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PREPARATION AND CHARACTERIZATION OF CHITOSAN-POLYETHYLENE 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

ĐSMAĐL DOĞAN GÜNBAŞ

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 CHITOSAN-POLYETHYLENE GLYCOL MICROSPHERES AND FILMS FOR

BIOMEDICAL APPLICATIONS

submitted by ĐSMAĐL DOĞAN GÜNBAŞ 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 Özgen

Dean, Graduate School of Natural and Applied Sciences

Assoc. Prof. Dr. Göknur Bayram

Head of Department, Polymer Science and Technology

Prof. Dr. Nesrin Hasırcı

Supervisor, Chemistry Dept., METU

Examining Committee Members:

Prof. Dr. Đsmet Deliloğlu Gürhan

Bioengineering Dept., Ege University

Prof. Dr. Nesrin Hasırcı

Chemistry Dept., METU

Assoc. Prof. Dr. Göknur Bayram

Chemical Engineering Dept., METU

Prof. Dr. Leyla Aras

Chemistry Dept., METU

Prof. Dr. Đnci Eroğlu

Chemical Engineering Dept., METU

Date:

iii

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: Đsmail Doğan GÜNBAŞ

Signature :

iv

ABSTRACT

PREPARATION AND CHARACTERIZATION OF CHITOSAN-

POLYETHYLENE GLYCOL MICROSPHERES AND FILMS FOR

BIOMEDICAL APPLICATIONS

Günbaş, Đsmail Doğan

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

v

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.

vi

ÖZ

KĐTOSAN-POLĐETĐLEN GLĐKOL MĐKROKÜRE VE

FĐLMLERĐNĐN BĐYOMEDĐKAL UYGULAMALAR ĐÇĐN

HAZIRLANMASI VE KARAKTERĐZASYONU

Günbaş, Đsmail Doğan

Yüksek Lisans, Polimer Bilimi ve Teknolojisi Bölümü

Tez Yöneticisi: Prof. Dr. Nesrin HASIRCI

Eylül 2007, 102 sayfa

Son yıllarda, biyobozunur polimerik sistemler cerrahi aletler, yapay organlar,

farklı yollarla uygulanan ilaç salım sistemleri, immobilize enzim ve hücre

taşıyıcıları, biyosensörler ve ortopedik uygulamalarda kullanılan malzemelerin

tasarımlanmasında büyük önem kazanmıştır. Agaroz, aljinat, dekstran ve

kitosan gibi polisakkarit bazlı polimerler biyomalzemelerin büyük bir sınıfını

oluşturmaktadır. Çeşitli hastalıkların tedavisinde kullanılan in vivo ilaç salım

sistemlerinin modüle edilmesi için birçok yol açmış olan kitosan,

biyouyumluluğu, düşük toksisitesi ve vücut içinde biyobozunur olması nedeniyle

doku mühendisliği dahil birçok biyomedikal uygulamada kullanılmaktadır.

Kitosan bazlı bu ilaç taşıyıcı sistemler, mikropartikül, nanopartikül, jel ve film

gibi çok farklı şekilde hazırlanabilmektedir.

Bu çalışmada, kitosan (CH) ve farklı kompozisyonlarda kitosan-polietilen glikol

(CH-PEG) mikroküreler yağ/su emülsiyon metodu kullanılarak ve gluteraldehit

ile çapraz bağlanarak hazırlanmıştır. Bazı mikrokürelere model bir kanser ilacı,

methotraxate (MTX), yüklenmiştir. SEM, partikül boyutu ve in vitro ilaç salım

analizleri gerçekleştirilmiştir. In vitro ilaç salım çalışmaları, kitosan mikroküreler

vii

ile karşılaştırıldığında MTX salımının CH-PEG mikrokürelerinde daha hızlı

olduğunu göstermiştir.

Çalışmanın ikinci aşamasında, CH-PEG mikroküreleri monoklonal antikor,

ımmunoglobulin G (IgG), ile konjuge edilmiştir. Yüklenen ilacın sitotoksik

etkinliği MCF-7 ve MCF-7/MDA-MB kanser hücreleri kullanılarak tayin edilmiştir.

Çalışmanın üçüncü aşamasında, mikroküreler ile aynı kompozisyonlarda CHF-

PEG filmler solvent uçurma yöntemiyle hazırlanmıştır. Filmlerin IR, DSC,

mekanik ve yüzey analizleri yapılmıştır. Filmlerin mekanik özelliklerinin uygun

miktarda PEG kullanılarak arttırılabileceği ancak fazla miktardaki PEG özelliklerin

bozulmasına neden olmuştur.

