Polímeros: Ciência e Tecnologia (Polimeros), vol.26, n.4, 2016 by Polímeros: Ciência e Tecnologia (Polimeros)

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http://dx.doi.org/10.1590/0104-1428.2604

Polímeros, 25

years sharing scientific

k n o w l e d g e w i t h i n t h e p o ly m e r c o m m u n i t y

Since its trembling firsts steps 25 years ago Polímeros has passed by many different experiences, which have taught all of us a great deal. Today we can say, without hesitation, that it has matured to a well-recognized journal which has the goal to share the scientific knowledge in Polymer Science and Technology developed by the work of their community members back to the community itself. Following the drive done by the leading world scientific journals towards a greater acceptance and visibility among the scientific community, 2016 was a year in which Polímeros have taken many improvements. Since the beginning of this year we have moved to a new platform to handle the submitted articles. The chosen one was ScholarOne Manuscripts, a web-based, submission and peer review workflow tool for scholarly publishers and societies from Thomson Reuters (www.thomsonreuters.com/ScholarOne). It is one of the leading scientific platforms in the world, and recommended by SciELO, the Scientific Electronic Library Online (www.scielo.org), which holds a selected list of the most important Brazilian scientific journals. If we can say that the scientific part of Polímeros is doing well the same cannot be said to its funding’s. They are getting lower every year and the expectations for 2017 are not favorable at all. Foreseen that the members of the Council Board have taken last year the hard decision to ask a page-charge to the authors of the accepted articles, to help funding the journal. So please, help maintain your journal, budgeting funds to publication when applying to grant your future projects. Opening this issue the president of the Scientific Committee Prof. De Paoli addresses his views to the polymeric community, highlighting the time-consuming and tiring work done by many anonymous people in order to maintain Polímeros as it stands today. Let us all do our part in order to keep Polímeros leading and strong for the next years to come ….

Sebastião V. Canevarolo Editor-in-Chief

Polímeros, 26(4), 2016

E1 1

E E E E E E E E E E E E E E E E E E E E E E E E E

E E E E E E E E E E E E E E E E E E E E E E E E E E E E

P o l í m e r o s - ISSUE 4 - V o l u m e X X V I - O c t / N o v / D e c - 2 0 1 6 - ISSN 0 1 0 4 - 1 4 2 8 - ISSN 1 6 7 8 - 5 1 6 9

( ELECTRONIC V ERSION )

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 Editorial Council

Editorial Committee

Marco-Aurelio De Paoli (UNICAMP/IQ) - President

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

Members

A s s o c i at e E d i t o r s

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) 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) Osvaldo N. Oliveira Jr. (USP/IFSC) Raquel S. Mauler (UFRGS/IQ) Regina Célia R. Nunes (UFRJ/IMA) Richard G. Weiss (GU/DeptChemistry) Rodrigo Lambert Oréfice (UFMG/DEMET) Sebastião V. Canevarolo Jr. (UFSCar/DEMa) Silvio Manrich (UFSCar/DEMa)

Adhemar C. Ruvolo Filho Alain Dufresne Bluma G. Soares César Liberato Petzhold João B. P. Soares José António C. Gomes Covas José Carlos C. S. Pinto Regina Célia R. Nunes Richard G. Weiss Rodrigo Lambert Oréfice

Desktop Publishing

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 2016

Financial support:

Polímeros / Associação Brasileira de Polímeros. vol. 1, nº 1 (1991) -.- São Carlos: ABPol, 1991Available online at: www.scielo.br

Quarterly v. 26, nº 4 (Oct./Nov./Dec. 2016) ISSN 0104-1428 ISSN 1678-5169 (electronic version)

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

1. Polímeros. l. Associação Brasileira de Polímeros. E2

Polímeros, 26(4), 2016

Editorial Section Editorial................................................................................................................................................................................................E1 News....................................................................................................................................................................................................E4 Agenda.................................................................................................................................................................................................E5 Funding Institutions.............................................................................................................................................................................E6

Technical Section Influence of sorbitol on mechanical and physico-chemical properties of soy protein-based bioplastics processed by injection molding Manuel Felix, Valme Carpintero, Alberto Romero and Antonio Guerrero...................................................................................................... 277

Adsorption of BSA (Bovine Serum Albuminum) and lysozyme on poly(vinyl acetate) particles Dirceu Pereira dos Santos, Tito Lívio Moitinho Alves and José Carlos Pinto................................................................................................ 282

Kinetic behavior of the reaction between silica and epoxidized liquid rubber Marcus Vinícius Braum and Marly Antônia Maldaner Jacobi........................................................................................................................ 291

Preparation of novel magnetic polyurethane foam nanocomposites by using core-shell nanoparticles Mir Mohammad Alavi Nikje, Sahebeh Tamaddoni Moghaddam and Maede Noruzian.................................................................................. 297

α-Tocopherol loaded thermosensitive polymer nanoparticles: preparation, in vitro release and antioxidant properties

Cirley Quintero, Ricardo Vera and Leon Dario Perez..................................................................................................................................... 304

Simulation of temperature effect on the structure control of polystyrene obtained by atom-transfer radical polymerization Roniérik Pioli Vieira and Liliane Maria Ferrareso Lona................................................................................................................................ 313

Oat fibers modification by reactive extrusion with alkaline hydrogen peroxide Melina Aparecida Plastina Cardoso, Gizilene Maria Carvalho, Fabio Yamashita, Suzana Mali, Juliana Bonametti Olivato and Maria Victoria Eiras Grossmann................................................................................................................................................................................ 320

Isolation of whiskers from natural sources and their dispersed in a non-aqueous medium Mauro Vestena, Idejan Padilha Gross, Carmen Maria Olivera Muller and Alfredo Tibúrcio Nunes Pires.................................................... 327

Effect of surface finishing on friction and wear of Poly‑Ether-Ether-Ketone (PEEK) under oil lubrication Thiago Fontoura de Andrade, Helio Wiebeck and Amilton Sinatora............................................................................................................... 336

Waterborne hyperbranched alkyd-acrylic resin obtained by miniemulsion polymerization Edwin Murillo and Betty López....................................................................................................................................................................... 343

Avaliação das propriedades da blenda de poli(3-hidroxibutirato)/quitosana após esterilização térmica ou radiolítica Grasielly Souza, Andrelina Santos e Glória Vinhas......................................................................................................................................... 352

Hidrogéis a base de ácido hialurônico e quitosana para engenharia de tecido cartilaginoso Mônica Helena Monteiro do Nascimento e Christiane Bertachini Lombello.................................................................................................. 360

Cover: Typical measurements for superficial roughness of discs on the three test repetitions; (a) turning; (b) grinding; (c) honing and (d) polishing. Optical micrograph of obtained PVAc microparticles. Arts by Editora Cubo.

Polímeros, 26(4), 2016

I I I I I I I I I I I I I I I I I

Braskem takes biobased plastic to the International Space Station

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Green Plastic, which is made from sugarcane, is now being used to fabricate parts in space, thanks to a partnership between Braskem, the largest thermoplastic resin producer in the Americas, and U.S.-based Made In Space, the leading developer of zero gravity 3D printers and an official supplier to NASA. The technology allows astronauts to fabricate tools and spare parts in space using the biobased resin, which effectively increases the autonomy of space missions. The first part made from the raw material outside of Earth was a pipe connector for a vegetable irrigation system, which was fabricated by the Additive Manufacturing Facility (AMF), the first commercial 3D printer permanently allocated in space. The equipment, which will fabricate various types of parts using I’m greenTM plastic, is located on the International Space Station (ISS) and was developed by Made In Space with the support of the Center for the Advancement of Science in Space (CASIS). For over a year, Braskem’s Innovation & Technology team has been working with Made In Space to develop a Green Plastic solution especially for 3D printing in zero gravity. The partnership will enable astronauts to receive by e-mail digital designs of the parts and then print them, which means dramatic savings in terms of time and costs. “Through this partnership, we combined one of the greatest innovations in polymers, Green Plastic, with advanced space technology to print 3D objects in zero gravity. Putting a renewable polymer in space for printing applications represents an important milestone in our history,” said Patrick Teyssonneyre, director of Innovation & Technology at Braskem. Polyethylene made from sugarcane was the material chosen for the project because of its combination of properties, such as flexibility, chemical resistance and recyclability, and also because it is made from a renewable resource. There are great expectations surrounding the project’s benefits, since 3D printing in space was defined by NASA as one of the advances essential for a future mission to Mars. “The ability to print parts and tools in 3D on demand increases the reliability and safety of space missions. This partnership with Braskem is fundamental for diversifying the raw materials used by the AMF and for making this technology more robust and versatile,” said Andrew Rush, CEO of Made In Space. Braskem’s technology is also present in the structure of the actual printer. The equipment’s printing bed is made of Braskem’s ultra-high molecular weight polyethylene (UHMW-PE), which is marketed under the brand UTEC. The resin provides increased tack for printing with Green Polyethylene and offers mechanical properties, such as superior abrasion and impact resistance. The project should drive the development of solutions that go beyond manufacturing in space to create opportunities for innovations in polyolefin applications. Braskem’s innovation team is ready to create, together with its Clients, solutions in Green Plastic and to make them specific for 3D printing. “The technology has E4

the potential to impact the plastics chain by enabling new applications and mass personalization made with a renewable resource,” said Gustavo Sergi, director of Renewable Chemicals at Braskem. Reinforcing the relevance of its environmental aspect, a new Life Cycle Assessment (LCA) of Green Plastic indicated the removal of 2.78 tons of CO2 for each ton of biobased resin produced. The study was conducted by the consulting firm ACV Brasil and subjected to a technical review by a panel formed by the Institute for Energy and Environmental Research GmbH (IFEU) and Michigan State University. Source: Sustainable Life Media Inc.

Research and Markets - Global Polymer Bearing Market Value of USD 12.89 Billion by 2026 - Trends, Technologies & Opportunities Report 2016-2026 Research and Markets has announced the addition of the “Polymer Bearing Market by End-Use Industry, Type of Material, and Region - Global Forecast to 2026” report to their offering. The global polymer bearing market is projected to reach USD 12.89 Billion by 2026, at a CAGR of 4.4% between 2016 and 2026. This growth is mainly attributed to the increasing demand for polymer bearings in the Asia-Pacific region. Increasing applicability of polymer bearings in the automobile, medical & pharmaceutical, textile, food processing, chemical, office products, and semiconductor industries further propels the growth of the global polymer bearing market. Polymer bearings are preferred for use on metal surfaces. Polymers offer several advantages over other common bearing materials. Polymer bearings are corrosion and chemical resistant. These bearings do not transfer heat to other areas of the mechanical assembly, as they are self-lubricating and thus, eliminate the possibility of failure due to lack of maintenance. The phenolics material type segment contributed the largest share to the global polymer bearing market in 2015. This segment is projected to grow at the highest CAGR from 2016 to 2026, owing to the increasing demand for phenolic polymer bearings from varied end-use industries such as automobile, textile, food packaging, and semiconductors. Phenolic polymer bearings also exhibit properties such as excellent strength and shock resistance, coupled with resistance to water, acid, alkali solutions. Phenolic polymeric materials act as self-lubricating materials and replace metal bearings in various applications. Among all end-use industries, the automobile segment accounted for the largest share of the global polymer bearing market in 2015. The automobile end-use industry segment is projected to grow at the highest CAGR from 2016 to 2026. Polymer bearings are widely used in the automobile industry, owing to their properties such as lightweight, low maintenance, lubrication-free, corrosion & chemical resistant, and high wear & fatigue resistant. Source: Newswire Association LLC. Polímeros, 26(4), 2016

May

August

Polymer Foam – 2017 Date: May 2-3, 2017 Location: Pittsburgh - United States Website: www.amiplastics.com/events/event?Code=C804

Annual Meeting on Biopolymers Date: August 02-04, 2017 Location: Melbourne, Australia Website: www.meetingsint.com/chemical-engineeringconferences/biopolymers

PLASTEC New England Date: May 3-4, 2017 Location: Boston - United States Website: plastec-new-england.plasticstoday.com Polymer Sourcing & Distribution - 2017 Date: May 16-17, 2017 Location: Hamburg - Germany Website: www.amiplastics.com/events/event?Code=C801 Plast-Ex Date: May 16-18, 2017 Location: Ontario - Canada Website: plastex.plasticstoday.com 5th International Symposium Frontiers in Polymer Science Date: May 17–19, 2017 Location: Seville - Spain Website: www.frontiersinpolymerscience.com

June Performance Polyamides - 2017 Date: June 6-7, 2017 Location: Cologne - Germany Website: www.amiplastics.com/events/event?Code=C803 PLASTEC East Date: June 13-15, 2017 Location: New York - United States Website: plastec-east.plasticstoday.com Polymers in Cables – 2017 Date: June 20-21, 2017 Location: Pittsburgh - United States Website: www.amiplastics.com/events/event?Code=C814 Additive Manufacturing and Functional Polymeric Materials Conference Date: June 23-26, 2017 Location: Albufeira – Portugal Website: www.zingconferences.com/conferences/additivemanufacturing-and-functional-polymeric-materialsconference-2017 Europe/Africa Polymer Processing Society Conference Date: June 26-29, 2017 Location: Dresden - Germany Website: www.pps2017dresden.de Conductive Plastics - 2017 Date: June 27-28, 2017 Location: Cologne - Germany Website: www.amiplastics.com/events/event?Code=C792

July 5th International Caucasian Symposium on Polymers and Advanced Materials Date: July 2-5, 2017 Location: Tbilisi - Georgia Website: www.icsp.tsu.ge

Plastech 2017 Date: August 22-25, 2017 Location: Caxias do Sul - Brazil Website: www.plastechbrasil.com.br

September 6th World Congress on Biopolymers Date: September 7-9, 2017 Location: Paris - France Website: bioplastics.conferenceseries.com Physical Aspects of Polymer Science Date: September 13, 2017 Location: Swansea - United Kingdom Website: paps17.iopconfs.org 2nd International Conference on Sustainable Bioplastics Date: September 25-27, 2017 Location: Berlin - Germay Website: bioplastics.conferenceseries.com

October Performance Polyamides USA Date: October 3-4, 2017 Location: Pittsburgh - USA Website: www.amiplastics.com/events/event?Code=C824 Polyolefin Additives Date: October 10-12, 2017 Location: Cologne - Germany Website: www.amiplastics.com/events/event?Code=C820 14° Congresso Brasileiro de Polímeros Date: October 22-26, 2017 Location: Águas de Lindóia - Brazil Website: www.cbpol.com.br 7th International Conference and Exhibition on Biopolymers and Bioplastics Date: October 19-21, 2017 Location: San Francisco - USA Website: biopolymers-bioplastics.conferenceseries.com

November Long-Fibre Thermoplastics 2017 Date: November 7-8, 2017 Location: Cologne - Germany Website: www.amiplastics.com/events/event?Code=C822 Plastimagen 2017 Date: November 7-10, 2017 Location: Mexico City - Mexico Website: www.plastimagen.com.mx PLASTEC Minneapolis Date: November 8-9, 2017 Location: Minneapolis - USA Website: plastecminn.plasticstoday.com

3rd Functional Polymeric Materials Date: July 7-10, 2017 Location: Rome - Italy Website: www.fusion-conferences.com/conference66.php

Polímeros, 26(4), 2016 E5

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

ABPol Associates Sponsoring Partners

Institutions UFSCar/ Departamento de Engenharia de Materiais, SP SENAI/ ServiĂ§o Nacional de Aprendizagem Industrial Mario Amato, SP UFRN/ Universidade Federal do Rio Grande do Norte, RN

PolĂmeros, 26(4), 2016

ABPol Associates Collective Members A. Schulman Plásticos do Brasil Ltda. Aditive Plásticos Ltda. Avamplas – Polímeros da Amazônia Ltda. CBE – Grupo Unigel Colorfix Itamaster Indústria de Masterbatches Ltda. Cromex S/A Cytec Comércio de Materiais Compostos e Produtos Químicos do Brasil Ltda. Formax Quimiplan Componentes para Calçados Ltda. Imp. e Export. de Medidores Polimate Ltda. Innova S/A Instituto de Aeronáutica e Espaço/AQI Jaguar Ind. e Com. de Plásticos Ltda Johnson & Johnson do Brasil Ind. Com. Prod. para Saúde Ltda. Master Polymers Ltda. Milliken do Brasil Comércio Ltda. MMS-SP Indústria e Comércio de Plásticos Ltda. Nexo International Ltda. Nitriflex S/A Ind. e Com. Politiplastic Politi-ME. Premix Brasil Resinas Ltda. QP - Químicos e Plásticos Ltda. Radici Plastics Ltda. Replas Comércio de Termoplásticos Ltda. Uniflon - Fluoromasters Polimeros Ind .Com. Imp. Export.Ltda

Polímeros, 26(4), 2016 E7

POLÍMEROS is celebrating its 25th birthday

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“A proposta de se ter uma revista como meio de divulgação dos trabalhos técnicos e científicos na área de polímeros foi o estopim que acendeu a grande ideia de se criar a ABPol” (The proposal to edit a journal to publish scientific and technical works in the area of polymers was the ignition of the fuse that started the great idea of creating ABPol). With those words, the first president of ABPol, Silvio Manrich, began the first editorial of the first issue of Polímeros, published in November 1991. This historic fact tells us that the creation of ABPol was a consequence of the idea of editing a journal and not the opposite. At that time the Brazilian polymers community was small and there were few Brazilian scientific journals, none of them exclusively dedicated to the area of polymers. This small community reacted promptly, submitting their manuscripts to Polímeros, from 6 in the first year to 164 in 2015. In total, 1832 manuscripts have been submitted to Polímeros since 1991 and 1229 were published. Manuscripts also came from abroad; the majority from Colombia and France (22 each), followed by USA (19), Portugal (14) and Germany (13). After 2013 the number of papers published in English increased with a consequent decrease in the number of papers in Portuguese and Spanish. English is the actual official language for submissions and, after 2017, Polimeros will be 100% in English. This encouraged submissions by a large number of scientists from Colombia, China, India, Iran and Turkey. Since 1991, many members of ABPol and of the Direction Board contributed for the shaping and consolidation of this journal. Many took part at the Editorial Committee or Editorial Council in these 25 years. All, with no exception, contributed to our journal. It is also important to remember those members of the ABPol staff, who worked hard to help editing the issues and in its dissemination. They played a paramount role in the whole process. Several steps are involved in publishing a scientific journal. Perhaps the most important is the evaluation and selection of the manuscripts for publication. For this task, the Editorial Committee always had the commitment of the Brazilian scientific community members and of many foreign scientists. In the last years, we also have foreign scientists actively participating to the Editorial Committee and in the revision of submitted manuscripts. Apart from the scientific level, gathering financial support for the journal was also part of the hard work. For a long time, funding from governmental agencies supplied the main costs. Preparing the funding applications, writing reports and account reports, relayed mainly upon individual initiative of the Editorial Committee members. In addition, private companies, who advertised in the covers and internal pages of Polímeros, also played an important financial role. Those who most contributed to Polímeros were: DPUnion, SABIC/GE Plastics South America, Braskem, AXPlásticos and Lanxess. In the last years, there was a gradual reduction of the official financial support and now authors of accepted papers cover part of the costs in the form of a page charge. Another consequence of the financial support depletion was the discontinuation of the printed version in 2015. Following an international tendency, the journal’s internet site publishes the accepted articles, with their DOI numbers. This shortened the publication time, increased the Brazilian and foreign access to the articles and will probably increase the number of citations. Presently, the foreign countries that most access our articles are: Portugal, China, USA, Russia and Colombia. Nowadays, Polímeros is indexed in several international databases and in SciELO. However, the biggest step in the history of Polímeros was, with no doubt, its inclusion at the ISI Web of Knowledge- Web of Science and the evaluation of its impact factor after 2008. After 2015, English became the official language for submission to Polímeros and ScholarOne™ the platform used for the submission process. This motivated an increased number of accesses and submissions from foreign scientists. In the near future, we expect a tendency to increase the submission of high scientific level manuscripts, internet accesses, citations and the impact factor of Polímeros. We congratulate all the polymeric community for these 25 years of dissemination of the polymer science worldwide and wish long life to Polímeros.

Marco Aurelio De Paoli President of the Editorial Council of Polímeros

CMY

Polímeros, 26(4), 2016

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http://dx.doi.org/10.1590/0104-1428.0044

Influence of sorbitol on mechanical and physico-chemical properties of soy protein-based bioplastics processed by injection molding Manuel Felix1*, Valme Carpintero1, Alberto Romero1 and Antonio Guerrero1 Department of Chemical Engineering, Faculty of Chemistry, University of Seville, Seville, Spain

*mfelix@us.es

Abstract Soy Protein Isolate (SPI) has been evaluated as useful candidate for the development of protein-based bioplastic materials processed by injection molding. The influence of sorbitol (SB) as plasticizer in mechanical properties and water uptake capacity was evaluated in SPI-based bioplastics. A mixing rheometer that allows monitoring torque and temperature during mixing and a small-scale-plunger-type injection molding machine were used to obtain SPI/Plasticizer blends and SPI-based bioplastics, respectively. Dynamic measurements were carried out to obtain mechanical spectra of different bioplastics. Moreover, the mechanical characterization was supplemented with uniaxial tensile tests. Additionally, the influence of SB in water uptake capacity was also evaluated. The introduction of SB leads to increase the rigidity of bioplastics as well as the water uptake capacity after 24h, however it involves a decrease in strain at break. Final bioplastics are plastic materials with both adequate properties for the substitution of conventional petroleum plastics and high biodegradability. Keywords: bioplastics, DMTA, plasticizer, sorbitol, soy protein.

1. Introduction The use of petroleum-based materials involves serious environmental damage. Each year, over 300 million tons of petroleum-based or gas-based polymers are produced worldwide for a wide variety of applications in almost all areas of daily life as well as in the process industry[1,2]. However, recently concerns about the decrease in new fossil resources, together with the lack of biodegradability of plastic materials, have encouraged the replacement of conventional oil-based plastics by others based on hydrocarbons derived from renewable resources[3,4]. Proteins are one the most promising renewable source for obtaining bio-based materials. These tend to form three-dimensional macromolecular networks, which are stabilized by hydrogen bonds, hydrophobic interactions, and disulﬁde bonds[5]. The diversity in protein availability, as well as in their assembling, a big amount of biodegradable materials can be obtained, offering a wide range of techno-functional properties[6].

been previously used for the elaboration of crayfish-based bioplastics[10], pea-based bioplastics[11], albumen-based[8] bioplastics or soy-based bioplastics[12], among others. However, glycerol has been used as the only plasticizer in these works and sorbitol has not previously tested. Before injecting the protein into the mould, it is necessary to obtain a protein/plasticizer blends[13]. Proteins themselves do not have sufficient plasticity to be handled, for this reason a plasticiser is required. The plasticiser reduces intermolecular forces and increase polymeric chains mobility[7]. Moreover, the plasticiser reduces the glass transition temperature of the thermoplastic proteins[14]. The most common plasticisers include water and polyols. Some of the most used plasticizer are the glycerol (GL) and the sorbitol (SB). These hydrophilic compounds have been used, among other biopolymers, in starch films[5,13,15] in order to improve their mechanical and barrier properties.

Protein-based bioplastics can be processed by using existing processing technologies, from the physicochemical[7] to thermomechanical methods (compression molding, thermomolding and extrusion)[8]. However, injection molding, which is the most common processing method used with synthetic polymers, has been poorly used for protein-based bioplastic applications[9]. This technique would suffer a remarkable demand if the feasibility of performing protein‑based materials were demonstrated. The use of injection-molding technique to produce protein-based bioplastics enable the manufacture of many kinds of shaped products, which entails new arguments in favor of considering these biodegradable polymers as an alternative to synthetic polymers. This technique has

The overall objective of this work was to evaluate the feasibility of using SB for developing high quality biodegradable soy-protein bio-based plastic materials (bioplastics) processed by injection molding with desirable thermo-mechanical properties and high biodegradability. To achieve this objective, soy protein concentrate (SPI) was processed with GL and SB (dissolved in glycerol or water). The mixing process was monitored using a mixing rheometer that allows the torque and temperature to be recorded during mixing. Mechanical properties of the final bioplastic materials were obtained by means of dynamic measurements (DMTA) and tensile-strength tests. Finally, the water uptake capacity, the loss of soluble matter and the swelling were determined.

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2. Materials and Methods

2.3.2 Tensile-strength measurements

2.1 Materials Soy protein isolate (SPI) was supplied by Protein Technologies International (Leper, Belgium). The protein content was 91.0 ± 0.2 wt. %. Both plasticizers, glycerol (GL) and sorbitol (SB), were purchased from Panreac Química, S.A. (Spain).

Tensile-strength tests were performed by using the Insight 10 kN Electromechanical Testing System (MTS, Eden Prairie, MN, USA), according to by ISO 527-2[17] for Tensile Properties of Plastics. Young’s Modulus (E), maximum stress (σmax) and strain at break (εmax) were evaluated from at least five duplicates for each product using type IV probes and an extensional rate of 1 mm·min−1.

2.2 Sample preparation

2.3.3 Water uptake capacity

Blends with constant protein/plasticizer ratio were manufactured by a thermomechanical procedure which includes two stages: Initially, blends were mixed in a two‑blade counter-rotating batch mixer Haake Polylab QC (ThermoHaake, Germany) at 25 °C and 50 r.p.m. for 10 min., monitoring torque and temperature. The protein/plasticizer ratios selected were 50 wt. % and 50 wt. % concentration for the protein and plasticizer, respectively (denoted as 50/50), and this ratio was kept constant in any case. Initially, the GL was used as the plasticizer of reference. After that, the influence of the SB as a plasticizer was evaluated (maintaining constant protein/plasticizer ratio). One of the most obvious constraints was the use of a solid plasticizer (SB). Initially, SB was mixed directly with the protein powder, however the blend was not suitable for the injection due to its lack of processability. For this reason, the SB was introduced dissolved in either GL or water (W) at the saturation concentration (50 and 70 wt. %, respectively). This SB saturated solution was used as plasticizer, and was introduced in a ratio of 50/50 (SPI/plasticizer). The specific mechanic energy (SME) of mixing (Equation 1) may be defined as follows[16]:

Water uptake capacity of bioplastics were measured according to the standard ASTM D-570[18]. The specimens were subjected to drying (conditioning) in an oven at 50 ± 2 º C for 5-6 hours to determine dry weight, then introduced into distilled water and weighed at 2 and 24 hours of immersion. Finally, it is subjected to drying (reconditioning) again and weighed to determine the loss of soluble material. All the experiments were performed in triplicate at room temperature. Water absorption capacity and loss of soluble material are determined by the following equations:

SME =

w tmix ∫ M ( t ) dt m 0

Secondly, the dough-like materials selected after mixing process were processed by injection molding using a MiniJet Piston Injection Molding System II (ThermoHaake, Germany) to obtain bioplastic specimen, the injection conditions were: cylinder temperature: 40 °C, mold temperature: 70 °C, injection pressure: 500 bar (20s) and post-injection pressure: 200 bar (200s)[14]. Two types of molds were used: a 60×10×1 mm rectangular shape mold for both DMTA experiments and water uptake, and a Dumpbell type probe defined by ISO 527-2[15] for Tensile Properties.

2.3 Characterization of biocomposites 2.3.1 Dynamic Mechanical Temperature Analysis (DMTA) DMTA tests were carried out with a RSA3 (TA Instruments, New Castle, DE, USA), on rectangular specimens using dual cantilever bending. All the experiments were carried out at constant frequency (1 Hz) and strain (between 0.01 and 0.30%, within the linear viscoelastic region). The selected heating rate was 3 °C·min−1 and the temperature interval was from -10 to 75 °C.

Wet Weight − Initial Dry Weight ·100 (2) Initial Dry Weight

Initial Dry weight − Final Dry weight % Loss of ·100 soluble material = Initial Dry weight

(3)

The swelling capacity was carried out as follows: the thickness of the rectangular probes was measured before the water immersion. After 24h, probes were carefully dried and their thickness was again measured. The swelling ratios of the SPI-based bioplastics were obtained by the following equation.

(1)

where w (in rad/s) is the mixing speed, m (in g) is the sample mass, M(t) (in N·m) is the torque and tmix (in s) is the mixing time.

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% Water uptake =

% swelling =

Initial thikcness − Final thikcness ·100 (4) Initial thickness

2.4 Statistical analysis At least three replicates of each measurement were carried out. Statistical analyses were performed using t-test and one-way analysis of variance (ANOVA, p < 0.05). Standard deviations were calculated.

3. Results and Discussions 3.1 Preparation of blends by thermoplastic mixing Figure 1 shows torque and temperature profiles as a function of mixing time for different blends. These results indicate the relevant dependence of torque and temperature on the plasticizer used. Thus, all profiles are characterized by a rapid increase in torque up to reach a maximum value (specially marked for SPI/GL system), followed by a decrease which is asymptotical towards a plateau value for SPI/GL and SPI/W/SB, and is subsequently followed by a moderate increase (reaching a value plateau) in SPI/GL/SB system. Temperature profiles are characterized by an almost constant value around 25 °C for SPI/GL and SPI/W/GL systems and by a moderate increase in the SPI/GL/SB system. The temperature profiles are in accordance with torque profiles, constant Polímeros, 26(4), 277-281, 2016

Influence of sorbitol on mechanical and physico-chemical properties of soy protein-based bioplastics processed by injection molding torque values at middle-time lead to constant temperature profiles. Moreover, the increase in torque is a consequence of the shear-induced crosslinking events which take place over the mixing stage of SPI/GL/SB system. As a consequence of the above-mentioned differences in torque profile, the energy employed for mixing (SME) is also quite different. The values for the SME for these three systems are included in Figure 1. A remarkable increase in this parameter can be observed in the SPI/W/SB system. This effect is related to the increase in torque caused by a structuration of the system, which leads to decrease the processability of blends.

3.2 Mechanical characterization of bioplastics 3.2.1 Dynamic Mechanical Temperature Analysis (DMTA) Figure 2 shows the values of the storage modulus (E’), the loss modulus (E”) (Figure 2A) and the loss tangent, tan δ (Figure 2B), as a function of temperature, obtained from DMTA measurements for different plasticizer. As may be observed in Figure 2A, all the specimens show similar profiles for E’ and for E”, undergoing a

remarkable decrease with increasing temperature. The higher structuration found in SPI/W/SB blends, is also found for probes at this protein/plasticizer composition. This system yields probes with higher viscoelastic modulus (E’ and E”), which indicates the higher protein-network formation during the injection molding stage. In any case, the use of SB as plasticizer seems to favor the protein-crosslinking of the final probes, which is factually the probe containing SB dissolved in water. The higher moduli found can be related to the fact that some of the water is lost during the injection stage, increasing the rigidity of the final probes. All the probes studied display similar loss tangent profiles (Figure 2B), which is characterized by an almost constant increase towards a maximum value. This behavior has been previously related to a glass-like transition[9]. The unimodal profiles indicate a good compatibility, between protein isolate and the different plasticizers, for all the systems after the injection molding process, regardless of the plasticizer used (GL, GL/SB or W/SB). Tan δ values are always lower than 0.5, which indicates the marked solid character of all systems studied, however the increase in temperature leads to increase the tan δ in any case, indicating that the solid character of probes is reduced.

3.2.2 Uniaxial tensile-strength measurements

Figure 1. Evolution of mixing torque and temperature as a function of time for systems SPI/GL, SPI/GL/SB and SPI/W/SB, as well as SME for all the protein-plasticizer studied (table inset).

Figure 3A displays the stress-strain curves obtained from tensile-strength measurements for all systems studied containing different plasticizers. The mechanical responses consist of an initial linear elastic interval, characterized by a constant stress-strain slope, which yields high values for the Young’s Modulus (E), followed by a deformation stage with a continuous decrease in the stress-strain slope. A second constant slope is reached over the plastic deformation stage. Before the end, all the curves reach a maximum value for the stress (σmax), followed by a decrease in σ and the strain at break (εmax) is reached. Probes containing GL seems to lead higher εmax, however probes containing SB have higher initial slope, which denotes higher elastic modulus. In order to carry out a proper comparison of all parameters from stress-strain curves, the values of these parameters (E, σmax and εmax) and their corresponding standard deviations

Figure 2. DMTA temperature ramp measurements, at 1 Hz and 3 °C·min−1, all the studied systems (SPI/GL, SPI/GL/SB and SPI/W/SB): (A) storage modulus (E′) and (B) tan δ. Polímeros, 26(4), 277-281, 2016

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Figure 3. Stress-Strain curves from tensile strength measurements (A) and parameters from tensile strength measurements: Maximum stress (σmax), strain at break (εmax) and Youngs’ Modulus (E) for the different probes studied (SPI/GL, SPI/GL/SB and SPI/W/SB) (B).

are plotted in Figure 3B for all the systems studied. This figure puts forward that the probe in which the GL is the only plasticizer, is the one that exhibits the highest εmax. On the contrary, SB-containing probes exhibit higher Young`s Modulus (elastic response), regardless of the plasticizer in which the SB is dissolved (GL or W). These results evidence the SB decreases the sliding ability of the different protein chains when the SB plays the role of plasticizer. Moreover, SPI-based probes do not experiment a noticeable decrease in σmax when after adding SB an increase in E takes place. Interestingly, this mechanical response is different to that one previously found by Felix et al.[10,19] and Rombouts et al.[20], however are in consonance with the mechanical properties found by Schmid et al.[21] and Tummala et al.[22] In any case, these elastic responses are consistent with the results from DMTA that showed lower elastic modulus (E’) for the system without SB (Figure 2A). These results indicate the feasibility of modulating mechanical properties of bioplastics where mechanical properties obtained after changing the plasticizer used are suitable for different applications.

3.3 Water uptake capacity Figure 4 shows the results from water uptake measurements. As it may be observed, all probes have a remarkable a high water uptake ability, which is related to the abundance of hydrophilic group in the SPI system. In fact, the value reached is much higher than those previously reported for albumen‑based bioplastics or soy[9,12]. The use of SB as plasticizer seems to give raises an increase in water uptake after 24h, which is probably related to the high hydrophilicity of this solid sugar alcohol. As regards the water-soluble loss matter the systems studied show a value of around 40%. These results suggest that the loss of soluble matter corresponds basically to the hydrophilic character of GL and W (liquid plasticizer used), which are easily release into the medium. These results are similar to other previously obtained, and also were attributed to the loss of plasticizer[9,11]. Finally, the swelling ratio is very similar for the systems containing GL, regardless of the presence of SB. However, when the SB is dissolved in W, the measurement of the swelling is impossible, because the probe is very irregular after the immersion in water for 24h. 280

Figure 4. Evolution of water uptake capacity (%) after immersion for 24 h in water, loss of soluble matter (%), and swelling for all the studied systems (SPI/GL, SPI/GL/SB and SPI/W/SB).

4. Conclusions The use of SB as plasticizer gives rise SPI- based bioplastics with different mechanical properties, which can be suitable for their use in different applications. All probes exhibit similar DMTA profiles, which are characterized by a decrease in both moduli (E’ and E”), however, the sorbitol seems to yield probes more rigid (higher values of E’ and E). This higher elastic response is also observed in tensile-strength measurements. In addition, the change of plasticizer allows the manufacturing of probes with different mechanical properties (both the σmax and εmax can be modulated). Results from water uptake capacity reveal a remarkable capacity of these SPI-based probes to absorb water, which is much higher than that one found for other protein-based bioplastics. The use of sorbitol increases this ability, which can contribute to obtain highly-demanded new super-absorbent materials.

5. Acknowledgements This work is part of a research project sponsored by Andalousian Government, (Spain) (project TEP-6134) and by “Ministerio de Economía y Competitividad” from Spanish Government (Ref. CTQ2015-71164-P). Polímeros, 26(4), 277-281, 2016

Influence of sorbitol on mechanical and physico-chemical properties of soy protein-based bioplastics processed by injection molding

6. References 1. DiGregorio, B. E. (2009). Biobased performance bioplastic: mirel. Chemistry & Biology, 16(1), 1-2. PMid:19171300. http:// dx.doi.org/10.1016/j.chembiol.2009.01.001. 2. Rocha, G. O., Farias, M. G., Carvalho, C. W. P., Ascheri, J. L. R., & Galdeano, M. C. (2014). Biodegradable composite films based on cassava starch and soy protein. Polímeros: Ciência e Tecnologia, 24(5), 587-595. http://dx.doi.org/10.1590/01041428.1355. 3. Thiré, R. M. S. M., Simao, R. A., Araújo, P. J. G., Achete, C. A., & Andrade, C. T. (2004). Reduction of hydrophilicity of biodegradable starch-based films by plasma polymerization. Polímeros: Ciência e Tecnologia, 14(1), 57-62. http://dx.doi. org/10.1590/S0104-14282004000100015. 4. Macea, R. B., De Hoyos, C. F., Montes, Y. G., Fuentes, E. M., & Ruiz, J. I. R. (2015). Synthesis and film properties of chitosan and whey. Polímeros: Ciência e Tecnologia, 25(1), 58-69. http://dx.doi.org/10.1590/0104-1428.1558. 5. Winkworth-Smith, C., & Foster, T. J. (2013). General overview of biopolymers: structure, properties, and applications. In S. Thomas, D. Durand, C. Chassenieux & P. Jyotishkumar (Eds.). Handbook of biopolymeric materials. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA. http://dx.doi. org/10.1002/9783527652457.ch2. 6. Verbeek, C. J. R., & van den Berg, L. E. (2010). Extrusion Processing and Properties of Protein-Based Thermoplastics. Macromolecular Materials and Engineering, 295(1), 10-21. http://dx.doi.org/10.1002/mame.200900167. 7. Genadios, A. (2002). Proteins based films and coting. New York: CRC Press. http://dx.doi.org/10.1201/9781420031980. 8. Jerez, A., Partal, P., Martinez, I., Gallegos, C., & Guerrero, A. (2005). Rheology and processing of gluten based bioplastics. Biochemical Engineering Journal, 26(3), 131-138. http://dx.doi. org/10.1016/j.bej.2005.04.010. 9. Felix, M., Martin-Alfonso, J. E., Romero, A., & Guerrero, A. (2014). Development of albumen/soy biobased plastic materials processed by injection molding. Journal of Food Engineering, 125, 7-16. http://dx.doi.org/10.1016/j.jfoodeng.2013.10.018. 10. Felix, M., Romero, A., Cordobes, F., & Guerrero, A. (2015). Development of crayfish bio-based plastic materials processed by small-scale injection moulding. Journal of the Science of Food and Agriculture, 95(4), 679-687. PMid:24909425. http:// dx.doi.org/10.1002/jsfa.6747. 11. Perez, V., Felix, M., Romero, A., & Guerrero, A. (2016). Characterization of pea protein-based bioplastics processed by injection moulding. Food and Bioproducts Processing, 97, 100-108. http://dx.doi.org/10.1016/j.fbp.2015.12.004. 12. Fernández-Espada, L., Bengoechea, C., Cordobés, F., & Guerrero, A. (2016). Protein/glycerol blends and injectionmolded bioplastic matrices: Soybean versus egg albumen. Journal of Applied Polymer Science, 133(6), n/a. http://dx.doi. org/10.1002/app.42980.

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13. Suyatma, N. E., Tighzert, L., Copinet, A., & Coma, V. (2005). Effects of Hydrophilic Plasticizers on Mechanical, Thermal, and Surface Properties of Chitosan Films. Journal of Agricultural and Food Chemistry, 53(10), 3950-3957. PMid:15884822. http://dx.doi.org/10.1021/jf048790+. 14. Irissin-Mangata, J., Bauduin, G., Boutevin, B., & Gontard, N. (2001). New plasticizers for wheat gluten films. European Polymer Journal, 37(8), 1533-1541. http://dx.doi.org/10.1016/ S0014-3057(01)00039-8. 15. Adebiyi, A. P., Adebiyi, A. O., Yamashita, J., Ogawa, T., & Muramoto, K. (2008). Purification and characterization of antioxidative peptides derived from rice bran protein hydrolysates. European Food Research and Technology, 228(4), 553-563. http://dx.doi.org/10.1007/s00217-008-0962-3. 16. Jerez, A., Partal, P., Martinez, I., Gallegos, C., & Guerrero, A. (2007). Protein-based bioplastics: effect of thermo-mechanical processing. Rheologica Acta, 46(5), 711-720. http://dx.doi. org/10.1007/s00397-007-0165-z. 17. International Organization for Standardization – ISO. (2012). ISO 527-2: plastics: determination of tensile properties: part 2: test conditions for moulding and extrusion plastics. Geneva: ISO. Retrieved in 26 May 2016, from http://www. iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail. htm?csnumber=56046 18. American Society for Testing and Materials – ASTM. (2001). ASTM D-571: standard test method for water absorption of plastics. West Conshohocken: ASTM. http://dx.doi.org/10.1520/ A0570_A0570M-98. 19. Felix, M., Romero, A., Martín-Alfonso, J. E., & Guerrero, A. (2015). Development of crayfish protein-PCL biocomposite material processed by injection moulding. Composites. Part B, Engineering, 78, 291-297. http://dx.doi.org/10.1016/j. compositesb.2015.03.057. 20. Rombouts, I., Lagrain, B., Brunnbauer, M., Koehler, P., Brijs, K., & Delcour, J. A. (2011). Identification of Isopeptide Bonds in Heat-Treated Wheat Gluten Peptides. Journal of Agricultural and Food Chemistry, 59(4), 1236-1243. PMid:21235244. http:// dx.doi.org/10.1021/jf103579u. 21. Schmid, M., Müller, K., Sängerlaub, S., Stäbler, A., Starck, V., Ecker, F., & Noller, K. (2014). Mechanical and barrier properties of thermoplastic whey protein isolate/ethylene vinyl acetate blends. Journal of Applied Polymer Science, 131(23), n/a. http://dx.doi.org/10.1002/app.41172. 22. Tummala, P., Liu, W., Drzal, L. T., Mohanty, A. K., & Misra, M. (2006). Influence of Plasticiczers on Thermal and Mechanical Properties and Morphology of Soy-Based Bioplastics. Industrial & Engineering Chemistry Research, 45(22), 7491-7496. http:// dx.doi.org/10.1021/ie060439l. Received: May 26, 2016 Revised: July 21, 2016 Accepted: Aug. 03, 2016

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Adsorption of BSA (Bovine Serum Albuminum) and lysozyme on poly(vinyl acetate) particles Dirceu Pereira dos Santos1, Tito Lívio Moitinho Alves1 and José Carlos Pinto1* 1

Programa de Engenharia Química, Instituto Alberto Luiz Coimbra de Pós-Graduação e Pesquisa em Engenharia – COPPE, Universidade Federal do Rio Janeiro – UFRJ, Rio de Janeiro, RJ, Brazil *pinto@peq.coppe.ufrj.br

Abstract Poly(vinyl acetate) (PVAc) particles find many uses in the biomedical field, including the use as particle embolizers. Particularly, embolizing particles can combine physical and chemical effects when they are doped with pharmaceuticals. For this reason, the adsorption of bovine serum albuminum (BSA) and lysozyme (used as model biomolecules) on PVAc particles produced through suspension polymerization is studied in the present manuscript in a broad range of pH values. It is shown that significant amounts of BSA and lysozyme can be adsorbed onto PVAc particles in the vicinities of the isoelectric point of the biomolecules (0.65mg of BSA and 1.0mg of lysozyme per g of PVAc), allowing for production of chemoembolizers through adsorption. Particularly, it is shown that lysozyme still presents residual activity after the adsorption process, which can constitute very important characteristic for real biomedical applications. Keywords: poly(vinyl acetate), lysozyme, Bovine Serum Albuminum (BSA), suspension polymerization, adsorption.

