TOPIC ION EXCHANGE CHROMATOGRAPHY IN FOOD ANALYSIS & INJECTABLE NANOPARTICLES FOR DRUG DELIVERY

HCM UNIVERSITY OF TECHNOLOGY FACULTY OF CHEMICAL ENGINEERING --&&&--

ESSAY TO SUBJECT OF APPLICATION OF CHROMATOGRAPHIC METHODS IN FOOD ANALYSIS

TOPIC ION EXCHANGE CHROMATOGRAPHY IN FOOD ANALYSIS

Supervisor:

Dr. Nguyễn Thị Lan Phi

Performer:

NGUYỄN VĂN TÚ

- 51305919

Ho Chi Minh City, 2016

TABLE OF CONTENTS

INTRODUCTION ..................................................................................................... 3 CONTENTS............................................................................................................... 4

I. ION EXCHANGE CHROMATOGRAPHY (IEC or IC) ....................... 4 1. Ion exchange chromatography............................................................. 4 2. Classification ......................................................................................... 5 II. PRINCIPLE OF IC ................................................................................. 5 1. Net surface charge and pH ................................................................... 5 2. Principle in IC separation .................................................................... 7 2.1. Equilibration.................................................................................... 7 2.2. Sample application and wash........................................................... 7 2.3. Elution ............................................................................................. 7 2.4. Regeneration.................................................................................... 9 3. Resolution.............................................................................................. 9 4. IC system ............................................................................................. 10 III. PROS AND CONS OF IC ................................................................... 11 IV. APPLICATIONS ................................................................................. 11 CONCLUSION ........................................................................................................ 15 REFERENCES ........................................................................................................ 16

2

INTRODUCTION

There are a number of chromatographic methods utilized in food analysis, including Gas Chromatography, High Performance Liquid Chromatography, Size Exclusion

Chromatography,

etc.

Ion

Exchange

Chromatography

or

Ion

Chromatography is a technique rather commonly used in many fields in industry, and it is of many applications in food analysis in particular.

Together with the current developments of continuous detectors, Ion Chromatography is more and more useful in food analysis, especially corporated with modern chromatographic techniques. Although there have been only limited applications of Ion Chromatography in food industry, today it appears to be a hopeful technique as a simple, unexpensive method for food industry, also for other ones.

3

CONTENTS I. ION EXCHANGE CHROMATOGRAPHY (IEC or IC) 1. Ion exchange chromatograph chromatography Ion

exchange

chromatograph chromatography

is

a chromatography technique

that

separates ions and polar molecules moleculesbased based on their affinity to the ion exchanger.

Analytes often used in IC are large proteins, smallnucleotides, and amino acids. acids IC is often used in protein purification, water analysis, and quality control. The water watersoluble and charged molecules such as proteins, amino acids, and pe peptides bind to oppositely charged stationary phase by forming covalent bonds. The equilibrated stationary phase consists of an ionizable functional group where the targeted molecules of a mixture to be separated and quantified can bind while passing through the column.[1] The history of IC primarily began between 1935 1935-1950 1950 through the Manhattan project that applications and IC were significantly extended. IC was originally introduced by two English researchers, agricultural Sir Thompson and chemist J T Way. It was in the fifties and sixties that theoreti theoretical cal models were developed for further understanding and it was not until the seventies that continuous detectors we were utilized, giving the way to the development from low-pressure pressure to high high-performance chromatography. Not until 1975 was "ion chromatography" established as a name of a technique,, and was thereafter used as a name for marketing purposes.

Fig 1. Some critical timelines in the history of IC

4

2. Classification Acording to the analytes, IC is often categorized into two main types, namely Anion and Cation Chromatography. Cation exchange chromatography is used when the desired molecules to separate are cations, and an anion exchange chromatography is to separate anions meaning that the beads in the column contain positively charged functional groups to attract the anions.

Similarly, the stationary phase of IC (often called IC media or IC exchanger) is also divided into Anion and Cation Exchager. Anion Exchanger is used for Anion Chromatography whreas Cation Chromatography uses Cation Exchanger for separating the targeting molecules.

Fig 2. Commonly used IC media[2]

II. PRINCIPLE OF IC 1. Net surface charge and pH IC separates molecules on the basis of differences in their net surface charge.

Molecules vary considerably in their charge properties and will exhibit different degrees of interaction with charged chromatography media according to differences in their overall charge, charge density, and surface charge distribution. The charged groups within a molecule that contribute to the net surface charge possess different

5

pKavalues (acid ionization constant) depending on their structure and chemical microenvironment.