Anahtar Kelimeler: Kitosan, polietilen glikol, mikroküre, film

viii

To the great memory of my father

ix

ACKNOWLEDGEMENTS

I would like to express my appreciation to Prof. Dr. Nesrin Hasırcı 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

Kızıltay, Tuğba Endoğan, Cantürk Özcan, Taylan Özerkan and Eda Ayşe Aksoy

for their valuable help, friendship and moral support.

I would like to extend my thanks to Prof. Dr. Đsmet Deliloğlu Gürhan for her help

especially for the cell culture studies. Special thanks to research assistant Sultan

Gülce for the cell culture studies.

Finally, my special appreciation and great gratitude is devoted to my mother

Leyla Günbaş, my father Münip Günbaş, my grandmother Gülfiye Günbaş and

my sisters Demet Günbaş and Duygu Deniz Günbaş for their endless love,

patience, moral support and encouragement in every moment of my life.

x

TABLE OF CONTENTS

ABSTRACT ................................................................................................. iv

ÖZ .................................................................................................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 Biomaterials...................................................................................1

1.2 Polymeric Biomaterials ....................................................................2

1.3 Controlled Drug Delivery .................................................................2

1.3.1 Conventional Drug Therapy versus Controlled Release..................3

1.3.2 Controlled Release Mechanisms .................................................4

1.4 Biodegradable Polymers for Drug Delivery .........................................6

1.5 Chitin and Chitosan.........................................................................8

1.6 Important Characteristics of Chitosan................................................9

1.6.1 Physicochemical Properties of Chitosan.......................................9

1.6.2 Solubility ..............................................................................11

1.6.3 Chemical properties ...............................................................12

1.6.4 Biological Properties ...............................................................13

1.7 Application Areas of Chitosan .........................................................14

1.7.1 Pharmateceutical and Biomedical Uses of Chitosan.....................15

1.8 Poly(ethylene glycol).....................................................................17

1.9 Microparticulate Systems for Controlled Release Applications .............19

1.10 Chitosan Microparticulate Drug Delivery Systems ..........................19

1.11 Release of Anticancer Drug from Chitosan Microspheres .................22

1.11.1 Chemical structure and Mechanism of Action of Methotrexate ......22

1.12 Drug Targeting..........................................................................23

1.13 Drug Targeting in Cancer Therapy ...............................................23

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1.13.1 Currently Available Therapeutics ..............................................24

1.13.2 Strategies to Deliver Drugs to Targets within the Tumour ...........24

1.13.3 Site Specific Drug Delivery Using Monoclonal Antibodies .............25

1.14 Aim of the study .......................................................................28

2.MATERIALS AND METHODS ......................................................................29

2.1 Materials .....................................................................................29

2.2 Methods ......................................................................................30

2.2.1 Preparation of Microspheres ....................................................30

2.2.1.1 Preparation of Drug-loaded Microspheres ..................................31

2.2.2 Characterization of Microspheres..............................................32

2.2.2.1 Morphological Analysis............................................................32

2.2.2.2 Particle Size Analysis ..............................................................32

2.2.3 Preparation of Chitosan and Chitosan-PEG Films ........................33

2.2.4 IR Analysis............................................................................34

2.2.5 Differential Scanning Calorimetry (DSC) Analysis .......................34

2.2.6 Mechanical Tests....................................................................34

2.2.7 Contact Angle Measurement ....................................................35

2.2.8 Conjugation of IgG to Microspheres..........................................35

2.2.9 Degradation of Microspheres ...................................................36

2.2.10 In-vitro Release Studies..........................................................36

2.2.11 Cell Studies...........................................................................37

3.RESULTS & DISCUSSION .........................................................................39

3.1 Chitosan and Chitosan-PEG Microspheres ........................................39

3.1.1 Effect of Crosslinker on Size and Shape of the Chitosan

Microspheres ...................................................................................39

3.2 Particle Size Analysis of Microspheres..............................................42

3.3 Drug Loading to Microspheres ........................................................44

3.4 In Vitro Release Studies ................................................................45

3.5 Conjugation of IgG to Microspheres ................................................53

3.6 Cell Culture and Coculture Studies ..................................................54

3.7 Degradation Studies......................................................................60

3.8 Chitosan and Chitosan-PEG Films....................................................63

3.8.1 Infrared Analysis....................................................................63

3.8.2 Differential Scanning Calorimetry Analysis.................................67

3.8.3 Mechanical Tests....................................................................69

xii

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

4.CONCLUSIONS........................................................................................79

REFERENCES.............................................................................................82

APPENDICES .............................................................................................91

xiii

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 kınetıcs ..........................................................................50

