. C. M. particles using water in supercritical carbon dioxide medium and its antimicrobial activity. The Journal of Supercritical Fluids, 55(3), 1089-1094. http://dx.doi.org/10.1016/j. supflu.2010.09.027. 28. Denyer, S., & Stewart, G. S. A. B. (1998). Mechanisms of action of disinfectants. International Biodeterioration & Biodegradation, 41(3-4), 261-268. http://dx.doi.org/10.1016/ S0964-8305(98)00023-7. 29. Popa, A., Davidescu, C. M., Trif, R., Ilia, G., Iliescu, S., & Dehelean, G. (2003). Study of quaternary ‘onium’ salts grafted on polymers: antibacterial activity of quaternary phosphonium salts grafted on ‘gel-type’ styrene-divinylbenzene copolymers. Reactive & Functional Polymers, 55(2), 151-158. http://dx.doi. org/10.1016/S1381-5148(02)00224-9. 30. Liang, J., Chen, Y., Barnes, K., Wu, R., Worley, S. D., & Huang, T. S. (2006). N-halamine/quat siloxane copolymers for use

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in biocidal coatings. Biomaterials, 27(11), 2495-2501. http:// dx.doi.org/10.1016/j.biomaterials.2005.11.020. 31. Pal, S., Tak, Y. K., & Song, J. M. (2007). Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the Gram-negative bacterium Escherichia coli. Applied and Environmental Microbiology, 73(6), 1712-1720. http://dx.doi.org/10.1128/AEM.02218-06. PMid:17261510. 32. Thiel, J., Pakstis, L., Buzby, S., Raffi, M., Ni, C., Pochan, D. J. & Shah, S. I. (2007). Antibacterial Properties of SilverDoped Titania. Nano Micro Small , 3(5), 799-803. https:// doi.org/1.1002/smll.200600481 Received: Mar. 07, 2019 Revised: Sept. 21, 2019 Accepted: Oct. 11, 2019

Polímeros, 29(4), e2019052, 2019


ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.06519

Synthesis and performance of AM/SSS/THDAB as clay hydration dispersion inhibitor Wei-Chao Du1,2* , Xiang-Yun Wang1, Man Liu3, Tai-Fei Bi4, Shun-Xi Song2, Jie Zhang1 and Gang Chen1 College of Chemistry and Chemical Engineering, Xi’an Shiyou University, Xi’an, People’s Republic of China 2 Key Laboratory of Auxiliary Chemistry and Technology for Chemical Industry, Ministry of Education, Shaanxi University of Science and Technology, Xi’an, People’s Republic of China 3 Oil Production No.11, Changqing Oilfield Company, Qingyang, People’s Republic of China 4 Oil Production No.1, Changqing Oilfield Company, Yan’an, People’s Republic of China

1

*duweichao@xsyu.edu.cn

Abstract In this paper, a novel zwitterionic copolymer AM/SSS/THDAB clay hydration dispersion inhibitor was synthesized by copolymerization of tris hydroxyethyl diallyl ammonium bromide (THDAB), sodium p-styrene sulfonate (SSS) and acrylamide (AM) initiated in an aqueous solution. The copolymer was characterized by FT-IR, GPC, TGA-DSC and SEM. Results demonstrated that molecular weight of AM/SSS/THDAB was 43674 g/mol and its temperature resistance ability was up to 225 °C. Evaluation experiments showed that AM/SSS/THDAB has an excellent clay hydration inhibitive performance. Methods including particle size analysis and SEM were utilized to study its dispersion inhibition mechanism by using sodium montmorillonite (Na-MMT). Results indicated that the micro-structure of Na-MMT has been successfully changed by AM/SSS/THDAB. In a word, the superior inhibition property makes the novel clay hydration dispersion inhibitor promised in water-based drilling fluids. Keywords: clay hydration inhibitor, low molecular weight, shale gas, water-based drilling fluids. How to cite: Du, W.-C., Wang, X.-Y., Liu, M., Bi, T.-F., Song, S.-X., Zhang, J., & Chen, G. (2019). Synthesis and performance of AM/SSS/THDAB as clay hydration dispersion inhibitor. Polímeros: Ciência e Tecnologia, 29(4), e2019053. https://doi.org/10.1590/0104-1428.06519

1. Introduction Drilling fluids are multicomponent systems used to aid the removal of cuttings from a borehole, and subject to a number of requirements to ensure a safe drilling operation[1]. The exploitation of unconventional reservoir such as shale gas has raised the attention of the world while the excessive depletion of conventional reservoir. However, the wellbore instability will be the major problem associated with the drilling operation of shale gas formations[2]. Generally, oil based muds (OBMs) is always the primary choice due to its superior clay hydration inhibition ability so as to avoid wellbore instability problem. Unfortunately, the environmental restrictions and high costs have largely limited the wide application of OBMs[3]. At the same time, water-based drilling fluids (WBDFs) is often employed in drilling of unconventional reservoir due to its simple formulation and low cost. Because the wellbore instability is due to hydration dispersion of clay minerals in shale. The development of clay hydration dispersion inhibitor and high performance WBDFs are the timely pursuit of drilling engineering[4-7]. Various large molecular weight copolymer inhibitors, such as HPAM, hyperbranched polyglycerols, FA-367, PDADMAC and polyamine have been investigated over the past decades[8-10]. The copolymer inhibitors will be wrapped and form a coating film in the surface of clay which can effectively prevent the wellbore instability of

Polímeros, 29(4), e2019053, 2019

shale. However, high molecular weight copolymer shows a great influence on the rheological property of WBDFs[8]. Besides, the high molecular weight copolymer chain is easy to curl and lose itself function in the environment of high salt, high calcium and high temperature[9]. Works have proved that copolymer with low molecular weight showed excellent salt and temperature resistance ability in WBDFs, and low molecular weight polymers such as polyamine, hyperbranched polymer has greatly aroused the interest of oilfield researcher in drilling operations in the last ten years. Zwitterionic polymers own anionic and cationic group, and have great advantages for maintaining the stability of wellbore[11]. Zhao et al.[12] has synthesized a zwitterionic copolymer AM/DMC/AMPS as a low-molecular-weight encapsulator in deep-water drilling fluid which have showed strong clay hydration inhibition performance. However, there are still few reports about low molecular weight zwitterionic copolymer as clay hydration inhibitor so far. In the present work, SSS and AM were copolymerized with a novel cationic monomer THDAB to synthesize a low molecular weight zwitterionic copolymer clay hydration dispersion inhibitor AM/SSS/THDAB for WBDFs. Where, AM acts as the backbone, THDAB acts as a functional monomer which ensure the adsorption of AM/SSS/THDAB

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O O O O O O O O O O O O O O O O


Du, W.-C., Wang, X.-Y., Liu, M., Bi, T.-F., Song, S.-X., Zhang, J., & Chen, G. onto the clay surface, SSS acts as a temperature resistant monomer to ensure the copolymer with outstanding salt and temperature resistance performance. The copolymer structure was characterized by FT-IR, GPC, TGA-DSC and SEM. The inhibition performance was evaluated by hot rolling recovery rate experiments and the inhibition mechanism of AM/SSS/THDAB was discussed via particle size analysis and SEM in the text.

2. Experimental

50 °C. After cooling to room temperature, the solution was concentrated under reducing pressure and then redissolved in ethyl acetate and ethanol (ethyl acetate: ethanol = 7:3). Product was isolated in high yield as a rod-like crystals or white powder after placed in room temperature for 24h. Yield: 94.3%. 1H NMR (400 MHz, D2O): 5.62-5.75 (m, 1H, CH=C), 4.89∼5.07 (m, 2H, C=CH2), 3.75∼3.76 (d, 6H, CH2-O), 3.60 (t, 2H, -CH-C=C), 3.33 (t, 3H, -OH), 3.04∼3.06(t, 6H, N-CH2-C-OH); IR (KBr), /cm–1:3360, 2920, 1630, 1400, 1080, 900, 520.

2.1 Materials

2.3 Synthesis of AM/SSS/THDAB

SSS, AM, trolamine, allyl bromide, (NH4)2S2O8, NaHSO3, ethanol, ethyl acetate and isopropanol were all of analytical pure and purchased from Kelon Co., Ltd, Chengdu, China. Poly-ECH-DMA with molecular weight 32352 g/mol was supplied by Maikeba Mud Co., Ltd, USA. PF-CMJ with molecular weight 2.3×105 g/mol and XY-27 with 4362 g/mol was received from Engineering Technology Research Institute Co., Ltd., CNPC, Beijing, China. PAM with molecular weight 1.8×105 g/mol and FA-367 with molecular weight 1.2×104 g/mol were supplied by Sichuan Guangya polymer technology Co., Ltd, Chengdu, China. Na-MMT with cation exchange capacity of 81.3 mmol/g was obtained from Xia Zijie Bentonite Technology Co., Ltd, Xinjiang, China. The shale samples were obtained from Longmaxi shale gas field, Chongqing, China, and the mineral compositions of shale samples were illustrated in Table 1.

AM/SSS/THDAB was synthesized by redox free radical copolymerization in aqueous solution, the synthesis route of AM/SSS/THDAB is shown in Figure 2. Appropriate 7.2g SSS was dissolved in 10 mL deionized water, the pH was adjusted to the indicated value at 7.0 by using 30 wt % NaOH solutions. Then, 2g AM and 4g THDAB were added to flask with stirring at constant temperature under nitrogen atmosphere for 20 min. Hereafter, the initiator K2S2O8 and NaHSO3, which the initiator concentration was 1.5 wt% relative to the total monomer amount were added. The polymerization was carried out at 65 °C for 0.5h while stirring, and then isopropanol was added to the solution, polymerization was preceded at 65 °C for another 7 h. The target product was obtained by repeatedly washing with ethanol to remove monomers, isopropanol and initiator, AM/SSS/THDAB was further dried under vacuum oven at 65 °C for 24 h.

2.2 Synthesis of polymeric monomer THDAB

2.4 Characterizations of AM/SSS/THDAB

THDAB was prepared based on a method previously reported[13,14], as shown in Figure 1. Briefly, trolamine (0.20 mol), allyl bromide (0.20 mol), and ethanol (150 mL) were placed in a round-bottom flask equipped with a reflux condenser and refluxed for 24 h with magnetic stirring at

FT-IR spectra were recorded via a WQF-520 Fourier transform infrared spectrometer in the wave number range of 4000-500 cm−1. GPC was utilized to measure the molecular weight by using an Alliance e2695 instrument (Waters, USA). The injection volume and operation hours were 50 μL and 90 min, respectively. TGA-DSC test was acquired on a simultaneous TGA-DSC (METTLER TOLEDO, Swiss) instrument under nitrogen atmosphere flow (40 mL min−1) with the heating rate of 10 °C min−1. SEM analysis of AM/SSS/THDAB solution was obtained with a FEI Quanta 450 instrument, in the magnifying multiple ranges from 500× to 10000×.

Figure 1. Synthesis route of THDAB.

2.5 Inhibition performance evaluation Hot rolling recovery tests were used to study the inhibition ability of AM/SSS/THDAB, and the tests were conducted at 100 °C for 16h.

2.6 Inhibition mechanism study 2.6.1 Particle size distribution analysis 8 g AM/SSS/THDAB was added into 400 mL WBDFs and stirred for 2h. Then, particle size analysis was measured with a laser diffraction technique (HORIBA, Japan) at room temperature.

Figure 2. Synthesis route of AM/SSS/THDAB. Table 1. The mineral compositions of shale sample. Mineral compositions Content/%

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Kaolinite

Chlorite

Illite

Sodium bentonite

Illite / Sodium bentonite

0.0

26.3

65.1

8.6

10.0

Polímeros, 29(4), e2019053, 2019


Synthesis and performance of AM/SSS/THDAB as clay hydration dispersion inhibitor 2.6.2 SEM analysis SEM was investigated with a FEI Quanta 450 instrument, range of the magnifying multiple was from 500× to 10000×, and the samples were trimmed from the bottom of the API filter cake after the room temperature and room pressure drilling fluids tests.

3. Results and Discussion 3.1 Characterizations of AM/SSS/THDAB 3.1.1 FT-IR analysis Figure 3 shows the FT-IR spectra of AM, SSS, THDAB and zwitterionic copolymer AM/SSS/THDAB. For AM/SSS/THDAB, the absorption peak at 3353 cm-1 was due to the -OH stretching of THDAB, the strong absorption peak recorded at 1663 cm-1 was assigned to the stretching vibration of C=O, the peaks at approximate 1219 cm-1 was assigned to the -SO3H stretching vibration, which indicated that SSS was involved in the copolymerization. The peak at 1400cm-1 and 1025 cm-1 was attributed to the characteristic absorption peak of C-N and C-O-C, respectively[15]. FT-IR characterization result demonstrates that AM/SSS/THDAB contains characteristic functional group absorption peaks of each monomer, indicating that AM, SSS and THDAB were successfully copolymerized to the target product.

Figure 3. FT-IR spectra of AM, SSS, THDAB and AM/SSS/THDAB.

3.1.2 Molecular weight measurement The average molecular weight of AM/SSS/THDAB was determined by GPC and the result is shown in Figure 4. Figure  4 shows the molecular weight measurement of AM/SSS/THDAB. AM/SSS/THDAB has a certain width of molecular weight distribution and the Mp of AM/SSS/THDAB is 43674 g/mol. With a wide molecular weight distribution, the low molecular weight of AM/SSS/THDAB (800-5000 g/mol) could enter the layer of tetrahedral crystal and compress the diffusion electric double layer of clay[16]. In addition, the large molecular weight part could be packed on the clay surface, thereby, to effectively inhibit the hydration dispersion of clay.

Figure 4. Molecular weight measurement of AM/SSS/THDAB.

3.1.3 TGA-DSC measurement Thermogravimetry (TGA) differential scanning calorimetry (DSC) was utilized to investigate the thermal stability of AM/SSS/THDAB, and the thermal gravimetric curve displayed the four stages for the weight loss. As shown in Figure 5, the first stage with a loss of 6.92 mass % in the temperature range of 40-153 °C was due to the combination of evaporation moisture and AM/SSS/THDAB. The second stage taken place in the temperature range of 153-225 °C with a loss of 4.73 mass % was mainly probably due to the decompositions of amide groups and quaternary ammonium groups in the copolymer. The third decomposition temperature occurred in 225-309 °C with a prodigious loss of 8.33 mass % was mainly ascribed to the decomposition of AM/SSS/THDAB, which due to the degradation of C-C in the side chain[15]. The final loss stage in the temperature range of 309-500 °C with a prodigious loss of 46.86 mass % indicated that the main structure of AM/SSS/THDAB was destroyed. The result of TGA-DSC demonstrated that AM/SSS/THDAB shows an excellent thermal stability ability. Polímeros, 29(4), e2019053, 2019

Figure 5. TGA-DSC measurement of AM/SSS/THDAB.

3.1.4 SEM characterizations The SEM characterizations of AM/SSS/THDAB solutions are shown in Figure 6. From Figure 6, we have observed that the cross-linking structure of AM/SSS/THDAB was not obvious, and the polymer was mainly stretched on the main chain. Due to 3/7


Du, W.-C., Wang, X.-Y., Liu, M., Bi, T.-F., Song, S.-X., Zhang, J., & Chen, G.

Figure 6. SEM images of AM/SSS/THDAB: 1000×, scale bar 200 μm (a) 10000×, scale bar 50 μm (b). Table 2. Inhibition performance evaluation of AM/SSS/THDAB aqueous solution. Concentration (wt %) 0.5 1.0 1.5 2.0 2.5 3.0

AV/mPa·s 5.0 7.5 8.0 9.5 10.0 12.5

PV/mPa·s 5.0 5.0 5.0 7.0 8.0 10.0

YP/Pa

Φ6/Φ3

0 2.5 3 2.5 2 2.5

1/1 1/1 1/1 1/1 2/1 2/1

Rolling recovery rate /% 34.6 53.7 79.3 83.6 83.9 84.1

Rolling condition: 100°C×16h

the sulfonamide structures of SSS and amide groups of AM in AM/SSS/THDAB, cross-linking interaction and intramolecular interaction were occurred, and the speculation has also been confirmed by SEM characterization. However, the rope-like molecular structure still can guarantee the covering of AM/SSS/THDAB on the clay surface.

3.2 Inhibition performance evaluation The hot-rolling recovery tests were carried out to study the inhibition property of AM/SSS/THDAB and several clay hydration inhibitors which are commonly used in oilfield. Table 2 shows the effect of AM/SSS/THDAB on its solution viscosity and clay rolling recovery rate. From the results, we can see that with the increasing of AM/SSS/ THDAB concentration, the viscosity of polymer aqueous solutions and shale hot rolling recovery rate have increased. When the polymer concentration was 2 wt%, the rolling recovery has reached 83.6%, and with the increasing of polymer concentration, the change of shale rolling recovery rate was not obvious yet. The clay inhibition property of AM/SSS/THDAB and several clay hydration inhibitors commonly used in oilfield were compared in this work, as shown in Table 3. As shown in Table 3, zwitterionic inhibitor XY-27 shows the limited inhibition performance leading to the rolling recovery rate of clay in its solutions was only 56.8%. The coating agent FA-367 demonstrated a nice clay hydration dispersion inhibitive performance and the clay rolling recovery rate has reach to 82.6%. As a film-forming plugging agent, Poly-ECH-DMA and PF-CMJ can adsorb on the clay surface because there are a large amount of absorbable groups on them[17-19], and the excellent clay hydration inhibition ability with the rolling 4/7

Table 3. Inhibition performance study of inhibitors. Inhibitor 2.0 wt% AM/SSS/THDAB 0.5 wt% XY-27 0.5 wt% FA-367 2.0 wt% Poly-ECH-DMA 0.5 wt% PAM 2.0 wt%PF-CMJ

Rolling recovery rate/% 83.6 56.8 82.6 79.2 62.3 72.6

recovery rate of clay was 79.2% and 72.6%, respectively. From the above results we can conclude that for all cases, copolymer inhibitors evaluated in this work all showed superior clay hydration dispersion inhibition performance.

3.3 Inhibition mechanism analysis 3.3.1 Particle distribution tests It is well known that the negative charge surface of clay is trendy to be neutralized by the positive charge of cations, and flocculation will be happened when positive polymer was added to the WBDFs[20-22]. The inhibition mechanism was evaluated by particle distribution tests, and the results are shown in Figure 7. As shown in Figure 7, for the case in distilled water, Na-MMT illustrated tiny average particle size of 33.06 μm, and the average particle size of Na-MMT has been increased to 425.37μm in 2.0 wt% AM/SSS/THDAB solutions. For the reason that zwitterionic AM/SSS/THDAB can be coated on the surface of clay to inhibit the hydration dispersion of clay and in that way to make the particle size obviously increased. Particle distribution tests have shown that AM/SSS/THDAB can form an effective coating on the clay surface. Polímeros, 29(4), e2019053, 2019


Synthesis and performance of AM/SSS/THDAB as clay hydration dispersion inhibitor 3.3.2 SEM observations SEM is a convenient technology to observe the morphological changes of material surface. The surface morphologies of Na-MMT without AM/SSS/THDAB and with AM/SSS/THDAB were analyzed by using SEM and the images are showed in Figure 8. During the preparation process of samples, there was a light pressure (0.69 MPa) on the surface of the muds cake. For the Na-MMT without AM/SSS/THDAB, there were large holes in the muds cake, which indicated the poor bonding force between Na-MMT particles[23,24]. In contrast, after treated with 2 wt% AM/SSS/THDAB, there were thin polymer films on the muds cake surface, which indicating

that AM/SSS/THDAB can inhibit the clay dispersion by wrapping in the clay surface. Through the inhibition mechanism analysis of AM/SSS/THDAB by the above two methods, and reference to swelling inhibition mechanism analysis reported by other work, we have proposed the clay hydration dispersion inhibition mechanism of AM/SSS/THDAB, which is shown in Figure 9. In the hot-rolling recovery tests, we found that AM/SSS/THDAB showed excellent hydration inhibition property, a possible explanation to the result might be that the larger molecular weight part of AM/SSS/THDAB can be wrapped on clay surface because there are hydroxyl and quaternary ammonium functional groups in the side chain of copolymer. What’s more, dispersion was not easy

Figure 7. Particle distribution tests.

Figure 8. SEM observations of Na-MMT composites (×5000): (a) basic muds cake; (b) treated with 2 wt% AM/SSS/THDAB.

Figure 9. Inhibition mechanism analysis of AM/SSS/THDAB. Polímeros, 29(4), e2019053, 2019

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Du, W.-C., Wang, X.-Y., Liu, M., Bi, T.-F., Song, S.-X., Zhang, J., & Chen, G. to occur while clay particles fixed on the long chain of AM/SSS/THDAB.

4. Conclusions In conclusion, a zwitterionic copolymer clay hydration dispersion inhibitor AM/SSS/THDAB for WBDFs was successfully prepared by THDAB, SSS and AM. The copolymer was characterized by FT-IR, GPC, TGA-DSC and SEM, results showed the molecular weight of AM/SSS/THDAB was 43674 g/mol and showed an excellent temperature resistance ability. Its inhibition performance and mechanism were systematically investigated by a range of methods, evaluation experiments indicated that AM/SSS/THDAB possessed superior inhibition properties compared with several inhibitors. Through the analysis of inhibition mechanism, three key points such as the multiple driven forces, wide molecular weight distribution and the long chain of copolymer could ensure the perfect inhibition performance of AM/SSS/THDAB. All these features indicate that AM/SSS/THDAB could be a potential clay hydration dispersion inhibitor for wellbore stability in drilling engineering.

5. Acknowledgements The authors would like to thank the Open Fund (KFKT2019-13) of the Key Laboratory of Auxiliary Chemistry and Technology for Chemical Industry, Ministry of Education Shaanxi University of Science and Technology for financial support.

6. References 1. Fagundes, K. R. S., Luz, R. C. S., Fagundes, F. P., & Balaban, R. C. (2018). Effect of carboxymethylcellulose on colloidal properties of calcite suspensions in drilling fluids. Polímeros: Ciência e Tecnologia, 28(4), 373-379. http://dx.doi. org/10.1590/0104-1428.11817. 2. Chen, S. Y., Shi, Y. P., Yang, X. Y., Xie, K. Z., & Cai, J. H. (2019). Design and evaluation of a surfactant–mixed metal hydroxide-based drilling fluid for maintaining wellbore stability in coal measure strata. Energies, 12(10), 1862-1881. http:// dx.doi.org/10.3390/en12101862. 3. Wilson, M. J., & Wilson, L. (2014). Clay mineralogy and shale instability: an alternative conceptual analysis. Clay Minerals, 49(2), 127-145. http://dx.doi.org/10.1180/claymin.2014.049.2.01. 4. Anderson, R. L., Ratcliffe, I., Greenwell, H. C., Williams, P. A., Cliffe, S., & Coveney, P. V. (2010). Clay swelling: a challenge in the oilfield. Earth-Science Reviews, 98(3-4), 201-216. http://dx.doi.org/10.1016/j.earscirev.2009.11.003. 5. Gholami, R., Elochukwu, H., Fakhari, N., & Sarmadivaleh, M. (2018). A review on borehole instability in active shale formations: Interactions, mechanisms and inhibitors. EarthScience Reviews, 177, 2-13. http://dx.doi.org/10.1016/j. earscirev.2017.11.002. 6. Du, W. C., Wang, X. Y., Chen, G., Zhang, J., & Slaný, M. (2020). Synthesis, property and mechanism analysis of a novel polyhydroxy organic amine shale hydration inhibitor. Minerals, 10(2), 128-142. http://dx.doi.org/10.3390/min10020128. 7. Rana, A., Arfaj, M. K., & Saleh, T. A. (2019). Advanced developments in shale inhibitors for oil production with low environmental footprints-A review. Fuel, 247, 237-249. http:// dx.doi.org/10.1016/j.fuel.2019.03.006. 6/7

8. Ahmed, H. M., Kamal, M. S., & Al-Harthi, M. (2019). Polymeric and low molecular weight shale inhibitors: a review. Fuel, 251, 187-217. http://dx.doi.org/10.1016/j.fuel.2019.04.038. 9. Ghaderi, S., Ramazani S.A, A., & Haddadi, S. A. (2019). Applications of highly salt and highly temperature resistance terpolymer of acrylamide/styrene/maleic anhydride monomers as a rheological modifier: rheological and corrosion protection properties studies. Journal of Molecular Liquids, 294, 111635111646. http://dx.doi.org/10.1016/j.molliq.2019.111635. 10. Abdollahi, M., Pourmahdi, M., & Nasiri, A. (2018). Synthesis and characterization of lignosulfonate/acrylamide graft copolymers and their application in environmentally friendly water- based drilling fluid. Journal of Petroleum Science Engineering, 171, 484-494. http://dx.doi.org/10.1016/j.petrol.2018.07.065. 11. Jia, H., Huang, P., Wang, Q. X., Han, Y. G., Wang, S. Y., Zhang, F., Pan, W., & Lv, K. H. (2019). Investigation of inhibition mechanism of three deep eutectic solvents as potential shale inhibitors in water-based drilling fluids. Fuel, 244, 403-411. http://dx.doi.org/10.1016/j.fuel.2019.02.018. 12. Zhao, X., Qiu, Z. S., Zhang, Y. J., Zhong, H. Y., Huang, W. A., & Tang, Z. C. (2017). Zwitterionic polymer P (AM-DMCAMPS) as a low-molecular-weight encapsulator in deepwater drilling fluid. Apply. Science, 7(6), 594-810. http://dx.doi. org/10.3390/app7060594. 13. Pu, X. L., Du, W. C., Sun, J. S., Luo, X., & Zhang, H. D. (2016). Synthesis and application of a novel polyhydroxy amine clay anti-swelling agent. Petrochemical Technology, 45, 595-600. 14. Silva, F. A., Siopa, F., Figueiredo, B. F. H. T., Gonçalves, A. M. M., Pereira, J. L., Gonçalves, F., Coutinho, J. A. P., Afonso, C. A. M., & Ventura, S. P. M. (2014). Sustainable design for environment-friendly monoand dicationic cholinium-based ionic liquids. Ecotoxicology and Environmental Safety, 108, 302-310. http://dx.doi.org/10.1016/j.ecoenv.2014.07.003. PMid:25108510. 15. Paz, R. A., Leite, A. M. D., Araújo, E. M., Medeiros, V. N., Melo, T. J. A., & Pessan, L. A. (2016). Mechanical and thermomechanical properties of polyamide 6/Brazilian organoclay nanocomposites. Polímeros: Ciência e Tecnologia, 26(1), 52-60. http://dx.doi.org/10.1590/0104-1428.1748. 16. Jain, R., & Mahto, V. (2015). Evaluation of polyacrylamide/ claycomposite as a potential drilling fluid additive in inhibitive water based drilling fluid system. Journal of Petroleum Science Engineering, 133, 612-621. http://dx.doi.org/10.1016/j. petrol.2015.07.009. 17. Costa, L. P., Jr., Silva, D. B. R., Aguiar, M. F., Melo, C. P., & Alves, K. G. B. (2019). Preparation and characterization of polypyrrole/organophilicmontmorillonite nanofibers obtained by electrospinning. Journal of Molecular Liquids, 275, 452462. http://dx.doi.org/10.1016/j.molliq.2018.11.084. 18. Salles, F., Douillard, J.-M., Bildstein, O., Gaudin, C., Prelot, B., Zajac, J., & Van Damme, H. (2013). Driving force for the hydration of the swelling clays: case of montmorillonites saturated with alkaline-earth cations. Journal of Colloid and Interface Science, 395, 269-276. http://dx.doi.org/10.1016/j. jcis.2012.12.050. PMid:23352873. 19. Boek, E. S., Coveney, P. V., & Skipper, N. T. (1995). Monte carlo molecular modeling studies of hydrated Li-, Na-,and K-smectites: understanding the role of potassium as a clay swelling inhibitor. Journal of the American Chemical Society, 117(50), 12608-12617. http://dx.doi.org/10.1021/ja00155a025. 20. Caglar, B., Çırak, Ç., Tabak, A., Afsin, B., & Eren, E. (2013). Covalent grafting of pyridine-2-methanol into kaolinite layers. Journal of Molecular Liquids, 1032, 12-22. http://dx.doi. org/10.1016/j.molstruc.2012.08.004. 21. Du, W. C., Pu, X. L., Sun, J. S., Luo, X., Zhang, Y. N., & Li, L. (2018). Synthesis and evaluation of a novel monomeric amine Polímeros, 29(4), e2019053, 2019


Synthesis and performance of AM/SSS/THDAB as clay hydration dispersion inhibitor as sodium montmorillonite swelling inhibitor. Adsorption Science and Technology, 36(1-2), 655-668. http://dx.doi. org/10.1177/0263617417713851. 22. Caglar, B., Topcu, C., Coldur, F., Sarp, G., Caglar, S., Tabak, A., & Sahin, E. (2016). Structural, thermal, morphological and surface charge properties of dodecyltrimethylammonium-smectite composites. Journal of Molecular Liquids, 1105, 70-79. http://dx.doi. org/10.1016/j.molstruc.2015.10.017. 23. PĂŠrez, A., Montes, M., Molina, R., & Moreno, S. (2014). Modified clays as catalysts for the catalytic oxidation of ethanol.