Since all molecules with ionizable groups can be titrated, their net surface charge is highly pH dependent. In the case of proteins, which are built up of many different amino acids containing weak acidic and basic groups, net surface charge will change gradually as the pH of the environment changes, that is, proteins are amphoteric. Each protein has its own unique net charge versus pH relationshipwhich can be visualized as a titration curve. This curve reflects how the overall net charge of the protein changes according tothe surrounding pH.[2]

Fig 3. Theoretical titration curves, showing how net surface charge varies with pH

IC takes advantage of the fact that the relationship between net surface charge and pH is unique for a specific analyte. In an IC separation, reversible interactionsbetween chargedmolecules and oppositely charged IC media are controlled in order to favor binding or elution of specific molecules and achieve separation. A certain molecule that has no net charge at a pH equivalent to its isoelectric point (pI)will not interact with a charged medium. However, at a pH above its pI, an analyte will bind to a positively charged medium or anion exchangerand, at a pH below its pI, ananalyte will bind to a negatively charged medium or cation exchanger. In addition to the ion exchange interaction, other types of binding can occur, but these effects are very small and mainly due to van der Waals forces and nonpolar interactions.

6

2. Principle in IC separation An IC medium comprises a matrix of spherical particles substituted with ionic groups that are negatively or positively charged. The matrix is usually porous to give a high internal surface area. The medium is packed into a column to form a packed bed.

The bed is then equilibrated with buffer which fills the pores of the matrix and the space among the particles. [2] 2.1. Equilibration

The first step is the equilibration of the stationary phase to the desired start conditions. When equilibrium is reached, all stationary phase charged groups are bound with exchangeable counterions, such as chloride or sodium. The pH and ionic strength of the start buffer are selected to ensure that, when sample is loaded, analytes bind to the medium and as many impurities as possible do not bind.

2.2. Sample application and wash

The second step is sample application and wash. The goal in this step is to bind the target molecules and wash out all unbound one. The sample buffer should have the same pH and ionic strength as the start buffer in order to bind all charged target molecules. Oppositely charged items bind to ionic groups of the IC medium, becoming concentrated on the column. Uncharged items, or those with the same charge as the ionic group, pass through the column at the same speed as the flow of buffer, eluting during or just after sample application, depending on the total volume of sample loaded.

2.3. Elution

When all the sample has been loaded and the column washed with start buffer so that all nonbinding molecules have gone out of the column, conditions are altered in order to elute the bound analytes. Most frequently, analytes are eluted by increasing the ionic strength (salt concentration) of the buffer or, occasionally, by changing the pH. As ionic strength increases the salt ions (typically Na+or Cl-) compete with the bound components for charges on the surface of the medium and one or more of the bound species begin to elute and move down the column. The molecules with the lowest net charge at the selected pH will be the first ones eluted from the column as ionic strength increases. Similarly, the components with the highest charge at a certain

7

pH will be most strongly retained and will be eluted thereafter. The higher the net charge of the target molecules, the higher the ionic strength that is needed for elution.

By controlling changes in ionic strength using different forms of gradient, components are eluted differently in a purified, concentrated form.

Fig. 4. Description of steps in IC separation

8

2.4. Regeneration

A final wash with high ionic strength buffer regenerates the column and removes any molecules still bound. This ensures that the full capacity of the stationary phase is available for the next run. The column is then re-equilibrated in start buffer before starting the next run.

Alternatively, conditions can be chosen to maximize the binding of contaminants to allow the target analytes to first pass through the column to be collected.

3. Resolution The resolution of an IC separation is a representation of the degree of separation between the peaks eluted from the column (the selectivity of the medium), the ability of the column to produce narrow, symmetrical peaks (efficiency) and, of course, the amount (mass) of sample applied. These factors depend upon practical issues such as matrix properties, binding and elution conditions, column packing, and flow rates.

Resolution (Rs) is defined as the distance between peak maxima compared with the average base width of the two peaks. Rscan be determined from a chromatogram, as shown in Figure 5.[2]

Fig. 5. Theoretical determination of Rs in IC separation[2]

9

Rsgives a measure of the relative separation between two peaks and can be used to determine if further optimization of the chromatographic procedure is necessary.

If Rs= 1.0 then 98% purity has been achieved at 98% of peak recovery, provided the peaks are symmetrical and approximately equal in size. Baseline resolution requires that Rs≼1.5. At this value, peak purity is 100% (Fig. 6).

Fig. 6. Separation result with different Rs, showing if further optimization is needed

4. IC system Similar to some other chromatography systems, IC has some fundamental components as the following figure:

Fig. 7. A typical IC system

10

The eluent generator is used to generate the proper buffer for analysis, follwed by the separation collumn with the charged stationary phase. The analysis data is obtained by combination with the detector connected to a screen.

III. PROS AND CONS OF IC IC is a very powerful separation technique that is used not only for preparative chromatography but also for analytical chromatography. However, like all other chromatography modes, IC does have some limitations.

One of the main disadvantages of IC is its buffer requirement: because binding to IC media is dependent on electrostatic interactions between analytes of interest and the stationary phase, IC columns must be loaded in low-salt buffers. For some applications, this restriction may require a bufferexchange step prior to IC analysis.

