PREPARATION AND CHARACTERIZATION OF CHITOSANPOLYETHYLENE GLYCOL MICROSPHERES AND FILMS FOR BIOMEDICAL APPLICATIONS

A THESIS SUBMITTED TO THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF MIDDLE EAST TECHNICAL UNIVERSITY

BY

SMAL DOAN GNBA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN POLYMER SCIENCE AND TECHNOLOGY

SEPTEMBER 2007

Approval of the thesis:

PREPARATION AND CHARACTERIZATION OF CHITOSANPOLYETHYLENE GLYCOL MICROSPHERES AND FILMS FOR BIOMEDICAL APPLICATIONS

submitted by SMAL DOAN GNBA in partial fulfilment of the requirements for the degree of Master of Science in Polymer Science and Technology, Middle East Technical University by,

Prof. Dr. Canan zgen Dean, Graduate School of Natural and Applied Sciences Assoc. Prof. Dr. Gknur Bayram Head of Department, Polymer Science and Technology Prof. Dr. Nesrin Hasrc Supervisor, Chemistry Dept., METU

Examining Committee Members: Prof. Dr. smet Delilolu Grhan Bioengineering Dept., Ege University Prof. Dr. Nesrin Hasrc Chemistry Dept., METU Assoc. Prof. Dr. Gknur Bayram Chemical Engineering Dept., METU Prof. Dr. Leyla Aras Chemistry Dept., METU Prof. Dr. nci Erolu Chemical Engineering Dept., METU

Date:

I hereby declare that all information in this document has been obtained and presented in accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all material and results that are not original to this work.

Name, Last name: smail Doan GNBA Signature :

iii

ABSTRACT

PREPARATION AND CHARACTERIZATION OF CHITOSANPOLYETHYLENE GLYCOL MICROSPHERES AND FILMS FOR BIOMEDICAL APPLICATIONS

Gnba, smail Doan

M.S., Department of Polymer Science and Technology Supervisor: Prof. Dr. Nesrin HASIRCI

September 2007, 102 pages

In recent years, biodegradable polymeric systems have gained importance for design of surgical devices, artificial organs, drug delivery systems with different routes of administration, carriers of immobilized enzymes and cells, biosensors, ocular inserts, and materials for orthopedic applications. Polysaccharide-based polymers represent a major class of biomaterials, which includes agarose, alginate, dextran, and chitosan. Chitosan has found many biomedical

applications, including tissue engineering, owing to its biocompatibility, low toxicity, and degradation in the body, which has opened up avenues for modulating drug release in vivo in the treatment of various diseases. These chitosan-based delivery systems range from microparticles to nanoparticles and from gels to films.

In this study, chitosan (CH) and chitosan-polyethylene glycol (CH-PEG) microspheres with different compositions were prepared by oil/water emulsion method and crosslinked with gluteraldehyde. Some microspheres were loaded with a model chemotherapeutic drug, methotrexate (MTX). SEM, particle size

iv

and in vitro release analysis were performed. In vitro drug release studies showed that the release of MTX from CH-PEG microspheres was faster compared to CH microspheres.

In the second part, CH-PEG microspheres were conjugated with a monoclonal antibody which is immunoglobulin G (IgG). The cytotoxicity efficiencies of entrapped drug were determined by using MCF-7 and MCF-7/MDA-MB breast cancer cell lines.

In the third part, CHF-PEG films with the same compositions as in microspheres were prepared by solvent casting method. IR, DSC, mechanical and surface analysis were performed. The mechanical properties of films were improved by the presence of proper amount of PEG but higher amounts of PEG caused the deteriotion in the properties.

Keywords: Chitosan, polyethylene glycol, microsphere, film.

KTOSAN-POLETLEN GLKOL MKROKRE VE FLMLERNN BYOMEDKAL UYGULAMALAR N HAZIRLANMASI VE KARAKTERZASYONU

Gnba, smail Doan

Yksek Lisans, Polimer Bilimi ve Teknolojisi Blm Tez Yneticisi: Prof. Dr. Nesrin HASIRCI

Eyll 2007, 102 sayfa

Son yllarda, biyobozunur polimerik sistemler cerrahi aletler, yapay organlar, farkl yollarla uygulanan ila salm sistemleri, immobilize enzim ve hcre tayclar, biyosensrler ve ortopedik uygulamalarda kullanlan malzemelerin tasarmlanmasnda byk nem kazanmtr. Agaroz, aljinat, dekstran ve kitosan gibi polisakkarit bazl polimerler biyomalzemelerin byk bir snfn oluturmaktadr. eitli hastalklarn tedavisinde kullanlan in vivo ila salm sistemlerinin modle edilmesi iin birok yol am olan kitosan,

biyouyumluluu, dk toksisitesi ve vcut iinde biyobozunur olmas nedeniyle doku mhendislii dahil birok biyomedikal uygulamada kullanlmaktadr.

Kitosan bazl bu ila tayc sistemler, mikropartikl, nanopartikl, jel ve film gibi ok farkl ekilde hazrlanabilmektedir.

Bu almada, kitosan (CH) ve farkl kompozisyonlarda kitosan-polietilen glikol (CH-PEG) mikrokreler ya/su emlsiyon metodu kullanlarak ve gluteraldehit ile apraz balanarak hazrlanmtr. Baz mikrokrelere model bir kanser ilac, methotraxate (MTX), yklenmitir. SEM, partikl boyutu ve in vitro ila salm analizleri gerekletirilmitir. In vitro ila salm almalar, kitosan mikrokreler

vi

ile

karlatrldnda

MTX

salmnn

CH-PEG

mikrokrelerinde

daha

hzl

olduunu gstermitir.

almann

ikinci

aamasnda,

CH-PEG

mikrokreleri

monoklonal

antikor,

mmunoglobulin G (IgG), ile konjuge edilmitir. Yklenen ilacn sitotoksik etkinlii MCF-7 ve MCF-7/MDA-MB kanser hcreleri kullanlarak tayin edilmitir.

almann nc aamasnda, mikrokreler ile ayn kompozisyonlarda CHFPEG filmler solvent uurma yntemiyle hazrlanmtr. Filmlerin IR, DSC, mekanik ve yzey analizleri yaplmtr. Filmlerin mekanik zelliklerinin uygun miktarda PEG kullanlarak arttrlabilecei ancak fazla miktardaki PEG zelliklerin bozulmasna neden olmutur.

Anahtar Kelimeler: Kitosan, polietilen glikol, mikrokre, film

vii

To the great memory of my father

viii

ACKNOWLEDGEMENTS

I would like to express my appreciation to Prof. Dr. Nesrin Hasrc for her valuable guidance and encouragement.

I also wish to give my special thanks to all my friends in our research group in Biomedical Materials Research Laboratory. I am especially grateful to Aysel Kzltay, Tuba Endoan, Cantrk zcan, Taylan zerkan and Eda Aye Aksoy for their valuable help, friendship and moral support.

I would like to extend my thanks to Prof. Dr. smet Delilolu Grhan for her help especially for the cell culture studies. Special thanks to research assistant Sultan Glce for the cell culture studies.

Finally, my special appreciation and great gratitude is devoted to my mother Leyla Gnba, my father Mnip Gnba, my grandmother Glfiye Gnba and my sisters Demet Gnba and Duygu Deniz Gnba for their endless love, patience, moral support and encouragement in every moment of my life.

ix

TABLE OF CONTENTS

ABSTRACT ................................................................................................. iv Z ................................................................................................. vi

ACKNOWLEDGEMENTS ................................................................................ ix TABLE OF CONTENTS ...................................................................................x LIST OF TABLES........................................................................................ xiii LIST OF FIGURES...................................................................................... xiv LIST OF SYMBOLS AND ABBREVIATIONS ..................................................... xvi 1.INTRODUCTION ........................................................................................1 1.1 1.2 1.3 Biomaterials...................................................................................1 Polymeric Biomaterials ....................................................................2 Controlled Drug Delivery .................................................................2 Conventional Drug Therapy versus Controlled Release ..................3 Controlled Release Mechanisms .................................................4

1.3.1 1.3.2 1.4 1.5 1.6

Biodegradable Polymers for Drug Delivery .........................................6 Chitin and Chitosan.........................................................................8 Important Characteristics of Chitosan................................................9 Physicochemical Properties of Chitosan .......................................9 Solubility ..............................................................................11 Chemical properties ...............................................................12 Biological Properties ...............................................................13

1.6.1 1.6.2 1.6.3 1.6.4 1.7

Application Areas of Chitosan .........................................................14 Pharmateceutical and Biomedical Uses of Chitosan .....................15

1.7.1 1.8 1.9 1.10 1.11 1.11.1 1.12 1.13

Poly(ethylene glycol).....................................................................17 Microparticulate Systems for Controlled Release Applications .............19 Chitosan Microparticulate Drug Delivery Systems ..........................19 Release of Anticancer Drug from Chitosan Microspheres .................22 Chemical structure and Mechanism of Action of Methotrexate ......22 Drug Targeting..........................................................................23 Drug Targeting in Cancer Therapy ...............................................23

1.13.1 1.13.2 1.13.3 1.14

Currently Available Therapeutics ..............................................24 Strategies to Deliver Drugs to Targets within the Tumour ...........24 Site Specific Drug Delivery Using Monoclonal Antibodies .............25 Aim of the study .......................................................................28

2.MATERIALS AND METHODS ......................................................................29 2.1 2.2 Materials .....................................................................................29 Methods ......................................................................................30 Preparation of Microspheres ....................................................30 Preparation of Drug-loaded Microspheres ..................................31 Characterization of Microspheres..............................................32 Morphological Analysis............................................................32 Particle Size Analysis ..............................................................32 Preparation of Chitosan and Chitosan-PEG Films ........................33 IR Analysis ............................................................................34 Differential Scanning Calorimetry (DSC) Analysis .......................34 Mechanical Tests....................................................................34 Contact Angle Measurement ....................................................35 Conjugation of IgG to Microspheres ..........................................35 Degradation of Microspheres ...................................................36 In-vitro Release Studies..........................................................36 Cell Studies ...........................................................................37

2.2.1 2.2.1.1 2.2.2 2.2.2.1 2.2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.7 2.2.8 2.2.9 2.2.10 2.2.11

3.RESULTS & DISCUSSION .........................................................................39 3.1 Chitosan and Chitosan-PEG Microspheres ........................................39 Effect of Crosslinker on Size and Shape of the Chitosan

3.1.1

Microspheres ...................................................................................39 3.2 3.3 3.4 3.5 3.6 3.7 3.8 Particle Size Analysis of Microspheres..............................................42 Drug Loading to Microspheres ........................................................44 In Vitro Release Studies ................................................................45 Conjugation of IgG to Microspheres ................................................53 Cell Culture and Coculture Studies ..................................................54 Degradation Studies......................................................................60 Chitosan and Chitosan-PEG Films....................................................63 Infrared Analysis....................................................................63 Differential Scanning Calorimetry Analysis.................................67 Mechanical Tests....................................................................69

3.8.1 3.8.2 3.8.3

xi

3.8.4

Contact Angle Measurements ..................................................77

4.CONCLUSIONS........................................................................................79 REFERENCES .............................................................................................82 APPENDICES .............................................................................................91

xii

LIST OF TABLES

Table 1.1 Requirements for biomedical polymers ............................................2 Table 1.2 Commercially available biodegradable drug delivery systems..............7 Table 1.3 Solution properties of chitosan......................................................12 Table 1.4 Chemical properties of chitosan ....................................................13 Table 1.5 Principal applications for chitosan..................................................14 Table 1.6 Principal properties of chitosan in relation to its use in biomedical applications................................................................................15 Table 2.1 Materials and Manufacturers.........................................................29 Table 2.2 Prepared Microspheres.................................................................32 Table 2.3 Prepared chitosan and chitosan-PEG films ......................................33 Table 2.4 Prepared samples for cell culture experiments ................................38 Table 3.1 Sizes of different microspheres .....................................................42 Table 3.2 Particle size analysis results .........................................................44 Table 3.3 Release rates of MTX from different type of microspheres ................48 Table 3.4 Amount of MTX entrapped in different type of the microspheres .......48 Table 3.5 Release knetcs ..........................................................................50 Table 3.6 Mechanical properties of CHF-PEG films .........................................69 Table 3.7 Contact angles of prepared films...................................................77

xiii

LIST OF FIGURES

Figure 1.1

Drug levels in the blood plasma (a) traditional drug dosing, (b) controlled-delivery dosing ............................................................4

Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5

Structures of cellulose, chitin and chitosan...................................10 Protonation of chitosan..............................................................11 Chemical structure of PEG .........................................................17 Schematic represantation of the suspension crosslinking

technique ................................................................................20 Figure 1.6 Figure 1.7 Figure 1.8 Figure 2.1 Figure 3.1 Figure 3.2 Methods for preparation of chitosan microspheres ........................21 Chemical structure of MTX .........................................................22 Schematic diagram of an immunoglobulin (IgG) ...........................26 Schematic representation of water-oil emulsion method ................31 Crosslinking reaction of chitosan and glutaraldehyde.....................39 SEM micrographs of microspheres (A) U-CH 1.25, (B) U-CH 2.5, (C) U-CH 5 microspheres ...........................................................40 Figure 3.3 SEM micrographs of microspheres (A) U-CH-PEG 1-0.5, (B) UCH-PEG 1-1, (C) U-CH-PEG 1-2 microspheres ..............................41 Figure 3.4 Figure 3.5 Size distribution of unloded microspheres ....................................42 SEM micrographs of drug loaded microspheres (A) CH 5, (B) CH-PEG 1-0.5, (C) CH-PEG 1-1, (D) CH-PEG 1-2 ..........................45 Figure 3.6 Figure 3.7 Figure 3.8 Release of MTX from CH and CH-PEG microspheres ......................46 Release of MTX from different type of the microspsheres ...............47 Percent MTX release from microspheres by taking total released MTX as 100%...........................................................................49 Figure 3.9 Zero-order release kinetic model plot for various microspheres ......51

