Professional Documents
Culture Documents
CARBOXYMETHYL CHITOSAN--CYCLODEXTRIN
NANOPARTICLES AS A DRUG DELIVERY SYSTEM: EVALUATION
AGAINST MCF-7 BREAST CANCER CELLS
Asamoah-Asare Joseph
VDC
--
MCF-7
():
(ASAMOAH-ASARE JOSEPH)
(BIOMEDICAL ENGINEERING)
CMC--CM -CD
CMC
CM -CD 1 - -3 - 3 -
EDC N-NHS CM -CD CMC
124-298nmZeta DOX
DOX MCF-7
DOX MCF-7
II
Abstract
Abstract
The aim of this study was to generate a new type of nanoparticles made of carboxymethyl
chitosan (CMC) and carboxymethyl -cyclodextrin (CM -CD) and to characterize it. This
nanoparticulate system should have a potential for the association and delivery of hydrophilic
and hydrophobic drugs as well as undergo further conjugation to other macromolecules such as
antibodies for targeted delivery especially in cancer therapy. Various CMC concentrations and a
fixed concentration of CM -CD were processed to nanoparticles via the ionic gelation technique
by grafting the CM -CD onto CMC using water-soluble 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as the condensing agents. The resulting
nanoparticles were spherical in shape as shown by atomic force microscopy with an average size
range of 124298 nm and showed a negative zeta potential. Doxorubicin hydrochloride (DOX), a
water soluble anticancer drug, was loaded in the nanoparticles with a high encapsulation
efciency. The in vitro drug release showed that the release of DOX from the nanoparticles could
be effectively sustained. The anti-tumor activity of the released DOX was assessed using a
MCF-7 breast cancer cell line. The cytotoxicity evaluation showed the drug loaded nanoparticles
inhibition on MCF-7 breast cancer cells.
Key words:
Chitosan; Cyclodextrin; Nanoparticles; Drug Delivery system; Targeted Drug Delivery; Cancer.
III
Declaration
Declaration
I hereby declare that this dissertation entitled Carboxymethyl Chitosan--Cyclodextrin
Nanoparticles as a Drug Delivery System; Evaluation Against MCF-7 Breast Cancer Cells,
submitted by me to the Central South University in partial fulfillment of the requirement for the
degree of Master of philosophy in Biomedical Engineering, is the result of the work carried out
by me, under the supervision of Prof. Zhang Yang-de, Prof. Chen Yuxiang and Prof. Wu
Fongming, Professors of the National Hepatobiliary and Enteric Research Center of Xiangya
Hospital of Central South University, between September 2011 to May 2014.
I further declare that no part of the results of this study has been submitted for any other degree
or for publication.
Signature:
Date:
ASAMOAH-ASARE JOSEPH
IV
Acknowledgement
Acknowledgement
My first and most important acknowledgement goes to my Lord Jesus who has served as a
comforter and has been the strength I needed to help me excel throughout my graduate career. I
would like to acknowledge my major professor Prof. Zhang Yang-de for his knowledge, patience,
and support. I thank my other supervisors Prof. Chen Yuxiang and Prof. Wu Fongming, and
other teachers in the institute who supported me in this research especially, Teacher Chen Wei. I
would also like to extend my gratitude to Professor Ren Caiping of the Key Laboratory for
Carcinogenesis & Cancer Invasion of the Chinese Ministry of Education, Central South
University for her resourceful contribution towards this dissertation.
I thank my colleagues of the National Hepatobiliary and Enteric Surgery Research Center, the
Institute of Biomedical Engineering and the State Key Laboratory of Nabiotechnology of
Xiangya Hospital of Central South University, China especially Yue Chunyun for their
enlightening ideas and suggestions which have contributed towards the knowledge I have
acquired during my period of study.
I will conclude by taking the time to thank my parents Mr. Johnson Asare and Grace Anim, Mrs.
Monica Asare, a phenomenal woman who has been supportive of me all these years, my siblings
Adwoa, Kweku and Dzifa, and finally to all my friends especially Linda and Carl. Your prayers,
love, and patience have given me the drive to work hard, stay focused, and overcome obstacles
encountered not only in my graduate career, but in many areas of my life.
Table of Content
Table of Contents
TITLE PAGE
DECLARATION (CHINESE).. I
ABSTRACT (CHINESE). II
ABSTRACT..... III
DECLARATION...... IV
ACKNOWLEGEMENT... V
TABLE OF CONTENT.... VI
ABBREVIATION......... IX
CHAPTER 1
Introduction
1.1 Polymers. 1
1.2 Biodegradable polymers. 4
1.3 Polymer-drug conjugates........ 5
1.4 Chitosan..... 18
1.5 Beta Cyclodextrin.......... 20
1.6 Cancer........ 21
1.7 Targeted Drug Delivery. 26
1.8 Doxorubicin... 28
1.9 Nanodrug Delievry Systems.. 29
CHAPTER 2
Materials and Method
VI
Table of Content
2.1 Materials.................... 31
2.2 Method....... 31
2.2.1 Preparation of Carboxymethyl Chitosan (CMC).......... 31
2.2.2 Preparation of Carboxymethyl -Cyclodextrin (CM -CD). 31
2.2.3 Preparation of Carboxymethyl Chitosan--Cyclodextrin Nanoparticles
(CMC -CD Nps)...... 32
2.2.4 Preparation of Doxorubicin-Carboxymethyl Chitosan--Cyclodextrin Nanoparticles
(DOX-CMC -CD Nps)... 32
2.3 Physiochemical Characterization... 33
2.4 Drug Encapsulation and Loading Studies..... 34
2.5 In vitro drug release studies .. 34
2.6 Cell Culture........ 35
2.7. In vitro anticancer activity analysis...... 35
2.8 Statistical Analysis............................................................................................................. 35
CHAPTER 3
Results
3.1 Size and Zeta Potential Studies..... 37
3.2 FTIR Studies...................... 38
3.3 AFM Studies.. 39
3.4 Drug Release Studies. 39
3.5 Anticancer Activity.... 40
VII
Table of Content
CHAPTER 4
Discussion
4.1 Synthesis of Nanoparticles.... 41
4.2 Size and Zeta Potential.. 42
4.3 AFM Studies.. 44
4.4 FTIR Analysis.... 45
4.5 Drug Encapsulation and Loading Studies.. 48
4.6 Drug Release Studies.. 52
4.7 Anticancer Activity......... 54
CHAPTER 5
Conclusion.... 57
REFERENCES.. 58
APPENDIX 69
VIII
Abbreviation
List of Abbreviations
Nps. Nanoparticles
DOX Doxorubicin Hydrochloride
FTIR... Fourier Transform Infra Red
AFM. .. Atomic Force Microscopy
IX
Chapter 1 Introduction
Chapter 1 Introduction
1.1 Polymers
Research in polymer science has led to the development of several novel drug-delivery systems.
The use of natural polymers in drug delivery is of great interest due to their desirable
biocompatible, biodegradable, hydrophilic and protective properties [1]. Polymers are
macromolecules having very large chains, contain a variety of functional groups, can be blended
with other low- and highmolecular-weight materials, and can be tailored for many applications.
They are synthesized from smaller molecules called monomers which are joined together by
covalent bonds which unlike metallic bonds are highly directional but not very strong. Most
polymers are organic compounds with carbon as the base element and their properties can be
modified to suit specific purposes by;
The classification of polymers may be based on their structures, the types of reactions by which
they are prepared, their physical properties, or their technological uses.
Based on their physical properties, there can be three different types of solid polymers; these are
elastomers, plastics and fibers. Plastics can be further categorized into thermoplastic polymers
and thermosetting polymers. Elastomers are rubber like solids with elastic properties. In these
elastomeric polymers, the polymer chains are held together by the weakest intermolecular forces.
1
Chapter 1 Introduction
These weak binding forces permit the polymer to be stretched. Some examples are buna-S, bunaN and neoprene. Fibres are the thread forming solids which possess high tensile strength and
high modulus. These characteristics can be attributed to the strong intermolecular forces like
hydrogen bonding. Some examples are polyamides and polyesters. Thermoplastics soften on
heating and can be converted into any shape that they can retain on cooling. The process of
heating, reshaping and retaining the same on cooling can be repeated several times.
Thermoplastic polymers include Polyethylene, PVC, nylon and sealing wax. Thermosetting
polymers normally are made from relatively low molecular weight, usually semi-fluid substances,
which when heated in a mold become highly cross-linked, thereby forming hard, infusible, and
insoluble products having a three-dimensional network of bonds interconnecting the polymer
chains [3]. Some common examples are Bakelite and urea-formaldelyde resins.
Polymers may be classified as linear, branched or cross-linked based on their structural architect.
Linear polymers consist of long and straight chains. Examples are high density polythene,
polyvinyl chloride, etc. Branched polymers contain linear chains having some branches such as
low density polythene. Cross-linked polymers are usually formed from bi-functional and tri-
Chapter 1 Introduction
functional monomers and contain strong covalent bonds between various linear polymer chains.
They include vulcanized rubber and urea-formaldehyde resins.
Polymers usually are prepared by two different types of polymerization reactions; addition and
condensation. In addition polymerization all of the atoms of the monomer molecules become part
of the polymer and on the other hand in condensation polymerization some of the atoms of the
monomer are split off in the reaction as water, alcohol, ammonia, or carbon dioxide, and so on.
The addition polymers are formed by the repeated addition of monomer molecules possessing
double or triple bonds, e.g., the formation of polythene from ethene and polypropene from
propene. However, the addition polymers formed by the polymerization of a single monomeric
species are known as homopolymer. The condensation polymers are formed by repeated
condensation reaction between two different bi-functional or tri-functional monomeric units.
Some polymers can be formed either by addition or condensation reactions. An example is polyethylene glycol, which, in principle, can form either by dehydration of1,2-ethanediol (ethylene
glycol), which is condensation, or by addition polymerization of oxacyclopropane (ethylene
oxide) [3].