IC, unlike some other chromatography methods, also permits high flow rates, which in some cases can be crucial to the recovery of active protein. Finally, a limitation of weak ion exchangers is their pH dependence. When working outside oftheir optimal pH range, these resins rapidly lose capacity, andmore importantly, resolution as table below.[3] PROS

CONS

- Permits high flow rate

- Sample must be loaded at low ionic strength

- Concentrates samples

- Clusters of positively charged residues can cause a net negatively charged protein to bind a cation exchanger, and vice versa

- High yield

- Small changes in pH can greatly alter binding profile of IC resin

-Buffers are non-denaturing

- Particle size greatly influences resolution

IV. APPLICATIONS IC is a powerful technique in a number of field, such as environment analysis, water treatment, pharmaceutical and drugs analysis and food analysis. IC has been utilized in environment and water treatment which the charged resins was used to separate metallic ion from the natural resources. Though applications in

11

pharmaceutical, drugs and food analysis are less than two fields above, IC gradually becomes hopeful technique corporated to other chromatography methods. [4-6] To illustrate, a remarkable investigation conducted in 2005 by C. Gu´erinDubiard et al. separated useful proteins in hen egg white into separate fractions. Due to the dependence of IC upon pH, researchers changed the pH to get individual components that have the different pI value. This separation procedure was depicted as following figure:

Fig. 8. Procedure of hen egg white separation[7]

12

The analytical results and the confirmation of analytes was determined by HPLC to delight the separation efficiency and the accuracy of this method (Fig. 9).

Fig. 9. HPLC analysis of hen egg white separation, showing the method efficiency

13

Based upon the HPLC analysis, proteins in hen egg white was separated successfully with the high efficiency. This paper also showed the importance of changing pH in IC in which flexibly changing pH of buffer can completely purify the analytes of interest in a mixture.

14

CONCLUSION

Ion exchange chromatography is a technique that separates charged molecules from the mixture basing upon their net surface charge at specific pH of the surroundings. Anion exchange chromatography uses the anion exchagers formed by functionalized the stationary phase with cationic groups for separation of anion analytes. By contrast, cation exchage chromatography separates the cationic molecules from the mixture while they pass through the collumn filled by negatively charged matrices.

IC is a useful technique used in a wide number of fields, especially in environment and water treatment. In food industry, together with the developments of continuous detectors currently, IC has been utilized with other chromatographic methods for separation of charged molecules such as proteins, amino acids and peptides. Like other modes of chromatography, IC has advantages and disavantages which can be noted to enhance the efficiency and resolution of this method.

15

REFERENCES

1.

2.

3.

4.

5.

6.

7.

Wikipedia. Ion Chromatography. Available from: https://en.wikipedia.org/wiki/Ion_chromatography.

Healthcare, G., Ion Exchange Chromatography: Principles and Methods, GE Healthcare: GE Healthcare.

BioRad, Ion Exchange Chromatography: Applications & Technologies BioRad: BioRad. Jackson, P.E., Ion Chromatography in Environmental Analysis.

Encyclopedia of Analytical Chemistry, 2000: p. 2779–2801.

Rohrer, L.B.a.J.S., Application of IC for pharma and biological products.

A JOHN WILEY & SONS, INC., PUBLICATION, 2012.

MICHALSKI, R., Industrial applications of ion chromatography.

CHEMIK, 2014. 68: p. 478-485. al, C.G.e.-D.e., Hen egg white fractionation by ion-exchange chromatography. Journal of Chromatography A, 2005. 1090: p. 58-67.

16

Injectable nanoparticles for drug delivery

TABLE OF CONTENTS INTRODUCTION ..................................................................................................... 2 CONTENTS ............................................................................................................... 3 I. GENERALITY OF INJECTABLE ROUTES ...................................................... 3

1. Intravenous (IV) ....................................................................................... 5 2. Intramuscular (IM) .................................................................................. 5 3. Subcutaneous (SC).................................................................................... 5 II. BIOLOGICAL BARRIERS IMPOSED BY THE MONOCYTE PHAGOCYTIC SYSTEM (MPS) ............................................................................. 6

1. Entrapment: Phagocytosis ....................................................................... 6 2. Escape ....................................................................................................... 7 3. Targeting or Evasion ................................................................................ 8 4. Avoidance.................................................................................................. 8 III. FUNCTIONALITY FOR TARGETING DELIVERY...................................... 9

1. Coating Functionality ............................................................................... 9 2. External assistance in targeting ............................................................. 11 IV. TYPES OF INJECTABLE NANOMATERIAL CARRIERS ........................ 12

1. Liposomes ............................................................................................... 12 2. Polymeric based carriers ........................................................................ 13 3. Inorganic based carriers ........................................................................ 15 V. CLINICAL TRIALS........................................................................................... 16 CONCLUSION ........................................................................................................ 19 REFERENCES ........................................................................................................ 20

Performer: Nguyễn Văn Tú - PhD Student

Bach Khoa University

Injectable nanoparticles for drug delivery INTRODUCTION

Organic Nanoparticle Technology has been concerned than ever before recent years due to its application to drug delivery, common to a number of therapeutic areas and targets. Earlier researches on liposomes and emulsions were the examples of enhancements that drug delivery could confer on established agents such as doxorubicin and amphotericin. The disposition of nanoparticles was changed in vivo, but the drug molecular structure was not transformed. For broader applicability, nanoparticles have been sticked with additional features in order to enhance their ability to targeting organs. Unlike microparticulates, nanoparticulates are sufficiently small to avoid embolism related to intravenous (i.v) delivery, and can also be used for the less invasive parenteral routes.[1] A large proportion of i.v. drugs in development are antineoplastic agents or antiinflammatory compounds. While they are fewer in number, there is a need for improved antimicrobial agents as well, although many companies are exiting this area.