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Applied Clay Science, 95, 18-24. http://dx.doi.org/10.1016/j. clay.2014.02.029. 24. Gang, C., Gao, L., Sun, Y., Gu, X., Hu, W., Du, W., Zhang, J., & Qu, C. (2019). A green shale inhibitor developed from lignin sulfonate and a mechanism study. Journal of Biobased Materials and Bioenergy, 13(6), 778-783. http://dx.doi. org/10.1166/jbmb.2019.1908. Received: Aug. 27, 2019 Revised: Oct. 25, 2019 Accepted: Feb. 03, 2020

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ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.08619

Effect of nanoclay addition and chemical treatment on static and dynamic mechanical analysis of jute fibre composites Seetharaman Arulmurugan1*  and Narayanan Venkateshwaran1 Department of Mechanical Engineering, Rajalakshmi Engineering College, Tamilnadu, Chennai, India

1

*arulmurugan.s@rajalakshmi.edu.in

Abstract In this article, the influence of alkali treatment and addition of montmorillonite nanoclay as filler on mechanical and visco-elastic behaviour of jute fibre polymer composite were investigated. The composites are fabricated using 5wt% of nanoclay, untreated and chemically treated jute fibre of various percentage by handlayup method. The static mechanical properties like tensile, flexural, impact and inter laminar shear strength are studied as per respective ASTM standard. The dynamic mechanical analysis was carried out to evaluate storage modulus and damping factor of the prepared composite. The composition and structure of the functional groups of modified fibres were examined by Fourier transform infrared spectroscopy. The results showed that the interaction of filler addition and NaOH+KMnO4 treatment of fibres have significantly improved the tensile, flexural and impact properties to 47.12, 201.13, 172.61MPa respectively. Dynamic mechanical analysis results revealed that the incorporation of filler increases the storage modulus and glass transition temperature. The incorporation of 5wt% clay and 25wt% jute fiber increase the glass transition temperature of the composite material from 109 to 115 °C. Keywords: chemical treatment, glass transition temperature, mechanical properties, nanoclay, natural fibre. How to cite: Arulmurugan, S., & Venkateshwaran, N. (2019). Effect of nanoclay addition and chemical treatment on static and dynamic mechanical analysis of jute fibre composites. Polímeros: Ciência e Tecnologia, 29(4), e2019054. https://doi.org/10.1590/0104-1428.08619

1. Introduction Natural fibre reinforced polymer composites offer more advantage over conventional material owing to low cost, low density and high specific properties and these hybrid composites are one of the promising fields in polymer science that makes attention for application in a variety of sectors ranging from aircraft to the building industry[1,2]. Renewable nanomaterials are used as reinforcements in the field of nanoscience and nanotechnology in order to have advanced materials with novel properties[3]. Nanoclay/natural fiber hybrid composites have great attention recently due to their wide variety of properties in automotive, biomedical, food packaging and other consumer applications with better mechanical, thermal, optical and barrier properties[4]. Natural fibres like jute, bamboo and coir with varying span length subjected to mechanical and physical properties are studied and it was found that the young’s modulus value increases gradually on increasing the span length. On investigating the SEM images of fiber, jute shows smoother surface when compared to bamboo and coir. This smooth surface with less porosity provides higher tensile strength when compared to coir fiber which has higher porosity[5]. A comprehensive literature review on various aspects of biocomposites and natural fibres with reference to physical modifications such as corona, plasma treatment and chemical modifications like alkaline, acetylation, silane, maleated coupling, enzyme

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treatment are discussed[6,7]. The jute fabrics reinforced with polypropylene matrix with various chemical treatments like hydroxyethyl methacrylate, urea, and KMnO4 were studied and it was found that the improved mechanical properties are achieved in the combinations of all the above mentioned treatments rather than the individual treatment[8]. Fibres like hemp and banana are treated with chemicals like acetyl, silane, alkali with various concentrations and are examined by using SEM, FTIR spectroscopy. Treatment with chemical decreases the loosely linked hydroxyl group of hemicellulose, lignin and breakdown the covering surface which exposes the cellulose surface for better adhesion. This makes fiber less hydrophilic and improved mechanical properties[9,10]. The mechanical and thermal properties of nanocomposite i.e., epoxy resins filled with nanosilica are investigated and it was found that the decrease in the glass transition temperature was noted with higher silica loading due to decrease in cross-link density of high mobility regions but enhanced mechanical property with increasing filler content[11,12]. Addition of organo modified and montmorillonite clay as filler in the matrix and fiber significantly improves the mechanical properties that encourage to use of structural purposes[13,14]. The interlaminar shear strength and free vibration characteristics of hybrid nanocomposite plates by reinforcing glass fibre mat, coconut sheath and organically

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O O O O O O O O O O O O O O O O


Arulmurugan, S., & Venkateshwaran, N. modified nanoclay in the polymer matrix was analysed. They observed that the second phase nanoscale filler in the matrix and fibre considerably improves the damping of the composites and shear strength property[15,16]. The dynamic mechanical analysis of particulate filled fibre reinforced polymer composites were investigated and it was found that the addition of filler shows better viscoelastic behaviour and higher glass transition temperature. The heterogeneity of the composite samples was analysed by using cole-cole plot[17-21]. The effect of silane, nano hybrid coatings on mechanical properties of basalt fibres are studied and it was found that the treated cum coated fibre shows 23% higher tensile strength than non-coated fibres[22]. The effect of water absorption on the mechanical properties of the hybrid composites was studied and it was found that the tensile and flexural properties were significantly reduced under wet condition. Similarly, in the clay filled hybrid composites the rate of moisture absorption decreases because the dispersal of nanoclay in the composites acts as an obstacle and restricts the flow of water in all direction of the composites[23,24]. The  woven kevlar- kenaf hybrid composites with various volume fractions are prepared and subjected to ballistic impact properties. From the results, it is found that the ballistic properties of the composites increase with the increase in thickness and areal density of the hybrid composites[25]. The nanomechanical properties of different loading levels of clay filled polyester composites through Vickers hardness test was studied and it was found that inclusion of 5wt.% clay into the polymer matrix results in an improvement in hardness of 26.52% and the fracture toughness depends on the montmorillonite clay content[26]. From the above literatures, it is found that addition of fillers, fibre parameters and chemical treatments improves the mechanical properties of the composite to a greater extend. Hence, in this work, optimum jute fibre parameters are predicted by carrying out mechanical tests. Further, to this an optimum clay percent is added with it to form hybrid composite. Finally, fibres are treated with alkalis, the combined effect of chemical treatment, filler addition on mechanical and dynamic mechanical properties are analysed.

2. Materials and Methods 2.1 Materials Jute fibres of yarn type was used as natural fibre reinforcement and it was procured from National Jute Board, Chennai, India. The montmorillonite nanoclay was obtained from Sigma Aldrich, Bangalore and India, it was used as filler in the polyester resin. The unsaturated polyester resin along with Cobalt napthanate as accelerator and Methyl Ethyl Ketone Peroxide are act as a catalyst, which are purchased from Vasavibala Resins, Chennai, India.

2.2 Nanocomposite preparation Jute fibres of varying fibre content (i.e.,5%,10%,15%,20%,25% & 30wt.%) are used as primary reinforcement whereas montmorillonite nanoclay is used as secondary reinforcement in the polyester resin. Initially, jute fibre of 20 mm length with various percent are mixed with resin mixer (i.e., polyester and optimum clay) to fabricate composite. The hand layup

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method is followed for it. The optimum weight percentage of nanoclay (5wt %) obtained earlier[27] was mixed with unsaturated polyester resin by sonification technique at the optimal frequency of 10 kHz and 60 minutes duration. This nanocomposite laminates are allowed to cure for one day at room temperature. Then the specimens are cut from the laminate according to ASTM standards for different tests.

2.3 Short beam shear testing The short beam shear test was conducted to find out the interlaminar shear strength (ILSS) by using Instron universal testing machine. The test was carried out according to ASTM standard D-2344 with a test speed of 2 mm/min. Composite plates were placed on two supports of diameter 3 mm and it was made free to rotate in order to have free lateral motion. Load was applied at the centre of the plate by using steel dowel of diameter 6 mm. The plate was loaded until fracture, and the fractured load was used to determine the apparent shear strength of the material. The ILSS was calculated for composite samples based on the Equation 1. F sbs = 0.75 X Pmax / ( b X h )

(1)

where, Fsbs = short beam strength, MPa; Pmax = max. load observed during the test, N; b = specimen width, mm; h = specimen thickness, mm.

2.4 Tensile, flexural and impact testing Tensile and flexural tests (three-point bending) are carried out by using the Instron universal testing machine while impact test is carried out by using an Izod impact test set-up in Tinius Olsen machine. The Tensile test was carried out with a loading speed of 2 mm/min as per ASTM standard D-638. Three point bending test is carried out by using ASTM standard D-790. Impact test is carried out according to ASTM standard D-256 without notch. Each test is carried out on five samples and the average value is taken.

2.5 Fibre surface treatments Untreated & chemically treated jute fibers were used for preparing the composites. Chopped Jute fibers (20 mm) were immersed in a vessel separately which contains 2% of NaOH aqueous solution, 2% of KMnO4 and NaOH+KMnO4 each for 1 hour at room temperature. The fibers were then washed with distilled water to remove the excess of sodium hydroxide and KMnO4 on the fibers. Afterward, the fibers were dried out at 50 °C in an oven for 3hrs. Chemically treated jute fibres are then reinforced in polyester/ montmorillonite nanocomposites with optimum fibre (i.e., 15 wt. % obtained earlier[27]) content by using hand lay-up process. The reaction of jute fibre with potassium permanganate in the presence of sodium hydroxide is shown below (2). O  OH Cellulose − OH + KMnO4 → Cellulose − H − O − Mn − OK +  O −

(2)

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Effect of nanoclay addition and chemical treatment on static and dynamic mechanical analysis of jute fibre composites 2.6 Fourier Transform Infrared (FTIR) spectroscopy The FTIR analysis was carried out by using a JASCO FT/IR-6300 spectrometer for the analysis of functional group. About 5 mg of untreated and treated fibers were milled into a tiny particle and mixed with potassium bromide then placed on a disc. An average scanning speed of 2 mm/sec was recorded for each spectrum at the wave numbers which ranging from 399.19 to 4000.6 cm-1 with a resolution of 4 cm-1.

2.7 Dynamic Mechanical Analysis (DMA) testing Dynamic mechanical analyser (SEIKO, Model DMAI/DMSC 6100) was used for find out the dynamic properties such as storage modulus, loss modulus and tan δ of the nanocomposites with the specimen size of 50 mm × 50 mm × 3 mm. DMA test was carried out at a temperature range of 30-175 °C at 5 °C/minute with different frequencies of 0.1 Hz, 1 Hz, 2 Hz, 5 Hz and 10Hz under the tensile mode.

on the surface of the fibre. Increase in the peak at 1026 cm-1 shows that the alkali treatment increases the hydroxyl group on the fibre surface[32]. The alkali and KMnO4 treatment reduce the hydrophilic nature of the fibre and increases the fiber matrix adhesion[33].

3.3 Mechanical properties of composites with surface treated fibre The chemically treated Jute fibre reinforced polyester/ montmorillonite hybrid nanocomposites fabricated are cut into required dimension as per ASTM standards and is tested for various mechanical properties. The results are shown in the Figures 3 to 5. From the results, it is found that the composite reinforced with both NaOH and KMnO4

3. Results and Discussions 3.1 Interlaminar Shear Strength (ILSS) The short beam test specimens are cut into required dimensions as per ASTM standards D-2344. Figure 1 shows the ILSS properties of the various composite tested. From the figure, it is found that the ILSS value for 5% nanoclay along with 5wt% jute fibre is 18.15MPa. On further addition of jute fibre i.e. 10, 15, 20wt% along with the 5wt% clay, the ILSS value increases by 10.3, 30.47, 40.72% respectively. From the Figure 1, it is found that the addition of 5% nanoclay with 25wt% jute fibre hybrid nanocomposite have enhanced the shear strength to 28.03 MPa with 54.44% increase when compared with 5% clay and 5wt% jute fibre and it was achieved due to enhanced bond characteristics, better interfacial areas and distinctive phase morphology of the fibre and nanoclay[28]. Further, it is clear that the addition of jute fibre in polyester matrix reduces the shear strength due to clustering of fibre in the polymer composite which attributing the non-uniform distribution of stresses[29].

Figure 1. Effect of nanoclay content on interlaminar shear strength of Jute fibre reinforced polyester/ montmorillonite clay nanocomposites.

3.2 FTIR analysis From the FTIR spectroscopy the chemical structure of the cellulose, hemicellulose and lignin constituents for the untreated and chemically treated jute fibres were studied and shown in the Figure 2. From the figure, the peaks at 902 cm-1 and 1440 cm-1 were designated as C-H bending of amorphous and crystalline cellulose[9]. These peaks remain unchanged for NaOH and KMnO4 treated fibres. This shows that the chemical treatments didn’t affect the cellulose arrangement of the fibre. The peak near 1742 cm-1 was due to C-O stretching of the acetyl and carboxyl groups in hemicelluloses of the raw jute fibre. This peak was not present for alkali treated fibres. This can be due to the elimination of acetyl group present in hemicelluloses after chemical treatment. The peak near 1440 cm-1 was assigned to CH3 deformation in lignin. The loss of lignin was found after alkali treatment and this is the reason for decrease in the absorption intensity ratio[30]. The absence of peaks at 1250 and 1550 cm-1 shows the removal of lignin and hemicellulose[31]. After potassium permanganate treatment, the aromatic band of lignin and cellulose intensity decreases and increase in the peak at 1720 cm-1 was found, which confirms the quinine formation Polímeros, 29(4), e2019054, 2019

Figure 2. FTIR spectra curve for untreated and chemically treated NaOH and KMnO4 fibre.

Figure 3. Comparison of the tensile properties of chemically treated jute fibre reinforced polymer composites with 5wt% montmorillonite clay as filler. 3/8


Arulmurugan, S., & Venkateshwaran, N. treated fiber shows an increase on the tensile strength from 40.38 to 47.12 MPa with respect to the composite made with the untreated fibers[30] as shown in Table 1. Similarly, the flexural and impact strength raises from 166.23 to 201.13 MPa and 151.58 to 172.61 MPa respectively with respect to untreated fibres. The fibres modified with sodium hydroxide aqueous solution along with the treatment of KMnO4 enhances the property of the hybrid composite material as compared with the fibres without surface treatment. The results show that the chemical treatment improves the crystallinity of the fibres, which improves the adhesion between the polymer matrix and the fibre surface thereby increases the mechanical properties of the composite materials[33].

3.4 Dynamic mechanical analysis test result 3.4.1 Effect of clay and fibre content on storage modulus (E’) The stiffness property of the composite material can be measured by using storage modulus value[34]. Figure 6 to 10 shows the effect of temperature and montmorillonite

Figure 4. Comparison of the flexural properties of chemically treated jute fibre reinforced polymer composites with 5wt% montmorillonite clay as filler.

Figure 5. Comparison of the impact properties of chemically treated jute fibre reinforced polymer composites with 5wt% montmorillonite clay as filler.

nanoclay loading on the storage modulus of polyester hybrid nanocomposite at various frequencies. The Figure 7 depicts that the storage modulus value at 1Hz frequency for 5wt % clay and 5, 10, 15, 20, 25wt % of jute fibres added hybrid composites are 2.5, 3.3, 4.1, 4.8 and 5.2 GPa respectively. The result shows that the energy accumulation capability of the nanocomposites can be enhanced by the addition of fibre content. The storage modulus drops near the glass transition temperature (Tg), which was due to the softness of the composite. The storage modulus of the composite material can be improved by the incorporation of nanoclay as filler material in the composites. These filler act as a stiffening agent by reducing the movement of the polymeric molecules[35]. From Figure  7 it is found that addition of nanoclay enriched the modulus considerably because of better interaction between polyester, nanoclay and fibre. Elevated storage modulus is observed in both the regions (i.e., glassy and rubbery) and also the jute fibre addition increases the Tg value of composite material from 109 to 115 °C. This is considerable increment of storage modulus of the hybrid

Figure 6. Storage Modulus of Jute fibre reinforced polyester/clay nanocomposites at 0.5Hz frequency.

Figure 7. Storage Modulus of Jute fibre reinforced polyester/clay nanocomposites at 1Hz frequency.

Table 1. Experimental mechanical properties of untreated jute reinforced polymer composites with 5wt% montmorillonite clay as filler. Serial No. 1 2 3 4 5 6

Jute Fiber Content (%) 5 10 15 20 25 30

Tensile Strength (MPa) 30.30 32.91 40.38 31.15 26.56 20.55

Flexural Strength (MPa) 91.77 121.39 166.23 203.10 234.93 212.46

Impact Strength (kJ/mm2) 41.41 100.00 151.58 130.30 103.03 84.85

Mechanial properties data from reference[27].

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Effect of nanoclay addition and chemical treatment on static and dynamic mechanical analysis of jute fibre composites composite material due to the incorporation of nanoclay in a polymer which enhances the stress transfer by acting as a secondary reinforcement in the composite[16]. Another observation observed from the figure is, nanoclay up to 5wt% in the polyester along with 25wt% jute fibre content increases the storage modulus extremely than the other weight percentage of montmorillonite nanoclay. This is because of increasing nanoclay content in the composite forms cluster formation. Due to this it forms heterogeneity property and it creates weak bonding between matrix and fibre. The ratio of loss modulus to storage modulus is called as damping factor and it measures energy dissipation through material when it is loading. The variation of Tan δ peak is

associated with molecular movement and amount of energy dissipation. In a composite material the energy dissipation is dependent on the molecular movement in the polymer chain, fibre/matrix interaction, strength of the fibre, fibre breakage and crack propagation[35]. From the Figures 11 to 15 it is found that composite with 5wt% nanoclay and 25wt% jute content has lower Tan δ peak, which is due to better fibre/matrix adhesion, decrease in molecular mobility and enhanced load carrying capacity. These are due to the addition of jute fibre as primary reinforcement and nanoclay as secondary produces positive hybrid effect in the composite. This hybrid effect carries more loads and observes large amount of energy and thus will increase the performance of composite material. The inclusion of nanoclay allows a lesser amount of energy at the interface

Figure 8. Storage Modulus of Jute fibre reinforced polyester/clay nanocomposites at 2Hz frequency.

Figure 11. Effect of Tanδ at 0.5Hz frequency on Jute fibre reinforced polyester/clay nanocomposites.

3.4.2 Effect of clay and fibre content on damping factor (tan δ)

Figure 9. Storage Modulus of Jute fibre reinforced polyester/clay nanocomposites at 5Hz frequency.

Figure 10. Storage Modulus of Jute fibre reinforced polyester/clay nanocomposites at 10Hz frequency. Polímeros, 29(4), e2019054, 2019

Figure 12. Effect of Tanδ at 1Hz frequency on Jute fibre reinforced polyester/clay nanocomposites.

Figure 13. Effect of Tanδ at 2Hz frequency on Jute fibre reinforced polyester/clay nanocomposites. 5/8


Arulmurugan, S., & Venkateshwaran, N.

Figure 14. Effect of Tanδ at 5Hz frequency on Jute fibre reinforced polyester/clay nanocomposites.

Figure 16. Cole-Cole plot of the Jute fibre reinforced polyester/ clay nanocomposites at 10Hz frequency. Table 2. Values of Tg and Peak height from tan δ at 10Hz frequency. Sample 5wt% JF NC 10wt% JF NC 15wt% JF NC 20wt% JF NC 25wt% JF NC 30wt% JF NC

Tg (Tan δ) (°C) 109 110 112 114 115 113

Peak height 0.34 0.33 0.31 0.25 0.23 0.28

Peak width at half height 4.86 4.92 5.03 5.07 5.13 6.22

Table 3. Experimental properties of Tan δ value of chemically treated jute reinforced polymer composites with 5wt% montmorillonite clay as filler. Figure 15. Effect of Tanδ at 10Hz frequency on Jute fibre reinforced polyester/clay nanocomposites.

which means better interface will dissipate lower energy and hence it will enhance the stiffness of the material and also the Tg value of composite material. Table 2 shows the data’s of Tg, peak height and peak width at half height obtained from tan δ curve at 1 Hz frequency. The peak height data is associated with the energy dissipation at the composite interface and peak width at half height relates the relaxation time. The higher value of peak height indicates the higher dissipation energy and from Table 2 it is found it for 5wt % jute fibre reinforced nanoclay filled composite material. The higher value of peak width is obtained for 30wt % jute fibre reinforced nanoclay filled composite material. It indicates that the higher dynamic heterogeneity is associated with that composite as mentioned by Saiter et al.[36]. Table 3 shows the tan delta of chemically treated with jute fibre composite study. 3.4.3 Cole-Cole plot Figure 16 shows the Cole-Cole plot of 5, 25 and 30 weight percentage of Jute fibre reinforced polyester/montmorillonite nanocomposites. The homogeneity property of the composite material can be indicated by cole-cole plot[20] and it is plotted as loss modulus (E”) vs. storage modulus (E’) at a particular frequency. From the Figure 16, it can be noted that the addition of 5wt% clay and 25wt% jute fibre in the polymer shows the perfect semi-circle which indicates the uniform distribution of clay and fibre with polyester and thus maintain the homogeneity nature. On further addition of fibre 6/8

5wt% NC + 15wt% JF Untreated NaOH KMnO4 Both NaOH & KMnO4

Dynamic Mechanical Analysis Tg (Tan δ) (°C) 112.0 112.3 112.5 113.0

i.e. 30wt%, the smooth and perfect semi circle changes to imperfect shape. This indicates that the material behaviour changes from homogeneous to heterogeneous[37]. From the results, it is concluded that the optimum percentage of clay and fiber enhances the dynamic mechanical properties of the composite material.

4. Conclusions The following observations were made during this study of montmorillonite nanoclay as a filler agent in unsaturated polyester resin along with chemically treated jute fibre as reinforcement. • The inclusion of montmorillonite nanoclay with jute fibre reinforced polyester composite enhances the mechanical properties in all the cases of investigation; • The maximum increase in the inter laminar shear strength is found as 28.03 MPa with the inclusion of 5wt% clay and 25wt % jute fibre which is 54.44% increase when compared to 5wt% clay and 5wt% jute fiber. This shows the existence of enhanced bond characteristics, better interfacial areas and distinctive phase morphology of the fibre reinforced polyester clay nanocomposites; Polímeros, 29(4), e2019054, 2019


Effect of nanoclay addition and chemical treatment on static and dynamic mechanical analysis of jute fibre composites • The surface treatment of fibre by alkali and KMnO4 shows better mechanical and thermal property which aids in improved interphase adhesion between reinforcements and matrix. Hence, the mechanical properties namely tensile, flexural and impact are improved by 16.70%, 21%, and 13.87% respectively when compared to untreated fibre; • From the dynamical mechanical analysis, it is found that the addition of nanoclay increases the storage modulus due to the imparting effect of filler reinforcements which are better rigid than the polymer matrix[38]. The increase in the glass transition temperature was found from 109 to 115 °C, however it reduces the damping factor. It may be due to the requirement of high thermal energy to induce uniform motion of molecules.

Based on the experimental analysis, it was found that the jute fibre reinforced clay filled polyester composite can be used as a substitute material for the medium and light weight applications like automotive industries, biomedical equipments, food packaging industries and other consumer applications with enhanced mechanical, thermal and damping properties.

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of Polymers and the Environment, 26(4), 1520-1527. http:// dx.doi.org/10.1007/s10924-017-1060-z. 32. Gumel, S. M., & Tijjani, A. A. (2015). The effect of fiber treatment on the water absorption of piliostigma reinforced Epoxy. ChemSearch, 6(2), 1-7. 33. Li, X., Tabil, L. G., & Panigrahi, S. (2007). Chemical treatments of natural fiber for use in natural fiber-reinforced composites: a review. Journal of Polymers and the Environment, 15(1), 25-33. http://dx.doi.org/10.1007/s10924-006-0042-3. 34. Dewan, M. W., Hossain, M. K., Hosur, M., & Jeelani, S. (2013). Thermomechanical properties of alkali treated jute-polyester/ nanoclay biocomposites fabricated by VARTM process. Journal of Applied Polymer Science, 128(6), 4110-4123. http://dx.doi. org/10.1002/app.38641. 35. Jesuarockiam, N., Jawaid, M., Zainudin, E. S., Hameed Sultan, M. T., & Yahaya, R. (2019). Enhanced thermal and dynamic mechanical properties of synthetic/natural hybrid composites with graphene nanoplateletes. Polymers, 11(7), 1085. http:// dx.doi.org/10.3390/polym11071085. PMid:31247898. 36. Saiter, A., Devallencourt, C., Saiter, J. M., & Grenet, J. (2001). Thermodynamically strong and kinetically fragile polymeric glass exemplified by melamine formaldehyde resins. European Polymer Journal, 37(6), 1083-1090. http://dx.doi.org/10.1016/ S0014-3057(00)00242-1. 37. Gheith, M. H., Aziz, M. A., Ghori, W., Saba, N., Asim, M., Jawaid, M., & Alothman, O. Y. (2019). Flexural, thermal and dynamic mechanical properties of date palm fibres reinforced epoxy composites. Journal of Materials Research and Technology, 8(1), 853-860. http://dx.doi.org/10.1016/j.jmrt.2018.06.013. 38. Palanivel, A., Veerabathiran, A., Duruvasalu, R., Iyyanar, S., & Velumayil, R. (2017). Dynamic mechanical analysis and crystalline analysis of hemp fiber reinforced cellulose filled epoxy composite. Polímeros: Ciência e Tecnologia, 27(4), 309-319. http://dx.doi.org/10.1590/0104-1428.00516. Received: Oct. 12, 2019 Revised: Feb. 04, 2020 Accepted: Feb. 11, 2020

Polímeros, 29(4), e2019054, 2019


ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.06618

Kraft lignin and polyethylene terephthalate blends: effect on thermal and mechanical properties Lívia Lazzari1, Eloilson Domingos1,2, Letícia Silva3,4, Alexei Kuznetsov3, Wanderson Romão1,2 and Joyce Araujo3*  Laboratório de Química Analítica, Instituto de Química, Instituto Federal de Educação, Ciência e Tecnologia do Espírito Santo – IFES, Vila Velha, ES, Brasil 2 Laboratório Petroleômico e Forense, Departamento de Química, Universidade Federal do Espírito Santo – UFES, Vitória, ES, Brasil 3 Divisão de Metrologia de Materiais, Instituto Nacional de Metrologia, Qualidade e Tecnologia – Inmetro, Duque de Caxias, RJ, Brasil 4 Universidade Federal do Rio de Janeiro – UFRJ, Duque de Caxias, RJ, Brasil 1

*jraraujo@inmetro.gov.br

Abstract In this work, bottle-grade poly(ethylene terephthalate) (PETR), kraft lignin (KL), and chemically modified lignin (ML) were used to form blends to improve the mechanical and thermal properties of pure PET. The PET/KL and PETR/ML blends were produced with 0.5, 1, 3, and 5 wt.% of lignin via melt extrusion and injection molding. The produced blends and PETR were characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetry (TGA), differential scanning calorimetry (DSC) and mechanical properties testing. The FTIR measurements confirmed the chemical modifications of the ML samples, while the TGA results showed KL to be thermally more stable than ML. The glass transition temperature of PETR changed as a function of the amount of lignin, as revealed by the DSC measurements. The PET/KL blends demonstrated their potential for use as an engineering material due to their improved thermal and mechanical properties compared to those of PETR. Keywords: blends, kraft lignin, mechanical properties, poly(ethylene terephthalate). How to cite: Lazzari, L., Domingos, E., Silva, L., Kuznetsov, A., Romão, W., & Araujo, J. (2019). Kraft lignin and polyethylene terephthalate blends: effect on thermal and mechanical properties. Polímeros: Ciência e Tecnologia, 29(4), e2019055. https://doi.org/10.1590/0104-1428.06618

1. Introduction Synthetic or petroleum-based polymers have many practical uses; however, their low biodegradability causes serious environmental problems. Consequently, several strategies to replace or reduce the use of synthetic polymers have been developed[1-4]. Sustainable development requirements open new perspectives for products obtained from polymer recycling processes, as they significantly contribute to a reduction in plastic waste[5]. Polyethylene terephthalate (PET) is a semi-crystalline thermoplastic aromatic polyester known for its mechanical properties, lightness, strength, and high transparency, which ensure its widespread use in food and cosmetic packaging materials[2,6-10]. Currently, recycled PET is often mixed with other polymers or fillers to produce polymer blends or composites with different mechanical and thermal properties compared to neat polymers, thus adding value to raw materials. For added-value processes to be consistently efficient, the compatibility between mixture components is highly essential to achieve satisfactory thermal and mechanical properties for a specific application[11-13]. Mechanical enhancements may be useful to the automobile and civil construction industries,

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while improvements of thermal stability would be useful for applications in the packaging and electronics industries. Lignin is one of the three main constituents of a plant. It is the second most abundant polymer in the world after cellulose. Generally, its structure depends on the species of wood and the processing conditions. Kraft, sulfite, and soda are the main processes used in chemical wood pulping for extracting cellulose from wood by dissolving the lignin that binds the cellulose fibers together[14,15]. The kraft pulping process involves digesting wood chips and moiled paper at elevated temperature and pressure in a water solution of sodium sulfide and sodium hydroxide, called “white liquor.” The white liquor chemically dissolves the lignin that binds the cellulose fibers together. Spent “white liquor,” containing suspended particulate solids and organic compounds, is concentrated to a mostly solid pulp, called “black liquor,” which contains between 10 to 50 wt.% of dissolved lignin[14,16,17]. Today, most of the lignin produced by the pulp and paper industry as a constituent part of the black liquor by-product is burned to provide heat for electric power generation. However, as lignin is a complex polyfunctional

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O O O O O O O O O O O O O O O O


Lazzari, L., Domingos, E., Silva, L., Kuznetsov, A., Romão, W., & Araujo, J. macromolecule composed of a large number of polar functional groups, it has the potential for use in several technological applications and can be used to produce high-added-value products[18-20]. Blending lignin with a polymeric matrix is a secure way to develop polymer-based products with desirable properties. However, the eventual incompatibility on a chemical level between the components may require the chemical modification of lignin before mixing to improve its dispersion in plastic or to increase interfacial adhesion[14,17,21,22]. This work aimed to develop a new engineering material with enhanced mechanical and thermal properties using waste PET bottles and lignin, which was obtained as a by-product of the kraft process. The PET/lignin blends were produced by melt extrusion and injection molding and contained chemically modified and unmodified lignin. The thermal and mechanical properties of the blends were compared with those of the PETR matrix.