Table 1.1 Physical Properties of Polymers
Properties of Plastics
Thermoplastics
PVC rigid
Polystyrene
PTFE
Polypropylene
Nylon
Cellulose nitrate
Cellulose acetate
Acrylic (methacrylate)
Polyethylene (high density)
Thermosetting plastics
Epoxy resin (glass filled)
Melamine formaldehyde
(fabric filled)
Urea formaldehyde
(kg m3)
Tensile Strength
(N mm2)
Elongation
(%)
E
(GN m2)
1330
1300
2100
1200
1160
1350
1300
1190
1450
48
48
13
27
60
48
40
74
2030
200
3
100
200700
90
40
1060
6
20100
3.4
3.4
0.3
1.3
2.4
1.4
1.4
3.0
0.7
16002000
18002000
68200
6090
1500
3890
BHN
Machinability
20
25
10
10
10
12
34
2
Excellent
Fair
Excellent
Excellent
Excellent
Excellent
Excellent
Excellent
Excellent
20
7
38
38
Good
Fair
710
51
Fair
(cellulose filled)
Phenol formaldehyde
(mica filled)
Acetals (glass filled)
Chapter 1 Introduction
16001900
3850
0.5
1735
36
Good
1600
5875
27
27
Good
Source: reference 3
The importance of polymers in our life is almost breathtaking. Proteins and carbohydrates, which
constitute two of the three principal classes of human foodstuffs, are natural polymers of high
molecular weight. Nucleic acids are responsible for transmission of genetic characteristics in
living organisms. Natural rubber, synthetic elastomers (synthetic rubbers), plastics, synthetic
fibers, and resins are all polymers having uses that reach into every part of our lives. Most of the
structural tissues of living things are composed of polymers. In plants these are chiefly cellulose
(a polysaccharide) and lignins. In animals the main structural polymers are proteins, which take
different forms as skin, hair, muscle, etc. In recent years, polymer has found its use in many
biomedical applications such as design of biomedical devices and in drug delivery systems.
Both natural and synthetic polymers have been widely studied as biodegradable polymers.
Biodegradable polymers are polymers that can undergo cleavage of bonds in the polymeric
backbone, either hydrolytically or enzymatically [4]. Unlike non degradable polymers which
remains in tissue after cell death or is eliminated by endocytosis, biodegradable polymers
undergo biodegradation to form smaller units which are then absorbed into the biochemical
pathway of the body [5]. A good example of the biodegradation of such polymers is as seen in
the breaking down of dextran by enzymes found in the kidney, liver and spleen of mammals.
Examples of biodegradable polymers are dextran, chitosan, dendrimers, collagen, cyclodextrin,
hyaluronic acid, etc.
4
Chapter 1 Introduction
Biodegradable polymers offer tremendous potential either as a drug delivery system alone or in
conjunction with other polymers. The most desirable biodegradable system is a completely
degradable system which leaves no residual polymer following the release of the drug in a
reasonable period of time [6]. Some biodegradable polymers used in drug delivery are vinyl
polymers such as N-(2-hydroxypropyl) methacrylamide [7], synthetic poly(-amino acids) such
as poly(L-glutamic acid), polysaccharides such as chitosan and dextrans [8], proteins such as
albumin, polyesters such as polylactide and polyglycolide [9], etc. A polymer may be grafted on
a different polymer; such co-polymers possess the cumulative favorable properties of its
constituent polymers. Table 1 shows some biodegradable polymers for biomedical applications.
Polymer
Tm (0C)
Tg (0C)
Tensile modulus
(MPa)
Degradation time
(months)
Polyglycolic acid
225 230
35 40
6 12
L-Polylactic acid
173 178
60 65
2.7
>24
DL-Polylactic acid
Amorphous
55 60
1.9
12 16
Polycaprolactone
58 63
0.4
>24
85/15 poly(DL-lactide-coglycolide)
Amorphous
56
65) (
50 55
60)
Source: reference 10
The need for polymer-drug conjugates became necessary because most conventional low
molecular weight anticancer drugs have an inherent character to transverse in and out of blood
vessels freely and this causes a non-selective distribution in both normal and tumor cells causing
undesirable side effects disastrous to patients [11]. Cancer-selective targeting became an
5
Chapter 1 Introduction
important goal for scientists [12] hence binding a drug to a polymer with its resultant increase in
molecular weight significantly altered the biodistribution of the drug into the cells by
endocytosis, achieving targeted delivery at both tissue and cellular levels [11]. Linking these
drugs to a macromolecule such as a polymer (polysaccharides, proteins, poly amino acids) makes
it possible for tumor accumulation [8] and confers on them some selectivity as well as enhancing
the drugs therapeutic effects.
The concept of polymeric macromoleculedrug conjugates was first proposed by Ringsdorf for
delivery of hydrophobic small drug molecules to their sites of action [7][8]. Since Ringsdorfs
proposal, polymer-drug conjugates or polymeric prodrugs as a special type of drug delivery
system have attracted considerable attention due to their particular therapeutic properties, such as
prolonged half-life, enhanced bioavailability, lower immunogenicity and antigenicity, and often
targeting specific cells, tissues or organs [13][14][15][16][17].
Chapter 1 Introduction
Chapter 1 Introduction
the size, shape, surface charge, and decorations, and mechanical properties of the conjugate play
key roles in biodistribution, vascular dynamics, targeting, clearance, uptake, drug release kinetics
and degradation [4][11].
Polymer-drug conjugates stay longer in circulation than low molecular drugs used in
chemotherapy since the endothelium of normal blood cells is typically impermeable to
macromolecules [4]. It is necessary that the polymer-drug conjugate is stable during circulation
in the bloodstream and the cytotoxic drug should only be released from the conjugate
intracellularly or intratumorally [19].
As opposed to low molecular weight drugs which diffuse through normal vasculature to any
tissue and internalize, macromolecules such as drug-polymer conjugates gain entry into tissues
only through gaps in angiogenic vessels [4][12]. This mechanism of entry by low molecular
drugs accounts for its high toxicity. Polymer bound drugs enter tumor cells through fluid phase
pinocytosis, dependent on the particle morphology and surface charge [4][19]. Pinocytosis
results in the internalization of solubilized material either contained in the extracellular fluid or
adherent to the plasma membrane into the endosomes, which are membrane bound vesicles
coated by the protein clathrin [11]. The internalized conjugate is then carried to the early
endosome which serves as a sorting compartment after which molecules obtained are recycled
into the plasma membrane or the late endosome and lysosome for enzymatic degradation.
Lysosomal degradation is an important step in delivery systems, the low pH of about 5 in the
lysosome and lysosomal enzymes play a crucial role in this regard as this creates the optimum
environment for acid-labile linkers or spacers linking the drug to the polymer in some conjugates
to degrade and subsequently release the conjugated drug. A number of studies have shown that
Chapter 1 Introduction
extracellular pH in tumors is consistently acidic and can reach pH values of 6.0 this being the
result of the accumulation of tumor derived metabolic acids and hypoxia because of poor
circulation within the tumor [4][19]. This relatively low extracellular pH environment also plays
an important role in hydrolyzing the acid-liable linker or spacer for drug release, a phenomenon
known as pH controlled drug release [19][22]. Macromolecules are retained longer and more
efficiently in the tumor cells due to the EPR effect [11][23] a major rationale for using polymeric
prodrugs. Upon internalization into the cells, the drug from the polymer carrier can exert its
therapeutic effect either from within the endosomal compartment or upon release into the
cytoplasm [11].
In vitro drug release studies for analyzing polymer-drug conjugates include the dialysis method
[24], high-performance liquid chromatography method [25] and the dispersion method [19].
Equipment such as a USP dissolution apparatus V from Pharmatest Germany can also be used
in vitro drug release studies [26]. In the mechanism of drug release, a too fast release can abolish
the advantages of polymer conjugation and yield conjugates with the same toxicity of free drugs
whereas a too slow release can impair drug activity [27][28]. An efficient polymeric prodrug
must therefore demonstrate a controlled release over a specific period of time.
Biodegradable polymers in polymer drug conjugates will undergo biodegradation after drug
release either hydrolytically or enzymatically with the degradation products absorbed in the
biochemical pathways. Semi-degradable polymers are degraded into smaller blocks during
lysosomal degradation and excreted by the kidney. Elimination of non-degradable polymers is
hampered by their high molecular weight and hence remain in the tissue after cellular death or
undergo exocytosis and then via the lymphatic circulation into the bloodstream for elimination
Chapter 1 Introduction
Drug polymer conjugates have also shown to overcome drug resistance. Resistance to
chemotherapeutic drugs in cancers is mainly due to the active transport of these drugs out of the
tumor cell mediated by certain proteins such as ATP-binding cassette transporters and multidrug
resistance-associated proteins [23]. Since drug conjugated polymers changes the path of drug
internalization from diffusion to endocytosis bypassing the efflux pumps(altering absorption
pathway from transcellular to paracellular or transcytosis routes), it minimizes the drug
interactions with multidrug resistance transporters leading to increased intra-cellular
accumulation and enhanced efficacy of the drug in resistant cells as realized in a number of
studies using HPMA-copolymer doxorubicin conjugates in resistant ovarian carcinoma cells [4].
Studies with Pluronic, an inert block copolymer comprising of hydrophilic ethylene oxide and
hydrophobic propylene oxide blocks, has demonstrated noteworthy improvement in the
cytotoxicity of daunomycin with Pluronic as compared to free drug in multidrug resistant cell
lines [11].
Polymeric drugs may also elicit the bystander effect a phenomenon whereby non targeted
neighboring tumor cells also perish due to the diffusion or gap junction transfer of the active
drug after its dissociation from the carrier within the targeted cells [4]. This phenomenon leads to
a high therapeutic effect which plays a crucial role in the elimination of drug resistance.
i.
they require less administrations due to increased tissue and blood half life which is
of great importance to patients, allowing for patience compliance and quality of life
10
Chapter 1 Introduction
as well as cost effective [12]. Example is in the treatment of patients with hepatitis C
and cirrhosis, the use of native interferon- which requires more than 2 to 3 injections
per week whiles pegylated interferon- (Pegasys) requires once a week injection
[29].
ii.
they enhance a drugs solubility, cellular uptake, intestinal absorption and physical
stability. For instance, Taxol and caplitaxel are converted to highly soluble drugs
[12].