Opportunities for enhancement in these specific therapeutic areas will be considered from a biological barrier perspective. Additionally, medical benefits arising from the ability to target to specific organs will also be shown. The limitations of predicate dosage form platforms need noted, which define the opportunities of nanoparticulates to address unmet needs.[1]

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2 Bach Khoa University

Injectable nanoparticles for drug delivery CONTENTS I. GENERALITY OF INJECTABLE ROUTES The goal of drug therapy is to prevent, cure, or control various disease states.

To achieve this goal, adequate drug doses must be delivered to the target tissues so that therapeutic yet nontoxic levels are obtained. Pharmacokinetics examines the movement of a drug over time through the body. Pharmacological as well as toxicological actions of drugs are primarily related to the plasma concentrations of drugs. Thus, the clinician must recognize that the speed of onset of drug action, the intensity of the drug's effect, and the duration of drug action are controlled by four fundamental pathways of drug movement and modification in the body (Figure 1).

First, drug absorption from the site of administration (Absorption) permits entry of the therapeutic agent (either directly or indirectly) into plasma. Second, the drug may then reversibly leave the bloodstream and distribute into the interstitial and intracellular fluids (Distribution). Third, the drug may be metabolized by the liver, kidney, or other tissues (Metabolism). Finally, the drug and its metabolites are removed from the body in urine, bile, or feces (Elimination). This chapter describes how knowledge of these four processes (Absorption, Distribution, Metabolism, and Elimination) influences the clinician's decision of the route of administration for a specific drug, the amount and frequency of each dose, and the dosing intervals.[2]

Figure 1. Four processes of drug inside the body

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Injectable nanoparticles for drug delivery The route of administration is determined primarily by the properties of the drug (for example, water or lipidsolubility, ionization, etc.) and by the therapeutic objectives (for example, the desirability of a rapid onset of action or the need for longterm administration or restriction to a local site). There are two major routes of drug administration, enteral and parenteral. (Figure 2 illustrates the subcategories of these routes as well as other methods of drug administration.)[2]

Figure 2.Commonly used routes of drug administration. IV = intravenous; IM = intramuscular; SC = subcutaneous

Enteral administration, or administering a drug by mouth, is the simplest and most common means of administeringdrugs. When the drug is given in the mouth, it may be swallowed, allowing oral delivery, or it may be placed underthe tongue, facilitating direct absorption into the bloodstream.

The parenteral route introduces drugs directly across the body's barrier defenses into the systemic circulation orother vascular tissue. Parenteral administration is used for drugs that are poorly absorbed from the GI tract (forexample heparin) and for agents that are unstable in the GI tract (for example, insulin). Parenteral administration is also used for treatment of unconscious patients and under circumstances that require a rapid onset of action. Inaddition, these routes have the highest bioavailability and are not subject to first-pass metabolism or harsh GIenvironments. Parenteral

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Injectable nanoparticles for drug delivery administration provides the most control over the actual dose of drug delivered to the body. However, these routes are irreversible and may cause pain, fear, and infections.

The three major parenteralroutes are intravascular (intravenous or intra-arterial), intramuscular, and subcutaneous (see Figure 1.2). Eachroute has advantages and drawbacks.[2] 1. Intravenous (IV) Injection is the most common parenteral route. For drugs that are not absorbed orally, suchas the neuromuscular blocker atracurium, there is often no other choice.

With IV administration,the drug avoids the GI tract and therefore, first-pass metabolism by the liver. Intravenous delivery permits arapid effect and a maximal degree of control over the circulating levels of the drug. However, unlike drugs inthe GI tract, those that are injected cannot be recalled by strategies such as emesis or by binding to activatedcharcoal. Intravenous injection may inadvertently introduce bacteria through contamination at the site ofinjection. IV injection may also induce hemolysis or cause other adverse reactions by the too-rapid delivery ofhigh concentrations of drug to the plasma and tissues. Therefore, the rate of infusion must be carefullycontrolled. Similar concerns apply to intra-arterially injected drugs.[2] 2. Intramuscular (IM) Drugs administered IM can be aqueous solutions or specialized depot preparations often asuspension of drug in a nonaqueous vehicle such as polyethylene glycol. Absorption of drugs in an aqueoussolution is fast, whereas that from depot preparations is slow. As the vehicle diffuses out of the muscle, thedrug precipitates at the site of injection. The drug then dissolves slowly, providing a sustained dose over anextended period of time. An example is sustained-release haloperidol decanoate, which slowlydiffuses from the muscle and produces an extended neuroleptic effect.

