-
Novel Polysaccharide Based Polymers and Nanoparticles for
Controlled Drug Delivery and
Biomedical Imaging
by
Alireza Shalviri
A thesis submitted in conformity with the requirements for the
degree of Doctor of Philosophy
Graduate Department of Pharmaceutical Sciences University of
Toronto
© Copyright by Alireza Shalviri (2012)
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ii
Novel Polysaccharide Based Polymers and Nanoparticles for
Controlled Delivery of Drugs and
Imaging Agents
Alireza Shalviri
Doctor of Philosophy
Graduate Department of Pharmaceutical Sciences
University of Toronto
2012
Abstract
The use of polysaccharides as building blocks in the development
of drugs and contrast agents
delivery systems is rapidly growing. This can be attributed to
the outstanding virtues of
polysaccharides such as biocompatibility, biodegradability,
upgradability, multiple reacting
groups and low cost. The focus of this thesis was to develop and
characterize novel starch based
hydrogels and nanoparticles for delivery of drugs and imaging
agents. To this end, two different
systems were developed. The first system includes polymer and
nanoparticles prepared by graft
polymerization of polymethacrylic acid and polysorbate 80 onto
starch. This starch based
platform nanotechnology was developed using the design
principles based on the
pathophysiology of breast cancer, with applications in both
medical imaging and breast cancer
chemotherapy. The nanoparticles exhibited a high degree of
doxorubicin loading as well as
sustained pH dependent release of the drug. The drug loaded
nanoparticles were significantly
more effective against multidrug resistant human breast cancer
cells compared to free
doxorubicin. Systemic administration of the starch based
nanoparticles co-loaded with
doxorubicin and a near infrared fluorescent probe allowed for
non-invasive real time monitoring
of the nanoparticles biodistribution, tumor accumulation, and
clearance. Systemic administration
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of the clinically relevant doses of the drug loaded particles to
a mouse model of breast cancer
significantly enhanced therapeutic efficacy while minimizing
side effects compared to free
doxorubicin. A novel, starch based magnetic resonance imaging
(MRI) contrast agent with good
in vitro and in vivo tolerability was formulated which exhibited
superior signal enhancement in
tumor and vasculature. The second system is a co-polymeric
hydrogel of starch and xanthan gum
with adjustable swelling and permeation properties. The
hydrogels exhibited excellent film
forming capability, and appeared to be particularly useful in
controlled delivery applications of
larger molecular size compounds. The starch based hydrogels,
polymers and nanoparticles
developed in this work have shown great potentials for
controlled drug delivery and biomedical
imaging applications.
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Acknowledgments
I would like to thank my supervisor Dr. Shirley Wu for believing
in me as well as for her
kindness, leadership, patience, and continuous support. She
always believed in my abilities and
pushed me to be the best I could be; for this I will always
remain thankful to her. Without her
mentorship this thesis would have not been possible.
I am greatly appreciative to Drs. Ping Lee, Tigran Chalikian and
Edgar Acosta for taking the
time to attend my committee meetings and giving me insightful
guidance and recommendations.
I also thank Dr. Warren Foltz, Dr. Andrew Rauth, and Dr. Heiko
Heerklotz for their continuous
support and guidance throughout my thesis.
My sincere thanks to all my colleagues who helped me, especially
Ping Cai. I also extend my
gratitude to those not mentioned here who have taken part, small
or large, in making this work
possible.
I am grateful to the Ontario Graduate Scholarship Program,
Natural Sciences and Engineering
Research Council of Canada, BioPotato network (Co-leaders: Drs.
Helen Tai and Yvan
Pelletier), Agricultural Bioproducts Innovation Program (ABIP)
of Agriculture & Agri-Food
Canada, CIHR/CBCRA, University of Toronto and Leslie Dan Faculty
of Pharmacy for
scholarships and research funding.
I thank my wife, Mana, for always being there for me. Without
her love, care and undivided
attention I would not have achieved any of this.
I thank my parents and my sister for their belief in my
abilities and constant moral and financial
support. I owe all my success to them. Finally, I thank the
mice. Without them meaningful
innovation would not be possible.