1. Introduction Different methods have been used to load drugs (or biological active substances) into polymer matrices, including co-precipitation, in-situ incorporation and adsorption[1-15]. In co-precipitation processes, the doped polymer beads are prepared through precipitation from a solution that contains both the drug and the polymer resin. When the drug incorporation is performed in situ, the drug is solubilized in the reaction medium before the synthesis of the polymer powder, which is formed in the presence of the drug. When adsorption processes are used, the drug is added to a medium that contains the suspended polymer material after production of the polymer matrix. When the adsorption technique is carried out, the drug is expected to be predominantly on the surface of the polymer bead. Vascular embolization is a medical procedure that consists in occluding a blood vessel intentionally by injecting a fine material (the embolic agent or embolizer) into the blood vessel[16-18]. Embolization techniques have been used to treat several clinical problems, including treatment of malignant tumors and arteriovenous malformation[16-20]. Transarterial chemoembolization is a medical technique that combines the application of a drug with an embolic agent, leading to local chemical and physical actions during treatment[21-24]. This encourages the development of techniques intended to load biological molecules onto embolizing polymer microparticles. Several commercial products are available for embolization and many of them are based on poly(vinyl alcohol) (PVA) and poly(vinyl acetate) (PVAc) microparticles[13-15,25-27]. Although PVAc is well known for its excellent adhesive properties[28], PVAc is also used in a broad range of applications, including medical applications, due to its biocompatibility[29-31]. For this reason, Pinto and coworkers[13-15,25,30,32-34] developed a sequential two-stage process to allow for production of

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spherical PVAc/PVA particles with core-shell morphology to be used as embolizers. Based on this process, additional studies were carried out to modify the final properties of the particles[32-34]. In order to load PVAc microparticles with bioactive compounds for posterior use as chemoembolizers, it is important to analyze how PVAc microparticles interact with model biological molecules. Bovine Serum Albuminum (BSA) is a model biological molecule that finds widespread use in the biotechnological and biomedical fields. Particularly, BSA has been used as a drug delivery agent, due to its capacity to bond covalently to different drugs[35-37]. BSA is a protein composed of 583 aminoacid residues, has molar mass of 66430g/gmol, is very soluble in water (it can be precipitated in high concentrations of a neutral salt, such as ammonium sulphate), has isoelectric point in the pH range of 4.60-5.70, and presents spheroidal shape, characteristic sizes of 4nm × 4nm × 14nm and Stoke radius of 3.48nm[38-40]. Lysozyme is another model biological molecule that finds many uses in the biotechnological and biomedical fields. Particularly, lysozyme has been employed as an antibiotic in films intended for food packaging[41-42]. For this reason, immobilization of lysozyme has been performed in different polymer materials, including cellulose acetate, nylon, chitosan and alginates[43]. Lysozyme is an enzyme produced by bacteria, fungi, plants and animals, presenting 129 aminoacid residues, molar mass of 14400g/gmol and isoelectric point in the pH 11.00[44-46]. It is important to emphasize that PVAc microparticles have not been used as supports for adsorption of enzymes in previous studies. However, the use of PVAc-based particle embolizers loaded with enzymes can be very advantageous in real medical applications, as the physical benefits provided by the occlusion of blood vessels can be combined with

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Adsorption of BSA (Bovine Serum Albuminum) and lysozyme on poly(vinyl acetate) particles the chemical benefits provided by the enzyme activity in the local embolized tissue. This certainly encourages the analysis of the enzymatic adsorption process over PVAc microparticles. Based on the previous remarks, the main objective of the present manuscript is analyzing for the first time how PVAc microparticles interact with BSA and lysozyme during adsorption experiments performed in aqueous suspensions. BSA and lysozyme are used here as model biological molecules in order to evaluate the efficiency of the adsorption process for the eventual preparation of chemoembolyzers through adsorption. It is shown in both cases that immobilization was more efficient in the vicinities of the isoelectric point of the protein, leading to loads of 0.65mg of BSA and 1.0mg of lysozyme per g of PVAc, which are comparable to the amounts required for some of the standard chemoterapic procedures. Particularly, it is shown that lysozyme still presents residual activity after the adsorption process, which can constitute very important characteristic for real biomedical applications.

2. Materials and Methods 2.1 Reagents All chemical reagents used for production of PVAc microparticles were purchased from Vetec (Rio de Janeiro, Brazil) in analytical grade, with minimum purity of 99.5wt%, and used as received. BSA and lysozyme were purchased from Sigma-Aldrich (Rio de Janeiro, Brazil) as pure liofilized proteins and used as received. Protein concentrations and enzymatic activities were characterized as described below prior to adsorption experiments. Water was distilled and demineralized prior to use.

2.2 Suspension polymerization reactions PVAc microparticles were prepared through suspension polymerization, as described elesewhere[13-15,25,30,32-34]. Reaction runs were performed in an 1-liter jacketed glass reactor, equipped with a stainless steel lid and a standard six-blade stainless steel turbine impeller. Initially, a suspending agent solution containing 0.2g of PVA (weight average molecular weight of 78x103g/gmol and degree of hydrolysis of 88%, as reported by the producer) in 420g of distilled water was prepared at room temperature. Afterwards, the monomer solution was prepared by dissolving 4g of the free-radical initiator benzoyl peroxide (BPO, containing 25% of humidity) in 200g of vinyl acetate (monomer, with minimum purity of 99.9%). The PVA solution was added to the reactor first and heated to 85 ºC, when the monomer solution was fed into the reactor. The reacting mixture was kept under continuous agitation of 1000 rpm for 4 hours. Finally, polymer particles were separated through vacuum filtration, washed with abundant cold distilled water and dried until constant weight in a vacuum oven at 40oC.

2.3 Polymer characterization The molecular weight distribution and molecular weight averages of the produced PVAc microparticles were determined through standard gel permeation chromatography (GPC) at room temperature and using tetrahydrofuran Polímeros, 26(4), 282-290, 2016

(THF, chromatographic grade) as the mobile phase. Analyses were performed in a Waters 600E equipment (Waters, US) equipped with a Waters 2414 refractive index detector and Ultrastyragel columns with porosity of 103, 104 and 105 Å. Calibration was carried out with polystyrene standards with weight-average molecular weights ranging from 5 × 103 to 3.0 × 106g/mol. The characteristic transition temperatures of the produced PVAc microparticles were determined through standard differential scanning calorimetry (DSC) analyses. Experiments were performed with help of a DSC7 equipment (Perkin‑Elmer, US), using empty aluminum pans as references and heating rates of 10 °C/min in the range of 0 to 200 °C. In all cases, data were collected during the second heating scan in order to erase the previous thermal history of the polymer samples. The morphology of obtained PVAc microparticles was characterized through optical microscopy, using a SMZ 800 microscope (Nikon, Japan) equipped with a Coolpix 995 digital camera (Nikon, Japan). Zeta potential analyses of the produced PVAc microparticles were performed through streaming potential measurements in a SurPASS Electrokinetic Analyzer (Anton-Parr, US). Measurements were conducted in a background electrolyte solution containing 10 mM of KCl over the pH range of 2 to 12 range at 25 °C, as recommended in the literature[47]. The solution pH was adjusted by addition of NaOH or HCl. Hydrophobicity analyses of the obtained PVAc product were performed with polymer films produced through slow solvent evaporation in vacuum ovens kept at room temperature until constant weight, from initial polymer solutions containing 4wt% of polymer and 96wt% of THF. The film was placed in a SCA 20 goniometer (Dataphysics, US) and contact angles were measured in triplicates at five different regions of the film. Particle size distributions (PSD) of the final PVAc microparticles were determined through standard dynamic light scattering analyses performed in a Hydro 2000S equipment (Malvern Instruments, US). Measurements were performed in dilute aqueous dispersions at room temperature.

2.4 Protein immobilization (adsorption) Protein immobilization was performed through physical adsorption, keeping 10 ml of aqueous protein solution (in sodium phosphate buffer of 0.05M) with known protein concentration in contact with 1 or 2g of PVAc microparticles for different periods of time under agitation of 250rpm at 30 °C in an orbital shaker (New Brunswick, US). The masses of polymer powder were defined to allow for effective mixing of the solid-liquid suspension and provide sufficient area for protein adsorption, as discussed in the next section. Adsorption experiments were carried out at pH values of 3.0, 5.0, 6.8, 8.6 and 10.8. The solution pH was adjusted by addition of NaOH or HCl.

2.5 Protein concentration Standard Bradford analyses were performed to determine protein concentrations in the BSA and lysozyme aqueous solutions[48]. Initially, 1 ml of the Bradford solution was 283

Santos, D. P., Alves, T. L. M., & Pinto, J. C. added to an Eppendorf, followed by addition of 0.2 ml of the sample solution or of the standard solution (used for calibration) at room temperature. After 10 minutes, light absorbance was measured with help of a UV-Mini spectrometer (Shimadzu, Japan) at the wavelength of 600nm. Calibration was performed with pure liofilized BSA samples.

2.6 Enzymatic lisozyme activity Enzymatic lysozyme activity was determined as described by the Sigma-Aldrich protocol. According to this procedure, 2.5 ml of an aqueous solution (66 mM potassium phosphate buffer at pH 6.22) containing 0.01wt% of liofilized Micrococcus powder (Sigma-Aldrich, Rio de Janeiro) were placed in a cuvette with optical path of 1 cm. Afterwards, 0.1 ml of the sample lysozyme solution was added to the cuvette. Light absorbance readings (at least five) were then performed within the first minute of reaction, using absorbance of the original potassium phosphate buffer as base line. The enzymatic activity is proportional to the angular coefficient of absorbance readings, as function of the reaction time. Light absorbance was measured with help of a UV-Mini spectrometer (Shimadzu, Japan) at the wavelength of 450nm. Calibration was performed with pure liofilized lysozyme samples. All experiments were performed at constant temperature of 25 °C. In order to evaluate the effect of pH on the enzymatic lisozyme activity, aqueous lysozyme solutions (0.05 M sodium phosphate buffer and 0.1g/l of enzyme) were prepared at pH values of 3.0, 5.0, 6.8, 8.6 and 10.8 and submitted to enzymatic activity tests. Similarly, in order to evaluate the effect of vinyl acetate on the enzymatic lisozyme activity, aqueous lysozyme solutions (0.05M sodium phosphate buffer, pH of 6.8 and 0.1g/l of enzyme) were prepared, put in contact with different amounts of vinyl acetate (0, 1, 5, 10, 25, 50 and 95wt% of VAc) and submitted to enzymatic activity tests. In all cases, the solution pH was adjusted by addition of NaOH or HCl.

3. Results and Discussions 3.1 Polymer characterization Obtained PVAc microparticles had the usual spherical shape of suspension polymer powders, as seen in Figure 1. Particles presented smooth surfaces and nonporous structure, as also characterized in previous works[13-15,25,30,32-34]. Besides, number-average molecular weight, weight-average molecular weight and polydispersity of the produced resins were equal to 33 × 103g/gmol, 84 × 103g/gmol and 2.55, respectively, as in typical PVAc materials produced in similar conditions[13-15,25,30,32-34]. The glass transition temperature (Tg) was equal to 42 °C, which is also characteristic of PVAc powders[13-15,25,30,32-34]. Monomer conversion was very high and close to 100%, so that residual monomer could not be detected by standard gas chromatograph and nuclear magnetic resonance analyses, after washing and vacuum drying of the polymer particles. Figure 2 shows the particle size distribution of the obtained PVAc microparticles, which present volume‑average diameter of 200µm and standard deviation of 90μm, with 95% of the mass contained in the diameter range of 50-550μm. As broad 284

Figure 1. Optical micrograph of obtained PVAc microparticles.

Figure 2. Particle size distribution of obtained PVAc microparticles.

particle size distributions is a characteristic of polymer powders produced through suspension polymerization, particle sieving and classification may be eventually necessary for some applications, including embolization. However, particle classification was not conducted here for execution of adsorption studies because polymer particles were nonporous, spherical and compact, with characteristic dimensions that were many orders of magnitude higher than the dimensions of the molecules involved in the experimental study, rendering the flat surface assumption valid for analysis of the obtained data. According to the distribution shown in Figure 2 and assuming that the obtained microparticles were nonporous and spherical, it was possible to calculate the specific area of the microparticles as equal to 0.026 m2/g. Similar results have been reported previously with help of standard BET analyses[25,33]. As one might already expect for suspension powders, the specific area of the microparticles can be regarded as very low (below the detection limits of most experimental techniques used to characterize specific areas of solid powders), so that the analyzed PVAc microparticles would barely be recommended as supports for preparation of enzymatic catalysts. Despite that, as these microparticles Polímeros, 26(4), 282-290, 2016

Adsorption of BSA (Bovine Serum Albuminum) and lysozyme on poly(vinyl acetate) particles can be used as embolizers, the supporting of small amounts of bioactive molecules on the external microparticle surface can exert beneficial effects on the performance of the final product. The contact angle of the obtained PVAc product was equal to 79°, indicating that particles presented a hydrophilic character, probably enhanced by the PVA suspending agent, which is expected to form a very thin film around the particle surfaces. The zeta potential of the obtained PVAc microparticles is shown in Figure 3. It can be observed that the isoelectric point of the obtained PVAc microparticles is placed at the vicinities of the pH value of 3.0, indicating that the surfaces of the microparticles are charged negatively (and weakly) in almost the entire range of analyzed pH values. Again, the weak accumulation of negative charges on the PVAc microparticle surfaces can be due to the presence of the PVA suspending agent, but also due to the spontaneous hydrolysis of the ester groups of PVAc molecules, which is enhanced in basic aqueous media[25]. It is important to emphasize that the accumulation of negative charges on the surfaces of the microparticles can allow for efficient immobilization of proteins when they are charged positively, if it is assumed that charge balance controls the adsorption process.

Figure 3. Zeta potential of obtained PVAc microparticles at different pH values.

3.2 Enzymatic lisozyme activity Figure 4 shows how the enzymatic lisozyme activity changes with the pH of the prepared aqueous solution. According to Figure 4, the maximum enzymatic activity of lysozyme is observed at the pH of 6.80, which is the average pH of body liquids and is very close to the neutral pH. In Figure 4 one can also observe that the enzymatic lisozyme activity is not very sensitive to pH variations at the acidic region, but decreases pronouncedly in the alkaline region. (Complete denaturation of the enzyme was observed at the pH of 10.80 after 24 hours.) Similar results were reported in other studies and at different temperatures[49-52]. As monomer conversion never reaches 100%, small amounts of vinyl acetate can possibly be released during the immobilization process, despite the efforts to purify the produced PVAc microparticles. For this reason, the enzymatic lisozyme activity was characterized in presence of vinyl acetate. As lysozyme is not soluble in vinyl acetate and the solubility of vinyl acetate in water is very small (around 2.5wt% at 30 °C), some experiments were performed in presence of two phases: an aqueous phase and an organic phase. Figure 5 shows how the enzymatic lisozyme activity of aliquots of the aqueous phase changes with the amount of vinyl acetate added to the lisozyme solution. It can be observed that the presence of vinyl acetate can lead to significant increase (30%) of the observed lisozyme activity. As the vinyl acetate content increases (and saturation of the aqueous phase takes place), a constant activity value is attained (although the experimental errors also increase possibly because of the unavoidable presence of very small organic droplets in the aqueous aliquots). As discussed in the literature[53], the presence of small amounts of organic solvents in the enzyme solution can stabilize the protein structure (conformation) and allows for enhanced enzymatic Polímeros, 26(4), 282-290, 2016

Figure 4. Enzymatic activity of lisozyme at different pH values.

Figure 5. Enzymatic activity of lisozyme in presence of vinyl acetate.

activity, as observed here when small amounts of vinyl acetate are added to the aqueous lisozyme solution.

3.3 Adsorption experiments A summary of the experimental design used to perform the adsorption experiments for both BSA and lisozyme is presented in Table 1. All experiments were performed at least three times in order to guarantee the statistical significance of the obtained results. 285

Santos, D. P., Alves, T. L. M., & Pinto, J. C. Figure 6 shows how the concentrations of BSA in the aqueous phase changed when the protein solution was kept in contact with the PVAc microparticles for 24 hours at 30 °C. As one can observe, significant changes of BSA concentrations could only be observed when the pH was equal to 5.0, which is close to the isoelectric point of BSA. It is important to note that similar results were obtained when different amounts of PVAc (1 or 2g) were used, indicating that the obtained results were not controlled only by the small specific areas of the produced PVAc powder. When similar experiments were performed at 10 °C, no significant change of the BSA concentrations could be observed in the aqueous phase, indicating that temperature exerts a pronounced effect on the efficiency of BSA adsorption onto the PVAc microparticles. When temperature was equal to 35 °C, results were similar to the ones reported for 30 °C. Additional increase of temperature was not possible because coagulation of PVAc particles takes place, due to the low Tg value of the polymer resin. Although lower temperatures usually tend to favor physical adsorption processes[54], the immobilization of BSA onto PVAc microparticles was favored by the higher temperatures, which were closer to the glass transition temperature of PVAc. This seems to indicate that the mobility of the polymer chains can be important for explanation of the obtained results. Apparently, the higher the mobility of the chains, the higher the amounts of BSA adsorbed on the PVAc microparticles. This can also explain why the adsorption process was more effective when the protein was close to its isoelectric point, as the absence of electrical charge can facilitate the interaction of the protein with the polymer surface. This result is also supported by previous results, which indicate that adsorption of BSA onto different supports is controlled by hydrophobic interactions and not by electrical charges[55-57]. It is also important to observe that the total amount of BSA adsorbed onto the PVAc microparticles at 30 °C and pH of 5.0 was equal to 0.65mg/g of polymer, much smaller than values reported previously for BSA adsorption onto inorganic supports of very high specific area (25mg/g of support in the vicinities of the isoelectric point)[57]. Assuming

that a single monolayer of BSA was formed over the particle surfaces, it is possible to conclude that the area occupied by each BSA molecule over the PVAc particles was equal to 5.2nm2, which is much lower than areas reported previously for BSA over other supports (from 35 to 44nm2)[38-40,56]. This clearly indicates that multiple BSA layers (as many as seven) can be formed over the PVAc particles or that BSA molecules can interact with PVAc chains and penetrate (through mixing) in the particle. It is important to observe that the PVAc microparticles that were subjected to BSA adsorption experiments at 30 °C and pH of 5.0 after 8 hours and containing 0.65mg of BSA per g of polymer were filtrated, dried at room temperature in vacuum oven and resuspended in 10 ml of aqueous medium at 30 °C and pH of 5.0. As BSA could not be detected in the aqueous medium after 8 h, desorption could be regarded as negligible at the analyzed conditions. Figure 7 shows how the concentrations of lisozyme in the aqueous phase changed when the protein solution was kept in contact with the PVAc microparticles for 24 hours at 30 °C. As one can observe, significant changes of lisozyme concentrations could only be observed after 8 hours when the pH was equal to 8.6, which is also close to the isoelectric point of lisozyme, as observed previously

Figure 6. Concentration of BSA in the aqueous phase after 24 hours at 30 °C.

Table 1. Experimental design for adsorption experiments. Code PVAc-1 PVAc-2 PVAc-3 PVAc-4 PVAc-5 PVAc-6 PVAc-7 PVAc-8 PVAc-9 PVAc-10 Control-1 Control-2 Control-3 Control-4 Control-5

286

Mass of PVAc (g) 1 1 1 1 1 2 2 2 2 2 0 0 0 0 0

pH 3.0 5.0 6.8 8.6 10.8 3.0 5.0 6.8 8.6 10.8 3.0 5.0 6.8 8.6 10.8

Figure 7. Concentration of lisozyme in the aqueous phase after 24 hours at 30 °C. Polímeros, 26(4), 282-290, 2016

Adsorption of BSA (Bovine Serum Albuminum) and lysozyme on poly(vinyl acetate) particles for BSA. After 24 hours of experiment, significant changes of concentration could also be observed for the control experiments, indicating the protein denaturation in basic aqueous media, as described previously and confirmed independently through the enzymatic activity tests shown in Figure 8. As a consequence, lisozyme adsorption experiments performed in basic aqueous media must be shorter, if protein denaturation must be avoided. Once more, it is important to note that similar results were obtained when different amounts of PVAc (1 or 2g) were used, indicating that the obtained results were not controlled only by the small specific areas of the produced PVAc powder. As in the previous case, when similar experiments were performed at 10 °C, no significant change of the lisozyme concentrations could be observed in the aqueous phase, indicating that temperature exerts a pronounced effect on the efficiency of lisozyme adsorption onto the PVAc microparticles, as also observed previously for BSA. Figure 9 shows the evolution of protein concentration and enzymatic lisozyme activity in the aqueous phase during the first ten hours of experiment, depicting that both dynamic trajectories are not proportional to each other and indicating that protein denaturation takes place at the pH value of 8.6. As done in the case of BSA, it is important to observe that the total amount of lisozyme adsorbed onto the PVAc microparticles at 30 °C and pH of 8.6 after 8 hours was equal to 1.0mg/g of polymer (or 25000 IU, in terms of activity), much smaller than values reported previously for lisozyme adsorption onto inorganic supports of very high specific area (385 to 450mg/g of support in the vicinities of the isoelectric point)[52,58,59]. Assuming that a single monolayer of lisozyme was formed over the particle surfaces, it is possible to conclude that the area occupied by each lisozyme molecule over the PVAc particles was equal to 3.3nm2, which is not compatible with the characteristic dimensions of the molecule and also indicates that multiple lisozyme layers can be formed over the PVAc particles or that lisozyme molecules can interact with PVAc chains and penetrate (through mixing) in the particle. It is important to observe that the PVAc microparticles that were subjected to lisozyme adsorption experiments at 30 °C and pH of 8.6 after 8 hours and containing 1mg of lisozyme per g of polymer were filtrated, dried at room temperature in vacuum oven and used for characterization of residual enzymatic activity. Enzymatic activity tests were performed with 100mg of PVAc microparticles in 10 ml of Micrococcus solution. Residual enzymatic activity was detected, but the consumption rates of the Micrococcus powder decreased at least two orders of magnitude, indicating the very slow release of the enzyme from the particle (Bradford tests could not detect significant release of protein in the aqueous phase, indicating that desorption was negligible at the analyzed experimental conditions) or denaturation of the enzyme. Based on the results presented in the previous paragraphs for the model biomolecules BSA and lisozyme, the adsorption of proteins onto PVAc microparticles seems to be controlled by hydrophobic interactions, depending on the availability of interfacial surface (as usual), but also depending significantly on the mobility of the polymer chains. Besides, as adsorption efficiency was also higher in Polímeros, 26(4), 282-290, 2016

the vicinities of the isoelectric point of the protein, it seems that interaction can be maximized in the absence of charges, as also observed for other support/protein pairs[60]. Based on these results, it can be conjectured that as much as 1mg of protein can be loaded into 1g of PVAc-based embolic agents through adsorption, if the adsorption process is performed

Figure 8. Enzymatic lisozyme activity in the aqueous phase after 24 hours at 30 °C.

Figure 9. (a) Concentration of lisozyme and (b) enzymatic lisozyme activity in the aqueous phase at 30 °C and pH of 8.6. 287

Santos, D. P., Alves, T. L. M., & Pinto, J. C. in the vicinities of the isoelectric point of the protein of interest, which is comparable to the amounts required for some of the standard chemoterapic procedures.

4. Conclusions The adsorption of bovine serum albuminum (BSA) and lysozyme (used as model biomolecules) on PVAc particles produced through suspension polymerization was studied in the present manuscript in a broad range of pH values. It was shown that significant amounts of BSA and lysozyme could be adsorbed onto PVAc particles in the vicinities of the isoelectric point of the biomolecules (0.65mg of BSA and 1.0mg of lysozyme per g of PVAc), allowing for production of chemoembolizers through adsorption. Besides, it was shown that the adsorption of the analyzed proteins onto PVAc microparticles seemed to be controlled by hydrophobic interactions, depending significantly on the mobility of the polymer chains and on the net charges of the analyzed protein. Based on these results, it can be conjectured that as much as 1mg of protein can be loaded into 1g of PVAc-based embolic agents through adsorption, if the adsorption process is performed in the vicinities of the isoelectric point of the protein of interest.

5. Acknowledgements The authors thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil) and FAPERJ (Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro) for scholarships and financial support.

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PolĂmeros, 26(4), 282-290, 2016

http://dx.doi.org/10.1590/0104-1428.2180

Kinetic behavior of the reaction between silica and epoxidized liquid rubber Marcus Vinícius Braum1* and Marly Antônia Maldaner Jacobi1 Programa de Pós-graduação em Ciência dos Materiais, Instituto de Química, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, RS, Brazil

*marcus.braum@pirelli.com

Abstract The use of epoxidized rubber as compatibilizer in silica-filled rubber compounds has been proposed in literature. However, the investigation of the reaction kinetics between the epoxy groups and the hydroxyl groups at the silica surface is not yet described. It is a difficult task, mainly because of the high dilution of the system due the difficulty of incorporating high levels of silica in a matrix containing epoxidized rubbers of high molecular weight. In this work, a mixture of precipitated silica and an epoxidized liquid rubber (EpLHPB) was prepared and the reaction between silanols and epoxy groups was followed by DSC under isothermal conditions, at 150, 160 and 170 °C. An autocatalytic data treatment was applied to determine the kinetic parameters of the reaction. Furthermore, it was possible to estimate the amount of epoxy groups required for the saturation of the external surface of the silica, resulting in 5.4 epoxy groups/nm2. Keywords: composites, epoxidized rubber, kinetics, silanols, silica.

1. Introduction High-performance rubber materials are usually reinforced by colloidal filler such as silica or carbon black, in order to improve their mechanical properties. Due to the presence of siloxane and silanol polar groups on its surface, silica exhibits a high specific component of surface free energy. Consequently, the interactions between these polar groups and the nonpolar hydrocarbon rubbers are very weak compared with the hydrogen bonds that occur between the silanols, resulting in weaker polymer-filler interaction and greater tendency to agglomerate than carbon black. For this reason, bifunctional silanes such as bis-(triethoxysilylpropyl) tetrasulfide (TESPT) are commonly used to enhance compatibility of nonpolar rubbers with silica and improve mechanical properties of silica-filled elastomers, since such coupling agents are capable of establishing covalent bonds between polymer chains and silica surface[1,2]. Despite its undeniable benefits, the use of silanes is not devoid of drawbacks. These include the formation of ethanol (a byproduct of the silanization reaction) and the processing difficulties of such compounds, whose mixing temperature range is narrow (145-155 °C) due to the need to ensure a sufficient silanization rate, while avoiding the risk of scorch. Because of such difficulties, rubber compounds containing silica in a high content often require the use of specially-designed mixers and three or more mixing stages[3,4]. In order to avoid the drawbacks of the silanization process, the use of epoxidized rubber as an alternative to the use of silanes has been investigated[5-7]. Jacobi et al.[6] showed that lightly-epoxidized cis-BR and silica can react during a reactive mixing process. However, attempts to study the kinetics of this reaction by DSC were unsuccessful, probably due to the high degree of dilution of the silica/epoxidized rubber system in such compounds[8]. Also using DSC technique, Wasantakorn[9] successfully observed the occurrence of reaction

Polímeros, 26(4), 291-296, 2016

between the epoxy groups of an epoxidized natural rubber (ENR) and hydroxyls of silicic acid, but without attempting to clarify any aspects of the kinetics of this reaction. In this paper, a kinetic study with emphasis on phenomenological modeling is presented for the reaction between epoxy groups and silica, involving the use of an epoxidized liquid rubber. The low viscosity of this rubber allows the intrusion of some chains in the macro- and mesopores of the silica, favoring the impregnation of the filler and thus enhancing the contact between epoxidized polymer and silica surface. Thus the reaction kinetics of mixtures of highly dispersible silica (HD) with a liquid hydroxylated polybutadiene epoxidized at 8.8 mol% (EpLHPB) was studied through isothermal experiments in DSC, from which it is possible to evaluate autocatalytic and nth order reactions[10,11]. In addition to the kinetic parameters of the reaction, the content of epoxide groups required for the saturation of the external surface of the silica could also be estimated.

2. Materials and Methods 2.1 Materials The commercial silica utilized in this study, Zeosil 1165MP (Rhodia), was used without further purification. The silica presented a total specific surface of 139 m2/g, determined by the BET method[12], and an external specific surface of 113 m2/g, determined by the t-plot method using the Lippens-de Boer equation[13]. Liquid hydroxylated polybutadiene (LHPB) was supplied by Petroflex, now known as Lanxess (Brazil), and used in the condition it was received in. The sample presented a Mn = 5400 g/mol, determined via GPC using THF as solvent, conventional calibration using PS, and detection

291

S S S S S S S S S S S S S S S S S S

Braum, M. V., & Jacobi, M. A. M. by refractive index. The microstructure was determined via FTIR analysis by liquid film on KBr[14], resulting in 20% of 1,2-vinyl conformation, 24% of 1,4-cis and 56% of 1,4-trans. EpLHPB was obtained and characterized in our laboratory, according to procedures similar to those described elsewhere[15]: LHPB was epoxidized in a toluene solution at 44% (w/w) at 50 °C for 6 hours via the method of the performic acid generated in situ, using stoichiometric molar ratio of the reactants (H2O2:C=C:HCOOH). The epoxy content was determined by 1H NMR (300 MHz, CDCl3).

2.2 Mixing procedure The mixing of the liquid rubber with the precipitated silica was performed by manual grinding in mortar and pestle for five minutes. EpLHPB/sílica samples were prepared in different proportions in order to obtain a different concentration of epoxide groups (available for reaction with silanol groups) per unit external surface of sílica, QEPOX, calculated from Equation 1: Qepox =

(

mEpLHPB × α epox

)

α epox × M epox + 100 − α epox × M olef  × msilica × S EXT  

(1)

where mEPLHPB is the weight of epoxidized oligomer in the mixture, in grams, αEPOX is the degree of epoxidation of the polymer, in mol%, MEPOX and MOLEF are the molar masses of the epoxidized and olefinic units, respectively, SEXT is the specific external surface of the silica, in m2/g, and mSILICA is the mass, in grams, of silica in the mixture. An example is given in Equation 2. By mixing 1.28 g of silica (SEXT = 113 m2/g) and 1.00 g of EpLHPB 8.8 mol% epoxidized, we have: 1.0 × 8.8 = = 11.0 ×10−6 mol / m 2 (2) Qepox [8.8 × 70 + 91.2 × 54] ×1.28 ×113

2.3 DSC analysis The differential scanning calorimetry studies were performed with a MDSC 2920 (TA Instruments, New Castle, DE, USA), using aluminum pans, under nitrogen flow. The dynamic scan was performed at a heating rate of 10 °C/min from 25 to 250 °C. Studies in isothermal scan mode were performed at temperatures of 150, 160 and 170 °C. The Standard E 2070 (Test Method A) was used in order to determine the kinetic parameters of the reaction. Differential scanning calorimetry has been widely used to develop cure kinetic models for thermoset matrices, assuming proportionality between the heat of the reaction completed (ΔHC) and the fraction converted (α) through the Equation 3: α = ∆H c / ∆H

The fractional rate of reaction dα/dt (min-1) was determined from these data using Equation 4: d α= / dt (dH / dt ) / ∆H

(4)

When an isothermal process is characterized by a heat flow curve that reaches a maximum within seconds then slowly decays, an nth order reaction is probable. If the thermal curve instead shows a maximum after tens of seconds, as shown in Figure 1, an autocatalyzed reaction is likely to occur. An autocatalyzed reaction follows the relationship of Equation 5[16,17]: dα= / dt k (T ) α m (1 − α)n

(5)

where k(T) is the specific rate constant at temperature T (min-1) and m, n are the partial reaction order terms. Also known as Sesták-Berggren equation, Equation 5 was originally applied to the kinetic of reactions in the solid state.[16] In order to determine the kinetic parameters k(T), m and n for each temperature, Equation 5 was cast in its logarithmic form (Equation 6) and solved with a multiple linear regression using Data Analysis Tool of Microsoft Excel. ln [= d α / dt ] ln  k (T )  + m ln [ α ] + n ln [1 − α ]

(6)

The change of k(T) with temperature is described with the Equation 7, of Arrhenius[17]: k (T ) = Ze(

− E / RT )

(7)

where Z (min-1) is the pre-exponential factor, E (J/mol) is the activation energy, R (8.314 J/K.mol) is the gas constant and T (K) is the absolute temperature. Thus, the plot of ln[k(T)] versus 1/T is linear, with the slope equal to –E/R and the intercept equal to ln[Z].

(3)

where ΔH is the total heat of the reaction. For each isothermal experiment, a linear baseline was constructed between the beginning and the end on the exotherm peak. The heat of the reaction ΔH (J/g) was then determined by integrating the total area of the peak bounded by the peak itself and the baseline constructed. After identifying the times corresponding to 10 and 90% of the peak area, at least 10 equally spaced time values were selected between 292

these limits. Then, the rate of reaction dH/dt (W) and the heat of the reaction completed ΔHC (J/g) were obtained for each time interval, as illustrated in Figure 1.

Figure 1. Typical DSC isotherm used to determine the total heat reaction, ΔH, the rate of reaction dH/dt and heat of the reaction completed ΔHC. Polímeros, 26(4), 291-296, 2016

Kinetic behavior of the reaction between silica and epoxidized liquid rubber

3. Results and Discussions In a preliminary experiment, a sample of silica impregnated with EpLHPB (1:1.28 by weight) was heated from 25 to 250 °C at 10 °C/min. In this experiment the appearance of an exothermic event was observed, whose onset temperature was 170 °C. Thus, this temperature was selected to carry out the isothermal tests. Figure 2 shows the isothermal heating runs at 170 °C for: a LHPB/silica mixture, pure silica, pure EpLHPB (8.8 mol% epoxidized), and a sample of silica impregnated with EpLHPB (1:1.28 by weight, QEPOX = 11.0 x 10-6 mol/m2) in the first and second heating runs. The appearance of an exothermic event is clearly observed in the first heating run of the mixture EpLHPB/silica, which disappears in the second run (a new heating program, after a fast cooling until 25 °C). Moreover, this thermal event is

not detected in the traces of the pure components and in the mixture of LHPB/silica in the same proportion. This is a strong indication that a reaction occurs between the epoxy groups of the EpLHPB and OH groups present on the silica surface. As mentioned in the introduction, previous studies[9] discuss this reaction without quantifying their kinetic parameters. Figure 3 shows the DSC measurements in isothermal scan mode for silicas impregnated with EpLHPB containing different amounts of epoxy groups per unit of silica external surface. As can be seen, when QEPOX is increased, the rate of the reaction silanol-epoxy is decreased and its onset is progressively retarded. Most likely this occurs as increasing the amount of liquid polymer in the mixture makes expelling the water adsorbed on the silica surface more difficult, hindering contact between the reactive groups. The results shown in Figure 4 validate this hypothesis, since pre‑heating the impregnated silica at 105 °C for 120 min led to a considerable decrease in onset time and a narrowing of the heat release curve. In order to verify if this reaction may occur at lower temperatures, or if the moisture present in the silica could interfere with the process, the sample EpLHPB at QEPOX = 9.2 x 10-6 mol/m2 was pre-heated under vacuum at 105 °C for 120 min and then analyzed. Figure 4 shows the behavior of the mixture with and without pre-heating. Notably, there is a substantial reduction in the time required for the occurrence of the maximum heat release: from 26 to 8 min, while there was no substantial change in the heat of reaction with the pre-heating process. It is inferred that partial reaction during this process should not have occurred, or even the formation of intermediates. Thus, the phenomenon can be attributed to an improvement in the contact between polymer and silica, i.e. a better wettability facilitated by the elimination of the air and water physically adsorbed on the silica surface.

Figure 2. DSC curves of the mixture EpLHPB/silica (QEPOX = 11.0 x 10-6 mol/m2 or weight ratio 1:1.28), pure ingredients and mixture LHPB/silica (weight ratio 1:1.28). The thermograms were obtained under nitrogen flow and isothermal conditions, at 170 °C.

The heat of reaction (ΔH) is an extrinsic process and can be used as a parameter to monitor the extent of the reaction, allowing the stoichiometric ratio of the reaction system to be determined[18]. In Figure 5 the heats of reaction were plotted against the respective values of QEPOX, using the data determined in the Figure 3. It is noted that ΔH

Figure 3. DSC curves of the mixture EpLHPB/silica in different proportions (curves are identified by the value of QEPOX). The thermograms were obtained under N2 flow and isothermal conditions, at 170 °C. Polímeros, 26(4), 291-296, 2016

293

Braum, M. V., & Jacobi, M. A. M. initially rises, it reaches a maximum and then it decreases again. From the trend line drawn it was possible to obtain an estimated value for the stoichiometric ratio epoxy/silica, or point of saturation of the silica surface with epoxy groups, being approximately 9.0 x 10-6 mol/m2. Below this value there is probably an excess of silica in the mixture, while higher values indicate an excess of epoxy groups. The value determined for the point of saturation of the silica surface may also be expressed as 5.4 oxirane rings per nm2 of silica external surface, which is very close to the physicochemical constant of Kiselev-Zhuravlev[19] (4.9 ± 0.5 OH/nm2), which describes the maximum density of silanol groups on a silica surface, regardless of its origin. Shown in Figure 6 are the isothermal scans for the silica impregnated with EpLHPB (QEPOX = 9.2 x 10-6 mol/m2), at 150, 160 and 170 °C. The samples were pre-heated under

Figure 4. Isothermal DSC runs at 170 °C of a mixture silica/EpLHPB at QEPOX = 9.2 x 10-6 mol/m2, with and without pre-heating of the sample.

vacuum at 105 °C for 120 min before being introduced into the DSC pans. Observing the shapes of the thermal curves can be inferred that an autocatalyzed reaction is likely, since the heat flow curves build to a maximum after 8 to 22 min. Instead, in an nth order reaction the heat flow curve would show a maximum within few seconds[17]. In autocatalytic processes the reaction product serves as an additional catalyst in the reaction, and the term αm in Equation 5 account for this extra effect[20]. The kinetic parameters obtained by applying the autocatalytic model are given in Table 1. The mechanism of the reaction between silica and epoxidized rubber does not seem to be the same over the entire temperature range, since the reaction orders are not constant[21]: The kinetic exponent n increases as a function of temperature, while the exponent m remains almost constant. A hypothetical mechanism of the silanol/epoxy reaction may involve nucleophilic attack on a carbon of the epoxy ring by a silanol group. At the beginning of the reaction, the reactive sites on the silica surface (silanols) are plentiful and the reaction of an epoxy group facilitates the approximation of neighboring groups, which could explain the observed autocatalytic effect. At this stage the reaction is kinetically controlled. In the course of the reaction, the reactive sites become scarce and sterically hindered, while the chain mobility gradually decreases due to the immobilization of chain segments on the silica surface. Similarly to the epoxy curing reactions, a diffusion-controlled process will probably occur at the onset of this vitrification process, when the kinetic reaction ends[20]. At low temperatures, the process is expected to start with the reaction of a few epoxy groups per chain, before spreading. However, the rise in temperature can cause the process to be started with the reaction of many groups per chain. Thus, an increase in temperature should preferably

Figure 5. Heat of reaction (ΔH) as function of epoxy groups content per unit external surface of silica (QEPOX), in μmol/m2. Drawn from the data in Figure 3.

Figure 6. Isothermal DSC runs at 150, 160 and 170 oC of a mixture silica/EpLHPB at QEPOX = 9.2 x 10-6 mol/m2. The sample was pre‑heated under vacuum at 105 °C for 120 min.

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Kinetic behavior of the reaction between silica and epoxidized liquid rubber Table 1. Kinetic parameters of the autocatalytic model obtained from isothermal analysis performed in DSC and data treatment according to ASTM E 2070[17]. T (K) 423 433 443

ln[k(T)] (ln[min-1]) -5.946 ± 0.016 -5.493 ± 0.014 -4.888 ± 0.015

m 0.65 ± 0.01 0.62 ± 0.01 0.56 ± 0.01

n 0.73 ± 0.01 0.98 ± 0.01 1.15 ± 0.01

E (kJ/mol)

ln[Z] (ln[min-1])

82.4 ± 8.0

17.4 ± 2.2

Figure 7. Fraction converted (a) and conversion rate (b) versus elapsed time after reaction onset for silica impregnated with EpLHPB (QEPOX = 9.2 x 10-6 mol/m2) at 150, 160 and 170 °C.

promote the initial process, kinetically controlled, resulting in faster chain immobilization and shifting the maximum in dα/dt to lower values of α, as indicated in Figure 7. Such an increase of the contribution of the diffusional process to the overall kinetics can explain the increase in the exponent n with temperature. The experimental data and theoretical curves for the fraction converted and fractional rate of reaction are shown in Figure 7. The theoretical curves of dα/dt were calculated using the parameters m, n and k presented in Table 1, while the theoretical curves of α were obtained through the integration of the theoretical curves of dα/dt using the Origin software. There is a very good agreement between experimental data and theoretical fittings, supporting the interpretation proposed above. The kinetic study showed that the process is very fast at 170 °C, which is very suitable as this temperature is within the usual range adopted for mixing processes (step of addition of fillers) and vulcanization in the rubber industry.

4. Conclusions The kinetics of the chemical reaction occurred between EpLHPB and silica could be studied by DSC. It was possible to estimate the content of epoxy groups required for the saturation of a silica external surface: 5.4 oxiranes/nm2, a value in close to the constant of Kiselev-Zhuravlev. The system presents features of an autocatalytic reaction and a reaction enthalpy of 1645 kJ/mol at 443K. The kinetic parameters could be determined through isothermal scanning, making possible the generation of predictive curves of the reaction. This is very useful for the optimization of the reactive mixing process between silica and epoxidized rubber. Polímeros, 26(4), 291-296, 2016

5. References 1. Byers, J. T. (2002). Fillers for balancing passenger tire tread properties. Rubber Chemistry and Technology, 75(3), 527-548. http://dx.doi.org/10.5254/1.3547681. 2. Vilgis, T., Heinrich, G., & Klüppel, M. (2009). Reinforcement of polymer nano-composites: theory, experiments and applications. Cambridge: Cambridge University Press. 3. Dierkes, W. (2005). Economic mixing of silica-rubber compounds (Doctoral thesis). University of Twente, Enschede. 4. Luginsland, H.-D. (1999). Processing of the organo silane Si 69. In Proceedings of the RubberChem ’99. Antwerp: Rapra. 5. Rocha, T. L. A. C., Schuster, R. H., Jacobi, M. M., & Samios, D. (2004). Influence of epoxidation on physical properties of SBR and its interaction with precipitated silica. Kautschuk und Gummi, Kunststoffe, 57(12), 656-661. Retrieved in 17 April 2015, from http://www.kgk-rubberpoint.de/ai/resources/7bfb4c001c9. pdf 6. Jacobi, M. M., Braum, M. V., Rocha, T. L. A. C., & Schuster, R. H. (2007). Lightly epoxidized polybutadiene with efficient interaction to precipitated silica. Kautschuk und Gummi, Kunststoffe, 60(9), 460-466. Retrieved in 17 April 2015, from http://www.kgk-rubberpoint.de/texte/anzeigen/718 7. Saramolee, P., Sahakaro, K., Lopattananon, N., Dierkes, W. K., & Noordermeer, J. W. M. (2014). Comparative properties of silica- and carbon black-reinforced natural rubber in the presence of epoxidized low molecular weight polymer. Rubber Chemistry and Technology, 87(2), 320-339. http://dx.doi. org/10.5254/rct.13.86970. 8. Braum, M. V. (2006). Melhoria da interação polímero-carga através do uso de borracha de polibutadieno epoxidada (Master’s dissertation). Universidade Federal do Rio Grande do Sul, Porto Alegre. 9. Wasantakorn, A. (2006). A Study on reaction of ENR with silicic acid. Journal of Research in Engineering and Technology, 3(1), 1-20. Retrieved in 17 April 2015, from http://anchan.lib. ku.ac.th/kukr/handle/ 003/19595 295

Braum, M. V., & Jacobi, M. A. M. 10. Berglund, L. A., & Kenny, J. M. (1991). Processing science for high performance thermoset composites. SAMPE Journal, 27(2), 27-37. 11. Nam, J., & Seferis, J. C. (1993). Application of the kinetic composite methodology to autocatalytic-type thermoset prepreg cures. Journal of Applied Polymer Science, 50(9), 1555-1564. http://dx.doi.org/10.1002/app.1993.070500909. 12. Brunauer, S., Emmett, P. H., & Teller, E. (1938). Adsorption of gases in multimolecular layers. Journal of the American Chemical Society, 60(2), 309-319. http://dx.doi.org/10.1021/ ja01269a023. 13. Lippens, B. C., & de Boer, J. H. (1965). Studies on pore systems in catalysts: V. The t method. Journal of Catalysis, 4(3), 319323. http://dx.doi.org/10.1016/0021-9517(65)90307-6. 14. Takahashi, M. F. K., & Polito, W. L. (1997). Aplicações da espectroscopia de infravermelho com Transformada de Fourier para especiação isomérica de polibutadienos hidroxilados utilizados na síntese de polímeros PU-Propelentes. Polímeros: Ciência e Tecnologia, 7(1), 37-43. http://dx.doi.org/10.1590/ S0104-14281997000100007. 15. Rocha, T. L. A. C., Jacobi, M. M., Samios, D., Meier, J., & Schuster, R. H. (2004). Study of the epoxidation of polydiene rubbers part III: Influence of epoxidation on viscoelastic behavior of SBR and BR melts. Kautschuk und Gummi, Kunststoffe, 57(7-8), 377-384. Retrieved in 17 April 2015, from http:// www.kgk-rubberpoint.de/texte/anzeigen/412 16. Sesták, J., & Berggren, G. (1971). Study of the kinetics of the mechanism of solid-state reactions at increasing temperatures.