3. Subcutaneous (SC) This route of administration, like that of IM injection, requires absorption and is somewhatslower than the IV route. Subcutaneous injection minimizes the risks associated with intravascular injection.[Note: Minute amounts of epinephrine are sometimes combined with a drug to restrict its area of action.Epinephrine acts as a local vasoconstrictor and decreases removal of a drug, such as lidocaine, from the site

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Injectable nanoparticles for drug delivery ofadministration.] Other examples of drugs utilizing SC administration include solids, such as a single rod containing the contraceptive etonogestrel that is implanted for long-term activity, and alsoprogrammable mechanical pumps that can be implanted to deliver insulin in diabetic patients.[2] II.

BIOLOGICAL

BARRIERS

IMPOSED

BY

THE

MONOCYTEPHAGOCYTIC SYSTEM (MPS) Based upon an understanding of compromised vasculature, the requirements of a drug delivery system intended for targeting to sites of tumor, infection, or inflammation can be specified. There is an upper limit placed upon the size of the particle, permitting diffusion through the vascular pores. The range of pore sizes is 300–700 nm, depending upon the tumor type, and therefore targeting particles should be substantially smaller, preferably <250 nm. The particles should be designed to target the pores rather than suffer less productive competitive encounters, the major one being that of entrapment by the monocyte phagocytic system (MPS).[1] 1. Entrapment: Phagocytosis The MPS system consists of fixed macrophage cells in key tissues, such as liver, kidney, lung, bone marrow, and spleen, as well as circulating monocytes, macrophages, and PMN cells. These are designed to rid the body of bacterial, viral, and particulate waste. The first step in the MPS removal process involves deposition of specific circulating blood proteins onto the particle, termed

opsonization, which

subsequently signal receptors on the macrophages and PMN for particle uptake (Figure 3).

Figure 3. The entrapment of Phagocytosis towards particulates

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Injectable nanoparticles for drug delivery Following opsonin docking on the receptors, an intracytoplasmic process is activated, reorganizing actin filaments, causingthe extension of pseudopodia to project from the phagocyte, surrounding the particle. The pseudopodia follow the contours of the particle as guided by further receptor docking onto the opsonized particle.

Provided the particle is smaller than approximately 8 mm, the spreading pseudopodia will eventually meet, totally engulfing the particle. The particle is then encased in an intracytoplasmic vacuole, termed a phagosome, formed from a remnant of the spreading pseudopodia.

While this process of phagocytosis is applicable to particles as small as 500 nm, a similar receptor-mediated endocytosis is more generally available to many different kinds of cells. This extends to particles as small as 100 nm and robably smaller. Non– receptor-mediated pincocytosis also becomes more prominent as particle size decreases from 1100 down to 100 nm.[1] 2. Escape Over the course of 15–30 minutes, the pH of the phagosome decreases from 7.4 to 4–5, as digestive enzymes are also added by docking vacuoles. Eventually, the phagosome unites with a lysosome, emptying its contents into the low pH environment. If the particle is not metabolizable or soluble, it will remain in the phagocyte. There are several ways in which phagocytized particles may escape the lysosome to enter the cytoplasm, and from there, the extracellular milieu. If the pH– solubility characteristic of the particle is such that it simply dissolves in the low pH environment of the lysosome, then the particle will dissolve.

If additionally, the solubilized constituents are soluble in phospholipid membranes,they may then dissolveinto the lysosomal membrane and enter the cytoplasm, diffusing down a concentration gradient. By the same process, the dissolved constituents may dissolve into the cytoplasmic membrane and diffuse intotheextracellularspace.Itraconazolenanosuspensionexhibits this behavior, and is able to vacate the phagolysosomal compartment, as from a depot, to provide sustained release to thesystemiccirculation.

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Injectable nanoparticles for drug delivery Alternatively,theparticlecoating may feature endosomolytic agents, which cause the lysosomal membrane to rupture, thus emptying the contents of the lysosome, including the particle, into the cytosol.[1] 3. Targeting or Evasion Depending on the pharmacokinetic and targeting needs, nanoparticulate dosage forms may be engineered to either target or evade the MPS. Targeting may be accomplished passively, simply by ensuring that the nanoparticulate remains intact to be phagocytized minutes after i.v. infusion. Alternatively, targeting motifs may be intentionally added to the coating of the particle, for the purpose of actively docking with particular macrophage receptors, thus triggering phagocytosis. Evasion of the MPS is most commonly performed by inhibiting the initial opsonization process. This is accomplished by coating the nanoparticles with a molecular layer that prevents deposition of the opsonizing proteins. The result is a significantly prolonged circulation time, than would otherwise occur. This affords sufficient time for the particle to encounter and diffuse through vascular pores, resulting in higher ratios of drug concentration in sites of tumor, infection, or inflammation, relative to normal tissue. This increases the therapeutic index by increasing local site efficacy and decreasing systemic toxicity.[1] 4. Avoidance Optimization of coating for minimizing MPS uptake has been exceedingly well studied, and utilizes predominantly hydrophilic polymers that are attached by various means to the particle surface. There is precedent for this from nature, where a strain of Pseudomonas aeruginosa is known to elaborate a viscous polyuronic acid polysaccharide, which interferes with phagocytosis by virtue of its hydrophilicity. The coating most often used in drug delivery applications features polymersof ethylene oxide. These may be adsorbed onto preexisting nanoparticulates, by using triblock copolymers, containing a centralhydrophobic polyoxypropylene segment, flanked by hydrophilic polyoxyethylene chains on either side. The hydrophobicportion permits physical adsorption onto hydrophobic surfaces of nanoparticles enabling the hydrophilic chains to project into the aqueous medium. The steric barrier inhibitsopsonic protein deposition. Consistent with this concept, it hasbeen found that