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Table of Contents
Acknowledgments..........................................................................................................................
iv
Table of Contents
.............................................................................................................................v
List of Tables
................................................................................................................................
xii
List of Figures
..............................................................................................................................
xiii
List of Abbreviations
...................................................................................................................
xxi
Chapter 1. Introduction
....................................................................................................................1
1.1 Breast Cancer
.......................................................................................................................2
1.1.1 Epidemiology
...........................................................................................................2
1.1.2 Breast Cancer Cells and Tumor Microenvironment
................................................2
1.1.3 Doxorubicin: a Potent Drug for Breast Cancer Chemotherapy
...............................5
1.1.4 Barriers to Cancer Chemotherapy
............................................................................7
1.1.5 Nanoparticulate Systems in Cancer Therapy
.........................................................13
1.1.6 Nanoparticles as Theranostics in Cancer
...............................................................19
1.2 Polysaccharides in Drug Delivery
.....................................................................................22
1.2.1 Starch
.....................................................................................................................23
1.2.2 Xanthan gum
..........................................................................................................26
1.3 Biomedical Imaging
...........................................................................................................27
1.3.1 In vivo Fluorescence Imaging
................................................................................27
1.3.2 Magnetic Resonance Imaging
................................................................................30
1.4 Drug Delivery to the Brain
................................................................................................40
1.4.1 Brain Anatomy
.......................................................................................................41
1.4.2 Strategies to Enhance Drug Delivery to the Brain
.................................................43
1.5 Goal for this work
..............................................................................................................47
1.6 Synopsis
.............................................................................................................................48
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Chapter 2. Design of pH-responsive Nanoparticles of Terpolymer
of Poly(methacrylic acid),
Polysorbate 80 and Starch for Delivery of Doxorubicin
...........................................................51
2.1 Abstract
..............................................................................................................................52
2.2 Introduction
........................................................................................................................53
2.3 Materials and Methods
.......................................................................................................55
2.3.1 Materials
................................................................................................................55
2.3.2 Synthesis of PMAA-PS 80-g-St Nanoparticles
.....................................................55
2.3.3 FTIR and 1H NMR Spectroscopy
..........................................................................56
2.3.4 Examination of the Nanoparticles with TEM
........................................................57
2.3.5 Determination of Particle Size and Surface Charge
..............................................57
2.3.6 Titration Studies
.....................................................................................................57
2.4 Results and Discussion
......................................................................................................58
2.4.1 PMAA-PS 80-g-St Nanoparticles were Synthesized Using a
Simple One-pot Method
...................................................................................................................58
2.4.2 Polymer Composition of the Nanoparticles
...........................................................60
2.4.3 Nanoparticles Size and Morphology
......................................................................64
2.4.4 PMAA-PS 80-g-St Nanoparticles Show pH-responsive Swelling
in Physiological pH Range
.........................................................................................67
2.4.5 Properties of Carboxylic Acid Groups in the Nanoparticles
.................................68
2.4.6 Effect of Processing Parameters on Particle size and pH
Sensitivity ....................71
2.5 Conclusions
........................................................................................................................74
2.6 Acknowledgements
............................................................................................................74
Chapter 3. pH Dependent Doxorubicin Release by Nanoparticles
Based on Terpolymer of
Poly(Methacrylic Acid), Polysorbate 80, and Starch for
Overcoming Multi-drug
Resistance in Breast Cancer Cells
.............................................................................................75
3.1 Abstract
..............................................................................................................................76
3.2 Introduction
........................................................................................................................77
3.3 Materials and Methods
.......................................................................................................80
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3.3.1 Materials
................................................................................................................80
3.3.2 Cell Maintenance
...................................................................................................80
3.3.3 Synthesis of PMAA-PS 80-g-St Nanoparticles
.....................................................81
3.3.4 Fourier Transform Infrared Spectroscopy
.............................................................81
3.3.5 Isothermal Titration Calorimetry (ITC)
.................................................................81
3.3.6 Dynamic Light Scattering
......................................................................................82
3.3.7 Transmission Electron Microscopy
.......................................................................82
3.3.8 Drug Loading Studies
............................................................................................83
3.3.9 X-ray Powder Diffraction (XRPD)