So far several polymer drug conjugates have been approved for clinical application or clinical
trials. Poly(styrene-co-maleic acid)-neocarzinostatin is used for the treatment of liver cancer in
Japan [16][19] and PGA-Paclitaxel conjugate (Paclitaxel poliglumex, CT-2103) is already in
phase II clinical trials [4] and expected enter the market in the near future [20]. Table 1.1 shows
some polymeric prodrugs currently in the market, Table 1.2 are examples of polymeric prodrugs
undergoing clinical trials and Table 1.3 shows a list of polysaccharides used in polymer drug
conjugates.
11
Chapter 1 Introduction
Trade Name
Technology
Company
Indication
Adagen
Enzon
Severe
immunodeficiency
Disease
Oncasper
PEGylated L-asparaginase
Enzon
1994
PEG-intron
Schering
Chronic hepatitis C
2001
PEG-ASYS
Roche
2002
Neulasta
Amgen
Febrile neutropenia
2002
Pfizer
Acromegaly
2003
combined
1990
Macugen
Osi-Eyetech
2004
Mircera
Amgen
2007
Cimzia
UCB Pharma
Crohn's disease
2008
Krystexxa
Savient Pharma.
Gout
2010
Omontys
PEGylated peginesatide
Takeda Pharma.
2012
Zinostatin
Stimaler
Styrene
Maleic
Neocarzinostatin
(SMANCS)
Yamanouchi
Hepatocellular carcinoma
1990
XYOTAX
Polyglutamic acid-paclitaxel
Cell Therapeutics
Anhydride-
Source: U S Food and Drug Administration website and websites of pharmaceutical companies supplying these
drugs
12
Filed EMEA
Chapter 1 Introduction
Technology
Company
Indication
Clinical
Phase
CALAA-01
Polymer-cyclodextrin nanoparticle-
Calando Pharma.
Solid tumors
Phase I
siRNA
NKTR-105
PEG-docetaxel
Nektar
Phase I
XMT-1001
Biodegradable polymer-camptothecin
Mersana
Solid tumors
Phase I
IT-101
Polymer-cyclodextrin nanoparticle-
Calando Pharma.
Solid tumors
Phase I / II
Camptothecin
NKTR-102
PEG-irinotecan
Nektar
Phase II
ProLindac
Access Pharma.
Ovarian cancer
Phase II
NKTR-118
PEG-naloxone
PEGylated-anti
VEGFR2
fragments
as an angiogenesis inhibitor
Nektar
Opoid-induced constipation
Phase II
UCB Pharma
Phase II
CDP 791
Fab
Paclical
Micelles xr17
Oasmia
Pharmaceutical
AB
Ovarian cancer
Phase III
GenexolPM
Micelles
Samyang
Phase II
13
Chapter 1 Introduction
Polymer
Drug
Linker
Hyaluronic
acid
DOX
Succinate/adipic
dihydrazide
Type of bond
(name and
cleavability)
Amide with
drug,
amide with
polymer
Selective uptake by
CD44
targeting (HBL-100,
SKOV-3, HCT-116)
2
Hyaluronic
acid
Hyaluronic
acid
DOX
PTX
Adipic
dihydrazide
Imine
(hydrazone)
with drug,
amide with
Slightly lower
toxicities
Polymer
(MDA-MB-468LN,
Succinate/adipic
dihydrazide
with polymer
MDA-MB-231, MCF7)
Selective uptake,
targeted
but lower toxicities
than
(MDA-MB-468LN,
s.c.)
Antitumor activity
in
human ovarian
carcinoma
Xenografts (NMP-1
and
SKOV-3ip, i.p.)
Higher anticancer
efcacy
relative to the
conventional
Hyaluronic
acid
Hyaluronic
acid
PTX
PTX
4Hydroxybutanoic
acid derived
with polymer
Direct
conjugation
Ester
Much stronger
inhibitory
effect than
conventional
PTX (RT-4 and RT112/84)
More cytotoxicity for
Hyaluronic
acid
Hyaluronic
acid
Sodium butyrate
Direct
conjugation
Curcumin
Direct
conjugation
Ester
The MW difference
did not
inuence the
biological
activity of the
compounds
No
Improvement in
No
cytotoxicity due to
the
water solubility and
cell
internalization ability
of
the conjugate (L929)
8
Hyaluronic
acid
MTX
Peptide-linkers
(egPhePhe-Alkyl)
14
Amide with
drug,
amide with
polymer
Not applicable
(arthritis
chemotherapy
purposes
model)
Chapter 1 Introduction
Dextran,
7-Ethyl-10-
carboxymethyl
aminopropyloxy-
Gly-Gly-Gly
Amide with
drug,
amide with
polymer
CPT
(T-2513)
11
Dextran,
Exatecan
carboxymethyl
(DX-8951, CPT
polyalcohol
analog)
Dextran,
PTX
Gly-Gly-Phe-Gly
Gly-Gly-Phe-Gly
carboxymethyl
12
Dextran,
oxidized
Amide with
drug,
amide with
polymer
DOX
Glycine
Not available
Not available
Efcacy of T-0128
was
10-fold superior to
that of
T-2513 against
Walker-256
carcinoma,
signicant
antitumor activity
against
the panel of
human tumor
xenografts (i.v.)a
A single-dose of the
conjugate (DE-310)
The conjugate
(AZ10992)
was inactive in vitro
(cell
lines are not
mentioned)
Not available
exhibited similar
or greater
antitumor activity
than
multiple
administrations of
DX-8951f (various
human
tumor xenografts
and murine solid
tumors)a
Signicant
antitumor
activity (colon26,
MX-1,
LX-1, HT-29, i.v.)
The conjugate (AD70)
showed higher
activity,
higher plasma
concentration,
lower acute
toxicity and
accumulation
in the heart in rats
(Walker256, i.v.)a
13
Dextran,
oxidized
Cytarabine (Arac)
N4-(4-
Amide
Not available
carboxybutyryl)-
ethylene
diamine
14
Dextran,
oxidized
MTX
Diaminohexane
or
5-amino-1pentanol
Amide amide or
amide
ester
(respectively)
Modest but
signicant
increases in
survival after
intracranial
polymeric
delivery of MTX
or
MTX-amidedextran (rats
brain tumors)
with intracranial 9L
Cytotoxicity of
MTX-ester-dextran
and
MTX-amide-dextran
gliosarcoma,
inserted
15
Chapter 1 Introduction
pellets)
15
Dextran
MTX
Direct
conjugation
Ester
Greater toxicity in
antiproliferative
effects
compared to free
MTX
comparison with
the
16
Dextran,
Jeffamine-ProVal-
carboxymethyl
released)
Gly-Leu-Ile-Gly
Amide with
drug,
amide with
polymer
No signicant
difference in
drug accumulation
at the
tumor site
between the
MMP-sensitive and
the
MMP-insensitive
conjugates which
shows
that the tumor
targeting
via EPR. (HT-1080
bearing
mice, overexpresses
MMP,
i.p.)
17
Chitosan,
MMC
Direct
conjugation
Amide
Not mentioned
N-succinyl
18
Chitosan,
MMC
Glutaryl
MMC
Glutaryl
Amide with
drug,
amide with
polymer
N-succinyl
19
Chitosan,
glycol
Amide with
drug,
amide with
polymer
Not mentioned
Not mentioned
Good antitumor
activities
against various
tumors
(P388 and L1210
i.p.,
M5076 i.v. and i.p.,
MH134
i.p.)
Same efcacy and
less
toxicity of the
conjugate
than the glycolchitosan
counterpart see
below and
signicant
periority over
the counteraprt in
i.v.
administration
(P388 i.p.,
Sarcoma 180 i.p.
and i.v.)
Same efcacy but
more
toxicity than the
succinyl-chitosan
counterpart (P388
i.p.,
16
Chapter 1 Introduction
Sarcoma 180 i.p.
and i.v.)
20
21
Chitosan
Chitosan,
glycol
DOX
DOX
Succinate
Amide with
drug, amide with
polymer
Reduced cytotoxicity
compared to of free
drug (SKOV-3,
MCF-7)
Amide with
drug,
Not mentioned
Not mentioned
DOX-loaded GCDOX
nanoaggregates
exhibit
lower systemic
toxicity but
comparable antitumor
activity via EPR
(B16F10
cis-Aconityl
amide with
polymer
II45, i.v.)
22
Chitosan, low
MW
PTX
Succinate
with polymer
Conjugate
signicant
(NCIH358, SK-OV-3,
growth/42% of
bioavailability
after oral
administration
(B16F10,
p.o. admin of the
conjugate
MDA-MB-231)
inhibition of tumor
Chitosan, low
MW
DTX
Succinate
Comparable
antitumor efcacy
and higher
bioavailability
(p.o.) than
i.v. DTX at the same
dose
along with
prolonged
blood circulation
time
blood and less
sub-acute
toxicity ((NCI-H358
and
U87MG)
24
Chitosan,
stearic
DOX
cis-Aconityl
acid grafted
25
Heparin, low
MW
ATRA
Ethylenediamine
17
Amide with
drug,
amide with
polymer
DOXChitosan
Stearic acid
micelles could
reverse the
drug resistant by the
Suppressed the
tumor
growth and
reduced the
toxicity better than
commercial DOX
HCl
injection (QGY,
i.v.)