2. Materials and Methods 2.1 Materials Lignin from the kraft process, referred to here as KL, was supplied by Fibria Celulose S/A (São Paulo, Brazil). Chemical modification of the lignin was accomplished using ethylic alcohol 95% (v/v) (CAS 64-17-5), sodium hydroxide 97% (wt/wt) (CAS 1310- 73-2), and monochloroacetic acid 99% (wt/wt) (CAS 79-11-8), all supplied by Sigma Aldrich. Lignin was extracted from the black liquor via a precipitation method that consisted of the following steps: reduction of the liquor pH value (start solution was pH >13) with CO2 injection, filtration of the precipitated lignin, suspension of the filtered lignin in a H2SO4 solution (pH 2.5), and filtration and washing of the lignin with an acidic solution (pH 2.5 and 60 °C). The bottle-grade PET (PETR) was obtained by grinding colorless PET bottles in a Retsch mill (model SM300) with a 2 mm sieve and 1500 rpm rotation. Prior to milling, the bottle labels were removed, and the areas with glue residue were cleaned.

2.2 Lignin modification The lignin modification was performed as described by Silva et al.[23]. Briefly, 10.0 g of lignin was suspended in 270 mL of 95% ethanol (v/v) under continuous stirring in a mechanical stirrer (Ethik Technology, model 105) to

which 27 mL of a 30% NaOH aqueous solution (wt/v) was added at a rate of 1 mL min-1 using an electronic pipette (Transferpette S) at room temperature. After the addition of the NaOH solution, the final solution was stirred for a further 60 min. Subsequently, 12.0 g of monochloroacetic acid was gradually added over the course of 30 min, without further agitation of the solution. The mixture was then stirred for an additional 210 min at 55 °C. The residue was suspended in 670 mL of 95% ethanol solution (v/v), which was neutralized with glacial acetic acid, and the separated product was filtered. After filtration, the product was washed three times with approximately 50 mL of 95% ethanol (v/v) to remove the impurities and by-products and then dried at 60 °C in an oven until a constant mass was achieved. The modified lignin is denominated in this work as ML[23].

2.3 Specimen preparation Milled PETR, KL, and ML samples were dried in a vacuum oven at 60 °C for 24 h prior to melting extrusion and injection molding. The PETR/lignin blends were fabricated by extrusion using a Thermo Scientific Haake MiniLab II extruder (processing temperature 275 °C and screw rotation speed 100 rpm). The reference specimens (PETR) and its blends were injected into a Thermo Scientific Haake MiniJet II injector (injection temperature 275 °C, injection pressure 450 bar, injection time 4 s and molding temperature 25 °C). Table 1 displays the formulations of the specimens.

2.4 FTIR The attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were obtained on a Frontier spectrometer from Perkin Elmer. Each spectrum was recorded as the mean of 16 consecutive scans, with a resolution of 4 cm-1 in a working range of 4000 to 630 cm-1.

2.5 Thermogravimetry (TGA) Thermogravimetry analysis (TGA) was performed on an SDT Q600 from TA Instruments. Approximately 10 mg of the sample was heated in the alumina crucible (25 °C to 900 °C) at a heating rate of 10 °C min-1 under a nitrogen flow of 20 mL min-1.

2.6 Differential Scanning Calorimetry (DSC) Differential scanning calorimetry (DSC) was carried out using a TA Instruments Q200. Approximately 5 mg of the

Table 1. Composition of the formulations used to fabricate the tensile specimens. Sample

PETR (wt.%)

KL (wt.%)

ML (wt.%)

1 100 0 0 2 99.5 0.5 0 3 99 1.0 0 4 97 3.0 0 5 95 5.0 0 6 99.5 0 0.5 7 99 0 1.0 8 97 0 3.0 9 95 0 5.0 * PETR : bottle-grade poly(ethylene terephthalate), KL: kraft lignin, and ML: chemically modified lignin.

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Formulation* PETR PET/KF 0.5 wt.% PET/KF 1.0 wt.% PET/KF 3.0 wt.% PET/KF 5.0 wt.% PET/ML 0.5 wt.% PET/ML 1.0 wt.% PET/ML 3.0 wt.% PET/ML 5.0 wt.%

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Kraft lignin and polyethylene terephthalate blends: effect on thermal and mechanical properties sample (injected material) was heated in the alumina crucible (25 °C to 400 °C) at a heating rate of 10 °C min-1 under a nitrogen flow of 50 mL min-1.

2.7 Tensile measurements The tensile tests were performed according to the ISO 527-1:2012 (type 5A specimen) standard. The tests were conducted at the facilities of the Federal University of Espírito Santo (UFES)/LABPETRO using a Lloyd Instruments LR5K Plus universal testing machine. The measurement conditions were a load cell of 5 kN, a 50 mm strain gauge with a measurable deformation of 25 mm, and a crosshead speed of 1 mm min-1.

3. Results and Discussion 3.1 ATR-FTIR Figure S1a (see Supplementary Material) shows the ATR-FTIR spectra of the KL and ML samples, and the main bands and respective assignments are listed in Table 2 and Table 3. Figure S1a shows significant differences between

the ATR-FTIR spectra of ML and KL samples – one of them is the absence of the band at 1710 cm-1 in the ML spectrum, exhibited by unmodified lignin (KL), which is attributed to the vibration of the carbonyl group conjugated to the aromatic ring. Instead, ML has two high-intensity bands, at 1598 and 1416 cm-1, assigned to the carboxylate anion. The appearance of these intense bands demonstrates the efficiency of the carboxylation process in the modification reaction. The KL and ML samples both exhibited bands related to the stretching and in-plane bending of the CH2 group, at 2938 and 1453 cm-1, respectively, (Tabs. 2 and 3), indicating that these groups did not participate in the chemical reaction. The main bands of the PETR polymer matrix as well as its PETR/KL and PETR/ML lignin blends are assigned in Table S1.

3.2 TGA Figure 1a depicts the TGA curves of KL and ML. Mass losses of approximately 6 wt.% for KL and 22 wt.% for ML were observed at around 100 °C. This weight drop can be attributed to moisture loss. The chemical decomposition of KL and ML occurs over a wide temperature range, with the most intense mass loss being observed between 200 °C and

Figure 1. (a) TGA curves of the unmodified lignin (KL), modified lignin (ML), and PETR and their own blends of (b) PETR/KL and (c) PETR/ML. Table 2. Assignments of the vibrational bands of the lignin (KF)[23-25]. Wavenumber (cm-1) 3413 2934 1710 1603 1505 1454 1211 1110 1019

Assignment OH group (hydroxyl) aliphatic C-H asymmetric stretching C=O stretch of carboxylic acids, ketones, and aldehydes C=C aromatic stretching C=C aromatic stretching CH2CH3 bending C-O phenolic stretching C-O stretching of the aliphatic secondary alcohol chain C-O stretching of the primary alcohol aliphatic chain

Table 3. Assignments of the vibrational bands of the modified lignin (ML)[23-25]. Wavenumber (cm-1) 3254 2938 1598 1416 1507 1453 1249 1123 1031

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Assignment OH group (hydroxyl) aliphatic C-H asymmetric stretching asymmetric carboxylate stretching symmetrical carboxylate stretch C=C aromatic stretches CH2CH3 bending C-O phenolic stretching C-O stretching of the aliphatic secondary alcohol chain C-O stretching of the primary alcohol aliphatic chain

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Lazzari, L., Domingos, E., Silva, L., Kuznetsov, A., Romão, W., & Araujo, J. 700 °C. In this temperature range, KL loses approximately 72 wt.%, while ML loses about 34 wt.%[23,26,27]. The wide range of thermal degradation of lignin can be explained by the different oxygenated functional groups in their structure, which provides different thermal stabilities[28]. The thermal degradation of lignin generally occurs in three main steps: 0 to 120 °C, related to the evaporation of absorbed water, 180 to 350 °C, associated with the degradation of carbohydrates, which are converted into volatile gases such as CO, CO2 and CH4, and above 350 °C, related to the degradation of lignin-derived products together with the removal of produced gases[28]. Due to the complexity of lignin’s structure, the chemical decomposition of this material involves several competing reactions. Lignin contains several chemical subunits within its macromolecule. During the thermal degradation process, chemical bonds of different bond energies are broken[23,26,27]. The dehydration of lignin prevails in its thermal degradation pathways. Dehydration of the lignin structure results in pyrolysis products with unsaturated side chains[28]. Carbon monoxide, carbon dioxide, and methane are also formed during these processes[29,30]. The decomposition of aromatic rings occurs above 400 °C[31]. Prolonged heating above 400 °C leads to saturation of aromatic rings, the disruption of C-C bonds, and the release of smaller molecules, such as water, CO2, and CO, accompanied by the rearrangement and condensation of the aromatic rings within the lignin structural units[29]. Figure 1a shows l a plot of the first derivative of the mass loss versus temperature for the KL and ML samples. One can conclude that the KL sample is thermally more stable than the ML sample due to the highest temperature of maximum weight loss related to the lignin carbohydrate degradation appeared at 320 °C for the ML sample but appeared in the interval between 320 °C to 380 °C for the KL sample. According to Kindsigo and Kallas[32], at ambient temperature in the presence of oxygen and water, it is possible to observe the degradation of lignin via wet oxidation, which is increased with elevated temperatures. In the analyzed temperature range of 110-190 °C, the lignin degradation was 20% at 110 °C and 53% at 190 °C; thus, it is expected that at higher temperatures the degradation rate would continue to increase. In this way, the chemical modification

of lignin through carboxylate anion incorporation favors the incorporation of water molecules into its structure due to the higher hydrophilicity of the carboxylated lignin sample (ML sample), making it more unstable and resulting in faster degradation. The total mass losses of the KL and ML samples when heated to 900 °C were 96 wt.% and 88 wt.%, respectively. One can also see from Figure 2a that the lignin continued to decompose at temperatures higher than 900 °C. Figures 1b and 1c compare the TGA curve of PETR with those of the PETR/KL (Figure 1b) and PETR/ML (Figure 1c) blends. The results of the TGA measurements demonstrate that the admixing of KL and ML into the PETR to form a blend caused a shift in the onset temperature of thermal degradation (450 °C), whereas the PETR sample exhibited two mass loss events: the first one in the temperature range of 50 °C to 100 °C (mass loss of 12.5 wt.%) and the second one in the temperature range of 400 °C to 450 °C (mass loss of 75 wt.%). As a consequence, these blends presented a higher residual mass than PETR (20-25 wt.% versus 12.5 wt.%) at 450 °C.

The increase in the onset temperature of thermal degradation of the PETR/lignin blends in relation to neat PETR may be attributed to the chemical compatibility between the PETR and KL and ML fillers due to the aromatic structure present in both sample types. However, the moisture present in the lignin blends increases the mass loss content in this temperature range, as our results show (see Figure 1a) with degradation temperatures above 450 °C.

3.3 Differential Scanning Calorimetry (DSC) Figure 2a shows the DSC curves of KL and ML. Typically, the Tg values of unmodified lignins vary from 90 to 180 °C[33,34]. The significant enthalpy relaxation process that usually occurs in polymers during DSC scanning makes it challenging to determine the Tg value of lignin from the DSC measurements. Complex hydrogen bonding interactions and the highly amorphous structure of kraft lignin favor enthalpy relaxation. This is detected in the interval where a slope change occurs in the heating curve[35-37]. The Tg values of KL and ML are 100 °C and 127 °C, respectively (Figure  2a). Table  4 summarizes the thermal properties of the studied samples obtained from the DSC measurements, such as the glass transition temperature (Tg), crystallization temperature (Tc), melting

Table 4. DSC results for PETR and PET/KL and PET/ML blends. Tg (°C)*

Tc (°C)*

Tm (°C)*

ΔHm (J/g)*

Xc (%)*

PETR

Sample

63.30

119.50

253.79

-4.45

3.18%

PET/KF 0.5wt.% PET/KF 1.0wt.% PET/KF 3.0wt.% PET/KF 5.0wt.% PET/ML 0.5wt.% PET/ML 1.0wt.% PET/ML 3.0wt.% PET/ML 5.0wt.%

70.29 69.70 68.34 68.80 74.96 74.76 74.77 74.98

117.37 116.42 116.89 117.99 113.61 113.05 110.46 109.45

257.18 256.59 256.49 253.09 255.02 258.26 258.15 256.34

-4.90 -5.49 -5.89 -5.94 -5.71 -5.26 -5.66 -5.70

3.52% 3.96% 4.34% 4.46% 4.10% 3.80% 4.17% 4.29%

* Tg, Tc, Tm, ΔHm and Xc correspond to glass transition temperature, crystallization temperature, melting temperature, melting enthalpy, and degree of crystallinity, respectively.

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Polímeros, 29(4), e2019055, 2019


Kraft lignin and polyethylene terephthalate blends: effect on thermal and mechanical properties temperature (Tm), melting enthalpy (Î&#x201D;Hm), and degree of crystallinity (Xc).

The variation in the values of Tg, Tc, and Tm of the blends in relation to those of PETR show a clear dependence on both the type and the amount of lignin added. The PETR sample exhibited Tg, Tc, and Tm values of 63.3, 119.5, and 253.8 °C, respectively, which are typical for this polymer. The DSC curves (Figure 2b, c) for the blends also showed well-defined endothermic peaks of glass transition, exothermic cold crystallization, and endothermic melting peaks, which is characteristic of PET. In general, PETR/KL blends exhibited higher Tg and Tm values (approximately 70 °C and 257 °C, respectively) compared with those of PETR. This shift of the glass and melting transitions to higher temperatures was somewhat larger in the case of PETR/ML for Tm (approximately 258 °C) and was notably larger for Tg (about 75 °C), except for the PETR/ML 5.0 wt.% sample. On the whole, the PETR/ML blends showed more stable melt characteristics (melting temperatures and melting enthalpies) compared to those of the PETR/KL blends. The value of Tc presented a maximum decrease in three units (119.5 â&#x2020;&#x2019; 116.4 °C) of temperature for the PETR/KL 1.0 wt.% blend, while in the PETR/ML blends, the most significant variation was Tc = 114 â&#x2020;&#x2019; 110 ° C (Table 5). The cold crystallization temperatures of the blends showed an opposite trend from the melting temperatures, where the cold crystallization temperatures of the PETR/KL and PETR/ML samples were decreased in comparison to that of PETR. This temperature reduction was notably larger in the PETR/ML blends.

The lignin macromolecule contains polar groups capable of producing chemical interactions to become closer to the PETR chains. These secondary forces may have contributed to the increase of Tg and Tm for the studied blends. The PETR/ML blends presented higher increases in these two parameters in relation to the PETR/KL blends, probably due to the incorporation of carboxylate groups in the lignin, favoring its chemical interaction with the PETR matrix, and consequently, increasing the Tg and Tm values[38,39]. Miscibility is a crucial parameter to be achieved in polymer blends to improve the properties of homopolymers. In our study, a single Tg was observed in the DSC curves of the blends, which is indicative of miscibility. The polarity of the lignin molecules results in strong interactions between them that hinder their miscibility with other polymers. To maintain miscibility, interaction forces between the polymer matrix (PET in our case) and lignin are required. Generally, PET has the ability to interact with lignin through Ď&#x20AC; electronic interactions favoring the miscibility between them. Hydrogen bonds that eventually form between PET and lignin polar groups also facilitate miscibility. For this reason, lignin surface chemical modification is typically performed to reduce the interaction forces between the lignin molecules, attaching them to hydrophilic polymer matrices, such as PETR. The degree of crystallinity is another important property of semi-crystalline thermoplastics that is directly related, among others, to the mechanical properties of plastics. The degree of crystallinity by DSC of the studied blends was assessed using the following Equation 1: Xc =

â&#x2C6;&#x2020;H m â&#x2C6;&#x2019; â&#x2C6;&#x2020;H c 0 â&#x2C6;&#x2020;H m

* 100%

(1)

Table 5. Tensile test results for PETR and PETR/KL and PETR/ML blends. Sample PETR PET/KF 0.5 wt.% PET/KF 1.0 wt.% PET/KF 3.0 wt.% PET/KF 5.0 wt.% PET/ML 0.5 wt.% PET/ML 1.0 wt.%

E (GPa)**

Î&#x201D;E (GPa)**

Ď&#x192; (Mpa)**

Î&#x201D;Ď&#x192; (Mpa)**

Îľrup %**

Î&#x201D;Îľrup %**

1.33 1.47 1.55 1.71 1.76 1.66 1.43

Âą0.04 Âą0.06 Âą0.05 Âą0.05 Âą0.12 Âą0.29 Âą0.22

57.31 60.20 61.62 39.56 37.77 20.59 20.41

Âą0.60 Âą0.85 Âą0.67 Âą1.28 Âą2.80 Âą3.60 Âą1.78

NB* NB* NB* 2.0 2.1 0.9 1.1

NB* NB* NB* Âą0.54 Âą0.29 Âą0.16 Âą0.40

*did not break; **E: modulus of elasticity; Î&#x201D;E: standard deviation of modulus of elasticity; Ď&#x192;: maximum tensile strength; Î&#x201D;Ď&#x192;: standard deviation of maximum tensile strength; Îľđ?&#x2018;&#x;đ?&#x2018;˘đ?&#x2018;?: strain at break; Î&#x201D;Îľđ?&#x2018;&#x;đ?&#x2018;˘đ?&#x2018;?: standard deviation of strain at break.

Figure 2. DSC plots for KL and ML (a) lignin; (b-c) PETR and their respective blends of (b) PETR/KL and (c) PETR/ML. PolĂ­meros, 29(4), e2019055, 2019

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Lazzari, L., Domingos, E., Silva, L., Kuznetsov, A., RomĂŁo, W., & Araujo, J. In this equation, the difference between the measured heats of melting, Î&#x201D;Hm, and the cold crystallization, Î&#x201D;Hc, define the fusion enthalpy of a sample. The term Î&#x201D;Hm0 is a reference value and represents the heat of melting if the polymer were 100% crystalline. The value Î&#x201D;Hm0 = 140 J g-1 was used[38]. All samples exhibited low values of Xc (see Table 4). Two different factors could have affected the overall crystallinity of the samples. The first one is related to the production process of the specimens, which was by injection molding. Significant differences between the injection and molding temperatures resulted in a predominantly amorphous sample structure because the polymer chains did not have enough time for periodic ordering. On the other hand, the presence of KL and ML lignin favored the crystallization of PETR, indicating that lignin acted as a nucleating agent to the PET domains[39].

3.4 Tensile tests The results of the mechanical tensile tests, including the modulus of elasticity (đ??¸), maximum tensile strength (Ď&#x192;), and strain at break (Îľđ?&#x2018;&#x;đ?&#x2018;˘đ?&#x2018;?), are shown in Table 5. The Ď&#x192; parameter (maximum tensile strength) of a polymer blend indicates the tension transfer capacity from the matrix to the filler, where the higher the interaction between phases, the higher the maximum tensile strength. The chemical modification of lignin did not produce efficient interfacial molecular interactions between the PET and ML components or, consequently, an enhancement in the mechanical properties of the PETR/ML blends, as is seen from the experimental data (Table  5). Moreover, higher contents of KL in polymer blends (<3.0 wt%) also seemed to be unfavorable for the strength and deformability of the PETR/KL blends. Formulations containing 3 wt.% and 5 wt.% of ML could not be tested at all, as the mixture of ML with PETR in these proportions resulted in specimens forming cracks even before the mold was withdrawn. In contrast, Youngâ&#x20AC;&#x2122;s modulus of all PETR/KL blends followed the fundamental law of mixtures and was nearly the additive function of the composition. On the other hand, lignin, which is more rigid than PETR, has the capacity to support the applied tension transferred from the polymeric matrix to itself, resulting in a higher Youngâ&#x20AC;&#x2122;s module of the respective blends in comparison with PETR[40]. Distinct from Youngâ&#x20AC;&#x2122;s modulus, the strength, and deformability of blends show more complex behavior. It is generally thought that these characteristics primarily depend on the strength of interfacial adhesion of the components, which is determined, in turn, by the mutual contact surface and the respective strength of interaction[41]. Water molecules can act as plasticizers to increase the mobility of ligninâ&#x20AC;&#x2122;s polymer chains during its dispersion in the PETR matrix. There was a much finer dispersion of the KL phase than the ML phase in the respective blends, resulting in a higher contact surface between the KL and PETR molecules. However, the positive effects of KL on the tensile properties of the PETR/KL blends seemed to diminish after the content of stiff KL chains exceeded a critical limit. We believe that the analogous critical limit was already attained for the lowest content of ML in the PETR/ML samples with respect to Youngâ&#x20AC;&#x2122;s modulus of the 6/9

material. As a result, a decreasing trend in the stiffness of PETR/ML with the content of ML was observed.

4. Conclusions In this study, kraft lignin (KL) and chemically modified kraft lignin (ML) were used to produce PETR/lignin blends. The results of ATR-FTIR, TGA, and DSC analyses verified the presence of chemical modifications in the ML samples. The TGA measurements indicated that KL was thermally more stable than ML, which is intrinsically linked to the ability of ML to absorb water, thus, increasing this materialâ&#x20AC;&#x2122;s susceptibility to degradation. Additionally, the water molecule absorption impacts the mechanical properties of the PETR/lignin blends, making the PETR/ML blends more fragile than the PETR/KL blends. The ATR-FTIR spectra of the blends showed no significant differences between them, while the DSC curves exhibited higher glass transition temperatures for the PETR/lignin blends compared with the PETR material. PET R/KL blends with small amounts of KL (up to approximately 1%) showed improved mechanical properties. Both the modulus of elasticity and the maximum tensile strength of PETR benefited from the addition of lignin in this situation; this is the expected behavior of compatible polymer blends.

5. Acknowledgements The authors thank FAPES for a scholarship to LĂ­via Lazzari, Fibria Celulose for supplying the lignin samples, and UFES/LABPETRO for the provision of laboratories and equipment. LetĂ­cia Silva and Joyce Araujo thank FAPERJ (grants E-26/201.978/2017 and E-26/202.746/2018) and CNPq (grant 311900/2017-8) for the fellowships.

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Received: Dec. 04, 2018 Revised: Aug. 03, 2019 Accepted: Oct. 03, 2019

Polímeros, 29(4), e2019055, 2019


Kraft lignin and polyethylene terephthalate blends: effect on thermal and mechanical properties

Supplementary Material Supplementary material accompanies this paper. Figure S1. (a) ATR-FTIR spectra of lignin obtained by the kraft process (KL) and chemically modified lignin (ML); (b) PETR/kraft lignin (KL) blends; (c) PETR/modified lignin (ML) blends. Table S1. Assignments of FTIR bands for PETR and their respective PETR/KL and PETR/ML blends [2-4]. This material is available as part of the online article from http://www.scielo.br/po

PolĂ­meros, 29(4), e2019055, 2019

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ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.08918

Synthesis of immobilized biocatalysts for wastewater decontamination Thâmara Machado e Silva1, Leonardo Luiz Borges1, Eli Regina Barboza e Souza2 and Samantha Salomão Caramori1*  Laboratório de Biotecnologia, Universidade Estadual de Goiás – UEG, Anápolis, GO, Brasil 2 Escola de Agronomia, Universidade Federal de Goiás – UFG, Goiânia, GO, Brasil

1

*sscaramori@gmail.com

Abstract The use of biodegradable polymers arouses biotechnological interest. This use allows applications in health and environment. Here is present the characterization and a proposition for the use of cashew (Anacardium othonianum Rizz.) polysaccharide including peroxidase immobilization for wastewater bioremediation. From the cashew gum exudate, the polysaccharide was extracted by precipitation in ethanol at 4 °C. This material is able to immobilize Horseradish peroxidase by physical adsorption and via sodium periodate with 75% and 93% of efficiency, respectively. These systems have a storage and operational stability, and removed phenolic compounds above 50% in industrial effluent samples. The bioassays in the presence of Artemia salina and Allium cepa root not only revealed no toxicity to this polysaccharide, but also presented the ability to reduce the toxicity of the industrial effluent by 50%. Immobilized cashew polysaccharide complexes are potential alternatives for waste treatment and decontaminant agents for water treatment applications. The polysaccharide is a low-cost natural matrix for environmental-technological applications. Keywords: bioremediation, ecological toxicity, natural polymers, peroxidase immobilization. How to cite: Silva, T. M., Borges, L. L., Souza, E. R. B., & Caramori, S. S. (2019). Synthesis of immobilized biocatalysts for wastewater decontamination. Polímeros: Ciência e Tecnologia, 29(4), e2019056. https://doi.org/10.1590/0104-1428.08918

1. Introduction Natural polymers generate a significant interest in research and industrial communities because their renewable origins reduce the accumulation of waste and cause less environmental impacts. They easily degrade throughout the industrial processes[1]. Such materials have also been used as thickeners, gelling agents, emulsifiers, stabilizers, binders, and biodegradable products[2], and for enzyme immobilization[3]. The Cerrado, the second largest Brazilian biome in area (approximately 22% of the country area), is home to the Cerrado arboreal cashew (Anacardium othonianum Rizz.)[4]. The water solubility of the polysaccharide from A. othonianum (PEJU) makes it an attractive support for enzyme immobilization, since its recovery can easily be achieved by simple precipitation with polar organic solvents, such as ethanol[3]. An alternative for the treatment of phenolic wastewater is the use of peroxidase (E.C.1.11.1.7). Such use was first proposed by Klibanov and collaborators[5], and has been continually improved to optimize technical and economic factors. This enzyme successfully eliminates phenol from aqueous solutions, thus being the most researched peroxidase. For the treatment of large volumes of pesticides[6], antibacterial[7] and textile effluents[8], reactors containing immobilized peroxidase are desirable because it enables

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enzyme recovery/recycling, thus decreasing costs of bioremediation[7]. The immobilization technique allows enzyme recovery and reusability, and admits a continuous conversion process[9]. The microenvironment in which immobilization occurs protect the enzyme chemical structure, sometimes offering charged groups (if the support has an ionic matrix), sometimes establishing multiple covalent bonds in order to avoid denaturation[6,7]. Depending on the nature of the support and the chemical method adopted to obtain the immobilized-enzyme system, it is possible to enhance catalytic efficiency, even using recalcitrant molecules as substrates[8]. The literature on peroxidase immobilization involves functionalizing steps with toxic agents, such as glutaraldehyde[3,7,10], or uses not suitable large-scale materials, such as bioaffinity chromatography[9]. This paper proposes the immobilization of horseradish peroxidase in Cerrado-arboreal cashew (Anacardium othonianum Rizz.) polysaccharide as an alternative to remove phenols and reduce biological toxicity (assays by Artemia salina and Allium cepa) of wastewater. This bio-based treatment represents an eco-friendly strategy to treat wastewater using immobilized peroxidase. Two immobilization methods are presented: the first is based on direct enzyme adsorption for the support; the second is based on binding via sodium periodate.