........................................................................84
3.3.10 In vitro Drug Release
.............................................................................................84
3.3.11 Cell Uptake Studies Using Fluorescence Microscopy
...........................................84
3.3.12 Cellular Uptake of Nanoparticles by Flow Cytometry
..........................................85
3.3.13 In vitro Assessment of Anticancer Efficacy of Dox-loaded
Nanoparticles ...........86
3.4 Results
................................................................................................................................86
3.4.1 Properties of PMAA-PS 80-g-St Nanoparticles and Their High
Capability of Efficiently Loading Dox without Loss of Colloidal
Stability ................................86
3.4.2 FTIR, XRD and ITC Experiments Revealed Strong Ionic
Interaction between the Nanoparticles and Dox
.....................................................................................88
3.4.3 The Nanoparticles Exhibited Sustained and pH Dependent
Release of Dox in vitro
........................................................................................................................95
3.4.4 Substantial Cellular Uptake of the Nanoparticles Evidenced
by Fluorescence Microscopy, TEM and Flow Cytometry
................................................................96
3.4.5 The Dox Loaded Nanoparticles Were Significantly More
Effective Against
MDR1 Cells than Free Dox
.................................................................................101
3.5 Discussion
........................................................................................................................103
3.6 Conclusions
......................................................................................................................105
3.7 Acknowledgements
..........................................................................................................105
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Chapter 4. Evaluation of New Multifunctional Nanoparticles based
on Terpolymer of
Polymethacrylic acid, Polysorbate 80, and Starch as a
Theranostic Nanoplatform for
Simultaneous in vivo Imaging and Treatment of Breast Cancer
.............................................106
4.1 Abstract
............................................................................................................................107
4.2 Introduction
......................................................................................................................108
4.3 Materials and Methods
.....................................................................................................110
4.3.1 Materials
..............................................................................................................110
4.3.2 Preparation of Dual Mode Nanoparticles
............................................................111
4.3.3 Dynamic Light Scattering, Electrophoretic Mobility
Measurements, and Transmission Electron Microscopy
.....................................................................113
4.3.4 Drug Release Studies
...........................................................................................113
4.3.5 Cell Lines
.............................................................................................................114
4.3.6 Animal Model and In vivo Treatment Protocol in Tumor
Bearing Mice ............114
4.3.7 Real Time In vivo and Ex-vivo Near-infrared Fluorescent
Imaging ....................115
4.3.8 Ex-vivo Tumor Fluorescence Microscopy
...........................................................117
4.4 Results
..............................................................................................................................118
4.4.1 Properties of the Nanoparticles
............................................................................118
4.4.2 Distribution and Tumor Accumulation of the PF-NPs and
SA-NPs in Whole Animals In vivo
....................................................................................................122
4.4.3 Real Time Pharmocokinetics of Nanoparticles in Tumor
Tissue ........................125
4.4.4 Organ Distribution of the Nanoparticles Determined by Ex
Vivo Imaging ........126
4.4.5 Microscopic Imaging of Tumor tissue Demonstrated
Extravasation of the Nanoparticles in the Tumor
.................................................................................128
4.4.6 Anti-tumor Efficacy of the Nanoparticles in a Murine
Breast Cancer Tumor Model
...................................................................................................................130
4.4.7 Preliminarily Assessment of Toxicity of the Nanoparticles
................................132
4.5 Discussion
........................................................................................................................134
4.6 Conclusions
......................................................................................................................136
4.7
Acknowledgments............................................................................................................137
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Chapter 5. A New Starch-based Polymeric MRI Contrast Agent with
Superior Signal
Enhancement in Blood and Tumor
.........................................................................................138
5.1 Abstract
............................................................................................................................139
5.2 Introduction
......................................................................................................................140
5.3 Materials and Methods
.....................................................................................................142
5.3.2 Cell line and Maintenance
...................................................................................142
5.3.3 Preparation of the Gd3+
Loaded PMAA-PS 80-g-St-DTPA Polymer (PolyGd) ..143
5.3.4 Confirmation of DTPA Conjugation to Starch
....................................................144
5.3.5 Determination of DTPA Content and Binding Affinity of
Gd3+
to St-DTPA .....145
5.3.6 Determination of Cytotoxicity of
PolyGd............................................................145
5.3.7 Comparison of the Relaxivity Properties of PolyGd and
Omniscan® in vitro ....146
5.3.8 Experimental Animals and Induction of Subcutaneous Breast
Tumors ..............146
5.3.9 Determination of In vivo MRI Contrast Enhancement of
PolyGd in Mice .........147
5.3.10 Validation of Whole-body Gd3+ Distribution by Inductively
Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)
........................................................148
5.4 Results and Discussion
....................................................................................................148
5.4.1 Confirmation of DTPA Conjugation to Starch
....................................................148
5.4.2 Determination of DTPA Content and Binding Affinity of Gd3+
to St-DTPA .....154
5.4.3 PolyGd Exhibited Lower Cytotoxicity than Free Gd3+
........................................155
5.4.4 PolyGd showed much higher relaxivity than
Omniscan®...................................156
5.4.5 In vivo MRI Contrast Enhancement of PolyGd in Mice