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Injectable nanoparticles for drug delivery the hydrophilic chains should be sufficientlylong (98 or more units of ethylene oxide) to create a corona ofsufficient thickness to prevent protein deposition. And thehydrophobic section should be sufficiently long (greater than 67 propylene oxide units) to resist shear detachment followingadministration in the blood. Certain inconsistencieswith

the

brush-like

theory

have

been

raised,

namely

that

theexperimentally effective grafting density,polymer chainlength, and poly(ethylene oxide) (PEO) molecular weight aretoo low compared with required theoretical values.

It is arguedthat surface bonding is at least as important as steric barriereffects, as shown by studies with phenoxy-substituted dextran polymers.Despite success in this area, much remains to be done. [1] Thepolymers that have proved most effective for prolonging circulation time, poloxamine-908, poloxamer-407, etc., are notapproved for use in injectable drugs.

Furthermore, althoughthey work well with polystyrene model nanoparticles, poloxamers and poloxamines do not prolong circulation time for a widevariety of nanoparticles with more hydrophilic surfaces such as albumin and PLGA. For this reason graft copolymers, primarily involving poly(ethylene glycol) (PEG), have been studied.PEG coating employs the same ethylene oxide repeat unitfound to be effective in poloxamer, but is covalently bonded tothe polymer comprising the bulk of the nanoparticle. Becauseit is tethered to the surface of the nanoparticle it is thereforeexpected to avoid the desorption issues found with the freesurfactants. PEG– PLGA copolymer was found to extend thehalf-life of incorporated albumin from 14 minutes, found withnon-PEGylated PLGA nanoparticles, to 4.5 hours. Thesystematic variation of both components of the polymer wasstudied. The PEG moiety was shown to repel the depositionof the opsonizing protein complement, as shown withWestern blot using antiopsonin antibodies, but was less effective in repelling Immunoglobulin G (IgG).[1] III. FUNCTIONALITY FOR TARGETING DELIVERY 1. Coating Functionality One of the more elaborate examples of coated nanoparticleselegantly incorporates features designed to accomplish allof the drug loading, MPS avoidance,

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Injectable nanoparticles for drug delivery active targeting, endocytotic uptake, and endosomal escape processes. A cyclodextrincontaining polycation of imidazole was designed toelectrostatically complex with a catalytic oligonucleotide, aDNAzyme, forming sub-100-nm particles termed ‘‘polyplexes.’’ The positively charged particles can interact withthe negatively charged cell surface proteoglycans for endocytotic uptake. Further, imidazole had been demonstrated toenhance endosomal escape. However, neutralization of theexcess charge was required for minimizing nonspecificuptake, to enhance efficiency of active targeting. This wasaccomplished with addition of the anionic glutamate functionality to adamantane–PEG conjugates, which forms inclusioncomplexes with the exposed cyclodextrins (Figure 4).[1, 3]

Figure 4. Assembly of Polyplex fomulations.

(A)-Polyplex (B)-PEG-Polyplex (C)-Tf-PEG-Polyplex

The exposed PEGchains confer long circulation in biological fluids.

Becausetransferrin is often upregulated in rapidly growing cells,active targeting was considered by adding transferrin–PEG–adamantane conjugates. Biodistribution in an HT-29, hightransferrin uptake, tumor xenograft mouse model was followed subsequent to different routes of administration. Intraperitoneal injection indicated high levels remaining in theperitoneum; presumably mobility was limited by their

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Injectable nanoparticles for drug delivery size,even at 30–50 nm. Subcutaneous injection did not result influorescence outside of the injection site. But i.v. deliveryshowed high levels in tumor, liver and kidney, all organs

richin

transferrin

receptor

activity.

Polyplexes

delivered

by

i.v.

wereinternalized by the tumor cells.[3] 2. External assistance in targeting An alternate approach that has been explored to providetargeting functionality to nanoparticles is via the use of anexternal energy source. For example, Rudge et al.described a nanoparticulate system that was responsive toan external magnetic field.