Figure 3.10 First order release kinetic model plot for various microspheres .......51 Figure 3.11 Higuchi kinetic model plot for various microspheres ......................52 Figure 3.12 Korsmeyer kinetic model plot for various microspheres .................52 Figure 3.13 Conjugation of microspheres ......................................................53 Figure 3.14 Confocal microscopy images of microspheres (A) non-

conjugated, (B) conjugated........................................................54

xiv

Figure 3.15 Pictures of MDA-MB and MCF-7 coculture.....................................54 Figure 3.16 The number of cells of 0.25 mg/mL and 2.5 mg/mL MTX incubated groups ......................................................................55 Figure 3.17 Pictures of cell plates containing (A) MTX-0.25 mg/mL after 144 hours, (B) MTX-2.5 mg/mL after 144 hours, (C) MTX-0.25 mg/mL after 240 hours, (B) MTX-2.5 mg/mL after 240 hours.........56 Figure 3.18 Pictures of cell plates after (A) 144 hours control, (B) 144 hours MTX-2.5, (C) 144 hours MTX-0.25, (D) 144 hours with U-CHPEG 1-1, (E) 144 hours with L-CH-PEG 1-1, (F) 144 hours CLCH-PEG 1-1, (G) 144 hours CU-CH-PEG 1-1.................................57 Figure 3.19 The number of cells of control group and U-CH-PEG 1-1 ................58 Figure 3.20 The number of cells of control and PEG .......................................59 Figure 3.21 The number of cells of control and MTX loaded CH-PEG 1-1...........59 Figure 3.22 SEM micrographs of microspheres (A) CH after 2 days, (B) CHPEG 1-1 after 2 days, (C) CH after 15 days, (D) CH-PEG 1-1 after 15 days, (E) CH after 60 days SEM, (F) CH-PEG after 60 days .......................................................................................61 Figure 3.23 SEM Micrographs of microspheres (A) CH after 2 days, (B) CHPEG 1-1 after 2 days, (C) CH after 60 days, (D) CH-PEG 1-1 after 60 days ...........................................................................62 Figure 3.24 IR spectra of (A) CHF, (B) CHF 0.1, (C) CHF 1.0 ...........................64 Figure 3.25 IR Spectra of (A) CHF-PEG 1-0.5, (B) CHF-PEG 1-1, (C) CHFPEG 1-1.5, (D) CHF-PEG 1-2 ......................................................65 Figure 3.26 Chitosan-PEG interaction............................................................66 Figure 3.27 DSC curves of (A) chitosan (DDA=85%), (B) PEG .........................67 Figure 3.28 DSC curves of (A) CHF-PEG 1-0.5-0.1, (B) CHF-PEG 1-1-0.1,

(C)CHF-PEG 1-1.5-0.1, (D) CHF-PEG 1-2-0.1 ...............................68 Figure 3.29 The effect of crosslinker on UTS values of chitosan films................70 Figure 3.30 Chemical reaction between chitosan and gluteraldehyde................71 Figure 3.31 The effect of crosslinker on UTS values of CHF-PEG films...............72 Figure 3.32 The effect of PEG on UTS of CHF-PEG films ..................................73 Figure 3.33 The effect of PEG on UTS of CHF-PEG films ..................................74 Figure 3.34 Effect of crosslinker on modulus of CHF films ...............................75 Figure 3.35 Modulus of CHF-PEG films..........................................................76 Figure 3.36 The effect of PEG on contact angles of CHF-PEG films....................78

xv

LIST OF SYMBOLS AND ABBREVIATIONS

CH PEG CH-PEG PEO DA DDA LDL HDL Ig IgG MAb Fab GA MTX PBS SEM EDC NHS FBS MMD VMD SMD UTS E SAB

Chitosan Polyethylene glycol Chitosan-polyethylene glycol Polyethylene oxide Deacetylation Degree of deacetylation Low density lipoproteins High density lipoproteins Immunoglobulin Immunoglobulin G Monoclonal antibody Antigen-binding fragment Gluteraldehyde Methotrexate Phosphate buffer solution Scanning electron microscopy 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide N-hydroxyl succinimide Foetal bovine serum Mass median diameter Volume mean diameter Surface mean diameter Ultimate tensile strength Modulus of elasticity Strain at break

xvi

CHAPTER 1

INTRODUCTION

1.1

Biomaterials

A biomaterial is used to make devices to replace a part or a function of the body in a safe, reliable, economic, and physiologically acceptable manner [1]. Various definitions of the term biomaterials have been proposed over the years. For example, biomaterial is a nonviable material used in a medical device, intended to interact with biological systems [2]. Other definitions have included any substance or combination of substances which are synthetic or natural in origin and can be used for any period of time, as a whole or as a part of a system which treats, augments, or replaces any tissue, organ, or function of the body [3] and synthetic as well as natural materials in contact with tissue, blood, and biological fluids, and intended for use for prosthetic, diagnostic, therapeutic, and storage applications without adversely affecting the living organism and its components [4].

Biomaterials are used for the production of various biomedical systems including pacemakers, sutures, heart valves, bone plates, intraocular lenses, controlled drug delivery systems etc. Certain metal alloys, polymers, ceramics and composites are used as biomaterials in the design and production of biomedical devices [5].

1.2

Polymeric Biomaterials

Polymeric materials have been widely used in medical disposable supplies, prosthetic materials, dental materials, implants, dressings, extracorporeal devices, encapsulants, polymeric drug delivery systems, tissue engineered products. The main advantages of polymeric biomaterials are ease of manufacturability to produce various shapes (latex, film, sheet, fibers, etc.), ease of secondary processability, reasonable cost, and availability with desired mechanical and physical properties. Biocompatibility, sterilizability, adequate mechanical and physical properties, and manufacturability are required

properties of polymeric biomaterials similar to other biomaterials as shown in Table 1.1 [5].

Table 1.1

Requirements for biomedical polymers Property Description

Biocompatibility

Sterilizability

Noncarcinogenesis, nonpyrogenicity, nontoxicity, and nonallergic response Autoclave, dry heating, ethylenoxide gas, and radiation Strength, elasticity, and durability

Physical property

Manufacturability

Machining, molding, extruding, and fiber forming

1.3

Controlled Drug Delivery

Controlled drug delivery occurs when a polymer, whether natural or synthetic, is combined with a drug or other active agent in such a way that the active agent is released from the material in predesigned matter. Therefore, the goal of all drug delivery systems is to deploy medications intact to specifically targeted parts of the body through a medium that can control the therapys

administration by means of either a physiological or chemical trigger. Controlled drug delivery can be the most important at times when traditional oral or injectable drug formulations cannot be used. These situations include requiring drug delivery to specific sites, drug delivery using nanoparticulate systems, the slow release of water-soluble drugs, the fast release of low-solubility drugs, delivery of two or more agents with the same formulation, and systems based on carriers that can dissolve or degrade and be readily eliminated [6].

1.3.1 Conventional Drug Therapy versus Controlled Release

The purpose of controlled-release systems is to achieve a delivery profile that would yield a high blood level of the drug over a long period of time. The drug level in the blood follows the profile shown in Figure 1.1 a, in which the level rises after each administration of the drug and then decreases until the next administration. With traditional drug administration, the blood level of the drug exceeds toxic level immediately after drug administration, and decreases below effective level after some time. At times, the drug concentration is very high, contributing to adverse side effects. At other times, the concentration is too low to provide therapeutic benefit. Controlled drug delivery systems are designed for long-term administration and the drug level in the blood follows the profile shown in Figure 1.1 b, remaining constant, between the desired maximum and minimum, for an extended period of time [6]. The drug delivery system should be designed such that a preferential accumulation of the drug is reached at the site of action, whereas the drug concentration elsewhere in the body should be as low as possible. The reason for this need of targeting is that a high concentration of the drug in tissues or cells other than those being targeted may cause problems related to side effects. The advantages of these systems are reproducible and achieves prolonged constant delivery rate, reduces side effects because the dose does not exceed the toxic level [7].

Figure 1.1

Drug levels in the blood plasma (a) traditional drug dosing, (b) controlled-delivery dosing

1.3.2 Controlled Release Mechanisms

There are four primary mechanisms by which active agents can be released from a polymeric delivery system: diffusion controlled, solvent activated, chemically controlled and magnetically controlled systems. In diffusion

controlled systems, there are two main types: reservoir and matrix. A reservoir consists of a drug core in powdered or liquid form and is generally spherical, cylindrical, or disc-like in shape. The drug slowly diffuses through a layer of nonbiodegradable polymeric material. The diffusion rate of the drug depends on the properties of drug and polymer. One of the problems with the reservoir

system is that such a system must be removed from the body after the drug is depleted because the polymer remains intact. Another potential problem is that if the reservoir membrane accidentally ruptures, a large amount of drug may be suddenly released into the bloodstream (known as drug dumping). In the matrix type of diffusion-control system, the drug is uniformly distributed throughout the polymer matrix and is released from the matrix at a uniform rate as drug particles dislodge from the polymer network. In such a system, unlike the reservoir, there is no danger of drug dumping in case of an accidental rupture of the membrane [8].

Solvent-activated systems are also of two types: swelling-controlled systems and osmotically controlled systems. In the swelling-controlled systems, the system consists of hydrophilic macromolecules cross-linked to form three dimensional network. The important characteristics of such systems is their permeability for low molecular weight solutes at a controlled rate. In the osmotically controlled system, a drug with low concentration in an external fluid moves across a semi-permeable membrane to a region inside the device where the drug concentration is high [8].

Chemically controlled systems also have two types: the bioerodible or biodegradable, system and the pendant-chain system. In the bioerodible or biodegradable system, the controlled release of the drug involves polymers that decompose gradually. As the polymer decomposes, the drug is dispersed throughout the polymer and is released slowly. The bioerodible systems have two important advantages. The first one is that after the drug supply is decomposed, polymers do not have to be removed from the body. The second is that it is not needed for drug to be water-soluble. In the pendant-chain system, the drug molecule is chemically linked to the backbone of the polymer. Chemical hydrolysis, or enzymatic cleavage occurs with the release of the drug at a controlled rate in the presence of enzymes and biological fluids in the body. The drug may be linked directly to the polymer or via spacer group [8].

Magnetically responsive drug carrier systems, composed of albumin and magnetic microspheres, have been developed for use in cancer chemotherapy because conventionally used systemic antineoplastic agents are unable to achieve ideal tumor specificity. These microspheres are theoretically capable of enhanced area-specific localization because of their magnetic characteristics. Two major advantages of the magnetically responsive carrier system over other drug delivery systems are its high efficiency for in vivo targeting and its controllable release of a drug at the microvascular level [8].

Due to rapid advances in recent years, the application of polymers to drug delivery has grown considerably. Polymers which are used for drug delivery can be divided into three categories, namely biodegradable or bioerodible polymers, soluble polymers and mucoadhesive polymers.

1.4

Biodegradable Polymers for Drug Delivery

Polymers that are degradable in vivo, either enzymatically or nonenzymatically, to produce biocompatible or nontoxic by-products are defined as biodegradable polymers [8]. Interest in biodegradable polymers which are used for drug delivery systems developed for two reasons. First, it was recognized that surgical removal of a drug-depleted delivery system was difficult, leaving nondegradable foreign materials in the body for an indefinite time period, which caused an undesirable toxicological hazard. Second, while diffusion-controlled release is an excellent means of achieving predefined rates of drug delivery, it is limited by polymer permeability and the characteristics of the drug [9].

Biodegradable polymers are classified into three groups based on their sources, namely natural, semisynthetic, and synthetic. Examples of commonly used natural biodegradable polymers are gelatin, alginate, albumin, collagen, starch, dextran, chitosan, and chitin, whereas examples of synthetic biodegradable polymers are polylactic acid, polyglycoloc acid, poly(lactide-co-glycolide), polyhydroxyvalerate, and polyanhydride [8].

However, most commonly used biodegradable polymers in drug delivery systems have natural origin. Table 1.2 shows some commercially available biodegradable drug delivery systems [8].

Table 1.2 Name of product

Commercially available biodegradable drug delivery systems Dosage form Active ingredient Leuprolide Octreotide Biodegradable polymera,b PLGA PLGA

Lupron Depot Sandostatin LAR Neutropin Depot Trelstar Depot Gliadel Zoladex Atridox
a

Microspheres Microspheres

Microspheres

Somatropin

PLGA

Microspheres Waffer Rod Gel

Triptorelin Cumustin Goserelin Doxycycline

PLGA Polyanhydride PLGA PLGA

PLGA: poly(lactic-co-glycolic acid) Polyanhydride: poly[bis(p-carboxyphenoxy) propane: sebacic acid] in a 20:80 molar ratio

Modifications can be made to naturally occurring biodegradable polymers, such as chitosan, alginate, and hyaluronic acid, to produce result semisynthetic in altered

biodegradable

polymers.