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Thermochimica Acta, 3(1), 1-12. http://dx.doi.org/10.1016/00406031(71)85051-7. 17. American Society for Testing and Materials – ASTM. (2000). ASTM E2070-00: standard test method for kinetic parameters by differential scanning calorimetry using isothermal methods. West Conshohocken: ASTM International. 18. González-Garcia, F., Miguez, E., & Soares, B. G. (2005). Characterization of diglycidyl ether of bisphenol A / aliphatic polyamines systems. Polímeros: Ciência e Tecnologia, 15(4), 261-267. http://dx.doi.org/10.1590/ S0104-14282005000400010. 19. Zhuravlev, L. T. (2000). The surface chemistry of amorphous silica. Colloids and Surfaces. A, Physicochemical and Engineering Aspects, 173(1-3), 1-38. http://dx.doi.org/10.1016/S09277757(00)00556-2. 20. Bilyeu, B., Brostow, W., & Menard, K. P. (2001). Epoxy thermosets and their applications III. Kinetic equations and models. Journal of Materials Education, 23(4-6), 189-204. Retrieved in 17 April 2015, from http://www.unt.edu/ LAPOM/ publications/pdf%20articles/Lisa/epoxyJME3.pdf 21. Kenny, J. M. (1994). Determination of autocatalytic kinetic model parameters describing thermoset cure. Journal of Applied Polymer Science, 51(4), 761-764. http://dx.doi.org/10.1002/ app.1994.070510424. Received: Apr. 17, 2015 Revised: Feb. 15, 2016 Accepted: Mar. 13, 2016

Polímeros, 26(4), 291-296, 2016

http://dx.doi.org/10.1590/0104-1428.2193

Preparation of novel magnetic polyurethane foam nanocomposites by using core-shell nanoparticles Mir Mohammad Alavi Nikje1*, Sahebeh Tamaddoni Moghaddam1 and Maede Noruzian1 1

Department of Chemistry, Faculty of Science, Imam Khomeini International University, Qazvin, Iran *drmm.alavi@gmail.com

Abstract Iron oxide magnetic nanoparticles (NP’s) converted to the core- shell structres by reacting with by n-(2-aminoethyl)-3aminopropyl trimethoxysilane (AEAP) incorporated in polyurethane flexible (PUF) foam formulations. Fourier transform spectra, thermal gravimetric analysis, scanning electron images, thermo-mechanical analysis and magnetic properties of the prepared nanocomposites were studied. Obtained data shown that by the increasing of the amine modified magnetic iron oxide NP’s up to 3% in the polymer matrix, thermal and magnetic properties improved in comparison with pristine foams. In addition, due to the presence of functional groups on the magnetic NP’s surface, hard phases formation decrease in the bulk polymer and cause decreasing of glass transition temperature. Keywords: magnetic iron oxide, magnetic nanoparticles, N-(2-aminoethyl)-3-aminopropyl trimethoxysilane (AEAP), Polyurethane flexible (PUF) foam.

1. Introduction Polyurethane’s (PU’s) as a well-known class of versatile polymeric materials prepare by a simple condensation reactions and because of their unique properties have been used in various applications namely, adhesives, coatings, elastomers and foams[1,2]. In the foam categories, flexible families are the largest product family by quantity by having more than 40% of all PU foams production capacity. Flexible PU foam because of its lightness and strength is used as cushioning namely, car seats, mattresses and packaging, but having their main own merits and drawbacks[3,4]. The main drawbacks referee to the low load bearing properties and low thermal stabilities in comparing to the other polymeric materials. In order to overcoming these drawbacks, in the recent years some novel nanocomposites are prepared[5]. Nanocomposites display prior properties when compared with their microcomposites counterparts, due to the much stronger interactions between the dispersed nanoparticles (NP’s) domains and the polymer matrix[6]. In this case and in flexible PU foams, the incorporation of nanoparticles increase foam density as well as improve compression and tear properties[7]. Literature survey reveals the application of well-known NPs in the flexible foam formulations namely, fumed silica[8], calcium carbonate[7], nanofibers[9], carbon nanotubes[9,10], zinc borate, phosphorous and expandable graphite[11] in order to improving of thermal, mechanical, acoustic and flame retardancy properties, respectively because of the high performances of nanoparticles in improving of target properties[12]. Above all, the roles of inorganic such as magnetic iron oxide NP’s (MNP’s) by having unique and high performances are undeniable. For example, application of MNP’s in the polymer matrix leads to the improvements in the thermal and magnetic properties and open new windows for the preparation of the new magnetic materials namely, magnetic polyurethane foams[13]. Among all of merits, the surface energy of MNP’s is very high and tends to agglomerate and is very difficult dispersed uniformly in to the polymer matrix[14]. Surface modification of MNP’s is used to improve the performance of nanoparticles and create

Polímeros, 26(4), 297-303, 2016

a good linkage between inorganic filler and organic polymer matrices[15]. In this cage, coupling agents has been used for modification of magnetic iron oxide via non-magnetic shell formation of silica what can reduce the agglomerating, enhances thermal resistance of iron oxide nanoparticles and improve the compatibility between magnetic Fe3O4 NP’s and PU matrices[16,17]. Magnetic nanoparticles incorporated in to the polymer matrix by some methods, such as melting, solution and insitu polymerization which the later is the most common and well known method. The in-situ polymerization of MNP’s in the polymer matrix is an excellent method to control the mean size and size dispersion of a nanoparticle population, which are crucial factor in determination the properties of the nanocomposites[18]. In our previous work, magnetic polyurethane rigid foam nanocomposite were prepared by incorporation of Fe3O4@SiO2 NP’s in polymer matrix. The results indicate the performance of MNP’s in enhancing of the thermal resistances, storage modulus, and magnetic properties of filled rigid foam in comparison with pure PU[5]. In this study, the super paramagnetic Fe3O4@AEAP NP’s incorporated in to the PU flexible foam. In order to improve the dispersion of Fe3O4 NP’s in PU matrix and compatibility between Fe3O4 NP’s and PU matrix, NP’s were modified with AEAP and Fe3O4@AEAP-PU flexible foam nanocomposites were prepared via in-situ polymerization. Our data showed superior and significant thermal stability and magnetic properties of resultant foams when MNP’s incorporated up to 3.0%.

2. Experimental 2.1 Materials Daltoflex EC 20240 formulated virgin polyol. The polyol (propylene oxide–ethylene oxide copolyether) as a colorless viscous liquid, having viscosity 1.250 Pa s at 208 °C, specific gravity 1.035 g/cm3 at 208 °C, fire point 240 °C, Mw 1900,

297

S S S S S S S S S S S S S S S S S S

Nikje, M. M. A., Moghaddam, S. T., & Noruzian, M. functionality 2 and hydroxyl numbers 59 mg KOH/g. Isocyanate was Suprasec 2027 diphenylmethanediisocyanate (MDI)-based prepolymer (dark brown liquid, having viscosity 0.220 Pa s at 258 °C, specific gravity 1.23 g/cm3 at 258 °C, NCO value 30.9% by weight of NCO groups analysis (group weight: 42 g/mol), average functionality 2.7, flash point 233 °C, and fire point 2458C). Daltoflex EC 20240 and Suprasec 2027 are chlorofluorocarbon (CFC) free systems, purchased from Huntsman Company with starting formulation as Daltoflex EC 20240: 100 pbw and Suprasec 2027: 65 pbw. The following reagents were purchased from Merck and used as received without further purification: Iron (II) chloride tetrahydrate (FeCl2·4H2O, 99.7%), iron (III) chloride hexahydrate (FeCl3·6H2O, 99.0%), ammonia (NH3.H2O, 25%-28%), ethanol (C2H5OH 99.7%), n-(2‑aminoethyl)-3-aminopropyl trimethoxysilane (AEAP) and citric acid.

2.2 Instruments Morphology studies and particle size of magnetic nanoparticles (Fe3O4) were done on a field emission scanning electron microscopy (Hitachi model Se 4160). Fourier transform infrared spectroscopy (FT-IR) spectra were done on a Bruker Tensor 27 spectrophotometer. The thermogravimetric analysis (TGA) of Fe3O4 and Fe3O4@AEAP NP’s and magnetic nanocomposite were performed on a Perkin-Elmer Paris Diamond TG/DTG under N2 and O2 atmosphere at a heating rate of 10 °C/min. Thermal mechanical analysis (TMA) was carried out by using a Linseis TMA instrument (TP 1000, Germany) over a temperature range from -100 to 250 °C and in compression mode. Magnetic hysteresis loops of magnetic foams were measured via a vibration sample magnetometer (VSM). To disperse modified magnetic nanoparticles in the polymer matrix, an ultrasonic homogenizer (Hielscher, Up 200S, Germany) was used.

2.3 Synthesis of Fe3O4@AEAP nanoparticles The magnetic NP’s were prepared through a co‑precipitation method by the reaction of ferric and ferrous (2/1 in mol/mol) in ammonia solution as reported by elsewhere[5]. Fe3O4@ AEAP NP’s was synthesized in two steps which in the first step, magnetic NP’s (100mg) was dispersed in ethanol/water (5/1) (110 ml) and sonicated for 20 min by drop wise addition of acetic acid and adjusting of pH at 4. Then AEAP (0.3 ml) was added to the solution and the mixture was stirred mechanically at room temperature for an additional 2 h. Finally, the core-shell NP was separated and washed with distilled water (2× 100 ml), collected and dried at 50 °C in an oven overnight and characterized.

mixture turns creamy and starts to expand. Gel time is the first point of stable network formation by intensive allophanate cross-links as well as urethane. Rise time is the time between the start of the final mixing and the time of complete expansion of the foaming mass. At the tack-free time, the outer surface of the foam losses its adhesiveness and the foam can be removed from the mold. 2.4.2 Synthesis of Fe3O4@AEAP-PU flexible foam nanocomposite In the first step, modified MNP was dispersed in the polyol matrix in weight percents of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 by vigorous stirring for 3min and the pre-mixture was sonicated for 4 min via an ultrasonic probe to the formation of a homogeneous mixture. In the next step, nano ‑particulated polyol was hand mixed with MDI in a 250 ml paper cup at 10:6 (polyol/MDI) weight ratios. Finally the sample was kept at room temperature for 24h for complete post curing and further testing[5]. The reaction pathway of PU-flexible foam nanocomposite formation is shown in Scheme 1. As shown in scheme, by incorporation of AEAP modified magnetic nanoparticles in polyurethane matrix, the interaction between isocyanate group and amino‑group with formation of urea fragments have been created.

3. Result and Discussion 3.1 Characterizations of magnetic and Fe3O4@AEAP nanoparticles (FT-IR, TGA and SEM analysis) The AEAP coating on the Fe3O4 NP’s was confirmed by FT-IR spectroscopy (Figure 1). The stretching vibration frequencies at 480 and 582 Cm-1 are attributed to the Fe-O functional groups of magnetic nanoparticles. After the coating of AEAP to Fe3O4 NP’s, the Fe-O-Si band stretching vibration appeared at 584 Cm-1 and overlaps with Fe-O bands[19]. In addition, the bands at 1091 Cm-1 corresponded to the stretching vibration of the Si-O bond. The absorption band at 3479 Cm-1 in the spectrum of the Fe3O4@AEAP NP is attributed to the –NH group introduced from the AEAP. Furthermore, the presence of band at 2896 and 2972 Cm-1 are corresponded to the stretching vibration of C-H groups on AEAP[6,20]. The obtained results from FTIR studies resulted that the surface of the Fe3O4 NP’s was successfully modified with AEAP.

2.4.1 Foam processing

Another method for confirmation of surface modification is thermo-gravimetric analysis method. Figure 2 shows the TGA of pure MNP’s (Fe3O4) and Fe3O4@AEAP NP’s. The weight loss of pure MNP’s take place below 120 °C and calculated as 5% which attributed to the evaporation of water molecules[21]. In addition, at 700 °C the total weight losses of Fe3O4@AEAP NP are assigned as 22%. Taking in to account the weight loss of pure MNP’s, it could be expected that the content of AEAP moiety on the magnetic NP’s surface was about 17%.

When the isocyanate is mixed with the polyol, the exothermic chemical reactions start. The foam processing is followed by the cream time, gel time, rise time and tack‑free time[15]. The cream time is the first step and corresponds to the start of bubble rise and the time at which the clear

For morphology studies of Fe3O4@AEAP NP, SEM images has been used and data shown in Figure 3. It can be observed from the images that magnetic and Fe3O4@ AEAP particulates have uniform spherical shapes with the size in the range of 30-40 nm and 50-60 nm, respectively.

2.4 Synthesis of polymer

298

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Preparation of novel magnetic polyurethane foam nanocomposites by using core-shell nanoparticles

Scheme 1. The formation of PU flexible foam nanocomposites.

Figure 1. FT-IR spectra of (a) Fe 3O4 and (b) Fe 3O4@AEAP nanoparticles.

Figure 2. TGA curves for (a) Fe 3O 4 and (b) Fe 3O 4@AEAP nanoparticles.

3.2 Characterization of PU-flexible foams nanocomposite

groups have not completely reacted[22]. Finally, the bands at 2889 and 2976 Cm-1 are assigned to the asymmetrical C-H stretching and symmetrical stretching of polyether groups in the aliphatic chains, respectively[23,24].

3.2.1 FT-IR analysis The ATR spectra of Fe3O4@AEAP/PUF nanocomposite containing Fe3O4@AEAP NP’s (1.5 to 3.0%) are shown in the Figure 4. As shown in the spectra, the absorption at 1105 Cm-1 is related to –C-O functional group. Similarly, band at 1234 Cm-1 corresponded to the C-N functional groups (FG) of the urethane. Furthermore, it can be seen that all PU samples have similar vibration bond at 3348 Cm-1 (hydrogen-bonded stretching vibration of urethane groups), 1595 Cm-1 (N-H bending vibrations), and 1714 Cm-1 (C=O) what are corresponded to the urethane functional groups. In the meantime and as shown in figure, the –NCO groups appeared at 2293 Cm-1 and indicated that the isocyanate Polímeros, 26(4), 297-303, 2016

3.2.2 Morphology studies (SEM) Surface morphological analysis of the magnetic nanocomposite was done by scanning electron microscopy (SEM) and shown in Figure 5 and cell density (Nf) is calculated by using Equation 1[12]. In this equation, n is the number of cells, A the area of the micrograph in Cm2, and M is the magnification factor. As shown in the Table 1, by increasing of modified MNP’s from 1.5 to 3%, cell density was increased and cell size was reduced. These results indicate that the nature of the dispersion plays a fundamental role in controlling the cell size during foaming. 299

Nikje, M. M. A., Moghaddam, S. T., & Noruzian, M.

Figure 3. FE-SEM images of synthesized (a) Fe3O4 and (b) Fe3O4@AEAP nanoparticles.

to aid the bubble nucleation process during cell formation and enhances the cell densities. 3.2.3 Thermo-gravimetric analysis (TGA) In order to evaluate the role of MNP’s on thermal properties of PUF’s, TGA experiments were done on nanocomposite samples and data compared with pristine one data. As shown in Figure 6 and Table 3, by incorporation of Fe3O4@AEAP NP’s from 0.5 to 3.0%, the performed thermal stability was observed for 3% filled sample. This behavior can be interpreted that, MNP’s have high specific thermal capacity that caused to heat preservation, acts as a thermal insulator, delay the degradation process and reduces the heat conduction to the PU matrix. 3.2.4 Magnetic properties analysis (VSM)

Figure 4. IR-ATR spectra of Fe3O4@AEAP-PUF foams, with different content of Fe3O4@AEAP: (a) 0; (b) 1.5 and (c) 3.0%. Table 1. Cell densities of PUF foams. NP’s (%) Nf (Cells/Cm3)×105

a(0.0) 0.198

b(1.5) 0.298

c (3.0) 0.364

3.2.5 Thermo-mechanical analysis (TMA)

 nM 2  2 N f =   A   

(1)

In addition, the foam density (D) is calculated using the Equation 2: D=

M V

(2)

In this equation, M is mass (gr) and V is the volume (Cm3) of the foam, respectively. As shown in Table 2, by increasing of the nanoparticles content, the foam density is increased. In the other words, foam densities is controlled by the competitive process between the cell nucleation, its growth, and coalescence and reveal that the nucleation process occurred in the well-dispersed modified MNP’s in the polymer matrix. In addition, MNP’s act as nucleation site 300

Figure 7, presented the magnetic hysteresis loops of the Fe3O4@AEAP-PU flexible foams nanocomposite with different content of Fe3O4@AEAP NP’s. From magnetic hysteresis loops, when the content of MNP’s varied from 1.5 to 3.0%, the saturation magnetization (Ms) is raised from 0.52 to 0.64 emu/g, respectively which indicate nanocomposite reveals super paramagnetic behavior, because no remanence magnetization is observed. Furthermore, magnetic nanocomposite could be magnetized and modulated via an external magnetic field. Linear thermal expansion coefficient (α) and glass transition temperature (Tg) are two important measurements in the thermal analysis of the polymers and expected by TMA results. In this study, α is calculated according to Equation 3, where dL is length changes, dT is temperature changes and L0 is initial length of the sample. Thermo-mechanical behaviors of the nanocomposite samples are shown in the Figure 8. As shown in Figure 8 and 9, by incorporation of modified MNP’s from 1.5 to 3.0% in PU matrix, the Tg value was decreased in comparison with pure foam. Additional, reduction of Tg in modified MNP’s samples affected by some factors, such as low cross link density, restriction of hard phase domain and enhance in chain mobility. ∝=

dL dT × L0

(3)

Polímeros, 26(4), 297-303, 2016

Preparation of novel magnetic polyurethane foam nanocomposites by using core-shell nanoparticles

Figure 5. FE-SEM images of PUF foams nanocomposite with different contents of Fe3O4@AEAP: (a) 0.0; (b) 1.5 and (c) 3.0%.

Figure 6. TGA curves of PUF foam nanocomposites with different contents of Fe3O4@AEAP nanoparticles: (a) 0.0; (b) 0.5; (c) 1.0; (d) 1.5; (e) 2.0; (f) 2.5 and (g) 3.0%. PolĂmeros, 26(4), 297-303, 2016

Figure 7. Magnetization hysteresis loops of Fe3O4@AEAP-PUF foams with different contenst of Fe3O4@AEAP nanoparticles: (a)Â 0.0; (b) 1.5 and (c) 3.0%. 301

Nikje, M. M. A., Moghaddam, S. T., & Noruzian, M. Table 2. Foam densities of prepared PUF foams. Samples (%) Density (g/cm3)

a(0.0) 0.027

b(0.5) 0.038

Table 3. T5%, T10%, Tmax and ash content (%).

c(1.0) 0.042

Fe3O4@AEAP

T5%

T10%

Tmax

Ash content

(%)

(°C)

(%)

a (0.0) b (0.5) c (1.0) d (1.5) e (2.0) f (2.5) g (3.0)

258.01 259.66 262.57 263.12 261.93 264.36 265.70

265.54 268.01 268.73 270.05 270.90 271.27 272.70

765.02 764.80 767.60 765.25 764.88 766.76 767.75

0.14 1.11 3.31 6.59 7.46 9.35 11.63

d(1.5) 0.047

e(2.0) 0.052

f(2.5) 0.055

g(3.0) 0.058

groups on MNP’s surface improved the interaction between inorganic nanofiller and polymer matrix. The results of the thermo-gravimetric analysis showed thermal stability of MNP’s nanocomposite enhanced because of MNP’s act as a thermal barrier. TMA results Tg decrements due to reduction of hard phases domain and limitation of soft segment mobility and freedom. The FE-SEM images showed that the modified MNP’s acted as the nucleation sites during cell formulation and led to the cell size decrements as well as cell density increments. Finally VSM results indicated super paramagnetic behavior for nanocomposites.

5. Acknowledgements The authors thank Imam Khomeini International University (IKIU) for the financial supporting of Dr. Alavi Nikje.

6. References

Figure 8. TMA curves of PUF foam nanocomposites with different contents of Fe3O4@AEAP: (a) 0.0; (b) 1.5 and (c) 3.0%.

Figure 9. Glass transition temperature (T g) of PUF foam nanocomposite with different percent of MNP’s (0.0, 1.5 and 3.0).

4. Conclusion In summary, magnetic polyurethane flexible foam nanocomposites are prepared by incorporation of synthesized Fe3O4@AEAP NP’s in polymer matrix via in-situ polymerization. The presence of reactive functional 302

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Preparation of novel magnetic polyurethane foam nanocomposites by using core-shell nanoparticles effects on polyurethane foam sound absorption. Journal of Polymer Research, 20(2), 1-10. http://dx.doi.org/10.1007/ s10965-012-0072-6. 10. Verdejo, R., Stämpfli, R., Alvarez-Lainez, M., Mourad, S., Rodriguez-Perez, M. A., Brühwiler, P. A., & Shaffer, M. (2009). Enhanced acoustic damping in flexible polyurethane foams filled with carbon nanotubes. Composites Science and Technology, 69(10), 1564-1569. http://dx.doi.org/10.1016/j. compscitech.2008.07.003. 11. Gao, X., Guo, Y., Tian, Y., Li, S., Zhou, S., & Wang, Z. (2011). Synthesis and characterization of polyurethane/zinc borate nanocomposites. Colloids and Surfaces. A, Physicochemical and Engineering Aspects, 384(1-3), 2-8. http://dx.doi.org/10.1016/j. colsurfa.2010.11.037. 12. Saha, M. C., Kabir, M. E., & Jeelani, S. (2008). Enhancement in thermal and mechanical properties of polyurethane foam infused with nanoparticles. Materials Science and Engineering A, 479(1-2), 213-222. http://dx.doi.org/10.1016/j.msea.2007.06.060. 13. Zhou, L., Li, G., An, T., & Li, Y. (2010). Synthesis and characterization of novel magnetic Fe3O4/polyurethane foam composite applied to the carrier of immobilized microorganisms for wastewater treatment. Research on Chemical Intermediates, 36(3), 277-288. http://dx.doi.org/10.1007/s11164-010-0134-5. 14. Cao, H., He, J., Deng, L., & Gao, X. (2009). Fabrication of cyclodextrin-functionalized superparamagnetic Fe3O4/aminosilane core–shell nanoparticles via layer-by-layer method. Applied Surface Science, 255(18), 7974-7980. http://dx.doi. org/10.1016/j.apsusc.2009.04.199. 15. Chi Hao, L., Haji Badri, K., & Haji Ahmad, S. (2011). Mechanichal and thermal properties of palm-based polyurethane composites filled with Fe3O4, PANI and PANI/Fe3O4. Sains Malaysiana, 40(4), 373-378. 16. Morel, A. L., Nikitenko, S. I., Gionnet, K., Wattiaux, A., LaiKee-Him, J., Labrugere, C., Chevalier, B., Deleris, G., Petibois, C., Brisson, A., & Simonoff, M. (2008). Sonochemical approach to the synthesis of Fe3O4@SiO2 core-shell nanoparticles with tunable properties. ACS Nano, 2(5), 847-856. PMid:19206481. http://dx.doi.org/10.1021/nn800091q. 17. Lu, Y., Yin, Y., Mayers, B. T., & Xia, Y. (2002). Modifying the surface properties of superparamagnetic iron oxide nanoparticles

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throuth a sol-gel approach. Nano Letters, 2(3), 183-186. http:// dx.doi.org/10.1021/nl015681q. 18. Ramos, J., Millán, A., & Palacio, F. (2000). Production of magnetic nanoparticles in a polyvinylpyridine matrix. Polymer, 41(24), 8461-8464. http://dx.doi.org/10.1016/S00323861(00)00272-X. 19. Shen, M., Cai, H., Wang, X., Cao, X., Li, K., Wang, S. H., Guo, R., Zheng, L., Zhang, G., & Shi, X. (2012). Facile one-pot preparation, surface functionalization, and toxicity assay of APTS-coated iron oxide nanoparticles. Nanotechnology, 23(10), 105601. http://dx.doi.org/10.1088/0957-4484/23/10/105601. 20. Nikje, M. M. A., & Tehrani, Z. M. (2010). Thermal and mechanical properties of polyurethane rigid foam/modified nanosilica composite. Polymer Engineering and Science, 5(3), 468-473. http://dx.doi.org/10.1002/pen.21559. 21. Feng, B., Hong, R. Y., Wang, L. S., Guo, L., Li, H. Z., Ding, J., Zheng, Y., & Wei, D. G. (2008). Synthesis of Fe3O4/ APTES/PEG diacid functionalized magnetic nanoparticles for MR imaging. Colloids and Surfaces. A, Physicochemical and Engineering Aspects, 328(1–3), 52-59. http://dx.doi. org/10.1016/j.colsurfa.2008.06.024. 22. Narine, S., Kong, X., Bouzidi, L., & Sporns, P. (2007). Physical Properties of Polyurethanes Produced from Polyols from Seed Oils: II. Foams. Journal of the American Oil Chemists’ Society, 84(1), 65-72. http://dx.doi.org/10.1007/s11746-006-1008-2. 23. Jalilian, M., Yeganeh, H., & Haghighi, M. N. (2008). Synthesis and properties of polyurethane networks derived from new soybean oil-based polyol and a bulky blocked polyisocyanate. Polymer International, 57(12), 1385-1394. http://dx.doi. org/10.1002/pi.2485. 24. Piszczyk, Ł., Strankowski, M., Danowska, M., Haponiuk, J. T., & Gazda, M. (2012). Preparation and characterization of rigid polyurethane–polyglycerol nanocomposite foams. European Polymer Journal, 48(10), 1726-1733. http://dx.doi. org/10.1016/j.eurpolymj.2012.07.001. Received: June 19, 2015 Revised: Nov. 06, 2015 Accepted: Mar. 15, 2016

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http://dx.doi.org/10.1590/0104-1428.2324

S S S S S S S S S S S S S S S S S S

α-Tocopherol loaded thermosensitive polymer nanoparticles: preparation, in vitro release and antioxidant properties Cirley Quintero1, Ricardo Vera1 and Leon Dario Perez2* Departamento de Química, Pontificia Universidad Javeriana, Bogotá, D.C., Colombia Grupo de Macromoléculas, Departamento de Química, Universidad Nacional de Colombia – UNAL, Bogotá, D.C., Colombia 1

*ldperezp@unal.edu.co

Abstract α-Tocopherol is the most bioavailable and active compound found in vitamin E with potential application in pharmaceutical, alimentary and cosmetic industries. However, its low solubility in aqueous medium and environmental instability limit its dosage. In this paper, we report the preparation of α-tocopherol loaded nanoparticles (TOC-NP) based on amphiphilic thermosensitive triblock copolymers PNIPAM-b-PCL-b-PNIPAM. The nanoparticles exhibited a core – shell structure, were positively charged and presented average diameters below 300 nm. TOC-NP presented controlled release of α-tocopherol at room temperature along 140h, and exhibited antioxidant properties in aqueous medium. Keywords: α-tocopherol, polycaprolactone, thermosensitive nanoparticles, triblock copolymer.

1. Introduction α-tocopherol (TOC), the most studied and bioavailable component of vitamin E, presents antioxidant activity, its consumption is widely considered to help to reduce risk of many chronic diseases associated to oxidative stress[1]. Thus, TOC is a widely used component in functional food, cosmetic and pharmaceutical industries. However, the design of proper dosage forms is still challenging due to its hydrophobicity and well-known sensitivity to oxygen and light. In order to overcome its environmental susceptibility, and improve its solubility in aqueous medium, encapsulation has been recently exploited by several authors[2-4]. The use of nanoparticles besides improving solubility in water, also provides controlled releasing. Some materials such as gliadin nanoparticles[5], self-assembled nanoparticles of tocopheryl monoesters modified-chitosan conjugates[6], and chitosan nanoparticles coated with zein[7] have been reported. Polymer nanoparticles (NP) have recently received great attention in the encapsulation and controlled release of bioactive substances[8]. They are obtained by the aggregation of water insoluble polymers in aqueous medium, assisted by different techniques such as solvent evaporation, salting‑out, dialysis and supercritical fluids[9]. Nevertheless, these aggregates are in most of the cases colloidally unstable, and require the use of stabilizers such as surfactants and hydrophilic polymers which making their preparation a complex process dependent on many parameters such as stabilizer concentration, ionic strength, the co-solvent characteristics, and mixing speed. Analogous to low molecular weight surfactants, amphiphilic block copolymers composed of hydrophilic and hydrophobic segments self-assemble in aqueous medium via hydrophobic association, which enables the preparation of NP[10,11]. The presence of hydrophobic domains confers

304

the resulting nanoparticles the ability of encapsulating hydrophobic substances. On the other hand, the hydrophilic block besides conferring colloidal stability to the NP, can also lead the particles to exhibit stimuli sensitive responses. For example, there are several reports about polymer nanostructures containing PNIPAM which endows them thermosensitivity, since it exhibits a lower critical solution temperature at 32 °C[12]. The implementation of technologies based in block copolymers is stimulated by advances in controlled radical polymerizations. Atom transference radical polymerization (ATRP) is one of the most versatile and powerful techniques that allows obtaining materials with controlled composition, and molecular weight and dispersity. The essential feature of ATRP is the equilibrium between a low concentration of active propagating species, and a larger number of dormant chains ended, via an inner sphere electron transfer process promoted by a transition metal complex. The main drawback of using ATRP is the removal of oxygen traces that requires tedious degasification process to avoid the catalyst deactivation. The development of Activator ReGenerated by Electron Transfer (ARGET) ATRP, which can be conducted in the presence of oxygen, enables the synthesis of novel structures such as bioconjugated materials and functional surfaces[13]. In the present work, we report the preparation and characterization of thermosensitive α-Tocopherol-loaded polymer nanoparticles (TOC-NP). The nanostructures were obtained from triblock copolymers PNIPAM-b-PCL-bPNIPAM synthesized via ARGETATRP. Polycaprolactone (PCL), is a semicrystalline aliphatic polyester, considered as a biocompatible, and bioresorbable material with high permeability to drugs approved by the FDA for biomedical

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α-Tocopherol loaded thermosensitive polymer nanoparticles: preparation, in vitro release and antioxidant properties applications[14-17]. On the other hand, PNIPAM is the most widely used thermosensitive polymer, the proximity of its transition temperature to physiological values have encourage its application in the fabrication of several biomedical devices[18].

2. Materials and Methods 2.1 Materials N-Isopropylacrylamide (NIPAM, 98%) was recrystallized from hexane, α,ω-dihydroxy-Poly(ε-caprolactone) of MN =10 and 45 kDa was purified by precipitation from a mixture of THF and methanol. 2-Bromoisobutyryl Bromide (BIBB, 98%), triethylamine (>99%), cupper(II) bromide (CuBr2, 98%) N,N,N’,N”,N”-Pentamethyldiethylenetriamine (PMDETA, 99%), anisole and α-tocopherol were used without any additional purification. All the reagents including solvents (hexane, acetone, methanol and tetrahydrofuran) were supplied by Sigma - Aldrich.

2.2 Synthesis procedures

2.4 Preparation of α-tocopherol loaded Nanoparticles (TOC-NP) The preparation of α-tocopherol loaded nanoparticles was as follows: 10 mg of α-tocopherol and 50 mg of the corresponding copolymer were dissolved in 5 mL of acetone. The resulting solution was slowly droplet (100µL/min) into 10.0 mL of an aqueous HCl solution pH 5.0. The resulting dispersion was gently stirred during 1 hour under reduced pressure to eliminate acetone residuals. Finally, the aqueous suspensions were lyophilized, rinsed with hexane at room temperature to eliminate non-encapsulated α-tocopherol. The amount of encapsulated α-tocopherol was determined dissolving the polymer samples in acetone, and analyzing the content of this substance by HPLC. The loading-efficiency (DLE%) as well as the drug content (DLC%) were estimated using as follows:

Encapsulation Amount of TOC inthe NP = ×100 (1) Eficience ( % ) Amount of TOC used inthe preparation

Synthesis of dibromide ended PCL: Br-PCL-Br was synthesized using a protocol previously published[19]. In a typical procedure for PCL Mn=10 KDa, 5 g of diol-ended PCL (0.35 mmol) was dissolved in 50 mL of anhydrous dichloromethane with 500 μL of triethylamine (3.5 mmol) whereas stirring under argon atmosphere. After that, 431μL of BIBB (3.5 mmol) is added drop wise to the above‑mentioned solution, previously cooled using an ice-water bath. The reaction mixture was stirred during 24h at room temperature. The product was precipitated by the addition of an excess of methanol, recovered by filtration, and finally submitted to three precipitation cycles. The same procedure was used to modify PCL of 45KDa. Synthesis of PNIPAM-b-PCL-PNIPAM: A typical protocol for the synthesis of copolymer 18:88:18 via ARGET-ATRP of NIPAM using Br-PCL-Br (Mn=10 kDa) as macroinitiator, and Cu(II)Br2/ PMDETA was as follows: In a reaction vessel, Br-PCL-Br (0.5 g, 36 μmol) was dissolved in 5 mL of anisole to which NIPAM (0.384 g, 3.4 mmol) and PMDETA (12.5 mg, 72 μmol) and CuBr2 (5.2 mg, 36μmol) were added. The system was purged with argon during 15 min. Then, Tin(II) 2-ethylhexanoate (Sn(EH)2) (72 mg, 180μmol) was added, the system was degassed by 5 min, and the polymerization reaction was allowed to proceed at 80 °C under argon atmosphere during 4h. The reaction product was passed through a column packed with basic alumina to eliminate cupper and tin residuals, and purified by three successive precipitations from a THF solution by the addition of methanol.

2.3 Copolymers characterization 1 HNMR spectra were collected in a Bruker Avance III spectrometer operated at 300 MHz. Samples were dissolved in CDCl3 and the spectra were recorded at 303 K. Chemical shifts (δ) were expressed in ppm respect to the CDCl3 signals. Molecular weight and distribution were measured by GPC in a Waters HPLC equipped with a differential refraction

Polímeros, 26(4), 304-312, 2016

index detector. The analyses were performed in THF at a flow rate of 0.8 mL/min using a HR 4E column.

Amount of TOC inthe NP Encapsulation of AT ( % ) = (2) mass of polymer + TOC

2.5 Nanoparticles characterization Particles average diameter was determined by Dynamic Light Scattering (DLS) using a Horiba LB 550 equipment. The measurements were carried out at 23 °C in aqueous dilutions of the samples (≈1/20) prepared using deionized water (~18 MΩ cm), in order to avoid particle – particle interactions, and multiple scattering effects, each dispersion plot corresponds to an average of 128 measurements acquired during 2 seconds. ζ potential was measured using a zeta potential analyzer Malvern Zetasizer Nano ZS. ζ potential was determined three times for each sample. For TEM analysis, 2 µL of the diluted samples (0.1 mg/mL) was spilled into a cupper grid Formvar coated, and dried at room temperature during 24h, the images were obtained in a Jeol 1400 plus microscope.

2.6 In vitro release of de α-tocopherol In vitro release of TOC from TOC-NP’s was studied based on a protocol published by Wang et al.[20]. Briefly, 4.0 mL of the α-tocopherol loaded nanoparticles dispersed in PBS pH 7.4 (4 mg of TOC-PNP´s/1 mL of buffer) were transferred to a dialysis bag (MWCO 12 kDa), and placed in 50.0 mL PBS pH 7.4 supplemented with SDS 5wt% to guarantee sink conditions. At selected time intervals, 2 mL of the release medium (outside the dialysis bag) was extracted and analyzed by HPLC to determine the concentration of α-tocopherol, and replace with equal volume of fresh releasing medium.

2.7 In vitro antioxidant activity evaluation The antioxidant activity of TOC-PNP’s was determined using 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) based on methods previously reported[21,22]. 305

Quintero, C., Vera, R., & Perez, L. D. Briefly, a stock solution of ABTS radical cation was prepared dissolving 20 mg of ABTS in 10 mL of deionized water, and then 2.5 mg of potassium persulfate K2S2O8 was dissolved. The resulting solution was stored at room temperature during 16 h in a dark bottle. Dilutions of ABTS with absorbance equal to 0.70± 0.02 at 740 nm containing different concentrations of the nanoparticles were prepared. The absorbance of ABTS+⋅ was monitored at 740 nm each 60 s using a microplate spectrometer FluorStar omega. The radical scavenging activity of TOC-NP was estimated as the inhibition percentage as shown in Equation 3. Inhibition ( % ) =

A0 − At x100 A0

(3)

Where A0 and At correspond to the initial and stationary absorbance (at 60 min), respectively. The trolox equivalent antioxidant capacity (TEAC) value represents the ratio between the slope of the plot inhibition (%) vs concentration of TOC-NP under investigation, compared with the slope of this plot for ABTS+⋅ scavenging by the water-soluble vitamin E analogue Trolox, used as an antioxidant standard.

radical polymerization (ATRP). NIPAM was polymerized using a dibromide-ended polycaprolactone (Br-PCL-Br) as macroinitiator and CuBr2/ PMDETA as a catalyst, as shown in Scheme 1. Two α,ω-dihydroxy-PCL samples with different Mn values of 10 and 45 kDa were transformed into Br-PCL‑Br by reacting with an excess of BIBB. The reaction was assessed by 1HNMR; the anchorage of bromoisobutyryl groups was indicated by the absence of the signal at 3.62 ppm of the hydroxymethylene terminal groups of PCL, and also by the presence of a new signal at 1.96 ppm due to methyl groups of BIBB. Triblock copolymers PNIPAM-b-PCL-b-PNIPAM containing PCL of 10 and 45 kDa and PNIPAM were synthesized using the fee reagent molar ratios and reaction times listed in Table 1. The block copolymerization was confirmed by 1HNMR and GPC. The complete signal assignation is presented in Figure 1. The average number molecular weight of the copolymers (Mn), and the polymerization degree of NIPAM (XNIPAM) relative to PCL were estimated from the 1 HNMR, using equations showed below: X NIPAM =

3. Results and Discussions 3.1 Synthesis of triblock copolymers PNIPAM-b-PCL-bPNIPAM

If 3

M n PCL 114

(4)

M n = M n PCL + (113.16 × X NIPAM )

Amphiphilic triblock copolymers PNIPAM-b-PCL-bPNIPAM were synthesized by using an activator regenerated by electron transfer (ARGET) process for atom transfer

(5)

Where If is the ratio of the intensity of signals at 1.6 ppm that corresponds to methylene (CH3)2-C groups of PNIPAM, and 2.40 ppm assigned to –CH2- of PCL.

Scheme 1. Synthesis of Polycaprolactone ended in Bromide groups and its block copolymerization with N-isopropylacrylamide via ATRP. Table 1. Molar ratio of the monomers in the fee and copolymers, Mn of the copolymers estimated by 1HNMR and molecular weight dispersity obtained by GPC. Sample

Br-PCL-Br:NIPAM in the feed

Copolymers Composition*

27:88:27 17:88:17 108:394:108

1:246 1:123 1:320

54:88 34:88 394:216

Mn (kDa) 16.03 13.90 69.36

PDI 1.21 1.32 1.25

*molar ratio of the monomers in the copolymers CL:NIPAM.

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α-Tocopherol loaded thermosensitive polymer nanoparticles: preparation, in vitro release and antioxidant properties

Figure 1. 1HNMR spectra for copolymer 108:394:108 showing the signal assignation.

3.2 Characterization of the nanoparticles In aqueous medium, amphiphilic block copolymers self-assemble by association of the hydrophobic blocks giving several types of nanoparticles. Although numerous morphologies have been reported, the most simple and investigated situation is the formation of hydrophobic cores and hydrophilic shells. Analogous to low molecular weight, amphiphilic block copolymers self-assemble forming polymer micelles, although PM’s can also exhibits a dynamic character resembling micelles obtained from low molecular weight surfactants. PM’s are often not dynamic depending on the hydrophobic/hydrophilic ratio and also the molecular weight of the copolymer. When the molecular weight of the hydrophilic block exceeds that of the hydrophobic block, the copolymer is easily dispersed in water and will self-assemble into small, relatively monodisperse micelles which are dynamic. However, as the molecular weight of the hydrophobic block approaches or exceeds the molecular weight of the hydrophilic block the polymer chains are insoluble limiting unimers exchange equilibrium that characterizes micellar dispersions[23]. In this work, NP were obtained using amphiphilic block copolymers PNIPAM-b-PCL-b-PNIPAM as precursors at pH 5.0 and 20 °C at which PNIPAM displays hydrophilic behavior. For all the samples, even at concentration of the polymer lower than 1 mg/L, and using pyrene as fluorescent probe, dissolution of the particles was not detected indicating that unimers exchange is quite slow, and therefore not observed under the experimental conditions. This behavior can be due to the fact the copolymer chains are poorly soluble in aqueous medium, as deduced from the composition of the samples presented in Table 2 where it is observed that the hydrophobic segment (PCL) exceeds the length of the hydrophilic ones. The size of NP’s obtained by nanoprecipitation was studied by DLS. Figure 2A-D shows the particle distribution and the corresponding TEM images as insets, the average diameter and dispersity index (PDI) values are summarized in Table 2. It is observed that the average particle diameter and its PDI depend on the composition of the copolymers. Polímeros, 26(4), 304-312, 2016

Table 2. Average diameter of the particles measured by DLS and the corresponding ζ potential values. Sample 27:88:27 17:88:17 108:394:108

Average diameter (nm) 281 178 44.4

Polydispersity 0.14 0.68 0.07

ζ potential (mV) +5.5 +3.5 +10.5

The smallest particles correspond to the copolymer containing the longest PCL block. Also, the largest PNIPAM segment leads to larger particles. The relationship between the size of the particles and the composition of the block copolymers can be understood based on the mechanism for particle formation. It is that first individual molecules rapidly associate via nucleation, and growth until the particles have reached a size where further growth increases the free energy of the system[23]. Copolymer 108:394:108 is quite insoluble in water, therefore its chains aggregate at very low concentration, leading to the formation of small particles. TEM images indicate that the nanoparticles obtained at 20 °C are spherical and exhibits a core-shell structure. For all the samples, primary particles smaller than 100 nm are forming aggregates. Particle size distribution of PN of copolymer 108:394:108 prepared at 40 °C is shown in Figure 2D. Compared to the corresponding nanoparticles obtained at 20 °C, the particles obtained at higher temperature exhibited a broader distribution, and also a significantly larger particle average size. TEM image (inset Figure 2D) indicates that the sample is composed by amorphous particles. This behavior obeys to the thermosensitive nature of PNIMAN, at 20°C it is hydrophilic and provides the particles stability. The surface charge of the particles was determined by measuring its ζ potential, the corresponding values are listed in Table 2. At all the composition the ζ potential was positive, which indicates that at the conditions at which the measurement was carried out, the PNIPAM segments are protonated. The results also indicate that ζ potential also depends on length of the hydrophilic segment. 307

Quintero, C., Vera, R., & Perez, L. D. Figure 3A shows the temperature-dependent optical transmittance at 540 nm obtained for nanoparticles dispersions in the range of 20 to 40 °C. It is observed that as the temperature increases, the transmittance of light decreases, which indicates that the turbidity of the samples increases, this behavior agrees with the lower critical solution temperature (c.a. 32°) of PNIPAM widely reported. These results probe that the nanoparticles are

thermosensitive and also corroborate the previously describe core shell morphology. As a consequence of the thermosensitive behavior of TOC-NP endowed by the presence of PNIPAM, the solubility of the nanoparticles can be altered by increasing the temperature. According to Figure 3B, at 20 °C the particles disperse well in water, but at 40 °C the particles are become more compatible with an organic non-polar phase such as decane.