Table 3.6 Mechanical properties of CHF-PEG films .........................................69

Table 3.7 Contact angles of prepared films...................................................77

xiv

LIST OF FIGURES

Figure 1.1 Drug levels in the blood plasma (a) traditional drug dosing, (b)

controlled-delivery dosing............................................................4

Figure 1.2 Structures of cellulose, chitin and chitosan...................................10

Figure 1.3 Protonation of chitosan..............................................................11

Figure 1.4 Chemical structure of PEG .........................................................17

Figure 1.5 Schematic represantation of the suspension crosslinking

technique ................................................................................20

Figure 1.6 Methods for preparation of chitosan microspheres ........................21

Figure 1.7 Chemical structure of MTX .........................................................22

Figure 1.8 Schematic diagram of an immunoglobulin (IgG) ...........................26

Figure 2.1 Schematic representation of water-oil emulsion method ................31

Figure 3.1 Crosslinking reaction of chitosan and glutaraldehyde.....................39

Figure 3.2 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) U-

CH-PEG 1-1, (C) U-CH-PEG 1-2 microspheres ..............................41

Figure 3.4 Size distribution of unloded microspheres ....................................42

Figure 3.5 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 Release of MTX from CH and CH-PEG microspheres ......................46

Figure 3.7 Release of MTX from different type of the microspsheres ...............47

Figure 3.8 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

xv

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-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

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) CH-

PEG 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) CH-

PEG 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) CHF-

PEG 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

xvi

LIST OF SYMBOLS AND ABBREVIATIONS

CH Chitosan

PEG Polyethylene glycol

CH-PEG Chitosan-polyethylene glycol

PEO Polyethylene oxide

DA Deacetylation

DDA Degree of deacetylation

LDL Low density lipoproteins

HDL High density lipoproteins

Ig Immunoglobulin

IgG Immunoglobulin G

MAb Monoclonal antibody

Fab Antigen-binding fragment

GA Gluteraldehyde

MTX Methotrexate

PBS Phosphate buffer solution

SEM Scanning electron microscopy

EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide

NHS N-hydroxyl succinimide

FBS Foetal bovine serum

MMD Mass median diameter

VMD Volume mean diameter

SMD Surface mean diameter

UTS Ultimate tensile strength

E Modulus of elasticity

SAB Strain at break

1

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].

2

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 Noncarcinogenesis, nonpyrogenicity, nontoxicity, and nonallergic response

Sterilizability Autoclave, dry heating, ethylenoxide gas, and radiation

Physical property

Strength, elasticity, and durability

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 therapy’s

3

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].

4

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

5

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].

6

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].

7

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 Commercially available biodegradable drug delivery systems Name of product

Dosage form Active ingredient

Biodegradable polymera,b

Lupron Depot

Microspheres

Leuprolide

PLGA

Sandostatin LAR

Microspheres

Octreotide

PLGA

Neutropin Depot

Microspheres

Somatropin

PLGA

Trelstar Depot

Microspheres

Triptorelin

PLGA

Gliadel

Waffer

Cumustin

Polyanhydride

Zoladex

Rod

Goserelin

PLGA

Atridox

Gel

Doxycycline

PLGA

a PLGA: poly(lactic-co-glycolic acid) b 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 semisynthetic

biodegradable polymers. These modifications can result in altered

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.

8

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 years because chitosan meets the most important

requirements for excipients in modern drug delivery systems, namely

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

1.5 Chitin and Chitosan

Chitin is a straight homopolymer composed of β-(1,4)-linked N-acetyl-

glucosamine 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’’.

9

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 180ºC 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 (30-

50%) 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-b-

D-glucose through a β 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.

10

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.

O

CH2OH

H

OHH

H

OH

H

H

O

O

CH2OH

H

OHH

H

OH

H

H

O

n

Cellulose

O

CH2OH

H

OHH

H

NHCOCH3

H

H

O

O

CH2OH

H

OHH

H

NHCOCH3

H

H

O

n

Chitin

O

CH2OH

H

OHH

H

NH2

H

H

O

O

CH2OH

H

OHH

H

NH2

H

H

O

n

Chitosan

Figure 1.2 Structures of cellulose, chitin and chitosan

11

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].

O

CH2OH

H

OHH

H

NH2

H

H

O

+ H+

O

CH2OH

H

OHH

H

NH3+

H

H

O

Figure 1.3 Protonation of chitosan

12

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) Cationic Amine (-NH3

+)

-Soluble in acidic solutions -Soluble at pH’s ≤6.5

-Insoluble at pH’s ≥6.5 -Forms viscous solutions

-Insoluble in H2SO4 -Forms gel with polyanions

-Limited solubility in H3PO4 -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].