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Silva, T. M., Borges, L. L., Souza, E. R. B., & Caramori, S. S. The hypothesis of this work is that the Cerrado-arboreal cashew polysaccharide is able to immobilize peroxidase and the polysaccharide-HRP complex can be applied to treat industrial effluents.

2. Materials and Methods 2.1 Material Horseradish peroxidase Type IV (E.C. 1.11.1.7), ethanol P.A., pyrogallol, hydrogen peroxide (H2O2) 35% (v/v), sodium phosphate monobasic, potassium dichromate, sodium periodate, nitrophenol, phenol, bromophenol, catechol, Giemsa stain, sodium azide, and the Folin-Ciocalteu reagent were purchased from Sigma-Aldrich (USA). The in natura gum (G) and polysaccharide (PEJU) characterizations were carried out according to Silva et al.[11]. Briefly, the gum in natura (G) was obtained through incisions in the trunks of 24 individuals of cashew trees implanted in an arboretum in Goiânia, Goiás state (Brazil), at the geographical coordinates 16°35’59.1” S, 49°16’ 47.1” W, and 730 m of altitude, totaling 6,400 m2 of area. The incisions, 10 cm long and 2 cm deep, were performed in triplicate on each tree branch on February (26 °C and 13 mm of rainfall) and September (25.7 °C, without rainfalls) of 2016 (Evaporimetric Station, 2016). After 15 days, the exudate nodes were collected with a spatula and stored in an amber bottle. The exsiccate of the species Anacardium othonianum Rizz. is deposited at the Herbarium of the Universidade Estadual de Goiás under the number 10,993. The exudate nodules were ground and dissolved in 20% (w/v) distilled water. The mixture was kept at room temperature for 24 h for complete dissolution. This mixture was filtered through nylon (90 threads), and then added to absolute ethanol at a ratio of 1:3. The suspension was kept at room temperature for 24 h. After decantation, the supernatant was discarded, and the precipitate was washed with absolute ethanol and filtered through nylon (110 threads). This procedure yielded 75% polysaccharide, named as PEJU-GO, which was dried outdoors and protected from sunlight exposure, crushed and then stored in hermetically sealed bottles at 4 °C until use.

2.2 Acquisition and characterization of the sodium periodate-treated arboreal cashew polysaccharide (PEJUp) A mixture containing of 0.05 g PEJU, 2.0 mL of 0.1 mol L-1 sodium periodate and 10 mL of 0.1 mol L-1 sodium phosphate buffer pH 7.0 was stirred for 30 min at 23 °C following the method proposed by Pappas et al.[12] Then, the complex sodium periodate-treated polysaccharide (PEJUp) was precipitated using absolute ethanol (1:3, v/v) at 10 °C for 24 h. The supernatant was discarded and the precipitate (PEJUp) was dried at 23 °C. The powder obtained was ground and stored in amber flasks at 4 °C until use. The morphological analysis of PEJUp was performed by scanning electron microscopy (Shimadzu, model SSx 550, Japan), with magnifications from 50 to 1,000x. 2/8

PEJUp samples were analyzed by infrared spectrometry (FTIR) using KBr pellets (Bomn FT-IR model MB100, USA), and scanned within the range of 4,000 to 500 cm-1. The determination of the crystalline phases of the samples was evaluated by X-ray diffraction (XRD) measurements on a D8 Discover diffractometer (Bruker, Germany), under rotation of 15 rpm and a range of 2° from 5° to 70° and a step of 0.02o [13]. The thermal stability of the PEJUp was evaluated by thermogravimetric analysis using the methodology of Lomonaco[14]. The samples were subjected to heating ramps of 25 °C to 500 °C at a rate of 3 °C min-1 using DTG-60H (Shimadzu, China).

2.3 Measurement of peroxidase activity (Horseradish peroxidase, HRP) and enzyme immobilization The enzyme activity was determined by the method of Halpin and Lee[15]. In test tubes, 2.4 mL of pyrogallol (1.6 mg mL-1), prepared in 0.1 mol L-1 sodium phosphate buffer pH 6.0, were incubated at 23 °C with 0.1 mL of the enzyme solution (29 U) prepared in the same buffer. Hydrogen peroxide (0.5 mL) at 0.05 mol L-1 was added and after one minute, the absorbance was measured at 420 nm. One enzyme unit (U) was considered as the amount of peroxidase that increases 0.1 absorbance min-1 of reaction under the assay conditions. HRP was immobilized by physical adsorption and by attachment using sodium periodate, obtaining more active points between the support and the enzyme. The best retention of enzymatic activity was conducted at different immobilization pH, days of storage and reuse using 29 U of enzymatic solution in 10 mg PEJU and 15 mg PEJUp under gentle agitation (720 rpm) for 2 h at 4 °C. Then, the PEJU-HRP and PEJUp-HRP were precipitated using absolute ethanol at 4 °C, centrifuged, and tested for immobilized enzymatic activity. The activity test consisted of adding 1.4 mL of 0.1 mol L-1 sodium phosphate buffer pH 6.0, 0.5 mL of hydrogen peroxide 0.05 mol L-1 and 1.0 mL of 0.07 mol L-1 pyrogallol to the PEJU-HRP and to the PEJUp-HRP complex. After one minute of reaction, readings were taken according to Halpin and Lee[15].

2.4 The potential use of free and immobilized peroxidase for phenolic waste degradation The tests with phenolic compounds and agroindustrial effluents (Wastewater Treatment Center and Textile effluent) were carried out based on the methodology of Akhtar and Husain[16] and Ramalho et al.[17]. The effluent samples were collected at the Wastewater Treatment Station (WTS) in the Anápolis Agroindustrial District (DAIA) (16°30’ S and 49°00’ W), and at Cia Hering in São Luís de Montes Belos, Goiás state, Brazil (16°52’ S and 50°32’ W). The organic load after 24 h of collection in the raw sewage, according to the Department of the Environment of the DAIA, is 3,750 kg BOD.day-1. The free HRP, the PEJU-HRP and the PEJUp-HRP complexes were incubated with the following phenolic compounds: pyrogallol, catechol, phenol, bromophenol, and nitrophenol (1.0 mmol L-1), as well as effluents (WTS and Polímeros, 29(4), e2019056, 2019


Synthesis of immobilized biocatalysts for wastewater decontamination Textiles), with addition of hydrogen peroxide (0.05 mol L-1). The mixture remained at 23 °C for 10 min, and the residual concentration of phenols were measured using the methodology of Lowry et al.[18]. For the best-immobilized complexes against the removal of phenols, toxicity tests on Artemia salina[19] and Allium cepa roots[20] were performed. The mitotic index (MI) and the frequency of mitotic cycle abnormalities (ACM) in the slides were evaluated. MI was determined using the Equation 1:

= MI NCM ÷ TNC x100

(1)

where NCM corresponds to the number of cells in mitotic division, and TNC corresponds to the total number of cells analyzed.

2.5 Statistical analyses Statistical analyses were performed using the R Studio program (package MASS)[21]. A two-way ANOVA was used to determine the statistical significance of the effects of the treatments on the immobilization processes with a posteriori Tukey test (p <0.05). The two-way ANOVA determined the statistical significance of pH effects on both treatments (represented in Figure 1 by different letters). In this analysis, the immobilization pH was used as a treatment with two levels: PEJU and PEJUp, representing physical adsorption and weak bond interactions (such as hydrogen bonds), respectively. The possible results are (1) the effect of the chemical treatment alone (immobilization pH) in case of a statistical significant size effect: it means that there is a difference between the tested chemical treatments; (2) the effect of the immobilization treatment alone in case of a statistical significant size effect: it means that there is a difference between the immobilization treatments tested; and (3) the joint effect for both treatments in case of a statistical significant size effect: it means that the effect observed depends on each level of variables tested.

3. Results and Discussions 3.1 Characterization of PEJUp

Figure 1. Immobilization of horseradish peroxidase on PEJU and PEJUp. Effect of pH. Black columns represent physically adsorbed HRP (PEJU-HRP). Gray columns show immobilization after sodium periodate treatment (PEJUp-HRP). Significant differences appear by distinct letters when p<0.05 by test Tukey.

The scanning electron microscopy (Figure 2) of the PEJUp presented a characteristic aspect for the treatment of polysaccharides with sodium periodate: irregular and amorphous fragments and dispersed mass, strongly influencing its stability in the aqueous system[22]. The reaction with sodium periodate results in the appearance of aldehyde carbonyls in vicinal carbons. It is widely used as a routine method for the elucidation of complex carbohydrate structures. This recent application has helped to interpret the fundamental

Figure 2. Electron micrograph of Cerrado-arboreal cashew (A. othonianum Rizz.) polysaccharide, PEJU (A: 200x and B: 1000x) and treated in sodium periodate, PEJUp (C: 200x and D: 1000x). Polímeros, 29(4), e2019056, 2019

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Silva, T. M., Borges, L. L., Souza, E. R. B., & Caramori, S. S. structures of some polysaccharides, such as cellulose, starch, xylan, and glycogen[23]. The infrared spectra of the PEJU[11] have polysaccharide bands, such as those within the range of 3,000 to 2,840 cm-1 (OH stretch) and close to 2,936 cm-1. This indicates that this material contains sugars such as galactose, arabinose, and rhamnose. It is different from that presented for PEJUp (Figure 3A), a region in which such vibrations did not appear due to the cleavage process of glucosidic bonds. The absorbance at about 1,740 cm-1 is characteristic of carbonyl groups, while the band around 880 cm-1 is generally assigned to the formation of hemiacetal bonds between the aldehyde groups and neighbor hydroxyl groups. Absorptions within the 1,500 to 600 cm-1 regions are associated with axial and angular deformations of C-O, C-N, C-C, and C-X bonds. In the presence of sodium periodate, the oxidation of some biopolymers (e.g., xanthan gum) is characterized by the specific cleavage of the C2 and C3 bond of residues, resulting in the formation of aldehyde groups[12]. Observing both materials, we found sharp bands in the regions between 1,400 and 800 cm-1 in the FTIR spectra of the oxidized periodate (PEJUp) due to OH groups exposure after oxidation. In the X-ray diffraction pattern of PEJUp (Figure 3B), the presence of these semi-defined peaks can be caused by a high sugar content in the sample and by the fragmentation process after treatment with sodium periodate[24]. The patterns showed the highest peak at 15.22o (100% relative intensity (RI)), followed by 22.4o (81% RI), 25.9o (80% RI) and 12.08o (65.9% RI), indicating the amorphous phase of the material. The increase in the amorphous percentage may be related to a change in the chain configuration. During the oxidation, there are breaks in chemical bonds and

chain breaks[25]. The crystalline and amorphous material forms may show differences in particle size and shapes, physical-chemical properties, chemical stability, water solubility and hygroscopicity[26]. The predominance of large and diffuse peaks in X-ray diffractograms indicates amorphous and crystalline materials with a considerable and semi-defined noise[9]. The thermogravimetric analysis (TGA) performed to evaluate the thermal stability of the polysaccharide (PEJUp) is shown in Figure 3C. The decomposition of PEJUp occurred in two stages: the first between 50 and 100 °C (maximum at 80 °C), which, in line with the characteristic of sugar chain breakdown caused by sodium periodate, suggests the beginning of carbon skeleton decomposition for the polysaccharide with this treatment. The second peak was observed between 320 and 500 °C (maximum at 500 °C), probably due to depolymerization of CO and CH4 bonds. Guo et al.[27] found a similar process in a study with xanthan gum. Silva et al.[11] reported a decomposition behavior for PEJU with a higher thermostability, the first between 280 and 300 °C (maximum 300 °C) and the second between 300 and 490 °C (maximum 430 °C). Mothé and Rao[28] verified only a decomposition step with the maximum temperature around 300 °C for the polysaccharide of A. occidentale. Residual masses (around 600 °C) were verified in some polysaccharides of galactoxyloglucans [29], guar gum [30] and carboxymethylated cashew gum[31].

3.2 Peroxidase immobilization in different ways The immobilization assays of Cerrado arboreal polysaccharides by HRP physically adsorbed resulted in 72.4% of immobilization yield (21 U) and, by using sodium

Figure 3. Characterization of Cerrado-arboreal cashew polysaccharide (A. othonianum Rizz.) treated in sodium periodate. (A) Infrared spectrum of (4000 to 500 cm-1); (B) Diffractogram; (C) Thermogravimetric analysis. 4/8

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Synthesis of immobilized biocatalysts for wastewater decontamination periodate as a treatment, in 93.1% of immobilization yield (27 U), when there were optimal conditions. Figure 1 shows the influence of pH on the immobilization process for PEJU-HRP and PEJUp-HRP. Using the physical adsorption method, the pH 7.0 was the best condition for HRP immobilization, followed by pH 4.0, 5.0 and 6.0. On the other hand, if the choice is periodate-treated PEJU (PEJUp), there are two suitable pH conditions for HRP immobilization: at 4.0 and 7.0. According to Pappas et al.[12], sodium periodate oxidases the pyranosidic structure, opening the ring to form two adjacent carbonyl groups. This chemical attack on PEJU probably destabilized the polysaccharide chain, resulting in such randomized behavior. Gomez-Estaca  et  al.[32] studied the effects of pH on A. occidentale polysaccharide-protein interactions. According to the analysis of potential zeta, the authors found PEJU as the negatively charged polymer in the pH range 2.0 to 8.0. Similarities between A. ocidentale and A. othonianum polysaccharides in composition and structure may suggest that, from pH 4.0 to pH 7.0, there are negative charges along the polymer that favors the immobilization via adsorption in a large pH range[11]. Depending on the load properties of the immobilization carrier, the ideal pH may undergo significant changes. The ideal pH of an anionic support-bound enzyme shifts to higher values (however, immobilization in a cationic matrix may exert the opposite effect, which justifies the result found for immobilization via sodium periodate-treated PEJUp: 24.3 U at pH 4.) The monitoring of pH in industrial processes (feed, pharmaceutical, water and effluent treatments) is essential for the effectiveness of the process[33]. Thus, PEJU as a support is able to retain HRP activity in both acidic and neutral media, which allows its application for different reaction purposes. The stability of PEJU-HRP and PEJUp-HRP was analyzed for 30 days of storage at 4 °C without addition

of any enzymatic stabilizer in either of the two complexes (Table 1). For immobilization by physical adsorption, over 70% of enzyme activity remained after 30 days. Bayramoğlu and Arıca[34] obtained over 84% of remaining peroxidase activity, but the authors used magnetic methacrylate beads activated using glutaraldehyde. The modified magnetic support, according the authors, was responsible for the enhance in the enzyme stability in comparison to free HRP. Otherwise, polysaccharides can play fundamental roles in enzymatic stability through electrostatic, dipole-ionic or hydrophobic interactions with the protein structure[3]. For PEJUp, after 15 days, the remained activity was only 6% under the same storage conditions as PEJU-HRP. Considering the degradation and instability (Figure 3B and 3C) of the PEJUp structure caused by sodium periodate oxidation, the physical peroxidase adsorption was the best choice for storage conditions in the immobilization of HRP using PEJU. Table  1 also shows the applicability and reuse of immobilized complexes. After three repeated runs, the PEJU-HRP without the addition of any stabilizer retained about 18% of its initial activity. The PEJUp-HRP, prepared in the same condition as the the adsorbed enzyme, had the double of activity retention (35%), totaling eight use cycles (retention of 9% of the initial activity). The remaining activity is lower than that reported for macroporous glucosidic polymers (50% activity retained after four cycles)[35] or microgels (50% retained activity after five cycles)[36] or even through the sodium periodate-treated support (9% of retained activity after eight uses), all of which are covalent strategies.

3.3 Industrial wastewater treatment using immobilized HRP and evaluation of residual toxicity The ability of HRP, PEJU-HRP and PEJUp-HRP complexes to remove phenols is evidenced by the data in Figure 4A and 4B. The immobilized complexes removed

Table 1. Stability – shelf life and reuse - of PEJU-HRP and PEJUp-HRP complexes.

Time (days of storage)

Cycles of use

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1 3 6 9 12 15 18 21 24 27 30 1 2 3 4 5 6 7 8

Remained activity (%) PEJU-HRP PEJUp-HRP 100 100 100 71.4 100 25.6 100 20.6 82 10.4 82.9 6.0 82 1.1 81.7 0.9 75.1 0.9 76.3 0.9 70.5 0.6 100 100 65 82 18 48 6 35 0 16 0 13 0 9 0 2.2

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Silva, T. M., Borges, L. L., Souza, E. R. B., & Caramori, S. S. Table 2. Evaluation of toxicity, citotoxicity and genotoxicity over Allium cepa roots by different PEJU complexes and phenolic waste. Treatments Distilled water Sodium azide (0.02g mL-1) PEJU (10mg mL-1) WTS PEJU-HRP-WTS

Toxicity (%) 0 63.5 0 90 0

Figure 4. Phenol efficiency removal (%) of horseradish peroxidase (HRP) and immobilized complexes PEJU-HRP (A) and PEJUp-HRP (B) in the phenolics effluent samples. The removal efficiency of free HRP are the closed symbols and the PEJU-HRP or PEJUp-HRP are the open symbols. --○-- pyrogallol; -- ∇-- phenol; --□-- bromophenol; --◊-- textile effluent; --△-- WTS effluent treatment.

phenolic compounds and presented a similar or higher performance compared to the free enzyme. Catechol and nitrophenol were not removed by any of the tested enzyme forms. For the phenolic prepared solutions, free HRP and PEJU-HRP removed 26% of pyrogallol up to 120 min (Figure 4A). After this time, the free peroxidase decreases its capacity, while the PEJU-HRP continues the same profile of degradation. This might be explained due to inactivation and/or inhibition on free enzyme caused by interactions between phenoxyl radicals or phenolic polymers produced during catalysis[37]. Approximately 38% of bromophenol could be removed by free HRP and by PEJUp-HRP up to 120 min, but the immobilized complex remained more stable than the free enzyme (Figure 4B). 6/8

Citotoxicity (%) 0 25.9 27.2 32.4 31.5

Genotoxicity (%) 0 47.0 9.4 34.0 18.9

All enzyme forms degraded the industrial effluents (Figure 4A  and  4B). PEJU-HRP (Figure  4A) showed a removal efficiency of 94% from the WTS, and 100% of the textile effluent. If the incubation time is considered, the immobilized form of peroxidase was more stable and continuously removed phenols from complex mixtures over time. This is in accordance to Tatsumi et al.[38], who used immobilized HRP on cellulose via oxidation through periodate, and removed more than 80% of phenol from aqueous solutions using continuous reactors. Figure 4 also shows that the maximum reaction time for phenol removal by immobilized complexes was up to 30 min. The free enzyme needed 90 min to achieve the maximum of phenol removal capacity. The advantage of an immobilized enzyme in such processes lies not only in its stability: the preparation maintains a remarkable discoloration activity when compared to the free form[11]. Table 2 shows the results of toxicity tests by incubating the roots of Allium cepa in PEJU complexes and controls. Bilal et al.[8], evaluating the performance of immobilized MnPeroxidase for the treatment of wastewater, discussed the biological toxicity of degraded compounds after waste treatment. Toxic agents must reduce more than 50% the germination index of Allium cepa in relation to the negative control[20]. This indicates that the effluent from WTS is a toxic material with a reduction effect (p<0.05) higher than the negative control (sodium azide 0.02 g mL-1 (63.5%)). The non-toxicity of PEJU is evident by growth averages: it is contained in the immobilized enzyme complex, and the treated effluent reflects this non-toxic material characteristic. The cytotoxic evaluation is based on the change in the mitotic index (MI) of the different treatments. Based on Table 2, the solutions containing PEJU did not promote cytotoxic alterations when compared to the negative control (water) (mitotic index lower than 22%). In the literature, cytotoxic interference occurs when the substance is capable of inhibiting 22% of the mitotic index in relation to the negative control and, at a greater amount of inhibition, over 50%[19]. Normal mitosis is noted when both negative control and PEJU were incubated within Allium cepa roots. By using the wastewater or only free HRP replacing PEJU, there is a genotoxic effect of those substances (Table  2). PEJU, as a support for HRP immobilization, was able to reduce the genotoxicity of the industrial effluent by 15.1% (Table 2). The genotoxicity test aims to identify if the analyzed substance affects key cell processes, such as duplication and gene transcription[39]. .This provided efficiency not only to the immobilization itself, but also to its contribution to the process of environmental restoration as an agent of bioremediation. The results of atoxicity of this material at the investigated concentrations against Artemia salina are promising Polímeros, 29(4), e2019056, 2019


Synthesis of immobilized biocatalysts for wastewater decontamination and corroborate the potential of this material for future biotechnological applications of these polysaccharides (in the food industry as thickening agents, in the pharmaceutical industry, and as adjuvants in the manufacture of drugs).

4. Conclusions The information obtained in this study indicates the Cerrado-arboreal cashew (Anacardium othonianum Rizz.) polysaccharide as a promising source for biotechnological application, especially the use of this material as a support for the immobilization of peroxidases in two ways: physical adsorption and chemical, non-covalent bond. Due to the instability of the support during the sodium periodate oxidation process, the PEJU-HRP showed a better action. It is stable in storage conditions. It can be repeatedly used for the enzymatic activity in laboratory practice. It has a homogeneous behavior in different pH conditions. Finally, it is a low-cost, non-toxic substance and extracted in a sustainable way. In addition to these applications, the removal of phenolic compounds through the immobilized enzyme system showed a high efficiency for both immobilization methods proposed. This material plays a significant role as a more stable, recoverable industrial biocatalyst with a low or no toxicity in tests with bioindicators, such as Artemia salina and Allium cepa roots. It is reusable in various other industrial, biotech, pharmaceutical and medical complexes. The technological interest in cashew polysaccharides, especially this Cerrado species, is related to its rheological characteristics and biodegradability. The possibility of using it as a low-cost substitute for other polysaccharides is attractive, and may expand the possibilities of using this material and promote its commercial exploitation by communities associated with cashew cultivation.

5. Acknowledgements The authors are grateful to the Regional Centre for Technological Development and Innovation (CRTI). They thank the employees of the DAIA Waste Treatment Station and the Cia Hering Work Safety Engineer (Alessandro de Paula Cardoso) for the effluent concession for analysis. Finally, the authors thank The Coordination for the Improvement of Personnel Higher Education (CAPES) and the program of research incentive grants (PROBIP/UEG).

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30. Varma, A. J., Kokane, S. P., Pathak, G., & Pradhan, S. D. (1997). Thermal behavior of galactomannan guar gum and its periodate oxidation products. Carbohydrate Polymers, 32(2), 111-114. http://dx.doi.org/10.1016/S0144-8617(96)00155-5. 31. Silva, F. E. F., Batista, K. A., Di-Medeiros, M. C. B., Silva, C. N. S., Moreira, B. R., & Fernandes, K. F. (2016). A stimuliresponsive and bioactive film based on blended polyvinyl alcohol and cashew gum polysaccharide. Materials Science and Engineering C, 58, 927-934. http://dx.doi.org/10.1016/j. msec.2015.09.064. PMid:26478388. 32. Gomez-Estaca, J., Comunian, T. A., Montero, P., FerroFurtado, R., & Favaro-Trindade, C. S. (2016). Encapsulation of an astaxanthin-containing lipid extract from shrimp waste by complex coacervation using a novel gelatin–cashew gum complex. Food Hydrocolloids, 61, 155-162. http://dx.doi. org/10.1016/j.foodhyd.2016.05.005. 33. Khodadoust, S., Kouri, N. C., Talebiyanpoor, M. S., Deris, J., & Pebdani, A. A. (2015). Design of an optically stable pH sensor based on immobilization of giemsa on triacetylcellulose membrane. Materials Science and Engineering C, 57, 304-308. http://dx.doi.org/10.1016/j.msec.2015.07.056. PMid:26354268. 34. Bayramoğlu, G., & Arıca, M. Y. (2008). Enzymatic removal of phenol and p-chlorophenol in enzyme reactor: horseradish peroxidase immobilized on magnetic beads. Journal of Hazardous Materials, 156(1-3), 148-155. http://dx.doi.org/10.1016/j. jhazmat.2007.12.008. PMid:18207637. 35. Xu, R., Chi, C., Li, F., & Zhang, F. (2013). Immobilization of horseradish peroxidase on electrospun microfibrous membranes for biodegradation and adsorption of bisphenol. Bioresource Technology, 149, 111-116. http://dx.doi.org/10.1016/j. biortech.2013.09.030. PMid:24096278. 36. Zhang, Y. P., Liu, T. H., Wang, Q., Zhao, J. H., Fang, J., & Shen, W. G. (2012). Synthesis of novel poly (N,N-diethylacrylamideco-acrylic acid) (P(DEA-co-AA)) microgels as carrier of horseradish peroxidase immobilization for pollution treatment. Macromolecular Research, 20(5), 484-489. http://dx.doi. org/10.1007/s13233-012-0044-z. 37. Vineh, M. B., Saboury, A. A., Poostchi, A. A., Rashid, A. M., & Pariva, K. (2018). Stability and activity improvement of horseradish peroxidase by covalent immobilization on functionalized reduced graphene oxide and biodegradation of high phenol concentration. Journal of Biological Macromolecules, 106, 1314-1322. http://dx.doi.org/10.1016/j.ijbiomac.2017.08.133. PMid:28851646. 38. Tatsumi, K., Wada, S., & Ichikawa, H. (1996). Removal of chlorophenols from wastewater by immobilized horseradish peroxidase. Biotechnology and Bioengineering, 51(1), 126-130. http:// dx.doi.org/10.1002/(SICI)1097-0290(19960705)51:1<126::AIDBIT15>3.0.CO;2-O. PMid:18627096. 39. Luz, A. C., Pretti, I. R., Dutra, J. C. V., & Batitucci, M. C. P. (2012). Evaluation of the cytotoxic and genotoxic potential of Plantago major L. in tests systems in vivo. Revista Brasileira de Plantas Medicinais, 14(4), 635-642. http://dx.doi.org/10.1590/ S1516-05722012000400010. Received: Jan. 30, 2019 Revised: Sept. 06, 2019 Accepted: Oct. 19, 2019

Polímeros, 29(4), e2019056, 2019


ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.02119

Design of a polymeric composite material femoral stem for hip joint implant Romeu Rony Cavalcante da Costa1* , Fellipe Roberto Biagi de Almeida1, Amanda Albertin Xavier da Silva1, Sandra Mara Domiciano1 and André Ferreira Costa Vieira2 Laboratório de Materiais Compósitos, Departamento de Engenharia Mecânica, Universidade Tecnológica Federal do Paraná – UTFPR, Cornélio Procópio, PR, Brasil 2 Departamento de Engenharia Eletromecânica, Universidade da Beira Interior, Colvilhã, Portugal 1

*romeu@utfpr.edu.br

Abstract Hip joint prosthesis are structural components that still have some challenging problems such as the interaction of physical and biological properties between the stem and the human femur. Composite materials allow to obtain high strength structures with a large variety of modulus of elasticity and favorable characteristics in the context of orthopedic implants. Therefore, the objective of this work was the development of a prosthesis model with biopolymeric matrix, namely the polyurethane (PU) derived from castor oil, reinforced with fiberglass. The implants were made of pure PU, PU with fiberglass, and PU with glass fiber and calcium carbonate. The reinforcement was constructed in the form of a core to be inserted into the hip prosthesis. The core and stem prototypes were produced using three-dimensional printing techniques, and subsequently used in the manufacture of flexible silicone molds. The results showed good mechanical potentialities of this material for orthopedics applications. Keywords: calcium carbonate, composite, femoral stem, fiberglass, polyurethane. How to cite: Costa, R. R. C., Almeida, F. R. B., Silva, A. A. X., Domiciano, S. M., & Vieira, A. F. C. (2019). Design of a polymeric composite material femoral stem for hip joint implant. Polímeros: Ciência e Tecnologia, 29(4), e2019057. https://doi.org/10.1590/0104-1428.02119

1. Introduction Fiber reinforced composites have been used in many industrial applications such as, aerospace, automotive, and military, due to advantageous mechanical properties when compared to metallic materials, namely specific strength and stiffness[1,2]. Associated to this, excellent ratio between fatigue resistance and weight, as well as fatigue resistance and wear ratio[3]. One of the segments that are considering the advantages of composite materials is the bioengineering, more specifically in the development of joint prosthesis. The composite materials allow the achievement of high strength structures with a wide range of elasticity modulus, which seems benefit from the standpoint of an orthopedic implant[4]. Prostheses made of metallic materials presents some disadvantages regarding biocompatibility issues and discrepancy of stiffness compared to the human bone. The Young modulus of human cortical bone varies from 12 to 20 GPa. On the other hand, the Young modulus of some special titanium alloys used in orthopedics components vary between 60 and 80 GPa[5]. A femoral stem for hip joint implants is a structural component that still presents challenging problems with no solutions in the field of orthopedic surgery. Among the complications of this process, stands out the inadequate combination amid the femoral stem and the cortical bone of the human femur. The application of materials that present

Polímeros, 29(4), e2019057, 2019

the Young’s modulus and strength close to that of the cortical bone is the goal to be achieved[6]. The optimization of femoral prostheses involves, essentially, the determination of the geometry and the elasticity’s module of materials that attenuate the stress shielding effect to acceptable levels of interface micro movements. Stress shielding is a process that occurs because the stresses sensed by bone are lower than those sensed by the healthy joint without a prostheses, which leads to bone resorption in a given region[4]. The mechanical properties is the principal determinant of functional compatibility of polymeric structures. The adequate interface between the artificial structure and the host tissue is only ensured if mechanical properties of this structure are similar to those of the tissue that is desired to recover or to replace[7]. The biopolymer used at this research is a PU, which is presented in the form bicomponent, being constituted of polyol and prepolymer. The polyol is synthesized from castor oil and the prepolymer is synthesized from the diphenylmethane diisocyanate. Since 1996 the PU have been used as bone cement for prosthetic implants and reparative substance in bone loss[8]. Currently, PU’s is the class of polymers most consumed in the world, being applied as, for example, paint components, coatings, foams, domestic,

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Costa, R. R. C., Almeida, F. R. B., Silva, A. A. X., Domiciano, S. M., & Vieira, A. F. C. and industrial components, as well as construction and biocomponents sectors[9-13]. Biocompability[14] studies using PU have been developed, as well as implant procedures for filling bone defects[15]. Souza[15] presents the application of PU derived from castor oil as filler to bone failures in legs affected by cancer. In almost all cases studied, the most important aspect to be satisfied was the perfect adaptation of bloody bone surface with the polymer surface, followed by a rigid stabilization, to allow the bone-polymer osseointegration.