......................................159
5.4.6 Biodistribution and Clearance of the PolyGd from the Body
..............................167
5.5 Conclusions
......................................................................................................................168
5.6
Acknowledgments............................................................................................................169
Chapter 6. Evaluating the Capability of Novel Starch Based
Nanoparticles for Controlled
Delivery of Drugs and Imaging Agents to the Brain
..............................................................170
6.1 Abstract
............................................................................................................................171
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6.2 Introduction
......................................................................................................................172
6.3 Materials and Methods
.....................................................................................................174
6.3.1 Materials
..............................................................................................................174
6.3.2 Synthesis and Preparation of the PMAA-Ps 80-g-St Polymer
and
Nanoparticles
.......................................................................................................175
6.3.3 Physicochemical Characterization of the PMAA-PS 80-g-St
Polymer and Nanoparticles
.......................................................................................................175
6.3.4 Time-of-Flight-Secondary Ion Mass Spectrometry
.............................................176
6.3.5 Animal Studies
.....................................................................................................176
6.3.6 In vivo Magnetic Resonance Imaging
(MRI).......................................................176
6.3.7 Ex-vivo Fluorescence Imaging of the Brain
.........................................................177
6.3.8 Fluorescence Microscopy
....................................................................................178
6.4 Results
..............................................................................................................................179
6.4.1 Properties of PMAA-PS 80-g-St Polymer and Nanoparticles
.............................179
6.4.2 Brain Accumulation of the PMAA-PS 80-g-St Nanoparticles at
Macroscopic
and Microscopic Levels
.......................................................................................184
6.5 Conclusions
......................................................................................................................189
Chapter 7. Novel Modified Starch-xanthan Gum Hydrogels for
Controlled Drug Delivery:
Synthesis and Characterization
...............................................................................................190
7.1 Abstract
............................................................................................................................191
7.2 Introduction
......................................................................................................................192
7.3 Materials and Methods
.....................................................................................................193
7.3.1 Chemicals
.............................................................................................................193
7.3.2 Synthesis of Cross-linked Fully Gelatinized Starch and
Xanthan Gum Hydrogels
.............................................................................................................193
7.3.3 Examination of Film Morphology
.......................................................................194
7.3.4 Confirmation of Cross-linking by Fourier Transformed
Infrared Spectroscopy .194
7.3.5 Solid State 31P NMR
Spectroscopy......................................................................194
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7.3.6 Study of the Swelling Behavior of Cross-linked
Starch-xanthan Gum Polymer .195
7.3.7 Determination of Gel Mesh
Size..........................................................................195
7.3.8 Permeability Studies
............................................................................................197
7.3.9 Statistical Analysis
...............................................................................................198
7.4 Results and Discussion
....................................................................................................198
7.4.1
Morphology..........................................................................................................198
7.4.2 Cross-linking of Starch-Xanthan Gum with STMP was
Confirmed ...................199
7.4.3 Swelling Kinetics and Equilibrium Swelling Ratio
.............................................205
7.4.4 Effect of STMP and Xanthan gum Concentration on the Film
Swelling ............206
7.4.5 Effect of Medium pH and Buffer salts on the Film Swelling
..............................209
7.4.6 Mesh Size of the Modified Starch-Xanthan Gum Gels
.......................................210
7.4.7 Permeability Studies
............................................................................................212
7.5 Conclusion
.......................................................................................................................215
7.6 Acknowledgements
..........................................................................................................216
Chapter 8. Conclusions and Future Perspectives
.........................................................................217
8.1 Overall Conclusions and Original Contributions of This
Thesis .....................................217
8.2 Limitation of the Work and Future Directions
................................................................222
8.2.1 Biodegradation, Body Clearance, and Biocompatibility of
the PMAA-PS 80-
g-St Nanoparticles
................................................................................................222
8.2.2 Utility of the PMAA-PS 80-g-St Polymer and Nanoparticles
as a Dual Model Imaging Probe
......................................................................................................223
8.2.3 Active Targeting
..................................................................................................224
8.2.4 Delivery of Dual Agents by the Nanoparticles
....................................................225
8.2.5 In vivo assessment of PMAA-PS 80-g-St for drug delivery to
the CNS .............226
8.2.6 Optimal Polymerization Method for Making More Uniform
Polymers .................227
8.2.6 In Vivo Assessment of Efficacy in Multidrug Resistant
Tumor Model ..............227
Bibliography
................................................................................................................................228
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List of Tables
Table 1.1 Examples of drug-nanoparticles system for delivery to
the brain ................................ 44
Table 2.1 Nanoparticle preparation recipes and the polymer
composition.. ................................ 60
Table 2.2 Intensity-weighted hydrodynamic diameter of
nanoparticles with different feed molar
ratio of MAA/St in 0.15 M PBS of various pH..