Figure 2. Particle size distribution obtained by DLS and the corresponding TEM image as inset obtained at 20 °C for copolymer (A) 17:88:17; (B) 27:88:27; and (C) 108:394:108; (D) corresponds to particles obtained from copolymer 108:394:108 at 40 °C.

Figure 3. Thermosensitive behavior of TOC-NP. (A) optical transmittance of aqueous dispersions of the NP in the range of 20 to 40 °C; (B) Photographs showing the behavior of aqueous dispersion of the NP at 20 and 40 °C. 308

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α-Tocopherol loaded thermosensitive polymer nanoparticles: preparation, in vitro release and antioxidant properties 3.3 Thermal characterization of the nanoparticles

From Figures 4C, D, it is observed that the intensity of the melting peak of PCL in the NP´s is minor compared with PCL homopolymer, indicating a lower crystallinity of the hydrophobic domains. The weight percentage of crystalline PCL in each one of the samples calculated from Equation 6 is listed in Table 3.

DSC experiments were performed heating the samples from room temperature to 150 °C at 20 °C/min to erase their thermal history and eliminate any volatile interfere. Then the samples were cooled at 10 °C/min to –50 °C, the Tc was taken as the temperature corresponding to the maximum of the exothermic crystallization peak (Figure 4A, C). ∆H m The melting temperature TM and the melting enthalpy= (6) χ ×100 0 X PCL × ∆H m were measure during a second heating at 10 °C/min up to 100 °C as the temperature corresponding to the minimum where ΔHm is the apparent heat of fusion per gram of the of the melting peak (Figure 4C, D), and the area under the nanoparticles, XPCL is the corresponding weight fraction peak, respectively. of PCL obtained from the sample compositions listed in Compared to bulk PCL, the melting temperature of PCL Table 1. ΔHm° is the thermodynamic heat of fusion per in NP is evidently low, which may be due to imperfections gram of completely crystalline PCL and was assumed to or reduction of the lamellar thickness of PCL due to the be 135.31 J/g[27]. [24,25] nanoconfinement . It agrees with the decrease of crystallization temperature (Tc) of PCL in the nanoparticles 3.4 Encapsulation of α-tocopherol and in vitro release compared to bulk PCL that characterizes crystallization in The loading-efficiency (TLE) and the TOC content confined structures. Hamley et al. studied the crystallization (TLC) determined by Equations 1 and 2, respectively, are of diblock copolymers composed by Polyethylene and either summarized in Table 4. It is observed that the nanoparticles a rubbery or glassy segment. In the last case, the authors obtained from the copolymer with the longest PCL segment found that the presence of rigid walls significantly retards the crystallization process[26]. presents larger TOC and TLC values. TOC-NPs were prepared

Figure 4. DSC thermograms obtained for the nanoparticles and PCL bulk (A, B) cooling and (C, D) heating. Polímeros, 26(4), 304-312, 2016

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Quintero, C., Vera, R., & Perez, L. D. Table 3. DSC characterization of the samples and their corresponding PCL precursors. Sample PCL-10 26:88:26 17:88:17 PCL-45 108:384:108

∆Hm

J/g 67.6 9.08 22.89 68.0 14.7

°C 57.5 53.3 52.8 56.1 52.0

°C 20.5 15.0 18.2 29.0 20.5

XPCL 1 0.61 0.72 1 0.64

χ wt% 50.0 11.0 23.5 50.3 17.0

Table 4. Encapsulation of TOC and antioxidant properties. TOC encapsulation Sample

Loading (%)

Efficiency (%)

8.1 8.5 10.1

40.5 42.5 50.5

26:88:26 17:88:17 108:384:108

Antioxidant Properties IC50 TEAC* (mg TOC-NP/L) 545 ± 54 0.69 ± 0.05 472 ± 21 0.71 ± 0.01 406 ± 18 1.15 ± 0.19

*Trolox Equivalent Antioxidant capacity expressed as mmol of trolox/weight (g) of TOC-NP.

by nanoprecipitation, both copolymer and tocopherol are dissolved in acetone, and then added dropwise into an aqueous solution at pH 5.0. Under these conditions, the encapsulation of TOC is due to its partition between the aqueous phase, and the nucleus of the forming particles. The low solubility of TOC in water facilitates its encapsulation in the hydrophobic PCL domains generated on the self‑assembly of the copolymers. In a previous research, it was found that hydrophobicity of cores of micelles obtained from block copolymers containing PCL, increases with me molecular weight of PCL. Assuming the same tendency, the larger values of encapsulation parameters obtained for copolymer 108:394:108 are due to a more hydrophobic particles core which summed to its low crystallinity enables the encapsulation of TOC. Nanoparticles based on PNIPAM‑b-PCL-b-PNIPAM allowed obtaining larger encapsulation percentages than the values early reported for polymer nanoparticles obtained from PCL homopolymer[28]. Release profiles of TOC from TOC-NP under sink conditions are shown in Figure 5. The plots show that these formulations exhibit a controlled release along 140 h. For each sample, the release profiles exhibits two different regions, an initial burst during the first 20 h, and then a minor release rate. After 150 h, the cumulative release was larger than 70% for all the formulations, but only NP obtained from copolymer 108:384:108 showed a cumulative release close to 100%.

3.5 In vitro antioxidant activity The antioxidant activity of TOC-NP’s was tested using the trolox equivalent antioxidant capacity (TEAC)[21]. In this test, the relative capacity of encapsulated TOC to scavenge the ABTS+⋅ radical - cation is compared to Trolox which is a hydrosoluble compound structurally analogous, and used as a standard. ABTS+⋅ radical cation in aqueous solution and in absence of light is stable for several hours. The strong absorption of this species at 732 nm permits sensing it residual concentration as it reacts with TOC. A typical plot of ABTS+⋅ inhibition percentage as function of time at different concentrations of TOC-NP is shown Figure 6A the 310

Figure 5. Release profiles of TOC from TOC-NP measured at room temperature under sink conditions.

inhibition at each concentration was taken at 60 min, at that time we obtained a better data correlation. The concentration of TOC-NP necessary to inhibit 50% of ABTS species was interpolated from the plots of inhibition percentage vs concentration. The corresponding values are listed in Table 4, the IC50 for all the samples was in the range of 400- 600 mg of TOC-NP/L. The antioxidant activity measurements of AT-NP dispersions, expressed as Trolox equivalent antioxidant capacity (TEAC) are presented in Table 4. TOC is known to be a lipophilic antioxidant with limited solubility in water. However, our results indicate that TOC-NP exhibits antioxidant properties in aqueous medium. Presumably, the small size of the particles and therefore their high surface area enables encapsulated TOC to react with ABTS+⋅. Two phenomena could account for the antioxidant properties of TOC-NP; first, free radicals could diffuse through the nanoparticles taking advantage of the low crystallinity of PCL hydrophobic domains, and also the surface characteristics of the particles promotes a fast Polímeros, 26(4), 304-312, 2016

α-Tocopherol loaded thermosensitive polymer nanoparticles: preparation, in vitro release and antioxidant properties

Figure 6. (A) Radical scavenging activity of TOC-NP expressed as ABTS inhibition percentage; (B) typical plot of inhibition (%) as function of the concentration of TOC-NPs.

migration of TOC to the aqueous phase. Slight differences among the antioxidant capacities obtained for the evaluated formulations agrees with their corresponding content of TOC.

4. Conclusions α- Tocopherol loaded nanoparticles were prepared by nanoprecipitation using amphiphilic triblock copolymers PNIPAM-b-PCL-b-PNIPAM as precursors. The nanoparticles presented average diameter smaller than 281 nm, were positively charged and exhibited thermosensitivity. TOC‑NP showed controlled released of TOC was achieved along 140 h, and radical scavenging activity in aqueous dispersion.

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6. Quiñones, J. P., Gothelf, K. V., Kjems, J., Yang, C., Caballero, A. M. H., Schmidt, C., & Covas, C. P. (2013). Self-assembled nanoparticles of modified-chitosan conjugates for the sustained release of dl-α-tocopherol. Carbohydrate Polymers, 92(1), 856-864. PMid:23218376. http://dx.doi.org/10.1016/j. carbpol.2012.10.005. 7. Luo, Y., Zhang, B., Whent, M., Yu, L., & Wang, Q. (2011). Preparation and characterization of zein/chitosan complex for encapsulation of α-tocopherol, and its in vitro controlled release study. Colloids and Surfaces. B, Biointerfaces, 85(2), 145-152. PMid:21440424. http://dx.doi.org/10.1016/j. colsurfb.2011.02.020. 8. Kumari, A., Yadav, S. K., & Yadav, S. C. (2010). Biodegradable polymeric nanoparticles based drug delivery systems. Colloids and Surfaces. B, Biointerfaces, 75(1), 1-18. PMid:19782542. http://dx.doi.org/10.1016/j.colsurfb.2009.09.001. 9. Rao, J. P., & Geckeler, K. E. (2011). Polymer nanoparticles: preparation techniques and size-control parameters. Progress in Polymer Science, 36(7), 887-913. http://dx.doi.org/10.1016/j. progpolymsci.2011.01.001. 10. Kataoka, K., Harada, A., & Nagasaki, Y. (2001). Block copolymer micelles for drug delivery: design, characterization and biological significance. Advanced Drug Delivery Reviews, 47(1), 113-131. PMid:11251249. http://dx.doi.org/10.1016/ S0169-409X(00)00124-1. 11. Yoon, H.-J., & Jang, W.-D. (2010). Polymeric supramolecular systems for drug delivery. Journal of Materials Chemistry, 20(2), 211-222. http://dx.doi.org/10.1039/B910948J. 12. Sierra-Martin, B., Retama, J. R., Laurenti, M., Fernández Barbero, A., & López Cabarcos, E. (2014). Structure and polymer dynamics within PNIPAM-based microgel particles. Advances in Colloid and Interface Science, 205(0), 113-123. PMid:24275613. http://dx.doi.org/10.1016/j.cis.2013.11.001. 13. Pintauer, T., & Matyjaszewski, K. (2008). Atom transfer radical addition and polymerization reactions catalyzed by ppm amounts of copper complexes. Chemical Society Reviews, 37(6), 1087-1097. PMid:18497922. http://dx.doi.org/10.1039/ b714578k. 14. Hutmacher, D. W., Schantz, T., Zein, I., Ng, K. W., Teoh, S. H., & Tan, K. C. (2001). Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling. Journal of Biomedical 311

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Polímeros, 26(4), 304-312, 2016

http://dx.doi.org/10.1590/0104-1428.2376

Simulation of temperature effect on the structure control of polystyrene obtained by atom-transfer radical polymerization Roniérik Pioli Vieira1,2* and Liliane Maria Ferrareso Lona2 Instituto Federal de Educação, Ciência e Tecnologia do Sul de Minas Gerais – IFSULDEMINAS, Pouso Alegre, MG, Brazil 2 Laboratory of Analysis, Simulations and Synthesis of Chemical Processes, School of Chemical Engineering, Universidade Estadual de Campinas – UNICAMP, Campinas, SP, Brazil 1

*ronierik@ymail.com

Abstract This paper uses a new kinetic modeling and simulations to analyse the effect of temperature on the polystyrene properties obtained by atom-transfer radical polymerization (ATRP). Differently from what has been traditionaly published in ATRP modeling works, it was considered “break” reactions in the mechanism aiming to reproduce the process at high temperatures. Results suggest that there is an upper limit temperature (130 °C), above which the polymer architecture loses the control. In addition, for the system considered in this work, the optimum operating temperature was 100 °C, because at this temperature polymer with very low polydispersity index is obtained, at considerable fast polymerization rate. Therefore, this present paper provides not only a tool to study ATRP processes by simulations, but also a tool for analysis and optimization, being a basis for future works dealing with this monomer and process. Keywords: ATRP, kinetic modeling, simulation, radical polymerization.

1. Introduction Atom-transfer radical polymerization (ATRP) is a powerful technique for controlled synthesis of polymers that provides several macromolecular architectures: polymers with narrow molecular weight distribution[1], block copolymers[2,3], random or gradient[4] and functionalized polymers[5-9]. ATRP has a great interest in the academic and industrial field because it can be used for various monomers, can be conducted in mild temperatures, and it is very resistant to impurities[10]. ATRP has been used industrially since 2005 with commercial products being manufactured in the US, Japan, and Europe. Some fundamental processes based on low catalyst, such as ARGET and ICAR ATRP, should soon be introduced to commercial sacales, but further scale-up will require synergistic input from process engineering, converting batch systems to continuous processes, transport phenomena, and accounting for complex stabilities such as temperature[10,11]. Profound mechanistic understanding is needed not only for optimization of the ATRP process but also to expand the range of polymerizable monomers, reduce the amount of catalyst, and allow synthesis of better defined polymers[11,12], which are related mostly with temperature. This is one of the most important variables in controlled polymerization systems, since it has a considerable influence on the polymerization rate and polymer properties. High temperatures accelerates the process, but provides high dispersity values which is not desirable. An analysis of temperature effect should provide directions for optimizations in ATRP, and also set a

Polímeros, 26(4), 313-319, 2016

temperature limit for a particular system. This limit should be a value that keep track of the formed polymer structure. As far as is known, modelling works available in literature did not address effects of temperature directly on the molecular weight and dispersities[12,13]. As a result, the polymerization control limit was also not explored properly[14]. Based on these observations, there is the need to demonstrate the effect of this variable on the polymer properties and the polymerization rate so that the readers may apply to other systems. The aim of this paper is to analyze the effect of temperature on the polymer properties and polymerization rate in ATRP by providing a limit for the styrene polymerization using CuBr/PMDETA as catalyst system. Therefore, this present research will be able to be used in future process optimization researches, which must have a value such that the polymerization control is not lost.

2. Kinetic Modeling Traditional researches on ATRP modeling have not been considered “break” reactions in the process such as chain transfer and terminations[13-19]. This current approach incorporates some reactions into the model aiming reproduce the sytem at high temperatures, which were usually considered in conventional polymerizations. This approach was validated in a previus research of our group[20,21], and the kinetic mechanism are shown in Equations 1-11.

313

S S S S S S S S S S S S S S S S S S

Vieira, R. P., & Lona, L. M. F. • Initiation: Keq RX + C ← → R• + XC (1) ki

R• + M → P1 (2) ktherm

3M  → 2 R• (3) k dim

2M → D (4)

• Propagation: Keq Pn X + C ← → Pn• + XC (5)

Pn• + M → Pn•+1 (6) kp

ktc

Pn• + Pm• → Pn + m (7) ktd

Pn• + Pm•  → Pn + Pm (8) •

d[M ] =−k p [ M ]µ0 − 2kdim [ M ]2 − ktr , M [ M ]µ0 − dt (12) ki [ R• ][ M ] − ktherm [ M ]3 d [ R• ] = −ki [ R• ][ M ] + ktherm [ M ]3 − ktp [ R• ]µ0 (13) dt

• Termination:

Pn•

and primary radicals. It was also used the well-known method of moments[18] to obtain average molecular weight (Mn and Mw) and polydispersity index (PDI). A system of 12 ordinary differential equations was generate, which was solved numerically for all simulations of this present paper. The system consists of mass balance for monomer, primary radicals and dimer (Equations 12, 13 and 14), plus the moment equations (Equations 15 to 23): moments of order “zero”, “one” and “two”, referring to “living” polymer, “dead” polymer and “dormant” polymer. The kinetic modeling was simplified for a batch system without considerable volume variation.

ktc

+ R → Pn (9)

d [ D] = +2kdim [ M ]2 − ktr , D [ D]µ0 (14) dt d µ0 = ka δ0 ([C ]0 − [ RX ]0 + δ0 ) − kdaµ0 ([ RX ]0 − δ0 ) − dt (15)

ktcµ02 − ktd µ02 − ktp [ R• ]µ0 − ktr , M [ M ]µ0 − ktr , D [ D]µ0

• Chain transfer: Pn•

ktr , M

•

+ M → Pn + R (10)

Pn• + D → Pn + P1 (11) ktr , D

Equations 1 through 11 are the chemical mechanism considered to represent the ATRP procces in this present paper. In such equations there are the some reactions traditionally used to represent the system: initiation step (Equation 1 and 2), the chemical equilibrium involving the propagation and dormant species (Equation 5), propagation of polymer chains (Equation 6), termination by combination (Equation 7), termination by disproportionation (Equation 8) and chain transfer to monomer (Equation 10). In addition to the reactions usually considered in ATRP, this paper proposed the inclusion of the following reactions: thermal initiation (Equation 3); dimerization reaction (Equation 4); termination caused by the reaction between the propagating species and a primary radical (Equation 9) and chain transfer to the dimer (Equation 11). The choice of these reactions was based on the conventional polymerizations in which these reactions are common at high temperatures. Such reactions have been considered in order to make the modeling more robust to represent the monomer conversion and the average properties at temperatures above 100 °C. Kinetic model was developed based on the mechanism proposed in Equations 1-11. From this mechanism, we carried out a material balance in order to account for the variation of the following species: “living” polymers, “dead” polymers, “dormant” polymers, monomer, dimer 314

d δ0 = kdaµ0 ([ RX ]0 − δ0 ) − ka δ0 ([C ]0 − [ RX ]0 + δ0 ) (16) dt

d λ0 1 = ktcµ02 + ktd µ02 + ktp [ R• ]µ0 + ktr , M [ M ]µ0 + dt 2 (17) ktr , D [ D]µ0 d µ1 = k pµ0 [ M ] + ka δ1 ([C ]0 − [ RX ]0 + δ0 ) − dt (18) kdaµ1 ([ RX ]0 − δ0 ) − ktcµ0µ1 − ktcµ0µ1 − ktp [ R• ]µ1 − ktr , M [ M ]µ1 − ktr , D [ D]µ1 d δ1 = −ka δ1 ([C ]0 − [ RX ]0 + δ0 ) + kdaµ1 ([ RX ]0 − δ0 ) (19) dt

d λ1 = ktcµ0µ1 + ktd µ0µ1 + ktp [ R• ]µ1 + dt (20)

ktr , M [ M ]µ1 + ktr , D [ D]µ1

d µ2 = k pµ0 [ M ] + ka δ2 ([C ]0 − [ RX ]0 + δ0 ) − dt kdaµ 2 ([ RX ]0 − δ0 ) − ktcµ0µ 2 − ktd µ0µ 2 − (21) ktp [ R• ]µ 2 − ktr , M [ M ]µ 2 − ktr , D [ D]µ 2 d δ2 = −ka δ2 ([C ]0 − [ RX ]0 + δ0 ) + (22) dt kdaµ 2 ([ RX ]0 − δ0 ) Polímeros, 26(4), 313-319, 2016

Simulation of temperature effect on the structure control of polystyrene obtained by atom-transfer radical polymerization d λ2 = ktcµ0µ 2 + ktd µ0µ 2 + ktp [ R• ]µ 2 + dt (23)

ktr , M [ M ]µ 2 + ktr , D [ D]µ 2

In Equations 12 a 23, [ M ] is the monomer concentration; [ R• ] is the primary radicas concentration; [ D] is the dimer

concentration; μ is the moment for the “living” polymers with orders 0, 1 and 2 as subscrites; δ is the moment for the “dormant” polymers with orders 0, 1 and 2 as subscrites; λ is the moment for the “living” polymers with orders 0, 1 and 2 as subscrites; kp is the kinetic rate coefficient of propagation; ki is the kinetic rate coefficient of initiation; ktherm is the kinetic rate coefficient of termal initiation; kdim is the kinetic rate coefficient of dimerization; ka is the kinetic rate coefficient of activation; kda is the kinetic rate coefficient of polymeric chain deactivation; ktc is the kinetic rate coefficient of termination by combination; ktd is the kinetic rate coefficient of despoportionation; ktp is the kinetic rate coefficient of termination by the reaction with a primar radical; ktr,M is the kinetic rate coefficient of chain transfer to moomer; and ktr,D is the kinetic rate coefficient of chain transfer to dimer.

3. Resolution of the Equations System For the ODE’s system solution it was developed a computer program in Fortran code with the aid of LSODE subroutine developed by Hindmarsh[22]. This subroutine uses the Adams-Moulton method to solve initial value problems. This subroutine was chosen because it is very efficient to solve problems with high numerical stiffness, which is quite common in polymer engineering[19,23,24].

As shown in Table 1, all population moments were assigned initial values equal to zero once the concentrations present in the process are negligible at time very close to zero. The system considered in this case study is the bulk styrene polymerization initiated by 1-phenylethyl bromide (1-PEBr), copper (I) bromide (CuBr) as catalyst and N,N,N’,N”,N”pentamethyldiethyllenetriamine as ligand. This system was chosen due to the wide availability of kinetic data as function of temperature, in addition to the fact that it is a widely used system. The kinetic parameters were calculated as temperature functions according Arrhenius’ expressions shown in Table 2. Table 2 provides the expressions to analyze the influence of temperature on the ATRP process. In addition to Arrhenius’ expression in Table 2, there is the traditional gel effect correlation proposed by Hui and Hamielec that affects the termination rate coefficient (kt). This constant depends on the monomer conversion and the parameters A1, A2 and A3 which in turn are related to the operating temperature.

4. Results and Discussions Using the kinetic model of this paper, computer simulations were performed considering different operating temperatures in order to analyze the influence of this parameter on the product. Figure 1 illustrates the monomer conversion as a function of polymerization time, considering an isothermal

To obtain the number-average molecular weight, we used values of moments of order “zero” and “one” for each species, as defined by Equation 24[18].  µ +λ +δ  Mn = MWM  1 1 1  (24)  µ 0 + λ 0 + δ0 

where Mn is the polymer number average molecular weight and MWM is the monomer molecular weight. The polymer weight-average molecular weight took into account the moments of order “one” and “two” for each species, and was calculated by Equation 25.  µ + λ 2 + δ2  (25) Mw = MWM  2   µ1 + λ1 + δ1 

where Mw is the polymer weight average molecular weight. After obtaining Mn and Mw, the polymer polydispersity

index (PDI) was obtained by Equation 26. PDI =

Mw Mn

(26)

The program input data refer to the initial concentrations of monomer, catalyst, ligand, initiator and operating temperature. Table 1 provides the set of initial conditions used to solve the equations system. Polímeros, 26(4), 313-319, 2016

Figure 1. Styrene conversion simulation as a function of reaction time using 1-PEBr as initiator (0.087 mol L–1), CuBr as catalyst (0.087 mol L–1) and dH-bipy (0.174 mol L–1) as binder six different temperatures (80, 90, 100, 110, 120 and 130 °C). Table 1. Initial conditions used in the program for solving the differential model. Parameter [M]0

Value (mol L–1) 8.7

[RX]0

0.087

[MtnY]0

0.087

[L]0

0.174

[D]0

[R*]0

All moments

315

Vieira, R. P., & Lona, L. M. F. Table 2. Expressions used to obtain the kinetic parameters for atom-transfer radical polymerization as functions of temperature. Parameter kp

Expression 4.226×107exp(-3910/T)

Reference Fu et al.[25]

1.63×106exp(-12020/T)

Fu et al.[25]

ktherm

2.19×10 exp(-13800/T)

Fu et al.[25]

kdim

188.97exp(-1947/T)

Belincanta-Ximenes et al.[26]

8.06×10 exp(-4694,51/T)

Seeliger and Matyjaszewski[27]

kda

3.860×10 exp(-2245/T)

Matyjaszewski[28]

kt0

3.820×10 exp(-958/T)

kt0×exp(-2×(A1X + A2X + A3X ))

Hui and Hamielec[29]

ktc

0.99kt

Fu et al.[25]

ktd

0.01kt

Fu et al.[25]

kt,p

Fischer and Paul[30]

ktr,M

2.310×106exp(-6377/T)

Fu et al.[25]

ktr,D

150

Fu et al.[25]

2.57 - (5.05×10-3T)

Hui and Hamielec[29]

9.56 - (1.76×10-2T)

Hui and Hamielec[29]

-3.03 + (7.85×10-3T)

Hui and Hamielec[29]

[2]

batch reactor operating at six different temperatures (80, 90, 100, 110, 120 and 130 °C). An analysis of Figure 1 makes it clear that the increase of temperature is accompanied by an increase in the monomer conversion, confirming the polymerization rate is higher at higher temperatures. This result fits the step of the radical propagation as an irreversible reaction, whose rate coefficient is strongly dependent on temperature (high activation energy). Matyjaszewski[11] discusses this strong influence of temperature on the propagation rate coefficient (kp). Moreover, the author also says that the activation step of dormant species (ka) helps to increase the rate since the chemical equilibrium in Figure 2 tends to be shifted towards the formation of propagation radicals. Figure 2 illustrates the equilibrium of polymer chains activation/deactivation in ATRP. The reagents are represented on the left side and the product on the right side. The energy associated with the products is greater than the energy associated with the reagents. There is an endothermic process for the activation of the polymer chains and thus increasing the system temperature favors the equilibrium constant displacement in this direction. As a result, there is a higher monomer conversion in Figure 1 because increasing the concentration of the propagating radicals caused an increase of reaction rate. Thus, considering optimize reaction time, it would be desirable to operate the system at the highest possible temperature in order to minimize the reactor size. However, the temperature increase influences the chemical equilibrium in Figure 2, making the polymer properties be affected (Mn and IPD). The process tends, at high temperatures, to behave such as a conventional radical polymerization, that is, with a large amount of “living” radicals susceptible to termination and chain transfer. This is the great challenge of ATRP: be conducted at a temperature such that the concentration of “dormant” polymers is high and the concentration of “living” polymers is low. In this case, the most important issue is to meet the ideal temperature for a specific polymerization be conducted in the shortest 316

Fu et al.[25]

[3]

Figure 2. Typical energy profile for the equilibrium of activation/ deactivation of the catalyst system in the ATRP processes, adapted from literature[10].

possible time without losing the controlled polymerization characteristics. Figure 3 shows a linear evolution of Mn as a function of monomer conversion, featuring a controlled polymerization process. Comparing both profiles of molecular weights (Mn and Mw), it can be observed that there is a great difference between these values in low and high conversions. This occurs because, at the beginning of the ATRP process, the chemical equilibrium between the propagating radicals and the deactivator agent has not been established. As a result, a high concentration of primary radicals is generated, raising the probability of terminations at low monomer conversions. In addition, there is also the same behavior that occurred at high monomer conversions, especially for the highest temperature analyzed (Figure 3c). This trend tends to be higher at elevated temperatures due to two factors: first because of the equilibrium displacement towards the formation of radicals in propagation (Figure 2) be favored. Second, due to the increase of kinetic rate coefficients of termination and chain transfer reactions, which led to the increase in the Polímeros, 26(4), 313-319, 2016

Simulation of temperature effect on the structure control of polystyrene obtained by atom-transfer radical polymerization

Figure 3. Simulations of number-average molecular weight (Mn) and weight-average molecular weight (Mw): (a) 80 °C; (b) 100 °C; (c) 120 °C and (d) comparison of the polydispersity ndexes in these temperatures.

concentration of “dead” polymers in the process (Figure 4 suggests there was a considerable increase in the concentration of “dead” polymer due to temperature increase). It is important to highlight the polydispersities indexes that are shown in Figure 3d present the some differences in their profiles. For example, at low monomer conversions, high PDI values were obtained for all three simulations. To temperatures of 80 and 100 °C, the PDI values presented no significant differences in the simulated conversion range, remaining with low values. With this result, clearly, it would be ideal to work in a temperature around 100 °C, since it provides low values of PDI, and also this temperature increases the polymerization rate. Moreover, for all simulated PDI profiles of Figure 3d, a coincident point was observed. This value is approximately 63% of monomer conversion. Finishing the polymerization in this range of conversion, the obtained polymers will have similar properties (Mn and PDI) for the three temperatures studied. This result suggests that it is possible to obtain polymers with a well-controlled structure at the highest temperature. The problem is related to the desired value of number-average molecular weight. In this case, the polymer obtained would show lower Mn values, around 6,500 g mol-1, since the polymerization will be stoped at Polímeros, 26(4), 313-319, 2016

Figure 4. Simulation of the “dead” polymer concentration profile as a function of polymerization time at 80, 100 and 120 °C in styrene ATRP (8.7 mol L–1) using 1-PEBr as initiator.

63% of conversion. Depending on the application, these characteristics would not be interesting. Figure 5a and b illustrate a linear increase of Mn as a function of monomer conversion, differentiating from Figure 5c, wherein Mn presented deviations from linearity, characterizing as an uncontrolled polymerization. This result suggests that the temperature of 130 °C may be a limit of the 317

Vieira, R. P., & Lona, L. M. F.

Figure 5. Simulations of number-average molecular weight (Mn) and weight-average molecular weight (Mw): (a) 90 °C; (b) 110 °C; (c) 130 °C and (d) comparison of the polydispersity ndexes in these temperatures.

system. At low temperatures (e.g. 90 °C), it is possible to obtain polymers with low PDI values in a large extension of the monomer conversion illustrated in Figure 5d. However, the polymerization rate is also very low. Thus, to obtain polymers with high molecular weight would be desirable to operate the reactor in a high residence time. Similarly to the result expressed in Figure 3d, for all temperature profiles, there is a conversion range (40 to 60%) that the PDI profiles are coincident, so it is possible to obtain similar polymer properties in this range. Comparing all PDI profiles of Figure 3 and 5 it can be seen that for the tested temperatures, 100 °C would be ideal, as they provide low PDI values at high monomer conversions. Moreover, such a reaction temperature provided a fast reaction rate without loss of controlled polymerization characteristics.

5. Conclusions The main objective of this paper was to stablished a temperature limit for styrene ATRP by the analysis of polymer properties and monomer conversions. Results suggested that 130 ºC is the process limit, because this temperature provided a nonlinear evolution of number-average molecular weight, i. e., there was a lose of the polymerization control. The results also indicate that for this system there is an optimum temperature of 100 ºC, which provides a relative fast 318

polymerization with a good arquitecture control. Moreover, the kinetic modeling proposed in this present work can be use to analyse every ATRP process, since it is general, and the user needs only to insert the reaction conditions plus the kinetic paramenters available in literature for several monomers and initiators.

6. References 1. Zhao, M., Zhang, H., Ma, F., Zhang, Y., Guo, X., & Zhang, H. (2013). Efficient synthesis of monodisperse, highly crosslinked, and “living” functional polymer microspheres by the ambient temperature iniferter-induced “living” radical precipitation polymerization. Journal of Polymer Science. Part A, Polymer Chemistry, 51(9), 1983-1998. http://dx.doi.org/10.1002/ pola.26579. 2. Lessard, B. H., & Marić, M. (2013). Water-soluble/dispersible carbazole-containing random and block copolymers by nitroxide-mediated radical polymerisation. Canadian Journal of Chemical Engineering, 91(4), 618-629. http://dx.doi. org/10.1002/cjce.21676. 3. Porras, C. T., D’Hooge, D. R., Van Steenberge, P. H. M., Reyniers, M. F., & Marin, G. B. (2013). A theoretical exploration of the potential of ICAR ATRP for one- and two-pot synthesis of well-defined diblock copolymers. Macromolecular Reaction Engineering, 7(7), 311-326. http://dx.doi.org/10.1002/ mren.201200085. Polímeros, 26(4), 313-319, 2016

Simulation of temperature effect on the structure control of polystyrene obtained by atom-transfer radical polymerization 4. Zhou, Y. N., Li, J. J., & Luo, Z. H. (2012). Synthesis of gradient copolymers with simultaneously tailor-made chain composition distribution and glass transition temperature by semibatch ATRP: from modeling to application. Journal of Polymer Science. Part A, Polymer Chemistry, 50(15), 30523066. http://dx.doi.org/10.1002/pola.26091. 5. Goldmann, A. S., Glassner, M., Inglis, A. J., & Barner-Kowollik, C. (2013). Post-functionalization of polymers via orthogonal ligation chemistry. Macromolecular Rapid Communications, 34(10), 810-849. PMid:23625725. http://dx.doi.org/10.1002/ marc.201300017. 6. Salian, V. D., & Byrne, M. E. (2013). Living radical polymerization and molecular imprinting: improving polymer morphology in imprinted polymers. Macromolecular Materials and Engineering, 298(4), 379-390. http://dx.doi.org/10.1002/ mame.201200191. 7. Yamago, S., Yamada, T., Togai, M., Ukai, Y., Kayahara, E., & Pan, N. (2009). Synthesis of structurally well-defined telechelic polymers by organostibine-mediated living radical polymerization: in situ generation of functionalized chaintransfer agents and selective omega-end-group transformations. Chemistry, 15(4), 1018-1029. PMid:19086048. http://dx.doi. org/10.1002/chem.200801754. 8. Hardy, C. G., Ren, L., Zhang, J., & Tang, C. (2012). Side-chain metallocene-containing polymers by living and controlled polymerizations. Israel Journal of Chemistry, 52(3-4), 230245. http://dx.doi.org/10.1002/ijch.201100110. 9. Badri, A., Whittaker, M. R., & Zetterlund, P. B. (2012). Modification of graphene/graphene oxide with polymer brushes using controlled/living radical polymerization. Journal of Polymer Science. Part A, Polymer Chemistry, 50(15), 29812992. http://dx.doi.org/10.1002/pola.26094. 10. Matyjaszewski, K. (2012). Atom transfer radical polymerization: from mechanisms to applications. Israel Journal of Chemistry, 52(3-4), 206-220. http://dx.doi.org/10.1002/ijch.201100101. 11. Matyjaszewski, K. (2012). Atom Transfer Radical Polymerization (ATRP): current status and future perspectives. Macromolecules, 45(10), 4015-4039. http://dx.doi.org/10.1021/ma3001719. 12. Vieira, R. P., & Lona, L. M. F. (2016). Optimization of reaction conditions in functionalized polystyrene synthesis via ATRP by simulations and factorial design. Polymer Bulletin, 73(7), 1795-1810. http://dx.doi.org/10.1007/s00289-015-1577-z. 13. Zhu, S. (1999). Modeling of molecular weight development in atom transfer radical polymerization. Macromolecular Theory and Simulations, 8(1), 29-37. http://dx.doi.org/10.1002/ (SICI)1521-3919(19990101)8:1<29::AID-MATS29>3.0.CO;2-7. 14. D’hooge, D. R., Reyniers, M. F., & Marin, G. B. (2009). Methodology for Kinetic Modeling of Atom Transfer Radical Polymerization. Macromolecular Reaction Engineering, 3(4), 185-209. http://dx.doi.org/10.1002/mren.200800051. 15. Shipp, D. A., & Matyjaszewski, K. (1999). Kinetic analysis of controlled/“living” radical polymerizations by simulations. 1. The importance of diffusion-controlled reactions. Macromolecules, 32(9), 2948-2955. http://dx.doi.org/10.1021/ma9819135. 16. Al-harthi, M., Cheng, L. S., Soares, J. B. P., & Simon, L. C. (2007). Atom-transfer radical polymerization of styrene with bifunctional and monofunctional initiators: experimental and mathematical modeling results. Journal of Polymer Science, 45, 2212-2224. http://dx.doi.org/10.1002/pola. 17. Bentein, L., D’hooge, D. R., Reyniers, M. F., & Marin, G. B. (2011). Kinetic modeling as a tool to understand and improve the nitroxide mediated polymerization of styrene. Macromolecular Theory and Simulations, 20(4), 238-265. http://dx.doi.org/10.1002/mats.201000081. 18. Ray, W. H. (1972). On the mathematical modeling of polymerization reactors. Journal of Macromolecular Polímeros, 26(4), 313-319, 2016

Science, Part C: Polymer Reviews, 8(1), 1-56. http://dx.doi. org/10.1080/15321797208068168. 19. Vieira, R. P., Ossig, A., Perez, J. M., Grassi, V. G., Petzhold, C. L., Costa, J. M., & Lona, L. M. F. (2013). Simulation of the equilibrium constant effect on the kinetics and average properties of polystyrene obtained by ATRP. Journal of the Brazilian Chemical Society, 24(12), 2008-2014. http://dx.doi. org/10.5935/0103-5053.20130251. 20. Vieira, R. P., Ossig, A., Perez, J. M., Grassi, V. G., Petzhold, C. L., Peres, A. C., Costa, J. M., & Lona, L. M. F. (2015). Styrene ATRP using the new initiator 2,2,2-tribromoethanol: experimental and simulation approach. Polymer Engineering and Science, 55(10), 2270-2276. http://dx.doi.org/10.1002/ pen.24113. 21. Vieira, R. P., & Lona, L. M. F. (2016). Kinetic modeling of atom-transfer radical polymerization: inclusion of break reactions in the mechanism. Polymer Bulletin, 73(8), 21052119. http://dx.doi.org/10.1007/s00289-015-1596-9. 22. Hindmarsh, A. C. (1983). ODEPACK, a systematized collection of ODE solvers. Scientific Computing, 1, 55-64. Retrieved in 9 November 2015, from https://computation.llnl.gov/casc/ nsde/pubs/u88007.pdf 23. Zapata-González, I., Saldívar-Guerra, E., Flores-Tlacuahuac, A., Vivaldo-Lima, E., & Ortiz-Cisneros, J. (2012). Efficient numerical integration of stiff differential equations in polymerisation reaction engineering: computational aspects and applications. Canadian Journal of Chemical Engineering, 90(4), 804-823. http://dx.doi.org/10.1002/cjce.21656. 24. Vieira, R. P., Mokochinski, J. B., & Sawaya, A. C. H. F. (2015). Mathematical modeling of the ascorbic acid thermal degradation in orange juice during industrial pasteurizations. Journal of Food Process Engineering, n/a. http://dx.doi.org/10.1111/ jfpe.12260. 25. Fu, Y., Mirzaei, A., Cunningham, M. F., & Hutchinson, R. A. (2017). Atom-transfer radical batch and semibatch polymerization of styrene. Macromolecular Reaction Engineering, 1(4), 425439. http://dx.doi.org/10.1002/mren.200700010. 26. Belincanta-Ximenes, J., Mesa, P. V. R., Lona, L. M. F., VivaldoLima, E., McManus, N. T., & Penlidis, A. (2007). Simulation of styrene polymerization by monomolecular and bimolecular nitroxide-mediated radical processes over a range of reaction conditions. Macromolecular Theory and Simulations, 16(2), 194-208. http://dx.doi.org/10.1002/mats.200600063. 27. Seeliger, F., & Matyjaszewski, K. (2009). Temperature effect on activation rate constants in ATRP: new mechanistic insights into the activation process. Macromolecules, 42(16), 60506055. http://dx.doi.org/10.1021/ma9010507. 28. Matyjaszewski, K., Paik, H., Zhou, P., & Diamanti, S. J. (2001). Determination of activation and deactivation rate constants of model compounds in atom transfer radical polymerization 1. Macromolecules, 34(15), 5125-5131. http://dx.doi.org/10.1021/ ma010185+. 29. Hui, A. W., & Hamielec, A. E. (1972). Thermal polymerization of styrene at high conversions and temperatures. an experimental study. Journal of Applied Polymer Science, 16(3), 749-769. http://dx.doi.org/10.1002/app.1972.070160319. 30. Fischer, H., & Paul, H. (1987). Rate constants for some prototype radical reactions in liquids by kinetic electron spin resonance. Accounts of Chemical Research, 20(5), 200-206. http://dx.doi.org/10.1021/ar00137a007. Received: Nov. 09, 2015 Revised: Mar. 11, 2016 Accepted: Mar. 21, 2016 319

http://dx.doi.org/10.1590/0104-1428.2316

S S S S S S S S S S S S S S S S S S

Oat fibers modification by reactive extrusion with alkaline hydrogen peroxide Melina Aparecida Plastina Cardoso1, Gizilene Maria Carvalho2, Fabio Yamashita1, Suzana Mali3, Juliana Bonametti Olivato1 and Maria Victoria Eiras Grossmann1* Departamento de Ciência e Tecnologia de Alimentos, Universidade Estadual de Londrina – UEL, Londrina, PR, Brazil 2 Departamento de Química, Universidade Estadual de Londrina – UEL, Londrina, PR, Brazil 3 Departamento de Bioquímica, Universidade Estadual de Londrina – UEL, Londrina, PR, Brazil

*victoria@uel.br

Abstract The modification of lignocellulosic fibers can enhance their interaction with other materials and alkaline hydrogen peroxide (AHP) is a reagent widely used to promote such modification. This work aimed to modify oat hulls fibers by reactive extrusion using AHP (7 g 100 g-1 of hulls). The modified oat hulls displayed performances comparable to those observed by other researchers using conventional AHP method (without extrusion). The AHP treated oat hulls showed increased luminosity compared to the extruded ones. Fourier transform infrared spectroscopy showed differences between the modified and unmodified structures. The removal of surface compounds resulted in a more open morphology, with greater surface area and greater porosity. Reactive extrusion can be an alternative method for fiber modification with several advantages, such as short processing time and no wastewater generation. Keywords: chemical modification, lignocellulosic fibers, microstructure, thermal stability.