13

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].

14

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 Principal applications for chitosan Agriculture -Defensive mechanisms in plants

-Stimulation of plant growth -Seed coating -Time release of fertilizers and nutrients into the soil

Water & waste treatment -Flocculant to clarify water (drinking water, pools) -Removal of metal ions -Ecological polymer (eliminate synthetic polymers) -Reduce odors

Food & beverages -Not digestible by human (dietary fiber) -Bind lipids (reduce cholesterol) -Thickener and stabilizer for sauces -Protective, fungistatic, antibacterial coating for fruit

Cosmetics & toiletries -Maintain skin moisture -Treat acne -Improve suppleness of hair -Reduce static electricity in hair -Tone skin -Oral care (toothpaste, chewing gum)

Biopharmaceutics -Immunologic, antitumoral -Hemostatic and anticoagulant -Healing, bacteriostatic

15

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 Principal properties of chitosan in relation to its use in biomedical applications

Potential Biomedical Applications

Principal Characteristics

Surgical sutures

Biocompatible

Dental implants

Biodegradable

Artificial skin

Renewable

Rebuilding of bone

Film forming

Corneal contact lenses

Hydrating agent

Time release drugs for animals and humans

Nontoxic, biological tolerance

Encapsulating material

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.

16

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].

17

Chitosan and its derivatives have been used for gene transfection; for N-

alkylated 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.

HOO

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

18

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 molecular weight distribution (low

polydispersity). Depending on the catalyst type the mechanism of

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].

19

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.

20

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

21

Interaction with anions (sulphate,tripolyphosphate

,hydroxide,molbdate) Thermal

crosslinking with citric acid

Solvent evaporation

Interfacial acylation

Coating on preformed

microparticles

Crosslinking with

chemicals

CHITOSAN MICROSPHERES

Gluteraldehyde crosslinking

Formaldehyde crosslinking

Genipin

crosslinking

Single emulsion

Multiple emulsion

Onotropic gelation

Wet phase inversion

Co-acervation

Precipitation

Precipitation chemical

crosslinking

Complex Co-acervation

Modified emulsification and onotropic

gelation

Emusification and onotropic

gelation

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.

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].

22

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.

N

N

N

N

N

HN

O

OHO

H2N

NH2 O

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 dose-

dependent.

23

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.

24

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.

25

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 enzyme–MAb 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.

26

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 N-

terminal 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 antibody’s function is to

recognize an antigen and, by cross reaction with other immune proteins, to

27

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 complement-

fixing 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.

28

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 MTT-

based 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.

29

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 and Manufacturers Materials Manufacturers

Chitosan (DDA=85%) Sigma, USA

Poly(ethylene glycol) (Mw=14000) Aldrich, USA

Acetic Acid (99-100%) J.T. Baker, Netherlands

Gluteraldehyde (50%) BDH, UK

Methotrexate Pharmachemie B.V., Netherlands

Tween 80 Acros Organics, USA

Immunoglobulin G (IgG) Jackson Immuno Research, USA

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide (EDC)

Sigma, USA

N-hydroxyl succinimide (NHS) Sigma, USA

Acetone Merck, Germany

Lysozome Datex Applichem, Germany

Dialysing Tube Sigma, USA

Corn Oil Sayınlar, Turkey

Millipore Millex, France

30

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-2-

hydrate in 1 L distilled water.

2.2 Methods

2.2.1 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 50ºC 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 50ºC for 12 hours. A schematic representation of the technique was

given in Figure 2.1.

31

Mechanicalstirrer

Chitosan or Chitosan-PEG solution in acetic acid Gluteraldehyde

Tween 80Corn oilMicrosphere formation

5 h stirring

Chitosan orChitosan-PEGmicrospheres

Filtering and washing

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.

32

Table 2.2 Prepared microspheres

Sample Concentration

of Chitosan in

total mixture

(%w/v)

Concentration

of PEG in total

mixture

(% w/v)

Concentration

of

Gluteraldehyde

(% v/v), 2 mL

Amount of

Methotrexate

(mg)

U-CH 1.25 3.0 - 1.25 -

U-CH 2.5 3.0 - 2.5 -

U-CH 5 3.0 - 5.0 -

U-CH-PEG 1-0.5 3.0 1.5 5.0 -

U-CH-PEG 1-1 3.0 3.0 5.0 -

U-CH-PEG 1-2 3.0 6.0 5.0 -

CH 5 3.0 - 5.0 5.0

CH-PEG 1-0.5 3.0 1.5 5.0 5.0

CH-PEG 1-1 3.0 3.0 5.0 5.0

CH-PEG 1-2 3.0 6.0 5.0 5.0

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.