These

modifications

can

physicochemical properties, such as thermogelling properties, mechanical strength, and degradation rates.

Biodegradation of polymer devices or drug delivery systems usually undergoes four steps: hydration, mechanical strength loss, integrity loss, and mass loss. The hydration is determined by the hydrophilicity/hydrophobicity or crystallinity of the polymer [10, 11]. Natural biodegradable polymers, such as collagen are hydrophilic and undergo degradation by hydrolysis.

Biodegradable

polymers

which

are

hydrophobic

can

undergo

surface

degradation which means degradation occurs on the outer layer exposed to the aqueous body fluid.

The factors which affect the degradation rate of the polymer involve chemical properties, physical properties, such as hydrophilicity and crystallinity,

geometric factors of the polymer devices, such as size, shape, and surface area; and additives, molecular weight of the polymers; and environmental factors, such as pH and ionic strength [12].

Chitosan and its derivatives have been used as excipients in drug delivery systems in recent for years because in chitosan drug meets the most important namely

requirements

excipients

modern

delivery

systems,

biodegradability, biocompatibility, bioadhesiveness and non-toxicity [13, 14].

1.5

Chitin and Chitosan

Chitin is a straight homopolymer composed of -(1,4)-linked N-acetylglucosamine units while chitosan comprises of copolymers of glucosamine and N-acetyl-glucosamine [15, 16] present in most of the families of living species. Thus, it constitutes the structure polymer of the cuticles of all the arthropods and the endoskeletons of all the cephalopods [3]. Also, It is very often present at the cell wall and in the extracellular matrix of most fungi. It is encountered in numerous microorganisms, in some algae, etc. However, chitosan is much less present in living media and to date it has only been observed in some microorganisms, particularly of fungal nature [17].

The origin of chitin and chitosan from historical point of view is also interesting. Chitin was discovered in 1811 by H. Braconnot during his studies on mushrooms and was termed fungine. He stated that fungine seems to contain more nitrogen than wood and concluded that it is a quite distinct substance among those identified in plants.

The term chitin was first proposed by C. Odier in 1823. He ignored the works of Braconnot and established for the first time a relationship between the insect cuticle and plant tissue. In 1859, C. Rouget treated chitin in hot and concentrated KOH and discovered chitosan. He proposed to name modified chitin to this new product. However, in 1894, F. Hoppe-Seyler, ignored the works of Rouget and proposed to term this derivative as chitosan [18]. In his work, he treated chitin with potassium hydroxide at 180C and obtained a product with no acetyl groups.

Chitosan is obtained from the N-deacetylation of chitin. All the methods are derived from the descriptions given in two patents [19, 20]. These methods consist of the using of highly concentrated solutions of sodium hydroxyde (3050%) at temperatures over 90 C for times over 1 hour. These methods allow to reach in one step deacetylation (DA) close to 10-15% in mild conditions. However, the deacetylation can be within 90-95% if the process is repeated more times.

The most important function of chitin is the amino groups. Therefore, most of the research is carried out on the amino groups of chitin. The amino groups in chitin are acetylated, thus chitin is a primary amine. It is difficult to sharply distinguish chitin from chitosan because fully acetylated or fully deacetylated chitins do not normally occur in nature and are difficult to prepare.

1.6

Important Characteristics of Chitosan

1.6.1 Physicochemical Properties of Chitosan

Chitin, naturally abundant mucopolysaccharide and the supporting material of crustaceans, insects, etc., is well known to consist of 2-acetamido-2-deoxy-bD-glucose through a linkage. Its immunogenicity is exceptionally low, in spite of the presence of nitrogen. It is a highly insoluble material resembling cellulose in its solubility and low chemical reactivity. It may be regarded as cellulose with hydroxyl at position C-2 replaced by an acetamido group.

Like cellulose, it functions naturally as a structural polysaccharide. Chitin is a white, hard, inelastic, nitrogenous polysaccharide and is the major source of surface pollution in coastal areas. Chitosan is the N-deacetylated derivative of chitin, although this N-deacetylation is almost never complete.

A sharp nomenclature with respect to the degree of N-deacetylation has not been defined between chitin and chitosan [21, 22]. The structures of cellulose, chitin and chitosan are shown in Figure 1.2. Chitin and chitosan are of commercial interest due to their high percentage of nitrogen (6.89%) compared to synthetically substituted cellulose (1.25%). This makes chitin a useful chelating agent [21]. As most of the present-day polymers are synthetic materials, their biocompatibility and biodegradability are much more limited than those of natural polymers such as cellulose, chitin, chitosan and their derivatives. However, these naturally abundant materials also exhibit a limitation in their reactivity and processability.

CH2OH O H OH H H H OH

H O

CH2OH O H OH H H H OH

n Cellulose

CH2OH O H OH H H

H O

CH2OH O H OH H H

H NHCOCH3

H NHCOCH3 n

Chitin

CH2OH O H OH H H H NH2

H O

CH2OH O H OH H H H NH2

n Chitosan

Figure 1.2

Structures of cellulose, chitin and chitosan

10

The word chitosan refers to a large number of polymers which differ in their degree of N-deacetylation (65-95%) and molecular weight (3800-2.000.000 daltons). These two characteristics are very important to the physicochemical properties of the chitosans and, hence, they have a major effect on the biological properties [21, 22].

1.6.2 Solubility

Chitosan is a weak base with a pKa value of about 6.2 - 7.0 which can be attributed to the D-glucosamine residue. Therefore, it is insoluble at neutral and alkaline pH values. However, it makes salts with inorganic organic acids such as hydrochloric acid, glutamic acid lactic acid and acetic acid. In acidic medium, the amino groups of chitosan are protonated (Figure 1.3) because these amino groups are weak basic groups capable of taking up hydrogen ions and consequently the chitosan molecule becomes a positively charged

polysaccharide that has a high charge density [13, 23-26].

CH2OH H O O H OH H H H NH2

+ H+

CH2OH H O O H OH H H + H NH3
Protonation of chitosan

Figure 1.3

11

Some important solution behaviors of two amine forms of chitosan are given in Table 1.3 [27].

Table 1.3

Solution properties of chitosan

Free Amine (-NH2)

-Soluble in acidic solutions -Insoluble at pHs 6.5 -Insoluble in H2SO4 -Limited solubility in H3PO4

Cationic Amine (-NH3+)

-Soluble at pHs 6.5 -Forms viscous solutions -Forms gel with polyanions -Soluble in some alcohol-water mixtures

-Insoluble in most organic solvents

-Solutions shear thinning

In fact, the solubility is a very difficult parameter to control because

it is

related to the degree of deacetylation (DDA), the nature of the acid used for protonation, the ionic concentration and the distribution of acetyl groups along the chain, the pH and conditions of isolation and drying of the polysaccharide [28].

1.6.3 Chemical properties

The amino groups on the chitosan chain has a lone electron pair with strong nucleophilic characteristics and the possibility of many reactions with other chemical groups. Also, the free amino, hydroxyl and carbonyl groups of chitosan are responsible for interactions with metal ions through different mechanisms including cation chelation (Table 1.4) [29]. Therefore, it is useful in chelating iron, magnesium, copper and can also be used to remove toxic heavy metal ions such as cadmium, silver, lead, nickel and chromium [30-32].

12

Table 1.4

Chemical properties of chitosan

-Linear polyamine (poly D-glucosamine) -Have reactive amino groups -Have reactive hydroxyl groups (C3-OH, C6-OH)

1.6.4 Biological Properties

The first interesting biological property of chitosan is its ability to be biodegradable and bioresorbable. Chitin deacetylases and enzymes hydrolyzing chitosan, such as chitinases, chitobiases, chitosanases, as well as

glucosaminidases and N-acetyl-glucosaminidases, are now well known [33, 34]. However, these enzymes seem to be completely absent in mammals. Lysozyme which can hydrolyze chitosan is a nonspecific proteolytic enzyme widespread in animals but when chitosan has a degree of deacetylation (DDA) below 30%, this activity disappears rapidly [35]. It has been shown that the biodegradation of chitosan is a phenomenon depending on several factors, especially the degree of acetylation, the degree of crystallinity, the molecular weight, the water content, and also the shape and the state of the surface of the material [36].

The other biological property of chitosan is that its biocompatiblity. Studies showed that in the case of the oral delivery of chitosan in rabbits, no particular adverse response of the host was noticed in normal conditions of administration [37]. Chitosan possesses no toxicity and can be applied onto the nasal epithelium [38]. Additionally, it exhibits biological offers such as a hemostatic, bacteriostatic, fungistatic, spermicidal, and anti-carcinogenic effects [39].

13

1.7

Application Areas of Chitosan

The principal applications of chitosan that they imply are given in Table 1.5 [40]. The great current interest in medical applications of chitosan and some of its derivatives is readily understood. The cationic character of chitosan is unique and it is the only pseudo-natural cationic polymer. Its film forming property and biological activity invite new application areas.

Table 1.5 Agriculture

Principal applications for chitosan -Defensive mechanisms in plants -Stimulation of plant growth -Seed coating -Time release of fertilizers and nutrients into the soil -Flocculant to clarify water (drinking water, pools) -Removal of metal ions -Ecological polymer (eliminate synthetic polymers) -Reduce odors -Not digestible by human (dietary fiber) -Bind lipids (reduce cholesterol) -Thickener and stabilizer for sauces -Protective, fungistatic, antibacterial coating for fruit -Maintain skin moisture -Treat acne -Improve suppleness of hair -Reduce static electricity in hair -Tone skin -Oral care (toothpaste, chewing gum) -Immunologic, antitumoral -Hemostatic and anticoagulant -Healing, bacteriostatic

Water & waste treatment

Food & beverages

Cosmetics & toiletries

Biopharmaceutics

14

The most important fields where the specificity of chitosan must be recognized are cosmetics and the pharmaceutical and biomedical applications which probably offer the greatest promise.

1.7.1

Pharmateceutical and Biomedical Uses of Chitosan

Chitosan has attracted great attention in pharmaceutical and biomedical fields because it exhibits favorable biological properties. Principal properties of chitosan in relation to its use in biomedical applications is shown in Table 1.6.

Table 1.6 applications

Principal properties of chitosan in relation to its use in biomedical

Potential Biomedical Applications Surgical sutures Dental implants Artificial skin Rebuilding of bone Corneal contact lenses Time release drugs for animals and humans Encapsulating material

Principal Characteristics

Biocompatible Biodegradable Renewable Film forming Hydrating agent Nontoxic, biological tolerance

Hydrolyzed by lyzosyme Wound healing properties Efficient against bacteria, viruses, fungi

Chitosan can be used in powder, solution, film, fiber forms and applied as sutures, bandages, synthetic skin grafts and eye bandages in wound healing.

15

Chitosan based wound dressing reduced scar tissue (fibroplasias) by inhibiting the formation of fibrin in wounds and it was hemostatic and formed a protective film coating [41]. The chitosan membrane showed controlled evaporative water loss, excellent oxygen permeability and effectively inhibiting invasion of exogenous microorganisms [42]. Wound covered with such membrane was hemostatic and healed quickly.

Enzyme immobilization is a technique to enhance the catalytic potential, resistance to pH and temperature. Chitosan is an excellent base material for immobilization of several carbohydrate degrading enzymes because it exhibits increased thermostability compared to the free enzyme. Urease has been immobilized covalently on to glutaraldehyde crosslinked chitosan membrane to provide resistance to the influence of inhibitors, such as boric acid, thioglycolic acid, sodium fluoride and acetohydroxamic acid [43].

Chitosan has become a useful dietary ingredient because of its beneficial plasma cholesterol level lowering effect. The hypocholesterolemic action of chitosan has been explained to be due to decreased cholesterol absorption and interference with bile acid absorption [44]. The antimicrobial property of chitosan has received considerable attention in recent years because of imminent problems associated with synthetic chemical agents. Chitosan showed a broad-spectrum antimicrobial activity against both gram-positive and gram-negative bacteria and fungi. This property of chitosan is useful in food preservation and food protection. To enhance the antibacterial potency of chitosan, thiourea chitosan was prepared by reacting chitosan with ammonium thiocyanate followed by its complexing with Ag+ [45].

Mucoadhesivity of chitosan and cationic derivatives is recognized and has been proved to enhance the adsorption of drugs especially at neutral pH; N-trimethyl chitosan chloride interacts with negatively charged cell membranes [46, 47].

16

Chitosan and its derivatives have been used for gene transfection; for Nalkylated chitosan, it has been shown that transfection efficiency increases upon elongating the alkyl side chains and levels off when the number of carbons in the side chain exceeds 8 [48].

An interesting application concerns a self-setting calcium phosphate cement; chitosan glycerophosphate mixed with calcium phosphate and citric acid forms an injectable self-hardening system for bone repair or filling [49].

1.8

Poly(ethylene glycol)

Poly(ethylene glycol) (PEG) is a simple polymer containing C-O-C bonds along the chain as shown in Figure 1.4.

