Transcript
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:
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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
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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.
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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
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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
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To the great memory of my father
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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.
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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
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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
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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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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
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