.......................................................... 65
Table 3.1 Characterization of the drug-loaded nanoparticles. The
effect of drug loading on the
particles size and surface charge is investigated..
......................................................... 87
Table 4.1 Summary of physicochemical properties of SA-NPs and
PF-NPs ............................. 118
Table 4.2 Tumor associated pharmacokinetic data derived from the
tumor average fluorescence
intensity versus time curve for SA-NPs and PF-NPs..
............................................... 126
Table 5.1 Gd3+
content, molecular weight, and r1 for Omniscan® and PolyGd. The
r1 were
measured in saline at 3 and 7 T..
.................................................................................
158
Table 7.1 Equilibrium swelling ratio of films with 10% xanthan
gum and various cross-linker
STMP levels..
..............................................................................................................
206
Table 7.2 Equilibrium volume swelling and gel mesh size of
starch-xanthan gum gels containing
10% xanthan gum and varying concentrations of cross-linker
(STMP).. ................... 212
Table 7.3 Equilibrium swelling ratio of films in 0.15M phosphate
buffer of pH=7.4 and
permeability of vitamin B12 across modified starch-xanthan gum
films of various
compositions.
..............................................................................................................
213
Table 7.4 Permeability of macromolecules and drugs of various
molecular weights and charges
across the starch-xanthan gum gel film containing 10% XG and 5%
STMP in 0.15 M
phosphate buffer of pH=7.4..
......................................................................................
214
Table 7.5 Permeability of two weakly acidic drugs across
starch-xanthan gum films containing
10%XG and 5% STMP in pH 2 and 7.4 buffer solutions with ionic
strength of 0.15M.
....................................................................................................................................
215
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List of Figures
Figure 1.1 Chemical structure of Doxorubicin
...............................................................................
6
Figure 1.2 Factors leading to MDR in cancer. One of the leading
causes of treatment failure in
cancer is the onset of resistant disease. MDR can be divided
into two broad categories:
Cellular resistance and non-cellular resistance.
............................................................ 11
Figure 1.3 Passive targeting of the tumor by EPR. The EPR effect
is the balance of enhanced
tumor permeability with poor tumor interstitial fluid drainage,
resulting in the selective
uptake and retention of nanoparticles in the tumor tissue.
........................................... 14
Figure 1.4 Drug loaded nanoparticles can overcome MDR cancer
cells. Endocytosis of the drug
loaded nanoparticles in membrane bound vesicles protects the
drug from the action of
the membrane efflux pumps. The nanoparticles release the drug
deep inside the cell
and the drug can gain access to its cellular target site (e.g.
DNA) ............................... 14
Figure 1.5 Chemical structure of starch
........................................................................................
24
Figure 1.6 Xanthan gum chemical structure
.................................................................................
27
Figure 1.7 Schematic comparison of fluorophores in the visible
spectrum versus the near infrared
for deep in vivo fluorescent imaging. Fluorophores in the
visible regions are limited by
poor penetration of the excitation photon or the poor
penetration of the emission
photons preventing the detection of fluorescent signal in vivo.
Near infrared photons
can provide deep tissue penetration.
.............................................................................
29
Figure 2.1 Schematic reaction of starch grafting and terpolymer
formation. FTIR spectra of
Starch, PMAA-PS 80, and PMAA-PS 80-g-St.
............................................................................
61
Figure 2.2 1H NMR spectra of A) PS 80, B) Starch, C) PMAA-PS 80,
D) PMAA-PS 80-g-St-2 in
0.05M NaOD
................................................................................................................
63
Figure 2.3 A) Intensity-weighted hydrodynamic diameter of the
PMAA-PS 80-g-St-2
nanoparticles in 0.15 M pH 7.4 PBS. The particles showed a
narrow size distribution.
B) TEM images of PMAA-PS 80-g-St-2 in 0.15 M PBS of pH=7.4..