(b) Core mold (core on PU reinforced fiberglass used to improve mechanical properties of the stem). It opens in two parts to extract the core after curing. It has two holes at the tips to remove resin excess; (c) Centering jigs open mold.

The first step in obtaining the stem is the manufacturing of the PU composite core reinforced with fiberglass (Figure 3a) to give the necessary shape and the fibers alignment (Figure 3b). Eight (8) and sixteen (16) fiberglass

Dontos[16] evaluated the biocompatibility of a wire knurled of castor oil PU (Biologic Lifting Wire) applied at the facial rejuvenation. Through his study, it was revealed an excellent Biologic Lifting Wire biocompability when implanted in subdermal tissue of mice, with the presence of small inflammatory reaction and quick collagen synthesis. This work aims to present the design and the fabrication methodology of a femoral stem built in polymeric composite material reinforced with fiberglass and calcium carbonate, also to get the approximated value of the theoretical elasticity’s module of the stem’s core calculated according to the rule of mixture. And, to experimentally verify the properties of the fabricated stems, there were static tests made according to the standards of ABNT NBR ISO 7206-04[17] e ABNT NBR ISO 7206-06[18].

2. Manufacturing Process of Femoral Stem The composite material used to produce the femoral stem consists of a PU matrix, which is obtained by mixing prepolymer (synthesized from diphenylmethane diisocyanate) [329 L (60% m/m)] and polyol (derived from castor oil) [471 (40% m/m)] supplied by Poliquil Araraquara Polímeros Químicos Ltda., in conjunction with glass fiber roving type E (TEX 2400) supplied by TEXIGLASS Ind. and Com. Têxtil Ltda., and Calcium Carbonate P.A. supplied by Synth. These last are used as reinforcement, in order to improve the mechanical strength and rigidity of the stem. The femoral stem drawing was developed in SolidWorks, based on references[6,19-22]. In this production process, the dimensional parameters and shape that would satisfy the bio-mechanical requirements in relation to the request of the weight and the rigidity of the remaining bone.

Figure 1. Plastic models of stem and necessary parts for stem manufacturing process obtained by 3D printing.

After obtaining the drawing of the femoral stem, which consists of core made of a fiber glass and PU composite, the plastic model and the centering jigs (Figure 1) were printed on a 3D printer (Stratasys Objet 24). The plastic model was then used to produce a silicon mold (the negative of the model) to produce the stem. The centering jigs were used to center the core during the PU resin injection into the mold. These printed parts were then used to make silicone molds. Figure 2 shows the three silicone molds made by pour casting, for the different parts of the stem, namely: (a) Stem mold with channels to vent and PU injection. It opens in two parts to extract the stem after curing and also to allow the introduction of the core and its centering before the injection process; 2/8

Figure 2. Silicone molds obtained using the printed models for manufacturing of: (a) the core, (b) the femoral stem and (c) the jibs for positioning of the core. Polímeros, 29(4), e2019057, 2019


Design of a polymeric composite material femoral stem for hip joint implant Ec =E f V f +EmVm

(1)

where Ec, E f and Em are the composite, fiberglass, and matrix elastic modulus, respectively. V f and Vm are the fiber and matrix volume fractions, respectively, that can be calculated using the Equations 2 and 3: Vf =

Vm =

vf vc

vm vc

(2) (3)

where v f , vm, vc are, the fiberglass, matrix and composite respectively volumes. Remembering that the total composite volume vc is the sum of the fiber glass volume, v f , the matrix volume, vm, and the voids volume vv, according to Equation 4: Figure 3. Fabrication process of the core: (a) Fiber impregnation with PU, (b) Placement of the impregnated fiber into the mold.

vc =vm +v f +vv

(4)

To proceed with the proper fixation of the core (Figure 4a) in the stem mold, centering jigs were placed in a 90 degrees angle with the core axis, as shown in Figure 4b. The base mixture to obtain the matrix (prepolymer and polyol) was placed in a vacuum chamber to remove bubbles, therefore reducing voids volume. Those stems manufactured with calcium carbonate (30% in mass), this component was previously mixed in to the polyol before the addition of the prepolymer. The purpose of using calcium carbonate was to improve osseointegration at stem surface. Furthermore, Taguti has shown that PU reinforced with calcium carbonate presents increased elastic modulus and strength when compared to pure PU[24].

Figure 4. (a) Composite core composed of glass fiber and PU matrix; (b) core positioned in the silicon mold for the manufacturing of the stem.

bundles were placed along the length of the core, thereby obtaining, for each configuration, different mass fractions of fiber, matrix and void. Mechanical properties depend on these fractions. These were calculated by measuring samples mass before and after the complete degradation of the PU in an oven at the temperature of 600°C, since the melting temperature of fiberglass is higher than that. According Costa[23], the elastic modulus for PU is 1.43 GPa. This was verified in experimental tensile tests. The elastic modulus is a key parameter for the success of femoral stems design for hip joint implant. Since glass fibers have an elastic modulus of about 70 GPa, the idea of combining it with PU to produce a composite material, is to achieve an elastic modulus close to that of human femur bone, which has a value of 17.3 GPa[6]. According to the rule of mixtures, considering iso deformation hypothesis, it was possible to calculate the theoretical elastic modulus of the composite material following Equation 1: Polímeros, 29(4), e2019057, 2019

With the core properly aligned in the mold, it was sealed with tape to prevent leakage during injection of the mixture. Then the mixture was injected into the mold, with the aid of an embolus, until the total filling of the mold. During the curing process, the mold was kept inside a pressure chamber at 0.8 MPa for 8 hours. The stems were divided into four diferent types, stems without a center (PU), stems reinforced with 8 strands of fiberglass (PU8G), stems reinforced with 16 strands (PU16G) of fiberglass and stems with 30% of CaCo3 (calcium carbonate) e reinforced with 16 strands of fiberglass.

2.1 Quasi-static tests Quasi-static tests were made in accordance to the standards of ABNT NBR ISO 7206-04 e ABNT NBR ISO 7206-6, which give the specifications concerning the positioning of the stems for the tests. These specifications say how far the stem needs to be inserted into the setting, both to the respect of the depth and the angle, to represent how it would be placed in the human femur. According to the standard ABNT NBR ISO 7206-04, the angles must be from, α= 10˚ degrees and β = 9˚ degrees, with 1˚ (one degree) of tolerance in both cases, and the insertion of the stem, that is the distance between the center of the sphere to the surface where the setting is, there must be D=80 mm. For the tests in accordance with the standard ABNT NBR ISO 7206-6, the angles must be α=10º and β=9º, com 0.30º (zero point thirty degrees) and 1º (one degree) of tolerance respectively, and the level of insertion of the 3/8


Costa, R. R. C., Almeida, F. R. B., Silva, A. A. X., Domiciano, S. M., & Vieira, A. F. C. stem into the femur coinciding with the real case of a total or partial substitution of the pelvic joint. To assure that the stem would be positioned correctly, there were two templates of the position, made with a 3D printer. In the Figure 5a  and  5b, the templates are according to the standards ASTM 7206-04 AND ASTM 7206-06, respectively. The femoral stem is placed into the template and them placed into the interior of a tube filled with concrete (Figure 5c). After the positioning of the stems, the verification of the height and angles was made witha digital microscope, model Dino-Lite Digital Microscope Pro. To make the measurement, the software DinoCapture 2.0 was used.

The tests were conducted with an advanced velocity of 2mm/mm utilizing a device developed by Almeida[25], that guarantees the fixation of the concrete base and the free movement of the “head” of the prosthesis, by way of bearings and a smooth upper base. The device for the test can be seen in Figure 6. The tests were made in a machine of universal tests model WDW-100E, made by Time Group Inc.

3. Results and Discussions Figure 7a shows the central nucleus (core) of the stems that was manufactured in two configurations: reinforced with 8 or 16 fiber bundles. Moreover, Figure 7b shows the

Figure 5. Template for the positioning of the stems (a) ASTM 7206-04; (b) ASTM 7206-06; (c) stem placed in concrete[25].

Figure 6. The device used in the tests of flex-tortion.

Figure 7. (a) composite core PU reinforced with fiberglass, (b) positioning of the core on the stem, (c to e) stems using different portions of fiber and (f) draw view of stem. 4/8

Polímeros, 29(4), e2019057, 2019


Design of a polymeric composite material femoral stem for hip joint implant positioning of the core on the stem, Figure 7(c to e) shows the core with 8 fiber bundles coated with PU, 16 fiber bundles coated with PU, and 16 fiber bundles coated with PU plus calcium carbonate (30% in mass), respectively. Finally, Figure 7f shows the basic dimensions of the stem design, in millimeters. The average values found for fiber volumetric fractions, matrix and voids and the elastic modulus for the fiberglass E-type, and polyurethane derived from castor oil are shown in Table 1. With those values was possible, through the rule of the mixture, the calculation of the composite theoretical elastic modulus of with distinct volumetric fractions of fiber and matrix, according to the different cores using 8 and 16 fiber bundles. As mentioned, the appropriate value for the elastic modulus of the material that composes the stem should be similar to that of the bone, specifically the femur, according to the literature, is 17.3 GPa[4]. The value of 24.03 GPa, which was obtained with 16 fiber bundles, fits perfectly to the necessary mechanical requirements. It has a slightly higher value than the human bone.

3.1 Tests quasi-static of the stems After the template of the stems according to the standard ABNT NBR ISO 7206-4;2011, there was a verification of their height and the angles. The measurements were analyzed with software DinoCapture 2.0. Figure 8 shows the image of the microscope on a screen indicating the

height of the center of the sphere in relation to the surface of the concrete used in the the stem template. In Figure 8, the measurements of the height of the center of the sphere in relation to the surface of the concrete can be seen (L) and the angles α and β , showing the real fixation of the stem, the closest required by the standard used. There were three hip implant stems tested of the four different types for the analysis of mechanical behavior: PU, PU8G, PU16G and PUCa16G. The medium load and medium maximum displacement of each type of stem is seen in Table 2 It is evident that the increase of the maximum load supported by the stems conforms to the increase of the applied reinforcement. However, the values obtained appear to be promising, considering the values required for surgery of a femoral stem, according to literature. Because the tests made in this work were quase-static, thus, when a dynamic load is applied to test of the fatigue, the material will respond more intensely, being able to reach a level of force much higher than that obtained by the type of test used in the present work. In accordance with Bergmann et al.[26] and Ramakrishna et al.[27], the medium force applied in hip joint is around 238% of the body weight, being able to reach peaks up to ten times the weight the body weight during intense activities such as jumping and running Silvestre[6], in his work, also did quasi-static tests with a femoral stem made in polyurethane without reinforcement of

Table1. Fractions of mass and elastic modulus of the core calculated by the rule of mixture. Portions of glass fiber 8 16

Em [GPa] 1.43 1.43

Ef [GPa] 72 72

Vf [%] 0.14 0.32

Vm [%] 0.8 0.69

Vv [%] 0.06 0.02

Ec [GPa] 11.23 24.03

Em -Elasticity Modulus of the Matrix; Ef - Elasticity Modulus of the Fiberglass; Vf – fiberglass volumetric fraction; Vm – matrix volumetric fraction; Vv – volumetric void fraction; Ec - Elasticity Modulus of the composite.

Table 2. Values obtained in the tests of femoral stems – ISO 7206-4. Relation Load-Displacement PU PU8G PU16G PUCa16G

Results Test according to ISO 7206-4 Maximum Load [N] Displacement until the Maximum Load [mm] 615.03±71.02 10.86±1.63 767.18±104.62 14.34±1.02 871.46±79.43 7.72±1.44 922.54±42.26 9.42±2.09

Stems without a center (PU); Stems reinforced with 8 strands of fiberglass (PU8G); Stems reinforced with 16 strands (PU16G) of fiberglass and stems with 30% of CaCo3 (calcium carbonate) and reinforced with 16 strands of fiberglass (PUCa16G).

Figure 8. Measurement of the height of the template of the stem in relation to the center of the sphere and the angles of positioning. Polímeros, 29(4), e2019057, 2019

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Costa, R. R. C., Almeida, F. R. B., Silva, A. A. X., Domiciano, S. M., & Vieira, A. F. C. the center, with a positioning of the stem calculated according to the standard ABNT NBR ISO 7206-4. During the test, he saw in his results that the maximum load supported by this stem without a center was approximately 750 N and a displacement equal to 7.5, mm. In this present work, for the same composition of the stem, the values found for the maximum load and the maximum displacement were, respectively, around 615 N and 10,86 mm. These discrepancies can be justified, for example, by the differences of geometry between the proposed stems, variations in the angles α and β and by small weaknesses in the securing elements during the tests, to analyze the mechanical behavior of the stems, they presented a supple behavior, without apparent fracture. Following the same procedure during the tests of the stems in accordance with the standard ABNT NBR 7206-4, new stems with identical composition to those already cited were submitted to tests in accordance with the standard ABNT NBR ISO 7206-6. Thus, three hip transplant stems of the four different types were tested to analyze the mechanical behavior: PU, PU16G and PUCa16G. The Information of the medium load and medium displacement of each type of stem are seen in Table 3. At the end of the test, stem number 7 of pure PU was the only one in which a total fracture of the neck of the prosthesis (Figure  9). However, as was expected with this form of fixation (ISO 7206-6), it resulted in a load resistance of approximately 48% greater than the form of fixation of the ISO 7206-4 and the displacement reached approximately 43% of this. In these tests, all of the stems were submitted to the maximum displacement possible until there was no limitation of contact, therefore, the obtained data is referring to the beginning of the test until the point in which there would be the contact of the sphere with the concrete (Figure 10). Observing the results obtained, it can be assumed that a possible cause of the low values of the maximum load was the lack of rigidity of the joint of the fixing device, that is, the load applying device, the axial socket and the acetabulum, Figure 6. Table 3 presents the data referring to these tests. The stems of PU 16G demonstrated an increased load capacity of approximately 20%, in a comparison with a stem of the same composition testes according to the standard ABNT NBR ISO 7206-4. On the other hand, the displacement according to ISO 7206-6 went beyond the other standard at 8.03%. The stems of PUCa16G presented the best results in the analysis according to the standard ABNT NBR ISO 7206-6.

The value of the maximum load went above 88% of that which was obtained according to the first standard of the stem of the same composition, reaching a value of 1739.73 N.

Figure 9. Fracture of the neck of the femoral stem without a center.

Figure 10. Aspect of the behavior of the stem during the tests.

Table 3. Values found in the tests of the femoral stems – ISO 7206-6. Relation Load-Displacement PU PU16G PUCa16G

Results Test according to ISO 7206-6 Maximum Load [N] Displacement until the Maximum Load [mm] 911.90±82.01 4.67±0.71 1043.84±132.57 8.34±1.56 1739.73±79.68 5.07±1.12

Stems without a center (PU); Stems reinforced with 16 strands (PU16G) of fiberglass and stems with 30% of CaCo3 (calcium carbonate) and reinforced with 16 strands of fiberglass (PUCa16G).

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Design of a polymeric composite material femoral stem for hip joint implant

Figure 11. Stress-strain curve of monotonic tensile test at different strain rates.

The displacement was 5.07 mm until the maximum load, that is, it moved 53.8% of the earlier displacement. All the tests made according to the standard ABNT NBR ISO 7206-6 were made until a complete or partial fracture of the necks of the femoral stems, Figure 10. As stated, the stem of pure PU was the one to have a complete fracture of the neck. The fractures of the reinforced stems show that the centers are more resistant when compared to the external form of the stem and that there is a problem in the interface of the center with the covering that it involves, which is the form of the stem. Thus, the composition between the center and this form resulted in the stem being more rigid, in a way that total fracture of the neck was avoided. For all the stems that have a center, this remained true after the tests, only the displacement of the outer form occurred. Figure 10 shows the aspect of the stems after the test, as well as the breaking of the interface between the center and the outer form. The results obtained were promising and show that the process of production needs adaptions and improvements of the experimental volumetric portion between the fiber and the matix for all of the body of the stem. So, there should be encouraged an increase in the resistance of the material[28], principally the polymetric material, especially paying attention to the adhesive behavior, Figure 11. Thus, when the stem is submitted to a load with higher speed or fatigue, it should be expected that the limit of resistance similarly increases to that which occurs to polyurethane without reinforcement.

4. Conclusions This work discusses production of a composite stem made of a biocompatible PU derived from castor oil, glass fibers and calcium carbonate. Special attention in this discussion was the structural stiff composite core, using different fiber volume fractions, corresponding to either 8 or 16 fiber bundles. These stem prototypes were produced with and without addition of calcium carbonate exhibiting, in both cases, good homogeneity of the polymer, which resulted in no apparent bubbles. Polímeros, 29(4), e2019057, 2019

With the values of the volumetric fraction of the fiber and the matrix was possible to determine the theoretical elastic modulus of the composite material used in the cores. Considering that the core is the main structural part, responsible to support the majority of the loads developed during gait and transfer them to the femur, the whole stem (core and PU casing) will possibly be able to resist the them. The theoretical value of 24.03 GPa (presented by the core with 16 fiber bundles), calculated using the rule of mixture, is higher than the elastic modulus of human bone (femur), which is 17.3 GPa[4]. In future works, proposed to validate the functional compatibility validation, using numerical methods for the simulation of the mechanical behavior and mechanical tests based on appropriate standards[29,30] to analyze the fatigue strength of the stem. Further simulations are also needed to analyze the influence of the stem in the stress field around the stem, and the consequent stress shielding effect due to the presence of this stem. This analysis will be made by comparing the designed stem presented here with another stem with the same geometry made of titanium alloys. Since this stem solution is to be assemble into the bone by press fit, and later to be progressively fixed by the osseointegration process, relative micromovements between stem and bone should be limited to allow new bone to grow over the stem surface. According to literature, these relative micromovements should be below 150 µm[31]. These micromovements depend on the stem stiffness, and therefore on its geometry and material properties. Hence, in future design iterations, the stem and core geometries will probably be changed to this future analyses. However, the present work shows very promising results aiming the improve the stress shielding effect of current gold standard solutions in titanium alloys.

5. Acknowledgements Poliquil Araraquara Polímeros Químicos Ltda. CAPES – Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.

6. References 1. Liu, Y., Yang, J. P., Xiao, H. M., Qu, C. B., Feng, Q. P., Fu, S. Y., & Shindo, Y. (2012). Role of matrix modification on interlaminar shear strength of glass fiber/epoxy composites. Composites Part B, Engineering, 43(1), 95-98. http://dx.doi. org/10.1016/j.compositesb.2011.04.037. 2. Harizi, W., Chaki, S., Bourse, G., & Ourak, M. (2014). Mechanical damage assessment of glass fiber-reinforced polymer composites using passive infrared thermography. Composites Part B, Engineering, 59, 74-79. http://dx.doi. org/10.1016/j.compositesb.2013.11.021. 3. Yuanjian, T., & Isaac, D. H. (2007). Combined impact and fatigue of glass fiber reinforced composites. Composites Part B, Engineering, 39(3), 505-512. http://dx.doi.org/10.1016/j. compositesb.2007.03.005. 4. Vieira, A. F. (2004). Design of a femoral component of a hip prosthesis articulate in composite materials (Master’s thesis). University of Porto, Portugal. 5. Bougherara, H., Zdero, R., Dubov, A., Shah, S., Khurshid, S., & Schemitsch, E. H. (2011). A preliminary biomechanical study of a novel carbon-fiber hip implant versus standard 7/8


Costa, R. R. C., Almeida, F. R. B., Silva, A. A. X., Domiciano, S. M., & Vieira, A. F. C. metallic hip implants. Medical Engineering & Physics, 33(1), 121-128. http://dx.doi.org/10.1016/j.medengphy.2010.09.011. PMid:20952241. 6. Silvestre, G. D., Fo. (2006). Design and structural analysis of femoral stem hip implant in polymeric composite material (Doctoral dissertation). Universidade de São Paulo, São Carlos. 7. Parthasarathy, M., & Sethuraman, S. (2014). Hierarchical characterization of biomedical polymers. In S. G. Kumbar, C. T. Laurencin & M. Deng (Eds.), Natural and synthetic biomedical polymers (pp. 33-42). USA: Elsevier. http://dx.doi. org/10.1016/B978-0-12-396983-5.00002-8 8. Azevedo, E. C., Claro, S., No., Chierice, G. O., & Lepienski, C. M. (2009). Instrumented indentation applied to the mechanical characterization of polyurethane derived from castor oil. Polímeros: Ciência e Tecnologia, 19(4), 336-343. http://dx.doi. org/10.1590/S0104-14282009000400014. 9. Charlon, M., Heinrich, B., Matter, Y., Couzigné, E., Donnio, B., & Avérous, L. (2014). Synthesis, structure and properties of fully biobased thermoplastic polyurethanes, obtained from a diisocyanate based on modified dimer fatty acid, and different renewable diols. European Polymer Journal, 61, 197-205. http://dx.doi.org/10.1016/j.eurpolymj.2014.10.012. 10. Cornille, A., Dworakowska, S., Bogdal, D., Boutevin, B., & Caillol, S. (2015). A new way of creating cellular polyurethane materials: NIPU foams. European Polymer Journal, 66, 129138. http://dx.doi.org/10.1016/j.eurpolymj.2015.01.034. 11. Fu, C., Liu, J., Xia, H., & Shen, L. (2015). Effect of structure on the properties of polyurethanes based on aromatic cardanolbased polyols prepared by thiol-ene coupling. Progress in Organic Coatings, 83, 19-25. http://dx.doi.org/10.1016/j. porgcoat.2015.01.020. 12. Thakur, S., & Karak, N. (2013). Castor oil-based hyperbranched polyurethanes as advanced surface coating materials. Progress in Organic Coatings, 76(1), 157-164. http://dx.doi.org/10.1016/j. porgcoat.2012.09.001. 13. Trinca, R. B., & Felisberti, M. I. (2015). Segmented polyurethanes based on poly(L-lactide), poly(ethylene glycol) and poly(trimethylene carbonate): physico-chemical and morphology. European Polymer Journal, 62, 77-86. http:// dx.doi.org/10.1016/j.eurpolymj.2014.11.008. 14. Sousa, T. P. T., Costa, M. S. T., Guilherme, R., Orcini, W., Holgado, L. A., Silveira, E. M. V., Tavano, O., Magdalena, A. G., Catanzaro-Guimarães, S. A., & Kinoshita, A. (2018). Polyurethane derived from Ricinus Communis as graft for bone defect treatments. Polímeros: Ciência e Tecnologia, 28(3), 246-255. http://dx.doi.org/10.1590/0104-1428.03617. 15. Souza, A. M. G. (2002). Biopolymer of castor for reconstruction of failures bone after tumor-resection: clinical application (Doctoral dissertation). Universidade Federal de Pernambuco, Recife. 16. Dontos, C. A. (2005). Fio lifting biológico: avaliação de sua biocompatibilidade e eficácia no rejuvenescimento facial (Master’s thesis). Universidade de São Paulo, São Carlos. 17. Associação Brasileira de Normas Técnicas. (2011). ABNT NBR 7206-4: determination fatigue strength properties and performance of femoral components with stem. Rio de Janeiro: ABNT. 18. Associação Brasileira de Normas Técnicas. (2004). ABNT NBR 7206-6: determination of fatigue properties head and neck region of femoral stems. Rio de Janeiro: ABNT.

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19. Simões, J. A., & Marques, A. T. (2005). Design of a composite hip femoral prosthesis. Materials & Design, 26(5), 391-401. http://dx.doi.org/10.1016/j.matdes.2004.07.024. 20. Bougherara, H., Bureau, M., Campbell, M., Vadean, A., & Yahia, L. (2007). Design of a biomimetic polymer-composite hip prosthesis. Journal of Biomedical Materials Research: Part A, 82(1), 27-40. http://dx.doi.org/10.1002/jbm.a.31146. PMid:17265439. 21. Bae, J. Y., Farooque, U., Lee, K., Kim, G. H., Jeon, I., & Yoon, T. R. (2011). Development of hip joint prostheses with modular stems. Computer Aided Design, 43(9), 1173-1180. http://dx.doi.org/10.1016/j.cad.2011.05.004. 22. Ruben, R. B., Fernandes, P. R., & Folgado, J. (2012). On the optimal shape of hip implants. Journal of Biomechanics, 45(2), 239-246. http://dx.doi.org/10.1016/j.jbiomech.2011.10.038. PMid:22115063. 23. Costa, R. R. C. (2007). Applicability of constitutive models for analyzing the mechanical behavior of a biopolymer (Doctoral dissertation). Universidade de São Paulo, São Carlos. 24. Taguti, M. V. H. (2015). Characterization and modeling of the mechanical behavior of polyurethane enriched with calcium carbonate (Master’s thesis). Universidade Tecnológica Federal do Paraná, Cornélio Procópio. 25. Almeida, F. R. B. (2016). Estudo, dimensionamento e desenvolvimento de um modelo de prótese de quadril em material compósito com matriz biopolimérica (Master’s thesis). Universidade Tecnológica Federal do Paraná, Cornélio Procópio. 26. Bergmann, G., Deuretzbacher, G., Heller, M., Graichen, F., Rohlmann, A., Strauss, J., & Duda, G. N. (2001). Hip contact forces and gait patterns from routine activities. Journal of Biomechanics, 34(7), 859-871. http://dx.doi.org/10.1016/ S0021-9290(01)00040-9. PMid:11410170. 27. Ramakrishna, S., Mayer, J., Wintermantel, E., & Leong, K. W. (2000). Biomedical applications of polymer-composite materials: a review. Composites Science and Technology, 61(9), 1189-1224. 28. Costa, R. R. C., Vieira, A. F. C., Guedes, R., & Tita, V. (2017). A biopolymer derived from castor oil polyurethane: experimental and numerical analyses. In V. K. Thakur, M. K. Thakur & M. R. Kessler (Eds.), Handbook of composites from renewable materials (pp. 581-606). Hoboken: Wiley-Scrivener Publishing. http://dx.doi.org/10.1002/9781119441632.ch60 29. Associação Brasileira de Normas Técnicas. (2008). ABNT NBR 7206-1: classifies the dimensions of the prosthesis. Rio de Janeiro: ABNT. 30. Associação Brasileira de Normas Técnicas. (2004). ABNT NBR 7206-10: determination of resistance to static load of modular femoral heads. Rio de Janeiro: ABNT. 31. Turner, T. M., Sumner, D. R., Urban, R. M., Rivero, D. P., & Galante, J. O. A. (1986). A comparative study of porous coatings in a weight-bearing total hip-arthroplasty model. The Journal of Bone & Joint Surgery, 68(9), 1396-1409. http://dx.doi.org/10.2106/00004623-198668090-00013. PMid:3782212. Received: Mar. 18, 2019 Revised: Nov. 26, 2019 Accepted: Dec. 05, 2019

Polímeros, 29(4), e2019057, 2019


ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.04819

Mechanical characterization of HDPE reinforced with cellulose from rice husk biomass Mariane Weirich Bosenbecker1, Gabriel Monteiro Cholant1, Gabriela Escobar Hochmuller da Silva1, Oscar Giordani Paniz1, Neftali Lenin Villarreal Carreño1, Juliano Marini2 and Amanda Dantas de Oliveira1*  Centro de Desenvolvimento Tecnológico – CDTec, Universidade Federal de Pelotas – UFPel, Pelotas, RS, Brasil 2 Departamento de Engenharia de Materiais – DEMa, Universidade Federal de São Carlos – UFSCar, São Carlos, SP, Brasil 1

*amandaoliveira82@gmail.com

Abstract High-density polyethylene (HDPE) reinforced with cellulose from rice husk (RH) were prepared and studied. The RH biomass was submitted to acid extraction and bleaching process and then analyzed for its cellulose extraction efficiency by X-ray diffraction (XRD) and Fourier transformation infrared spectroscopy (FTIR). After that, the RH cellulose (RHC) was incorpored to the HDPE matrix by melt blending with different filler contents (5, 10 and 15 wt%), and then characterized in terms of mechanical properties and morphology. The RHC incorporation in the HDPE matrix resulted in an increase in elastic modulus regardless the filler content added; also, the impact resistance was maintained for RHC contents up to 10%. The morphological analysis of the composites showed that the cellulose was well dispersed in the matrix, which contributed to the improvement of the final rigidity of these materials, indicating the feasibility of incorporating this residue in the production of HDPE composites. Keywords: cellulose, biomass, polymeric composites, rice husk. How to cite: Bosenbecker, M. W., Cholant, G. M., Silva, G. E. H., Paniz, O. G., Carreño, N. L. V., Marini, J., & Oliveira, A. D. (2019). Polímeros: Ciência e Tecnologia, 29(4), e2019058. https://doi.org/10.1590/0104-1428.04819

1. Introduction Polymeric composites reinforced with vegetable fibers have been the target of great academic and industrial interest for replacing, generally with cost advantages and lightness, materials made of conventional polymeric composites. These composites are less aggressive and toxic, since they are reinforced with raw materials of plant origin, such as oils, starch and cellulose. Besides coming from renewable sources, meeting the requirements of biodegradability and preservation of the environment throughout its cycle of life[1], they can be an alternative for the reduction of inappropriate waste disposal when the utilized fibers are from agroindustrial waste[2]. Rice is the most popular food in the world, its importance can be noted by its numbers of consumption and production. The consumption of rice as food for 2017 was estimated at 53.7 kg/person. To meet this demand, rice production forecasts, for the same year, were estimated at 759.6 million tonnes according to the Food and Agriculture Organization (FAO)[3]. In this context, the Brazilian production of rice is in the order of 12 million tons/year and the state of Rio Grande do Sul represents 60% of this production[4]. For each ton of rice produced, it is estimated that 0.23 ton of RH are formed during the rice refining process, being the most voluminous by-product[5]. In this way, RH is an agroindustrial large quantity residue in Rio Grande do Sul.