Longer systemic
circulation
conjugate
nanoparticles
time relative to
that of PTX
Chapter 1 Introduction
exhibited higher
cytotoxic
activity than PTX
plus ATRA
solution
solution/no
signicant
effects on hemolysis
(HepG2)
26
Heparin, low
MW
PTX
Carbonate,
Amide with
polymer,
Higher toxicity of
heparin
ethylenediamine
PTX conjugate
No
nanoparticles (KB)
27
Heparin,
PTX
Single aminoacid
Ester
spacer (Val,
Leu, or
succinylated
Phe)
Similar ovarian
tumor
growth inhibition as
PTX
and induces no
obvious
body weight loss
for
leucine prodrugs
28
Heparin,
O-acetylated
PTX
Direct
conjugation
Ester
The anticoagulant
activity
or aminoacid
spacer (Val,
Leu, or
Phe)
No
Source: reference 8
1.4 Chitosan
Chitosan, a biomaterial obtained via alkaline N-deacetylation of chitin [28][32][33][34][35][19]
which is the second most abundant organic material after cellulose [33][34] has recently attracted
much attention from scientists across the globe. It is a copolymer that is primarily composed of
(14) linked 2-amino-2-deoxy-D-glucopyranose units, and residual 2-acetamido-2-deoxy-Dglucopyranose units [34][35][19]. It possesses the special properties such as biocompatibility,
biodegradability, and biological activities as a result it has been widely applied to biomedicals,
pharmaceuticals, cosmetics, etc [36]. Although the polymer backbone consists of hydrophilic
functional groups and is hydrophobic in nature, chitosan is normally insoluble in water and most
18
Chapter 1 Introduction
common organic solvents (e.g. DMSO, DMF, NMP, organic alcohols, pyridine) [37]. The
insolubility of chitosan in aqueous and organic solvents is a result of its crystalline structure,
which is attributed to extensive intramolecular and intermolecular hydrogen bonding between the
chains and sheets, respectively [38]. Chitosan has found application in many areas of drug
delivery [35][37], however its low solubility poses a challenge as a drug delivery system.
Chitosan has both reactive hydroxyl and amino groups [36] and hence can be modified to
enhance its solubility. For instance, the introduction of polar ionic groups to the polymer
backbone by exploiting the nucleophilicity of the electrons rich N-amino functional groups. This
enhances the solubility and drug delivery efficiency of chitosan. Such modifications include the
formation of graft copolymers by introduction of some monomers [36]. Such copolymers include
dextran sulphate-chitosan [37], 6-O-Cholestrol modified Chitosan [33], glycol chitosan [13] and
Chitosan -cyclodextrin [39].
19
Chapter 1 Introduction
Chapter 1 Introduction
relatively easy. However, the secondary side is shown to be the more important side of CD in
binding studies [50]. The stability of the CD-inclusion complex depends on the polarity of the
guest molecule and on the compatibility of the size of the host and that of the guest [51]. Recent
biotechnological advancements have resulted in dramatic improvements in CD production,
which has lowered their production cost. This has led to the availability of highly puried CDs
which are well suited as pharmaceutical excipients. CDs are widely used in basic research and
industrial processes for the microencapsulation of unstable or volatile substances [52].
Cyclodextrin has the merit of a hydrophobic cavity which is easy to assemble with other
molecules whiles chitosan has a merit of degradation slowly in organism without triggering any
immune response. Therefore grafting cyclodextrin molecules into chitosan reactive sites may
lead to a molecular carrier that possess the cumulative effects of inclusion, size specificity and
transport properties of cyclodextrin as well as a controlled release ability of the polymeric matrix
[53]. Chitosan grafted with cyclodextrin also have the ability to form complexes with a variety of
other appropriate compounds [54]. The products obtained by CD grafting to chitosan using
different methods and their inclusion ability, absorption and controlled release properties have
been studied extensively.
1.6 Cancer
Cancer known medically as malignant neoplasm is one of the most devastating diseases and it
involves various genetic alterations and cellular abnormalities [55]. The World Health
Organization estimates that 84 million people will die of cancer between 2005 and 2015 [56].
Cancer is basically a condition which involves unregulated cell growth in which cells divide
uncontrollably forming malignant tumors which may spread to other parts of the body through
21
Chapter 1 Introduction
the bloodstream or the lymphatic system. There are over 200 different known cancers that afflict
humans [57] the most common being breast, liver, lung and skin cancer. Worldwide, breast
cancer is the most common invasive cancer in women and accounts for more than 25% of
cancers in women [58].
Cancer is basically a condition which involves unregulated cell growth in which cells divide
uncontrollably forming malignant tumors which may spread to other parts of the body through
the bloodstream or the lymphatic system. Normal cells in the body follow an orderly path of
growth, division, and death. When the process of programmed cell death lais, apoptosis breaks
down, cancer begins to form. This leads to a mass of abnormal cells that grows out of control.
Cancer harms the body when these cells divide uncontrollably to form lumps or masses of tissue
(except in the case of leukemia where cancer prohibits normal blood function by abnormal cell
division in the blood stream). Tumors can grow and interfere with the digestive, nervous, and
circulatory systems and they can release hormones that alter body function. Tumors that stay in
one spot and demonstrate limited growth are generally considered to be benign. Malignant
tumors are termed cancerous and are more agile than non-malignant ones.
Although the mechanisms of formation and spreading of cancer are still not well understood,
both external factors (tobacco smoking, chemicals, radiation, and infections) and internal factors
(inherited metabolism mutations, hormones, and immune conditions) are believed to be relevant
[59]. A person is susceptible to cancers if there are damages or mutations to DNA which
damages the genes involved in cell division. There are four main types of gene responsible for
the cell division process: oncogenes tell cells when to divide, tumor suppressor genes tell cells
when not to divide, suicide genes control apoptosis and tell the cell to kill itself if something
22
Chapter 1 Introduction
goes wrong, and DNA-repair genes instruct a cell to repair damaged DNA. Cancer hence occurs
when a cell's gene mutations make the cell unable to correct DNA damage and unable to commit
suicide. It may also occur as a result of mutations that inhibit oncogene and tumor suppressor
gene function, leading to uncontrollable cell growth. Recent results of cancer genomics have
revealed that most human solid tumors are not only single gene-based events but also multiple
genomic alterations [12].
Carcinogens, a class of substances directly responsible for damaging DNA also predisposes a
person to cancer. Tobacco, asbestos, arsenic, radiation such as gamma and x-rays, the sun and
compounds in car exhaust fumes are all examples of carcinogens. When our bodies are exposed
to carcinogens, free radicals are formed that try to steal electrons from other molecules in the
body. Theses free radicals damage cells and affect their ability to function normally.
Cancer may also be the result of a genetic predisposition that is inherited from family members.
It is possible to be born with certain genetic mutations or a fault in a gene that makes one
statistically more likely to develop cancer later in life.
Aging increases the possibility of cancer-causing mutations in our DNA. This makes age an
important risk factor for cancer. Several viruses have also been linked to cancer such as: human
papilloma virus which causes cervical cancer, hepatitis B and C which causes liver cancer and
Human immunodeficiency virus (HIV) which causes Kaposi sarcoma. Factors that suppress or
weakens the immune system and inhibit the body's ability to fight infections also increases the
chance of developing cancer.
Cancer can be treated by surgery, radiation therapy, gene therapy, immunotherapy, chemotherapy
23
Chapter 1 Introduction
or monoclonal antibody therapy depending on the location and grade of the tumor as well as the
state of the disease and the condition of the patient. Surgery is the best known treatment for
cancer which has not metastasized by completely removing the cancer from the body.
Radiotherapy destroys cancer by focusing high-energy rays (gamma rays or x-rays) on the cancer
cells which causes damage to the molecules that make up the cancer cells and leads them to
commit suicide.
Chemotherapy agents are cytotoxic drugs used to treat cancer that function by targeting fast
growing cells and by blocking some critical elements of the cell division process, impairing
mitosis as well as promoting apoptosis [60]. Chemical drugs are generally considered to be one
of the most efficient forms of cancer therapy [61][62]. Chemotherapy utilizes chemicals that
interfere with the cell division process, damaging proteins or DNA so that cancer cells are self
destroyed. These treatments target any rapidly dividing cells (not necessarily just cancer cells),
but normal cells usually can recover from any chemical-induced damage while cancer cells
cannot. Chemotherapy is generally used to treat cancer that has spread or metastasized because
the medicines circulate throughout the entire body. Chemotherapy treatment occurs in cycles so
the body has time to heal between doses. However, there are still common side effects such as
hair loss, nausea, fatigue, and vomiting. Combination therapies often include multiple types of
chemotherapy or chemotherapy combined with other treatment options. At present, combinations
of different chemotherapeutic drugs in a chemotherapy regime are an attractive strategy for
effective anticancer treatment [63]. Different types of synthetic and natural anticancer drugs have
been used for chemotherapy [64]. Drugs for chemotherapy include mertansine, paclitaxel,
docetaxel, doxorubicin, cisplatin, gemcitabine and fluorouracil.
24
Chapter 1 Introduction
Immunotherapy aims to get the body's immune system to fight the tumor. Local immunotherapy
injects a treatment into an affected area, for example, to cause inflammation that causes a tumor
to shrink. Systemic immunotherapy treats the whole body by administering an agent such as the
protein interferon alpha that can shrink tumors [65][66]. Immunotherapy can also be considered
non-specific if it improves cancer-fighting abilities by stimulating the entire immune system, and
it can be considered targeted if the treatment specifically tells the immune system to destroy
cancer cells. These therapies are relatively young, but researchers have had success with
treatments that introduce antibodies to the body that inhibit the growth of breast cancer cells [65].
Several cancers have been linked to some types of hormones, most notably breast and prostate
cancer [67][68]. Hormone therapy is designed to alter hormone production in the body so that
cancer cells stop growing or are killed completely [68]. Breast cancer hormone therapies often
focus on reducing estrogen levels (a common drug for this is tamoxifen) [67][68] and prostate
cancer hormone therapies often focus on reducing testosterone levels [68]. In certain cancers,
administration of hormone agonists, such as progestogens may be therapeutically beneficial.
Contemporary methods for generating an immune response against tumours include intravesical
BCG immunotherapy for superficial bladder cancer, and use of interferons and other cytokines to
induce an immune response in renal cell carcinoma and melanoma patients.