The particles were comprised ofactivated carbon to allow adsorption and loading of drugand metallic iron to provide magnetically triggered targetingof the particles. Good loading efficiency could be obtained for anumber of drugs including doxorubicin, mitomycin C, methotrexate, and camptothecin (Figure 5).[4]

Figure 5.Administration of MTCs to patient

In vivo studies using magneticdoxorubicin particles showed that efficient targeting wasachieved by injecting the particles using an arterial catheter,and then homing the particles to a specific tissue, by using astrong magnetic field. In another study, a much higher concentration of mitoxantrone was obtained in the tumor area,by using only 50% and 20% of the normal dose by the use ofmagnetic drug targeting.

Ultrasound triggered drugdelivery has also been adopted to provide targeted release

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Injectable nanoparticles for drug delivery ofdrug to tumors. Nanoparticles and micelles accumulate intothe tumor as a result of passive targeting and the EPR effect.Ultrasound is then applied to trigger the release of the drugso that the entire drug load is delivered within the tumor (Figure 6).[5]

Figure 6. The four components of the ultrasound/micelle modality of drug targeting

IV. TYPES OF INJECTABLE NANOMATERIAL CARRIERS There are three main components to an effective drug delivery nanoparticle: the core material of the nanoparticle, the therapeutic payload, and surface modifiers.

Although a generalized structure does not accurately portray all nanomedicines, one may be used to aid in understanding the objective of each portion of a nanomedicine carrier. Nanomedicine carriers generally have the ability to load either hydrophobic or hydrophilic therapeutics. Thus, suitable carrier materials have to be thoughtfully selected for every therapeutic. However, some carrier materials have both hydrophobic and hydrophilic regions. These materials could be effectively used to design nanocarriers for delivery of multiple drugs. Additionally, the nanoparticle core material must be non-toxic, non-immunogenic, and should be easily eliminated from the body to avoid toxic accumulation and side effects. The core material must also possess a release mechanism for the therapeutic payload after the carrier has reached its destination.[6] 1. Liposomes Liposomes are composed of lipid or phospholipid molecules containing a hydrophilic head region and hydrophobic tail region that have aggregated together to form an enclosed bilayer particle with an aqueous center and lipid membrane (Figure 7).

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12 Bach Khoa University

Injectable nanoparticles for drug delivery

Figure 7. A type of liposomes

Liposomes have been receiving attention as therapeutic carriers for over 40 years and have been studied as carriers for anticancer drugs, antifungal drugs, analgesics, and gene therapies as well as for vaccines. They offer the ability to deliver both hydrophilic drugs (in the aqueous center) and lipid-soluble drugs (within the bilayer structure). This sort of therapeutic loading does not occupy surface functionality groups that may further be used to attach targeting ligands and/or biocompatibility agents such as PEG, chitosan, silk-fibroin, and polyvinyl alcohol (PVA). These agents create stealth liposomes—liposomes that avoid MPS uptake, thus having increased circulation times. Furthermore, the phospholipids being used to create the liposome may be changed or modified to customize the properties of the liposomal surface and membrane layer.[6] 2. Polymeric based carriers Polymer carriers offer a large versatility in both structure and physiochemical properties. A major reason for this versatility is the wide variety of monomers that may be used to form the polymer architectures. Some of the commonly used structures as injectable nanocarriers include polymersomes, dendrimers, and cyclodextrincontaining polymers (CDPs). Polymersomes are structurally similar to liposomes, but they are formed from amphiphilic block copolymers. Dendrimers are multi-branched

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Injectable nanoparticles for drug delivery polymer structures extending out from a core. CDPs are polymers that contain cyclodextrin molecules within the core structure or attached as side chains.[6] Furthermore, certain polymers contain chemical groups that have the ability to adapt accordingly to the current environment resulting in a change of properties in the overall polymer itself. These polymers are referred to as responsive or “smart polymers”. Some common environmental stimuli include pH, ionic strength, chemical agents, mechanical stress, temperature, electromagnetic radiation, and electric field.

Common corresponding changes include optical clarity, conductivity, surface chemistry, shape, permeability, mechanical properties, and phase separation. These corresponding changes in properties result in the release of therapeutics. Additionally, hydrophobic, hydrophilic, and electrostatic interactions are used between the polymers themselves, polymers and therapeutics, and polymers and attachment molecules to aid in targeting, biocompatibility, and to form more stable structures to increase the efficacy of the nanocarriers.[6]

Figure 8. Assembly of Polymersome, Dendrimer and Cyclodextrin

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Injectable nanoparticles for drug delivery 3. Inorganic based carriers Another material that is getting much attention in the field of nanomedicine is carbon nanotubes (CNTs). Although CNTs are not naturally water-soluble, they may be treated with acids to create terminal carboxylic groups and/or have covalent attachments of hydrophilic groups to their surface via functionalization chemistry to increase solubility and circulation time. In one particular study, the covalent attachment of PEG with an average 17 molecular weight of 1500 Dalton (PEGSWNTs) led to a circulation time of 22.5 hours in mice post-intravenous exposure (Figure 9). [7]

Figure 9. TEM image of PEG-SWNTs.