33

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 µm and 100 µm.

Table 2.3 Prepared chitosan and chitosan-PEG films

Sample Concentration of

Chitosan

(%w/v)

Concentration

of PEG

(% w/v)

Concentration of

Gluteraldehyde

(% v/v), 2 mL

CHF 0.1 1.0 - 0.1

CHF 0.5 1.0 - 0.5

CHF 1.0 1.0 - 1.0

CHF-PEG 1-0.5-0.1 1.0 0.5 0.1

CHF-PEG 1-0.5-0.5 1.0 0.5 0.5

CHF-PEG 1-0.5-1.0 1.0 0.5 1.0

CHF-PEG 1-1-0.1 1.0 1.0 0.1

CHF-PEG 1-1-0.5 1.0 1.0 0.5

CHF-PEG 1-1-1.0 1.0 1.0 1.0

CHF-PEG 1-1.5-0.1 1.0 1.5 0.1

CHF-PEG 1-1.5-0.5 1.0 1.5 0.5

CHF-PEG 1-1.5-1.0 1.0 1.5 1.0

CHF-PEG 1-2-0.1 1.0 2.0 0.1

CHF-PEG 1-2-0.5 1.0 2.0 0.5

CHF-PEG 1-2-1.0 1.0 2.0 1.0

34

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

10ºC/min using dry nitrogen flow. Heating curves with a rate of 10ºC/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 30±2 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 ρ=F/A where ρ 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.

35

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 µL from stock of 2.5 mg/mL 1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide (EDC), 250 µL immunoglobulin G (IgG) and

10 µL 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.

36

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 37°°CC.. At certain time intervals dissolution medium was

withdrawn and immediately replaced with equal volumes of fresh PBS. The

removed solutions were analyzed spectrophotometrically at λ=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 Korsmeyer–Peppas (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∞(1−e−k1t) (2)

37

where, Q∞ being the total amount of drug in the matrix and k1 the first-order

kinetic constant.

Qt=kHt1/2 (3)

where, kH is the Higuchi rate constant.

Furthermore, the Korsmeyer–Peppas (4) semi-empirical model was also

applied.

Qt/Q∞=ktn (4)

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 µL of polymer test specimens (0,1 mg

microspheres in 100 µL) .

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

38

MTT (in Dulbecco’s modified PBS) were added to each well and incubated at

37oC under 5% CO2 atmosphere for 4 h.

After that, 100 µL 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 µL

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

Control Only cell culture

MTX-0.25 O.25 mg/mL free drug

MTX-2.5 2.5 mg/mL free drug

U-CH-PEG 1-1 Unloaded microspheres

L-CH-PEG 1-1 Drug loaded microspheres

CU-CH-PEG 1-1 Conjugated unloaded microspheres

CL-CH-PEG 1-1 Conjugated drug loaded microspheres

39

CHAPTER 3

RESULTS & DISCUSSION

3.1 Chitosan and Chitosan-PEG Microspheres

3.1.1 Effect of Crosslinker on Size and Shape of the Chitosan

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.

O

HO N

HOH2C

O

O

O

HOH2C

O

HO N

HOH2C

HO N

CH

CH2

CH2

CH2

CH

NHO

O

O

O

O

O

HOH2C

HO N HO N

HOH2CHOH2C

O

CH

CH2

CH2

CH2

CH

HC

CH2

CH2

CH2

CH

Figure 3.1 Crosslinking reaction of chitosan and glutaraldehyde

40

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

A

B

C

41

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-CH-PEG 1-1, (C) U-CH-PEG 1-2

A

B

C

42

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

Type of the

Microspheres

Average Size

(µm)

U-CH 1.25

144.23

U-CH 2.5

97.06

U-CH 5

90.99

U-CH-PEG 1-0.5 107.53

U-CH-PEG 1-1

116.37

U-CH-PEG 1-2

162.90

0

20

40

60

80

100

120

140

160

180

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

Average S

ize of the M

icro

spheres

(µm)

Figure 3.4 Size distribution of unloaded microspheres

43

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 µm to 90.99 µm 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.