Antibody drug conjugates have also been developed in the treatment of cancers. The basic
strategy underlying antibody drug conjugate technology is to combine the target selectivity of
monoclonal antibodies with the potency of cytotoxic agents, such as certain natural products and
synthetic molecules, with the goal of generating therapeutic drugs that are highly efficacious but
also safe [62][69]. Improved understanding of tumor biology and advances in antibody
25
Chapter 1 Introduction
engineering have made it possible to identify better tumor targets for antibody-based therapies
and to generate less immunogenic humanized and human antibodies [70] which is then linked to
a cytotoxic drug for cancer therapy. Example of such antibody drug conjugates is trastuzumab
emtansine which targets HER2 antigen over expressed on metastatic breast cancer and
gemtuzumab ozogamicin for leukemia [69]. Trastuzumab emtansine is composed of the
humanized IgG1 anti-HER2 Antibody trastuzumab (Herceptin) with maytansinoid DM1 via a
non-cleavable thioether linkage SMCC [62]. Most antibody drug conjugates rely on the release
of the drug from the antibody after it is in the endosome in order for it to exert its
pharmacological activity in the cytosol or nucleus [70][18]. These antibody drug conjugates have
higher efficacy with fewer side effects as compared to their non conjugated cytotoxic low
molecular weight drugs due to their specific targeting.
Since cancer drugs can cause enormous toxicity to normal tissues and cells [69][8][31][71] the
opportunity to deliver them locally creates the possibility of improving both the safety and
efficacy of cancer chemotherapy [72]. The need for the development of novel cancer therapies
and drug delivery strategies that provide specific targeting of tumor cells has been continually at
the forefront of medical science [73]. Drug delivery systems may either be active targeting or
passive targeting [18]. Passive targeting is achieved by exploiting the enhanced permeability
and retention (EPR) effect [4][11][19][74]. Tumor microvascular endothelium exhibits elevated
permeability as a result of various vascular mediators such as bradykinin and prostaglandins
which induce extensive vascular permeability, coupled with lack of lymphatic drainage,
macromolecules accumulate in tumor tissues for long periods compared to normal tissues
26
Chapter 1 Introduction
[19][74]. This is the EPR effect which is observed in almost all human cancers with the
exception of hypovascular tumors [12].
Targeted delivery is essential because of the narrow therapeutic index of most antitumor drugs
and hence the urgent medical need to achieve focused drug delivery [75].
27
Chapter 1 Introduction
target tissues or cells enhancing the therapeutic effect [21]. Targeted drug delivery and release
technology have the following advantages
i.
ii.
to decrease the amount of drugs needed to attain a desirable therapeutic dose in the
target cancer cells
iii.
to reduce the amount of drugs in non-cancerous cells and to reduce its side effects.
1.8 Doxorubicin
One of the most widely used cytotoxic drugs in chemotherapy is Doxorubicin Hydrochloride
(DOX). However, problems related to the development of drug resistance and acute
cardiotoxicity have led investigations into alternative forms of its administration for cancer
therapy [76]. Encapsulation of DOX in drug delivery systems have been well investigated for
decades and has seen great achievements in recent times with the approval and use of
Doxorubicin Hydrochloric acid liposome for the treatment of Kaposi Sarcoma (FDA) and some
others in clinical trials. Microencapsulation of DOX for its controlled release over extended
periods of time have shown to have demonstrated great benefits such as drug accessibility to
tumors, reduced cytotoxicity, prolonged plasma level of DOX as well as increased cytotoxicity
against multidrug resistance cell lines in vitro [76].
28
Chapter 1 Introduction
29
Chapter 1 Introduction
intercellular penetration and distribution [78]. Nano-sized particles accumulate in solid tumors at
much higher concentrations than in normal tissues or organs due to the EPR effect. Nanoscale
active targeted release drugs therefore have a great potential in clinical tumor therapy.
In this study, Carboxymethyl Chitosan--Cyclodextrin Nanoparticles filled with DOX which has
the possibility of encapsulating both hydrophilic and hydrophobic drugs and further conjugation
to a ligand such as an antibody for a more targeted delivery is synthesized. The nanoparticless
were then characterized, drug release studies carried out and anticancer effect determined.
30
Acetonitrile,
Isopropyl
alcohol,
Acetone,
HCl,
Methanol,
1-ethyl-3-(3-
2.2 Method
2.2.1 Preparation of Carboxymethyl Chitosan (CMC)
Carboxymethyl chitosan (CMC) was prepared according to the method described by Yu et. al
[79]. Briefly, 10 g of chitosan dissolved in 200 mL of isopropanol was placed into a 500 mL ask.
After 1 h of stirring at 500C, 20 g of 50% sodium hydroxide solution was added, and the reactive
system was alkalized at this temperature for 1 h. Then a solution of 15 g of monochloroacetic
acid in 20mL of isopropanol was added dropwise, and the mixture was reacted at 600C for 4 h.
The resulting product was then removed from the reaction ask, dissolved in distilled water,
precipitated in a 10-fold amount of cold methanol, and collected on a lter. The obtained follow
product was puried by reprecipitation from water/methanol and then dried in vacuum at 400C
under reduced pressure for 12 hours.
31
a mixture of -CD (5.7 g, 5 mmol) and sodium hydroxide (5 g, 0.125) in water (20 ml) was
treated with 0.47 g monochloroacetic acid solution (5 mmol) for 5 hours at 500C. The reaction
mixture was then cooled and its pH adjusted to 7 by adding HCl. The obtained product was
precipitated with excess acetone, filtered and dried at 400C under vacuum to give the CM -CD.
32
solution. 1 mg of the prepared nps was added to 4 ml of the drug solution. The mixture was then
stirred for 48 hours and then dialyzed against deionized water for 24 hours. This was then freeze
dried to obtain the drug loaded nps. To avoid photodegradation of DOX procedures were
performed in the absence of light. DOX loaded in the nps was determined by UV absorption at
480 nm.
34
x 100
35
36
Chapter 3 Results
Chapter 3 Results
3.1 Size and Zeta Potential Studies
Size and zeta potential studies revealed that nps size and surface charge is dependent on the
concentration of CMC to CM -CD used.
CMC concentration (%
w/v)
0.05
Precipitation
0.15
Precipitation
0.30
306.0
-7.9
0.45
261.1
-10.2
0.60
211.7
-12.2
0.75
195.1
-19.4
0.90
167.8
-22.9
1.05
150.9
-25.9
1.20
133.0
-28.4
1.30
124.0
-33.7
1.50
Table 3.1 Size and zeta potential of CMC--CD nanoparticles using different CMC
concentrations.
37
Chapter 3 Results
Figure 3.2 FTIR spectra of (a) chitosan (b) -cyclodextrin (c) carboxymethyl chitosan (d)
38
Chapter 3 Results
2.500
2.000
Absorbance mean
1.500
Linear (Absorbance
mean)
1.000
0.500
0.000
0
0.005
0.01
0.015
39
Chapter 3 Results
Drug release shows a burst release followed by a gradual release at both pH 4.5 and pH 7.4.
Cummulative release of DOX (%)
80
70
60
50
Cummulative
drug release (%)
pH 4.5
Cummulative
drug release (%)
pH 7.4
40
30
20
10
0
0
20
40
60
Time (Hours)
80
100
Figure 3.5 Drug release studies for doxorubicin at pH 4.5 and 7.4
80.0
70.0
60.0
Free DOX
50.0
40.0
30.0
20.0
10.0
0.0
0
5
10
CDOX(g/mL)
15
(g/ml)
Figure 3.6 Cytotoxicity effect of free doxorubicin and doxorubicin loaded CMC--CD nps on
MCF-7 breast cancer cells after 24 hours.
40
Chapter 3 Results
Free DOX
92.4
75.7
54.3
47.8
42.9
38.3
30.8
26.4
23.6
21.3
Table 3.2 Cell viability studies data for free doxorubicin, doxorubicin loaded CMC--CD nps
and blank CMC--CD on MCF-7 breast cancer cells after 24 hours
120.0
100.0
80.0
Free DOX
60.0
40.0
20.0
0.0
0
10
15
Figure 3.7 Cytotoxicity effect of free doxorubicin and doxorubicin loaded CMC--CD nps on
ARPE-19 epithelial cells after 24 hours
41
Chapter 4 Discussion
Chapter 4 Discussion
4.1. Synthesis of nanoparticles
In order to investigate the feasibility of preparing CMC--CD nanoparticles by ionic gelation.
Ionic gelation technique is based on the ionic interactions between the positively charged
primary amino groups of chitosan or some of its derivatives and the negatively charged groups of
a polyanion. Ionic gelation technique has attracted considerable attention due to this process is
non-toxic, organic solvent free, convenient and controllable. In this research, various
concentrations of CMC were grafted onto a fixed concentration of CM -CD. This method
utilizes the ionic interaction between the positively charged amino groups of N,O methylated
chitosan and the negatively charged CM -CD, and the ability amino groups in the chitosan and
its methylated derivative to form a gel after contact with polyanions by forming inter- and
intramolecular linkages. The ionic gelation process is extremely mild and involves the mixture of
two aqueous solutions at room temperature.
The synthesis route of the nanoparticles is shown below in scheme 4.1.
Chapter 4 Discussion
The Stokes-Einstein relation that connects diffusion coefficient measured by dynamic light
scattering to particle size. where Dh is the hydrodynamic diameter (particle size!), Dt is the
translational diffusion coefficient, kB is Boltzmanns constant, T is thermodynamic temperature
and is dynamic viscosity.
The calculations are handled by instrument software. However, the equation does serve as
important reminder about a few points. The first is that sample temperature is important, as it
appears directly in the equation. Temperature is even more important due to the viscosity term
since viscosity is a stiff function of temperature. Finally and most importantly, it reminds the
analyst that the particle size determined by dynamic light scattering is the hydrodynamic size.
That is the determined particle size is the size of a sphere that diffuses the way as your particle.