1. Introduction In the last decades, discarded plastic materials are accumulated in the environment, as a consequence of the resistance of polymers derived from petroleum to natural degradation. The substitution by natural or synthetic biodegradable polymers has been extensively studied because they degrade more rapidly compared to traditional polymers. However, the majority of biodegradable polymers (polyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB), polylactic acid (PLA), polycaprolactones (PCL)) are higher in cost and possess inferior mechanical properties compared with, for example, high-density polyethylene (HDPE) and low-density polyethylene (LDPE)[1,2]. Starch, a natural, low-cost polymer from renewable sources, also produces materials with diminished mechanical properties and high moisture sensitivity, even when used in blends with other biodegradable polymers[3-7]. In this context, natural fibers have been studied as a good alternative for reinforcement being mixed with pure biodegradable polymers or their blends to form composites with reduced environmental impact[8]. Fibers of curaua, jute, sisal, sugarcane, and others have been tested for this purpose[4,8-15]. Oat hulls (a byproduct from the processing of oat grains, rich in fiber) are an interesting option for use as reinforcement in composites. Studies have been conducted with the inclusion of these fibers in trays of cassava starch (produced by extrusion followed by thermoforming) with good results[16]. However, despite their several advantages, such as low cost, easy accessibility and high biodegradability, cellulosic fibers present problems in terms of fiber - matrix adhesion and changes in water absorption, and the mechanical properties

320

of some composites can remain unsatisfactory[17]. Aiming to overcome these limitations, the modification of fibers has been proposed[18]. To improve the compatibility of natural fibers with polymers, several authors[17,19-25] proposed the surface modification of fibers from different sources. These modifications decrease fiber hydrophilicity and/or increase adhesion with the polymer matrix, enhancing the thermal and mechanical properties of the composites. Various reagents are used, including sodium hydroxide (NaOH); NaOH in combination with other reagents, such as hydrogen peroxide (H2O2); and hot water with the subsequent addition of neutral detergents. When alkaline hydrogen peroxide (AHP) is applied to the fiber, the components responsible for its natural color become oxidized (bleaching action) and the hydrolysis of the lignin macromolecule may occurs. The intensity of hydrolysis, as well as the modification degree, are dependent on the reaction conditions[19,26].Other reactions include the conversion of lignin phenolic groups in aldehyde, carbonyls and quinones and, in the most dramatic cases, the disruption of aromatic rings[27]. The modification of the fibers is typically achieved by employing conventional reactors, which require long reaction times (up to 48 h) and generate large volumes of effluent[28]. The advantages of using reactive extrusion are a drastic reduction in the reaction time and the lack of effluents. Although corrosion-resistant materials (more expensive) would have to be used in the manufacture of the screw and barrel to protect against possible corrosion caused by the use of chemicals, the process would be recommended. Additionally, reagents not consumed in the

Polímeros, 26(4), 320-326, 2016

Oat fibers modification by reactive extrusion with alkaline hydrogen peroxide reaction and/or degradation products will be present in the modified fiber and may affect, positively or negatively, its future applications. In a preliminary study, aiming the application in food products, our research group employed reactive extrusion with AHP to modify the fibers of oat hulls[29]. The characterization was limited to the hydration properties of the fibers. Now, looking the possible use of these fibers as reinforcing agents in biodegradable polymeric matrices, this work was carried out in order to promote its modification by a similar process, characterizing the modified fibers in relation to their physical, thermal and microstructural properties.

2. Materials and Methods 2.1 Materials The fiber source was micronized oat hulls (4.64 g 100 g–1 of ash, 3.95 g 100 g–1 of protein, 2.12 g 100 g–1 of lipid, 23.13 g 100 g–1 of cellulose, 26.25 g 100 g–1 hemicellulose and 3.80 g 100 g–1 of lignin) provided by SL Cereals Alimentos Ltda (Mauá da Serra, Brazil). Hydrogen peroxide 200 V, (Biotec, Pinhais, Brazil) and NaOH (Synth, Diadema, Brazil) were also used.

2.2 Oat fiber modification The micronized hulls (250 g) were modified by reactive extrusion with alkaline hydrogen peroxide according Galdeano and Grossmann[29]. Initially, the hulls were conditioned to 32% moisture by adding the required volume of water containing dissolved enough NaOH for the material to reach a pH of 11.5. The conditioned hulls were placed in a sealed polyethylene bag and allowed to equilibrate under refrigeration (7-10 °C) for 24 hours, for moisture balance. Next, the hulls were removed from the refrigerator and allowed to rest for 1 hour to attain the ambient temperature and 34 mL of hydrogen peroxide 200 V (7 g 100 g–1 of hulls) was then added. The material was extruded in a single screw extruder (AX Plasticos, Diadema, Brazil) with D = 1.6 cm, L/D = 40, four heating zones and a 0.8 mm die diameter. The processing temperatures in all zones were maintained at 110 °C, and the screw speed was 100 rpm with constant feeding. These conditions were established from preliminary tests. The steady state was controlled by maintaining constant amperage. After extrusion, the samples were dried (60 °C/24 h) and coarsely ground in a blender (Arno, São Paulo, Brazil) followed by a second ground in a table mill (IKA A-11 Basic Mill - São Paulo - Brazil). To assess the effect of the extrusion process, a sample was processed by the same procedure but without the addition of reagents (sodium hydroxide and hydrogen peroxide). All obtained materials were divided into two equal portions, one of which underwent washing with 10 volumes of distilled water and was then stored in a refrigerator for 6 hours for decantation. Subsequently, the supernatant was discarded, and the remaining material was dried in an oven at 60 °C for 24 h. This wash was performed to assess the removal of compounds that were solubilized by processing without using large amounts of water, as used in traditional processes. Thus, five different samples were obtained: F1untreated fiber, F2- fiber modified by extrusion, F3- fiber modified by extrusion and then washed, F4- fiber modified Polímeros, 26(4), 320-326, 2016

by AHP and F5- fiber modified by AHP and then washed. All assays were performed in duplicate.

2.3 Physical properties of the fibers The luminosity (L*) of the samples was measured with a Minolta CR-10 portable colorimeter (Sino Devices, Georgia, United States) using CIE illuminant D65 (natural daylight) placed at an angle of 8/d and a 10th CIE standard observer with an 8 mm diameter reading area. The fiber samples were placed in a plastic container (1 cm in height and 4 cm in diameter). The water absorption index (WAI) and the swollen volume (SV) were determined using the methods described by Seibel and Beleia[30]. The WAI was obtained by the ratio between the weight of the wet sample and the initial dry weight and the volume of the wet sample was considered SV. The analysis were performed in triplicate.

2.4 Scanning electron microscopy The fibers were analyzed for their microstructure by scanning electron microscopy (SEM) using an electron microscope (FEI Quanta 200, North Carolina, United States). Before the analysis, the samples were pre-dried at 60 °C for 24 hours and left in a desiccator with silica for 24 hours. The dried samples were then coated with a 15 to 20 nm layer of gold using a Sputter Coater BAL-TEC SCD 050 (Baltec, Balzers, Liechtenstein) prior to analysis.

2.5 Fourier transform infrared (FTIR) spectroscopy Only for this analysis, the samples were washed with large amount of distilled water, aiming to facilitate the detection of modifications promoted by the treatments. Therefore, only the samples F1, F2 and F4 were analysed. After washing, the samples were dried in an oven at 60 °C for 24 hours and maintained in a desiccator with silica for 24 hours. The FTIR spectra were obtained with a spectrophotometer (FT-IR/NIR Spectrmeter Spectrum Frontier, São Paulo, Brazil). The analyses were performed in the mid-infrared, covering wave numbers from 4000 to 400 cm–1, with 2 cm–1 resolution. A total of 32 scans were performed on each sample.

2.6 Thermogravimetric analysis (TGA) Samples of approximately 13.5 mg were used and subjected to the following analysis conditions: temperature range of 30 °C-800 °C, heating rate of 20 °C/min and nitrogen atmosphere. The equipment used was a TGA 4000 (Waltham, Massachusetts, USA).

2.7 Statistical analysis Statistical evaluation was performed by analysis of variance (ANOVA). Means were compared by the Tukey test at a 5% significance level.

3. Results and Discussions It is important to note that, while other studies characterized modified fibers that had been thoroughly washed, in the present work the samples were unwashed or were washed with only 10 volumes of water, aiming to take advantage of no waste production. 321

Cardoso, M. A. P., Carvalho, G. M., Yamashita, F., Mali, S., Olivato, J. B., & Grossmann, M. V. E. 3.1 Physical properties of fiber hulls The physical measurements of color (luminosity), water absorption indices (WAI) and swelling volumes (SV) of the oat hull samples are shown in Table 1. Regarding the luminosity, it was observed that the treatment of the husks (fiber) only by extrusion (F2) caused darkening (decreased L*), which could be related to the high temperature used in the process, which would induce caramelization and Maillard reactions[31]. When these extruded fibers were washed (F3), no whitening effect was observed, indicating that the formed compounds responsible for the darkening were insoluble. Furthermore, when the fibers were treated with AHP (F4), discoloration was noticed (increase in brightness) compared with F2 and F3, due to the oxidation of the pigments that impart dark color to the natural fibers. Thus, the darkening effect of extrusion was compensate by the bleaching effect of AHP treatment. After chemical treatment and subsequent washing (F5), the fibers showed an even greater increase in brightness, indicating that, in addition to the oxidation of the pigments, the hydrolysis of the pigments and/or of lignin also occurred forming soluble compounds that were removed in the wash. Similar effects were observed by other researchers[28,32]. It is well stablished the water absorption process of the lignocellulosic fibers shows two phases, requiring long times for the fully hydration. However, the first phase (corresponding to the Fick law diffusion) has a high absorption coefficient and is completed in 10 – 15 min. The water intake in this phase results from a capillary action (due to the porous structure) and this will be the phase directly impacted by any modification in the fiber promoted by the treatments. Thus, the WAI was determined by a methodology widely used by several researchers, in which the immersion time of fiber in water is 30 min aiming to identify the occurrence of modification, even though the fiber was not fully hydrated.

Only the oat fiber modified by AHP (F4) behaved differently from the untreated fiber (F1) in terms of water absorption (Table 1), indicating this treatment modified the fibers structure. A comparison of the smallest value (observed in the untreated fiber) with the highest value (observed in the fiber treated with peroxide) showed that the water uptake increased 36.12% after treatment. This can be attributed to the increased exposure of hydroxyls in the cellulose after the hydrolysis of some of the lignin fractions by chemical treatment with AHP. Gould, Jasberg and Cote[33] also observed increased water uptake in wheat straw treated with AHP. The effect of the possible removal of hemicelluloses[34] from the fiber structure should also be considered. Their removal exposes macromolecules of cellulose, causing large voids that would facilitate the water entry[35]. Thus, the increase of WAI in F4 must also have resulted from the retention of water in the interstitial spaces of the cell wall, which were more accessible after chemical treatment. The same factors that increased the WAI could have contributed to the higher swelling of the fibers, but this was not observed (Table 1). Statistically, there were no differences in the swollen volume values. This could be due to the saturation of the cell wall of the fiber with water, which would decrease the flexibility of the wet fiber and limit the differences among the samples[36]. The observed result is interesting for the application of the modified fibers in composites, in which changes in the volume of fibers motivated by water intake are undesirable.

3.2 Morphologic characterization (SEM) The images of untreated (F1) extruded (F2) and treated by AHP (F4) fibers are shown in Figure 1. The partially washed samples (F3 and F5) are not displayed because their similarity with the corresponding unwashed (F2 and F4, respectively). Analyzing the morphology of the untreated fiber (F1), beams oriented in the longitudinal direction that

Table 1. Luminosity, water absorption index and swollen volume of oat hulls under different treatments. Samples

Luminosity*

WAI*

SV*

(L) (g wet fibers g dry fiber-1) (mL g-1) F1 44.83 ± 0.07b 2.99 ± 0.08a 3.0 ± 0.2a F2 34.41 ± 0.37a 3.53 ± 0.14ab 3.1 ± 0.5a F3 35.27 ± 0.21a 3.72 ± 0.31ab 3.3 ± 0.5a b b F4 45.76 ± 0.21 4.07 ± 0.16 3.4 ± 0.4a F5 51.43 ± 0.17c 3.44 ± 0.30ab 3.5 ± 0.3a *Means (n = 3) ± standard deviation; WAI: water absorption index; SV: swollen volume. F1: untreated fiber; F2: fiber modified by extrusion; F3: fiber modified by extrusion and washed; F4: fiber modified by AHP; F5: fiber modified by AHP and washed. Different lowercase letters in the same column indicate significant differences between the means (p ≤ 0.05).

Figure 1. SEM of the fibers: untreated (F1); physically modified by extrusion (F2) and chemically modified with AHP (F4). 322

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Oat fibers modification by reactive extrusion with alkaline hydrogen peroxide were covered with a layer of material forming a smooth surface are observed, which might be a consequence of the presence of wax and other non-cellulosic materials (hemicelluloses, lignin and pectin), forming a compact structure. This surface layer is damaged in several regions, perhaps due to the milling of the oat hulls by the physically intensive micronization process. In F2, a more rugged surface was observed compared with F1. Due to the shear forces and high temperature and pressure during extrusion coupled with the additional grinding process, more cracks were opened, making the structure more porous. An increased damage of the structure was observed in the fibers modified with AHP (F4), which displayed an increase in the number of fiber bundles and deep valleys compared with the untreated fibers (F1). Thus, it can be observed that the chemical modification increases the exposure of the surface, increasing the area of holes and valleys present throughout the rough fiber surface. This effect can be ascribed to the action of AHP coupled with the physical action of the extrusion process. Campos et al.[17], Brígida et al.[19], and Teodoro et al.[37] also reported the efficient removal of waste wax and fatty acids from the surfaces of fibers after treatment with hydrogen peroxide in sisal and coconut fibers. The modifications promoted in the fiber morphology by AHP treatment can explain the higher WAI of the treated sample (Table 1).

3.3 FTIR The FT-IR spectra of samples are presented in Figure 2. The bands between 1739 cm–1 and 1022 cm–1 are enlarged (right figure) for better visualization of the region of interest concerning the changes occurring in the fibers. The characteristic bands of the fibers correspond to the absorption bands of lignin, hemicellulose and cellulose. The absorption bands observed in these components are: –OH (3500-3200 cm–1), C=O (~ 1739 cm–1) C-O-C (~ 1259 cm–1-1155 cm–1) and C-OH (~ 1084 cm–1-1022 cm–1)[38]. The band at 3455 cm–1 is attributed to OH stretching vibrations

related to the ring (CH-OH) and side chain (–CH2-OH) of cellulose. At 2920 cm–1 are the trademark stretching vibrations of CH2. At 1739 cm–1, it is possible to observe a shoulder in the sample from the untreated fiber (F1), which can be assigned to the C=O group of hemicellulose and/or the ester bonds of the carboxyl groups present in hemicellulose and lignin. An increase in this shoulder was observed after reaction with AHP (F4) when compared to the untreated fiber (F1), which could indicate slight changes in hemicellulose and lignin due to AHP. According to Liu et al.[39], the carbonyl number (C=O) initially present in the fiber can be increased by oxidation reactions or decreased by cyclization reactions, both of which are promoted by hydrogen peroxide. The bands at 1640 cm–1 to 1424 cm–1 originate from carboxyl-conjugated carbonyl stretches, and these are generally increased after significant oxidation by hydrogen peroxide[40]. These bands are similar for samples F2 and F4, indicating that extrusion, similar to AHP, promoted changes in these chemical groups. The bands at 1380 cm–1 represent the symmetric and asymmetric deformation of the C-H bond of cellulose and hemicellulose, which are most representative in the modified fibers (F2 and F4) relative to the untreated fiber (F1), highlighting the loss of lignin and indicating greater exposure of cellulose[19,40]. The bands at 1249 cm–1 and 1155 cm–1 represent the relative axial vibration of C-O groups. An increase in this band was observed in the F4 sample, which could be due to an increase in carboxylic acids and a decrease in the content of phenolic hydroxyl groups as a result of changes caused by AHP on lignin[40,41].

3.4 Thermogravimetric analysis (TGA) The thermal characterization of oat hulls that were untreated (F1), treated by extrusion (F2) and treated with alkaline hydrogen peroxide (F4) through TGA appears in Figure 3. The washed samples (F3 and F5) are not presented because there were not differences when compared with the correspondent non-washed (F2 and F4, respectively).

Figure 2. FTIR spectra of fibers: F1 (untreated); F2 (treated by extrusion) and F4 (treated with AHP). Polímeros, 26(4), 320-326, 2016

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Figure 3. Thermogravimetric parameters of fibers: untreated (F1); treated by extrusion (F2) and treated with AHP (F4).

For all samples, an initial and small loss peak can be observed at the temperature of 83 °C due to the loss of water remaining in the material after the drying process. The second mass loss occurred at temperatures of 316 °C, 313 °C and 322 °C for the F2, F1 and F4 samples, respectively, corresponding to the degradation of hemicelluloses and pectin[42]. The weight losses at temperatures of 354 °C, 368 °C and 370 °C (samples F4, F1 and F2, respectively) correspond to the decomposition of cellulose and lignin[43,44]. Comparing the thermograms of the untreated oat hulls (F1) with those of the physically and chemically treated hulls (F2 and F4, respectively), it can be observed that the extrusion process did not affect the stability of the fibers, as the temperatures of the different degradation fractions were similar in samples F1 and F2. The curves of F4 showed a slight increase in the thermal stability of the hemicelluloses and pectins (peak temperature of 322 °C compared to 316 °C for the untreated hulls) and a small decrease in the temperature of degradation of cellulose and lignin (from 368 °C in the untreated oat hulls to 354 °C in the hulls treated with AHP). These lower differences indicated the physical and chemical treatments used were not enough to promote noticeable effects in the thermal stability of fiber components. Brígida et al.[19] observed a slight increase in the thermal stability of all fractions of green coconut fiber after traditional treatment with H2O2.

4. Conclusions Reactive extrusion can be an alternative and effective method to modify fibers and exhibits multiple advantages compared to conventional methods, including a lower processing time and the absence of effluents. These advantages can overcome the additional cost of eventually required wear resistant corrosion screw and barrel for the processing. The extrusion process combined with hydrogen peroxide was shown to be a viable method for modifying the fiber of oat hulls, promoting bleaching, removing surface compounds and altering physical and chemical properties. FTIR revealed differences between the modified and unmodified structures, 324

proving the efficiency of the reactive extrusion. TGA analysis showed the AHP process has no noticeable effect on the thermal stability of oat fiber. The modified fibers, which possess differentiated properties, may be considered for several applications in paper, textile and plastic industries.

5. Acknowledgements The author thanks CNPQ, CAPES and UEL for the financial support given to this work.

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Polímeros, 26(4), 320-326, 2016

http://dx.doi.org/10.1590/0104-1428.2367

Isolation of whiskers from natural sources and their dispersed in a non-aqueous medium Mauro Vestena1,2*, Idejan Padilha Gross1, Carmen Maria Olivera Muller3 and Alfredo Tibúrcio Nunes Pires1 Polymeric Materials Research Group – POLIMAT, Chemistry Department, Universidade Federal Santa Catarina – UFSC, Florianópolis, SC, Brazil 2 Chemistry Academic Department, Universidade Tecnológica Federal do Paraná – UTFPR, Campus Pato Branco, Pato Branco, PR, Brazil 3 Department of Food Science and Technology, Universidade Federal Santa Catarina – UFSC, Florianópolis, SC, Brazil 1

*mauro@utfpr.edu.br

Abstract Whiskers have been used as a nanomaterial dispersed in polymer matrices to modify the microscopic and macroscopic properties of the polymer. These nanomaterials can be isolated from cellulose, one of the most abundant natural renewable sources of biodegradable polymer. In this study, whiskers were isolated from sugarcane bagasse and corn cob straw fibers. Initially, the cellulose fiber was treated through an alkaline/oxidative process followed by acid hydrolysis. Dimethylformamide and dimethyl sulfoxide were used to replace the aqueous medium for the dispersion of the whiskers. For the solvent exchange, dimethylformamide or dimethyl sulfoxide was added to the aqueous dispersion and the water was then removed by fractional distillation. FTIR, TGA, XRD, TEM, Zeta and DLS techniques were used to evaluate the efficiency of the isolation process as well as the morphology and dimensions of the whiskers. The dimensions of the whiskers are comparable with values reported in the literature, maintaining the uniformity and homogeneity in both aqueous and non-aqueous solvents. Keywords: whiskers, cellulose bleaching, agricultural waste.

1. Introduction Agricultural production has expanded in recent years and the lignocellulosic byproducts generated have great potential for exploitation as new raw materials. Agricultural byproducts, such as sugarcane bagasse (SCB) and corn cob straw (CCS), are residues produced on a large scale which, after treatment, can be added to a polymeric matrix in order to alter the physicochemical characteristics of the nanocomposites. Lignocellulosic materials consist mainly of cellulose, hemicellulose and lignin, the contents of which vary depending on the raw material and the physiological characteristics of the plant. SCB is comprised of 42 to 46% cellulose, 21 to 28% lignin and 27 to 29% of hemicellulose[1-3]. On the other hand, CCS contains 42 to 44% cellulose, 22 to 28% lignin and 27 to 28% hemicellulose[4-6]. Minor amounts of other polysaccharides (found in the cell walls) and minerals may also be present in these raw materials. Cellulose is an abundant biopolymer which is biodegradable and renewable and it can be obtained in an environmentally sustainable manner. It has a microfibrillar structure composed of β-1,4-linked anhydro-D-glucose (C6H10O5) units, and the repeat segment is a dimer of glucose, known as cellobiose. The spatial conformation and arrangement associated with intra- and intermolecular hydrogen bonds lead to the rigid characteristics of cellulose[7,8]. Six interconvertible cellulose polymorphs, namely I, II, III1, III2, IV1 and IV2 have been identified, each with its particular set of network parameter characteristics of unit cells, type I being referred to as native cellulose[7]. In order to isolate the cellulose from lignocellulosic

Polímeros, 26(4), 327-335, 2016

plant materials, the non-cellulosic components (hemicellulose, lignin, etc.) need to be removed from the fiber through a process known as bleaching. Different treatments have been applied in the bleaching of natural fibers, such as the use of an alkali, followed by an oxidizing/alkaline environment with the use of an oxidant based on chlorinated compounds[9,10]. Studies have been conducted using oxidants which generate very low levels of waste, such as hydrogen peroxide, thereby reducing the environmental impact[1,11]. Rosa et al.[12] has isolated cellulose from rice husk by cellulose extraction in two-step chlorine-free bleaching processes. Other processes are performed without the alkali treatment step, using systems such as nitric/acetic acid[11] and peracetic acid[13] and mixtures of hydrogen peroxide with manganese(II) sulfate and zinc oxide or manganese(II) sulfate and titanium dioxide[14]. In the bleaching process the control variables, such as temperature, time and concentration of reagents, play an extremely important role. After bleaching, the cellulose isolated by mechanical, chemical or enzymatic treatment can result in microcrystalline cellulose, microfibrillated cellulose or whiskers. The microfibrils which make up the cellulose fiber consist of amorphous and crystalline domains and their acid hydrolysis allows the isolation of the crystalline domains, denoted as whiskers or cellulose nanocrystals, due to the dimensional characteristics of the material. The aspect ratio of the whiskers is dependent on the variables involved in the process used to isolate the whiskers, such as the raw material, temperature, reaction

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Vestena, M., Gross, I. P., Muller, C. M. O., & Pires, A. T. N. time, reagent concentration and reagent/raw materials ratio. Habibi et al.[7] and Moon et al.[15] reported that whiskers have an acicular shape and length/diameter ratio of 10 to 30, with lengths of 35 to 500 nm and a diameter of 3 to 15 nm, depending on the raw material and treatment used to isolate the whiskers. The large surface area and crystallinity of the whiskers make these promising materials for incorporation into polymeric matrixes, where they increase the mechanical strength and modify the gas barrier properties[7,15-17]. On the other hand, the whiskers can be dispersed homogeneously in polymers which are soluble in aqueous solution due to their hydrophilic surface[18]. However, this restricts their use in the matrices of hydrophobic polymers due to difficulties related to their dispersion in these systems. Studies have been directed toward obtaining an adequate dispersion of whiskers in hydrophobic matrices, using approaches such as the addition of anionic surfactants or tert-butanol to the dispersing medium[19], the use of a cationic surfactant[20], the use of partially hydrolyzed poly(vinyl alcohol)[21], surface functionalization of the whiskers[22-24], the use of lyophilization and redispersion in a non-aqueous solvent. Of these proposed methods, the redispersion of the whiskers in non-aqueous solvents has proved to be a promising technique which allows homogeneous dispersion in hydrophobic polymers, it being necessary to assess the process conditions in relation to the polymer matrix. Yu et al.[25] isolated whiskers in an aqueous medium and then replaced the water with acetone and subsequently replaced the acetone with chloroform in several successive centrifugation steps and finally the whiskers were incorporated into the poly(3-hydroxybutyrateco-3-hydroxyvalerate) matrix. Pracella et al.[26] used an analogous procedure for the solvent exchange of the whiskers suspension and subsequent incorporation of the whiskers into poly(lactic acid). The whiskers yield from the isolation process, as well as the whiskers suspension in non-aqueous solvents and subsequent incorporation into the hydrophobic polymer matrix, are dependent on the characteristics of the raw material, the bleaching process and the procedure used for the isolation of the whiskers, in addition to the characteristics of the solvent used in the suspension and the polymer matrix employed to produce the nanocomposite. In this context, the aim of this study was to evaluate the processes used for the isolation of whiskers from sugarcane bagasse and corn cob straw, to promote the exchange of the whiskers dispersing medium and keep the whiskers homogeneously dispersed in non-aqueous solvents to facilitate the obtainment of a nanocomposite polymeric matrix containing whiskers.

2. Materials and Methods 2.1 Materials The sugarcane bagasse (SCB) was provided by regional ethanol producers (São Paulo, SP Brazil) and the corn cob straw (CCS) by regional organic producers (Pato Branco, PR Brazil). Poly(lactic acid) (Code 3251D) was purchased from NatureWorks, Cargill (Minnetonka, MN, USA). Sulfuric acid, sodium hydroxide and hydrogen peroxide were purchased 328

from Lafan Química Fina, and dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) were supplied by Nuclear (Brazil). Cellulose was purchased from Merck and lignin obtained by isolation from the raw materials (SCB and CCS) according to regulation TAPPI T222 om-88[24]. All of the chemical reagents were used without further purification.

2.2 Bleaching of fibers from raw materials The SCB and CCS raw materials were washed with distilled water, dried and milled and then passed through a 30-mesh sieve. In the next step, the fibers were dried in an oven with air circulation at 105 °C for 4 h prior to the bleaching process. For the bleaching process, three different procedures were applied: (i) treatment with NaOH aqueous solution; (ii) treatment with H2O2; and (iii) treatment with Ca(ClO)2 aqueous solution. (i) Treatment with NaOH aqueous solution - The fibers were maintained in a 5% aqueous solution of NaOH (1:20 w/v fiber/solution) at 85 ºC for 90 min, under mechanical stirring. In the next step, the dispersion was filtered and the fibers washed until neutral pH and dried at 105 °C for 4 h. The fibers obtained through this treatment are denoted as SCB/NaOH or CCS/NaOH, according to the raw material. (ii) Treatment with H2O2 - The fibers subjected to treatment (i) were then maintained in an aqueous solution of 5% NaOH and 11% H2O2 (1:20 w/v) at 55 °C for 90 min and the dispersion was filtered. The fibers were then washed with distilled water until neutral pH and dried at 105 °C in an oven with air circulation for 4 h. These treated fibers were denoted by SCB/H2O2 or CCS/H2O2, according to the raw material. (iii)Treatment with Ca(ClO)2 aqueous solution - Fibers subjected to treatment (i) were maintained in an aqueous solution of 2.5% Ca(ClO)2 and 5% NaOH (w/v), at 45 ºC for 240 min and the dispersion was filtered. The fibers were then washed until neutral pH and dried at 105 ºC for 4 h. These treated fibers were denoted by SCB/Ca(ClO)2 or CCS/Ca(ClO)2, according to the starting raw material.

2.3 Characterization of raw materials and bleached fibers To evaluate the effect of bleaching on the composition and crystallinity of the lignocellulosic materials, SCB and CCS samples before and after the bleaching processes were characterized by Fourier transformed infrared spectroscopy (FTIR), thermogravimetry analysis (TG) and X-ray diffraction (XRD). The cellulose, lignin and hemicellulose contents of the untreated SCB and CCS were determined in accordance with the respective recommendations of the Technical Association of the Pulp and Paper Industry (TAPPI standards). 2.3.1 Content of cellulose, lignin and hemicellulose in SCB and CCS raw materials The lignin, cellulose and hemicellulose of the raw materials SCB and CCS were determined according to the TAPPI Standard T222 om-88[27], the ashes according to the norm TAPPI T211 OM 93[28] and the extractive was Polímeros, 26(4), 327-335, 2016

Isolation of whiskers from natural sources and their dispersed in a non-aqueous medium determined according to the standard TAPPI T 204 om-88[29]. The holocellulose content (cellulose and hemicellulose) and the pulp were determined according to standard TAPPI T13 m-54[30], allowing the determination of the hemicellulose content. All measurements are expressed on a dry basis. 2.3.2 Fourier transform infrared spectroscopy (FTIR) The FTIR spectra for the cellulose, lignin, SCB and CCS samples before and after the bleaching process were obtained on a Shimadzu IR Prestige system, performing 20 scans, with a resolution of 4 cm–1, in the range of 4000 to 600 cm–1 at room temperature. The samples were prepared in the form of compressed KBr disks. 2.3.3 Thermogravimetric analysis (TGA) Thermal degradation measurements were taken on a Shimadzu TGA-50 thermogravimetric analyzer, from room temperature to 600 °C at a heating rate of 10 °C min–1. The flow rate of the nitrogen purge gas was 50 mL min–1. 2.3.4 X-ray diffraction (XRD) X-ray patterns of the specimens were obtained on a Philips X’Pert diffractometer (Netherlands), with Cu (Kα) radiation (λ = 1.5418 Å), operating at room temperature, 30 mA and 40 kV. The scanned region of 2 to 40° (2θ) and a pitch of 0.05° s–1 were applied to evaluate the crystallinity index of the SCB and CCS samples, before and after the bleaching processes. The samples were milled to yield material between 120 and 450 mesh sizes. The crystallinity index (CI) was determined using Equation 1, by comparing the corresponding areas of the crystalline and amorphous fractions, obtained by deconvolution of the diffractogram, where AA is the area of the amorphous phase and At the total area of  A  CI= 1 − a  x100 (1) At  

2.4 Extraction of whiskers To extract the whiskers bleached fibers obtained from CCS and SCB were selected, through treatments (ii) with H2O2 and (iii) with Ca(ClO)2, as described in section 2.2. Acid hydrolysis was carried out at 55 °C for 75 min with 60 wt % H2SO4 (1:20 w/v) under mechanical stirring. The fiber suspension was diluted with water (five times the volume of the reaction mixture) at room temperature to stop the reaction and after centrifugation the supernatant was removed. Distilled water was then added to the sediment, which was suspended under mechanical stirring, and the suspension was centrifuged until the appearance of the cloud point. This suspension of cellulose whiskers was collected and treated using dialysis bags in a cellulose acetate membrane with a cut off of 10,000 g mol–1 at neutral pH and stored at 5 °C.

2.5 Replacement of dispersive medium of whiskers The dispersive medium containing the cellulose whiskers was changed from water to DMF or DMSO, in order to solubilize the hydrophobic polymer matrix. The DMF or Polímeros, 26(4), 327-335, 2016

DMSO was added to the aqueous suspension of cellulose whiskers and the water eliminated through vacuum distillation at 60 °C, since the boiling point of a non-aqueous solvent is greater than the boiling point of water. In the exchange of the aqueous dispersion medium containing the whiskers the volumes of solvent before and after fractional distillation were gravimetrically monitored.

2.6 Characterization of whiskers 2.6.1 Whiskers in aqueous and non-aqueous media In order to estimate the yield of the isolation process of the whiskers and efficiency in the solvent exchange the quantity of whiskers dispersed in the aqueous and non-aqueous medium was determined using the gravimetric procedure. A sample of 2 mL of the whiskers suspension in aqueous medium was placed in Petri dishes and kept in an oven with air circulation at 80 °C to evaporate the solvent. The sample was then maintained at 105 °C until constant weight, and the quantity of whiskers was calculated and expressed as weight per volume of suspension. The yield of the hydrolysis reaction was estimated from the weight of the cellulose content of the bleached fibers. An analogous procedure was used to determine the efficiency of the solvent exchange from water to DMF or DMSO, maintaining the suspension at 130 °C until complete evaporation of the solvent. 2.6.2 Zeta potential analysis (ξ) and dynamic light scattering (DLS) The surface charges were measured by zeta potential analysis (ξ) from an aliquot of the neutral aqueous suspension of the whiskers with the concentration equalized at 0.005% (w/w). In order to evaluate the stability of the suspensions they were kept under refrigeration at 7 °C and analyzed at 6-month intervals, after sonication (UltraCleaner 1600A) for 30 min and centrifugation under a force of 1370 x g. Dynamic light scattering was carried out using a Zetasizer Nano ZS system (Malvern, UK), by detecting back-scattered laser-light (θ = 173°) and comparing the coherence of scattering patterns as a function of time, with 0.002 g mL–1 of whiskers in aqueous, dimethylformamide or dimethyl sulfoxide suspensions, at 25 °C. Measurements of the particles in suspension were carried out in triplicate, and each measurement was composed of 15 runs every 10 s. Analysis based on the diffusion rate of the particles in the fluid was conducted to calculate the autocorrelation function and deduce the particle size information. 2.6.3 Transmission electron microscopy (TEM) Transmission electron microscopy analysis of the whiskers was carried out using a JEOL-1011 TEM electron microscope operating at 80 kV. One drop (0.002 g mL–1) of the suspension of whiskers was diluted in 2 mL of isopropyl alcohol and deposited on a grid coated with copper film (FORMVAR) and after being almost dried it was stained by adding one drop (~ 4 μL) of 3% uranyl acetate (w/v). The excess liquid was removed and this was followed by drying at room temperature. 329

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3. Results and Discussions 3.1 Characterization of raw materials Table 1 shows the composition of cellulose, lignin, hemicellulose, ash and extractive for the sugarcane bagasse (SCB) and corn cob straw (CCS). As expected, for both raw materials cellulose predominates in relation to the other components. The percentages of each component are consistent with values reported in the literature[1-6]. SCB fibers have a higher content of lignin and lower content of hemicellulose than CCS fibers, which affects the physiological characteristics of these fibers.

3.2 Evaluation of the effect of bleaching process Figure 1 shows the FTIR spectra, in the range of 1400‑1850 cm–1, for cellulose and lignin obtained by isolation from the raw materials straw corn cob straw (CCS) and sugar cane bagasse (SCB) before and after the bleaching treatments. In the range of 1400 to 1850 cm–1 the cellulose shows absorption bands at 1645 and 1430 cm–1 related to the symmetrical angular deformations of the OH and CH2 (constituents of the chemical structure of the cellulose repeating unit), respectively[31]. Due to the phenolic groups present in the lignin, the infrared spectrum shows absorption bands at 1600 and 1510 cm–1, related to axial deformations of the aromatic rings[32]. Besides the characteristic absorption bands of cellulose and lignin, the untreated SCB and CCS present absorption bands at 1730 cm–1, characteristic of the axial stretching of the carbonyl and associated with hemicellulose[10,14]. The infrared spectra of the untreated SCB and CCS indicate the presence of cellulose, lignin and

hemicellulose, in agreement with the results of the quantitative analysis shown in Table 1. After the alkaline treatment (i) of the raw materials (SCB and CCS) there was a reduction in the intensity of the absorption bands as expected, which is associated with the functional groups present in the polymer chain of the lignin and hemicellulose. Although the NaOH treatment promoted the removal of lignin, the presence of shoulders at 1510 and 1600 cm–1 indicates that complete removal of lignin did not occur. In order to reduce the lignin content in the samples, we used the treatments (ii) or (iii), after which the infrared spectra suggest greater efficiency of the bleaching processes, with a reduction in the intensity of the absorption bands related to lignin. Figure 2 shows the TG curves obtained for the pure cellulose, the lignin obtained by isolation from the raw materials, the raw material and the fibers after the bleaching processes for (a) SCB and (b) CCS. The TG curves of the raw materials exhibited similar behavior with weight loss at 100 °C associated with the removal of water molecules and in temperature range of 250 °C to 400 °C related to the thermal degradation of the constituents of the sample, while at temperatures above 400 ºC only the solid waste residue remained. The residue of the lignin isolated from SCB and CCS is higher than the residue of cellulose and other studied samples. At 450 °C there was 23% of residue for the raw material samples and 63% and 71% of residue for lignin from SCB and CCS, respectively. The percentages of residue for the fibers after treatment with hydrogen peroxide were 19% and 13% for SCB and CCS, respectively. These values of residue percentage is related to higher quantity of lignin in SCB than in CCS samples after bleaching

Table 1. Composition mass percentage of sugarcane bagasse (SCB) and corn cob straw (CCS). Raw material SCB CCS

Cellulose 44.6 ± 1.2 47.3 ± 2.1

Lignin 24.0 ± 1.3 14.4 ± 0.9

Hemicellulose 26.0 ± 2.1 34.8 ± 2.3

Ash 2.8 ± 0.3 1.5 ± 0.1

Extractive 2.5 ± 0.3 2.0 ± 0.2

Figure 1. Infrared spectra for pure cellulose, lignin and the samples after the bleaching process (indicated in the figure) using (a) SCB and (b) CCS as raw materials. 330

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Isolation of whiskers from natural sources and their dispersed in a non-aqueous medium

Figure 2. TG curves and DTG curves for (a, c) SCB and (b, d) CCS related to pure cellulose, lignin and the samples after the bleaching processes (indicated in the figure).

processes, as can be seen by relative intensity at 1510 cm–1 and 1600 cm–1 in the FTIR spectra, since the lignin was not totally removed. The amount of residue observed after the calcium hypochlorite process can be included the presence of calcium compound. The DTG curves enable the visualization of the different stages of thermal degradation of the components in the samples, as shown in Figure 2c, d for samples derived from SCB and CCS, respectively. Pure cellulose presents a single-stage thermal degradation event with the maximum degradation rate at 365 °C, as described in the literature[10,33]. Besides the degradation stage associated with cellulose, the raw materials presented other degradation stages related to lignocellulosic material, such as lignin and hemicellulose[33]. After treatment (i) with NaOH, the behavior of the DTG curve with a single stage of thermal degradation tends to be similar to that of pure cellulose, indicating the removal of the lignocellulosic components. This tendency was noted for the sequence of treatments with hydrogen peroxide and calcium hypochlorite, indicating greater efficiency for these bleaching processes. This behavior was observed for CCS and SCB, in agreement with the residue levels observed in the thermal analysis and infrared spectroscopy. Figure 3a, b show the diffractograms for the SCB and CCS raw materials after treatments (ii) and (iii), respectively, with peaks at 14.7°, 16.6°, 22.5° and 34.7°, which relate to the families of crystalline planes: (101), (10 1 ), (002) and (004), Polímeros, 26(4), 327-335, 2016

respectively, characteristic of the cellulose I[34]. The peaks associated with the crystal structure suggest that the alkali treatment is not aggressive in relation to changes in the cellulose crystal lattice. According to Borysiak and Doczekalska[35], the crystalline structure of cellulose can change from cellulose I to cellulose II in an aqueous 15% NaOH (w/v) solution. The crystallinity index was 30% for both of the raw materials studied. After the bleaching process the fibers obtained from SCB showed values for the crystallinity index of 70% and 68%, after treatment with hydrogen peroxide and calcium hypochlorite, respectively. For the fibers obtained from CCS the crystallinity index was 67% after both treatments. For comparison, the crystallinity index determined by Rosa et al.[12] for rice husk cellulose and microcrystalline cellulose were 67% and 79%, respectively. An increase in the CI values indicates a greater quantity of crystalline cellulose in the samples compared to amorphous material, which is related to the effectiveness in the removal of hemicelluloses and lignin, in agreement with the results obtained in the infrared spectroscopy and thermogravimetric analysis.

3.3 Characterization of whiskers Table 2 shows the yields for the whiskers isolated from the bleached raw materials by acid hydrolysis. A value of 60% (w/w) was obtained for the bleached cellulose, suggesting that the process used for the isolation of the nanocrystals 331

Vestena, M., Gross, I. P., Muller, C. M. O., & Pires, A. T. N.

Figure 3. XRD spectra for (a) SCB and (b) CCS raw materials and after treatments with H2O2/NaOH or Ca(ClO)2/NaOH (as indicated in the figure).

had a good efficiency, regardless of the bleaching process and raw material used. The literature reports different yields for the whiskers according to the hydrolysis conditions applied for the bleaching of the fibers. Correa[36] studied the hydrolysis of curauá fibers with 60% H2SO4 (v/v) at 45 °C for 75 min and obtained a whiskers yield of 70% while Taipina[37] using the same kind of fiber obtained a yield of 65%. On the other hand, Oksman and Bondeson et al.[38] carried out hydrolysis with H2SO4 (63.5% w/w), using a 10:1 (v/w) solution/raw material ratio and a reaction time of 2 h and obtained whiskers with a yield of 30%. Dong et al.[39] reported yields of between 35% and 48% when varying the hydrolysis conditions (64% H2SO4 w/w) applying temperatures of 26 °C to 65 °C and reaction times of between 0.25 h and 18 h. Mathew et al.[40] studied wood waste from the production of ethanol, after bleaching and acid hydrolysis and recorded 46% yield for the whiskers. In this regard, we also conducted preliminary experiments under more severe conditions, with 65% H2SO4 (w/w), a temperature of 55 °C and 75 min of reaction and with 60% H2SO4 (w/w) at 55 °C for 300 min, and in both cases the yield of whiskers obtained was 30%. However, the suspensions had a dark color, indicating the carbonization of the whiskers. The stability of aqueous suspensions of whiskers was analyzed via the zeta potential (ξ). The values obtained at 6-month intervals are shown in Table 3. The potential of around –64 mV in neutral aqueous medium (pH between 6.0 and 7.0) is indicative of negative charges on the surface of the whiskers, due to the grafting of sulfate groups in the cellulose hydrolysis process[41,42]. These groups enable the formation of a colloidal suspension and its stability is due to the electrostatic repulsion of these surface charges[40]. According to Greenwood[43], potentials above 30 mV are sufficient for obtaining a suspension with good stability. The morphology and size of the whiskers were evaluated by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Figure 4a, b show the TEM micrographs for the whiskers dispersed in water. For both raw materials (SCB and CCS) the whiskers were rod shaped (acicular), which is characteristic of structural rigidity. For SCB the average length (L) was 230 ± 35 nm and the average diameter (D) determined by TEM was 16 ± 3 nm, with an 332

Table 2. Yields obtained for the whiskers after the hydrolysis processes. Raw material SCB CCS

Bleaching process

Yield (%)

SCB/H2O

SCB/Ca(ClO)2

CCS/H2O2

CCS/Ca(ClO)2

Table 3. Zeta potential (mV) of whiskers. Storage time (months) 0 6 12

SCB

CCS

–64.3 ± 6.9 –64.1 ± 1.5 –63.2 ± 1.2

–65.2 ± 7.8 –64.1 ± 0.7 –65.6 ± 4.5

aspect ratio (L/D) of 15. For the CCS the values determined by TEM were L = 288 ± 62 nm and D = 16 ± 4 nm, with an L/D ratio of 18. The acicular shape and dimensions are similar to those described in the literature for whiskers, with lengths of around 35 to 500 nm and diameters of between 3 and 15 nm[7,15]. More specifically, for sugarcane bagasse, Teixeira et al.[1] obtained average values for the length and diameter of 255 ± 55 nm and 8 ± 3 nm, respectively, while Mandal et al.[10] reported a shorter length (170 nm) and larger diameter (35 nm). Lima et al.[44] and Braun et al.[45] considered the Broersma relationships using the rotation and translational diffusion coefficients to obtain the dimensions of the whiskers. According to these authors, the spheroidal form factor contains more than one adjustable parameter, allowing the simultaneous determination of the diameter and length, which are related to the size of the whiskers. Thus, using the DLS technique the values obtained for the dimensions of the whiskers were 232 ± 5 nm and 242 ± 15 nm for SCB and CCS, respectively, which are in agreement with the values determined by TEM. On the other hand, for the whiskers obtained from SCB under more severe hydrolysis conditions, with 65% H2SO4 (w/w) at 55 °C for 75 min, the size obtained by DLS was 167 ± 2 nm, which indicates that the hydrolysis conditions Polímeros, 26(4), 327-335, 2016

Isolation of whiskers from natural sources and their dispersed in a non-aqueous medium can alter[39] the dimensions and surface characteristics of the whiskers, as well as their yield. Thus, it is possible to control the aspect ratio (L/D) and surface area, which are important characteristics of nanocrystals, making them of interest for different applications. The incorporation of whiskers, homogeneously distributed in matrices of water-insoluble polymers presents a great challenge. Approaches commonly applied to overcome this difficulty include the use of surfactants, the surface functionalization of the whiskers and/or the lyophilization and solvent dispersion of hydrophobic polymer matrices. However, a large quantity of surfactants is required to maintain the stability of the suspension due to the high specific surface area of the nanocrystals. Furthermore, as observed by Bondeson and Oksman[21] the surfactants may cause degradation of the polymer matrix. On the other hand, Peterson et al.[19] reports that when lyophilizing a suspension of whiskers in aqueous medium and after their dispersion in chloroform, undesirable flakes are formed in the solvent. Viet et al.[46] studied a suspension of lyophilized whiskers in aqueous medium and their subsequent dispersion in dimethylformamide and dimethyl sulfoxide, while maintaining the aggregates in the suspension. Kamal et al.[47] used the spray dried, freeze drying and spray freeze drying of whiskers

while maintaining agglomerates in suspension in molten polymer (PLA) at a minimum size of 1μm. In this study, we replaced the aqueous dispersion medium with dimethylformamide or dimethyl sulfoxide, as described in the experimental section, and evaluated the morphology and dispersion characteristics of the whiskers in these non-aqueous media. Figure 5a, b show the TEM micrographs of the whiskers obtained from SCB and CCS as raw materials, respectively, dispersed in dimethylformamide. A homogenous dispersion of the whiskers was maintained, and the lengths (L) were 240 ± 61 nm and 245 ± 80 nm and the diameters (D) were 16 ± 5 nm and 18 ± 6 nm for SCB and the CCS, respectively. These dimensions are in agreement with the values obtained through DLS of 240 (±26) for SCB and 250 (±8) for CCS in dimethylformamide and 224 (±5) for SCB and 244 (±8) for CCS in dimethyl sulfoxide. The solvent exchange procedure was effective and appropriate to maintain the dispersion of the whiskers. As discussed above, the method is simple and inexpensive, indicating that the solvent mixture is not azeotropic and since there was a considerable difference between the boiling temperatures of the solvents efficient water removal could be carried out by fractional distillation.