44

Table 3.2 Particle size analysis results

Type of the

microsphere

D (v, 0.1)

(µm)

D (v, 0.5)

(µm)

D (v, 0.9)

(µm)

VMD

(µm)

SMD

(µm)

CH 1.25 92.24 139.96 206.67 144.23 67.13

CH 2.5 53.43 87.45 157.35 97.06 43.50

CH 5 44.15 80.98 155.54 90.99 37.07

CH-PEG 1-0.5 48.37 102.02 178.35 107.53 39.62

CH-PEG 1-1 49.21 107.36 197.64 116.37 43.00

CH-PEG 1-2 82.34 156.84 252.83 162.90 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).

A

B

45

Figure 3.5 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

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.

A

B

C

D

46

Release of MTX from CH 5 Microspheres

0

100

200

300

400

500

600

700

800

0 200 400 600 800 1000

Time (hr)Amount of MTX released (µg)

Release of MTX from CH-PEG 1-0.5 Microspheres

0

100

200

300

400

500

600

700

800

900

0 200 400 600 800 1000

Time (hr)

Amount of MTX released (µg)

Release of MTX from CH-PEG 1-1 Microspheres

0

100

200

300

400

500

600

700

800

900

1000

0 200 400 600 800 1000

Time (hr)

Amount of MTX released (µg)

Release of MTX from CH-PEG 1-2 Microspheres

0

200

400

600

800

1000

1200

0 200 400 600 800 1000

Time (hr)

Amount of MTX released (µg)

Figure 3.6 Release of MTX from CH and CH-PEG microspheres

47

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.

0

200

400

600

800

1000

1200

0 200 400 600 800 1000

Time (day)

Amount of MTX released (µg)

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

48

Initial and late release rates are given in Table 3.3.

Table 3.3 Release rates of MTX from different type of microspheres

Type of the microsphere

Composition (CH/PEG)

(w/w)

Release Rates (µg.h-1)

Initial Late

CH - 1.86 0.19

CH-PEG 1-0.5 0.3/0.15 2.32 0.18

CH-PEG 1-1 0.3/0.3 2.58 0.24

CH-PEG 1-2 0.3/0.6 2.93 0.22

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

Type of the

Microspheres

Theoritical

amount of MTX

(µg)

Total entrapped

MTX (µg)

Encapsulation

efficiency

(%)

CH 5000 678.97 13.58

CH-PEG 1-0.5 5000 810.15 16.20

CH-PEG 1-1 5000 896.69 17.93

CH-PEG 1-2 5000 1077.38 21.55

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.

49

0

0,2

0,4

0,6

0,8

1

1,2

0 200 400 600 800 1000

Time (hr)

% Release

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

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.

50

Table 3.5 Release Kinetics

Zero Order Sample

Ko R2

CH 0.6144 0.8230

CH-PEG 1-0.5 0.7142 0.7712

CH-PEG 1-1 0.8542 0.8411

CH-PEG 1-2 0.9640 0.7475

First Order Sample

K1 R2

CH 0.0013 0.6854

CH-PEG 1-0.5 0.0013 0.6022

CH-PEG 1-1 0.0014 0.7096

CH-PEG 1-2 0.0013 0.5792

Higuchi Sample

KH R2

CH 0.4550 0.9612

CH-PEG 1-0.5 0.5380 0.9307

CH-PEG 1-1 0.6273 0.9646

CH-PEG 1-2 0.7304 0.9128

Korsmeyer Sample

KKP R2 n

CH 0.7795 0.9624 0.4398

CH-PEG 1-0.5 0.8730 0.9223 0.4540

CH-PEG 1-1 0.8574 0.9680 0.4684

CH-PEG 1-2 1.001 0.9120 0.4787

51

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

0

5

10

15

20

25

0 200 400 600 800 1000

Time (hr)

Cumulative (%) drug release

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

Figure 3.9 Zero-order release kinetic model plot for various microspheres

0

1

2

3

4

5

6

7

8

0 200 400 600 800 1000

Time (hr)

ln M

t

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

Figure3.10 First order relase kinetic model plot for various microspheres

52

0

5

10

15

20

25

0 5 10 15 20 25 30 35

t1/2

Cumulative (%) drug release

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

Figure3.11 Higuchi kinetic model plot for various microspheres

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

0 0,5 1 1,5 2 2,5 3 3,5

Log t

Log (Mt/M∞)

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

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.

53

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

H3CH2C N C N (CH3)3

H+

N

CH3

CH3

EDC

IgGThe reaction of the carbonyl group of IgG with EDC

forming an activated peptide intermediate

C O

C N (CH2)3H+

N CH3

CH3

IgG

NH2

Chitosan MS

The activated IgG reacting with amine group of chitosan to form IgG-chitosan conjugate

C O

NH

Chitosan MS

+ UREA

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.