HO

OH n

Figure 1.4

Chemical structure of PEG

PEG is one of the most frequently used water-soluble polymers in biomedical applications. Because of its high solubility in water, where it behaves as a highly mobile molecule, PEG is useful in biomedical applications. In addition, it has a large exclusion volume, occupying a larger volume in aqueous solution than other polymers of comparable molecular weight. Because of these properties, PEG molecules in aqueous solution tend to exclude or reject other polymers. It is unusual among the group of water-soluble polymers in that it is also soluble in a variety of organic solvents, including methylene chloride, ethanol, and acetone. These properties lead to a number of useful applications: (i) addition of PEG to aqueous solutions of proteins and nucleic acids frequently induces crystallization; (ii) addition of high concentrations of PEG to cell suspensions induces cell fusion; (iii) immobilization of PEG to polymer surfaces greatly reduces protein adhesion; and (iv) covalent coupling of PEG to proteins

17

decreases their immunogenicity and increases their half-life in plasma. PEG is non-toxic and biocompatible polymer [50]. They may have been co-polymerized with linear aliphatic polyesters like poly(lactic acid) (PLA) for use in drug delivery systems and tissue engineering and also for improving the

biocompatibility of polymers [51, 52].

Poly(ethylene glycol) is produced by interaction of calculated amount of ethylene oxide with water, ethylene glycol or ethylene glycol oligomers as shown below;

HOCH2CH2OH + n(CH2CH2O) HO(CH2CH2O)n+1H

The reaction is catalyzed by acidic or basic catalysts. Ethylene glycol and its oligomers are preferable as a starting material than water because it allows the creation of polymers with narrow on the molecular catalyst weight type the distribution mechanism (low of

polydispersity).

Depending

polymerization can be cationic or anionic. Anionic mechanism is more preferable because it allows one to obtain PEG with low polydispersity.

Low molecular weight PEGs (< 1000) are liquids at room temperature. Higher molecular weight PEGs are solids and, when the molecular weight is above 2.104, PEG is frequently referred to as poly(ethylene oxide) (PEO) or polyoxyethylene. In some cases this is a useful distinction, since PEG generally refers to molecules with terminal hydroxyl groups on each end of the molecule while PEO generally refers to units of sufficient molecular weight that the end groups can be neglected. PEG has been studied in great detail and its properties and applications have been reviewed [53].

18

1.9

Microparticulate Systems for Controlled Release Applications

Microencapsulation is defined as a technology of packaging solids, liquids, or gaseous materials in miniature, sealed capsules that can release their contents at controlled rates under specific conditions [54]. Biocompatible polymers are used as encapsulating materials for this purpose.

The size of microcapsules may range from submicrometer to several millimeters and they have a multitude of different shapes, depending on the materials and methods used to prepare them.

Microsphere-based drug delivery systems have occupied a unique position in drug therapy due to their attractive properties and advantages over

conventional drug delivery systems. The use of microsphere-based therapy allows drug release to be carefully tailored to the specific treatment site through the choice and formulation of various drug-polymer combinations [55, 56]. The total dose of medication and the kinetics of release are the variables and they can be manipulated to achieve the desired result. Microspheres can be developed into an optimal drug delivery system which will provide the desired release profile using innovative microencapsulation technologies, and by varying the copolymer ratio, molecular weight of the polymer, etc [56].

Microsphere based systems may increase the life span of active constituents and control the release of bioactive agents. Being small in size, microspheres have large surface to volume ratios and can be used for controlled release of insoluble drugs.

1.10

Chitosan Microparticulate Drug Delivery Systems

Chitosan microspheres are used to provide controlled release of many drugs. They improve the bioavailability of degradable substances such as protein or enhance the uptake of hydrophilic substances across the epithelial layers.

19

These microspheres are being investigated both for parenteral and oral drug delivery. Release of a drug from a drug delivery device is influenced by number of parameters. Therefore, many researches have been done to analyse these factors in order to control the release of drugs. Some important parameters are origin of chitosan, particle size, crosslinking density of the system, loaded drug concentration, degree of deacetylation, pH of the medium etc.

Reacting chitosan with controlled amounts of multivalent anion results in crosslinking between chitosan molecules. This crosslinking has been used extensively for the preparation of chitosan microspheres. Other crosslinking agents such as glutaraldehyde, formaldehyde and naturally occurring

crosslinking agent genipin have also been used for preparation of microspheres. A schematic representation of the suspension crosslinking technique is given in Figure 1.5 [57].

Figure 1.5

Schematic represantation of the suspension crosslinking technique

20

Apart from crosslinking, chitosan microparticulate drug delivery systems, chitosan microspheres have also been prepared by a number of other processes. Figure 1.6 shows various methods which have been used for the preparation of chitosan microspheres.

CHITOSAN MICROSPHERES

Interaction with anions (sulphate,tripolyphosphate ,hydroxide,molbdate)

Solvent evaporation

Coating on preformed microparticles

Crosslinking with chemicals

Thermal crosslinking with citric acid Interfacial acylation Onotropic Wet phase gelation inversion Co-acervation Gluteraldehyde Formaldehyde crosslinking crosslinking Genipin crosslinking Precipitation Emusification and onotropic gelation Modified emulsification and onotropic gelation Complex Co-acervation Single emulsion Precipitation chemical crosslinking

Multiple emulsion

Figure 1.6

Methods for preparation of chitosan microspheres

The use of chitosan in controlled drug delivery systems aims to prepare microparticulate systems kinetically controlling drug release in order to make the release more dependent on pharmaceutical formulation than

physicochemical characteristics of the drug [38].

21

Controlled released drugs from chitosan microparticulate delivery systems provide many advantages in comparison with conventional forms; reduced side effects, drug concentration kept at effective levels in plasma, improved utilisation of drugs and decrease in dosing times.

Various therapeutic agents such as anticancer [58], antiinflammatory [59], cardiac [60], antibiotics [61], antithrombotic [62], steroids [63], proteins [64], amino acids [65], antidiabetic [66] and diuretics [67] have been incorporated in chitosan microspheres to achieve controlled release.

1.11

Release of Anticancer Drug from Chitosan Microspheres

1.11.1 Chemical structure and Mechanism of Action of Methotrexate

Methotrexate, abbreviated MTX and formerly known as amethopterin, is an antimetabolite drug used in treatment of cancer and autoimmune diseases. It acts by inhibiting the metabolism of folic acid. The chemical structure of MTX is given in Figure 1.7.

H2N N

N N N O O O OH H N

NH2

Figure 1.7

Chemical structure of MTX

Methotrexate is a weak dicarboxylic acid with pKa 4.8 and 5.5, and thus it is mostly ionized at physiologic pH. Oral absorption is saturatable and thus dosedependent.

22

Methotrexate was first developed in the 1940s as a specific antagonist of folic acid. This drug inhibits the proliferation of malignant cells. Because

administration of high doses of reduced folic acid (folinic acid) or even folic acid itself can reverse the antiproliferative effects of methotrexate, it is clear that methotrexate does act as an antifolate agent [68].

1.12

Drug Targeting

Many scientists have dedicated their research to the development of drug targeting strategies for the treatment of disease since the early 1960s. In general, the aim of targeted therapies is to increase the efficacy and reduce the toxicity of drugs. The pharmacokinetics and cellular distribution of the drug is largely determined by the behaviour of the carrier molecules. Furthermore, selective delivery into the target tissue may allow a higher drug concentration at or in the target cells or even in specific compartments of the target cells. As a result, drug efficacy can be enhanced.

The choice of carrier system to be used in drug targeting strategies depends on which target cells should be reached and what drug needs to be delivered. Carriers can be divided into particle type, soluble and cellular carriers. Particle type carriers include liposomes, lipid particles (low and high density

lipoproteins, LDL and HDL, respectively), microspheres and nanoparticles, and polymeric micelles. Soluble carriers consist of monoclonal antibodies and fragments, modified plasma proteins, peptides, polysaccharides, and

biodegradable carriers consisting of polymers of various chemical composition.

1.13

Drug Targeting in Cancer Therapy

In most Western countries, cancer is the second most common cause of death among adults. Great progress has been made in the treatment of selected tumours and approximately 50% of all tumours can be cured by current treatment strategies.

23

Radiotherapy and chemotherapy have greatly improved the management of patients with a variety of solid and haematologic tumours. However, most metastatic solid tumours remain largely incurable because of insufficient tumour selectivity of anti-cancer agents and poor penetration in the tumour mass [69].

1.13.1 Currently Available Therapeutics

Radiation

therapy

and

chemotherapy,

non-surgical

methods

of

cancer

treatments, are procedures that kill cells. The main problem with these treatments is that they do not provide specificity for cancer cells. In the case of radiation therapy, the radiation is localized to the tumour in order to increase the specificity. For anti-cancer drugs, the rapid proliferation of many of the cancer cells makes them more sensitive to cell killing than normal cells. However, both therapeutic treatments are limited by their cytotoxic effects on normal cells. In radiotherapy, the radiation dose is limited by normal tissue surrounding the tumour. For anti-cancer drugs, the killing of rapidly dividing normal cells limits the dose that can be given.

1.13.2 Strategies to Deliver Drugs to Targets within the Tumour

Many approaches have been developed to increase the therapeutic index by improving the specificity and efficacy of the drug and reducing the toxicity. One example of these approaches is to target the cytotoxic agent to the tumour cells. The inherent features of cancer cells can be used in the development of targeting agents for tumour cells.

For targeting cytotoxic agents, various strategies have been developed. These include:

1) Monoclonal antibodies (MAb) against tumour-associated antigens or growth factors using their intrinsic activity or used as carriers to target cytotoxic drugs, radionuclides and toxins.

24

2) Bispecific monoclonal antibodies (BsMAb) which combine the specificity of two different antibodies within one molecule and crosslink an effector cell or a toxic molecule with the target cell.

(3) Pro-drugs in conjunction with enzymes or enzymeMAb conjugates.

(4) Synthetic copolymers as drug carriers.

(5) Liposomes as carriers for drug delivery.

1.13.3 Site Specific Drug Delivery Using Monoclonal Antibodies

Antibodies are complex proteins, consisting of multiple polypeptide chains that contain a variety of reactive chemical groups, such as amino, carboxyl, hydroxyl, and sulfhydryl. The basic structure of all antibody or immunoglobulin (Ig) molecules consists of 4 protein chains shaped like a capital letter "Y" and linked by disulphide bonds. There are two pairs of chains in the molecule as heavy and light chains.

The discovery of antibodies were first reported by Paul Ehrlich [70]. Most antibodies used in cancer diagnosis and therapy are derived from the IgG isotype. Its basic monomer structure is shown in Figure 1.8.

25

Figure 1.8

Schematic diagram of an immunoglobulin (IgG)

Each chain is divided into regions or domains consisting of around 110 amino acid residues. The light chain has two domains and the heavy has four. The Nterminal domain at the tip of the arms of the "Y" on both the heavy and light chain are known to be variable in amino acid sequence composition and are thus called variable domains (VL and VH). An IgG isotype antibody consists of two antigen-binding fragments (Fabs), which are connected via a flexible region (the hinge) to a constant (Fc) region. This structure comprises two pairs of polypeptide chains, each pair containing a heavy and a light chain of dissimilar sizes. Both heavy and light chains are folded into immunoglobulin domains. The variable domains in the amino-terminal part of the molecule are the domains that identify and bind antigens; the rest of the molecule is composed of constant domains that vary among immunoglobulin classes.

The Fc portion of the immunoglobulin serves to bind a variety of effector molecules of the immune system, as well as molecules that establish the biodistribution of the antibody [71]. In nature, an antibodys function is to recognize an antigen and, by cross reaction with other immune proteins, to

26

initiate an immunological response. This response should direct the removal of the antigen or the cell bearing the antigen as a result of the antigen/antibody recognition. In 1976, Kohler and Milstein generated continuous hybridoma cell line which is capable of producing monoclonal antibody (MAb) of a defined specificity [72]. This property makes MABs excellent candidates as carriers of therapeutic agents for delivery to specific sites.

Monoclonal antibodies (MAbs) possess a molecular polarity. This polarity is based on the joining of an antigen-binding fragment (Fab) to a complementfixing fragment (Fc). The Fab fragment is responsible for specific antigen binding, whereas the Fc fragment binds to effector cells, fixes complements [8].

When antibodies are used as a drug delivery system, either alone or when conjugated, size, charge, antigen specificity, and affinity of them are important. For example, some antibody molecules may be degraded rapidly and excreted while others may have longer half-lives [73-75]. For the production of Mabs, a wide range of animal species can be used to produce. At the present time, production of MAbs is predominantly limited to mice, rats, and, to some extent, humans [76].

Since there is the greatest need for target-site specificity in drug targeting and delivery using antibodies, this area has been most useful in the field of chemotherapy. Use of MAbs in targeting cytotoxic drugs to specific tissues has been studied for over 20 years. Antibodies have been found to have many applications in the management of human carcinomas, including colorectal, gastric, ovarian, endometrial, breast, lung, and pancreatic.

27

1.14

Aim of the study

The aim of this study was to prepare chitosan-polyethylene glycol (CH-PEG) matrices in the form of microspheres for drug delivery and targeting. For this purpose, CH-PEG microspheres were prepared in different compositions by water/oil emulsification method. The release experiments of a chemotherapatic drug, methotrexate (MTX), were studied in vitro by dialysis method and the amount of drug releases was analyzed by UV-spectrophotometry. Some microspheres were conjugated to IgG as tumor antibodies. The cytotoxicities of free drug and drug containing microspheres were determined by measuring the inhibation of cell growth in MCF-7 and MDA-MB breast cancer cell lines by MTTbased cytotoxicity assay.