Polímeros, 29(4), e2019058, 2019

The average chemical composition of RH is defined in 35% cellulose, 25% hemicellulose, 20% lignin and 20% of others [6]. It is important to note that these contents may vary significantly according to type of paddy, crop year, climatic and geographical conditions and sample preparation for analysis. These peels have low commercial value because the contained silica (SiO2) and the fibers do not have nutritional value, therefore they are not used in human or animal feeding. The destination found for the RH, in the last years, has been the discharge into crops and river bottoms, resulting in serious environmental impacts. In other cases, RH has been used as a source of energy for parboiling, but it is also a problem as it contributes to air pollution. Polymeric composites using cellulose as reinforcing agent have several advantages, generally these natural reinforcements are lighter, more economically attractive and provide greater resistance when compared with inorganic reinforcing agents [7]. But interestingly, there are no reports on the use of cellulose obtained from RH in HDPE matrix. Therefore, the objective of this work was to evaluate the use of cellulose extracted from RH in the obtention of composites with HDPE by melt blending, offering an alternative reuse for this agroindustrial residue. The work also aimed to promote an improvement in the mechanical properties of HDPE.

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Bosenbecker, M. W., Cholant, G. M., Silva, G. E. H., Paniz, O. G., Carreño, N. L. V., Marini, J., & Oliveira, A. D.

2. Materials and Methods

2.3 HDPE/Cellulose composites preparation

High density polyethylene - HDPE (GM9450F) obtained from Braskem S/A was used as polymeric matrix. The rice hulks were obtained from a cereal supply located in Pelotas/RS – Brazil. All reagents used in this work had analytical grade.

2.3.1 Melting blending pocess

2.1 Cellulose obtaining from RH Figure 1 presents a flowchart of the methodology used for the RHC obtention. Firstly, the rice hulks were milled in a knife mill (Marconi – Model MA 340). After that, the milled biomass was submitted to the first step treatment which consists in an acid treatment to remove the hemicellulose and lignin present in the RH . The milled RH was added in a solution of water, acetic acid and nitric acid at a ratio of 6:5:1 v/v, respectively, under mechanical stirring and reflux at 80ºC for 4h. The resulting material was washed and filtered with distilled water untill reached the neutral pH. The bleaching process was done by the imersion of the resulting material from the first step in a solution of sodium hypochlorite for 24h. After the steady time, the material was filtered using distilled water untill was neutralized. The non-passing material was the RHC, which was dried at 60ºC for 24h.

2.2 Cellulose characterization 2.2.1 X-ray Diffraction (XRD) The XRD analyzes were performed using a diffractometer (D8 Advance Bruker) with wavelength of ƛ = 1,541 Å, operating at 40 KV and 40 mA. The scans were performed in the range of 2θ between 10 and 50º, at a rate of 1/min. 2.2.2 Fourier Transformation Infrared Spectroscopy (FTIR) The analyses were done using a Spectrum 1000 spectrophotometer from Perkin Elmer, in the range between 400 and 4000 cm -1. Each spectrum corresponds to the average of 10 scans at a resolution of 4 cm-1. 2.2.3 Scanning Electron Microscopy (SEM) analysis The microscopy analyses of the RH in natura, the RH after the first treatment and the RH cellulose were performed by SEM using a Shimadzu, modelo SSX-550 Superscan equipament.

To produce the composites, a single screw extruder (CIOLA, model MEP-18) with L/D = 22 was used. The temperature profile used was 180/190 °C in the two heating zones and 190 °C for the matrix. The composites were obtained with 5, 10 and 15 wt% by mass of cellulose. The pure polymer also was obtained using the same processing conditions in order to have their results compared to those obtained to the composite materials. 2.3.2 Composites molding The materials obtained from the extrusion process were pelletized and then molded by compression at 200ºC in a hydraulic press (Marconi, MA098/A) using the following cycle: pre-heating (for 2 min) / pressing (3 ton for 2 min) / relief / pressing (5 tons for 2 min) / forced cooling in water at room temperature. This process results in plates with 10 cm of length, 10 cm of width and 3.2 mm of thickness. The specimens for tensile and impact mechanical tests were stamped from the plates obtained. Figure 2 shows a schematic of all the steps involved in processing of the composites. 2.3.3 Composites evaluation 2.3.3.1 Mechanical characterization The mechanical properties were determined by tensile and Izod impact tests. The dimensions of the samples were defined according to the American Society for Testing Materials (ASTM) standards. 2.3.3.2 Tensile strengh test The tensile tests were performed according to ASTM D-638 on a universal testing machine (INSTRON E3000) with a load cell of 5000 N, distance between claws of 115 mm and deformation rate of 5 mm.min-1. The widths and thicknesses of the specimens were measured using a caliper gauge (Mitutoyo, ± 0.001 mm). 2.3.3.3 Izod impact test For the Izod impact tests (RESIL 25, from Ceast) the specimens were notched with a depth of 2.54 ± 0.1 mm, with minimum notch speed in accordance with ASTM D256. The tests were performed at room temperature.

Figure 1. Flowchart of the RHC obtention process. 2/7

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Mechanical characterization of HDPE reinforced with cellulose from rice husk biomass 2.3.3.4 SEM analysis The fracture surfaces of the impacted specimens were observed by SEM, using a Jeol JSM- 6610LV equipament. A thin layer of gold was deposited on the material with metallizer.

3. Results and Discussions 3.1 Cellulose characterization The in natura RH color is brown and after the first chemical treatment a brown-orange coloration was observed. After the bleaching, the material appears completely white (see Figure 1). These color changes are directly associated with the removal of non-cellulosic materials[8]. Thus, the white color observed in the final product was an indication of a high purity of the cellulosic material. However, further analysis is required, such as chemical composition, morphology, crystallinity and functional groups for its characterization[9]. 3.1.1 X-ray Diffraction (XRD) X-ray diffraction was used to investigate the crystalline structure of RH before and after the chemical treatments used. In addition, microcrystalline cellulose (MCC) of commercial

origin was analyzed for comparison. The diffractograms are represented in Figure 3. It is reported in the literature, that cellulose have crystalline sctructure, due to Van der Waals’ bonds, while hemicellulose and lignin have an amorphous characteristic[10]. As expected, natural rice husk has low crystallinity. There is an increase in the crystallinity of the samples according to the chemical treatments used, which may be related to the partial elimination of hemicellulose and lignin, indicating the efficiency of the procedure used to obtain the cellulose from rice husk. The peaks at approximately 16°, 22° and 34° are characteristic of type I cellulose[9], corresponding to the (101), (002) and (004) planes, respectively. 3.1.2 Fourier Transformation Infrared Spectroscopy (FTIR) Figure 4 shows the spectra of the in natura rice husk, after acid extraction, RHC and MCC. It is possible to observe in all spectra the presence of two major regions of absorbance[11]. The first region at high wavenumbers (2700-3500 cm-1) can be atribuited to different vibrations in polysaccharides and the second region at lower wavenumbers (800 to 1800 cm-1) is typical assigned to cellulose[12]. The peak in the region between 3500-3000 cm-1 confirms the presence of hydroxyl groups (O-H). For the RHC, the

Figure 2. Flowchart of the composites processing.

Figure 3. X-ray diffractograms of RH, RH after acid treatment, RHC and MCC. Polímeros, 29(4), e2019058, 2019

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Bosenbecker, M. W., Cholant, G. M., Silva, G. E. H., Paniz, O. G., Carreño, N. L. V., Marini, J., & Oliveira, A. D. increase in the area of ​​this band indicates the presence of a greater number of OH groups[13] and, consequently, an increase in RHC hydrophilicity. The peak in 2921 cm-1 is observed in all spectra and it is due to the presence of C-H stretching vibration in polysaccharides, evidencing the presence of cellulose and hemicellulose[12,14]. The bands in the range of 1700-1730 cm-1 are attributed to the vibration of acetyl and uronic groups of hemicellulose, or even to the ester linkage of carboxyl and p-coumaric acids in lignin[15]. In this region a peak reduction was observed, according to the chemical treatment sequences, thus confirming the partial removal of hemicellulose and lignin[11]. A small band was observed in the range of 1629-1621 cm-1, which can be attributed to the water absorbed in cellulose[12]. In general, the FTIR spectra have confirmed that RHC have a more open structure related to their greater wettability as the hemicellulose is dissolved or removed[13]. The band at 1170 cm-1 corresponds to the asymmetric stretching of the C-O-C bond of cellulose, hemicellulose and lignin[16]. The bands observed at approximately 1030 cm-1 and at 800 cm-1 in the spectra correspond to the stretching bonds of C=O in guaiacyl rings[17] and CH deformations in cellulose[15], the latter one presents an increase in intensity

as the treatment is performed, indicating an increase in cellulose content. 3.1.3 SEM analysis Through the SEM images it was possible to investigate the changes in the structure of the RH, as well as the cellulose, as shown in Figure 5. The RH, Figure 5A, presented a more compact structure, with an irregular outer surface of hemicellulose, lignin, inorganic components and lumps or protuberances[18], when compared to the RHC, Figure 5B and C, in which the bleached fibers had a fibrous and rough structure, with a mean diameter of 5 μm[8]; also some particulates are observed, which can be related to residual material from the synthesis or even remained impurities. It was also observed the presence of some pores in the rough surface of the fibers, indicating that there may be an increase in the effective surface area for contact with the polymeric matrix, which contributes to the improvement of the mechanical properties.

3.2 Composites characterization 3.2.1 Mechanical characterization Figure 6 presents the graphs for the mechanical characterization.

Figure 4. FTIR spectra of HR biomass, HR after the acid extraction, RHC and MCC.

Figure 5. SEM micrographs of: (A) RH; (B) and (C) RHC. 4/7

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Mechanical characterization of HDPE reinforced with cellulose from rice husk biomass 3.2.1.1 Tensile strength test In Figure 6A it is possible to observe that the modulus of elasticity of the composites presented different behaviors in relation to the HDPE. The HDPE composites have higher elastic modulus than pure HDPE. On the other hand, when analyzing the results obtained for composite materials prepared with different filler contents, it is verified that the values of ​​ elastic modulus presented by these materials have no significant difference. It is known that when a composite is reinforced with cellulose, the tensile modulus of the polymer compound can improve considerably. However, this property change will also depend on the strength and effectiveness of the interfacial adhesion between cellulose and matrix [19], which can be improved with prior treatment and also of the amount of filler used. Low contents of cellulose can form regions of high mechanical stress, by a not so homogeneously dispersed mixture, resulting in a decrease in elastic modulus. Higher contents of cellulose, in its turn, can result in a better distribuition of stresses, and thus greater modulus [20]. This small increase in the values of elastic modulus indicates a low interaction between the polymer and the matrix, and this can be explained by its lack of chemical affinity, since the HDPE is apolar and the cellulose is known for its polarity, as can be confirmed by FTIR. 3.2.1.2 Izod impact test In Figure 6B it is observed that HDPE presented an average impact resistance of 178.65 J/m, higher than the composites with 5, 10 and 15 wt% of cellulose, with values of 172.99 J/m, 162.54 J/m and 126.36 J/m, respectively. Its already established in literature that HDPE has a high Izod impact strength, which is decreased when reinforced with vegetable fibers [21]. Considering the standard deviations, it can be affirmed that the addition of up to 10 wt% of RHC does not promote significant reductions in the impact resistance when compared to pure HDPE. The HDPE/15 wt% RHC presented a greater decrease impact resistance, breaking more easily, and also making it a material with greater rigidity lower tenacity. This

can be explained since the introduction of reinforcements can promote stress concentration, generating the presence of micro-interfacial voids, which make the microcracks to propagate easily in the composite, and then reducing impact resistance [22]. Also, RH is fragile and therefore, have a low capacity to absorb energy during the propagation of the fracture. Similar results of decrease in impact strength with increasing cellulose content was observed in literature [19], who reported an increase in its brittleness due to large amounts of cellulose in a HDPE matrix. This decrease in impact strength of the polymer matrix can also be reverted by the use of impact modifiers, which improve the matrix / reinforcement interface. 3.2.2 SEM analysis Its well established that interfacial adhesion matrix/filler and dispersion of the filler in the polymer matrix are determining factors in mechanical properties of any composite material [23]. In this way, the mechanical results can be confirmed to the observed in SEM analysis. Figure 7 presents SEM micrographs obtained from the fracture surface of the samples after the Izod impact tests were performed. The micrographs revealed a heterogeneous distribution of the RHC in HDPE matrix, with RHC presenting different morphologies, such as rods-like structures, particulates and also agglomerates. These different morphologies can be justified by the fragmentation of RHC during the composites extrusion and the agglomeraion due the greater affinity of cellulose with itself than with the matrix. Considering the amount of RHC used represents most of the composite volume, we can affirm that it was possible to achieve a good adhesion between the phases, with the RHC distributed in the polymer, contributing to the increase of the elastic modulus when comparing to the HDPE. However, it is possible to observe regions of detachment of the RHC from the matrix, forming voids. The more RHC was used, the more detachments could be perceived, due to the higher amount of RHC. In the same way, more voids are formed. The appearance of defects as voids with the increase in the amount of RHC used, justifies the results obtained by the impact resistance tests.

Figure 6. (A) Elastic modulus and (B) Impact strength for pure HDPE, HDPE 5wt% RHC, HDPE 10wt% RHC and HDPE 15wt% RHC composites. Polímeros, 29(4), e2019058, 2019

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Bosenbecker, M. W., Cholant, G. M., Silva, G. E. H., Paniz, O. G., Carreño, N. L. V., Marini, J., & Oliveira, A. D.

Figure 7. SEM micrographs of (A) HDPE 5wt% RHC, (B) HDPE 10wt% RHC and (C) HDPE 15wt% RHC.

All observed fracture surfaces are rough, indicating that the addition of RHC did not change the fracture behavior of the polymeric matrix, which remained plastic.

4. Conclusions Considering that the results of FTIR, XRD and SEM are in agreement with the literature, we can say that the treatment used was efficient for obtaining cellulose from RH biomass. The addition of RHC in the HDPE polymer matrix had good effect on the elastic modulus of the composites, as we can confirm by the improvement in the rigidity of the composites when compared to the pure matrix, but without significant changes for higher contents of RHC. The impact strength have decreased as the RHC content increase in the polymeric matrix. Micrographs obtained by MEV from the fractured surface obtained by impact test of the composites, revealed some adherence between RHC‑matrix. In this context, the results obtained for the HDPE composites with the addition of RHC present a superiority over the pure matrix and thus create the possibility of its use in composites and an alternative to use this residue.

5. Acknowledgements The authors would like to acknowledge the CAPES (Coordination for the Improvement of Higher Education Personnel), the Centro de Microscopia Eletrônica do Sul (CEMESUL) of Federal University of Rio Grande, the Department of Materials Engineering of Federal University of São Carlos (DEMa/UFSCar) and the Laboratório de Materiais Poliméricos (LaPol) of Federal University of Rio Grande do Sul (UFRGS) for the use of infrastructure.

6. References 1. Ligowski, E., Santos, B. C., & Fujiwara, S. T. (2015). Materiais compósitos a base de fibras da cana-de-açúcar e polímeros reciclados obtidos através da técnica de extrusão TT - Composite materials based on fibers from sugar cane and recycled polymers obtained by extrusion technique. Polímeros: Ciência e Tecnologia, 25(1), 70-75. http://dx.doi. org/10.1590/0104-1428.1605. 2. Yang, H. S., Kim, H. J., Park, H. J., Lee, B. J., & Hwang, T. S. (2006). Water absorption behavior and mechanical properties 6/7

of lignocellulosic filler-polyolefin bio-composites. Composite Structures, 72(4), 429-437. http://dx.doi.org/10.1016/j. compstruct.2005.01.013. 3. Food and Agriculture Organization of the United Nations – FAO. (2018). Seguimiento del mercado del arroz de la FAO (Informe FAO de actualización de precios del arroz, Vol. 21, No. 1, 10 p.). Rome: FAO. Retrieved in 2019, June 22, from http://www.fao.org/3/I9243ES/i9243es.pdf 4. Santos, P., & Costa, A. C. S. (2013). X-ray diffraction and thermal analysis of kaolins particle size fractions. Semina. Ciências Exatas e Tecnológicas, 34(1), 9-22. http://dx.doi. org/10.5433/1679-0375.2013v34n1p9. 5. Chandrasekhar, S., Satyanarayana, K. G., Pramada, P. N., Raghavan, P., & Gupta, T. N. (2003). Processing, properties and applications of reactive silica from rice husk - an overview. Journal of Materials Science, 38(15), 3159-3168. http://dx.doi. org/10.1023/A:1025157114800. 6. Wu, G., Qu, P., Sun, E., Chang, Z., Xu, Y., & Huang, H. (2015). Physical, chemical, and rheological properties of rice husks treated by composting process. BioResources, 10(1), 227-239. Retrieved in 2019, June 22, from https://bioresources.cnr.ncsu. edu/resources/physical-chemical-and-rheological-propertiesof-rice-husks-treated-by-composting-process/ 7. Campos, A., Teodoro, K. B. R., Marconcini, J. M., Mattoso, L. H. C., & Martins-Franchetti, S. M. (2011). Efeito do tratamento das fibras nas propriedades do biocompósito de amido termoplástico/policaprolactona/sisal. Polímeros, Ciência e Tecnologia, 21(3), 217-222. http://dx.doi.org/10.1590/s010414282011005000039. 8. Johar, N., Ahmad, I., & Dufresne, A. (2012). Extraction, preparation and characterization of cellulose fibres and nanocrystals from rice husk. Industrial Crops and Products, 37(1), 93-99. http://dx.doi.org/10.1016/j.indcrop.2011.12.016. 9. El Halal, S. L. M., Colussi, R., Deon, V. G., Pinto, V. Z., Villanova, F. A., Carreño, N. L. V., Dias, A. R. G., & Zavareze, E. R. (2015). Films based on oxidized starch and cellulose from barley. Carbohydrate Polymers, 133, 644-653. http:// dx.doi.org/10.1016/j.carbpol.2015.07.024. PMid:26344323. 10. Prado, K. S., & Spinacé, M. A. S. (2015). Characterization of Fibers from Pineapple’s Crown, Rice Husks and Cotton Textile Residues. Materials Research, 18(3), 530-537. http:// dx.doi.org/10.1590/1516-1439.311514. 11. Jonoobi, M., Harun, J., Shakeri, A., Misra, M., & Oksmand, K. (2009). Chemical composition, crystallinity, and thermal degradation of bleached and unbleached kenaf bast (Hibiscus cannabinus) pulp and nanofibers. BioResources, 4(2), 626-639. Retrieved in 2019, June 22, from https://bioresources.cnr.ncsu. edu/resources/chemical-composition-crystallinity-and-thermalPolímeros, 29(4), e2019058, 2019


Mechanical characterization of HDPE reinforced with cellulose from rice husk biomass degradation-of-bleached-and-unbleached-kenaf-bast-hibiscuscannabinus-pulp-and-nanofibers/ 12. Hospodarova, V., Singovszka, E., & Stevulova, N. (2018). Characterization of cellulosic fibers by FTIR spectroscopy for their further implementation to building materials. American Journal of Analytical Chemistry, 09(06), 303-310. http://dx.doi. org/10.4236/ajac.2018.96023. 13. Rong, M. Z., Zhang, M. Q., Liu, Y., Yang, G. C., & Zeng, H. M. (2001). The effect of fiber treatment on the mechanical properties of unidirectional sisal-reinforced epoxy composites. Composites Science and Technology, 61(10), 1437-1447. http:// dx.doi.org/10.1016/S0266-3538(01)00046-X. 14. Adel, A. M., Abd El-Wahab, Z. H., Ibrahim, A. A., & Al-Shemy, M. T. (2010). Characterization of microcrystalline cellulose prepared from lignocellulosic materials. Part I. Acid catalyzed hydrolysis. Bioresource Technology, 101(12), 4446-4455. http:// dx.doi.org/10.1016/j.biortech.2010.01.047. PMid:20185300. 15. Pelissari, F. M., Sobral, P. J. A., & Menegalli, F. C. (2014). Isolation and characterization of cellulose nanofibers from banana peels. Cellulose (London, England), 21(1), 417-432. http://dx.doi.org/10.1007/s10570-013-0138-6. 16. Rosa, M. F., Medeiros, E. S., Malmonge, J. A., Gregorski, K. S., Wood, D. F., Mattoso, L. H. C., Glenn, G., Orts, W. J., & Imam, S. H. (2010). Cellulose nanowhiskers from coconut husk fibers: effect of preparation conditions on their thermal and morphological behavior. Carbohydrate Polymers, 81(1), 83-92. http://dx.doi.org/10.1016/j.carbpol.2010.01.059. 17. Silvério, H. A., Flauzino Neto, W. P., Dantas, N. O., & Pasquini, D. (2013). Extraction and characterization of cellulose nanocrystals from corncob for application as reinforcing agent in nanocomposites. Industrial Crops and Products, 44, 427-436. http://dx.doi.org/10.1016/j.indcrop.2012.10.014.

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18. Oliveira, J. P., Bruni, G. P., Lima, K. O., Halal, S. L. M. E., Rosa, G. S. D., Dias, A. R. G., & Zavareze, E. D. R. (2017). Cellulose fibers extracted from rice and oat husks and their application in hydrogel. Food Chemistry, 221, 153-160. http:// dx.doi.org/10.1016/j.foodchem.2016.10.048. PMid:27979125. 19. Boran, S. (2016). Mechanical, morphological, and thermal properties of nutshell and microcrystalline cellulose filled high-density polyethylene composites. BioResources, 11(1), 1741-1752. http://dx.doi.org/10.15376/biores.11.1.1741-1752. 20. Spadetti, C., Silva, E. A., Fo., Sena, G. L., & Melo, C. V. P. (2017). Propriedades térmicas e mecânicas dos compósitos de polipropileno pós-consumo reforçados com fibras de celulose. Polímeros, Ciência e Tecnologia, 27(spe), 84-90. http://dx.doi. org/10.1590/0104-1428.2320. 21. Morais, J. A., Gadioli, R., & De Paoli, M.-A. (2016). Curaua fiber reinforced high-density polyethylene composites: effect of impact modifier and fiber loading. Polímeros, Ciência e Tecnologia, 26(2), 115-122. http://dx.doi.org/10.1590/01041428.2124. 22. Poletto, M., & Zattera, A. J. (2017). Mechanical and dynamic mechanical properties of polystyrene composites reinforced with cellulose fibers : coupling agent effect. Journal of Thermoplastic Composite Materials, 30(9), 1242-1254. http:// dx.doi.org/10.1177/0892705715619967. 23. Mathew, A. P., Oksman, K., & Sain, M. (2005). Mechanical properties of biodegradable composites from poly lactic acid (PLA) and microcrystalline cellulose (MCC). Journal of Applied Polymer Science, 97(5), 2014-2025. http://dx.doi. org/10.1002/app.21779. Received: June 22, 2019 Revised: Nov. 20, 2019 Accepted: Feb. 11, 2020

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ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.05519

Acoustic approach of weldability for nanocomposite (nanosilica/PA6) welded by ultrasonic welding Anderson Ribeiro1* , Jaime Casanova2, Sérgio Duarte Brandi3 and Diego de Moura Pinheiro3 Faculdade de Tecnologia do Estado de São Paulo – FATEC, Campi Itaquera, São Paulo, SP, Brasil 2 Departamento de Engenharia de Materiais, Universidade Federal do Amazonas – UFAM, Manaus, AM, Brasil 3 Departamento de Engenharia Metalúrgica e de Materiais, Universidade de São Paulo – USP, São Paulo, SP, Brasil

1

*andersoncnribeiro@gmail.com

Abstract Polymer matrix nanocomposites (NMP) have attracted a great interest mainly in the automotive, aerospace and medical industries since they have good mechanical properties, dimensional, thermal and chemical stability, as well as interesting electrical conductivity and cost reduction in the manufacturing process. However, welding of this class of materials presents serious challenges such as improving weldability of the joint and understanding the mechanisms responsible for coalescence. The objective of this work was to evaluate the coalescence of an NMP joint (comprising a PA6 matrix and with nanosilica of different percentages of silicon) using ultrasonic welding, as well as to perform an acoustic approach of the energy dissipation during the welding process. It is concluded that the NMP samples tend to show better coalescence as the percentage of nanosilica increases, up to a certain limit. On the other hand, the higher the content of nanoparticle the smaller the energy absorption. Keywords: ultrasonic welding, nanocomposite, acoustics, sound spectral. How to cite: Ribeiro, A., Casanova, J., Brandi, S. D., & Pinheiro, D. M. (2019). Acoustic approach of weldability for nanocomposite (nanosilica/PA6) welded by ultrasonic welding. Polímeros: Ciência e Tecnologia, 29(4), e2019059. https://doi.org/10.1590/0104-1428.05519

1. Introduction Ultrasonic welding is a widely used fusion joining process in the manufacture of polymer parts that can be used in various industrial sectors such as in the automotive, medical and aerospace industries. Studies have shown the advantages of the ultrasonic welding process in polymer‑based matrix composite materials since they present better mechanical resistance and low cost when compared to other thermoplastics[1]. In ultrasonic welding, the high‑frequency mechanical vibration is converted into heat by the combination of the contact surface and the intermolecular friction. In this way, the coalescence between the parts to be welded occurs[2]. Ultrasonic welding is considered as a favorable process to join thermoplastic composite materials[3]. On the other hand, polymer matrix nanocomposites have attracted a great interest mainly from industry (such as automotive, aerospace and medical industries) and researchers. The intentions are aimed at improving properties, such as mechanical, thermal and chemical properties as well as interesting electrical conductivity and cost reduction in the manufacturing process[4-6]. Ultrasonic welding for thermoplastics is well understood, however the literature has few papers on the ultrasonic welding of nanocomposite materials[7,8] For instance, ultrasonic welding of HDPE (Hight density polyethylene) nanocomposites with 0, 3, 6 and 9 wt% nanoclay was investigated. It was evaluated the effect of welding parameters on the weld strength. Results showed

Polímeros, 29(4), e2019059, 2019

that the maximum weld strength decreases with increasing nanoclay content[9]. Likewise, Shiu-Hung Hung evaluated some welding parameters (such as welding time, welding pressure, vibration amplitude, pressure time and joint geometry) responsible for the strength of the welded joint of a PP based composite material (glass fibre). In this research, it was observed that the composite material requires less welding energy to obtain a strong and better joint strength compared to the unfilled polymer. Also, it was shown that a higher percentage of loads contributed to intensifying the mechanical properties of the joint[2]. Similarly, Benatar and Gutowski characterized and modelled the process of ultrasonic welding for fibre reinforced composite[10]. This work aims to evaluate the coalescence of a joint of a nanocomposite material (PA6 matrix reinforced with spherical nanoparticles of silica) with different percentages of silica using ultrasonic welding, as well as to perform an acoustic energy dissipation approach.