It should also be noted that higher molecular weight PEG chain attachment resulted in lower RES uptake, longer circulation times, and lower amounts of SWNTs present in both the liver and spleen. Although preliminary studies show some promising results for CNTs, much more biocompatibility information is needed to see the full extent of effects for CNTs throughout the body.

In addition, Gold nanoparticles (AuNps) have recently emerged as an attractive candidate for delivery of small drug molecules or biomolecules, such as proteins, or RNA or DNA into target cells. Advantages of AuNps over polymeric nanoparticle gene delivery agents include (i) ease of preparation of mono-dispersed particles of size ranging from 1 to 150nm; (ii) non–toxicity of the gold core; (iii) easy functionalization of small molecules or nucleic acids via covalent or noncovalent interactions; and (iv)

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Injectable nanoparticles for drug delivery ability to release the attached drug at remote places using their photo-physical properties (Figure 10).[8]

Figure 10. Synthesis scheme for the preparation of DOX conjugated Au NPs.

V. CLINICAL TRIALS Of all the nanocarriers above, liposomes are the most developed and currently possess the greatest amount of clinical trials with some formulations currently in the marketplace. This is probably due to the fact that the other materials have not been investigated for the same duration and are relatively new in comparison. For this reason, polymer based materials, CNTs and AuNps should not be overlooked as nanomedicine delivery materials just because of the relatively low number of recent clinical trials; however, CNTs do still have much to prove with respect to safety and long-term biodistribution in order to be considered viable nanocarriers. Recent clinical trials are shown in Figure 11.

Some of the more promising trials include Genexol-PM, which is an amphiphilic diblock copolymer (PEG-(D,L-lactic acid)) forming a micelle that delivers Paclitaxel for various types of cancers. Clinical trials are currently in phase 4 using Genexol–PM for recurrent breast cancer and phase 3 for breast cancer. The liposomal formulation AmBiosome® consisting of the antifungal Amphotericin B is currently in phase 4 trials for fungal infections associated with acute leukemia and for central line fungal infections. ThermoDox, a thermally sensitive doxorubicin-loaded liposome is currently in phase 3 trials for hepatocellular carcinoma. Lastly, Caelyx, a doxorubicin

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Injectable nanoparticles for drug delivery HCL loaded liposome that is PEGylated, is currently in phase 4 trials for ovarian neoplasms.[6]

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Injectable nanoparticles for drug delivery

Figure 11. Some of prominent applications using nanoparticles in drug delivery

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Injectable nanoparticles for drug delivery CONCLUSION Injectable routes for drug delivery have a number of advantages as well as drawbacks. However, the usage of nanoparticulate in injectable delivery have been attended, especially in cancer therapy. For efficiency, nanoparticles can be functionalized depending upon the targets and the characteristics of human cell, it can be either coated by receptors which target the specific tumuors or used the assistance of external energy sources for helping targeting therapy. A number of systems of nanomaterials utilized in drug delivery including liposomes, polymeric based carriers and inorganic based carriers. Of all systems, liposomes have been developed and possess the greatest amount of the clinical trials currently in the market.

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CHAUBAL, B.R.a.M.V., Injectable Nanoparticles for Efficient Drug Delivery.

Vol. 159. 2006: Taylor & Francis Group, LLC. 222-255.

Finkel, R.C., Michelle A.; Cubeddu, Luigi X, Lippincott's Illustrated Reviews: Pharmacology, 4th Edition, R.C. Finkel, Michelle A.; Cubeddu, Luigi X, Editor 2009, Lippincott Williams & Wilkins. al, S.H.P.e., Targeted Delivery of RNA-Cleaving DNA Enzyme (DNAzyme) to Tumor Tissue by Transferrin-Modified, Cyclodextrin-Based Particles. Cancer Biology & Therapy, 2004. 3(7): p. 11.

S. Rudge , C.P., C. Vessely, J. Koda, S. Stevens, L. Catterall, Adsorption and desorption of chemotherapeutic drugs from a magnetically targeted carrier (MTC). Journal of Controlled Release, 2001. 74: p. 6.

N.Y. Rapoport a, D.A. Christensen a, H.D. Fain b, L. Barrows b, Z. Gao a, Ultrasound-triggered drug targeting of tumors in vitro and in vivo. Ultrasonics, 2004. 42: p. 8.

David M. Webster, P.S., Mark E. Byrne, Injectable Nanomaterials for Drug Delivery: Carriers, Targeting Moieties, and Therapeutics. European Journal of Pharmaceutics and Biopharmaceutics, 2012.

S.-T. Yang, J.-H.L., J. Wang, Dr. H.-F. Sun, Prof. Y. Liu, Dr. H. Wang, Covalently PEGylated Carbon Nanotubes with Stealth Character In Vivo.

Nanotubes, 2008. 4(7): p. 5.

Agha Zeeshan Mirza a, Hina Shamshadb, Preparation and characterization of doxorubicin functionalized gold nanoparticles. European Journal of Medicinal Chemistry, 2011. 46: p. 4.

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