54

Figure 3.14 Confocal microscopy images of microspheres (A) non-conjugated, (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).

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

MDA-MB

MCF-7

A

B

55

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-(4,5-

dimethylthiazol-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 Dulbecco’s modified

PBS) were added to each well and incubated at 37oC under 5% CO2 atmosphere

for 4 h. After that, 100 µL 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.

0

1

2

3

4

5

6

7

8

9

144 240

Time (hr)

Cell n

um

ber (x

1000)

Control 0.25 mg/mL MTX 2.5 mg/mL MTX

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

56

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.

A

B

C

D

57

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

B

D

A

C

E

F

G

58

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.

0

1

2

3

4

5

6

7

8

9

144 240

Incubation time (hr)

Cell n

um

ber (x

1000)

Control Group U-CH-PEG 1-1

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 µL 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).

59

0

2

4

6

8

10

12

14

Control PEG (0.5 mg/mL) PEG (1.0 mg/mL)

The n

um

ber of cells (x1000)

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 µL of polymer test specimens which contains 0.1 mg

microspheres/100 µL. The cell viability decreased when compared with control

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

0

1

2

3

4

5

6

7

8

9

144 240

Incubation time (hr)

Cell n

um

ber (x

1000)

Control Group L-CH-PEG 1-1

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

60

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.

A B

61

Figure 3.22 SEM micrographs of microspheres (A) CH after 2 days, (B) CH-PEG 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

A

F E

D C

B

62

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) CH-PEG 1-1 after 2 days, (C) CH after 60 days, (D) CH-PEG 1-1 after 60 days

B A

C D

63

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 µm to 0.10 µm.

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.

64

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

cm-1

A

B

C

4400 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600

%T

65

4400 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600

cm-1

%T

A

B

C

D

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.

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

66

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 C–O

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.

O

O

CH2

HH

OH

HNH2

H

OH

HO

CH2CH2 O CH2CH2 O CH2CH2 OHn

H

O

O

CH2OH

O

HH

H

NH2

H

H

H

O

CH2CH2OO CH2CH2HO CH2CH2

n

n

H

Figure 3.26 Chitosan-PEG interaction

67

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.

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 113.67ºC. 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

-50 0 50

100 150 200 250

Temperature (ºC)

Exothermic Heat Flow (W/g)

A

B

113.67 ºC

64.13 ºC

68

regions, the crystalline melting temparature (Tm) was not observed mostly

because of its rigid-rod polymer backbone having strong intra- and inter-

molecular 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 64.13ºC (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).

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

-50

0 50

100 150 200 250

Exothermic Heat Flow (W/g)

A

B

54.69 ºC D

61.74 ºC D

60.95 ºC D

61.04 ºC D

C

D

69

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)

UTS

(MPa)

E

(GPa)

SAB

(%)

CHF 0.1 0.1 136.38±8.07 1.99±0.09

39.10±1.62

CHF 0.5 0.5 109.85±4.61

1.56±0.07

19.11±2.61

CHF 1.0 1.0 102.06±4.00

1.46±0.16

20.65±5.60

CHF-PEG 1-0.5-0.1 0.1 75.80±2.06 1.02±0.08 27.84±1.62

CHF-PEG 1-0.5-0.5 0.5 83.23±3.99 1.30±0.11 26.38±4.35

CHF-PEG 1-0.5-1.0 1.0 90.60±2.83 1.52±0.16 27.45±6.00

CHF-PEG 1-1-0.1 0.1 83.29±3.46 1.30±0.12 18.65±5.68

CHF-PEG 1-1-0.5 0.5 92.15±4.96 1.39±0.23 18.87±3.39

CHF-PEG 1-1-1.0 1.0 100.19±4.77 1.40±0.05 18.96±2.35

CHF-PEG 1-1.5-0.1 0.1 56.93±2.85 1.00±0.08 11.00±2.47

CHF-PEG 1-1.5-0.5 0.5 67.86±1.98 1.09±0.11 11.43±1.67

CHF-PEG 1-1.5-1.0 1.0 58.85±2.29 1.04±0.05 9.59±0.77

CHF-PEG 1-2-0.1 0.1 42.39±3.01 0.87±0.10 8.89±2.47

CHF-PEG 1-2-0.5 0.5 52.75±5.15 0.86±0.07 9.41±2.41

CHF-PEG 1-2-1.0 1.0 41.35±3.33 1.11±0.16 6.40±0.01

70

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).

0

20

40

60

80

100

120

140

160

0.1 0.5 1.0

Concentration of GA (% v/v)

UTS(M

Pa)

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.