Figure 4. TEM micrographs for whiskers dispersed in aqueous medium obtained from (a) SCB and (b) CCS as raw materials.

Figure 5. TEM micrographs for whiskers obtained from (a) SCB and (b) CCS as raw materials dispersed in DMF. Polímeros, 26(4), 327-335, 2016

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4. Conclusions The procedures used for the isolation of whiskers and the oxidant/alkali bleaching processes followed by acid hydrolysis were found to be effective, providing good yields and dimensional characteristics as well as dispersity in a solvent medium. The isolated whiskers had nanometric dimensions and aspect ratio (L/D) values comparable with those reported in the literature. When the aqueous medium was replaced with a non-aqueous medium using a simple low cost technique, without the need for mechanical work, the whiskers remained dispersed and the morphological characteristics of the whiskers were maintained. The dispersion of the whiskers in a non-aqueous solvent facilitates future studies on the development of techniques to obtain nanocomposite whiskers homogeneously dispersed in matrices of polymers by dissolving them in a common solvent followed by solvent evaporation.

5. Acknowledgements The authors are grateful for the financial support provided by CAPES and CNPq and also for the characterization by Transmission Electron Microscopy performed by LCME – UFSC.

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http://dx.doi.org/10.1590/0104-1428.2183

S S S S S S S S S S S S S S S S S S

Effect of surface finishing on friction and wear of Poly‑Ether-Ether-Ketone (PEEK) under oil lubrication Thiago Fontoura de Andrade1*, Helio Wiebeck2 and Amilton Sinatora3 Centro de Excelência em Engenharia, Eaton Grupo Veículo, Valinhos, SP, Brazil Departamento de Engenharia Metalurgica e de Materiais, Escola Politécnica, Universidade de São Paulo – USP, São Paulo, SP, Brazil 3 Laboratório de Fenômenos de Superfície – LFS, Departamento de Engenharia Mecânica, Escola Politécnica, Universidade de São Paulo – USP, São Paulo, SP, Brazil 1

*thiagofandrade@eaton.com

Abstract The tribological properties of poly-ether-ether-ketone (PEEK) containing 30% of carbon fiber were studied in an oil‑lubricated environment and different surface finishing of the metallic counterbody. Four different finishing processes, commonly used in the automotive industry, were chosen for this study: turning, grinding, honing and polishing. The test system used was tri-pin on disc with pins made of PEEK and counterbody made of steel; they were fully immersed in ATF Dexron VI oil. Some test parameters were held constant, such as the apparent pressure of 2 MPa, linear velocity of 2 m/s, oil temperature at 85 °C, and the time - 120 minutes. The lubrication regime for the apparent pressure of 1 MPa to 7 MPa range was also studied at different sliding speeds. A direct correlation was found between the wear rate, friction coefficient and the lubrication regime, wherein wear under hydrodynamic lubrication was, on average, approximately 5 times lower, and the friction coefficient 3 times lower than under boundary lubrication. Keywords: tribology, PEEK, roughness, wear, friction.

1. Introduction A major challenge for the use of polymeric materials in the mobility industry is the replacement of metallic materials used in the power train, especially the engine and transmission[1-3]. The conditions of torque, temperature and friction in the components of those systems render impractical the use of most polymers, nonetheless, there are some alternatives that can be studied, among those which poly-ether-ether-ketone (PEEK). The combination of mechanical and wear resistance is one of the characteristics of PEEK which may allow its application in mechanical components such as gears, bearings, thrust washers, bushings, seals, forks, coupling elements, among others[4-6]. In order to enhance such properties even further; it is common to add carbon fiber to this polymer[7]. Thus, it is important to understand the tribological behavior of this material when in contact with metal parts with different roughness and in lubrication environments. Originally, it was thought that less roughness meant less wear, due to abrasion, in polymers[8]. However, recent research has shown that for certain polymers there is an optimal roughness, pointing to a complex effect of the roughness of the counterbody in polymer wear. Experiments conducted in dry condition with ultra‑high molecular weight polyethylene (UHMWPE) rubbing against metallic parts with extremely smooth surfaces, have shown wear rates comparable to wear rates of parts rubbing against relatively rough surfaces. It is believed that, due to the reduced number and height of the asperities of the smoother surface, a thin film irregularly transferred from the work piece is formed, which functions as debris and accelerates abrasive

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wear. However, at a certain level of roughness, detached polymeric debris adhere to the valleys of the roughness, thereby reducing wear rate[9]. Friedrich et al.[10] studied the influence of roughness direction and parameters on dry wear. The study found that, when surface roughness is perpendicular to slip, natural PEEK does not change its wear characteristics with the increase of roughness Ra, whereas, when surface roughness is parallel, there is a notable increase in the wear rate of PEEK without additives. For versions combined with fibers, the effects of counterbody roughness and relative slip direction were found to be less significant than that of regular PEEK. The effect of controlled surface topography of metallic counterbodies in non-lubricated transfer and wear of PEEK was also studied by Ramachandra and Ovaert[11]. For that investigation, specimens were manufactured with longitudinal, transverse, and angled grooves, but no correlation among the angle of the channel, wear and friction coefficient was observed. A well accepted model for polymer wear against very rough surfaces is simple abrasion, in which the metallic roughness penetrates the polymer up to a certain depth. The wear rate is determined by the depth of roughness penetration, shear angle and sliding distance. However, the wear rate changes with time because the asperities are covered by frayed polymer[12]. Although a large number of the sliding friction studies conducted with this material were done in non-lubricated environments; sliding friction studies in lubricated environments

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Effect of surface finishing on friction and wear of Poly-Ether-Ether-Ketone (PEEK) under oil lubrication are necessary. The objective of the present work was to evaluate friction and wear behavior of PEEK combined with 30% carbon fiber, in a lubricated environment, and with different roughness of metallic parts.

up to 12 m/s. Through this experiment it was possible to ascertain the friction coefficient for each of the lubricating regime.

2. Materials and Methods

Specific wear rate (WS) expressed by Equation 1, was calculated through material mass loss (Δm) obtained from the difference in pin mass before and after the test, divided by the load (F), sliding distance (L) and material density (ρ).

The present study was conducted using Victrex 150CA30‑type which is PEEK matrix combined with 30% carbon fiber [13]. The pins were injected with a diameter of 5 mm, as per ASTM G99-04[14], and at a temperature of approximately 380 °C. The friction surface was smoothly polished using a 0.5 µm sandpaper for 3 minutes in order to reduce remnant rough edges from injection molding, and correct friction surface planeness. The counterbody was manufactured with SAE 8620 steel, submitted to carburizing, quenching and tempering to a hardness range of 58-63 HRC. After heat treatment, the counterbody surface finish was achieved from four different processes: turning, grinding, honing and polishing.

2.1 Friction test Polymer friction and wear behavior were analyzed using a three-pin on metallic disc tribometer sliding unidirectionally. The three-point contact was used to give greater stability for the rotation speed and strength used in the test. Tribological results obtained on tri-pin on disk test machines are generally different from those obtained from single-pin test machines. Single-pin tests tend to display stick-slip and preferential wear of pin edges. Three-pin on disc systems are particularly more suitable for the study of roughness on wear as they maintain the contact surface fairly constant after the initial running-in[7]. The test was conducted in a lubricated environment with all the pins completely immersed in ATF Dextron VI oil and at a temperature of 85 ± 5 °C inside the test chamber. For each test, three new pins were placed onto the cylindrical device. The pins were positioned 120° apart from each other and moved on the same track. Normal force was applied via a piezo-actuator on a servo-controlled mechanism. Thus, the capacitive sensor enabled normal force to be continuously monitored and compared with the nominal force of approximately 118 N (equivalent to an apparent contact pressure of 2 MPa), so that any variation could be immediately corrected. Rotational speed was 125 rad/s, which corresponds to a linear speed of 2 m/s, also kept constant throughout the test. Test duration was defined after evaluation of wear over different sliding periods, until wear rate remained constant; this resulted in the adoption of a 120-minute period. It is known that result variability is inherent to pin‑on‑disc friction tests; therefore, each test was repeated at least three times. In order to obtain the friction coefficient curves versus sliding speeds, the samples slid for 60 minutes at a speed of 2 m/s and pressure of 2 MPa, and were then subjected to constant pressure of 1 MPa and 7 MPa. For each pressure, the pins were subjected to 10 different speeds for 10 minutes each. Initial speed was set at 1.2 m/s, and augmented, in arithmetic progression, at the rate of 1.2 m/s, for each step, Polímeros, 26(4), 336-342, 2016

2.2 Assessment of wear

Ws =

∆m  mm3    FLρ  Nm 

(1)

2.3 Roughness measurements Roughness measurements were conducted through white‑light interferometry (Zygo Nexview). Typical topography for each of the finishing studied is shown on Figure 1 and the average linear roughness parameters are shown on Table 1, these parameters were obtained perpendicular to the sliding direction. Four measurements were obtained for each sample, 90° apart.

2.4 Microscopic assessment The friction surfaces of the polymeric pins and metallic counterbodies were analyzed with a stereomicroscope in order to investigate the mechanisms responsible for wear in each test.

3. Results and Discussion 3.1 Definition of test duration The first factor to be considered was the definition of the duration of the test, in order to grantee that the system was in permanent wear regime. The test showed that, after 60 minutes, the mass loss remained stable for the two samples, when load, speed, surface finishing, lubrication and temperature conditions were maintained, as shown in Figure 2. Moreover, the mean friction coefficient for N=1 and N=2 did not vary significantly during the test (Figure 3), thus characterizing the permanent wear regime.

3.2 Definition of the lubrication regime Figure 4 shows friction coefficient variation as a function of the sliding speed, with honing finishing. In Figure 4 it can be observed that, when submitted to constant contact pressure, the friction coefficient decreases as sliding speed increases, for both higher and lower pressures, with a sharper decrease for the latter. Thus, the lubrication regimes were defined for pressures between 1 MPa and 7 MPa. For 1 MPa the mixed regime occurs in a range of 1.2 m/s to approximately 5 m/s, whereas for 7 MPa, the mixed regime only changes into hydrodynamic at 9 m/s. This occurs because in the sliding hydrodynamic regime the minimum lubrication film thickness is very sensitive to load. Base on the Stribeck curve, the friction coefficient range was defined for each lubrication regime, as follows: boundary regime 0.09 to 0.13, mixed regime 0.04 to 0.09 and hydrodynamic regime lower than 0.04[15]. 337

Andrade, T. F., Wiebeck, H., & Sinatora, A.

Figure 1. Typical measurements for superficial roughness of discs on the three test repetitions; (a) turning; (b) grinding; (c) honing and (d) polishing. Table 1. Values for superficial roughness for each steel counterbody finishing: roughness average (Ra), root mean square (RMS), and total roughness (Rz). Finishing Process Turning Grinding Honing Polishing

Ra (µm) 1.264 ± 0.010 0.825 ± 0.082 0.277 ± 0.064 0.048 ± 0.003

RMS (µm) 1.477 ± 0.013 1.022 ± 0.126 0.357 ± 0.082 0.063 ± 0.004

Rz (µm) 6.340 ± 0.149 4.622 ± 1.758 2.236 ± 0.499 0.575 ± 0.096

Figure 2. Specific wear rate (a) and mass loss (b) variation as a function of time for contact pressure of 2 MPa, speed of 2 m/s, turning, lubricated with ATF Dexron VI oil and temperature of 85 °C. N=1 and N=2 correspond to the two repetitions conducted. 338

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Effect of surface finishing on friction and wear of Poly-Ether-Ether-Ketone (PEEK) under oil lubrication 3.3 Influence of roughness on the friction coefficient Figure 5 illustrates the relationship between the friction coefficient and roughness of the metallic disc. For the same lubrication regime, surface finishing did not influence the

Figure 3. Friction coefficient (CoF) variation for test periods of 0-30 minutes (a), 30-60 minutes (b), 60-120 minutes (c) and 120‑180 minutes (d). N=1 and N=2 identify the two samples tested.

Figure 4. Change in the friction coefficient as a function of sliding speed for two different apparent contact pressures for a sample with honing finishing: (●) 1 MPa, (■) 7 MPa.

Figure 5. Influence of the metallic counterbody roughness on the friction coefficient. Polímeros, 26(4), 336-342, 2016

friction coefficient. For turning and grinding finishings, the friction coefficient observed was an average of 0.121 ± 0.007 and 0.109 ± 0.009, respectively; for polishing, it was approximately 0.038 ± 0.005, however for honing it displayed a greater dispersion of the results around 0.088 ± 0.026. Several authors[6,7,11,16,17] have investigated PEEK, as well as its composites, for dry sliding, with friction coefficient variations between 0.3 and 0.5. In the present experiment, the samples submitted to turning and grinding remained within a friction coefficient rate between 0.13 and 0.10, which was defined as boundary regime on Figure 4. A determining factor for the friction coefficient in a lubricated environment, to remain lower than the dry friction coefficient, under boundary regime, is that the lubricant ATF Dexron VI contains lubrication additives, whose efficiency was initially studied by Bowden and Tabor[18]. They observed that, by adding stearic acid to a stainless steel friction surface submitted to dry sliding, the friction coefficient was reduced to approximately 0.10 under experiment conditions. Since then, several different additives, both organic and inorganic have been studied[15]. Such additives form a film of molecular dimensions which can be absorbed either chemically or physically by the metallic surface, thus avoiding direct contact between the parts. The results of friction for honing, on the other hand, displayed greater dispersion of the data. By analyzing such results together, in Figure 4 and Figure 5, it was possible to state that for a pressure of 2 MPa and speed of 2 m/s, the lubrication regime for this type of finishing is on the threshold between boundary and mixed regimes. Polished samples displayed a typical friction coefficient of the hydrodynamic regime. In such regime, lubricating films are usually thick enough so as to not allow the contact between surfaces. This condition is often called optimal lubrication, as it warrants a low friction coefficient, and high wear resistance. Experimental results for friction as a function of time, for all four types of finishing, are shown in Figure 6. The friction coefficient tended to increase as test duration increased for turning, whereas for grinding and honing the friction coefficient decreased during the test. In the case of counterbodies submitted to grinding, the friction coefficient remained stable throughout the test. For samples submitted to turning, it is reasonable to state that the friction coefficient increases as a function of time, as explained by Greenwood and Williamson[19]. When the two surfaces come in contact during the sliding test, due to the deformation and/or cut of the polymeric material of the pins, the contact points will increase, thus increasing contact surface in comparison to the rougher surface. This results in greater shear force that will deform the material of the pin, whose hardness is lower, thus increasing the friction coefficient. Abrasion marks are detectable only on the surfaces of samples tested against discs submitted to turning, as shown on Figure 7. One hypothesis for the friction coefficient reduction, as seen for honing and grinding, is that in a lubricated environment, a continuous film of lubricant may not be fully formed on the friction surface during the “running‑in” stage, so the friction coefficient was higher during that stage. After running-in, a more continuous layer of lubricant 339

Andrade, T. F., Wiebeck, H., & Sinatora, A.

Figure 6. Results of all friction tests conducted for the four different finishings examined, as follows: (a) turning; (b) grinding; (c) honing and (d) polishing.

Figure 7. Contact surface of the (a) polymeric sample and (b) metallic counterbody observed through stereomicroscope.

gradually formed on the friction surface, and the direct contact between the asperities gradually diminished by the lubricating film[20]. For the polished surfaces, the friction coefficient remained stable throughout the test, as they were submitted to hydrodynamic regime, in which the friction characteristics are derived purely from the shearing of the lubricating (film).

3.4 Influence of roughness on the wear rate Figure 8 shows the effect of the four finishing options for the counterbody in relation to wear rate for the material studied. Specific wear rate varied approximately 4-fold, when 340

Figure 8. Influence of the metallic counterbody roughness on the wear rate of the PEEK pin.

finishings with lower Ra and higher Ra were compared. A different behavior was observed in dry wear conditions of the same material, as described by Friedrich et al.[10]. In that case, the average wear rate remained constant regardless of the roughness, which, to some extent, was also observed in lubricated environment, however, for each lubricating regime. The specific wear rate for dry conditions (pv ≈ 5 MPa.m/s) was 8 to 20 times greater than the wear in a lubricated environment (pv ≈ 4 MPa.m/s) under boundary regime. The mean wear rate generated by the counterbodies submitted Polímeros, 26(4), 336-342, 2016

Effect of surface finishing on friction and wear of Poly-Ether-Ether-Ketone (PEEK) under oil lubrication iii. Tribological characteristics of PEEK were shown to be much more sensitive to the lubrication regime than to superficial roughness; however the lubrication regime was also defined by roughness. iv. The average wear rates generated with counterbodies summited to turning, grinding, honing were very similar, whereas polished samples displayed a much lower wear, due to the fact that they were under hydrodynamic regime.

5. Acknowledgements The authors wish to thank Mr. Ricardo Elhke and Mr. Kida Kazuhiro from Victrex for kindly supplying the polymeric material, as well as the test benches, and for the invaluable technical discussions. Figure 9. Polymer wear rate versus friction coefficient for the different counterbody surface finishing. (a) hydrodynamic; (b) mixed and (c) boundary lubrication regimes.

to turning, grinding, and honing were very similar, while the polished samples displayed a markedly lower wear. This happens because under hydrodynamic lubrication regime, the lubricant film is thick enough to prevent the contact between the solid parts, thus significantly reducing wear rate between components. Figure 9 shows the wear rate as a function of the friction coefficient observed for different counterbody finishings. In this graph, the relationship between the friction coefficient and the specific wear rate for each finishing is clear. Although the wear increased with the increase of the friction coefficient, there does not seem to be a direct correlation with the roughness of the counterbody, but with the lubrication regime in which the system is operating. In these tests, neither debris nor thin film were observed by optical stereomicroscope in any of the counterbodies, not even on the samples submitted to turning or grinding, which operated under boundary regime. However, both debris and films adhered to the surface of the counterbody were observed in tests conducted under dry conditions, depending on the roughness [2]. Lubricant flow, besides significantly reducing the heat caused by friction and contact temperature, also removed the debris form the contact region, thus reducing wear caused by abrasion.

4. Conclusions The present work studied the tribological properties of PEEK combined 30% carbon fiber in an oil-lubricated environment for four different counterbody finishings. As a result we reached the following conclusions: i. The steady-state wear regime happened 120 minutes after the stabilization of the wear rate and the friction coefficient. ii. For the pressure range between 1 and 7 MPa, the friction coefficient for the boundary regime was 0.09 to 0.13, the mixed regime was between 0.04 and 0.09, and for the hydrodynamic regime it was below 0.04. Polímeros, 26(4), 336-342, 2016

6. References 1. America Chemistry Council. (2014). Plastics and polymer composites technology roadmap for automotive markets. Washington: America Chemistry Council. 2. Nunez, E. E., & Polycarpou, A. A. (2015). The effect of surface roughness on transfer of polymer films under unlubricated testing conditions. Wear, 326-327(15), 74-83. http://dx.doi. org/10.1016/j.wear.2014.12.049. 3. Gutiérrez, J. C., Rubio, J. C. C., & Faria, E. (2014). Usinabilidade de materiais compósitos poliméricos para aplicações automotivas. Polímeros. Ciência e Tecnologia, 24(6), 711-719. http://dx.doi. org/10.1590/0104-1428.1582. 4. Greco, A. C., Erck, R., Ajayi, O., & Fenske, G. (2011). Effect of reinforcement morphology on high-speed sliding friction and wear of PEEK polymers. Wear, 271(9-10), 2222-2229. http://dx.doi.org/10.1016/j.wear.2011.01.065. 5. Altstaedt, V., Werner, P., & Sandler, J. (2003). Rheological, mechanical and tribological properties of carbon-nanofibre reinforced poly (ether ether ketone) composites. Polímeros: Ciência e Tecnologia, 13(4), 218-222. http://dx.doi.org/10.1590/ S0104-14282003000400005. 6. Zhang, G., & Schlarb, A. K. (2009). Correlation of the tribological behaviors with the mechanical properties of poly-ether-etherketone (PEEKs) with different molecular weights and their fiber filled composites. Wear, 266(1-2), 337-344. http://dx.doi. org/10.1016/j.wear.2008.07.004. 7. Elliott, D. M., Fisher, J., & Clark, D. T. (1998). Effect of counterface surface roughness and its evolution on the wear and friction of PEEK and PEEK-bonded carbon fibre composites on stainless steel. Wear, 217(2), 288-296. http:// dx.doi.org/10.1016/S0043-1648(98)00148-3. 8. Birkett, A., & Lancaster, J. K.(1985). Counterface effects on the wear of a composite dry-bearing liner. In JSLE International Tribology Conference (pp. 8-10). Tokyo: Elsevier. 9. Stachowiak, G. W., & Batchelor, A. W.(2005). Engineering tribology. Oxford: Butterworth Heinemann. 10. Friedrich, K., Karger-Kocsis, J., & Lu, Z. (1991). Effects of steel counterface roughness and temperature on the friction and wear of PEEK composites under dry sliding conditions. Wear, 148(2), 235-247. http://dx.doi.org/10.1016/00431648(91)90287-5. 11. Ramachandra, S., & Ovaert, T. C. (1997). The effect of controlled surface topographical features on the unlubricated transfer and wear PEEK. Wear, 206(1-2), 94-99. http://dx.doi. org/10.1016/S0043-1648(96)07354-1. 341

Andrade, T. F., Wiebeck, H., & Sinatora, A. 12. Eiss, N. S., Wood, K. C., Herold, J. A., & Smyth, K. A. (1979). Model for the transfer of polymer to rough, hard surfaces. Journal of Lubrication Technology, 101(2), 212-218. http:// dx.doi.org/10.1115/1.3453326. 13. Victrex Materials Properties Guide. (2014, 20 november). Retrieved in 24 July 2015, from http://www.victrex.com 14. ASTM International. ASTM G-99-04: standard test method for wear testing with pin-on-disk apparatus metals test methods and analytical procedure (Vol. 03.02, Section 3). West Conshohocken: ASTM. 15. Hamrock, B. J., Schmid, S. R., & Jacobson, B. O.(2004). Fundamentals of fluid film lubrication (2nd ed.). New York: Marcel Dekker. 16. Lu, Z. P., & Friedrich, K. (1995). On sliding friction and wear of PEEK and its composites. Wear, 181-183(2), 624-631. http:// dx.doi.org/10.1016/0043-1648(95)90178-7. 17. Zhang, G., Rasheva, Z., & Schlarb, A. K. (2010). Friction and wear variations of short carbon fiber (SCF)/PTFE/graphite

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(10Â vol.%) filled PEEK: effect of fiber orientation and nominal contact pressure. Wear, 268(7-8), 893-899. http://dx.doi. org/10.1016/j.wear.2009.12.001. 18. Bowden, F. P., & Tabor, D. (1950). Friction and lubrication of solids. Oxford: Clearendon Press. 19. Greenwood, J. A., & Williamson, J. B. P. (1966). Contact of nominally flat surfaces. Proceedings of the Royal Society of London. Series A, Mathematical and Physical Sciences, 295(1442), 300-319. http://dx.doi.org/10.1098/rspa.1966.0242. 20. Zhang, Z. Z., Xue, Q. J., & Shen, W. C. (1997). Tribological properties of metal-plastic multilayer composites under oil lubricated conditions. Wear, 210(1-2), 195-203. http://dx.doi. org/10.1016/S0043-1648(97)00040-9. Received: July 24, 2015 Revised: Mar. 23, 2016 Accepted: Mar. 31, 2016

PolĂmeros, 26(4), 336-342, 2016

http://dx.doi.org/10.1590/0104-1428.2344

Waterborne hyperbranched alkyd-acrylic resin obtained by miniemulsion polymerization Edwin Murillo1* and Betty López2 Grupo de Investigación en Materiales Poliméricos – GIMAPOL, Departamento de Química, Universidad Francisco de Paula Santander, San José de Cúcuta, Colombia 2 Grupo de Investigación en Ciencia de los Materiales, Universidad de Antioquia, Calle, Medellín, Colombia

*edwinalbertomurillo@gmail.com

Abstract Four waterborne hyperbranched alkyd-acrylic resins (HBRAA) were synthesized by miniemulsion polymerization from a hyperbranched alkyd resin (HBR), methyl methacrylate (MMA), butyl acrylate (BA) and acrylic acid (AA), by using benzoyl peroxide (BPO) and ammonium persulfate (AP) as initiators. The reaction between HBR and acrylic monomers was evidenced by differential scanning calorimetric (DSC), nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The conversion percentage, glass transition temperature (Tg), content of acrylic polymer (determined by soxhlet extraction) and molecular weight increased with the content of acrylic monomers used in the synthesis. The main structure formed during the synthesis was the HBRAA. The analysis by dynamic light scattering (DLS) showed that the particle size distribution of HBRAA2, HBRAA3 and HBRAA4 resins were mainly monomodal. The film properties (gloss, flexibility, adhesion and drying time) of the HBRAA were good. Keywords: hyperbranched polymers, miniemulsion polymerization, alkyd-acrylic resins, properties.

1. Introduction The use of renewable resources in the preparation of various industrial materials has been revitalized due to the environmental concerns. Last year, research on coating (research) was conducted aiming to the reduction of volatile organic compounds (VOCs)[1]. These latters (the VOCs) are toxic, responsible for global warming and climate changes, and photochemical ozone layer deterioration[1]. Some alternatives have been adopted to obtain ecofriendly materials were, among others, waterborne coatings and hyperbranched polymers with high solid content are being developed for this purposes[1-6]. A hybrid polymer is composed of at least two kinds of materials, different in chemical nature, and which normally incompatible but bonded either by chemical covalent bonds or strong physical secondary intermolecular forces[7,8]. Recently, researches have been devoted to this topic[9-12]. The alkyd resins are a good alternative for obtaining environmentally friendly hybrid coatings (waterborne resins); since they can be modified by addition reactions with other materials through double bonds, offering the access to other structures and materials with novel and enhanced properties[13-15]. The HBR exhibit short time for chemical drying, high gloss as well as chemical resistance[16]. Acrylic resins are more resistant against ultraviolet light, show shorter drying time and better chemistry resistance than alkyd resins[15]. However, the properties of gloss and hardness in a porous substrate of acrylic resins are regular[15]. Therefore, the objective of the modification of HBR with acrylic monomers is to obtain a synergetic compromise between most needed properties. The study of alkyd-acrylic resins or oil-acrylic

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monomers has been only focused on materials synthesis with the lowest branching degree[17-22]. There are two methods usually employed to synthesize alkyd-acrylic resins, which are emulsion polymerization[22], and mini-emulsion polymerization[13,14]. The emulsion polymerization is not a suited method, because alkyd resins exhibit low hydrophilicity, since the diffusion of alkyd resins toward micelles is very slow, which produces colloidal instability (phase separation or secondary nucleation)[7,15]. The miniemulsion polymerization is the most adequate method, since it does not require the diffusion process. This is due to the polymerization occurs on the monomer droplets and the low hydrophilicity of the resin and the hydrophobicity of the co-surfactant help to avoid the Ostwald ripening process[7]. The hydrophobic nature of the alkyd resins makes it impossible to be accommodated by traditional emulsion polymerization due to mass-transfer limitations[14]. Despite of the disadvantages of emulsion polymerization, it has been investigated in several studies[7,15]. Alkyd/acrylic resin were prepared by solution polymerization by using BPO and 2,2-azobisisobutyronitrile (AIBN) as initiators. An alkyd resin was obtained from soybean oil, glycerol, phthalic anhydride, and tetrahydrophthalic anhydride and modified with acrylic monomers. The acrylic monomers employed were MMA, BA and methacrylic acid (MAA). NMR analysis shown that acrylic groups were grafted into the polyester backbone of the alkyd via the hydrogen abstraction of the glycerol. It was also determined that the choice of initiator has no effect on graft location, but the system initiated with BPO provided spectral evidence that the BPO can directly abstract the doubly allylic hydrogen of fatty acids presents

343

S S S S S S S S S S S S S S S S S S

Murillo, E., & López, B. in the alkyd[23]. Alkyd-acrylic hybrids having final solids content of 75-80% were prepared by dropping alkyd resin (75 wt% of fatty acid based on the total alkyd) into an acrylic dispersion. The resulting suspoemulsion structures and the alkyd droplet sizes in the hybrids were dependent on the preparation procedure as well as the interfacial tension between the two liquid phases. However, when the surfactant was added to the alkyd prior to the addition of the alkyd to the latex, a multi-emulsion (W/O/W) of alkyd was formed[20]. Alkyd resins were modified with acrylic monomers (BA, MMA, ethyl acrylate and ethyl methacrylate) by using a reversible-addition chain transference (soya based alkyd with a carboxy-functional trithiocarbonate). AIBN was the initiator employed. NMR spectra showed the presence of acrylic blocks the pendant fatty acids and the formation of homopolyacrylate[24]. Hybrid waterborne alkyd-acrylic dispersions with solid content of 40 wt%, free from any surfactant and organic solvent, were synthesized by a melt co-condensation reaction between an acrylic prepolymer bearing carboxylic groups and an alkyd resin by a phase inversion process. The acrylic prepolymer were prepared first by free radical polymerization of MMA, ethylhexyl acrylate, maleic anhydride and methacrylic acid. The alkyd resin was synthesized from soya fatty acid, phthalic anhydride, benzoic acid and pentaerythritol. The insertion of anhydride moieties within the acrylic prepolymer ensured the efficient coupling between the acrylic prepolymer and the alkyd resin and prevented the phase separation[25].

Alkyd-acrylic resins were synthesized by emulsion polymerization and these materials presented a synergic effect[22]. The properties of these resins were better than alkyd-acrylic blends. In the synthesis of alkyd-acrylic resins, several structures are present by the end of the reaction, which are alkyd resin, acrylic polymer and alkyd-acrylic resin[17,26]. The synthesis of waterborne alkyd-acrylic resins has been achieved from alkyd resins with low branching degrees, but so far, there has not been any research on the synthesis of the waterborne hyperbranched alkyd-acrylic resins (HBRAA) obtained by miniemulsion polymerization from HBR. In order to continue contributing to the development of new environmentally friendly materials in this work, four HBRAA were synthesized by the polyaddition reaction between acrylic monomers and a HBR (Figure 1). The effects of acrylic monomers on the structural, thermal and film properties of HBRAA resins were studied.

2. Experimental Section 2.1 Materials The synthesis and properties of the HBR were reported in an earlier publication, and it was named HBRA4[3,27]. The methyl methacrylate (MMA), acrylic acid (AA) and butyl acrylate (BA). Sodium dodecyl sulfate (SDS), AP, BPO, xylene, tetrahydrofurane (THF), sodium hydroxide, acetone, toluene, sodium chloride (NaCl), hexadecane (HD), toluene, chloroform and diethyl ether were purchased from

Figure 1. Schematic representation of the synthesis of a HBRAA. 344

Polímeros, 26(4), 343-351, 2016

Waterborne hyperbranched alkyd-acrylic resin obtained by miniemulsion polymerization Aldrich and all used as received. The purities of all reactive chemicals are higher than 99 wt%. Cobalt, calcium and zirconium octoates were supplied by Colorquímica S.A.

2.2 Synthesis of the HBRAA In order to synthesise the HBRAA, the corresponding amount of HBR, HD (4 wt% with respect to the amounts of acrylic monomers), BPO (0.5 wt% with respect to amounts of acrylic monomers) and acrylic monomers were mixed to obtain a homogeneous system (hydrophobic solution). Table 1 presents the proportions of HBR and acrylic monomers employed on the synthesis. In all cases the proportions of acrylic monomers (MMA-BA-AA) were 49:50:1. A solution of SDS (312 ml 0.02 M) and AP (40 ml 0.01 M) in water were prepared in different recipients. For preparing the miniemulsions, the hydrophobic and SDS solutions were mixed during thirty minutes using a homogenizer (UltraTurrax) at 24000 rpm in an ice bath to avoid an increase in the temperature, which may cause premature polymerization. The obtained solution was transferred to a reactor equipped with a mechanical stirrer, condenser, and a heating system. The reaction system was kept under nitrogen atmosphere and heated to 80 °C. The solution of initiator and the amount of sodium bicarbonate (0.5 wt% with respect to total mix) were added to the system. Finally the system was stirred at 200 RPM during five hours. In all cases, the solid content was kept constant (40 wt%). The reactions conversion was followed gravimetrically. The conversion study was done from extractions of small aliquots at different reaction times intervals. In all cases, two drops of an ethyl hydroquinone solution (1 wt%) were added to stop posterior polymerization. The samples were vacuum dried at 50 C for 48 hours. Finally, the samples were taken out and weighted.

2.3 Characterization of the HBRAA resins The studies of compositional analysis were performed by a soxleht extraction technique. The HBR and HBRAA are soluble in diethyl ether, but the free acrylic polymer (without grafting in HBR) is insoluble in this solvent. The free acrylic polymer is soluble in THF[17,28]. For the quantification, the HBRAA were first submitted to soxleht extraction with diethyl ether for 24 hours and then the residue was dried and weighed. The HBR and HBRAA were extracted as previously mentioned. For the extraction of the acrylic polymer, the above residue was kept on soxleht extraction with THF for 24 hours, and the final residue was then dried and weighted. Finally other extractions were done using toluene, chloroform and acetone, in order to determine the insoluble material content. The NMR analysis was carried

out in a Bruker AC 300 MHz spectrometer. The 1H NMR spectrum was obtained by using deuterated chloroform (HBR and HBRAA4) and tetrahydrofurane as solvents (HBRAA2 and HBRAA3). The DLS analysis was done at room temperature and at 50 °C by using a Zetasizer Nano Series machine from Malvern Instruments. A 633 nm wavelength and an incidence angle of 173º was used. GPC analysis was performed in Waters 600 equipment by using a styragel column with dimensions of 4.6 × 300 mm. The samples were dissolved in THF at 30 °C. For the quantification, polystyrene standards were used to obtain calibration curves. Millennium 2000 software was used for data acquisition. DSC analysis was performed using TA Instrument model Q100 equipment, employing heating and cooling rates of 30 °C/min, under nitrogen atmosphere. Minimum film formation temperature (MFFT) analysis of the HBRAA was performed on Rhopoint equipment. The temperature was between -5 and 13 °C and a pressure of 5 Psi. The HBRAA films, with a thickness of 75 μm, were applied in a plate, which had a temperature gradient (-5 and 13 °C) and a sensor that allows the calibration of intermediate temperature. For the film properties, the HBRAA were mixed with cobalt octoate (0.6%wt), calcium octoate (0.6%wt) and zirconium octoate (1.8%wt). The gloss, adhesion, drying time and flexibility of the films were then studied. By using a film applicator, the films were applied on steel surfaces and dried at 25 °C, under relative humidity of 40%. The analyses of adhesion (ASTM D 3359), flexibility (ASTM D 522), specular gloss at 60º and 85º (ASTM D 523), and drying time (ASTM D 1640).

3. Results and Discussion Figure 2 presents the results of the conversion degree of the HBRAA as a function of time. The conversion increased with the amount of polymerized acrylic monomers. This behavior has been already observed for conventional alkyd resins[17,28]. The conversion degree values of the HBRAA were the following: 95.03% for HBRAA1, 97.53% for HBRAA2, 98.99% for HBRAA3 and 99.65% for HBRAA4. The conversion after thirty minutes increased quickly with the content of acrylic monomers. The free radicals of BA have a preference for reacting though double bonds of the

Table 1. Proportions of HBR and acrylic monomers employed on the synthesis. HBRAA HBRAA1 HBRAA2 HBRAA3 HBRAA4

HBR (%) 50 40 30 20

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(MMA-BA-AA) (49:50:1) (%) 50 60 70 80

Figure 2. Conversion percentage of the HBRAA. 345

Murillo, E., & López, B. HBR[18]. It has been reported in the case of MMA, which has a sterically hindered radical center[18]. Therefore the BA is more reactive than MMA, it is possible that this increasing to conversion percentage (after thirty minutes) is related with the grafting of MMA onto HBR or the beginning of termination reactions. Another hypothetical reason of the increasing in conversion percentage at 30 min of reaction is that, the AA being soluble in water reacted with AP in the aqueous phase and the free radicals derived from this monomer inside the droplets, thus increasing the reaction conversion. Therefore the conversion at this time is higher with the content of this monomer. The required reaction time is around 180 min. For the synthesis of conventional alkyd-acrylic resins (structure with lowest branching) obtained by miniemulsion polymerization, the conversion degree was around 80%. This low conversion was due to the limiting conversion[18], which is produced by abstraction of allylic hydrogen (linolenic or linoleic acids). This produces a chain transfer to relatively inactive radical on the HBR with a reduction in the overall polymerization rate, and when approached by another active acrylic macroradical, it terminates with the formation of a grafted HBR[18]. It can be concluded that in our study this process was minimized or was not present at all, since the conversion degree values obtained in this study were higher than those reported by Hudda et al[18]. The grafting mechanism proposed to the synthesis of the HBRAA is grafting through chain transfer and addition through the double bond[18]. An important aspect that contributed to achieve a good conversion degree was the presence of double bonds of the HBR, which are on the periphery along with the use of two initiators, which increased the probability of grafting of the acrylic monomers onto HBR. The grating degree (GD) of the acrylic monomers in the HBR was calculated using the equation: GD =

A x100 B

form the acrylic polymer is high, which was more probable for the sample HBRAA4. To compute the grafting degree of the acrylic monomers onto HBR, this assumption was made; the amount of the free acrylic polymer (soluble in THF) is equal to non-grafted polyacrylate, since HBR and HBRAA are soluble in diethyl ether. Therefore the residues obtained after extraction operation with diethyl ether are acrylic polymer and insoluble material totally extracted in this process[28]. The grafting degree increased with the amount of HBR, it was due to the low probability of formation of the acrylic polymer and high number of double bonds available for grafting. Because the acrylic polymer is soluble in THF, it is deduced that the final residue or insoluble fraction corresponding to the crosslinking structure contains crosslinked HBRAA. A study concerning conventional alkyd resin, using the same acrylic monomers, did not show the formation of crosslinking[17]. The HBRAA crosslinking is attributed to the high density of double bonds in the periphery and initiator content. The insoluble materials increased with the proportion of HBR employed in the synthesis. Wang et al. reported[28] that the insoluble material is due to crosslinking of the HBR and also to the high number of acrylic chains grafted onto HBR. This corroborated the hypothesis that there are some grafted acrylic units in insoluble residue (solid that remain after the extraction process). Figure 3 shows the NMR spectra of the HBR, HBRAA2, HBRAA3 and HBRAA4. The signals that appeared in the spectra of the samples HBRAA2 and HBRAA3 at 1.73 and 3.58 ppm, were due to protons of the THF. The signals of HBR have been explained in a previous paper[3]. Among these signals the most important is the signal of the protons of -HC=CH- bonds (at 5.3 ppm), because this signal is evidence of the reaction between acrylic monomers and the HBR. In the NMR spectra of the HBRAA, it can be observed that the signal of the protons of -HC=CH- bonds decreased its intensity with the increase of the acrylic monomers proportion in the synthesis. This is a proof of the reaction between acrylic monomers and HBR. The HBRAA4 exhibited the lowest intensity of the signal due to protons of -HC=CH- bonds. This is attributed to the fact that the highest amount of acrylic monomers was used and that they reacted in high yield during the synthesis of this resin. The signal around 4.0 ppm was attributed to protons of -CH2 joined to ester groups (-CH2OCOR) and the signals of protons of the unreacted OH groups (-CH2OH) of linear and terminal units appeared between 3.4 y 3.6 ppm. The wide signal surrounding 2.7 ppm was possibly due to overlapping of the -CH2- protons of -CH2COOH and -CH2COOCH2- groups. Other signals in the HBRAA spectrum between 0.5 and 2.5 ppm, were attributed to aliphatic protons (-CH, -CH2 and -CH3).