2. Materials and Methods 2.1 Materials used A polymer matrix composite consisting of polyamide (commercial name PA6-B260) and silica nanoparticles (with the different percentages of nanosilica, namely: 1, 5 and 7%)

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O O O O O O O O O O O O O O O O


Ribeiro, A., Casanova, J., Brandi, S. D., & Pinheiro, D. M. was used. Table 1 presents the physicochemical characteristics of nanosilica, while Table 2 presents the physical properties of PA6. To obtain the test specimens, PA6 (in pellets form) was immersed in liquid nitrogen to avoid polymer melting and to increase the hardness, and then pulverised to small grains with a mean particle size of 1.0 mm. The powder obtained was then dried for 48 hours in an oven at 70 °C. Likewise, the nanosilica was dried under vacuum at 100 °C. Subsequently, mechanical mixing was carried out (with the addition of 1% by weight of zinc stearate as a lubricating agent) to obtain the samples with 1%, 5% and 7% by weight of nanosilica in PA6. After the extrusion process of the materials as mentioned above, the samples were cut into pellets. The next step was the injection of the samples into the DEMAG Ergotech pro 35115 injection moulding machine, in which 50 test bodies were injected for each percentage of nanosilica. Table  3 shows the mechanical properties for each sample group of the experiment.

2.2 Welding parameters The welding parameters used in the experiment were determined after a welding process involving 30 specimens (CPs) for each condition, for a total of 120 CPs. The defects in the welds were evaluated, and it was possible to construct the operating window for the process and to determine the appropriate parameters. Thus, a welding pressure of 15 psi, with varying welding time, from 0.2 to 0.6 seconds and a 0.1-second interval was employed for all the samples. Before the welding process, all the materials were stored in an oven at 60 °C for at least 48 hours. Figure 1 shows how the test bodies were attached to the welding equipment. The  measurement of the welded area was done using ImageJ software.

2.3 Tests The assay to evaluate shear strength was performed according to ASTM D 638 standard. 15 samples for each percentage of nanosilica were evaluated in the shear

Table 1. Physico-chemical characteristics of nano-silica. Properties Specific surface area (BET) Average particle size Compressed density (DIN EN ISO 787/11) Humidity Loss in fire 2h a 1000 °C, Based on a dried material by 2h a 105 °C pH dispersion of 4% SiO2 level

Unit m2/g Nm g/l % in weight % in weight % in weight

Value 200 ± 25 12 close to 50 ≤ 1.5 ≤ 1.0 3.7 4.7 ≥ 99.8

Table 2. Mechanical Properties of Polyamide (PA6). Properties Tensile stregth

B 260 s 75

Unit MPa

ASTM Standard D638

Tensile modulus

c 50 s 2900

MPa

D638

Elongation

c 1300 s 50

%

D638

Flexural strength

c >120 s 100

MPa

D790

Flexural modulus

c 40 s 2800

MPa

D790

Rockwell hardness

c 1000 s 120

R

D785

Impact resistance Izod

c 100 s 50

J/m

D256

c 90

Table 3. Mechanical properties of the nanocomposite. Percentage of nanosilica 0% 1% 5% 7%

Maximum load (N)

UTS (N/mm2)

YS (N/mm2)

Along. (%)

E (N/mm2)

2599.15 2604.65 2068.42 2565.42

59.21 59.35 47.15 58.45

29.44 22.38 20.69 23.75

424.62 72.12 84.30 36.44

189.07 573.53 255.21 629.78

UTS = Ultimate tensile strength; YS = Yield strength; E = modulus of elasticity.

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Acoustic approach of weldability for nanocomposite (nanosilica/PA6) welded by ultrasonic welding strength test. The speed and load for performing the test were 5 mm/min and 1.47 N, respectively.

2.4 Acoustic analysis Three recordings were made per sample group including three recordings with the sonotrode without pressing the sample. The recordings of the subharmonics were performed using a dynamic unidirectional (cardioid)

microphone, a rigid microphone stand, an analogue-digital converter and recording software. The microphone was positioned with an inclination of 45°, 10 cm from the point of interest (sonotrode and sample). The positioning was done empirically. Spectral results of the recording were analyzed in real time and the info were processed by commercial software named SpectraLAB: FFT - Spectral Analysis System[11] which is able to process data by fast Fourier transform analysis (FFT).

3. Results and Discussions The spectra corresponding to the recordings of the sound emitted for each welding condition can be observed in Figure 2. In this figure, the spectra were plotted using “amplitude intensity versus frequency” since this provides conditions to identify the time for a specific frequency in addition to estimating the energy of the signal in the spectrum.

3.1 Acoustic approach of the welding energy

Figure 1. Fixation of test bodies.

The quality of the welded joint of the polymer matrix composite material (CMP) is related to several factors such as the heat generated by the action of the tool, the incorporation of the load/polymer and the mechanical properties of the CMP, besides the melt area during the

Figure 2. Sound spectral emitted in each welding condition. Polímeros, 29(4), e2019059, 2019

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Ribeiro, A., Casanova, J., Brandi, S. D., & Pinheiro, D. M. welding process. Thus, ultrasonic welding is the result of several regimes in which acoustic contact drop, reduction in static pressure, and changes in amplitude and vibration frequency occur[12,13]. In order to evaluate the spectra corresponding to the ultrasonic oscillation generated by the sonotrode without sample (SA), with pure sample (AP), and other welding conditions, samples with 1% (A1), 5% (A5) and 7% (A7) nanosilica, the model that treats the level of intensity of spectrum (ISL - Intensity Spectrum Level), were used for several frequency bands. Thus, ISL is the sound wave intensity for 1 Hz frequency band and can be determined by Equation 1. ISL = 10log

I ( in1Hz band ) 1Hz I ref

(1)

On the other hand, the conditions analyzed in this present work resulted in spectra with several band levels (BL); this implies that the intensity of a BL is determined by Equation 2. BL 10log

I ( band de1Hz ) 1Hz ∆f + 10log I ref 1Hz

(2)

Otherwise, BL may be related to ISL as presented in Equation 3. BL = ISLave + 10log ( ∆f )

(3)

where: ∆f = f 2 − f1 , of ISLave corresponds to the average ISL. Finally, the integration of Equation 3 corresponds to the area of the spectra shown in Figure 2 (considering the limits of integration from 800 Hz to 20 kHz) in which it is possible to compare the eminent energy of the sound wave for each studied welding condition. The calculated energy is shown in Figure 3. From Figure 3 it can be seen that the energy intensity emitted in sound waveform during AP welding is 7% lower than the energy estimated for AS. In other words, the AP welding condition absorbed approximately 93% of

Figure 3. Estimated energy in the sound emissions in each welding condition. 4/7

the vibration energy emitted by the ultrasonic oscillation generated by the sonotrode (OUS). To explain the factors that contributed to this great disparity, the dissipation and absorption of energy will be evaluated. Considering the model presented in Figure 4, we see that the ultrasonic vibration is transmitted to the material by the sonotrode and induces the interface where the joint will occur (region 6)[14]. In the same way, the welding is influenced by the acoustic impedance of both the nanocomposite and the OUS. The intensity of the ultrasonic oscillation generated by the OUS is given by: I0 = 2π 2 f 2 A02 ρ0 c0

(4)

In the SA case, the absorption of I0 occurred only at the base (region 4 of Figure 4), disregarding the losses by the environment, so all the vibration dissipated by the system in the form of sound energy was captured and converted into ISL by the recording software. On the other hand, for AP welding, I0 suffered losses due to the acoustic impedance of the polymer (Z1 = ρ1c1 ), this resulted in I1 (intensity of the oscillation at interface 6) that was responsible for the vibration at the polymer/polymer interface. Thus, the increase in temperature occurred due to the absorption of the mechanical vibrations, a reflection of the vibration in the bound region and the friction at the surface. The combination of absorption and vibration reflections corresponds to the acoustic attenuation of the material. The calculation of the absorption coefficient of the wave can be done by using Equation 5. V  A ( f ) = −20 * log  2   V1 

(5)

Figure 4. Schematic representation of vibration through sonotrode, workpiece and anvil[14]. (1) working welding tool (sonotrodo); (2) and (3) acoustical impedance of the materialsZ ( Z1 = ρ1c1); Z 0 = ρ0 c0 - acoustical impedance of the ultrasonic oscillatory system; (4) support; (5) welded materials; (6) material/material interface; (7) reflection of a supersonic wave from an interface of medium; (8) energy absorption zone; X = thickness. Polímeros, 29(4), e2019059, 2019


Acoustic approach of weldability for nanocomposite (nanosilica/PA6) welded by ultrasonic welding Knowing that:

however, sample A5 presents the smallest value of α and

V2 =αz V1

(6)

where A(f) is the acoustic absorption, given in dB. α is the absorption coefficient in dB / mm, and z is the distance travelled by the wave, in mm. The relation

V2 can be considered as the acoustic V1

attenuation. Table 4 presents the results obtained in the determination of the acoustic attenuation and the absorption coefficient using transducers of 5 and 10MHz. Research has shown that acoustic attenuation in PMCs depends on the size of the charge of the particles, the degree of crystallinity (increasing the degree of crystallinity tends to reduce acoustic dissipation) and the incorporation of the charge in the matrix, i.e. the interaction between polymer/load[15]. It is summarized that CMPs with small particle sizes present better acoustic attenuation and higher volumetric fraction conditions for the same particle size, which results in higher wave attenuation compared to CMP with larger particles. This is because the increase in the number of particles in the matrix has a larger surface area for wave propagation[16]. In Table  4 we observe that the values of the absorption coefficients (α) and the acoustic attenuation

V2 for each studied condition are very close, V1

higher value of

V2 . These data justify the higher energy value V1

dissipated for A5 as seen in Figure 3. On the other hand, it can be seen (in Table 4) that sample A1 has similar α value with sample A7, but higher than sample A5. Some of the factors that contributed to the behaviour of α in A1 were the homogeneous distribution and the better incorporation of the nanoparticles in the CMP, this also explains the lower energy dissipated value for A1 (see Figure 3). However, for A7, a lower value of α was expected, since it had a higher fraction of nanosilica, but there was the formation of agglomerates and the inhomogeneous distribution of the particles.

3.2 Weld area and shear strength It is possible to observe in Figure 5 that samples with 5% and 7% nanosilica presented higher welded area compared to the other welding conditions for all the welding times analyzed. Analyzing Figure  5, it is possible to highlight the influence of the percentage of nanosilica on the welded area. In this way, it is observed that the shear strength increases with increasing load percentage. Figure 6 and 7 depict the shearing surface from each welded condition. It can be observed the size of the molten pool and their distribution in the weld. In Figure 6 the melted region grows up from the edge to the center, but not completely, while in Figure 6b

Figure 5. Weld area analysis for each welding condition. Table 4. Evaluation of sound attenuation. Transducer of 5 MHz

Transducer of 10 MHz

Sample

V1 (V)

V2 (V)

V2 V1

AP A1 A5 A7

1.80 2.10 1.86 2.23

0.262 0.275 0.212 0.325

0.15 0.13 0.11 0.15

Polímeros, 29(4), e2019059, 2019

α (dB/mm) 0.0221 0.0198 0.0173 0.0221

V1 (V)

V2 (V)

V2 V1

α (dB/mm)

0.65 1.24 0.825 1.24

0.075 0.181 0.093 0.168

0.12 0.15 0.11 0.14

0.0175 0.0221 0.0171 0.0205

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Ribeiro, A., Casanova, J., Brandi, S. D., & Pinheiro, D. M.

Figure 6. (a) SEM image of sheared surface for sample with 0% wt. nanosilica; (b) SEM image of sheared surface for sample with 1% wt. nanosilica.

Figure 7. (a) SEM image of sheared surface for sample with 5% wt. nanosilica; (b) SEM image of sheared surface for sample with 7% wt. nanosilica.

we can see small melted regions distributed inside the weld. However, the molten area tends to increase for samples with higher percentage of nanosilica (Figure 7). It is commonly accepted that the addition of nano-fillers into semi-crystalline polymers can accelerate their crystallization behavior and increase the thermal conductivity of polymer matrix[17-19]. Comparing the results presented in Figure 3 with those of Figure 5, we can see that the welded conditions with the highest emission energy (A5 and A7) were the ones with the best mechanical weldability (second percentage of the welded area - Figure 5). This is because the charge has a dissipative modulus higher than that of the polymer[20]. Thus, the energy was dissipated by vibration at the welded interface. Finally, the nanocomposite with higher load percentage requires less amount of vibrational energy to be welded (than the pure polymer), since when the load vibrates in phase with the sonotrode, more energy is transferred to the matrix[21]. On the other hand, the pure polymer is a more viscous material, with a high dissipative modulus (E”), and does not vibrate in phase with the sonotrode. Consequently, the energy rate transferred to the joint to be welded is lower[22]. 6/7

4. Conclusions When analyzing the results obtained in the acoustic energy dissipation approach associated with the shear strength test, it was possible to conclude that: • Pure samples (without the addition of nanosilica) welded by ultrasound absorbed about 90% of the vibration energy emitted by ultrasound oscillation by the sonotrode. This was due to the difference in acoustic impedance between the polymer, the sonotrode and the fixed sample base. Therefore, the main factors responsible for welding were the absorption of mechanical vibrations and friction at the surface interface; • The sample with 5% nanosilica (A5) presented low value of absorption coefficient and high value of acoustic attenuation due to the volumetric fraction of nanosilica, which causes a larger surface area for sound wave scattering. The sample with 7% nanosilica (A7) did not show the same behaviour as A5 because there was the formation of agglomerates and the distribution of inhomogeneous particles; Polímeros, 29(4), e2019059, 2019


Acoustic approach of weldability for nanocomposite (nanosilica/PA6) welded by ultrasonic welding • Samples A5 and A7 showed better weldability, although they absorbed less amount of vibrational energy; • The dissipative module of the nanosilica is superior to the modulus of the polymer, i.e. when the charge vibrates in phase with the oscillation transmitted by the sonotrode, a greater amount of energy is transmitted to the interface that will be welded.

5. References 1. Rashli, R., Bakar, E. A., Kamaruddin, S., & Othman, A. R. (2013). A review of ultrasonic welding of thermoplastic composites. Caspian Journal of Applied Sciences Research, 2(3), 1-16. Retrieved in 2019, July 11, from http://www.cjasr. com/images/manuscripts/2013/03/cjasr/01_CJASR-12-16-307. pdf 2. Liu, S. J., Chang, I.-T., & Hung, S.-W. (2001). Factors affecting the joint strength of ultrasonically welded polypropylene composites. Polymer Composites, 22(1), 132-141. http://dx.doi. org/10.1002/pc.10525. 3. Villegas, I. F., & Palardy, G. (2017). Ultrasonic welding of CF/ PPS composites with integrated triangular energy directors: melting, flow and weld strength development. Composite Interfaces, 24(5), 515-528. http://dx.doi.org/10.1080/09276 440.2017.1236626. 4. Lehmann, B., Schlarb, A. K., Friedrich, K., Zhang, M. Q., & Rong, M. Z. (2008). Modelling of mechanical properties of nanoparticle-filled polyethylene. International Journal of Polymeric Materials and Polymeric Biomaterials, 57(1), 81100. http://dx.doi.org/10.1080/00914030701337232. 5. Han, Z., & Fina, A. (2011). Thermal conductivity of carbon nanotubes and their polymer nanocomposites: a review. Progress in Polymer Science, 36(7), 914-944. http://dx.doi. org/10.1016/j.progpolymsci.2010.11.004. 6. Kim, H., Miura, Y., & Macosko, C. W. (2010). Graphene/ polyurethane nanocomposites for improved gas barrier and electrical conductivity. Chemistry of Materials, 22(11), 34413450. http://dx.doi.org/10.1021/cm100477v. 7. Arul Selvan, S. G., Rajasekar, R., Kalidass, M., & Selwin, M. (2017). Vibration and ultrasonic welding behaviour of polymers and polymer composites: a review. Journal of Chemical and Pharmaceutical Sciences, 2017(3), 55-61. Retrieved in 2019, July 11, from https://www.jchps.com/specialissues/2017%20 Special%20Issue%203/MKCE_MECH%2012.pdf 8. Lin, L., & Schlarb, A. K. (2015). Vibration welding of polypropylene-based nanocomposites – The crucial stage for the weld quality. Composites Part B, Engineering, 68, 193-199. http://dx.doi.org/10.1016/j.compositesb.2014.08.052. 9. Flowers, S., Thomas, J., Mokhtarzadeh, A., & Benatar, A. (2006). Study of ultrasonic welding of hdpe-based nanoclay composites. In ANTEC 2006 Plastics: Annual Technical Conference Proceedings (pp. 2189-2193). Charlotte: Society of Plastics Engineers.

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10. Benatar, A., & Gutowski, T. G. (1989). Ultrasonic welding of PEEK graphite APC-2 composites. Polymer Engineering and Science, 29(23), 1705-1721. http://dx.doi.org/10.1002/ pen.760292313. 11. SpectraLAB. (1998). FFT Spectral Analysis System. Version 4.32.14. Sound Technology. 12. Lyashko, F. E., & Sokolova, O. F. Method of ultrasound welding thermoplastic. Patent 2220917 kl. B 29 C 65/08. Russia. 13. Raza, S. F. (2015). Ultrasonic welding of thermoplastics (Doctoral thesis). The University of Sheffield, United Kingdom. 14. Khmelev, V. N., Slivin, A. N., & Abramov, A. D. (2017). Model of process and calculation of energy for a heat generation of a welded joint at ultrasonic welding polymeric thermoplastic materials. In 8th Siberian Russian Workshop and Tutorial on Electron Devices and Materials (pp. 316-322). Erlagol: IEEE. http://dx.doi.org/10.1109/SIBEDM.2007.4292995 15. Grewe, M. G., Gururaja, T. R., Shrout, T. R., & Newnham, R. E. (1990). Acoustic properties of particle/polymer composites for ultrasonic transducer backing applications. IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control, 37(6), 506-514. http://dx.doi.org/10.1109/58.63106. PMid:18285071. 16. Nargund, S. (2016). Evaluation of stress wave attenuation in a polymer matrix composite using finite element analysis technique. In ASME 2016 International Mechanical Engineering Congress and Exposition (pp. 1-9, Paper No: IMECE2016-67055, V010T13A018). Phoenix: ASME. http://dx.doi.org/10.1115/ IMECE2016-67055 17. Garcia, M., Vliet, G. V., Jain, S., Zyl, W. E. V., & Boukamp, B. (2004). Polypropylene/SiO2 nanocomposites with improved mechanical properties. Reviews on Advanced Materials Science, 6(2), 169-175. Retrieved in 2019, July 11, from https://research. tue.nl/en/publications/polypropylenesio2-nanocompositeswith-improved-mechanical-propert 18. Zhou, T. H., Ruan, W. H., Mai, Y. L., Rong, M. Z., & Zhang, M. Q. (2008). Performance improvement of nano-silicapolypropylene composites through in-situ cross-linking approach. Composites Science and Technology, 68(14), 28582863. http://dx.doi.org/10.1016/j.compscitech.2007.10.002. 19. Pflug, G., Gladitz, M., & Reinemann, S. (2009). Wärme besser leiten. Kunststoffe, 12, 54-60. Retrieved in 2019, July 11, from https://www.kunststoffe.de/_storage/asset/538981/ storage/master/file/5806046/download/W%C3%A4rme%20 besser%20leiten.pdf 20. Rosato, D. V. (1990). Plastics processing data handbook. New York: Van Nostrand Reinhold. http://dx.doi.org/10.1007/97894-010-9658-4. 21. Fitzgibbon, W. E., & Wheeler, M. F. (1992). Wave propagation and inversion. Philadelphia: SIAM. 22. Benatar, A., & Cheng, Z. (1989). Ultrasonic welding of thermoplastics in the far-field. Polymer Engineering and Science, 29(23), 1699-1704. http://dx.doi.org/10.1002/pen.760292312. Received: July 11, 2019 Revised: Jan. 16, 2020 Accepted: Feb. 19, 2020

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ISSN 1678-5169 (Online)

https://doi.org/10.1590/0104-1428.08118

Design of chitosan-alginate core-shell nanoparticules loaded with anacardic acid and cardol for drug delivery João Campos Paiva Filho1, Selene Maia de Morais1, Antonio Carlos Nogueira Sobrinho1, Gessica Soares Cavalcante1, Nilvan Alves da Silva2 and Flávia Oliveira Monteiro da Silva Abreu3*  Laboratório de Química de Produtos Naturais, Programa de Pós-graduação em Biotecnologia – RENORBIO, Universidade Estadual do Ceará – UECE, Itaperi Campus, Fortaleza, CE, Brasil 2 Laboratório de Quimica Analítica e Ambiental, Universidade Estadual do Ceará – UECE, Itaperi Campus, Fortaleza, CE, Brasil 3 Laboratório de Quimica Analítica e Ambiental, Programa de Pós-graduação em Ciências Naturais – PPGCN, Universidade Estadual do Ceará – UECE, Itaperi Campus, Fortaleza, CE, Brasil

1

*flavia.monteiro@uece.br

Abstract Anacardic Acid (AA) and Cardol (CD) are the main constituents of the cashew nut shell liquid, which presented several biological activities. In this study, a 23 factorial experimental design was employed in order to evaluate the influence of the reaction conditions in the nanoencapsulation of AA and CD using Chitosan (CH), Alginate (ALG) and Arabic Gum matrices. The nanoparticles (NPs) with higher stability and encapsulation efficiency were those with ALG as an outer coating and with lower content of surfactant. The NPs presented nanometric size with 90% of the distribution ranging from 70 to 250 nm. The in vitro kinetics revealed that CH-ALG/AA and CH-ALG/CD NPs followed zero-order kinetics model, showing a significantly slow release rate, with values of 33% and 63%, respectively, after 240h. Particularly, CH-ALG/CD NPs presented higher inhibitory capacity for all strains of dermatophytes due to their release rate, with promising results for antimicrobial control. Keywords: anacardic acid, cardol, drug delivery, chitosan, nanomaterials. How to cite: Paiva Filho, J. C., Morais, S. M., Sobrinho, A. C. N., Cavalcante, G. S., Silva, N. A., & Abreu, F. O. M. S. (2019). Design of chitosan-alginate core-shell nanoparticules loaded with anacardic acid and cardol for drug delivery. Polímeros: Ciência e Tecnologia, 29(4), e2019060. https://doi.org/10.1590/0104-1428.08118

1. Introduction ‘Anacardic acid and cardol, which are major constituents of the natural cashew nut shell liquid (CNSL), are part of a group of phenolic lipids that has anticancer and antimicrobial activity, among several other activities[1-3]. Anacardic acid is a mixture of 6-alkyl salicylic acids and cardol, which is a mixture of alkyl-resorcinols with variations in the unsaturation of their side chain[4]. Muroi and Kubo[5] reported antimicrobial activities against Staphylococcus aureus, Streptococus mutans and Helicobacter pylori, also acting in mechanisms that promote the physical rupture of bacterial cell membrane due to their surfactant action[6,7]. Anacardic acid displays antimicrobial and anthelmintic uses among others and can be used in large scale; however, it presents a certain instability related to the decarboxylation process, which happens when it is heated. In order to avoid this undesirable reaction, this active compound could be encapsulated using polymer matrixes which could preserve their physical-chemical characteristics. Biopolymers such chitosan and alginate are desirable for encapsulation and has been used for this purpose in the past 20 years[8,9]. According to Peniche and Arguelles-Monal[9] the ability of alginate and chitosan to form polyelectrolyte complexes allows the development of biomaterials, generating tridimensional

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matrices known as cross-linked gel. Chitosan (CH), a polycation, and alginate, a polyanion, are some examples of polysaccharides used in the preparation of nanoparticles (NPs)[9]. In this work, polysaccharide nanoparticles based on chitosan, alginate and arabic gum were prepared with anacardic acid and cardol. A 23 experiment design was applied in order to optimize the encapsulation efficiency (EE) of the nanoparticles, in vitro release profile and antimicrobial activity was also tested, aiming the production of devices with prolonged antimicrobial action.

2. Materials and Methods 2.1 Materials The polymers Chitosan (CH) (Polymar, 72.3% DD), Alginate (ALG, Dynamic), and Arabic gum (AG, Dynamic) were used. Sodium tripolyphosphate (TPP) (Dynamic) and Tween 80 (Dynamic) were also used. Natural CNSL was obtained by maceration of cashew nuts with hexane for one week at room temperature (28 °C). Anacardic acid (AA) and Cardol (CD) were isolated from the natural CNSL by precipitation of AA calcium salt and separation of cardol by solvents extraction[10].

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O O O O O O O O O O O O O O O O


Paiva Filho, J. C., Morais, S. M., Sobrinho Nogueira, A. C., Cavalcante, G. S., Silva, N. A., & Abreu, F. O. M. S. 2.2 Nanoparticles preparation The NPs were prepared according to the following methodology[11]: A solution containing 50 mL of 1% Chitosan and 100 uL of Tween 80 (Sigma-Aldrich) were subjected to mechanical stirring. Subsequently, AA or CD was added to the solution and submitted to the ultrasonic bath (Ultra 800, Ciencor Scientific Ltda, São Paulo, Brazil) for 15 min, using Tween 80:AA with a proportion of 1:3 or 1:1. Subsequently, it was added 0.1% TPP dropwise, using a mass ratio of chitosan:TPP (50:1) and the solution was subjected to magnetic stirring for 30 min. Subsequently 1% (w/v) ALG or AG gum was added in the solution using a mass ratio chitosan:ALG of 10: 1 ratio and the solution was subjected to magnetic stirring for another 30 min. At last, the solution was centrifuged (Kasvi K14-4000, Curitiba, Brazil) at 4000 rpm for 20 min. The NPs were frozen and lyophilized (L101, Liobras, Brazil).

2.3 Characterization of NPs The reaction yield of NPs production was determined by the ratio of the freeze-dried mass of the NPs regarding the total mass of reagents added. The concentration of compound in the NPs was determined by UV-Vis spectroscopy (Thermo Scientific – Genesys 102S) according to the following procedure: 5 mg of each sample were placed in 5.0mL of ethyl alcohol for 48 hours in a hermetic vial. The drug loading was calculated using calibration curves of anacardic acid and cardol at 312 nm and 275 nm, respectively, as indicated in Equation 1 and Equation 2: y = 0.0093x – 0.0209 y = 0.0008x – 0.0225

R 2 = 0.9999 R 2 = 0.9989

(1) (2)

where: y is the measured absorbance and x is the concentration of the compound in mg/L. The physical-chemical characterization was performed for the optimal formulations of NPs with each encapsulated product, chosen through ANOVA statistical analysis using p<0.05 due to their more satisfactory yield and EE results. Size distribution and Zeta Potential of the NPs were determined in a Nano Zeta Sizer (Malvern 3600). 5mg of the NPs were placed in 20ml of deionized water under magnetic stirring for 24h. Aliquots of 4 ml were withdraw and placed in the cubit for analyses at pH 6,0 at a temperature of 25 °C in collaboration with the Biomass Technology Laboratory located at the Brazilian Agricultural Research Corporation (Embrapa-CE). The NPs were characterized by FTIR using a Shimadzu Prestige-21 spectrophotometer, where the samples were prepared as potassium bromide pellets (KBr) in the ratio of 1:20 (m/m) (sample: KBr). The procedure was carried out at the Analytical Center of the Federal University of Rio Grande do Sul (UFRGS).