Also, CHF-PEG films with the same composition as the microspheres were prepared to search surface properties as well as mechanical properties for a possible design of a controlled release system.

28

CHAPTER 2

MATERIALS AND METHODS

2.1

Materials

Materials used in this study and their manufacturers are listed in Table 2.1. Table 2.1
Materials Chitosan (DDA=85%) Poly(ethylene glycol) (Mw=14000) Acetic Acid (99-100%) Gluteraldehyde (50%) Methotrexate Tween 80 Immunoglobulin G (IgG) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) N-hydroxyl succinimide (NHS) Acetone Lysozome Dialysing Tube Corn Oil Millipore Sigma, USA Merck, Germany Datex Applichem, Germany Sigma, USA Saynlar, Turkey Millex, France

Materials and Manufacturers

Manufacturers Sigma, USA Aldrich, USA J.T. Baker, Netherlands BDH, UK Pharmachemie B.V., Netherlands Acros Organics, USA Jackson Immuno Research, USA Sigma, USA

29

Phosphate buffer solution (0.01 M, pH 7.4) was prepared by dissolving 533.87 mg sodium phosphate and 1091.37 mg disodium hydrogen phosphate-2hydrate in 1 L distilled water.

2.2 2.2.1

Methods Preparation of Microspheres

Chitosan microspheres were prepared by using different concentrations of gluteraldehyde (GA). For this reason, chitosan solutions were prepared by dissolving chitosan in 5% (v/v) acetic acid to form 3% (w/v) chitosan solution. GA solutions (1.25%, 2.50% and 5.00% (v/v)) were used as crosslinker for each solution separately. Then 10 mL of these solutions were suspended in 50mL corn oil with addition of 0.5 mL tween 80 and were stirred at 1000 rpm for 30 minutes. 1 mL gluteraldehyde was added twice at 15th and 30th minutes (total 2 mL) by stirring at room temperature. The reaction was carried out for 5 hours at room temperature with 1000 rpm stirring. Then the microspheres were filtered off, washed several times with acetone and then with diethly ether and dried at 50C for 12 hours.

Chitosan-PEG semi-interpenetrated microspheres were prepared from chitosan and PEG solutions. Chitosan-PEG solutions were prepared by dissolving in 5% (v/v) acetic acid to form 3% (w/v) chitosan solution. 10 mL chitosan-PEG solution was dispersed in 50 mL corn oil containing 0.5 mL Tween 80, to form water-in-oil emulsions. Solution was stirred at 1000 rpm for 30 minutes and 1 mL 5% (v/v) gluteraldehyde solution was added at 15 minutes intervals twice by stirring at room temperature. Then the reaction was carried out for 5 hours at room temperature with 1000 rpm stirring. Then the microspheres were filtered off, washed several times with acetone and then with diethlyether and dried at 50C for 12 hours. A schematic representation of the technique was given in Figure 2.1.

30

Chitosan or Chitosan-PEG solution in acetic acid

Mechanical stirrer

Gluteraldehyde

Filtering and washing Microsphere formation 5 h stirring

Corn oil

Tween 80

Chitosan or Chitosan-PEG microspheres

Figure 2.1

Schematic representation of water-oil emulsion method

2.2.1.1

Preparation of Drug-loaded Microspheres

In order to prepare methotrexate (MTX) loaded microspheres, 5 mg MTX in 2.5 mL was added into the 10 mL of chitosan-PEG solution in 5% (v/v) acetic acid at the beginning of the microsphere preparation process. Then the same procedures were applied as described previously by adding this 12.5 mL solution into 50 mL corn oil. Various types of microspheres prepared in this study are given in Table 2.2.

31

Table 2.2

Prepared microspheres
Concentration of Chitosan in total mixture (%w/v) Concentration of PEG in total mixture (% w/v) 1.5 3.0 6.0 1.5 3.0 6.0 Concentration of Gluteraldehyde (% v/v), 2 mL 1.25 2.5 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Amount of Methotrexate (mg)

Sample

U-CH 1.25 U-CH 2.5 U-CH 5 U-CH-PEG 1-0.5 U-CH-PEG 1-1 U-CH-PEG 1-2 CH 5 CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2

3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0

2.2.2 Characterization of Microspheres

2.2.2.1

Morphological Analysis

The morphology of microspheres was examined by a scanning electron microscope (SEM, Jeol Model 6400). For this purpose, the samples were coated with gold under vacuum and their scanning electron micrographs were obtained.

2.2.2.2

Particle Size Analysis

Particle size analysis were performed on samples of microspheres suspended in acetone using Malvern Mastersizer S Version 2.15 equipment. The average size and size distribution curves were obtained.

32

2.2.3 Preparation of Chitosan and Chitosan-PEG Films

Chitosan-PEG solutions with the same compositions as in the microspheres were prepared by dissolving 300 mg chitosan (degree of deacetylation=85%) and different amounts of PEG (150, 300, 450, 600 mg) in 30 mL of 5%

aqueous acetic acid solution at ambient temperature with stirring. CHF-PEG films were crosslinked with different concentrations of gluteraldehyde to obtain films of various degrees of crosslinking (Table 2.3). The concentrations of gluteraldehyde solutions were; 0.1%, 0.5%, 1.0% (v/v). 3 mL of each solution was added to 30 mL CHF-PEG solution and stirred 30 minutes prior to putting into molds. Solutions (30 mL) were put into plastic petri dishes (diameter=9 cm) and films were obtained after evaporation of water at room temperature. The thickness of the films measured with micrometer demonstrated different thicknesses in the range of 30 Table 2.3 m and 100 m.

Prepared chitosan and chitosan-PEG films

Concentration of Chitosan (%w/v) Concentration of PEG (% w/v) 0.5 0.5 0.5 1.0 1.0 1.0 1.5 1.5 1.5 2.0 2.0 2.0 Concentration of Gluteraldehyde (% v/v), 2 mL 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0

Sample

CHF 0.1 CHF 0.5 CHF 1.0 CHF-PEG 1-0.5-0.1 CHF-PEG 1-0.5-0.5 CHF-PEG 1-0.5-1.0 CHF-PEG 1-1-0.1 CHF-PEG 1-1-0.5 CHF-PEG 1-1-1.0 CHF-PEG 1-1.5-0.1 CHF-PEG 1-1.5-0.5 CHF-PEG 1-1.5-1.0 CHF-PEG 1-2-0.1 CHF-PEG 1-2-0.5 CHF-PEG 1-2-1.0

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

33

2.2.4 IR Analysis

Structural changes of the prepared films were examined by using Perkin Elmer 1600 Series FTIR.

2.2.5 Differential Scanning Calorimetry (DSC) Analysis

DSC thermograms of the prepared films were obtained by using DuPond 2000 Differential Scanning Calorimeter. Samples were heated at a scanning rate of 10C/min using dry nitrogen flow. Heating curves with a rate of 10C/min were obtained.

2.2.6 Mechanical Tests

Tensile tests were carried out for the prepared CHF-PEG films crosslinked with different amount of gluteraldehyde (GA). Samples were cut as rectangular strips. The gauge length was 302 mm and the width was 10 mm for each sample. The thickness of each specimen was measured at two ends and at the middle by a micrometer and the average of these values was used in calculations. At least 5 experiments were carried out for each type of films and average values of mechanical properties were calculated.

LLOYD LRX 5K (LLOYD Instrument, ENGLAND), equipped with a 100 N load cell, was used for mechanical testing experiments (Figure 2.2). The mechanical test machine was under the control of a computer running program WindapR. During measurement, the film was pulled by top clamp at a rate of 3 mm/min. The tensile load applied on the specimen was continuously recorded by the computer. The tensile strength for each specimen was obtained from the equation of =F/A where is the tensile strength (in MPa), F is the maximum load (in N) applied just before rupture and A is the initial area (mm2) of the specimen.

34

The load deformation curve was converted to stress-strain curve, where stress is the load per unit area (F/A as pascal) and strain is deformation per unit length (l/l0, where l0 is the initial length and l is the change in the length). Slope of the straight line exist in elastic region of the stress-strain curve is accepted as the elastic modulus (in GPa) of the specimen. F versus l/l0 graphs are given in Appendix D.

2.2.7 Contact Angle Measurement

Control samples and crosslinked CHF-PEG film samples were used in contact angle measurements for the investigation of hydrophobicity-hydrophilicity change at the surface by the content of PEG and GA concentrations. Contact angles of the samples were obtained by goniometer (CAM 200, Finland) immediately after putting deionized distilled water droplets on the polymer surfaces taken at room temperature. At least 5 measurements were obtained for each sample and average values were calculated.

2.2.8 Conjugation of IgG to Microspheres

Conjugation

with

IgG

experiments

were

carried

out

for

CH-PEG

1-1

microspheres. For this purpose 3 mg of microspheres were incubated overnight at +40C in the presence of 250 L from stock of 2.5 mg/mL 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC), 250 L immunoglobulin G (IgG) and 10 L from stock of 0.92 mg/mL N-hydroxyl succinimide (NHS). After 24 h incubation, conjugated microspheres were washed with PBS (0.01M, pH 7.4) solution. Then microspheres left under vacuum to remove water. Confocal microscopy and cell studies were achieved in order to examine IgG binding.

35

2.2.9 Degradation of Microspheres

10 mg of microspheres were incubated in 10 mL PBS (0.01 M, pH 7.4) with 30 mg of lysozyme for 60 days. In certain periods little amount of samples were taken to observe the change in the shapes of the microspheres by stereo microscopy and SEM. Also, hydolytic degradation of microspheres were studied. For this purpose, 10 mg of micropsheres were placed into PBS buffer (0.01 M, pH 7.4) at 37oC under unstirred conditions for 60 days and then these microspheres were taken out and frozen in liquid nitrogen and examined by SEM.

2.2.10 In-vitro Release Studies

In-vitro MTX release profiles from microspheres were obtained by using dialysis method. For this purpose 100 mg microspheres, loaded with MTX, were placed into a dialysis tube (molecular weight cut off 12000 D), then soaked in 10 mL phosphate buffer solution (0.01 M, pH 7.4). The samples were put into a shaking water bath at 37C. At certain time intervals dissolution medium was withdrawn and immediately replaced with equal volumes of fresh PBS. The removed solutions were analyzed spectrophotometrically at =259 nm in order to determine the amount of released MTX by using a calibration curve (Appendix A).

For the investigation of release kinetics; zero-order (1), first-order (2), Higuchi (3) and the KorsmeyerPeppas (4) semi-empirical equations, which are given below, were used; Qt=Q0+k0t (1)

where, Qt is the amount of drug released at time t, Q0 the amount of drug in the solution at t=0, (usually, Q0=0) and k0 is the zero-order release constant. Qt=Q(1ek1t)

(2)

36

where, Q being the total amount of drug in the matrix and k1 the first-order kinetic constant. Qt=kHt1/2 where, kH is the Higuchi rate constant. Furthermore, the KorsmeyerPeppas (4) semi-empirical model was also applied. Qt/Q=ktn (4) (3)

where, Qt/Q is the fraction of drug released at time t; k is a constant comprising the structural and geometric characteristics of the tablet; and n is the release exponent where it is a parameter which depends on the release mechanism.

2.2.11 Cell Studies

MCF-7 cell line was routinely cultivated in RPMI 1640 supplemented with 10% FBS (Fetal bovine serum), penicillin (100 U/mL) and streptomycin (100 mg/mL) at 37oC, and under 5% CO2 atmosphere. 6x103 cells were seeded into each well of a 96-well plate and incubated for 24 h at 37oC. Then, each well of the cell cultures were exposed to 100 microspheres in 100 L) . L of polymer test specimens (0,1 mg

After, 144 h (6 days) incubation time and 240 h (10 days) incubation time, cells were microphotographed (in the wells in growth medium) by Olympus (CK 40, Japan with camera attachment).

After 144 h and 240 h incubation, exposure of the cells to polymers was stopped by discarding the medium. The numbers of cells survived determined by using MTT assay which measures reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide to a purple formazan product by using the calibration curve (Appendix C). This assay estimate cell viability and

proliferation as follows. After discarding the exposure medium, 0.5 mg/mL of

37

MTT (in Dulbeccos modified PBS) were added to each well and incubated at 37oC under 5% CO2 atmosphere for 4 h. After that, 100 L of dimethyl sulphoxide (DMSO) was added to each well to

dissolve the formazan salts. MCF-7 cells were cultured with microspheres and with free drug for 6 and 10 days.

MCF-7 (human breast adenocarcinoma) and MDA-MB (human causasian breast carcinoma) were routinely cultivated in RPMI 1640 supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 mg/mL) at 370C, and 5% CO2 atmosphere. 1.103 cells were seeded into each well of a 96-well plate and incubated for 24 hours at 37oC. Samples in each eppendorf tube were diluted with 1 mL cell culture medium. Then, the cell cultures were exposed to 100 L

of specimens. After, 144 hours incubation period cells were photographed by a microphotographer. MCF-7 and MDA-MB cells both were cocultured with the microsphere samples and with free drug as control for 6 days. Photographs of these cultured and cocultured samples were taken and then cell absorbance of all samples were measured at 570 nm by UV visible spectrophotometer (VersaMax, molecular device, USA) (Appendix C).