(1)

where, A and B are the amount of acrylic monomers grafted onto the HBR and the amount of acrylic monomers used in the synthesis, respectively. Table 2 shows the results of compositional analysis. The HBRAA content (hybrid resins) for all samples was high and this increased with the amount of acrylic monomers employed in the synthesis. The quantity of free acrylic polymer in the final product also increased with the content of acrylic monomers initially employed in the synthesis. These behaviors were expected because as the amount of acrylic monomers increase, the probability to Table 2. Compositional analysis of the HBRAA. HBRAA HBRAA1 HBRAA2 HBRAA3 HBRAA4

346

Alkyd-acrylic resins

Acrylic polymer

Grafting degree

Insoluble material

(%) 59.88 68.99 80.81 90.01

(%) 1.01 1.73 2.08 2.41

(%) 97.98 97.12 97.03 96.97

(%) 39.11 29.28 17.11 7.58

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Waterborne hyperbranched alkyd-acrylic resin obtained by miniemulsion polymerization Figure 4 presents the size distributions of the drops and the particles. Drop sizes of the HBRAA1 were not monomodal (Figure 4a), which was possibly due to higher difficulty to break the drops of the HBR, since the HBRAA1 presented the highest content of HBR employed in the synthesis, as a result, the miniemulsification process was less effective to this sample. The HBRAA2 exhibited higher monomodal behavior than that of theHBRAA1 (Figure 4b). HBRAA3 (Figure 4c) and HBRAA4 (Figure 4d) were monomodal, but the distribution was wide, which indicates that the highest amount of acrylic monomers facilitated the rupture of the drops to form miniemulsions with small drops, which is due to low viscosity of these monomers and change on surface tension.

the samples HBRAA2, HBRAA3 and HBRAA4 were higher than those of the average particle sizes; this was possibly caused by some change of the surface tension during the polymerization due to occurrence of homogeneous nucleation during the polymerization though of MMA and AA, since these monomers present certain solubility in water. Additionally the shelf life of the miniemulsions (before the polymerization process) was studied. This shelf life is defined as the time required for a cream phenomenon to appear in the samples[30]. This process is a product of the Ostwald Ripening process and may occur before or after the polymerization process[31]. A hexadecane, as co-surfactant, was used to avoid the above process mentioned. It is highly hydrophobic and prevents the diffusion of the monomers from small drops toward big drops. All resins exhibited excellent stability without cream exhibition after twelve months (Table 3). The results of this analysis are evidence that the amount of coagulum after polymerization is possibly low, since the drop and particle size of the HBRAA are almost similar. In order to evaluate the colloidal stability of the HBRAA, they were submitted to storage for three months at 50 °C. In the Figure 5, the particle size distributions of the HBRAA

All HBRAA presented nanometric particle sizes (50‑500 nm). Some works have reported that the final particle sizes are a one-to-one copy of the monomer droplets[29]. The HBRAA1 presented a small increase in particle size with respect to drop size (Figure 4 and Table 3), which might be attributed to: a) the amount of surfactant was not enough to stabilize the drops of this resin, b) occurrence of changes in surface tension and chain conformation, and c) coagulation between particles. The average drop sizes of

Figure 3. 1H RMN spectra (a) HBR; (b) HBRAA2; (c) HBRAA3 and (d) HBRAA4. Table 3. Drop and particle average size and shelf life of the HBRAA. HBRAA HBRAA1 HBRAA2 HBRAA3 HBRAA4

Drop size (nm)

Particle size (nm)

d1 (%)

d2 (%)

d1 (%)

d2 (%)

102.30 (18.10%) 50.27 (10.50%) 271.90 (100%) 206.80 (100%)

509.40 (81.90%) 361.80 (89.50%) -

164.00 (60.20%) 55.41 (4.80%) 200.20

662.00 (39.80%) 319.40 (95.20%) -

>12

160.60

>12

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Shelf life (Months)

>12 >12

347

Murillo, E., & López, B.

Figure 4. Drop and particle size distributions of the HBRAA (a) HBRAA1; (b) HBRAA2; (c) HBRAA3 and (d) HBRAA4.

Figure 5. Particle size distributions of the HBRAA at room temperature (0 months) and at 50 °C (one, two and three months). (a) HBRAA1; (b) HBRAA2; (c) HBRAA3 and (d) HBRAA4. 348

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Waterborne hyperbranched alkyd-acrylic resin obtained by miniemulsion polymerization are observed. During the storage process the flocculation, deflocculation, coagulation and migration of surfactant from the aqueous phase toward the particles can occur. All these processes affect the colloidal stability. The coagulation process produces an increase in particle size. This phenomenon was observed mainly in the HBRAA1. These resins at room temperature (0 month) presented two distributions, and then at storage at 50 °C, it showed a wide distribution and the presence of an additional peak at 3551 nm. The HBRAA2, after one month of storage at 50 °C, exhibited an additional peak at around 45.48 nm, and a particle size distribution more wide than at room temperature (0 month). The peak at 45.46 nm is possibly due to the deflocculation process. Furthermore, in the third month it presented another peak at around 4000 nm, which is probably due to the coagulation or flocculation process. The HBRAA3 after one month presented another small distribution with a particle size of 45.48 nm. However, the particle size distribution is very similar at different times (0, one, two and three months). The HBRAA4 was the unique sample that showed a monomodal distribution and a particle size distribution very stable with the time. Therefore, it exhibited the highest colloidal stability. The Brownian movement of the particles increased with temperature. This improves the coalescence of the particles, according to the results obtained in this study. It can be concluded that HBRAA3 and HBRAA4 exhibited higher colloidal stability than HBRA1 and HBRAA2 resins. The behavior showed by the HBRAA3 and HBRA4 are possibly related to the small particle size, and high covering of the particle surface by the surfactant. Table 4 presents the results of the gel permeation chromatography (GPC) analysis. The values of number average molar masses (Mn) increased with the proportion of acrylic monomers employed in the synthesis and these are higher than HBR (6611 g/mol)[27]. The great difference between the Mn values of the HBRAA and HBR was due to the synthesis process, since a polymer with high molar mass can be obtained by miniemulsion polymerization[19]. The polydispersity index (PI) of the HBRAA2 was similar to HBRAA1 and HBRAA3 and lower than HBRAA4. According to these results, PI was independent of the amount of acrylic monomers employed in the synthesis. The difference on PI was possibly due to the miniemulsion polymerization process, which is random, since the acrylic monomers have so many places where they can be grafted. Figure 6 shows the DSC thermograms of the all HBRAA, which presented higher glass transition temperature (Tg) than HBR (-49.8 °C)[27]. The Tg values of the HBRAA were as follow: HBRAA1, -24.7 °C, HBRAA2, -16.6 °C, HBRAA3, -14.5 °C and HBRAA4, -12.9 °C. All HBRAA

have an increased Tg values with respect to the amount of acrylic monomers employed in the synthesis. Despite that the polybutyl acrylate has a Tg value around -51.1 °C[32], polymethyl methacrylate confers high structural stiffness to the HBRAA, since, it has a Tg value around 105 °C[33]. A second Tg was observed for HBRAA2 at -0.5 °C, HBRAA3 at -0.90 oC and HBRAA4 at -2.1 oC, which are possibly due to free acrylic polymer[17]. MMFT is the minimum temperature that is required for the latex to form a clear optically and uniform film, once it is applied on a surface. At lower temperature than MMFT, the film is opaque or granular[34]. The MMFT values of the HBRAA were as follow: HBRAA1 < -5 °C, HBRAA2 -5 °C, HBRAA3 -3.8 °C and HBRAA4 3.0 °C. According to these results, all resins presented a good film formation, even at lower temperatures, which is very adequate for their use for room temperature coating. These results are very important since none of HBRAA require the use of a coalescent agent for drying, which is an advantage from environmental (reduction of volatile organic substances), economic (reduction in the cost of the formulation) and synthetic (another variable to control) aspects point of views. The MMFT values of the HBRAA presented the same behavior on Tg for all these resins and the which was lower than MFFT. For an application of a binder, it is very important that their(s) film properties should be determined, since a good film formation is required to ensure suitable properties for the coating. In a previous study[3], the film properties of HBR were evaluated, and these will be compared here with those of the HBRAA. Table 5 presents the results of the gloss, adherence, drying time and flexibility analyses. All films exhibited transparency. All HBRAA presented lower gloss than HBR (60°/85°=89.9/983)[3]. The gloss values of the HBRAA decreased with the increase of the acrylic monomers content. This behavior was expected, since the gloss value of acrylic resins is lower than alkyd resins[2,16]. When the gloss value of a resin, measured at 60º, is higher than 70º, the resin presents a high gloss[1]. Therefore, these resins exhibited a high gloss. The adhesion of the HBRAA was superior to the HBR (90%)[3]. The behavior

Table 4. Mn, Mw and PI of the HBRAA. HBRAA HBRAA1 HBRAA2 HBRAA3 HBRAA4

(g/mol) 45716 50061 65202 90826

(g/mol) 99812 108844 182123 413679

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PI 2.75 2.17 2.79 4.55

Figure 6. DSC thermograms of the HBRAA. 349

Murillo, E., & López, B. Table 5. Values of gloss, adhesion, drying time and flexibility of the HBRAA. HBRAA HBRAA1 HBRAA2 HBRAA3 HBRAA4

Gloss

Adhesion

600/850 85,5/92,5 83,7/89,0 79,5/88,9 71,9/87,8

(%) 100 100 100 100

was expected because the acrylic resins present a good adherence[35]. The flexibility of the HBRAA was good since none of the resins presented severe fracture or rupture after analysis. The drying time of the HBRAA was inferior to that of HBR (204 min.)[3], and decreased with the content of acrylic monomers. During the drying process, the solvent evaporation (physical drying) occurs at first and then the oxidative process (crosslinking through double bonds). When the HBR is modified with acrylic monomers, the drying process is mainly physical. Moreover, the oxidative drying is slower than physical drying, due to the low reactivity of the double bond (-HC=CH-).

4. Conclusions Different environmentally friendly HBRAA were obtained in this study, which contribute to the development of new materials. In all cases, the conversion percentage of the reaction was high. The particle size of the HBRAA was mainly nanometric. This is very good, since it confers a high superficial area, which is very important in the coating industry. All HBRAA presented better adhesion, flexibility and drying time than HBR, but HBR exhibited the higher gloss than HBRAA. This led us to conclude that all HBRAA exhibited a synergy in their properties. The HBRAA3 and HBRAA4 exhibited higher colloidal stability than the HBRAA1 and HBRAA2. An important result is that none of the HBRAA need coalescent agent to the drying process, since the HBRAA presented lower MFFT values than room temperature.

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Flexibility Pass Pass Pass Pass

Drying time (min) 90 80 72 45

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Waterborne hyperbranched alkyd-acrylic resin obtained by miniemulsion polymerization in hybrid miniemulsion polymerization. Polymer, 46(4), 9931001. http://dx.doi.org/10.1016/j.polymer.2004.11.035. 19. Wang, Q., Fu, S., & Yu, T. (1994). Emulsion polymerization. Progress in Polymer Science, 19(4), 703-753. http://dx.doi. org/10.1016/0079-6700(94)90031-0. 20. Jowkar-Deriss, M., & Karlsson, O. J. (2004). Morphologies and droplet sizes of alkyd–acrylic hybrids with high solids content. Colloids and Surfaces. A, Physicochemical and Engineering Aspects, 245(1-3), 115-125. http://dx.doi.org/10.1016/j. colsurfa.2004.07.003. 21. Quintero, C., Mendon, S. K., Smith, O. W., & Thames, S. F. (2006). Miniemulsion polymerization of vegetable oil macromonomers. Progress in Organic Coatings, 57(3), 195201. http://dx.doi.org/10.1016/j.porgcoat.2006.08.011. 22. Heiskanen, N., Jämsä, S., Paajanen, L., & Koskimies, S. (2010). Synthesis and performance of alkyd-acrylic hybrid binders. Progress in Organic Coatings, 67(3), 329-338. http://dx.doi. org/10.1016/j.porgcoat.2009.10.025. 23. Dziczkowski, J., Dudipala, V., & Soucek, M. D. (2012). Grafting sites of acrylic mixed monomers onto unsaturated fatty acids: Part 2. Progress in Organic Coatings, 73(4), 308-320. http:// dx.doi.org/10.1016/j.porgcoat.2010.12.006. 24. Dziczkowski, J., Chatterjee, U., & Soucek, M. D. (2012). Route to co-acrylic modified alkyd resins via a controlled polymerization technique. Progress in Organic Coatings, 73(4), 355-365. http://dx.doi.org/10.1016/j.porgcoat.2011.03.003. 25. Elrebii, M., Mabrouk, A. B., & Boufi, S. (2014). Synthesis and properties of hybrid alkyd–acrylic dispersions and their use in VOC-free waterborne coatings. Progress in Organic Coatings, 77(4), 757-764. http://dx.doi.org/10.1016/j.porgcoat.2013.12.016. 26. Tsavalas, J. G., Luo, Y., & Schork, F. J. (2003). Grafting mechanisms in hybrid miniemulsion polymerization. Journal of Applied Polymer Science, 87(11), 1825-1836. http://dx.doi. org/10.1002/app.11916. 27. Murillo, E. A., Vallejo, P. P., & López, B. L. (2011). Effect of tall oil fatty acids content on the properties of novel hyperbranched alkyd resins. Journal of Applied Polymer Science, 120(6), 3151-3158. http://dx.doi.org/10.1002/app.33502. 28. Wang, S. T., Schork, F. J., Poehlein, G. W., & Gooch, J. W. (1996). Emulsion and miniemulsion copolymerization of

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acrylic monomers in the presence of alkyd resin. Journal of Applied Polymer Science, 60(12), 2069-2076. http://dx.doi. org/10.1002/(SICI)1097-4628(19960620)60:12<2069::AIDAPP4>3.0.CO;2-K. 29. Schork, F. J., Luo, Y., Smulders, W., Russum, J. P., Butte, A., & Fontenot, K. (2005). Miniemulsion Polymerization. Advances in Polymer Science, 175, 129-255. http://dx.doi.org/10.1007/ b100115. 30. Schork, F. J., Poehlein, G. W., Wang, S., Reimers, J., Rodrigues, J., & Samer, C. (1999). Miniemulsion polymerization. Colloids and Surfaces. A, Physicochemical and Engineering Aspects, 153(1-3), 39-45. http://dx.doi.org/10.1016/S09277757(98)00424-5. 31. Landfester, K., Schork, F. J., & Kusuma, V. A. (2003). Particle size distribution in mini-emulsion polymerization. Comptes Rendus. Chimie, 6(11-12), 1337-1342. http://dx.doi.org/10.1016/j. crci.2003.07.019. 32. Merkel, M. P., Dimonie, V. L., El-Aasser, M. S., & Vanderhoff, J. W. (1987). Process parameters and their effect on grafting reactions in core/shell latexes. Journal of Polymer Science. Part A, Polymer Chemistry, 25(7), 1755-1767. http://dx.doi. org/10.1002/pola.1987.080250705. 33. Matsumoto, A., Kodama, K., Aota, H., & Capek, I. (1999). Kinetics of emulsion crosslinking polymerization and copolymerization of allyl methacrylate. European Polymer Journal, 35(8), 1509-1517. http://dx.doi.org/10.1016/S00143057(98)00216-X. 34. Esser, R. J., Devona, J. E., Setzke, D. E., & Wagemans, L. (1999). Water based crosslinkable surface coatings. Progress in Organic Coatings, 36(1-2), 45-52. http://dx.doi.org/10.1016/ S0300-9440(99)00019-3. 35. Kin, H., Hayashi, S., & Mizumachi, H. (1998). Miscibility and fracture energy of probe tack for acrylic pressure-sensitive adhesives: acrylic copolymer/tackifier resin systems. Journal of Applied Polymer Science, 69(3), 581-587. http://dx.doi. org/10.1002/(SICI)1097-4628(19980718)69:3<581::AIDAPP18>3.0.CO;2-W. Received: Sept. 18, 2015 Revised: Apr. 15, 2016 Accepted: Apr. 28, 2016

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http://dx.doi.org/10.1590/0104-1428.2215

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

Avaliação das propriedades da blenda de poli(3-hidroxibutirato)/quitosana após esterilização térmica ou radiolítica Evaluation of the properties poly(3-hidroxybutyrate)/ chitosan blend after radiolytic or thermal sterilization Grasielly Souza1, Andrelina Santos1 e Glória Vinhas1* Laboratório de Materiais Poliméricos e Caracterização – LMPC, Departamento de Engenharia Química, Universidade Federal de Pernambuco – UFPE, Recife, PE, Brasil

*gmvinhas@yahoo.com.br

Resumo Nesse trabalho foi desenvolvido filme de PHB/quitosana com aplicação promissora em embalagens de alimentos e medicamentos. Esses filmes foram preparados via casting solution e após esterilização térmica ou radiolítica suas propriedades foram avaliadas a partir da microscopia eletrônica de varredura, ensaio de tração, calorimetria exploratória diferencial e análise termogravimétrica. Foram constatadas alterações na morfologia dos filmes de PHB/quitosana após os processos de esterilização. As propriedades mecânicas da blenda se mantiveram aproximadamente constantes após esterilização térmica. Porém, após irradiação as blendas apresentaram-se quebradiças. As propriedades térmicas da blenda foram alteradas apenas para as doses de 50 e 75 kGy, em que foi observado uma redução no valor da entalpia de cristalização, grau de cristalinidade, cristalinidade relativa e taxa de cristalização da blenda. Foi observado que a degradação térmica dos filmes de PHB e das blendas, antes e após os processos de esterilização apresentaram um único estágio (250-300 °C). Palavras-chave: blendas poliméricas, esterilização térmica, esterilização radiolítica, PHB, quitosana. Abstract In this work was developed PHB/chitosan film with promising application in food and medicine packaging. These films were prepared by the casting solution and after radiolytic or thermal sterilization their properties were evaluated by scanning electron microscopy, tensile testing, differential scanning calorimetry and thermogravimetric analysis. PHB/chitosan films changed their morphology after sterilization processes. Mechanical properties of the blend remained approximately constant after thermal sterilization. But, after irradiation the blends presented brittle. Thermal properties of the blend were changed only for the doses of 50 and 75 kGy, in which case was observed a reduction in the crystallization enthalpy value, the degree of crystallinity, relative crystallinity and crystallization rate of the blend. It was observed thermal degradation of the PHB films and blends, before and after sterilization processes presented a single stage (250-300 °C). Keywords: polymers blends, thermal sterilization, radiolytic sterilization, PHB, chitosan.

1. Introdução Os produtos fabricados a partir de materiais plásticos têm estado cada vez mais presentes no cotidiano das pessoas. Isso se deve a algumas propriedades desses materiais, como leveza, atoxicidade, durabilidade, baixo custo, capacidade de serem reciclados, de serem moldados e de substituir produtos feitos de metais, madeira, vidro, papel, entre outros. Além disso, apresentam uma variedade de aplicações, desde plásticos rígidos altamente cristalinos a polímeros dúcteis[1,2]. Em contrapartida, o crescente uso desses produtos e seu descarte incorreto têm provocado uma série de danos ao meio ambiente, uma vez que, os mesmos são produzidos

352

a partir de polímeros sintéticos convencionais, bastante resistentes ao ataque de micro-organismos, e, portanto, levam mais tempo para se degradarem, contribuindo assim para o aumento da quantidade de resíduos plásticos presentes no meio ambiente[3]. Com a finalidade de minimizar o impacto ambiental causado pelo mau gerenciamento de resíduos plásticos, tem-se como principais alternativas: a incineração, a reciclagem ou a substituição dos polímeros sintéticos convencionais por polímeros biodegradáveis[4,5]. Os polímeros biodegradáveis são consumidos em semanas ou meses, sob condições favoráveis, pela ação de

Polímeros, 26(4), 352-359, 2016

Avaliação das propriedades da blenda de poli(3-hidroxibutirato)/quitosana após esterilização térmica ou radiolítica micro‑organismos de ocorrência natural como bactérias, fungos e algas, resultando em dióxido de carbono (CO2), metano (CH4), componentes celulares e outros produtos[4,6]. Os polímeros biodegradáveis podem ser provenientes de fontes naturais renováveis como milho, celulose, batata, cana de açúcar, ou serem sintetizados por bactérias como é o caso do poli(3-hidroxibutirato) (PHB), ou ainda, podem ser derivados de fonte animal, como a quitosana que é produzida a partir da carapaça de alguns crustáceos e também está presente em alguns insetos, fungos, cogumelos e minhocas[7-9]. Muitos pesquisadores vêm se dedicando ao estudo da modificação dos polímeros biodegradáveis para viabilizar o seu processamento e ampliar sua aplicabilidade. Para isso, blendas, compósitos e nanocompósitos estão sendo desenvolvidos[1,10-13]. Além disso, os polímeros biodegradáveis estão sendo cada vez mais utilizados em aplicações médico‑hospilares ou alimentícias. Porém, esses setores necessitam que seus produtos estejam estéreis, portanto tornar-se importante avaliar e relatar as possíveis degradações sofridas por estes materiais após processos de esterilização. Por esse motivo, o objetivo principal desse trabalho foi ampliar os estudos dos polímeros biodegradáveis, avaliando a estabilidade térmica ou radiolítica da blenda PHB/quitosana destinadas para aplicações médico-hospitalares ou alimentícias. A escolha do PHB está relacionada com suas propriedades semelhantes ao polipropileno (PP), já a quitosana por ser um polímero que apresenta atividade antimicrobiana, biocompatibilidade e baixa toxicidade[7,14].

2. Experimental 2.1 Materiais O PHB utilizado foi fornecido pela PHB Industrial S/A e a quitosana comercial de massa molar média com grau de desacetilação de 75-85% utilizada foi da marca Sigma‑Aldrich. O clorofórmio e o ácido acético utilizados foram da marca Vetec e Quimex, respectivamente.

2.2 Preparação dos filmes poliméricos Os filmes foram obtidos utilizando a técnica solution casting. 2.2.1 Filme de PHB puro O PHB foi peneirado e a granulometria utilizada variou entre 50 e 100 mesh. A solução de PHB foi obtida dissolvendo‑se 1,3 g do polímero em 50 mL de clorofórmio, num béquer de 250 mL. A solução foi agitada por 3 horas em agitador magnético a 60 °C. Após agitação, a solução foi deixada em repouso por 48 horas para que o PHB intumescesse. Após o tempo de 48 horas, a solução de PHB foi agitada em homogeneizador Turratec TE-102 por 15 minutos e foi filtrada utilizando chumaço de algodão para retirar possíveis impurezas da solução. Após a filtração, a solução foi vertida em placa de petri de vidro (20 cm de diâmetro) e os filmes foram secos em uma sala cuja temperatura ambiente era em torno de 30 °C. A proporção de massa de solução de polímero por cm2 de placa utilizada na produção dos filmes foi de 0,24 g/cm2. Polímeros, 26(4), 352-359, 2016

2.2.2 Filme de quitosana pura A solução de quitosana foi obtida dissolvendo-se 1,0 g de quitosana em 100 mL de solução de ácido acético (0,5% v/v) em um Erlenmeyer de 250 mL. A solução foi agitada em agitador magnético, a temperatura ambiente (~25 °C) até completa solubilização. A solução foi vertida em placa de petri de vidro e os filmes foram secos em estufa a aproximadamente 40 °C. A proporção de massa de solução de polímero por cm2 de placa utilizada na produção dos filmes foi de 0,32 g/cm2. 2.2.3 Blenda PHB/quitosana As soluções de PHB e quitosana foram preparadas separadamente como descritas nos itens anteriores. A blenda foi obtida de modo que a concentração de quitosana fosse 0,7% (m/m), para isso foi adicionada 1 mL da solução de quitosana a 50 mL da solução de PHB e a solução resultante foi agitada por 15 minutos em homogeneizador Turratec TE-102. A solução final foi vertida em placa de petri de vidro e os filmes foram secos em uma sala cuja temperatura ambiente era de aproximadamente 30 °C. A proporção de massa de solução de polímero por cm2 de placa utilizada na produção dos filmes foi de 0,24 g/cm2.

2.3 Esterilização térmica Os filmes de PHB puro, de quitosana pura e as blendas de PHB/quitosana foram submetidos à esterilização térmica em autoclave modelo 415 Fanem. O processo consistiu na obtenção de vapor a temperatura de 121 °C, sob pressão de 1 atm. Nestas condições, os filmes foram mantidos por 15 minutos. Após o tempo de esterilização, os filmes foram resfriados.

2.4 Esterilização radiolítica Os filmes de PHB puro, quitosana pura e as blendas de PHB/quitosana foram irradiados à temperatura ambiente (~25 °C) nas doses de 25, 50 e 75 kGy. A irradiação foi realizada com raios gama provenientes de uma fonte 60Co em equipamento “Gammacell”, modelo GC 220, localizado no Departamento de Energia Nuclear (DEN) da Universidade Federal de Pernambuco.

2.5 Técnicas de caracterização 2.5.1 Microscopia eletrônica de varredura (MEV) Os filmes de PHB puro e as blendas de PHB/Quitosana, antes e após esterilização térmica ou radiolítica, foram analisados em microscópio eletrônico de varredura (MEV) Shimadzu SS-550 Superscan. As amostras foram metalizadas com uma fina camada de ouro, utilizando fita de carbono como suporte antes de serem escaneadas. 2.5.2 Ensaio mecânico de tração Os ensaios de tração dos filmes de PHB puro, quitosana pura e da blenda PHB/quitosana, antes e após esterilização térmica ou radiolítica, foram realizados em triplicata e conduzidos em máquina universal, marca EMIC seguindo a norma ASTM D-882[15], a temperatura ambiente (~25 °C) e sem controle de umidade, nas seguintes condições: 353

Souza, G., Santos, A., & Vinhas, G. velocidade da garra: 5 mm/min, distância inicial entre as garras: 40 mm, dimensão do corpo de prova: 2,5 × 7,5 cm. 2.5.3 Análise Termogravimétrica (TGA) As amostras de PHB puro, de quitosana pura e de PHB/quitosana, antes e após esterilização térmica ou radiolítica, foram submetidas a testes termogravimétricos para obtenção das faixas de perda de massa. Foi utilizada uma termobalança Perkin Elmer, modelo STA 6000. Os testes foram realizados com taxa de aquecimento de 10 °C/min, sob atmosfera de nitrogênio (fluxo: 20 mL/min), em uma faixa de temperatura de 50-600 °C. 2.5.4 Calorimetria Exploratória Diferencial (DSC) As amostras dos filmes de PHB puro e das blendas PHB/quitosana, antes e após processos de esterilização térmica ou radiolítica, foram caracterizadas por calorimetria exploratória diferencial (DSC), em equipamento da marca Mettler Toledo, modelo DSC 1 STARe SYSTEM, utilizando cadinho de alumínio sob atmosfera de nitrogênio (fluxo: 50 mL/min) com massa entre 4 e 10 mg. Todos os testes foram conduzidos em três estágios: aquecimento de 25 °C a 185 °C, a uma taxa de aquecimento de 30 °C/min, resfriamento até 25 °C, a uma taxa de resfriamento de 16 °C/min e reaquecimento até 185 °C, a uma taxa de aquecimento de 30 °C/min. A partir deste método foi possível obter a temperatura de fusão (Tm), temperatura de cristalização (Tc) e entalpia de cristalização (ΔHc). A cristalinidade relativa (x), o grau de cristalinidade (Xc), a taxa de cristalização (c) e a entalpia de cristalização (∆Hc) foram calculadas utilizando o software INTEGRALTM, desenvolvido na UFCG. As fórmulas matemáticas utilizadas no programa para obtenção destes parâmetros estão apresentadas nas Equações 1, 2, 3 e 4, respectivamente. t2 1 t x= ∫ J ( t ′ ) − J 0 ( t ′ ) dt ′, E0 = ∫ J ( t ) − J 0 ( t ) dt E0 t1 t1

XC =

= c

(1)

(2)

dx 1 = J (t ) − J0 (t ) dt E0

(3)

w p ∆H m°

E0 ∆H c = ms

(4)

Onde J é o fluxo de calor medido no DSC, J0 é a linha de base virtual durante o evento de cristalização, t1 e t2 são os tempos inicial e final do evento, respectivamente, E0 é o calor latente de mudança de fase, wp é a fração mássica do PHB na amostra, ms é a massa da amostra e ∆Hm° é o calor latente de fusão por unidade de massa do PHB 100% cristalino, que é 146 J/g de acordo com a literatura[16].

3. Resultados e Discussão 3.1 Filmes poliméricos A Figura 1 mostra os filmes de PHB puro, quitosana pura e da blenda PHB/quitosana estudadas neste trabalho. Os filmes de PHB puro apresentaram-se homogêneos, opacos e sua espessura média foi de 0,07 ± 0,02 mm. Os filmes de quitosana pura apresentaram-se homogêneos, transparentes e sua espessura média foi de 0,06 ± 0,03 mm. As blendas de PHB/quitosana apresentaram-se homogêneas, opacas e sua espessura média foi de 0,08 ± 0,01 mm.

3.2 Avaliação das propriedades físicas das blendas e dos polímeros puros 3.2.1 Propriedades morfológicas A morfologia da superfície dos filmes foi estudada por MEV, como mostra a Figura 2. O filme de PHB puro apresentou uma superfície rugosa e irregular (Figura 2a), morfologia semelhante foi também constatada por Abdelwahab et al.[10]. Com a adição da quitosana, os filmes apresentaram mudanças em suas características morfológicas. Foram observados grânulos de diferentes diâmetros (Figura 2b) que podem ser associados à presença da quitosana. A formação desses grânulos possivelmente foi devido a imiscibilidade da solução de quitosana em água na solução de PHB em clorofórmio. A superfície da blenda após processo de esterilização térmica (Figura 3a) apresentou-se rugosa e irregular com a presença de grânulos (quitosana) de diâmetros menores comparados a blenda antes da esterilização térmica, também foi observado a presença de alguns vazios na matriz. Já os filmes irradiados a 25 kGy (Figura 3b) apresentaram-se lisos e homogêneos.

Figura 1. Filmes de PHB puro (a), de quitosana pura (b) e a blenda PHB/quitosana (c). 354

Polímeros, 26(4), 352-359, 2016

Avaliação das propriedades da blenda de poli(3-hidroxibutirato)/quitosana após esterilização térmica ou radiolítica 3.2.2 Propriedades mecânicas Foram realizados testes de resistência à tração para os filmes de PHB puro, PHB/quitosana e quitosana pura antes e após processos de esterilização térmica ou radiolítica. As propriedades mecânicas avaliadas neste trabalho

Figura 2. Micrografia dos filmes de PHB puro (a) e da blenda PHB/quitosana (b) com ampliação de 1000x.

Figura 3. Micrografia dos filmes de PHB/quitosana esterilizado termicamente (a) e a 25 kGy (b) com ampliação de 1000x. Polímeros, 26(4), 352-359, 2016

foram tensão máxima, deformação específica e módulo de elasticidade. A partir das Figuras 4, 5 e 6, é possível observar que as propriedades mecânicas dos filmes de quitosana após esterilização térmica diminuem drasticamente. Isso comprovou a alta sensibilidade térmica da quitosana que também foi observada por Corazzari et al.[17]. Apesar das grandes variações das propriedades mecânicas dos filmes de quitosana pura, após processo de esterilização térmica, as blendas mostraram-se mais estáveis, variando pouco as suas propriedades. Para a tensão máxima, o filme de quitosana pura sofreu uma redução de 92,4% nessa propriedade, enquanto a blenda praticamente não sofreu alteração. Para a deformação específica, o filme de quitosana pura sofreu uma redução de 62,5%, enquanto a blenda se manteve praticamente constante. Já para o módulo de elasticidade, essa redução foi de 63,4% para o filme de quitosana pura e 13,6% para a blenda. Esses fatos mostraram que a presença da quitosana não comprometeu as propriedades mecânicas estudadas da blenda após processo de esterilização térmica, isso faz com que este material seja promissor para possíveis aplicações médico-hospitalares ou alimentícias. Pelo teste de Duncan, os filmes de PHB puro e a blenda não esterilizada não diferiram significativamente as propriedades mecânicas estudadas. O mesmo ocorreu para os filmes de PHB puro e a blenda esterilizada termicamente. Comparando os filmes de PHB puro e a blenda antes e após esterilização térmica, pode-se afirmar que não houve alterações significativas em suas propriedades, já os filmes de quitosana sofreram alterações significativas. Todos os testes foram realizados ao nível de 5% de significância. As Figuras 7, 8 e 9 mostram uma comparação entre as propriedades mecânicas dos filmes de quitosana pura antes e após esterilização radiolítica. Foi observada uma queda brusca em suas propriedades mecânicas (tensão máxima e módulo de elasticidade), exceto a deformação específica que se manteve praticamente constante. Embora a deformação específica tenha variado pouco (Figura 8), um fato positivo foi a diminuição do desvio das amostras com o aumento da dose de radiação, o que supostamente indica uma maior homogeneidade das mesmas. Caso semelhante foi observado para o módulo de elasticidade (Figura 9).

Figura 4. Gráfico da tensão máxima × amostras dos filmes antes e após processo de esterilização térmica (ET). 355

Souza, G., Santos, A., & Vinhas, G. Pelo teste de Duncan, é possível afirmar que não houve diferença significativa para as amostras esterilizadas por radiação gama, independentemente da dose utilizada. Porém, houve uma diferença significativa entre os filmes de quitosana pura não esterilizado e esterilizado por radiação gama para a tensão máxima e para o módulo de elasticidade ao nível de 5% de significância. Já para a deformação específica não há uma alteração significativa. Não foi possível realizar os ensaios mecânicos dos filmes de PHB puro e das blendas após esterilização radiolítica, pois esses se apresentaram altamente quebradiços. Esse fato é um forte indício de que houve degradação do material após irradiação. 3.2.3 Propriedades térmicas 3.2.3.1 Calorimetria Exploratória Diferencial (DSC) Figura 5. Gráfico da deformação específica × amostras dos filmes antes e após processo de esterilização térmica (ET).

A Figura 10 mostra o fluxo de calor no DSC em função do tempo, no intervalo do teste 0 – 21 minutos para o PHB puro e para a blenda PHB/quitosana antes e após esterilização térmica ou radiolitica. Pode-se observar quatro eventos de mudança de fase: fusão (F1) da fração cristalina, com pico duplo, durante o primeiro aquecimento, cristalização a

Figura 6. Gráfico do módulo de elasticidade × amostras dos filmes antes e após processo de esterilização térmica (ET). Figura 8. Gráfico da deformação específica × amostras dos filmes de quitosana pura não irradiada e irradiada.

Figura 7. Gráfico da tensão máxima × amostras dos filmes de quitosana pura não irradiada e irradiada. 356

Figura 9. Gráfico do módulo de elasticidade × amostras dos filmes de quitosana pura não irradiada e irradiada. Polímeros, 26(4), 352-359, 2016

Avaliação das propriedades da blenda de poli(3-hidroxibutirato)/quitosana após esterilização térmica ou radiolítica partir do fundido (C1) durante o resfriamento, cristalização a frio (C2) e segunda fusão (F2) durante o reaquecimento. Esses eventos também foram observados por Machado et al.[18]. A cristalização a frio apenas é observada para as blendas irradiadas nas doses de 50 e 75 kGy. Com a adição da quitosana na matriz do PHB percebe-se que não houve uma modificação na cristalinidade deste, pois o grau de cristalinidade, a temperatura e a entalpia de cristalização não apresentaram diferenças significativas em relação ao Tabela 1. Parâmetros do DSC dos filmes de PHB puro e PHB/ quitosana. Amostras PHB pó PHB puro Blenda Blenda ET Blenda 25 kGy Blenda 50 kGy Blenda 75 kGy

Tc (°C) 114 113 110 113 114 72/61 71/57

Tm (°C) 170 167 166 165 165 166 164

∆Hc (J/g) 66,35 68,43 67,39 70,35 66,81 37,27 25,27

Xc (%) 45,45 46,87 46,16 48,19 45,76 25,53 17,31

ET – Esterilizado termicamente.

filme de PHB puro (Tabela 1). Esse resultado também foi observado por Matet et al.[13] que comprovaram que baixos percentuais de quitosana (2, 5 e 10%) na blenda PE/quitosana não alterou a temperatura de cristalização e fusão. As temperaturas características (cristalização e fusão), a entalpia de cristalização e porcentagem de cristalização obtidas por DSC estão relatadas na Tabela 1. A temperatura de cristalização e fusão do filme de PHB puro são, respectivamente, 113 e 167 °C, que é praticamente a mesma do PHB em pó (Tabela 1) e estas temperaturas estão de acordo com as encontradas na literatura[10,18]. A esterilização térmica e a irradiação a 25 kGy não alteraram significativamente a temperatura de cristalização (Tc), a temperatura de fusão (Tm), entalpia de cristalização (∆Hc) e o grau de cristalinidade (Xc) deste material. Porém, as blendas esterilizadas a 50 e 75 kGy apresentaram dois picos de cristalização que refere-se à cristalização a frio e no estado fundido, também apresentaram uma diminuição nos valores de entalpia de cristalização e grau de cristalinidade em relação aos outros filmes. Provavelmente, essas doses de radiação gama comprometeram a região cristalina da blenda[19]. A Figura 11 mostra as curvas de cristalinidade relativa, x, ou seja, a fração de material cristalizada, para o filme de PHB puro e para a blenda PHB/quitosana antes e após esterilização térmica ou radiolítica. Para o filme de PHB puro e para a blenda PHB/quitosana não esterilizada, o processo de cristalização é iniciado praticamente na mesma temperatura, aproximadamente 120 ºC, já para as blendas esterilizadas termicamente ou irradiadas na dose de 25 kGy percebe-se que o processo de cristalização ocorre em temperaturas mais altas. Para a indústria, isso significa que durante o resfriamento do material polimérico processado ocorrerá uma cristalização antecipada. As blendas que foram esterilizadas na dose de 50 e 75kGy, percebe-se que a fração de material que cristaliza apresentam um perfil próximo da linearidade, ou seja, as blendas esterilizadas nessas doses apresentam uma faixa de temperatura maior para finalizar a cristalização.

Figura 10. Fluxo de calor em função do tempo para o filme de PHB puro e para a blenda PHB/quitosana antes e após esterilização térmica (ET) ou radiolítica.

Quando se avalia a taxa de cristalização (min-1) (Figura 12), a cristalização do filme de PHB puro é mais rápida do que a cristalização das blendas, principalmente em relação a blenda

Figura 11. Cristalinidade relativa do filme de PHB puro e da blenda PHB/quitosana antes e após esterilização térmica ou radiolítica.

Figura 12. Taxa de cristalização em função da temperatura para o filme de PHB puro e para as blendas de PHB/quitosana antes e após esterilização térmica ou radiolítica.

Polímeros, 26(4), 352-359, 2016

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Souza, G., Santos, A., & Vinhas, G. não esterilizada. É notório a influência da radiação gama nas doses de 50 e 75 kGy na blenda referente a cristalinidade relativa e a taxa de cristalização. Na Figura 12, observa-se uma lenta cristalização dessas blendas, com intervalo maior de temperatura, além disso a taxa de cristalização máxima é muito baixa quando comparada com os demais filmes. 3.2.3.2 Análise termogravimétrica (TGA) A degradação térmica dos filmes de PHB puro antes e após esterilização térmica ou radiolítica está apresentada na Figura 13. As análises termogravimétricas dos filmes de PHB puro mostraram degradação total em um único estágio entre 250 e 300 ºC. Figura 13. Curvas de TGA para os filmes de PHB puro antes e após processos de esterilização térmica (ET) ou radiolítica.

Figura 14. Curvas de TGA para os filmes de quitosana pura antes e após processos de esterilização térmica (ET) ou radiolítica.

As análises termogravimétricas dos filmes de quitosana pura antes e após processos de esterilização térmica ou radiolítica estão apresentadas na Figura 14. A degradação térmica da quitosana aconteceu em três estágios: processo de desidratação, decomposição do biopolímero e geração de material carbonizado. Essas três etapas de degradação térmica da quitosana também foi observada por Corazarri et al.[17]. Na faixa de temperatura entre 50 e 150 ºC, a perda de massa pode ser associada à dessorção da água presente na superfície do polímero. Na faixa entre 150‑450 °C foi observado o processo principal de degradação térmica. Segundo Corazarri et al.,[17] nesta etapa ocorre a liberação de H2O, NH3, CO, CO2 e CH3COOH. Por fim, na faixa de 450-600 °C houve a liberação de CH4. A porcentagem da perda de massa das amostras de quitosana em cada estágio da degradação e a massa residual está relatada na Tabela 2. O comportamento térmico da blenda PHB/quitosana, antes e após processos de esterilização térmica ou radiolítica, foi semelhante ao filme de PHB puro. A degradação térmica aconteceu em um único estágio entre 250 e 300 °C (Figura 15). As amostras não apresentaram massas residuais.

4. Conclusões A avaliação das propriedades morfológicas, mecânicas e térmicas dos filmes de PHB puro, de quitosana pura e das blendas PHB/quitosana após processos de esterilização térmica ou radiolítica, realizada neste trabalho, forneceu algumas conclusões que estão apresentadas a seguir: Os filmes de quitosana pura apresentaram alterações significativas nas propriedades mecânicas estudadas após esterilização térmica ou radiolítica. Já para os filmes de PHB puro, a esterilização térmica não comprometeu as propriedades mecânicas estudadas, em contrapartida, a esterilização radiolítica degradou completamente os filmes, resultando em filmes altamente quebradiços. Figura 15. Curvas de TGA para as blendas PHB/quitosana antes e após processos de esterilização térmica (ET) ou radiolítica.

As blendas PHB/quitosana após os processos de esterilização estudados tiveram comportamento semelhante

Tabela 2. Dados obtidos das curvas de TG das amostras de quitosana em estudo. Amostras Quitosana 75 kGy Quitosana ET Quitosana pura

1º estágio (50-50 °C) 10,0 10,0 9,0

Perda de massa (%) 2º estágio (150-450 °C) 43,5 45,3 42,7

3º estágio (450-600 °C) 3,7 4,3 3,6

Massa residual (%) 42,8 38,3 44,7

ET – Esterilizado termicamente.

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Avaliação das propriedades da blenda de poli(3-hidroxibutirato)/quitosana após esterilização térmica ou radiolítica ao filme de PHB puro. Isso mostrou que a quitosana não alterou as propriedades mecânicas das blendas. Com base no MEV foi possível observar na blenda a presença de grânulos, referentes à quitosana, dispersos na matriz de PHB. Também foi observado que após esterilização térmica, a blenda apresentou uma superfície rugosa, com a presença de grânulos e alguns vazios na matriz. Já após a esterilização radiolítica, a blenda apresentou-se lisa e homogênea. A porcentagem de quitosana adicionada e o processo de esterilização térmica não alteraram a temperatura e entalpia de cristalização, a temperatura de fusão e o grau de cristalinidade das blendas, porém as blendas irradiadas com doses de 50 e 75 kGy apresentaram uma redução no valor da entalpia de cristalização, no grau de cristalinidade, na cristalinidade relativa e na taxa de cristalização do filme de PHB/quitosana, além disso, apresentaram dois picos de cristalização. O comportamento térmico das blendas, antes e após processos de esterilização térmica ou radiolítica, observado pelas curvas de TGA foi semelhante ao comportamento térmico do filme de PHB puro. Diante destas conclusões, foi notório que o PHB é muito mais determinante nas propriedades das blendas do que a quitosana e que a blenda estudada apresenta uma viabilidade comercial em processos que exijam apenas a esterilização térmica.

5. Agradecimentos Os autores agradecem à PHB industrial S.A. (Brasil) pelo fornecimento do PHB, à Agência Nacional do Petróleo, Gás Natural e Biocombustíveis – ANP e à Financiadora de Estudos e Projetos – FINEP – por meio do Programa de Recursos Humanos da ANP para o Setor de Petróleo e Gás – PRH-ANP/MCT, em particular ao PRH 28 pelo apoio financeiro.