43
Chapter 4 Discussion
The hydrodynamic radius is not the same as the radius of gyration. Hydrodynamic sizes are more
easily measured than radii of gyration and can be measured over a wider range of sizes. The
conversion from hydrodynamic radius to radius of gyration is a function of chain architecture.
The zetasizer works based on dynamic light scattering. In nanoparticle samples that contain a
distribution of particle sizes the obtained particle size, known as the z-average size, is a weighted
mean size. The z-average size increases as the particle size increases. And, it is extremely easy to
measure reliably. For these reasons, the z-average size has become the accepted norm for particle
sizing by dynamic light scattering.
Size and zeta potential results of this study are represented in Table 3.1 and Figure 3.1 in which it
can be observed that the formation of nanoparticles is possible for some specific concentrations
of CMC to CM -CD. It can also be noted that the particle size is dependent on the CMC to CM
-CD concentration ratio. Further experiments were conducted using a CMC concentration of 0.9%
and CM -CD concentration of 0.8mg/ml.
Chapter 4 Discussion
extremity of a fine tip and the atom(s) at the sample surface. In the contact mode, the user fixes
the value of the repulsive force between the tip and the sample which will be maintained constant
during the raster scan of the sample, providing an isoforce image of the surface. In the oscillating
mode, the tip oscillates at a high frequency, determined by the cantilever spring constant, and
interacts with the surface only at the lower end of each cycle.
The first most important step in AFM studies is the sample preparation for imaging. The AFM
can only image material which adheres or is fixed to a support or substrate so that samples would
not be pushed away by the scanning tip and cannot be imaged by this technique.
Mica, glass and silicon oxide have proved to be excellent substrates. The most widely used
substrate for imaging biological specimens is mica. The surface of mica can be modied with
silanes either to promote adsorption or to allow covalent binding of the biomolecules. Glass
represents another suitable substrate for biological AFM. Glass cover slips are at enough for
imaging cells and other large structures, but are usually too rough for imaging adsorbed
molecules. The glass surface is always coated with organic contaminants and particles that
should be removed before use.
After the selection of an appropriate substrate, the next is the immobilization of the sample on
the substrate. This procedure consists of depositing a drop of an aqueous solution containing the
macromolecule of interest on the substrate, and then allowing the drop to evaporate or blowing
the sample dry in nitrogen. Alternatively, after dropping the sample, the substrate can be spinned
for the even distribution of the sample and adsorption of the biomolecules or sample. After this
the substrate having the sample can be imaged using the AFM.
AFM micrographs established that the CMC--CD nanoparticles had a discrete spherical shape
45
Chapter 4 Discussion
with sizes in agreement with the measurements of the dynamic light scattering. An AFM
micrograph of CMC--CD nanoparticles prepared from the 0.9% CMC solution is shown in
Figure 3.3.
FTIR is the most recent technology that uses IR in quantitative analysis. It is used by organic
chemists to determine the components of organic compounds. Most of the organic compounds
reveal their distinguishing component when exposed to infrared radiation. The revealed
distinguishing component emits energy (wavelength) that is represented by a graph called
spectrum. To identify a component of certain compounds, they are exposed to high energy such
as Infrared Radiation (IR). The reaction results to emission of energy showing the reactions of
the molecules, which are automatically plotted to a graph by one of the programs embedded in
spectroscopic instruments. Using the generated graph, organic chemists analyze the plot and
detect distinctive peaks that can be attributed to the components of the compound. For instance, a
graph shows two distinctive peaks, and after analyzing the plot, you found out that one peak
corresponds to Hydrogen (H) and the other is Oxygen (O2); thus, you can safely say that it is H20
or water molecule. Molecules that react with IR always exhibit the same distinguishing peak of
energy so they can easily be identified from the graph.
In the FTIR studies the first step was the sample preparation. The standard method to prepare
solid sample for FTIR spectrometer was to use Potassium Bromide (KBr). About 2 mg of sample
and 200 mg KBr were dried and ground. The particle size was unified to be less than two
micrometers. Then, the mixture was squeezed to form transparent pellets which can be measured
directly. The second step was getting a background spectrum by collecting an interferogram and
46
Chapter 4 Discussion
its subsequent conversion to frequency data by inverse Fourier transform. The background
spectrum was obtained because the solvent in which the sample was placed will have traces of
dissolved gases as well as solvent molecules that contribute information that is not the sample.
The background spectrum will contain information about the species of gases and solvent
molecules, which may then be subtracted away from the sample spectrum in order to gain
information about just the sample. The background spectrum also takes into account several
other factors related to the instrument performance, which includes information about the source,
interferometer, detector, and the contribution of ambient water. Next, a single-beam spectrum of
the sample, which contained absorption bands from the sample as well as the background
(gaseous or solvent) was obtained. The ratio between the single-beam sample spectrum and the
single beam background spectrum gave the spectrum of the sample.
The FTIR spectrum of chitosan showed a broad -OH stretch absorption band between 3404 and
3100 cm-1 and the aliphatic CH stretch between 2990 and 2850 cm-1. The absorption peaks
between 1200 and 1020 cm-1 represented the free primary amino group (NH2) at C2 position.
The peak at 1647cm-1 represented acetylated amino group of chitin, which indicated that the
sample was not fully deacetylated. The peak at 1384 cm-1 represented the CO stretch of
primary alcoholic group (CH2OH). In the FTIR spectrum of CMC, the strong peaks at 1598
and 1406 cm-1 could be assigned to the asymmetry and symmetry stretch vibration of COO,
respectively. Also, the CO adsorption peak of the secondary hydroxyl group becomes stronger
and moves to 1070 cm-1. The results also indicated that the carboxymethyl substitution occurred
at the C6 position of chitosan.
The IR spectrum of -CD shows its characteristic strong OH absorption band at 3500 cm-1 and
47
Chapter 4 Discussion
a strong CO band around 1020 cm-1. In the IR spectrum of CM -CD, the strong peak at
1720 cm-1 could be assigned to the stretching vibration of carbonyl group. This peak is not
observed in -CD. The results indicate that the carboxymethylation occurs at C 6 position. The
spectra of CMC--CDs show not only the characteristic peaks of CMC, but also the
characteristic absorption bands of CM -CD. The relative intensity of the 3500 cm-1 band of
CMC--CD nps is much higher than the intensity of 3500 cm-1 of CMC. The increased intensity
could be attributed to the presence of more OH groups in CMC--CD nps resulting from the
coupling of CM -CD. Further, the characteristic peak of the -pyranyl vibration of CMC at
894.9 cm-1 and the characteristic peak of the -pyranyl vibration of CM -CD at 942.8 cm-1 both
appeared in CMC--CD nps. The absorption peak at 1644 cm-1, due to carbonyl stretching of
primary amide band, also conrms the grafting of CM -CD onto CMC. Results from the FTIR
studies is shown above in Figure 3.2.
Chapter 4 Discussion
transparent solvent. The other beam, the reference (colored blue), passes through an identical
cuvette containing only the solvent. The intensities of these light beams are then measured by
electronic detectors and compared. The intensity of the reference beam, which should have
suffered little or no light absorption, is defined as I0. The intensity of the sample beam is defined
as I. Over a short period of time, the spectrometer automatically scans all the component
wavelengths in the manner described. The ultraviolet (UV) region scanned is normally from 200
to 400 nm, and the visible portion is from 400 to 800 nm. If the sample compound does not
absorb light of a given wavelength, I = I0. However, if the sample compound absorbs light then I
is less than I0, and this difference may be plotted on a graph versus wavelength. Absorption may
be presented as transmittance (T = I/I0) or absorbance (A= log I0/I). If no absorption has occurred,
T = 1.0 and A= 0. Most spectrometers display absorbance on the vertical axis, and the commonly
observed range is from 0 (100% transmittance) to 2 (1% transmittance). The wavelength of
maximum absorbance is a characteristic value, designated as max. Different compounds may
have very different absorption maxima and absorbances. Intensely absorbing compounds must be
examined in dilute solution, so that significant light energy is received by the detector, and this
requires the use of completely transparent (non-absorbing) solvents. The most commonly used
solvents are water, ethanol, hexane and cyclohexane. Solvents having double or triple bonds, or
heavy atoms (e.g. S, Br & I) are generally avoided. Because the absorbance of a sample will be
proportional to its molar concentration in the sample cuvette, a corrected absorption value known
as the molar absorptivity is used when comparing the spectra of different compounds. This is
defined as:
Molar Absorptivity, = A/ c l
Where A= absorbance, c = sample concentration in moles/liter and l = length of light path
49
Chapter 4 Discussion
The proportionality approach to these kinds of problems focuses on the idea that the absorbance
of a solution is directly proportional to its concentration. When using this approach it is
necessary to be sure that the values given are for different concentrations of the same chemical
measured under the SAME conditions (both wavelength and the path length). For example in a
solution with a concentration of 0.14M is measured to have an absorbance of 0.43. Another
solution of the same chemical is measured under the same conditions and has an absorbance of
0.37. The solution to this problem can be set up using the equation shown below, which simply
says that the ratio of the concentrations is proportional to the ratio of absorbances.
We can use C1 to represent the unknown concentration. You can derive this equation from Beer's
law (Absorbance = E L c)
C1 / C2 = A1 / A2
Therefore,
C1 = (A1 / A2) * C2
50
Chapter 4 Discussion
Thus, C1 = 0.12M
In the graphical method, if you make a graph of absorbance versus concentration, if the
absorbing species obey's Beers lambert's Law, then you will get a straight line. You can then have
software of various types can give the equation representing that straight line in the form y = mx
+ b. If you plot the concentration on the Y axis and absorbance on the x axis .then the above
equation would be
Y (concentration in whatever units the graph was derived from) = slope x ( absorbance value ) +
intercept.
So if the predictive equation were y = 0.93 X + 0.1 for a Beer's Law line plotting absorbance
verses concentration in mg/ml and the absorbance value of your unknown sample were 0.5 then
the concentration would be
Alternatively a straight line can be plotted to determine the absorbance of your sample by
drawing a perpendicular from the straight line to the concentration based on the absorbance value
for your sample.