Table 2.4

Prepared samples for cell culture experiments

Sample Sample Content Only cell culture O.25 mg/mL free drug 2.5 mg/mL free drug Unloaded microspheres Drug loaded microspheres Conjugated unloaded microspheres Conjugated drug loaded microspheres

Control MTX-0.25 MTX-2.5 U-CH-PEG 1-1 L-CH-PEG 1-1 CU-CH-PEG 1-1 CL-CH-PEG 1-1

38

CHAPTER 3

RESULTS & DISCUSSION

3.1

Chitosan and Chitosan-PEG Microspheres of Crosslinker on Size and Shape of the Chitosan

3.1.1 Effect Microspheres

Chitosan microspheres were prepared by using water-oil emulsion method and glutaraldehyde (GA) was used as crosslinker. Aldehydes can react with amino groups of proteins. GA has two reactive aldehyde groups in one molecule and has been used as a crosslinker of collagen and other proteins and biological soft tissues [77]. Crosslinking reaction between chitosan and glutaraldehyde is shown in Figure 3.1.

HOH2C O O HO

HOH2C O O N CH CH2 CH2 CH2 CH HO

HOH2C O N CH CH2 CH2 CH2 CH HO N HC CH2 CH2 CH2 CH HO O O O HOH2C N

HO O

HO O

O HOH2C HOH2C

Figure 3.1

Crosslinking reaction of chitosan and glutaraldehyde

39

In the preparation of microspheres, 2 mL of GA solutions with different concentrations were used in order to obtain various crosslinking degree and the proper spherical shape of the microspheres. As the GA concentration was increased, color changed from pale yellow to brownish. This color change is due to the reaction between chitosan amino groups and aldehydes which involves the formation of a Schiff base, which is accompanied by color formation and is called maillard reaction [78]. SEM micrographs show the differences in the structures of chitosan microspheres prepared by using different concentration of GA (Figure 3.2).

Figure 3.2 SEM micrographs of microspheres (A) U-CH 1.25, (B) U-CH 2.5, (C) U-CH 5

40

For the chitosan microspheres prepared with 1.25 % GA, the obtained microspheres does not have properly spherical shape (Figure 3.2 A). When GA concentration was increased to 5 %, proper spherical microspheres with uniform size were obtained (Figure 3.2 C).

For the preparation of CH-PEG semi-IPN microspheres, 5% GA concentration was chosen and kept constant and the amount of PEG was altered. SEM micrographs of the prepared CH-PEG microspheres are shown in Figure 3.3.

Figure 3.3

SEM micrographs of microspheres (A) U-CH-PEG 1-0.5, (B) U-CHPEG 1-1, (C) U-CH-PEG 1-2

41

3.2

Particle Size Analysis of Microspheres

Particle size distribution curves of the microspheres were obtained in acetone and the obtained results are given in Appendix B. Average volume mean diameters are given in Table 3.1 and Figure 3.4.

Table 3.1

Sizes of different microspheres Average Size ( m) 144.23

Type of the Microspheres U-CH 1.25

U-CH 2.5

97.06

U-CH 5

90.99

U-CH-PEG 1-0.5 U-CH-PEG 1-1

107.53 116.37

U-CH-PEG 1-2

162.90

180 Average Size of the Microspheres ( m) 160 140 120 100 80 60 40 20 0 U-CH 1.25 U-CH 2.5 U-CH 5.0 U-CH-PEG 1-0.5 U-CH-PEG 1-1 U-CH-PEG 1-2 Type of Microsphere

Figure 3.4

Size distribution of unloaded microspheres

42

The particle size of the microspheres are affected by the preparation parameters such as stirring rate, crosslinking degree, degree of deacetylation of chitosan and chitosan solution concentration. Previously it was shown that as the concentration of chitosan solution increases, mean particle sizes of the microspheres increases and as the stirring speed increases, mean particle sizes of the microspheres decreases [32].

In this study, a constant concentration of chitosan and stirring rate was applied as 3.0% CH in 5.0% acetic acid and 1000 rpm, respectively. The one parameter that affect particle size of the microspheres is that the crosslinking degree. Increase in the concentration of crosslinker caused a decrease in the average mean diameters of the chitosan microspheres from 144.23 m to 90.99 m as

expected. The microspheres with higher concentration of crosslinker are more compact in comparison with lower concentration of crosslinker due to the high degree of crosslinking.

For CH-PEG semi-IPN microspheres, the amount of PEG is the other factor affecting the size of the microspheres. As PEG content increases, particle size of the microspheres increases causing the formation of bigger microspheres. When the amount of PEG increases in the reaction medium, this also increases the total amount of the polymer in the microspheres.

The sizes which were given up to now were the volume mean diameter of the microspheres. The size distribution which is given as percentage of the microspheres under a certain size are given in Table 3.2.

43

Table 3.2

Particle size analysis results

D (v, 0.1) ( m) D (v, 0.5) ( m) D (v, 0.9) ( m) VMD ( m) SMD ( m)

Type of the microsphere

CH 1.25 CH 2.5 CH 5 CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2

92.24 53.43 44.15 48.37 49.21 82.34

139.96 87.45 80.98 102.02 107.36 156.84

206.67 157.35 155.54 178.35 197.64 252.83

144.23 97.06 90.99 107.53 116.37 162.90

67.13 43.50 37.07 39.62 43.00 67.90

D (v, 0.1) is the size of particle for which 10% of the sample is below this size. D (v, 0.5) is the size of particle at which 50% of the sample is smaller and 50% is larger than this size. This value is also know as the mass median diameter (MMD). D (v, 0.9) gives a size of particle which 90% of the sample is below this size. D (4, 3) is the volume mean diameter (VMD). SMD is the surface mean diameter [D (3, 2)] also known as the Sauter mean.

3.3

Drug Loading to Microspheres

Methotrexate was loaded to microspheres during the preparation process. The size and shape of the microspheres were not affected by loading Methotrexate according to SEM micrographs (Figure 3.5).

44

Figure 3.5 SEM micrographs of drug loaded microspheres (A) CH 5, (B) CHPEG 1-0.5, (C) CH-PEG 1-1, (D) CH-PEG 1-2

3.4

In Vitro Release Studies

UV spectrum of methotrexate showed maximum absorption at 259 nm. This wavelength was used for the determination of methotrexate in the preparation of calibration curve and in the detection of the amount of methotrexate released from microspheres. The release behaviour of methotrexate for each sample (CH 5, CH-PEG 1-0.5, CH-PEG 1-1 and CH-PEG 1-2) are given in Figure 3.6.

45

Release of MTX from CH 5 Microspheres

Amount of MTX released ( g) 800 700 600 500 400 300 200 100 0 0 200 400 Time (hr) 600 800 1000

Release of MTX from CH-PEG 1-0.5 Microspheres

Amount of MTX released ( g) 900 800 700 600 500 400 300 200 100 0 0 200 400 Time (hr) 600 800 1000

Release of MTX from CH-PEG 1-1 Microspheres

1000 900 800 700 600 500 400 300 200 100 0 0 200 400 Time (hr) 600 800 1000 Amount of MTX released ( g)

Release of MTX from CH-PEG 1-2 Microspheres

Amount of MTX released ( g) 1200 1000 800 600 400 200 0 0 200 400 Time (hr) 600 800 1000

Figure 3.6

Release of MTX from CH and CH-PEG microspheres

46

It was observed that as PEG concentration increases in the microspheres, the release rate of drug also increases (Figure 3.7). This result can be explained by high solubility and diffusivity of PEG in aqueous media. Release from microspheres can take place via number of routes including surface, total sphere disintegration, microsphere hydration (swelling), drug diffusion and desorption with attack by enzymes mainly effecting in vivo microsphere breakdown. In order to permit a steady and controlled release of drug from matrix, the microsphere stability and microsphere biodegradability has to be balanced. In this study, microspheres were crosslinked with GA in order to achieve microsphere stability as well as controlled degradation and it is assumed that the drug release from microspheres occurred by microsphere hydration (i.e swelling), by a slow degradation of the microspheres, diffusion of the PEG and drug through the crosslinked chitosan matrix.

Amount of MTX released ( g)

1200 1000 800 600 400 200 0 0 200 400 600 800 1000 Time (day) CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2

Figure 3.7

Release of MTX from different type of the microspsheres

47

Initial and late release rates are given in Table 3.3. Table 3.3 Release rates of MTX from different type of microspheres Composition (CH/PEG) (w/w) 0.3/0.15 0.3/0.3 0.3/0.6 Release Rates ( g.h-1) Initial CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2 1.86 2.32 2.58 2.93 Late 0.19 0.18 0.24 0.22

Type of the microsphere

The amount of MTX that was entrapped, the maximum amount that was released from microspheres and the calculated encapsulation efficiencies are given in Table 3.4.

Table 3.4

Amount of MTX entrapped in different type of the microspheres

Theoritical amount of MTX ( g) Total entrapped MTX ( g) Encapsulation efficiency (%) 678.97 810.15 896.69 1077.38 13.58 16.20 17.93 21.55

Type of the Microspheres

CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2

5000 5000 5000 5000

Taking the total released amount of MTX as 100% for all microspheres, percent release of MTX is given in Figure 3.8. Encapsulation efficiencies were found to be quite low in the range of 13.58-21.55%, demonstrating parellel increase with PEG content. Initial release rates increases with increasing PEG content because of increasing in porosity of the microspheres.

48

1,2

0,8

% Release

0,6

0,4

0,2

0 0 200 400 Time (hr) CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2 600 800 1000

Figure 3.8

Percent MTX release from microspheres by taking total released MTX as 100%

In order to investigate the mode of release from microspheres, the release data were analyzed with zero-order kinetic, first order kinetic, square root of time equation (Higuchi equation) and Korsmeyer equation. The release rate kinetics data for all the systems are given in Table 3.5.

49

Table 3.5 Release Kinetics

Sample Ko CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2 Sample K1 CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2 Sample KH CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2 Sample KKP CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2 0.7795 0.8730 0.8574 1.001 0.4550 0.5380 0.6273 0.7304 Korsmeyer R2 0.9624 0.9223 0.9680 0.9120 n 0.4398 0.4540 0.4684 0.4787 0.0013 0.0013 0.0014 0.0013 Higuchi R2 0.9612 0.9307 0.9646 0.9128 0.6144 0.7142 0.8542 0.9640 First Order R2 0.6854 0.6022 0.7096 0.5792 Zero Order R2 0.8230 0.7712 0.8411 0.7475

50

The plots of all kinetic models are shown in Figures 3.9-3.12.

25 Cumulative (%) drug release 20 15 10 5 0 0 200 400 Time (hr) CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2 600 800 1000

Figure 3.9

Zero-order release kinetic model plot for various microspheres

8 7 6 5 ln Mt 4 3 2 1 0 0 200 400 Time (hr) CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2 600 800 1000

Figure3.10

First order relase kinetic model plot for various microspheres

51

25 Cumulative (%) drug release 20 15 10 5 0 0 5 10 15 t1/2 CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2 20 25 30 35

Figure3.11

Higuchi kinetic model plot for various microspheres

1,6 1,4 1,2 Log (Mt/M) 1 0,8 0,6 0,4 0,2 0 0 0,5 1 1,5 Log t CH CH-PEG 1-0.5 CH-PEG 1-1 CH-PEG 1-2 2 2,5 3 3,5

Figure3.12

Korsmeyer kinetic model plot for various microspheres

According to the highest correlation coefficient (R2) values, MTX release from chitosan microspheres have good correlation with Korsmeyer and also with Higuchi equation. CH-PEG 1-0.5 microspheres follows Higuchi equation. CH-PEG 1-1 microspheres showed linearity with Korsmeyer equation and CH-PEG 1-2 microspheres follows Higuchi equation and also Korsmeyer equation.

52

3.5

Conjugation of IgG to Microspheres

The prepared CH-PEG 1-1 microspheres were conjugated with immunoglobulin G antibody. The reaction is shown in Figure 3.13.

IgG C O OH

EDC H2 H+ H3C C N C N (CH3)3 N CH3 CH3 The reaction of the carbonyl group of IgG with EDC forming an activated peptide intermediate

IgG

C O H+ C N (CH2)3 N CH3 CH3 NH2 Chitosan MS IgG C O NH Chitosan MS + UREA The activated IgG reacting with amine group of chitosan to form IgG-chitosan conjugate

Figure 3.13 Conjugation of microspheres

EDC reacts carboxylic acid groups at the end of the attachment site of the IgG to form activated peptide intermediate. Then the activated IgG reacts with amine group of chitosan to form IgG-chitosan conjugate by yielding urea.

To investigate microspheres conjugated with IgG, confocal microscopy was used. However, we were not able to distinguish whether IgG moities were conjugated or not, because IgG and glutaraldehyde gives emission at the same wavelength. As can be seen from the Figure 3.14 conjugated microsphere is having all the same color. As a result of this, cell culture experiment was conducted.

53

Figure 3.14 Confocal microscopy images of microspheres (A) nonconjugated, (B) conjugated

3.6

Cell Culture and Coculture Studies

MCF-7 (human breast adenocarcinoma) and MDA-MB (human causasian breast carcinoma) both were breast carcinoma cell lines. MCF-7 cells were epithelial cells tightly attached to the flask surface MDA-MB cells are round shaped and loosely attached to the flask surface (Figure 3.15).