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http://dx.doi.org/10.1590/0104-1428.1987

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Hidrogéis a base de ácido hialurônico e quitosana para engenharia de tecido cartilaginoso Hyaluronic acid and chitosan based hydrogels for cartilage tissue engeneering Mônica Helena Monteiro do Nascimento1 e Christiane Bertachini Lombello2* Human and Natural Sciences Center, Universidade Federal do ABC – UFABC, Santo André, SP, Brazil Engineering, Modelling and Applied Social Sciences Center, Universidade Federal do ABC – UFABC, Santo André, SP, Brazil

*christiane.lombello@ufabc.edu.br

Resumo A Engenharia de Tecidos envolve o desenvolvimento de novos materiais ou dispositivos capazes de interações específicas com os tecidos biológicos, buscando a utilização de materiais biocompatíveis que devem servir como arcabouço para o crescimento de células in vitro, organizando e desenvolvendo o tecido que posteriormente será implantado no paciente. Uma variedade de arcabouços como hidrogéis poliméricos, sintéticos e naturais, têm sido investigados para a expansão de condrócitos in vitro, visando o reparo da cartilagem lesionada. Um hidrogel de interesse particular na regeneração de cartilagem é o ácido hialurónico (AH). Trata-se de um biopolímero atraente para a fabricação de arcabouços artificiais para Engenharia de Tecidos por ser biocompatível e biodegradável. A biocompatibilidade do AH deve-se ao fato de estar presente na matriz extracelular nativa, deste modo, cria-se um ambiente propício que facilita a adesão, proliferação e diferenciação celular, além da existência de sinalização celular específica, o que contribui para a regeneração do tecido. O uso de hidrogel composto de ácido hialurónico e quitosana (QUI) também tem sido investigado em aplicações de Engenharia de Tecidos de cartilagem, com resultados promissores. Baseando-se nestas informações, o objetivo este trabalho foi investigar as alternativas disponíveis para regeneração tecidual da cartilagem e conhecer mais detalhadamente as relações entre células e biomateriais. Palavras-chave: ácido hialurônico, biocompatibilidade, engenharia de tecidos, quitosana, cultura de células. Abstract Tissue Engineering involves the development of new materials or devices capable of specific interactions with biological tissues, searching the use of biocompatible materials as scaffolds for cell growth in vitro, organizing and developing tissue that is subsequently implanted into the patient. A variety of scaffolds such as polymeric hydrogels, natural and synthetic, have been investigated for the expansion of chondrocytes in vitro in order to repair the damaged cartilage. A hydrogel of particular interest in cartilage regeneration is hyaluronic acid (HA). HA are attractive biopolymers for manufacturing artificial scaffolds for Tissue Engineering, it is biocompatible and biodegradable. The biocompatibility of HA is due to the fact that it is present in native extracellular matrix, thus creates an environment, which facilitates the adhesion, proliferation and differentiation, in addition to the existence of specific cell signaling, which contributes to tissue regeneration. The use of hydrogel composed of hyaluronic acid and chitosan (CHI) has also been investigated for applications in Tissue Engineering of soft tissues, like cartilage, with promising results. Based on this information, this study aims to investigate the alternatives available for cartilage tissue regeneration and meet more detail the relationships between cells and biomaterials. Keywords: hyaluronic acid, biocompatibility, tissue engineering, chitosan, cell culture.

1. Introdução As patologias de tecidos cartilaginosos vêm sendo objeto de estudos relevantes nos últimos anos por representarem um importante problema de saúde mundial. Danos à cartilagem articular continuam sendo uma questão desafiadora na área da ortopedia. A cartilagem articular é conhecida por

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ter uma pobre capacidade intrínseca para o reparo, devido em grande parte à sua avascularidade e ao acesso limitado às células reparadoras. As células deste tecido apresentam baixo índice mitótico, o que contribui para a sua limitada capacidade de regeneração[1].

Polímeros, 26(4), 360-370, 2016

Hidrogéis a base de ácido hialurônico e quitosana para engenharia de tecido cartilaginoso Por esse motivo, pequenos ferimentos na cartilagem articular podem evoluir para danos progressivos e degeneração tecidual. Os tratamentos convencionais, cirúrgicos ou não, podem trazer uma importante melhora da sintomatologia do paciente, no entanto não garantem necessariamente o reparo do tecido lesado. Os resultados mais positivos para a regeneração da cartilagem, principalmente dos tecidos articulares, se referem ao uso de terapia celular[2].

Tendo em vista a necessidade de aprimorar as alternativas disponíveis para regeneração tecidual da cartilagem e conhecer mais detalhadamente as relações entre células e biomateriais, este estudo traz a revisão de tópicos relacionados à área da Engenharia de Tecidos, especificando a utilização de hidrogéis à base de AH e QUI como substratos para a cultura de células.

Atualmente, a Engenharia de Tecidos vem despontando como um ramo científico capaz de fornecer metodologias promissoras para o tratamento de patologias do tecido cartilaginoso. Segundo Langer e Vacanti[3], este é um campo científico-tecnológico interdisciplinar que aplica os conceitos de engenharia e ciências da vida com o intuito de desenvolver substitutos biológicos capazes de restaurar, preservar e restabelecer as funções de um órgão ou tecido lesado. Os três elementos constituintes da Engenharia de Tecidos são, fundamentalmente, o arcabouço (scaffold), as células, e o microambiente fisiológico.

2. Engenharia de Tecidos

Os arcabouços são componentes fundamentais na Engenharia de Tecidos, na medida em que fornecem o suporte estrutural à fixação e crescimento das células, criando um microambiente propício para a substituição ou reparação dos tecidos, e auxiliando estruturalmente o tecido recém‑formado[4]. Os arcabouços, ao mimetizar a matriz extracelular (MEC), devem interagir com as células de maneira específica e direcionada a fim de obter comportamentos específicos, relacionados à adesão, espalhamento, crescimento e diferenciação celulares. Uma variedade de arcabouços como hidrogéis, de polímeros sintéticos ou naturais, têm sido investigados para o cultivo de condrócitos in vitro, visando o reparo da cartilagem lesada. Um hidrogel de interesse particular na regeneração de cartilagem é o ácido hialurónico (AH). O AH é um polímero de origem natural, pertencente à família dos glicosaminoglicanos (GAGs), presente em abundância na matriz extracelular da cartilagem articular e desempenha papel importante numa variedade de processos celulares. Trata-se de um hidrogel atraente para a fabricação de arcabouços artificiais para Engenharia de Tecidos por ser biocompatível, biodegradável e apresentar benefícios no tratamento inicial em lesões osteoartríticas[5-7]. Contudo, deve-se utilizar o AH em relativa baixa quantidade, uma vez que a presença em excesso deste biopolímero pode promover a redução da adesão celular devido à carga negativa característica deste GAG. Esta complicação pode ser superada pela combinação entre o AH e hidrogéis de carga positiva (policátions) tais como a quitosana (QUI). Este biomaterial polimérico tem origem natural, apresenta importante biocompatibilidade e biodegradabilidade, além de ser um polissacarídeo com estrutura molecular bastante similar à de GAGs presentes na cartilagem articular[5]. Como as GAGs, a QUI está fortemente envolvida na modulação de processos celulares, como a condrogênese, por interagir com fatores de crescimento e citocinas. Além disso, estudos realizados com hidrogéis compostos por AH e QUI reportaram a maior adesão celular nas blendas, demonstrando que a QUI melhorou a bioestabilidade e a biocompatibilidade do arcabouço[8]. Polímeros, 26(4), 360-370, 2016

Quando a estrutura biológica de um órgão ou tecido não pode ser naturalmente reparada, a alternativa viável para o restabelecimento das funções normais do paciente é, muitas vezes, utilizar um implante feito de um biomaterial[9]. A regeneração de órgãos e tecidos vivos, ou o reparo da função destes, é o objetivo da Engenharia de Tecidos. Uma abordagem consiste no recrutamento de células do próprio paciente, que são dissociadas dos tecidos originais e cultivadas sobre arcabouços biológicos ou sintéticos, conhecidos como scaffolds (arcabouços, suportes, matrizes tridimensionais, ou estruturas), para então serem reinseridos no paciente. Como uma ciência multidisciplinar a Engenharia de Tecidos envolve conhecimentos das áreas de biologia, ciências da saúde, engenharia e ciência dos materiais[9]. O termo “Engenharia de Tecidos” foi citado pela primeira vez numa reunião patrocinada pela NSF (National Science Fundation) em 1987 e num Workshop a Engenharia de Tecidos foi definida como Aplicação de princípios e métodos de engenharia e ciências da vida com o propósito do entendimento fundamental das relações estrutura-função nos tecidos normais e patológicos dos mamíferos e o desenvolvimento de substitutos biológicos para recuperar, manter, ou incrementar a função do tecido[10]. No final da década de 80, a Engenharia de Tecidos começou a ser ministrada em centros de ensino com o objetivo de avançar nos conhecimentos e pesquisas no tratamento de doenças utilizando abordagens celulares[11]. Durante a década de 1990, a Engenharia de Tecidos progrediu rapidamente com o desenvolvimento de alguns substitutos biológicos de tecidos. Produtos biomédicos como pele artificial, com células viáveis, e terapia celular, com condrócitos autólogos cultivados, entraram prontamente no mercado[2,12,13]. Para criar um tecido substituto, um número pequeno de células, idealmente, pode ser colhido do paciente usando uma técnica de biopsia e as células são então cultivadas para a obtenção de um número apropriado em laboratório. Estas células podem então ser cultivadas em arcabouço (natural ou sintético), na presença de fatores de crescimento. Se providas com as condições e sinalização adequados, as células irão secretar vários componentes da matriz extracelular (MEC) para criar de fato um tecido vivo que pode ser usado como tecido substituto a ser reimplantado no paciente[9,14]. A avaliação dos progressos da Engenharia de Tecidos em suas mais diversas aplicações clínicas (pele, osso, cartilagem, nervos, medula óssea, vasos sanguíneos, córnea, 361

Nascimento, M. H. M., & Lombello, C. B. válvulas cárdicas e miocárdio) é francamente satisfatória. Ao longo de anos têm sido desenvolvidos, disponibilizados e comercializados inúmeros substitutos funcionais e estruturais para aplicação clínica[15].

2.1 Arcabouços para cultura de células A primeira etapa na Engenharia de Tecidos inicia-se com o desenvolvimento, seleção e processamento dos arcabouços. Os biomateriais, utilizados na Engenharia de Tecidos como arcabouços para cultura de células, podem ser definidos como um material não viável, ou seja, não vivo, utilizado em um dispositivo médico com intenção de interagir com sistemas biológicos[16]. Quanto ao tipo de material, estes biomateriais podem ser: polímeros, metais, cerâmicas e compósitos. É desejável que estes arcabouços sejam biocompatíveis, de maneira que apresentem a capacidade de induzir uma resposta apropriada do hospedeiro, minimizando as reações imunológicas; e biodegradáveis, onde os produtos de degradação não devam ser citotóxicos, tumorigênicos, ou causar qualquer outro efeito indesejável ao organismo. Os polímeros compõem uma classe bastante ampla de biomateriais, classificados como naturais (por exemplo: colágeno, agarose, alginato, fibrina, AH, gelatina, gel de plaquetas e glicosaminoglicanos) ou sintéticos (por exemplo: poli(α-hidróxi ácidos e poliHEMA)[9,17]. A biocompatibilidade de alguns polímeros naturais, como o colágeno e o AH, deve-se ao fato destes compostos estarem presentes na matriz extracelular nativa. Deste modo, cria-se um ambiente propício que facilita a adesão, proliferação e diferenciação celular, para além da existência de sinalização celular específica, o que contribui para a regeneração do tecido[16]. Em função da permanência no corpo, os biomateriais podem ser classificados como permanentes ou temporários. Os materiais permanentes são utilizados com a finalidade de substituir um tecido lesionado por tempo indeterminado, assim são produzidos de modo a reter as suas características mecânicas e físico-químicas por longos períodos. Já os biomateriais temporários são utilizados em situações onde se necessita de um suporte que preencha apenas temporariamente a região lesada, até que a recomposição tecidual se concretize, ou ainda que direcione o processo regenerativo[18]. As propriedades mecânicas e físico-químicas dos arcabouços utilizados na Engenharia de Tecidos são características importantes, que influenciam a sua aplicação final. Estes arcabouços não só apresentam uma função mecânica, como também apoiam a fixação, migração, proliferação e diferenciação celular para expressão de fenótipos desejáveis[16,19]. As propriedades mecânicas dos arcabouços como a resistência à tração, flexão e compressão, ductilidade (propriedade de suportar a deformação sem romper ou fraturar) e módulo de Young (razão entre tensão e deformação no regime elástico) são determinadas tanto pelas propriedades do material quanto pela estrutura do mesmo (macro, micro e nanoestrutura). A combinação das propriedades mecânicas do arcabouço com o ambiente do enxerto é de crucial importância para que a progressão da recuperação tecidual não seja limitada por falhas mecânicas do arcabouço[20]. Já propriedades 362

químicas como: hidrofilicidade, hidrofobicidade e cargas elétricas superficiais regulam o contato célula-tecido, e devem ser mimetizadas para obtenção de melhores resultados no desenvolvimento tecidual[21]. É importante também salientar que a estrutura química do biomaterial e o seu processamento determinam as propriedades funcionais e a interação das células com o arcabouço[22]. Além disso, estes arcabouços podem ser projetados de duas formas: porosos e não porosos ou densos. A porosidade é uma característica fundamental para que ocorra o alojamento adequado das células, propiciando a interação arcabouço-célula, bem como uma otimização do transporte de nutrientes e de gases através matriz tridimensional, por meio da vascularização deste. Em particular, a microporosidade é importante para ingresso capilar e interações entre células e matriz, enquanto a macroporosidade é relevante para o fornecimento de nutrientes e remoção de resíduos do metabolismo celular. A porosidade não deve ser excessiva, afim de não comprometer a estabilidade mecânica do arcabouço[15,21,23,24]. Além do tamanho dos poros, a sua morfologia pode significativamente influenciar o desempenho de uma matriz implantada e a taxa de crescimento interna do tecido. A porosidade ideal é estritamente ligada ao tipo de tecido, e várias arquiteturas de tecidos podem ser associadas com um microambiente diferente. Assim, o biomaterial deve ser moldado em diferentes geometrias, de forma a adequar-se às necessidades dos diferentes tipos de tecidos. Formas de disco ou cubo são usualmente utilizadas na regeneração de tecido ósseo, tubular aplicada na regeneração do tecido nervoso, vascular ou na regeneração da traqueia e achatada na engenharia de pele, cartilagem, intestino e fígado[15]. Estes arcabouços provêm um suporte biomecânico inicial até que as células produzam a MEC adequada. Durante a formação, deposição e organização da nova matriz, o ideal é que o arcabouço seja degradado e metabolizado, deixando que o órgão vital ou tecido seja reestabelecido, mantenha ou melhore sua função tecidual[25]. É importante sincronizar o tempo de degradação/absorção do biomaterial com o tempo de regeneração do tecido, possibilitando a transferência gradativa das funções do biomaterial para o tecido recém-formado, não devendo, contudo, originar produtos de degradação que possam interferir com o tecido em crescimento ou originar um processo inflamatório[15,19]. Na escolha do biomaterial, deve-se ter ainda em consideração, que há matrizes cujas características químicas definem a sua suscetibilidade à degradação aquosa ou enzimática[9,15]. Segundo Vert et al.[26] dentro da Engenharia de Tecidos os termos biodegradação, bioabsorção e biorreabsorção apresentam definições distintas: biodegradável é um termo utilizado para macromoléculas ou substâncias poliméricas susceptíveis à degradação por atividade biológica, com a redução das massas molares das macromoléculas que formam as substâncias; biorreabsorvível faz referência a materiais poliméricos e dispositivos sólidos que mostram degradação através da diminuição de tamanho e que são reabsorvidos in vivo (por exemplo materiais que são eliminados dos organismo por processos metabólicos, de secreção ou excreção); e bioabsorvível refere-se a materiais poliméricos Polímeros, 26(4), 360-370, 2016

Hidrogéis a base de ácido hialurônico e quitosana para engenharia de tecido cartilaginoso e dispositivos que podem se dissolver em fluidos corpóreos sem qualquer clivagem das cadeias macromoleculares ou diminuição de massa molecular.

2.2 Interação entre células e biomateriais O estudo das interações entre células e biomateriais na Engenharia de Tecidos é de grande importância para a determinação das propriedades biológicas dos implantes. A diversidade de respostas celulares a diferentes materiais evidencia a capacidade das células de discriminar quimicamente o arcabouço e de aderir ou não à sua superfície[11]. O conceito da biocompatibilidade refere-se à capacidade de um biomaterial, utilizado na concepção de arcabouços, desempenhar a sua função desejada no que diz respeito a uma terapia médica, sem suscitar quaisquer efeitos indesejáveis locais ou sistêmicos para o destinatário ou beneficiário daquela terapia, mas gerando a mais apropriada e benéfica resposta celular ou tecidual, numa situação específica. A chave para compreender este conceito é a determinação de quais mecanismos, químicos, bioquímicos, fisiológicos, físicos ou outros, tornam-se funcionais nas condições altamente específicas associadas com o contato entre os biomateriais e os tecidos do corpo, e quais são as consequências dessas interações[9,27]. Neste sentido pode-se citar quatro aspectos mais importantes com relação às interações entre biomateriais e tecidos[14]: • Fenômenos físicos-químicos de interface relacionados com os primeiros instantes de contato entre o biomaterial, tecido e ambiente de implantação; • Resposta do tecido e meio orgânico à presença do material; • Mudanças ocorridas nos materiais como resultados da ação do meio (tecido, fluidos orgânicos) sobre o material: degradação e corrosão; • Reação de alguma parte do organismo, não diretamente em contato com o implante.

A biocompatibilidade de um biomaterial está estreitamente relacionada ao comportamento das células em contato com a superfície, especialmente a adesão celular. Quando um implante é instalado em um defeito tecidual ou em um meio de cultura in vitro, inicia-se um processo de adsorção de proteínas em sua superfície. Essa camada de proteínas controla a interação das células do tecido adjacente com a superfície do implante[24,28]. O fenômeno de adsorção de proteínas na superfície de arcabouços é motivado pelo fato de que as proteínas possuem geralmente tanto grupamentos polares quanto apolares, favorecendo uma concentração destas numa interface que separa duas fases com distintas características (como entre uma fase líquida – fluidos corpóreos – e a superfície hidrofóbica de um biomaterial). Uma estratégia para se manipular o processo de adsorção de proteínas na superfície de biomateriais diz respeito ao enxerto de cadeias poliméricas especificamente escolhidas na superfície de materiais para controlar a adsorção[14,28]. Polímeros, 26(4), 360-370, 2016

Desta maneira, a associação dos arcabouços celulares com moléculas sinalizadoras, incluindo fatores de crescimento, citocinas, e os compostos químicos não-proteícos, é comumente utilizada para promover a regeneração do tecido lesado[29,30]. Essas moléculas, ao se ligarem a seus receptores na superfície celular, ativam vias de sinalização que irão induzir a proliferação celular, diferenciação e a síntese de proteínas da matriz extracelular. Algumas destas moléculas podem ser liberadas lentamente através de cápsulas poliméricas e podem estimular o crescimento do tecido danificado. Essas biomoléculas permitem também melhorar e orientar a organização e neovascularização de tecidos lesados[4]. Caso a superfície de um biomaterial não seja biorreconhecida pelas células, haverá imediatamente a sinalização para o desenvolvimento de um processo inflamatório resultando na formação de uma cápsula fibrosa que por sua vez isolará o biomaterial do corpo. Caso ocorra o biorreconhecimento, haverá a possibilidade de interação da superfície com células que poderão proliferar e estimular a regeneração dos tecidos, fixação de implantes e provocar menor grau de inflamação[27]. 2.2.1 Dinâmica celular: adesão, proliferação e migração celular Quando se trata de regeneração de órgãos e tecidos, torna‑se fundamental o estudo do comportamento e diferenciação celular induzidos pela estrutura, composição e presença de elementos biológicos dos arcabouços, para aperfeiçoar os substratos e avançar em técnicas de cultivo celular, que possam permitir a reprodução dos tecidos e órgãos em toda sua complexidade[9]. A interação celular com o substrato deve ser semelhante ao de condições in vivo. Idealmente as características físico-químicas dos arcabouços devem mimetizar a matriz extracelular e esta, por sua vez tem grande influência sobre a migração, proliferação e diferenciação das células[18,31]. Normalmente, para que ocorra uma boa interação arcabouço-célula é necessário que se estabeleça inicialmente a adesão celular ao substrato. O processo de adesão está relacionado com a adsorção de proteínas adesivas e é direcionado por características do substrato como hidrofilicidade, hidrofobicidade e disposição de cargas elétricas de superfície[17,31]. Somente após de aderidas, as células iniciam seu processo de espalhamento, proliferação e produção de matriz extracelular[32]. A matriz extracelular (MEC) animal é formada por vários tipos de moléculas (fibronectina, lamininas, elastina, proteoglicanas, colágenos) as quais associam-se entre si formando redes ou malhas. Estas, por sua vez, constituem estruturas morfofisiológicas bem definidas e conhecidas por lamina basal e tecido conjuntivo. Enquanto a algumas macromoléculas de MEC, como os colágenos, atribui-se função estrutural, a outras, como as GAGs, associa-se hidratação[33-35]. Malhas moleculares representam os ambientes naturais da maioria das células e tecidos de mamíferos. Assim sendo, células devem ser capazes de reconhecer e responder a cada uma das dimensões das malhas: química (tipos moleculares), mecânica (tensão e rigidez) e arquitetônica (forma e estrutura dos arranjos moleculares), além de também responderem à 363

Nascimento, M. H. M., & Lombello, C. B. hidrodinâmica e à físico-química inerentes à hidratação do meio. Tanto a formação quanto a funcionalidade do tecido resultante estarão, dessa forma, diretamente relacionadas à mecanoquímica da malha molecular onde as células formadoras do tecido em questão se encontram[36,37]. A adesão celular a um biomaterial está relacionada a dois fenômenos diferentes: (1) fase de anexação, que ocorre rapidamente, envolve eventos como ligações físico-químicas entre as células e o material por forças iônicas e forças de van der Walls; (2) fase de adesão, que ocorre posteriormente e envolve diversas moléculas biológicas como proteínas de matriz extracelular, proteínas de membrana celular e do citoesqueleto, que interagem conjuntamente para induzir a transdução do sinal, promovendo a ação de fatores de transcrição e consequentemente regulando a expressão gênica. A adesão celular é mediada por diferentes tipos de proteínas receptoras transmembrana conectadas ao citoesqueleto das células. Os aspectos da dinâmica da adesão celular necessitam de regulação destes receptores de adesão celular, que se encontram na superfície da célula. Este fenômeno é crucial para a união de células individuais em tecidos tridimensionais de animais[38]. Os sítios de adesão entre as células e o substrato são chamados de contatos focais ou placas de adesão, sendo a distância de união entre ambos em torno de 10-20 nm. As faces externas dos contatos focais apresentam proteínas receptoras específicas como as integrinas. Na face interna, proteínas como as talinas, paxilinas, vinculinas e tensinas são conhecidas por mediarem as interações entre filamentos de actina e integrinas. A formação de contatos focais geralmente ocorre em células com baixa motilidade e pode ser produzida in vitro através de proteínas da matriz extracelular como as fibronectinas ou vitronectinas[39]. As comunicações intercelulares podem ocorrer através da troca direta de íons via “gap junctions”, também denominadas de junções comunicantes, ou através de sinais produzidos pela ação das caderinas. Junções aderentes e desmossomos promovem uma ancoragem entre as células vizinhas. Já a adesão entre célula e substrato é realizada, preferencialmente, pela família das integrinas, que é composta por 22 heterodímeros de 2 tipos de sub-unidades e, ligadas não covalentemente entre si. Essa diversidade de estruturas ocorre devido às várias possibilidades de ligantes (colágeno, laminina, fibronectina, osteopontina, vitronectina, sialoproteína, trombospondina)[38]. A migração celular requer uma integração entre célula, substrato e citoesqueleto. Primeiramente, as células desenvolvem uma protrusão formando o lamelipódio e, em seguida, utilizam as interações adesivas para gerar tração e energia para o movimento. Por último, ocorre a liberação dos pontos de adesões, seguida pelo destacamento e retração. As integrinas são envolvidas nessa migração celular[38]. Mudanças nas formas dos tecidos, por exemplo, durante o desenvolvimento ou remodelação, frequentemente envolvem migrações celulares extensivas. As células podem migrar individualmente ou como uma parte aderente do tecido, acompanhando mudanças morfogenéticas nos tecidos. Para que a migração celular seja entendida como um processo integrado há a necessidade de pesquisas das propriedades físicas e químicas dos componentes celulares 364

que trabalham juntos como um sistema dinâmico, incluindo sua termodinâmica, cinética e características mecânicas, pois a migração é um processo coordenado tanto espacialmente quanto temporalmente[40]. Na Engenharia de Tecidos, a migração celular se torna crucial quando se trata de colonização dos arcabouços. Lo et al., (2000) mostraram em seus trabalhos que as células apresentam diferentes morfologias e taxas de motilidade de acordo com a rigidez, flexibilidade e deformação mecânicas do substrato[41].

2.3 Hidrogéis Devido à capacidade de simular a natureza da maioria dos tecidos moles, os hidrogéis são biomateriais altamente atraentes para o desenvolvimento de análogos sintéticos da MEC. Essas estruturas de cadeias poliméricas reticuladas possuem alto teor de água e fácil transporte de oxigênio, nutrientes e resíduos. Além disso, muitos hidrogéis podem ser facilmente modificados para apresentar ligantes de adesão celular, viscoelasticidade e degradabilidade[34]. Os hidrogéis são materiais poliméricos que podem ser usados como biomateriais, pois apresentam uma estrutura tridimensional e importante capacidade de intumescimento em água ou fluidos biológicos, que permitem a inoculação celular sobre estes arcabouços. A massa de água contida em um hidrogel após intumescimento pode variar de 10 a milhares de vezes de sua massa inicial[42]. Para que possam ser utilizados como arcabouços, os hidrogéis poliméricos devem sofrer processo de reticulação. Este processo pode ser realizado por métodos químicos, envolvendo ligações covalentes entre cadeias poliméricas, ou métodos físicos, como interações iônicas e interações por cristalização, que envolvem forças e interações intermoleculares[9,42,43]. Para a preservação estrutural do hidrogel no seu estado hidratado é necessário a formação de ligações intermoleculares que resultem no balanço entre o padrão de densidade de reticulação e capacidade de intumescimento[42]. Alguns protocolos de reticulação têm sido utilizados com êxito, permitindo que hidrogéis poliméricos sejam aplicados em Engenharia de Tecidos, e garantindo as suas propriedades de biocompatibilidade, biodegradação e bioatividade[44]. Estes retículos são formados por ligações intermoleculares de modo que evitam a dissolução das cadeias poliméricas e contêm domínios ou grupos hidrofílicos que são hidratados em meio aquoso para formar a estrutura tridimensional do hidrogel[43]. Os hidrogéis podem ser classificados de diversos modos. Quanto ao método de formação das cadeias, os hidrogéis podem ser classificados em homopolímeros, quando são utilizados apenas um tipo de monômero; copolímeros, quando são utilizados mais de um tipo de monômero; polímeros interpenetrantes, quando as cadeias poliméricas de um dado hidrogel penetram e se emaranham com as cadeias de um outro hidrogel, formando blendas de hidrogéis. Quanto à carga iônica, os hidrogéis podem ser classificados em neutros, quando os meros do hidrogel não apresentam grupamentos ionizávei, poli(metil metacrilato), por exemplo; catiônicos, quando os meros do hidrogel apresentam grupamentos capazes de formação de cátions por variação do pH do meio reacional; e aniônicos, quando os meros do hidrogel Polímeros, 26(4), 360-370, 2016

Hidrogéis a base de ácido hialurônico e quitosana para engenharia de tecido cartilaginoso apresentam grupamentos capazes de formação de ânions por variação do pH do meio reacional[45]. A capacidade dos hidrogéis de se ligar com moléculas de água surge principalmente devido à presença de grupos hidrofílicos tais como grupos aminos, carboxilas e hidroxilas. Esta propriedade dos hidrogéis é denominada grau de intumescimento que é diretamente proporcional à quantidade de água absorvida pelo hidrogel[42]. Os hidrogéis utilizados para a cultura de células podem ser formados a partir de uma vasta gama de materiais naturais e sintéticos, oferecendo um amplo espectro de propriedades mecânicas e químicas. Hidrogéis naturais para a cultura de células são tipicamente formados de proteínas e componentes da matriz extracelular, como colágeno, fibrina, AH, bem como de materiais de outras fontes biológica, tais como QUI, alginato ou de fibrilas de seda. Uma vez que são derivados de fontes naturais, estes hidrogéis são inerentemente biocompatíveis e bioativos[46]. Eles também promovem muitas funções celulares, devido a presença de fatores endógenos, o que pode ser vantajoso para a viabilidade, proliferação e desenvolvimento de muitos tipos de células[34]. Por outro lado, os hidrogéis podem ser formados por moléculas sintéticas, tais como poli (etileno glicol) (PEG), poli (álcool vinílico) e poli (2-hidroxi-etil-metacrilato)[34]. 2.3.1 Ácido hialurônico O AH é um hidrogel atraente para a fabricação de arcabouços artificiais para engenharia tecido porque é biocompatível, biodegradável, bioativo, não-imunogénico e não-trombogênico[7]. Trata-se de um polissacarídeo linear de alta massa molar pertencente à família dos GAGs e é composto por unidades dissacarídicas polianiônicas de ácido D-glicurônico (GlcUA) e N-acetilglicosamina (GlcNAc) unidas alternadamente por ligações β(1→3) e β(1→4)[47]. O AH produzido comercialmente é obtido de materiais ou estruturas de origem animal e/ou de bactérias, através do isolamento direto ou da fermentação. O AH está presente em todos os vertebrados e também na cápsula de algumas cepas de Streptococcus sp., mas está ausente em fungos, plantas e insetos[48]. O AH é encontrado principalmente na matriz do tecido conjuntivo e é produzido por células de origem mesenquimal, com função de organizar os elementos da matriz extracelular (MEC)[49]. O AH é sintetizado por três tipos de enzimas sintases (AHS1, AHS2 e AHS3) que estão localizadas na membrana celular, e é imediatamente secretado para a MEC onde interage com os demais constituintes para fornecer suporte mecânico ao tecido[50]. No ser humano, este mucopolissacarídeo está presente no líquido sinovial, na pele, nos tendões, no humor vítreo e no cordão umbilical. Na pele, bem como nas cartilagens, a função do AH é ligar-se à água, mantendo a tonicidade e a elasticidade desses tecidos. No líquido sinovial, sua função básica é o de manter um suporte protetivo e lubrificante para as células das articulações. No olho, atua como componente natural dos tecidos oculares, tais como córnea, esclera e corpo vítreo[44,51]. Em condições fisiológicas, o AH apresenta abundantes cargas negativas (devido a presença de radicais carboxila e N-acetila), podendo assim absorver grandes quantidades de Polímeros, 26(4), 360-370, 2016

água, formando uma estrutura hidratada em forma de rede, controlando o transporte de água e restringindo o movimento de agentes patogênicos, proteínas plasmáticas, e proteases[50]. O AH é também um composto reconhecido por sua importância em controlar e regular o comportamento das células e a interação célula-célula, especialmente no decurso do reparo de tecidos. Este último inclui a ativação e modulação das respostas imunes, promoção de angiogênese, bem como a proliferação e migração celular[52]. Desta maneira, o AH interage com receptores de superfície celular, por exemplo: CD44, ICAM-1 e RHAMM, para ativar várias vias de sinalização, tais como c-Src, Ras e proteínas quinase ativadas por mitógenos (MAPK). Estas vias regulam várias funções celulares, incluindo a adesão celular, rearranjo do citoesqueleto, migração, proliferação e diferenciação[52,53]. Especificamente, os receptores RHAMM e CD44 têm sido sugeridos como os principais fatores envolvidos na motilidade celular. Ambos os receptores de AH são bem conhecidos por estarem envolvidos no reparo de feridas e tem sido demonstrado em numerosos estudos que a regulação da motilidade celular, contribui para uma variedade de doenças, incluindo artrite e câncer[54]. O AH pode ser rapidamente degradado na MEC por espécies de oxigênio reativas ou pelas enzimas hialuronidases, e o tempo de meia-vida in vivo pode variar de horas a dias, dependendo do tipo de tecido[53]. Devido a sua biocompatibilidade, o AH vem sendo utilizado como biomaterial em diversas aplicações, as quais são divididas em cinco grandes grupos: viscocirurgia – para proteger tecidos delicados e fornecer espaço durante as manipulações cirúrgicas ou em cirurgias oftalmológicas; “viscoaugmentation” ou aumento de volume – para preencher e aumentar os espaços nos tecidos, como na pele, nos músculos esfíncter, nos tecidos vocais e na faringe; viscosseparação – para separar a superfície de tecidos conectivos traumatizados por processos cirúrgicos ou lesões, a fim de evitar adesões e formação excessiva de cicatrizes; viscossuplementação – para substituir ou suplementar fluidos de tecidos, como a substituição do fluido sinovial em artrites dolorosas, e para aliviar a dor; viscoproteção – para proteger superfícies de tecidos saudáveis, feridos ou lesionados, de securas ou agentes nocivos do ambiente, e para promover a cicatrização dessas superfícies[55]. Na medicina clínica o AH é usado como um marcador diagnóstico para várias doenças, como câncer, artrite reumatóide, patologias hepáticas e suplementação de fluido sinovial debilitado em pacientes com artrite. O AH também é usado em certas cirurgias oftalmológicas, na reconstrução de tecidos moles e em revestimentos hidrofílicos para dispositivos médicos. Além disso, o AH é utilizado em cosméticos devido à sua alta capacidade de retenção de água e em sistemas de liberação modificada de fármacos, devido à sua biodegradabilidade[44,48,56]. 2.3.2 Quitosana A quitina é um polissacarídeo nitrogenado composto por ligações β(14) 2 acetamino-2-desoxi- β -D-glicose, geralmente derivada do exoesqueleto de animais marinhos, tais como caranguejo, camarão, lagosta e krill. Estima-se que 2,3 milhões de toneladas de quitina são produzidas como 365

Nascimento, M. H. M., & Lombello, C. B. resíduos da indústria de alimentos a cada ano tornando-se um material econômico e renovável. No entanto, a quitina é considerada quimicamente inerte e é insolúvel em água e solventes orgânicos. A N-desacetilação da quitina conduz ao seu principal derivado, a QUI. O grau de desacetilação é um fator importante para a determinação das características da QUI[57]. A QUI é um polissacarídeo natural, estruturalmente similar às GAGs, atóxica e bioabsorvível[2]. Devido à sua biocompatibilidade e biodegradabilidade, a QUI tem sido amplamente aplicada na entrega de drogas, terapia gênica, tratamento de águas, produção de cosméticos, aditivos alimentícios, membranas semipermeáveis e no desenvolvimento de biomateriais para a Engenharia de Tecidos[58,59]. Como citado anteriormente, este biomaterial polimérico é comumente obtido por desacetilação de quitina, e seus meros permanecem unidos por ligações β(14). A presença de grupamentos amina na composição da QUI é responsável por sua natureza policatiônica em soluções ácidas. Sua natureza catiônica é responsável pelas interações eletrostáticas com GAGs, proteoglicanos e demais moléculas carregadas negativamente, tanto presentes na matriz extracelular, quanto na superfície celular[57]. Como um polímero natural, a QUI possui propriedades favoráveis em aplicações na Engenharia de Tecidos. Foi demonstrado que este tipo de substrato é capaz de interagir com células vivas, sem apresentar sinais de citotoxicidade ou desencadear uma resposta imunitária, O estudo realizado por Nwe et al.[60], mostrou que o número de células aderidas no arcabouço de QUI foi altamente dependente do grau de desacetilação e o tipo da linhagem celular utilizada. Menor grau de desacetilação favorece a formação de poros menores, desta forma é possível obter uma melhor resistência mecânica, absorção moderada de água e maior atividade celular comparado com arcabouços produzidos com altos graus de desacetilação[60]. A importância da desacetilação se dá por conta da limitada solubilidade da quitina[61], o preparo de filmes desse material se da melhor na forma de QUI que é solúvel em soluções aquosas de vários ácidos (ácido acético e clorídrico são as mais utilizadas). A QUI também apresenta a característica de biodegradabilidade. Quitinases, quitosanases e lisozimas em geral, degradam a QUI em oligômeros e monômeros de QUI e, finalmente, em um amino-açúcar comum, N – acetilglucosamina que, em seguida, entra no ciclo de glicoproteína e, eventualmente, é excretada na forma de dióxido de carbono. A taxa de degradação de QUI está relacionada com o peso molecular e grau de desacetilação[57]. 2.3.3 Aplicação de hidrogéis a base de ácido hialurônico e quitosana na Engenharia de Tecido de cartilagem O tecido cartilaginoso é um tipo especializado de tecido conjuntivo de consistência semi-rigida, avascular, sem inervação, contendo muita MEC esparsamente povoada por células (condrócitos). A matriz cartilaginosa é constituída por colágeno, ou colágeno mais elastina, em associação com macromoléculas de proteoglicanos, AH e diversas glicoproteínas. Os condrócitos presentes no tecido são altamente especializados e sua principal função é conferir as propriedades biomecânicas ao tecido, pela síntese dos 366

componentes da matriz extracelular[33]. Existem várias metodologias sendo empregadas na tentativa de reparar lesões focais da cartilagem articular. Tais metodologias incluem tratamentos cirúrgicos que vão desde métodos de estimulação da medula óssea como desbridamento, perfurações múltiplas, abrasões, microfaturas, até métodos biológicos modernos como transplantes periosteais e pericondrais, transplante autólogo de condrócitos e enxertos autólogos osteocondrais[2]. Vários destes métodos biológicos encontram‑se associados à Engenharia de Tecidos[62-64]. Como in vivo, os tecidos projetados in vitro devem fornecer transporte de nutrientes, estabilidade mecânica, coordenação de processos multicelulares e um microambiente celular que preserve a estabilidade fenotípica das células[65]. Dentre os hidrogéis aplicáveis à engenharia de tecido cartilaginoso, o AH é um GAGs presente na matriz extracelular do tecido cartilaginoso, que fornece um microambiente capaz de manter a homeostase e preservar o estado diferenciado dos tecidos. O AH é uma das moléculas presentes no mesênquima cujo papel é um dos mais fundamentais nas primeiras fases da condrogênese. Além disso, o AH é capaz de influenciar condrócitos a desencadear uma sofisticada sinalização de uma rota metabólica que melhorar as suas funções celulares[5]. Interações celulares entre condrócitos e o AH ajudam a organizar a MEC da cartilagem e manter as proteoglicanas no interior da cartilagem. O AH também estimula a diferenciação condrogênica de células-tronco mesenquimais e a produção de proteoglicanas através da sua interação com os condrócitos[53]. Estudos demonstraram que os condrócitos cultivados em um suporte de AH expressam colágeno tipo II e agregan e regulam negativamente a produção de colágeno tipo I. Mais recentemente, foi também demonstrado que o AH cria um ambiente em que os condrócitos regulam negativamente a expressão de fatores catabólicos e da apoptose. Estes resultados demonstraram uma capacidade potencial do AH em prevenir a cartilagem contra danos, além da possibilidade de ter benefícios no tratamento inicial em lesões osteoartríticas[6]. O uso de hidrogel composto de AH e QUI também tem sido investigado em aplicações de Engenharia de Tecidos de cartilagem, com resultados promissores. A natureza catiônica da QUI em meio aquoso ácido é a principal responsável pelas interações eletrostáticas com GAGs aniônicos, proteoglicanos e outras moléculas carregadas negativamente. Esta propriedade é de grande interesse, visto que um grande número de citocinas/fatores de crescimento está ligado aos GAGs (por exemplo, o AH). Um arcabouço incorporando um complexo de quitosana-glicosaminaglicana pode reter e concentrar fatores secretados através da colonização de células[66]. A associação de AH e QUI geralmente leva à formação de um complexo de polieletrólito (PEC)[67]. PECs são preparados geralmente através da mistura de dois polímeros de cargas opostas, o que leva a auto-montagem devido ao aumento de entropia[68]. Porém, estes hidrogéis compostos não possuem as propriedades de homogeneidade, transparência e estabilidade a curto prazo, que são necessárias no caso de uma formulação injetável. Assim, sistemas mais complexos Polímeros, 26(4), 360-370, 2016

Hidrogéis a base de ácido hialurônico e quitosana para engenharia de tecido cartilaginoso têm sido propostos para contornar estas limitações, nos quais AH e QUI são ligados através de uma reticulação química com agentes tais como carbodiimida, o glutaraldeído, genipina, e di-hidrazida adica[68,69]. Porém, muitas vezes o uso de agentes de reticulação química tem sido vistos como um obstáculo no preparo de hidrogéis, devido à sua toxicidade para células[70,71]. Uma alternativa para a associação de AH e QUI, sem a adição de um agente reticulante, é por meio de uma oxidação. Neste caso, o AH é oxidado, usando o periodato, para formar dialdeído (HDA) ou ácido hialurônico oxidado (A-AH). O HDA é posteriormente associado à quitosana por meios de reações de base de Schiff entre os grupos amino da quitosana e grupos aldeído no HDA. Estas modificações químicas, portanto, oferecem a vantagem da formação do gel e do arcabouço sem o uso de agentes de reticulação, podendo assim criar um microambiente com biocompatibilidade melhorada para a regeneração de tecidos[69,72]. O estudo de Muzzarelli et al., (2012) [73] demonstrou que esta blenda de AH e QUI é propícia para propagação e desenvolvimento de condrócitos que, localizados no interior do arcabouço, se re-diferenciaram e sintetizaram cartilagem do tipo hialina. A produção de matriz extracelular também é influenciada pelo arcabouço[5,73]. Estudos comparativos demonstram que a adesão e proliferação celulares são mais expressivos quando as blendas são utilizadas como arcabouços, se comparados aos polímeros não associados, AH e QUI[74]. Uma das vantagens do uso de blenda é a possibilidade de utilização clínica na forma injetável, para cirurgias minimamente invasivas. O estudo de Walker e Madihally[75] demonstraram que a incorporação de AH à hidrogéis à base de QUI melhorou a integridade estrutural do hidrogel injetável em relação a outras formulações. O hidrogel injetável, desenvolvido no trabalho de Tan et al.[69], conseguiu preservar o fenótipo de condrócitos e permitiu a adesão dessas células. Yan et al. [8] também reportaram a maior adesão de condrócitos nas blendas, mostrando que a QUI melhorou a bioestabilidade e a biocompatibilidade do biomaterial. Outra estratégia promissora para a regeneração do tecido cartilaginoso tem sido a combinação da Engenharia de Tecidos com sistemas de entrega de genes terapêuticos. Lu et al.[76] desenvolveram e avaliaram um arcabouço a base de QUI embutidos com nanopartículas de AH/QUI/pDNA (plasmídeo de DNA) que codificam o gene do fator de transformação de crescimento β1 (TGF-β1). O TGF-β1 é conhecido por promover a proliferação e diferenciação de condrócitos e consequentemente aumentar a síntese de componentes da MEC[29]. Os condrócitos cultivados neste arcabouço mostraram alta proliferação e aumento da expressão de TGF-β1[76].

3. Conclusão Arcabouços derivados de polissacarídeos naturais são promissores em aplicações de Engenharia de Tecidos, em que se assemelham aos glicosaminoglicanos da matriz extracelular. Nos tecidos, a MEC possui função mecânica e sinalizadora de diferentes comportamentos celulares. Os arcabouços ao mimetizar a MEC, devem interagir com Polímeros, 26(4), 360-370, 2016

as células de maneira específica e direcionada a fim de obter comportamentos específicos, relacionados à adesão, espalhamento, crescimento e diferenciação celulares. O desenvolvimento de arcabouços a base de hidrogéis poliméricos é objeto de vários estudos, uma vez que estes biomateriais apresentam propriedades cruciais para esta aplicação, tais como biocompatibilidade e biodegradabilidade. Dentre os arcabouços aplicáveis à engenharia de tecido cartilaginoso o hidrogel de AH e QUI tem sido investigado e apresentam resultados promissores. O uso da blenda composta pelos hidrogéis citados é propícia à adesão, crescimento, propagação diferenciação e desenvolvimento de condrócitos, além de estimular a produção de MEC. Estas características são de suma importância no âmbito da regeneração de tecidos e proporcionam, portanto, uma oportunidade potencial para usar hidrogéis compostos por AH e QUI em aplicações de Engenharia de Tecidos de cartilagem.

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