Chapter 4 Discussion
(A = Elc). Absorbance is unitless and usually a fraction like 0.2 or 0.1. Molar absorptivity is in
units of L/mol/cm whiles path length is in cm and concentration in this case would be mol/L.
In our study, doxorubicin hydrochloride (DOX) was chosen as a model drug. DOX can be
readily loaded into the nanospheres due to the interaction between the unreacted carboxyl groups
of CMC with the amino groups of DOX and inclusion complex formation between DOX and CD moiety.
For the drug encapsulation and loading efficiency studies, we used the Beers law of
proportionality in the calculations. For this purpose the concentration of the DOX solution used
for the encapsulation was 0.16mg/ml and had an absorbance of 3.085. After the encapsulation the
supernatant containing the remaining DOX obtained after centrifuging had an absorbance of
0.302. From Beers law of proportionality, C1 / C2 = A1 / A2.
Since 4ml of the 0.16mg/ml DOX solution was used, the amount of DOX in this solution will be
0.64mg and for the 0.0157mg/ml unencapsulated DOX solution, 0.0628mg.
From this studies, Encapsulation Efficiency was 90.2% and the Loading Efficiency was 57.7%.
It is well known that conventional micelles formed by amphiphilic copolymers have poor
performance to encapsulate water soluble drugs. Our method provides an efcient strategy to
load water soluble drugs and the nanospheres prepared exhibit a high encapsulation efciency
for DOX, which is comparable to that of nanospheres with a vesicular structure specially
designed for hydrophilic drug delivery
52
Chapter 4 Discussion
The drug release study was determined by first developing a concentration absorbance graph for
DOX. This was done by first making a stock solution of DOX using deionised water to dilute it
to different concentrations. The absorbance of these solutions are then determined by uv vis
spectrometry and using excel to plot a concentration absorbance graph from which the
concentration of the released DOX during the drug release at a particular time can be determined
when its absorbance is known. The concentration absorbance graph for DOX is shown above in
Figure 3.4.
The release of DOX from CMC--CD nanoparticles was investigated using a dialysis method at
370C in PBS with 2 pH values, 7.4 and 4.5 as shown in Figure 3.5. The two pH values were
selected to stimulate the drug release in blood and the tumor cells due to the extensive report that
the pH in the tumor cells is around 5.0, while it is 7.4 in the blood [60]
The release profile of DOX displayed a biphasic pattern, which was characterized by at first fast
release followed by much slower release which could be as a result of the inclusion
complexation between DOX and -cyclodextrin and chitosan moiety being relatively stable. At
different pH conditions, the DOX release reached a plateau at a similar incubation time that is
about 13 hours. It can be inferred that the protonation of the carboxyl groups in CMC--CD at
different pH conditions soon reached a balance, and so did the electrostatic interaction between
cationic DOX and anionic carriers, which contributed to a similar release plateau time. This
study also clearly showed that the pH value of the medium had a remarkable effect on the DOX
release rate from CMC--CD nanoparticles. At pH 7.4, only 30% of total drug released before
the release prole had virtually plateaued. This result suggests that DOX-NP maintained strong
53
Chapter 4 Discussion
This release profile can be explained on the basis that DOX was loaded into CMC--CD
nanoparticles by electrostatic interactions, and the degree of protonation of the carboxyl groups
in CMC--CD polymer was governed by environmental pH. After most of the carboxyl groups
in CMC--CD were protonated in low pH environment, the electrostatic interaction between
CMC--CD and DOX was weakened as nanoparticles form aggregation and precipitation, and
hence the accelerated release of hydrophilic DOX out from the nanoparticles inner core [80].
However, the release did not reach 100% in the test duration, even at pH 4.5, which might be
attributed to the electrostatic interaction between DOX and residual ionized carboxyl groups.
Additionally, stable nanoparticles enhance construct towards dissociation, and hydrophobic
interactions between DOX and CMC--CD polymers or DOX molecules themselves might also
contribute to incomplete drug release. The pH responsiveness is one of the most frequently used
biological stimuli exploited for triggered drug release, because pH values vary in the different
biological compartments and the cellular organelles. For example, the pH value at the tumor
extracellular environment is more acidic, that is 6.8 than that in blood, 7.4 and the pH values in
the endosomes and lysosomes are even lower [7]. The pH-sensitive drug carriers not only greatly
reduce the side effects to normal tissues in blood circulation by minimizing drug loss, but also
undergo fast release during the endocytosis process after take-up by the tumor cells, which could
improve the overall therapeutic efcacy.
54
Chapter 4 Discussion
In this study, MTT assay was used to evaluate the cytotoxicity of both blank nanoparticles and
drug loaded nanoparticles shown in Figure 3.6. Cytotoxicity studies were performed to
investigate the impact of drug loaded nanoparticles and blank particles loaded on proliferation of
a human breast cancer cell line, MCF-7 and ARPE-19 epithelial cells. Doxorubicin is among the
first generation anthracycline antibiotics which could intercalate into DNA and thus cause the
inhibition of DNA-dependent DNA and RNA synthesis. For the tumor cells treated by
doxorubicin, apoptosis, rather than necrosis, is the major mechanism of cell death. It is important
to determine the effective nanoparticle concentration as different amounts of the nanoparticles
contain different amounts of DOX. It can be observed that the cell cytoxicity for the cancer cell
lines depends on the amount of drug loaded nanoparticles used and hence the amount of drug in
the nanoparticle plays an important role in its cytotoxicity. Additionally, free DOX seems more
toxic than the loaded nanoparticles thus has a greater activity in inhibiting growth of the MCF-7
cells this is because DOX release from the nanoparticles is sustained whiles small molecular free
DOX diffuse into cells very fast, a phenomenon which poses toxicity to normal cells in
chemotherapy. The in vitro effect of the CMC--CD nanoparticles, without DOX, was evaluated
to ensure that the drug vehicle did not have an independent toxicity effect. Blank nanoparticles
demonstrated very low cytotoxic effects on the MCF-7 cells with cell viability almost a 100%.
This result showed that nanoparticles without DOX do not inhibit the proliferation of MCF-7
cells, even at a high concentration and indicates its good biocompatibility. It can be observed that
for the non cancerous ARPE-19 epithelial cells, cytotoxic levels for both drug loaded and blank
nanoparticles were significantely low. This study proved that the encapsulated or surface bound
55
Chapter 4 Discussion
DOX did not lose its bioactivity and still exhibited a considerable anti-proliferate activity, in
comparison to the effect of free DOX.
Therefore, more accurate and reproducible studies are needed to assist in the future design of
clinical formulations.
56
Chapter 5 Conclusion
Chapter 5 Conclusion
In the study we describe a novel nanoparticulate system which is composed of solely
carboxymethyl chitosan and carboxymethyl cyclodextrin and has the interesting features such
as being obtained spontaneously under exceptionally mild conditions without involving very
high temperatures, organic solvents. The nanoparticles exhibited;
(i)
appropriate size and shape and a negative zeta potential. It had a drug encapsulation
efficiency of over 90% and a drug loading efficiency of 57.7%.
(ii)
Drug release studies showed an initial burst release followed by a controlled release
of the drug over a period of time and was pH dependent.
(iii)
Cytotoxicity studies revealed that the nanospheres are effective against cancer cells
but less toxic to normal epithelial cells.
This showed that the nanoparticulate system in this study, was a stable, non-toxic
nanoparticle that efciently encapsulates DOX without diminishing its bioactivity. All these
special properties render these nanoparticles as very promising drug carrier for both
hydrophilic and hydrophobic drugs. The ability to attach tumor-specic ligands to this
scaffold by EDC/NHS conjugation chemistry makes it an ideal platform for development of
more selective anticancer therapy.
57
References
References
2. Brown BH, Smallwood RH, Barber DC, Lawford PV, Hose DR. Medical physics and
biomedical engineering. Institute of Physics Publishing, London 1999: 4.2.3.
3. Idol JD, Lehman RL. The CRC Handbook of Mechanical Engineering, 2nd Edition. CRC Press,
London 2004: 19.
6. Andrianov AK, Payne LG. Polymeric carriers for oral uptake of microparticulates, Advanced
Drug Delivery Reviews 1998; 34: 155-170.
58
References
9. Lao LL, Venkatraman SS, Peppas NA. Modeling of drug release from biodegradable polymer
blends. European Journal of Pharmaceutics and Biopharmaceutics 2008; 70: 796-803.
10. Lu Y, Chen SC. Micro and nano-fabrication of biodegradable polymers for drug delivery.
Advanced Drug Delivery Reviews 2004; 56: 1621-1633.
11. Nori A, Kopeek J. Intracellular targeting of polymer-bound drugs for cancer therapy.
Advanced Drug Delivery Reviews 2005; 57: 609-636.
12 Maeda H, Bharate GY, Daruwalla J. Polymeric drugs for efficient tumor-targeted drug
delivery based on EPR-effect. European Journal of Pharmaceutics and Biopharmaceutics 2009;
71: 409-419.
13. Lee S J, Koo H, Jeong H, Huh M S, Choi Y, Jeong SY, Byun Y, Choi K, Kim K, Kwon IC.
Comparative study of photosensitizer loaded and conjugated glycol chitosan nanoparticles for
cancer therapy. Journal of Controlled Release 2011; 152: 21-29.
14. Yang L, Zeng R, Li C, Li G, Qiao R, Hu L, Li Z. Novel synthesis and in vitro drug release of
polymeric prodrug: Chitosan-O-isopropyl-5-O-d4T monophosphate conjugate. Bioorganic
Medicinal Chemistry Letters 2009; 19: 2566-2569.
15. Canal F, Sanchis J, Vicent MJ. Polymer-drug conjugates as nano-sized medicines. Current
Opinion in Biotechnology 2011; 22: 894-900.
References
17. Zhang Y, Chan HF, Leong KW. Advanced materials and processing for drug delivery: The
past and the future. Advanced Drug Delivery Reviews 2013; 65: 104-120.