MCF-7

MDA-MB

Figure 3.15 Pictures of MDA-MB and MCF-7 coculture

54

Before testing the cytotoxic effects of microspheres, free drug was tested for its cytotoxicity effects on MCF-7 cell line. For that reason, 6x103 MCF-7 cells were seeded into each well of a 96-well plate and incubated for 24 h at 37oC. The concentrations of MTX are 0.25 mg/mL and 2.5 mg/mL. Then, each well of the cell cultures were exposed to test specimens. After 144 h and 240 h incubation, exposure was stopped by discarding the medium. Cell survival after exposure was determined using a MTT assay which measures reduction of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to a purple formazan product, was used to estimate cell viability and proliferation as follows. After discarding the exposure medium, 0.5 mg/mL of MTT (in Dulbeccos modified PBS) were added to each well and incubated at 37oC under 5% CO2 atmosphere for 4 h. After that, 100 L of dimethyl sulphoxide (DMSO) was added to each

well to dissolve the formazan salts. The number of cells alive 0.25 mg/mL and 2.5 mg/mL MTX incubated groups after 144 and 240 hours are given in Figure 3.16.

9 8 7 6 5 4 3 2 1 0 144 Time (hr) Control 0.25 mg/mL MTX 2.5 mg/mL MTX 240

Figure 3.16 The number of cells of control, 0.25 mg/mL and 2.5 mg/mL MTX incubated groups

As seen from Figure 3.14, the cell viability was decreased as expected as the concentration of MTX in the culture media was increased. After 240 hours of exposure, almost no live cells were observed for the samples which exposed to 2.5 mg/mL MTX while the samples which were treated with 0.25 mg/mL MTX had ~1.103 live cells. Light microscopy photographs of the cell plates with 0.25

C e ll n u m b er (x 10 00 )

55

mg/mL and 2.5 mg/mL MTX after 144 hours and 240 hours are given in Figure 3.17.

Figure 3.17 Pictures of cell plates containing (A) MTX-0.25 after 144 hours, (B) MTX-2.5 after 144 hours, (C) MTX-0.25 after 240 hours, (B) MTX-2.5 after 240 hours

U-CH-PEG 1-1 and CU-CH-PEG 1-1 microspheres that were crosslinked with GA (2 mL, 5% v/v) were studied with MCF-7/MDA-MB coculture because MCF-7 cells have estrogen receptors while MDA-MB do not have those receptors. The purpose of the coculture of MCF-7 and MDA-MB cells was to observe the specific activity of IgG conjugated micropsheres to the MCF-7 cells. Light microscopy photographs of the cell plates after 144 hours are given in Figure 3.18.

56

Figure 3.18 Pictures of cell plates after (A) 144 hours control, (B) 144 hours MTX-2.5, (C) 144 hours MTX-0.25, (D) 144 hours with U-CH-PEG 1-1, (E) 144 hours with L-CH-PEG 1-1, (F) 144 hours CL-CH-PEG 1-1, (G) 144 hours CU-CH-PEG 1-1

57

As seen from Figure 3.18, as the size of the micropsheres are bigger than the cells and the cells were attached to the culture plate surface before the addition of the microspheres, IgG conjugated CH-PEG 1-1 microspheres could not bind the IgG receptors of MCF-7 cells. It was almost impossible to discriminate the binding differences between the two cell lines. Therefore, we only used only MCF-7 cell line for our following experiments.

The MCF-7 cell absorbance of control group, U-CH-PEG 1-1 incubated group after 144 hours and 240 hours are given in Figure 3.19.

9 8 Cell nu m b er (x1000) 7 6 5 4 3 2 1 0 144 Incubation time (hr) Control Group U-CH-PEG 1-1 240

Figure 3.19 The number of cells of control group and U-CH-PEG 1-1

As can be seen from Figure 3.19, U-CH-PEG 1-1 microspheres which do not carry any drug demonstrated some toxicity and affected cell viability. It is know that chitosan and PEG are non-toxic but the decrease in cell numbers may be the result of the release of PEG from microspheres which causes a change in the viscosity of medium and blocks the transfer of nutrients and this decreases the cell viability. For that reason, cell cultures were exposed to only PEG to examine cytotoxicity. In this experiment, 6 samples of 100 L of 0.5 mg/mL and 1.0

mg/mL PEG solutions were prepared and results have shown that PEG does not have cytotoxic effect (Figure 3.20).

58

14 The n um ber of cells (x1000) 12 10 8 6 4 2 0 Control PEG (0.5 mg/mL) PEG (1.0 mg/mL)

Figure 3.20 The number of cells of control and PEG

The other reason for toxicity of unloaded microspheres might be caused by crosslinking agent which is gluteraldehyde because it is well known that gluteraldehyde is toxic.

In order to test the cytotoxicity of MTX loaded microspheres, the cell cultures were exposed to 100 microspheres/100 L of polymer test specimens which contains 0.1 mg

L. The cell viability decreased when compared with control

group. The obtained results after 144 and 240 hours are given in Figure 3.21.

9 8 Cell nu m b er (x1000) 7 6 5 4 3 2 1 0 144 Incubation time (hr) Control Group L-CH-PEG 1-1 240

Figure 3.21 The number of cells of control and MTX loaded CH-PEG 1-1

59

In general, cytotoxicity results indicated that the cell viability was decreased as the amount of drug in the culture medium was increased. Free drug showed a higher cytotoxicty than entrapped drug. Thus, it could be concluded that entrapment of this anticancer drug in CH-PEG microspheres produced a less cytotoxic effect. This was due to the sustained release of the drug from the microspheres.

3.7

Degradation Studies

Chitosan is degradable in vitro at a slow rate. In the presence of lysozyme, the degradation speed can be accelerated [79]. To mimic the in vivo degradation performance, 10 mg CH or CH-PEG microspheres were incubated in 10 mL PBS buffer (0.01M, pH 7.4) containing 30 mg lysozyme. In order to see the deformation in the structure of the microspheres, SEM analysis were performed. SEM micrographs of microspheres removed from the medium at certain time intervals are given in Figure 3.22.

60

Figure 3.22 SEM micrographs of microspheres (A) CH after 2 days, (B) CHPEG 1-1 after 2 days, (C) CH after 15 days, (D) CH-PEG 1-1 after 15 days, (E) CH after 60 days, (F) CH-PEG 1-1 after 60 days

61

As seen from Figure 3.22, when microspheres are placed into PBS buffer containing lysozyme, microspheres began to disintegrate slowly after 15 days. After 60 days, they do not maintain their original shapes. CH microspheres disintegrated more slowly than CH-PEG 1-1 microspheres. The extent of degradation and solubility of polymer depends upon the concentrations of crosslinkers used [80]. For that reason, because of crosslinking, complete disintegration could not be observed in 60 days since the degradation process appears to be very slow.

Also, hydrolytic degradation of the microspheres were examined (degradation by the hydrolysis of the amino/imine bonds present in microspheres). 10 mg of micropsheres were placed into PBS buffer (0.01 M, pH 7.4) at 37oC under unstirred conditions and freeze dried microspheres were examined by SEM in certain time periods. SEM micrographs of microspheres placed in PBS buffer (0.01 M, pH 7.4) are given in Figure 3.23.

Figure 3.23 SEM micrographs of microspheres (A) CH after 2 days, (B) CHPEG 1-1 after 2 days, (C) CH after 60 days, (D) CH-PEG 1-1 after 60 days

62

For hydrolytic degradation, the crosslinked CH and CH-PEG 1-1 microspheres when placed in PBS buffer (0.01 M, pH 7.4) at 37oC were found to maintain their shape and physical integrity for the studied period (Figure 3.23). This is due to the inherent hydrophobicity of the chitosan microspheres at high pH value.

3.8

Chitosan and Chitosan-PEG Films

CHF-PEG films were prepared by solvent casting method with the same composition as in microspheres. The thickness of CHF and CHF-PEG films were changing between 0.03 m to 0.10 m.

3.8.1 Infrared Analysis

Infrared spectra of chitosan films with different GA concentrations and CHF-PEG films with different PEG ratio are given in Figure 3.24 and Figure 3.25, respectively.

63

%T

4400

4000

3600

3200

2800

2400

2000

1800

1600

1400

1200

1000

800

600

cm-1

Figure 3.24 IR spectra of (A) CHF, (B) CHF 0.1, (C) CHF 1.0

Figure 3.24 shows the IR spectra of chitosan film and chitosan films crosslinked with different concentrations of gluteraldehyde. The chitosan film without crosslinker shows absorbtion bands 1014 cm-1 due to C-O strecthing. The O-H and N-H stretching bands of chitosan overlap in the 3000-3600 cm-1 region. For chitosan films crosslinked with GA, a peak forms at about 1630 cm-1 indicating the formation of C=N due to immine reaction between amino groups of chitosan and aldehydes groups of GA. The two peaks at about 2900 cm-1 indicates the

64

aldehydes C-H stretchings belonging to GA. These bands indicate the presence of both molecules in the film structure and thus proves that the crosslinking reaction occurs between them.

IR spectra of CHF-PEG films are given in Figure 3.25.

B
%T

4400

4000

3600

3200

2800

2400

2000 cm-1

1800

1600

1400

1200

1000

800

600

Figure 3.25 IR Spectra of (A) CHF-PEG 1-0.5, (B) CHF-PEG 1-1, (C) CHF-PEG 1-1.5, (D) CHF-PEG 1-2

65

Infrared spectroscopic study is often used to determine the interactions between chitosan and the counterpart polymers. The type of hydrogen-bonding within chitosan/counterpart polymer may be complicated because there are several groups that can form hydrogen bonds in chitosan [81]. The amine, residual amide, hydroxyl groups of chitosan can form hydrogen bonds with PEG (Figure 3.26). The peaks at 840, 961, 1248, 1271, 1460 cm-1 in films are the characteristic peaks of PEG. Peaks at 1100 cm-1 are assigned to the CO stretching of PEG. PEG has no amide carbonyl band. Nevertheless, we can get some information of hydrogen-bonding interactions from the change of the amide carbonyl band of chitosan itself. The amide carbonyl of chitosan shifted to lower wave number as PEG was added. The low wave number shift of the amide carbonyl band of chitosan can be attributed to its interaction with PEG.

HO CH2CH2 O H H CH2 OH HO

O CH2CH2 O NH2 H O HH O H

H O H

CH2 CH2 O H CH2OH O H H H n O NH2

H n

CH2CH2 O CH2CH2

O CH2CH2 OH

Figure 3.26 Chitosan-PEG interaction

66

3.8.2 Differential Scanning Calorimetry Analysis The thermal transition of chitosan, PEG and CHF-PEG films prepared with 0.1% GA were determined by DSC analysis. The DSC diagrams of chitosan and PEG are shown in Figure 3.27.

64.13 C
Exothermic Heat Flow (W/g)

113.67 C

-50

5 0

100

150

200

250

Temperature (C)

Figure 3.27 DSC curves of (A) Chitosan (DDA=85%), (B) PEG

The main feature in the chitosan curve is that there is a large endothermic peak at 113.67C. Similar remarkable endothermic peak has been reported by Chuang et al. [82], who attributed this peak to the dissociation process of interchain hydrogen-bonding of chitosan. Although chitosan has crystalline

67

regions, the crystalline melting temparature (Tm) was not observed mostly because of its rigid-rod polymer backbone having strong intra- and intermolecular bonding. This behavior is frequently detected in many polysaccharides such as cellulose and chitin derivatives. The semicrystalline PEG has a glass transition temperature (Tg) significantly below room temperature and a melting peak at 64.13C (estimated from DSC curve midpoint). The melting peak of PEG is affected by blending with chitosan, lower Tm and weaker melting peaks of PEG were observed within all CHF-PEG films prepared with 0.1% GA (Figure 3.28).

61.04 C

60.95 C Exothermic Heat Flow (W/g)

61.74 C

54.69 C

D
50 0 5 0 100 150 200 250

Figure 3.28 DSC curves of: (A) CHF-PEG 1-0.5-0.1, (B) CHF-PEG 1-1-0.1, (C)CHF-PEG 1-1.5-0.1, (D) CHF-PEG 1-2-0.1

68

3.8.3 Mechanical Tests CHF-PEG films, prepared at different crosslinking degree and different amount of PEG, were analyzed for their tensile properties. The mean ultimate tensile strength (UTS), modulus of elasticity (E) and strain at break (SAB) values of CHF and CHF-PEG films are given in Table 3.6.

Table 3.6 Mechanical properties of CHF-PEG films

SAMPLE Crosslinker Concentration (% v/v) CHF 0.1 CHF 0.5 CHF 1.0 CHF-PEG 1-0.5-0.1 CHF-PEG 1-0.5-0.5 CHF-PEG 1-0.5-1.0 CHF-PEG 1-1-0.1 CHF-PEG 1-1-0.5 CHF-PEG 1-1-1.0 CHF-PEG 1-1.5-0.1 CHF-PEG 1-1.5-0.5 CHF-PEG 1-1.5-1.0 CHF-PEG 1-2-0.1 CHF-PEG 1-2-0.5 CHF-PEG 1-2-1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 136.388.07 109.854.61 102.064.00 75.802.06 83.233.99 90.602.83 83.293.46 92.154.96 100.194.77 56.932.85 67.861.98 58.852.29 42.393.01 52.755.15 41.353.33 1.990.09 1.560.07 1.460.16 1.020.08 1.300.11 1.520.16 1.300.12 1.390.23 1.400.05 1.000.08 1.090.11 1.040.05 0.870.10 0.860.07 1.110.16 39.101.62 19.112.61 20.655.60 27.841.62 26.384.35 27.456.00 18.655.68 18.873.39 18.962.35 11.002.47 11.431.67 9.590.77 8.892.47 9.412.41 6.400.01 UTS (MPa) E (GPa) SAB (%)

69

The mean ultimate tensile strength (UTS) value of CHF 0.1 was found as 136,38 MPa. When GA concentration is increased from 0.1 % to 0.5 %, the UTS value decreased to 109.85 MPa. Further increase of GA concentration to 1 % decreases the UTS value to 102.06 MPa.