2.4 In Vitro release kinetics The release kinetics of the AA-loaded and CD-loaded NPs were obtained using a dialysis system. For each sample, 60 mg NPs were introduced into cellulose acetate membranes (14 KDa pore) within a beaker containing 68 mL of HCl solution at pH 3. The releasing system was 2/10

kept under magnetic stirring and constant temperature at 25 °C for 72 h. Three aliquots were taken at regular time intervals and analyzed by spectrophotometry in the Genesys 10S UV–vis (Thermo Scientific). The concentration of the AA and CD present in the medium were determined using Equation 1 and Equation 2. Kinetic mathematical modeling was applied to verify the mechanism of drug release for the studied formulations. The points obtained from the in vitro release curves were linearized according to the Order-zero, First-order, Higuchi and Hixson-Crowell kinetic models[12]. To further elucidate the mechanism of drug release, kinetics based on the Korsmeyer-Peppas model was also evaluated by applying the Equation 3: Mt = K tn M∞

(3)

Where: Mt is the amount of drug released over time, M∞ is the amount of drug released as time approaches infinity, Mt / M∞ represents the fractional release of the drug, t is the release time, and K is the release rate of the system, in which it incorporates structural and geometric characteristics of the polymer system under study. The diffusion coefficient n denotes the characteristic of the kinetic mechanism of the drug according to the assumed value. It was calculated the kinetic constants for each model, as well as the exponent n based on Equation 3. The correlation coefficient (R2) was used as an indication of the best fit for each release system.

2.5 Antimicrobial activity assay The minimum inhibitory concentration (MIC) for Candida spp was determined in accordance with the Clinical and Laboratory Standards Institute[13]. The broth microdilution assay for T. rubrum was performed as described on the literature[14,15]. The strains were obtained from the fungal collection of the Microbiology Laboratory of the State University Vale do Acaraú, the URM culture collection of the Mycology Department from Federal University of Pernambuco, and Hospital Santa Casa de Misericórdia de Sobral. Four strains of T. rubrum were tested, two of Candida albicans, and two of Candida tropicalis were included in this study. For the broth microdilution method, standardized inocula (2.5-5 x 103 CFU mL-1 for Candida spp. and 5.0 x 104 CFU mL-1 for T. rubrum) were prepared by turbidimetry. The suspensions were diluted to 1:2000 for Candida spp. and 1:500 for T. rubrum, both with RPMI 1640 medium (Roswell Park Memorial Institute – 1640) with l-glutamine without sodium bicarbonate (Sigma Chemical Co.). They were buffered to pH 7.0 with 0.165 M MOPS (Sigma Chemical Co.), to obtain inocula of 2.5-5 x 103 CFU mL-1 and 5.0 x 104 CFU mL-1, respectively. The minimum fungicidal concentrations (MFC) for both Candida spp. and T. rubrum were determined as follows: the samples were prepared in 4% DMSO. Amphotericin B (AMB) and ketoconazole (KTC) (Sigma, Chemical Co., USA) were prepared in distilled water. For the susceptibility analysis, the samples were tested in concentrations ranging from 0.002 to 2.5 mg/mL. The microdilution assay was performed in 96-well microdilution plates and the microplates were Polímeros, 29(4), e2019060, 2019


Design of chitosan-alginate core-shell nanoparticules loaded with anacardic acid and cardol for drug delivery incubated at 37 °C and read visually after 2 days for Candida spp. and 5 days for T. rubrum. The MFCs were determined as the lowest concentration resulting in no growth on the subculture after 2 days for Candida spp. and 5 days for T. rubrum[16]. All experiments were performed in triplicate.

2.6 Experimental design A factorial experimental design was applied in two levels in order to investigate the effect of three variables on the encapsulation process in the NPs. The studied independent variables were the coating Polymer Matrix (Mat), surfactant dosage (Sd) and type of drug, i.e., Anacardic acid AA and Cardol CD. The dependent variables (responses) were the reaction yield and the drug loading in the NPs. Eight experiments were performed for each dependent variable, grouped into blocks of 2 randomly selected experiments to minimize nuisance effects. The planning matrix containing A, B and C independent variables is shown in Table 1. The statistical treatment of the data was performed through ANOVA analysis in the Excel program (Microsoft 2010). A linear regression analysis was performed among the variables, finding an equation and a R2 value to point out the significance of reaction parameters, (p) > 0.05.

The factorial design and statistical analysis were conducted in order to select NPs with higher drug content and optimized yield, and then those were further investigated regarding in vitro release analysis, as well as characterized by FTIR spectroscopy, particle size, zeta potential and thermogravimetric analysis.

3.1 Influence of the reaction variables on the yield and drug loading The effect of the Polymer Matrix (A), Surfactant dosage (B) and type of drug (C), on the yield and drug loading were evaluated. Table 1 shows the dependent and independent variables. The results showed considerable variation in the reaction yield, with average values of 89% and 74% for NPs with AA and CD, respectively. Drug loading also showed a distinct pattern for each drug entrapped, with average values of 26% and 6% for NPs loaded with AA and CD, respectively. ANOVA and linear regression analysis identified that the factor C was the only factor that influenced on yield, verified by significance analysis. After linear regression, Equation 4 predicts the effect of the variables on yield: Y1 = 81.25 – 7.5 C

3. Results and Discussions NPs were produced based on CH and ALG polysaccharides with a neutral surfactant, in order to provide better solubilization of the compounds in aqueous medium under different reaction conditions. NPs preparation was performed using two distinguished stages. Initially, it was formed a central nucleus with CH and TPP, using few ionic crosslinking points between the CH amino groups with the TPP phosphate groups in the presence of surfactant and the active compound. A pre-gel is formed, evidenced by a slight clouding in the solution. After that, an anionic polymer was added into the system. This polymer entangles with the pre-nucleus using strong ionic electrostatic forces as well intermolecular forces, coating the inner core by polyelectrolyte complexation (PEC) [11] . In this study, the proposed structure attempts to entrap higher amount of AA or CD within the particles and provide a greater protection of these compounds against external environmental factors. Figure 1 shows the representation of the hypothetical structure for the CH-ALG-CD and CH-ALG-AA NPs.

R 2 = 0.70

(4)

The type of drug entrapped was responsible for 70% of the yield variation in the experiment, where AA presented a yield higher than CD. The average yield is the intersection

Figure 1. Hypothetical proposed structure of NPs CH-ALG loaded with Cardol (CD) or anacardic acid (AA). (TPP: Tripolyphosphate).

Table 1. 23 Factorial design with its drug loading and yield of the material. Nº 1 2 3 4 5 6 7 8 9

NP code CH-ALG/1sd/AA CH-AG/1sd/AA CH-ALG/3sd/AA CH-AG/1sd/AA CH-ALG/1sd/CD CH-AG/1sd/CD CH-ALG/3sd/CD CH-AG/1sd/CD CH-ALG/1.3sd/CD

Independent variables Matrix (A)1 Surfactant (B)2 -1 -1 1 -1 -1 +1 1 +1 -1 -1 1 -1 -1 +1 -1 +1 -1 0

Drug Type (C)3 -1 -1 -1 -1 +1 +1 +1 +1 +1

Drug loading

Yield (%)

42.6 (2.3) 38.9 (5.7) 12.6 (0.7) 9.9 (0.1) 11.6 (0.4) 1.5 (0.3) 5.6 (0.4) 5.7 (0.4) 22.3 (0.5)

92 89 85 89 84 66 72 73 82

Factor A: Coating Polymer Matrix (Mat), High level (+) = Alginate (ALG); low level (-) = Arabic Gum; 2Factor B: Surfactant dosage (Sd), High level (+) = 1:1 and low level (-) = 1:3; 3Factor C: Type of Drug, High level (+) = Cardol and low level (-) = Anacardic Acid. 1

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Paiva Filho, J. C., Morais, S. M., Sobrinho Nogueira, A. C., Cavalcante, G. S., Silva, N. A., & Abreu, F. O. M. S. value, (81.25). Higher yield was obtained when C = -1, reaching a theoretical yield of 88.75, close to the values found in reaction number 1 and 2 (NP CH-ALG/1sd/AA and NP CH-AG/1sd/AA). AA and CD are compounds, which were stabilized in the particle by the surfactant added in the system. However, in an acid medium, AA presents a carboxyl group in the structure, which may interact additionally by ionic forces with available amino groups of CH, becoming even more stable within the particle, and increasing the yield. Regarding drug loading, the factors B, C and the interaction effect BC were significant verified by ANOVA using p < 0.05. These factors correspond to the effect of surfactant dosage (B), type of drug (C) and to an interaction effect between surfactant dosage and type of drug (BC). Linear Equation 5 predicts the effect of the variables on the drug loading with 96% of correlation: Y2 = 16.1 - 7.6 B - 10 C + 7.2 BC

R 2 = 0,96

(5)

Replacing the variables B, C and BC in Equation 5 with normalized values (-1, +1), an optimization was achieved with the following combination of values for B, C and BC respectively: -1, -1 and +1, where the optimal drug loading (Y2) value is 40.8%. This combination matches lower surfactant dosage for anacardic acid (AA) and better interaction among the variables. This theoretical value is also close to the values found in reactions number 1 and 2 (NP CH-ALG/1sd/AA and (NP CH-AG/1sd/AA)). The drug type is the main factor that directly influences the formed NPs. At a lower level (C = -1), there was a significant increase on yield and drug loading, regarding the use of AA. In this sense, it is possible to conjecture that due to the acid carboxylic group present only in the chemical structure of AA, strong ionic interactions take place with the

amino chitosan groups, in an acid medium, which favors the entrapment into the nanoparticle system. There is also an effect of surfactant and an interaction effect of the surfactant and drug type. When the surfactant dosage is lower (B = -1), regarding the Sd:CH 1:3 ratio, in combination with AA as the drug type (C = -1), best results are obtained (Reaction number 1 and Reaction number 2, see Table 1). This is likely due to these ionic interactions between chitosan and AA, which favored the retention of the drug inside the particle dispensing the use of an excess of surfactant to stabilize the particle. For CD drug (C= +1), the surfactant dosage also has to be used at a lower level (1%, B= -1); however, the optimum theoretical maximum is lower due to negative interaction effect of CD with the surfactant. It seems that the optimum dosage of surfactant to stabilize cardol could be intermediate between the levels studied for this type of drug. Based on these results, a new reaction was produced (reaction number 9, Table 1), using 1.3% of surfactant dosage, aiming to increase drug loading. Results showed that the drug loading has increased from 11 to 22% maintaining a high yield, improving significantly the encapsulation system. In summary, the reaction conditions chosen after statistical analysis were the ones using low surfactant content (1% for AA and 1.3% for CD). Despite the type of external polymer matrix did not influenced statistically, ALG was chosen as external coating, and the NPs selected were the ones from reaction number 1 (CH/ALG-AA) and reaction number 9 (CH/ALG-CD). Both NPs were evaluated regarding physical‑chemical characterization and antimicrobial tests.

3.2 Infrared spectra Figure 2 shows the structural characterization obtained by FTIR spectroscopy for CH, ALG, AA, CD and NPs loaded with AA and CD. CH showed two strong vibrations

Figure 2. FTIR spectra of the materials Chitosan (CH). sodium alginate (ALG). Cardol (CD). anacardic acid (AA). NPs CH-ALG-CD and NPs CH-ALG-AA. 4/10

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Design of chitosan-alginate core-shell nanoparticules loaded with anacardic acid and cardol for drug delivery at 1654, assigned to amide I, and at 1596 cm-1 assigned to symmetrical and asymmetrical bending of amine and amide II. ALG has carboxymethyl groups in the structure, with an absorption peak at 1744 cm-1, assigned to the carboxymethyl dimer (O=COH…O=COH) intermolecular interaction which is in agreement with previously published results[17]. The anacardic acid exhibits a wide band of axial deformation of the hydroxyl OH bond (3400-2500 cm-1), referring to the symmetrical stretching of this group. It also presents a band at 1650 cm-1 of the C=O stretch of the carboxyl, and also a band at 1584 cm-1 relative to the asymmetrical and symmetrical stretching of the -COO groups. On the other hand, cardol exhibits axial deformation of the hydroxyl OH bond (3400 cm-1)[18]. The NPs showed the axial deformation of OH (3400 cm-1), C-O-C (1034 cm-1) and =C-H of the benzene ring in either cardol or anacardic acid (1610-1550 cm-1). The vibrations of the charged amino groups were observed near 3448 cm-1 after formation of the complex, meaning that the -COO- ions of the ALG bonded with –NH3+ of the CH forming the polyelectrolyte complex and modifying the vibrational modes of the polysaccharides[17,19]. Even with the overlapping of the vibrational modes of the drugs with those of the CH and ALG, it is possible to observe, in the spectrum of NPs with AA, the increase in intensity around 1700 cm-1, and it indicates the presence of the carboxylic acid groups of AA incorporation of the drug incorporated into the NPs.

3.3 Particle size and zeta potential The particle size was evaluated for CH-ALG NPs loaded with AA and CD. Figure 3 shows the particle size profile for the NPs, where the distribution pattern presented some variations according to the type of active encapsulated principle. NPs loaded with AA presented an average particle size of 220 nm, where 90% of the presented values are ranging from 90 to 250nm, with a unimodal distribution. NPs loaded with CD presented a discrete bimodal profile, where 95% of the NPs presented a size from 70 to 200 nm, and 5% of the NPs presented higher particle size, with values from 250nm to 530nm. Both systems presented a high fraction of particles below 250 nm, which are favorable applications

for use in in vivo release systems[20-22]. According to the literature, particles with this particle size are susceptible to a higher rate of mobility in controlled release system applications compared to larger materials[21,22]. The SEM micrographs of ALG-CHI hydrogels loaded with AA and CD are shown in supplementary information[23-26] (Figure S1). The morphology showed micro spheroid-shaped clusters, formed by the agglomeration of several spherical NPs. This morphology is in according to the literature[27] where aggregation of spherical NPs often is observed as a result of freeze-drying process. NPs loaded with CD (Figure S1 b) presented higher particle size than those loaded with AA (Figure S1 a). The Zeta Potential was investigated for the NPs in aqueous suspension at pH 6.0. NPs presented negative values of surface potential, respectively of –18.8 and –9.8 mV for those loaded with AA and CD. The NPs loaded with AA presented a negative charge density higher than those loaded with CD, being comparatively more stable and less predisposed to agglomerate, as observed in SEM micrographs. The negative zeta potential values detected are attributed to the ion charge (-COO) of the alginate on the outer surface of the particle and indicate that the CH-based core was successfully coated[28,29]. Therefore, NPs CH-ALG assumed moderate values of potential, with over 90% of the fraction with size lower than 250 nm (Figure 3a and 3b), with good probability to disperse successfully in future in vivo applications.

3.4 In Vitro release profiles The in vitro release profile of the CH-ALG NPs, performed at pH 3.0 to simulate the stomach acid condition, is displayed in Figure 4. It evidenced lower release rates in the first 48 hours, with corresponding release values of 10% and 6% for CD and AA, and even after 96h, NPs release was increased to 19% and 7% for CD and AA, respectively. After 240h, CH-ALG-AA NPs released only 33% of AA, showing a strong interaction to the surfactant-CH-ALG system, which prevented its releasing. In acid medium, the carboxyl groups present in the AA acid formed strong ionic and hydrogen intermolecular interactions with amine CH protonated groups, and the hydrocarbon chain portion of the AA were

Figure 3. Distribution of particle size based on volume for (a) CH-ALG-AA NPs and (b) CH-ALG-CD NPs. Polímeros, 29(4), e2019060, 2019

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Paiva Filho, J. C., Morais, S. M., Sobrinho Nogueira, A. C., Cavalcante, G. S., Silva, N. A., & Abreu, F. O. M. S. successfully stabilized by the surfactant. In this case, AA was retained inside the NP in acid medium for a prolonged time (over 10 days). On the other hand, CH-ALG-CD NPs presented a satisfactory controlled release profile. Table 2 shows the kinetic constant and the correlation coefficient for the studied Kinetic models. A kinetic study based on the conventional release models[12] from the in vitro release of AA and CD revealed that both systems were best fitted in the zero-order kinetic model, due to their higher correlation coefficient (R2). This model is based on the slow release of active substances from systems that gradually disintegrate in the dissolution medium[30]. In this case, the abrupt increase in the release profile after 180 h is attributed to the slowly dissolution of the NP particle, where the active drug was dispersed in the aqueous system. These NPs are intended to serve active substances in a controlled and prolonged way, as is the case with some of the transdermal systems, such as matrix tablets, coated forms, osmotic systems, drugs with low solubility, among others[29]. In our study, the release occurred only after a considerably prolonged period (240 h), and there was a maximum release of approximately 63% CD and 39% AA, in accordance with zero-order kinetic model. In this system, NPs were produced using CH and ALG as coatings; a possible explanation for this behavior can be associated to a two-step mechanism: initially, occurs the migration of the active principle by diffusion from the inner core to the interface of CH-ALG coating, with possible accumulation of a retention volume[29]. A concentration gradient is produced and the release rate is slow and controlled due to the affinity of AA and CD by the CH-ALG interfaces. Over time, is reached a saturation

concentration in the interface, followed by the rupture of CH-ALG barrier, causing a sudden increase in the release medium[26]. A similar pattern is found in the literature[31]. Alginatecalcium chloride microparticles loaded with eucalyptus oil presented a slow release rate, with 60% of eucalyptus oil release after 97h. Such a sustained released improve the product shelf life due to the protective effect of the nanoparticles. The Korsmeyer-Peppas model provides a constant (KKP), regarding the release rate, where CD presented a release rate (KKP) higher than AA, with values of respectively 4.15 and 2.87 h-1. Both systems presented diffusion coefficients outside the limits of the Korsmeyer-Peppas model (n ˂ 0.5). Reports in the literature suggest that a classical Fickian diffusion or “Less Fickian” behavior is characterized when the values of the diffusion coefficient are below the proposed limit[32,33]. A similar pattern was also found in other studies, where values below 0.5 were reported in Lippia sidoides release from ALG and Cashew gum NPs[34] and also ibuprofen release from polysaccharide matrices based on hydroxypropyl meticellulose[35].

3.5 Antimicrobial activity The encapsulation of natural products with previously characterized biological action is important in order to improve physical-chemical conditions and bioavailability, optimizing a controlled release. Alginate and chitosan are among the most widely polysaccharides in synthesis of nanoparticles. As they were prepared in aqueous medium

Figure 4. Controlled in vitro release of Nanoparticles with anacardic acid and cardol. Table 2. Kinetic constant (K) and the correlation coefficient (R2) for the Kinetic models studied: zero order, first-order, Higuchi, HixsonCrowell and Korsmeyer-Peppas NPs CH-ALG-AA CH-ALG-CD

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Zero-order

First-order K1(h-1)

R2

K0(h-1)

R2

0.9441 0.9786

0.0560 0.1392

0.9389 0.9641

0.0007 0.0007

R2

Higuchi KH(h-1/2)

0.9035 0.8950

0.7594 1.8879

Hixson-Crowell R2 KHC(h-1/3) 0.9408 0.9657

0.0009 0.0024

Korsmeyer-Peppas R2 KKP(h-n) n 0.9081 0.8195

2.8734 4.1514

0.2563 0.3194

Polímeros, 29(4), e2019060, 2019


Design of chitosan-alginate core-shell nanoparticules loaded with anacardic acid and cardol for drug delivery Table 3. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Strains C. albicans LABMIC 0105 C. albicans LABMIC 0407 C. tropicalis LABMIC 0110 C. tropicalis LABMIC 0111 T. rubrum LABMIC 5906 T. rubrum LABMIC 6205 T. rubrum LABMIC 6213 T. rubrum LABMIC 6753

Minimum Inhibitory Concentration/Minimum Fungicidal Concentration (mg/mL) AA NP/AA CD NP/CD AMB1 1.25/2.5 NI NI NI 0.125 NI NI NI NI 0.125 1.25/2.5 NI NI NI 0.125 2.5/2.5 2.5/2.5 2.5/2.5 2.5/2.5 0.125 0.075/0.039 0.625/0.312 0.625/0.312 0.625/0.312 NT 0.075/0.039 0.625/0.312 0.625/0.312 0.625/0.312 NT 0.075/0.039 0.625/0.312 0.625/0.312 0.625/0.312 NT 0.075/0.039 0.625/0.312 1.25/0.625 0.625/0.312 NT

KTC2 NT NT NT NT 0.25 0.25 0.25 0.25

AA: Anacardic Acid; NP/AA: Nanoparticles CH-ALG-AA; CD: Cardol ; NP/CD: Nanoparticles CH-ALG-CD; NI: no inhibition; NT: not tested; 1AMB: standard Amphotericin B; 2KTC: standard Ketoconaloze.

without the presence of harmful substances, it is considered a safe method, without the presence of harmful substances[36]. Regarding the in vitro antifungal activity of AA and CD in its free and encapsulated forms, the results are summarized in Table 3. The compounds showed no significant activity when tested against strains of Candida spp (C. albicans LABMIC 0105, C. albicans LABMIC 0407, C. tropicalis LABMIC 0110 and C. tropicalis LABMIC 0111), except for anacardic acid, which was active in inhibiting the microbial growth of C. albicans LABMIC 0105. However, the results showed MIC and MFC values from 0.625 and 0.312 mg/mL, respectively, for all strains of dermatophytes (T. rubrum LABMIC 5906, T. rubrum LABMIC 6205, T.  rubrum LABMIC 6212 and T. rubrum LABMIC 6753). The fungistatic (MIC) and fungicidal (MFC) action against dermatophyte strains showed that AA and CD, through an action on the fungal cell membrane, possibly inhibited the growth of the microorganisms in a dose-dependent concentration. Dermatophytes such as Trichophyton rubrum with a high affinity for keratinizined tissues are fungi responsible for dermatophytosis of human and veterinary skin infections[37]. The results demonstrate that the encapsulation of AA and CD decreased the MIC and MFC values, expressing the antifungal activity because it requires lower samples concentration to perform the same activity when compared to the non-encapsulated samples. In the antifungal assays, the incubation period of the sample with the dermatophytic fungi is at least 120 hours, and then the controlled release of the encapsulated samples allowed an antimicrobial action by inhibiting growth at lower concentrations than for the free samples. AA and CD are phenolic compounds present in Cashew nut shell liquid with antimicrobial action against Gram‑positive and negative bacteria strains proven by previous studies[1,35]. The antimicrobial action seems to be related to the amphipathic character of the phenolic lipids. The interaction of the hydroxyl groups of the aromatic ring with phospholipids by means of hydrogen bonds and the lipophilic side chain are characteristics responsible for the high affinity of the CNSL to the lipid bilayers present in the bacterial membranes[38]. AA and CD are entrapped within the NPs. However, based on the in vitro release profile, it is expected that CH-ALG-CD NPs present higher antimicrobial efficiency, due to the progressive release of CD over the 120h test. Evidently, both systems presented high content of the retained compound after the test period, Polímeros, 29(4), e2019060, 2019

with a potential effect of microbial inhibition in a period of over 10 days. This structural feature contributes to the greater antimicrobial activity of the encapsulated samples.

4. Conclusions Chitosan-alginate NPs can be used for encapsulation of cardol and anacardic acid using a two stage procedure of gelification followed by external coating. Statistical analysis showed that the characteristics of the NPs are strongly dependent on the surfactant dosage and the type of compound encapsulated. Low surfactant dosage is required to obtain with higher yield and higher loading. The negative zeta potential values suggested that the external coating of the particles was indeed composed of anionic ALG chains. The kinetic study showed a desirable profile, best fitted in zero-order model, probably through a two-step mechanism, caused by accumulation of the active principles in CH‑ALG interface by migration followed by the rupture of the interfacial barrier after saturation, with a steep increase in the release medium. Microbiological studies showed that CH-ALG-CD maintained the same moderate activity of free CD when compared to the standard, showing effectiveness in the encapsulation system.

5. Acknowledgements The authors thank Professor Dr. Men de Sá from Embrapa Agroindustria Tropical for zeta potential and particle size analysis. This work was supported by the Conselho Nacional de Desenvolvimento Científico - CNPq [Projeto Universal 442965/2014-1].

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Design of chitosan-alginate core-shell nanoparticules loaded with anacardic acid and cardol for drug delivery oil in ionically cross-linked alginate microcapsules and its controlled release. Carbohydrate Polymers, 131, 23-3. http:// dx.doi.org/10.1016/j.carbpol.2015.05.054. PMid:26256156. 32. Asare-Addo, K., Levina, M., Rajabi-Siahboomi, A. R., & Nokhodchi, A. (2010). Study of dissolution hydrodynamic conditions versus drug release from hypromellose matrices: the influence of agitation sequence. Colloids and Surfaces. B, Biointerfaces, 81(2), 452-460. http://dx.doi.org/10.1016/j. colsurfb.2010.07.040. PMid:20729043. 33. Wang, J., Wu, W., & Lin, Z. (2008). Kinetics and thermodynamics of the water sorption of 2-hydroxyethyl methacrylate/styrene copolymer hydrogels. Journal of Applied Polymer Science, 109(5), 3018-3023. http://dx.doi.org/10.1002/app.28403. 34. Oliveira, E. F., Paula, H. C. B., & Paula, R. C. M. (2014). Alginate/cashew gum nanoparticles for essential oil encapsulation. Colloids and Surfaces. B, Biointerfaces, 113(1), 146-151. http:// dx.doi.org/10.1016/j.colsurfb.2013.08.038. PMid:24077112. 35. Shoaib, M. H., Tazeen, J., Merchant, H. A., & Yousuf, R. I. (2006). Evaluation of drug release kinetics from ibuprofen matrix tablets using HPMC. Pakistan Journal of Pharmaceutical Sciences, 19(2), 119-124. PMid:16751122.

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36. Bouchara, J. P., Mignon, B., & Chaturvedi, V. (2017). Dermatophytes and dermatophytoses: A thematic overview of state of the art, and the directions for future research and developments. Mycopathologia, 182(1-2), 1-4. http://dx.doi. org/10.1007/s11046-017-0114-z. PMid:28138872. 37. Parasa, L. S., Tumati, S. R., Kumar, L. C. A., Chigurupati, S. P., & Rao, G. S. (2011). In vitro-antimicrobial activity of cashew (Anacardium occidentale, L.) nuts shell liquid against methicillin resistant Stephylococcus aureus (MRSA) clinical isolates. International Journal of Pharmacy and Pharmaceutical Sciences, 3(4), 436-440. Retrieved in 2019, April 1, from https://innovareacademics.in/journal/ijpps/ Vol3Issue4/2724.pdf 38. Kozubek, A., & Tyman, J. H. P. (1999). Resorcinolic lipids, the natural non-isoprenoid phenolic amphiphiles and their biological activity. Chemical Reviews, 99(1), 1-26. http:// dx.doi.org/10.1021/cr970464o. PMid:11848979. Received: Apr. 01, 2019 Revised: Feb. 17, 2020 Accepted: Feb. 19, 2020

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Paiva Filho, J. C., Morais, S. M., Sobrinho Nogueira, A. C., Cavalcante, G. S., Silva, N. A., & Abreu, F. O. M. S.

Supplementary Material Supplementary material accompanies this paper. Figure S1: SEM micrographs of (a) NPs CH-ALG-AA and (b) NPs CH-ALG-CD. Figure S2: Thermograms of (a) AA and (b) NPs CH-ALG-AA; (c) CD and (d) NPs CH-ALG-CD. Green Line: (%)Â weight change as function of the temperature; blue line: Derivative weight change as a function of increasing temperature This material is available as part of the online article from http://www.scielo.br/po

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PolĂ­meros, 29(4), e2019060, 2019


UBE lança ETERNATHANE®, pré-polímeros de poliuretano à base de policarbonato-diol para elastômeros de alto desempenho e durabilidade A UBE é uma indústria multinacional Japonesa que atua nos setores de químicos, máquinas, fármacos, energia e construção. Com escritórios ao redor do mundo e fábricas no Japão, Tailândia e Espanha, há um destaque na produção de caprolactama, poliamidas, fertilizantes e produtos químicos nos. O poliuretano para elastômeros tornou-se cada vez mais soosticado para atender às exigências do mercado atual. Neste contexto, a UBE desenvolveu o ETERNACOLL® e o ETERNATHANE®, uma grande plataforma de soluções que oferecem possibilidades personalizáveis aos materiais de poliuretano, bem como retenção de desempenho superior e a longo prazo, como estabilidade térmica, resistência a óleo, estabilidade hidrolítica, resistência à intempéries e resistência química.

retenção das propriedades mecânicas após exposição a altas temperaturas

redução da absorção de água

retenção das propriedades originais após severa agressão hidrolííca e química

redução da perda de volume quando exposto à abrasão extrema

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Polímeros VOLUME XXIX - Issue IV - Oct./Dec., 2019

October 27-31, 2019 Dall´Onder Grande Hotel Bento Gonçalves, RS, Brazil

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Polímeros: Ciência e Tecnologia 4th. issue, vol. 29, 2019  

The journal Polímeros: Ciência e Tecnologia is a quarterly publication of the Brazilian Polymer Association (Associação Brasileira de Políme...

Polímeros: Ciência e Tecnologia 4th. issue, vol. 29, 2019  

The journal Polímeros: Ciência e Tecnologia is a quarterly publication of the Brazilian Polymer Association (Associação Brasileira de Políme...

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