71

Figure 3.30 Chemical reaction between chitosan and gluteraldehyde

72

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

0

20

40

60

80

100

0.1 0.5 1.0

Concentration of GA (v/v)

UTS (MPa)

The Effect of Crosslinker on CHF-PEG

1-1 Films

0

20

40

60

80

100

120

0.1 0.5 1.0

Concentration of GA (v/v)

UTS (MPa)

The Effect of Crosslinker on CHF-PEG

1-1.5 Films

0

10

20

30

40

50

60

70

80

0.1 0.5 1.0

Concentration of GA (v/v)

UTS (MPa)

The Effect of Crosslinker on CHF-PEG

1-2 Films

0

10

20

30

40

50

60

70

0.1 0.5 1.0

Concentration of GA (v/v)

UTS (MPa)

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

73

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 1-2-

0.1 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)

0

20

40

60

80

100

0.15 0.3 0.45 0.6

Amount of PEG (g)

UTS (MPa)

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

0

20

40

60

80

100

120

0.15 0.3 0.45 0.6

Amount of PEG (g)

UTS (MPa)

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

0

20

40

60

80

100

120

0.15 0.3 0.45 0.6

Amount of PEG (g)

UTS (MPa)

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

74

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.

0

20

40

60

80

100

120

140

160

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7

Amount of PEG (g)

UTS (MPa)

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).

75

Modulus of CHF Films

0

0,5

1

1,5

2

2,5

0.1 0.5 1.0

Concentration of GA (%v/v)

E (G

Pa)

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.

76

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

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

0.15 0.3 0.45 0.6

Amount of PEG (g)

E (GPa)

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

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

0.15 0.3 0.45 0.6

Amount of PEG (g)

E (GPa)

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

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

0.15 0.3 0.45 0.6

Amount of PEG (g)

E (GPa)

Figure 3.35 Modulus of CHF-PEG films

77

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

Sample Concentration of GA

(v/v)

Contact Angle

CHF - 97.93±8.45

CHF 0.1 0.1 100.15±4.26

CHF 0.5 0.5 95.91±3.57

CHF 1.0 1.0 79.03±0.76

CHF-PEG 1-1-0.1 0.1 80.19±5.62

CHF-PEG 1-1-0.5 0.5 88.34±2.00

CHF-PEG 1-1-1.0 1.0 68.27±1.67

CHF-PEG 1-2-0.1 0.1 70.96±1.89

CHF-PEG 1-2-0.5 0.5 68.39±4.99

CHF-PEG 1-2-1.0 1.0 69.14±2.19

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.

78

0

10

20

30

40

50

60

70

80

90

100

0.1 0.5 1.0

Concentration of GA (% v/v)

Contact Angle

CHF-PEG 1-1 CHF-PEG 1-2

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

79

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 µm to 90.99 µm. 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

80

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 rigid-

rod polymer backbone having strong intra- and inter-molecular bonding.

PEG showed sharp melting peak at 64.13ºC. 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.

81

- 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 CHF-

PEG 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.

82

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91

APPENDIX A

y = 0,0559x - 0,0004

R2 = 0,9992

0

0,1

0,2

0,3

0,4

0,5

0,6

0 2 4 6 8 10 12

Conc. of MTX (µg/mL)

Abs. at 259 nm

Figure A.1 Calibration curve for methotrexate (259 nm)

92

APPENDIX B

Figure B.1 The histogram table and plot of CH 1.25

93

Figure B.2 The histogram table and plot of CH 2.5

94

Figure B.3 The histogram table and plot of CH 5

95

Figure B.4 The histogram table and plot of CH-PEG 1-0.5

96

Figure B.5 The histogram table and plot of CH-PEG 1-1

97

Figure B.6 The histogram table and plot of CH-PEG 1-2

98

APPENDIX C

0,300

0,350

0,400

0,450

0,500

0,550

0,600

0,650

0,700

0,750

0,800

0,850

0,900

0,950

1,000

1,050

1,100

1,150

1,200

1,250

1 5 10 50 100 500 1000

Cell Number (x1000)

Absorb

ance (570nm

)

Figure C.1 MTT calibtation curve

99

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

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

Cell A

bsorb

ance (570 n

m)

Figure C.2 Cell absorbance of MCF-7 cell culture after 6 days

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

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

Cell A

bsorb

ance (570 nm)

Figure C.3 Cell absorbance of MCF-7 cell culture after 10 days

100

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

101

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

102

Figure D.5 Tensile test graph of CHF-PEG-1-2-0.1

103