18. Ulbrich K, Subr V. Polymeric anticancer drugs with pH-controlled activation. Advanced
Drug Delivery Reviews 2004; 56: 1023-1050.
19. Anitha A, Deepagan VG, Rani VVD, Menon D, Nair SV, Jayakumar R. Preparation,
characterization, in vitro drug release and biological studies of curcumin loaded dextran
sulphate-chitosan nanoparticles. Carbohydrate Polymers 2011; 84: 1158-1164.
20. Greco F, Vicent MJ. Combination therapy: Opportunities and challenges for polymer-drug
conjugates as anticancer nanomedicines. Advanced Drug Delivery Reviews 2009; 61: 1203-1213.
23. Kopeek J. Polymer-drug conjugates: Origins, progress to date and future directions.
Advanced Drug Delivery Reviews 2013; 65: 49-59.
24. Mayaa S, Kumara LG, Sarmento B, Rejinolda S, Menona D, Naira SV, Jayakumara R.
Cetuximab
conjugated
O-carboxymethyl
chitosan
nanoparticles
for
targeting
EGFR
References
25. Arya G, Vandana M, Acharya S, Sahoo SK. Enhanced antiproliferative activity of Herceptin
(HER2)-conjugated gemcitabine-loaded chitosan nanoparticle in pancreatic cancer therapy.
Nanomedicine 2011; 7: 859-870.
26. Puttipipatkhachorn S, Nunthanid J, Yamamoto K, Peck GE. Drug physical state and drugpolymer interaction on drug release from chitosan matrix films. Journal of Controlled Release
2001; 75: 143-153.
27. Pasut G, Veronese FM. Polymerdrug conjugation, recent achievements and general
strategies. Progress in Polymer Science 2007; 32: 933-961.
28. Depan D, Kumar AP, Singh RP. Cell proliferation and controlled drug release studies of
nanohybrids based on chitosan-g-lactic acid and montmorillonite. Acta Biomaterialia 2009; 5:
93-100.
29. Heathcote EJ,Mitchell ML, Graganm W, Cooksley E, Dusheiko GM, Lee SS, Balart L,
Reindollar R, Reddy RK, Wright TL, Lin A, Hoffman J, De Pamphilis J. Peginterferon alfa-2a in
patient with chronic hepatitis C and cirrhosis, New England Journal of Medicine 2000; 343:
1673-1680.
31. Li C. Poly (1-glutamic acid) anticancer drug conjugates. Advanced Drug Delivery Reviews
2002; 54: 695-713.
References
33. Chen M, Liu Y, Yang W, Li X, Liu L, Zhou Z, Wang Y, Li R, Zhang Q. Preparation and
characterization of self-assembled nanoparticles of 6-O-cholestrol-modified chitosan for drug
delivery. Carbohydrate Polymers 2011; 84: 1244-1251.
34. Alves NM, Mano JF. Chitosan derivatives obtained by chemical modifications for biomedical
and environmental applications. International Journal of Biological Macromolecules 2008; 43:
401-414.
35. Zhu S, Qian F, Zhang Y, Tang C, Yin C. Synthesis and characterization of PEG modified Ntrimethylaminoethylmethacrylate chitosan nanoparticles. European Polymer Journal 2007; 43:
2244-2253.
36. Duan W, Chen C, Jiang L, Li GH. Preparation and characterization of the graft polymer of
chitosan with poly[rosin-(2-acryloyloxy)ethyl ester]. Carbohydrate Polymers 2008; 73: 582-586.
37. Kean T, Thanou M. Biodegradation, biodistribution and toxicity of chitosan. Advanced Drug
Delivery Reviews 2010; 62: 3-11.
62
References
40. Deshpande S, Varma A, Kennedy J. Metal complexation by chitosan and its derivatives: a
review. Carbohydrate Polymers 2004; 55: 77.
41. Liang KH, Wan Ngah WS. Adsorption of gold ions onto chitosan and n-carboxymethyl
chitosan: Equilibrium studies. Industrial & Engineering Chemistry Research 1999; 38:1411-1414.
43. Emanuelli M, Muzzarelli R, Tanfani F, Bolognini L. Aspartate glucan, glycine glucan, and
serine glucan for the removal of cobalt and copper from solutions and brines. Biotechnology and
Bioengineering 1985; 27: 11151121.
45. Mi F, Lin Y, Yu L, Chen S, Wu Y, Sung H. A novel ph-sensitive hydrogel composed of n,ocarboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery. Journal of
Controlled Release 2004; 96: 285300.
46. Buschmann HJ, Knittel D, Schollmeyer E. New textile applications of cyclodextrins. Journal
of Inclusion Phenomena and Macrocyclic Chemistry 2001; 40: 169-172.
63
References
49. Laine V, Sarguet CA, Gadelle A, Defaye J, Perly B, Pilard DF. Inclusion and solubilization
properties of 6-S-glycosyl-6-thio derivatives of -cyclodextrin. Journal of the Chemical Society,
Perkin Transactions1995; 2:14791485.
50. Pablo RSR, Isasi JR, Gaitano GG. Binding of dibenzofuran and its derivatives to watersoluble -cyclodextrin polymers. Journal of Photochemistry and Photobiology A: Chemistry
2005; 173: 248257.
51. Szejtli J. Cyclodextrin technology. Kluwer Academic Publishers, London 1988: 167.
52. Indra P, Bhesh B, Bruce D. Evaluation of various extraction methods of encapsulated oil
from -cyclodextrinlemon oil complex powder. Journal of Food Composition and Analysis
2000; 13: 5970
53. Prabaharan M, Mano JF. Chitosan derivatives bearing cyclodextrin cavities as novel
adsorbent matrices. Carbohydrate Polymers 2006; 63: 153-166.
54. Jayakumar R, Prabaharan M, Reis RL, Mano JF. Graft copolymerized chitosan-present status
and applications. Carbohydrate Polymers 2005; 62: 142-158.
64
References
56. Danhier F, Feron O, Preat V. To exploit the tumor microenvironment: Passive and active
tumor targeting of nanocarriers for anti-cancer drug delivery. Journal of Controlled Release 2010;
148: 135-146.
57. Cancer Research UK. How many different types of cancer are there?
http://www.cancerresearchuk.org/cancer-help/about-cancer/cancer-questions/how-manydifferent-types-of-cancer-are-there. Retieved 18 January 2013.
58. Chalasani P, Downey L, Stopeck AT. Caring for the Breast Cancer Survivor: A Guide for
Primary Care Physicians. The American Journal of Medicine 2010; 123: 489-495.
60. Danhier F, Feron O, Preat V. To exploit the tumor microenvironment: Passive and active
tumor targeting of nanocarriers for anti-cancer drug delivery. Journal of Controlled Release 2010;
148: 135-146.
61. Lee SJ, Koo H, Jeong H, Huh MS, Choi Y, Jeong SY, Byun Y, Choi K, Kim K, Kwon IC.
Comparative study of photosensitizer loaded and conjugated glycol chitosan nanoparticles for
cancer therapy. Journal of Controlled Release 2011; 152: 21-29.
62. Sapra P, Shor B. Monoclonal antibody-based therapies in cancer: Advances and challenges.
Pharmacology & Therapeutics 2013; 138: 452-469.
References
emerging approach for cancer therapy. Drug Discovery Today 2012; 17: 17-18.
65. Bremers AJA, Parmiani G. Immunology and immunotherapy of human cancer: present
concepts and clinical developments. Critical Reviews in Oncology & Hematology 2000; 34: 1-25.
67. Kenemans P, Stampfer M. Hormone replacement therapy and breast cancer. Eur J Obstet Gyn
Reproductive Biology 1996; 67: 1-4.
68. Stoke Z, Chan S. Principles of cancer therapy by hormone treatment. Surgery (Oxford) 2003;
21: 280-283.
70. Nielsen UB, Marks JD. Internalizing antibodies and targeted cancer therapy: direct selection
from phage display libraries. Pharmaceutical Science and Technology Today 2000; 3: 282-291.
72. Moses MA, Brem H, Langer R. Advancing the field of drug delivery:Taking aim at cancer.
66
References
73. Wojtyk JTC, Goyan R, Gudgin-Dickson E, Pottier R. Exploiting tumour biology to develop
novel drug delivery strategiesfor PDT. Medical Laser Application 2006; 21: 225-238.
75. Marcucci F, Lefoulon F. Active targeting with particulate drug carriers in tumor therapy:
fundamentals and recent progress. Drug Discovery Today 2004; 9: 219-228.
76. Janes KA, Fresneau MP, Marazuela A, Fabra A, Alonso MJ. Chitosan nanoparticles as
delivery systems for doxorubicin. Journal of Controlled Release 2001; 73: 255-267.
77. Xu X, Clarke P, Szalai G, Shively JE, Williams LE, Shyr Y, Shi E, Primus FJ. Targeting and
therapy of carcinoembryonic antigen-expressing tumors in transgenic mice with an antibodyinterleukin 2 fusion protein. Cancer Research 2000; 60: 447584.
78. Elzoghby AO, Samy WM, Elgindy NA. Albumin-based nanoparticles as potential controlled
release drug delivery systems. Journal of Controlled Release 2012; 157: 168-182.
67
References
80. Guo H, Zhang D, Li C, Jia L, Liu G, Hao L, Zheng D, Shen J, Li T, Guo Y, Zhang Q. Selfassembled nanoparticles based on galactosylated O-carboxymethyl chitosan-graft-stearic acid
conjugates for delivery of doxorubicin. International Journal of Pharmaceutics 2013; 458: 31-38.
68
Appendix
Publications
1. Joseph Asamoah-Asare, Yangde Zhang , Yuxiang Chen, Mo Fongming, Caiping Ren.
Novel Carboxymethyl Chitosan--Cyclodextrin Nanoparticles as a Drug Delivery System;
Preparation and Characterization. International Journal of Engineering Sciences and
Research Technology, ISSN 2277-9655. May 2014. 3(5); 141-146.
69