Tensile strength of polymers increases with the crosslinking degree, thus crosslinking polymers improves their mechanical properties. However, the presence of high amounts of crosslinker concentrations decreased UTS values of chitosan films (Figure 3.29).

160 140 120 UTS(MPa) 100 80 60 40 20 0 0.1 0.5 Concentration of GA (% v/v) 1.0

Figure 3.29 The effect of crosslinker on UTS values of CHF films

This situation can be explained by two reasons. The unreacted excess crosslinker if exist in the matrix acts as a plasticizer in the crystalline structure. Aldehyde polymers from homopolymerization of GA can exist in the structure. It is given in literature in the commercial products that these reactions occur [83]. Thus, as illustrated in Figure 3.30, some of the GA can be included in more complex graft polymers on the chitosan.

70

Figure 3.30 Chemical reaction between chitosan and gluteraldehyde

71

For CHF-PEG films, increasing GA concentration increases the UTS values of films as seen from Figure 3.31. However, presence of extra PEG caused a decrease in the mechanical properties.

The Effect of Crosslinker on CHF-PEG 1-0.5 Films

100 U S( P ) T Ma 80 60 40 20 0 0.1 0.5 Conce ntra tion of GA (v/v) 1.0

The Effect of Crosslinker on CHF-PEG 1-1 Films

120 100 U S( P ) T Ma 80 60 40 20 0 0.1 0.5 Conce ntra tion of GA (v/v) 1.0

The Effect of Crosslinker on CHF-PEG 1-1.5 Films

80 70 60 50 40 30 20 10 0 0.1 0.5 Concentration of GA (v/v) 1.0

U S( P ) T Ma

The Effect of Crosslinker on CHF-PEG 1-2 Films

70 60 U S( P ) T Ma 50 40 30 20 10 0 0.1 0.5 Concentration of GA (v/v) 1.0

Figure 3.31 The effect of crosslinker on UTS values of CHF-PEG films

72

Mechanical properties are also affected by the addition of PEG. The mechanical properties of CHF-PEG 1-0.5-0.1 and CHF-PEG 1-1-0.1 are 75.80 and 83.29 MPa, respectively and the addition of PEG increases the mechanical properties. However, the mechanical properties of CHF-PEG 1-1.5 -0.1 and CHF-PEG 1-20.1 were much lower as 56.93 and 42.39, respectively and it means that addition of more PEG decreases the mechanical properties as expected. Figure 3.32 illustrates the effect of PEG on CHF-PEG films. The same trend was observed for all CHF-PEG films prepared with 0.5 %GA and CHF-PEG films prepared with 1.0 % GA.

The Effect of PEG on Films 0.1 % GA (v/v)

100 80 UTS (MPa) 60 40 20 0 0.15 0.3 0.45 0.6 Amount of PEG (g)

The Effect of PEG on Films 0.5 % GA (v/v)

120 100 UTS (MPa) 80 60 40 20 0 0.15 0.3 0.45 0.6 Amount of PEG (g)

The Effect of PEG on Films 1.0 % GA (v/v)

120 100 UTS (MPa) 80 60 40 20 0 0.15 0.3 0.45 0.6 Amount of PEG (g)

Figure 3.32 The effect of PEG on UTS of CHF-PEG films

73

Due to attractive interaction between the hydroxly groups of chitosan and the hydroxyl groups of PEG, the mechanical properties of CHF-PEG films were improved. The hydrogen bond formed by the interaction between the two kinds of hydroxyl groups would be maximized for the proper proportion of chitosan to PEG (Figure 3.33). However if the PEG is added additonally which provides too many hydroxyl groups, these extra groups could also interact with other OH groups that would reduce the attractive force [84]. This decreases the mechanical properties of the films.

160 140 120 UTS (MPa) 100 80 60 40 20 0 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 Amount of PEG (g) 0.1 % GA 0.5 % GA 1.0 % GA

Figure 3.33 The effect of PEG on UTS of CHF-PEG films

Mean Elastic Modulus value of CHF 0.1 was found to be 1.99 GPa. Increasing GA concentration to 0.5 % and 1 %, the mean elastic modulus values decreased to 1.56 GPa and 1.46 GPa, respectively (Figure 3.34).

74

Modulus of CHF Films

2,5 2 E (G P a ) 1,5 1 0,5 0 0.1 0.5 Concentration of GA (%v/v) 1.0

Figure 3.34 Effect of crosslinker on modulus of CHF films

Mean Elastic Modulus values of CHF-PEG films with different PEG amounts and different GA concentrations are given in Figure 3.35. The modulus of CHF-PEG 1-0.5-0.1 is 1.02 GPa and the modulus of CHF-PEG 1-1-0.1 is 1.30 GPa. Inceasing amount of PEG inceases modulus of films like UTS values. However, the modulus of CHF-PEG 1-1.5-0.1 and CHF-PEG 1-2-0.1 are 1.0 GPa and 0.87 GPa, respectively. It means that modulus of films increased for the proper

proportion of chitosan to PEG, similar to UTS values and then decreased with increasing PEG concentration.

75

Modulus of CHF-PEG with 0.1 % GA (v/v)

1,6 1,4 1,2 E (GPa) 1 0,8 0,6 0,4 0,2 0 0.15 0.3 0.45 0.6 Amount of PEG (g)

Modulus of CHF-PEG with 0.5 % GA (v/v)

1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 0.15 0.3 0.45 0.6 Amount of PEG (g)

E (GPa)

Modulus of CHF-PEG with 1.0 % GA (v/v)

1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 0.15 0.3 0.45 0.6 Amount of PEG (g)

E (GPa)

Figure 3.35 Modulus of CHF-PEG films

76

3.8.4 Contact Angle Measurments

In

order

to

see

the

effect

of

crosslinker

and

PEG

on

the

surface

hydrophilicity/hydrophobicity of the prepared films, the static contact angles of films were measured by using a contact angle goniometer. The results of water contact angles of films are given in Table 3.7.

Table 3.7

Contact angles of prepared films Concentration of GA (v/v) Contact Angle

Sample

CHF CHF 0.1 CHF 0.5 CHF 1.0 CHF-PEG 1-1-0.1 CHF-PEG 1-1-0.5 CHF-PEG 1-1-1.0 CHF-PEG 1-2-0.1 CHF-PEG 1-2-0.5 CHF-PEG 1-2-1.0

0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0

97.938.45 100.154.26 95.913.57 79.030.76 80.195.62 88.342.00 68.271.67 70.961.89 68.394.99 69.142.19

It can be seen from the data obtained that there is no exact relation between the contact angle and crosslinker concentration. The contact angles of CHF-PEG films decreased by adding PEG. The enhanced hydrophilicity can be attributed to the presence of PEG chains on the material surfaces. PEG has hydrophilic polymer chains which would improve wettability. The result may also be attributed to the availability of the terminal hydroxyl groups of PEG since OH may possibly improve the hydrophilicity of the biomaterials. However, excess PEG content in the membranes did not significantly change the water contact angles, it even inceased slightly CHF-PEG films prepared with 0.1 % and 1.0 % GA (Figure 3.36). The increased contact angle suggests additional interactions that influence wettability of films.

77

100 90 80 70 60 50 40 30 20 10 0 0.1 0.5 Concentration of GA (% v/v) CHF-PEG 1-1 CHF-PEG 1-2 1.0

Figure 3.36 The effect of PEG on contact angles of CHF-PEG films

Contact Angle

78

CHAPTER 4

CONCLUSIONS

In this study, chitosan (CH) and chitosan-polyethylene glycol (CH-PEG) microspheres with different compositions were prepared by oil/water emulsion method and crosslinked with gluteraldehyde. A model chemotherapeutic drug, methotrexate (MTX), was loaded into microspheres. SEM, particle size and in vitro release analysis were performed. Then, CH-PEG microspheres were conjugated with a monoclonal antibody which is immunoglobulin G (IgG). The cytotoxicity efficiency of entrapped drug were determined by using MCF-7 breast cancer cell line along with MCF-7/MDA-MB cocultures to search for the specific efficiency of the drug loaded microspheres to MCF-7 cells. In the third part, CHF-PEG films with the same compositions as in microspheres hardened with gluteraldehyde films were prepared by solvent casting method. IR, DSC, mechanical and surface analysis were performed.

For the microspheres; Increase in the concentration of crosslinker caused more spherical CH microspheres and decreased the size of the CH microspheres from 144.23 m to 90.99 m. Amount of PEG is the other factor affecting the

size of the microspheres. As PEG content increased, particle size of the microspheres increased causing the formation of larger microspheres.

The amount of released MTX was analyzed spectrophotometrically at 259 nm. It was observed that the release trend of MTX slightly depended on the amount of PEG. Maximum release was increased as the amount of PEG in the structure increased. Encapsulation efficiencies

79

were found to be quite low in the range of 13.58-21.55%. According to the highest correlation coefficient (R2) values, MTX release from chitosan microspheres have good correlation with Korsmeyer and also with Higuchi equation, CH-PEG 1-0.5 microspheres follows Higuchi equation, CH-PEG 1-1 microspheres showed linearity with Korsmeyer equation and CH-PEG 1-2 microspheres follows Higuchi equation and also Korsmeyer equation.

Cytotoxicity results of empty microspheres indicated that even the microspheres which do not carry any drug, demonstrated some toxicity and affect the cell viability. This might be caused by crosslinking agent which is glutaraldehyde.

Degradation of microspheres in the presence of lysozyme and hydrolytic degradation were examined by SEM. After 60 days in PBS buffer containing lysozyme, microspheres do not maintain their original shapes but disintegration was not observed since the degradation process appeared to be very slow because of crosslinking. For hydrolytic degradation, the crosslinked microspheres were found to maintain their shape and physical integrity for the studied period.

For the films; From DSC analysis, although chitosan has crystalline regions, the crystalline melting temparature (Tm) was not found because of its rigidrod polymer backbone having strong intra- and inter-molecular bonding. PEG showed sharp melting peak at 64.13C. The melting peak of PEG is affected by blending with chitosan, lower Tm and weaker melting peaks of PEG were observed within all CHF-PEG films.

In the case of mechanical analysis, the results showed that CHF-PEG films had the required strength for biomedical applications. The

mechanical properties of films could be improved by the proper amount of PEG and additional PEG caused the properties to deteriorate.

80

The contact angles of CHF-PEG films was lowered by adding PEG. However, extra amounts of PEG in the membranes did not change the water contact angles significantly or even caused slight increase for CHFPEG films prepared with 0.5 % and 1.0 % GA. The increased contact angle suggests additional interactions that influence wettability of films.

This study demonstrated that the chitosan based systems can be modulated by changing the compositions for biomedical applications.

81

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APPENDIX A

0,6 0,5 A bs. at 259 nm 0,4 0,3 0,2 0,1 0 0 2 4 6 8 10 12 Conc. of MTX ( g/mL) y = 0,0559x - 0,0004 R2 = 0,9992

Figure A.1

Calibration curve for methotrexate (259 nm)

91

APPENDIX B

Figure B.1

The histogram table and plot of CH 1.25

92

Figure B.2

The histogram table and plot of CH 2.5

93

Figure B.3

The histogram table and plot of CH 5

94

Figure B.4

The histogram table and plot of CH-PEG 1-0.5

95

Figure B.5

The histogram table and plot of CH-PEG 1-1

96

Figure B.6

The histogram table and plot of CH-PEG 1-2

97

APPENDIX C

1,250 1,200 1,150 1,100 1,050 1,000 0,950 0,900 0,850 0,800 0,750 0,700 0,650 0,600 0,550 0,500 0,450 0,400 0,350 0,300 1 5 10 50 100 500 1000

Ab so rb an ce (570n m )

Cell Number (x1000)

Figure C.1

MTT calibtation curve

98

1,6

C e ll A b s o rb a n c e (5 7 0 n m )

1,4 1,2 1 0,8 0,6 0,4 0,2 0

Blank Control MTX-2.5 MTX-0.25 U-CH-PEG 1-1 L-CH-PEG 1-1 CL-CH-PEG 1-1 CU-CH-PEG 1-1

Figure C.2

Cell absorbance of MCF-7 cell culture after 6 days

1,6

Cell Absorbance (570 nm)

1,4 1,2 1 0,8 0,6 0,4 0,2 0

Blank Control MTX-2.5 MTX-0.25 U-CH-PEG 1-1 L-CH-PEG 1-1

Figure C.3

Cell absorbance of MCF-7 cell culture after 10 days

99

APPENDIX D

Figure D.1

Tensile test graph of CHF 0.1

Figure D.2

Tensile test graph of CHF-PEG-1-0.5-0.1

100

Figure D.3

Tensile test graph of CHF-PEG-1-1-0.1

Figure D.4

Tensile test graph of CHF-PEG-1-1.5-0.1

101

Figure D.5

Tensile test graph of CHF-PEG-1-2-0.1

102

103