......................... 66
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xiv
Figure 2.4 A) Relative diameter vs. pH for the nanoparticles
with different feed molar ratio of
MAA/St in 0.15 M PBS. DpH/D4 represents particles diameter at
different pH relative
to pH 4. B) Effect of pH on surface charge for particles of
various MAA/St molar
ratio..
.............................................................................................................................
68
Figure 2.5 A) Potentiometric titration curves. Empty triangles
represent the uncorrected
potentiometric titation curve for PMAA-PS 80-g-St-2 latex
dispersion. Filled circles
represent the titration curve after correction. Empty circles
show the blank titration
curve. The arrow represents the equivalence point. The
equivalence points are used to
calculate the MAA contents in various nanoparticle batches. B)
Variation in the
apparent dissociation constant (pKa) as a function of the degree
of ionization (α) for
nanoparticles of different starch and MAA contents.
................................................... 70
Figure 2.6 Effect of A) SDS, B) PS 80, C) total monomer
concentration, D) cross-linker molar
ratio on particle size and pH sensitivity..
......................................................................
73
Figure 3.1 A) Number-weighted Gaussian distribution of PMAA-PS
80-g-St nanoparticles
loaded with doxorubicin (LC=33%) in 0.15 M phosphate buffer at
pH 7.4, B)
Transmission electron micrograph of doxorubicin loaded
nanoparticles (LC=33%)... 88
Figure 3.2 FTIR spectra of A) PMAA-PS 80-g-St nanoparticles, B)
Dox, C) Dox loaded PMAA-
PS 80-g-St nanoparticles..
.............................................................................................
89
Figure 3.3 XRD spectrum of A) Doxorubicin in native form, B)
PMAA-PS 80-g-St
nanoparticles, C) Doxorubicin loaded nanoparticles (LC=50%), D)
doxorubicin loaded
nanoparticles (LC=50%) after 6 months storage at room
temperature.. ....................... 91
Figure 3.4 A) The blank differential enthalpy curves of
titrating 8.5 mM doxorubicin into buffers
of various pH. B) Differential enthalpy curves of titrating 8.5
mM doxorubicin into
0.1mg/ml PMAA-PS 80-g-St nanoparticles in buffers at different
pH. The ionic
strength was kept constant at 0.15M by addition of NaCl. C) The
blank differential
enthalpy curves of titrating 8.5 mM doxorubicin into DDIW with
different NaCl
contents, D) Differential enthalpy curves of titrating 8.5 mM
doxorubicin into
0.1mg/ml PMAA-PS 80-g-St nanoparticles in DDIW with different
NaCl contents. .. 94
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xv
Figure 3.5 Effect of pH on kinetics of doxorubicin release from
the naoparticles with drug
loading content of 33% at 37 ºC. The release of free doxorubicin
from the dialysis bag
was used as control. For each buffer system the ionic strength
was kept constant at
0.15 M by adding NaCl.
................................................................................................
95
Figure 3.6 A) Fluorescence microscopy image of MDA-MB435/LCC6
cells with and without
(control) 4 hr incubation with fluorescent nanoparticles at
final concentration of 0.25
mg/ml. Nuclei were stained with Hoechst 33342 and visualized
with DAPI filter, cell
membranes were stained with Vybrant™DiI and visualized with Cy3
filter, and NPs
were labelled fluoresceinamine isomer I and visualized with FITC
filter. Optical slices
were taken every 2 µm from the uppermost and lowermost regions
of the cell, allowing
for selection of an image at approximately the midpoint of the
nucleus. B) TEM
micrographs of MDA-MB435/LCC6 cells treated with 0.25 mg/ml
PMAA-PS 80-g-St
NPs for 4 hrs. The nanoparticles were loaded with gadolinium
(metal) and appear as
electron dense deposits.
................................................................................................
98
Figure 3.7 Flow cytometry histograms for MDA-MB435/LCC6 cells
showing the effect of
incubation time and temperature on particle uptake. The cells
were incubated with
fluorescent labelled naoparticles at the final nanoparticle
concentration of 0.25 mg/ml.
A) MDA-MB435/WT (1) untreated cells at 37 ºC, (2) 1 hr
incubation at 37 ºC, (3) 4
hrs incubation at 37 ºC, (4) 24 hrs incubation at 37 ºC. B)
MDA-MB435/WT (1)
untreated cells, (2) 1 hr incubation at 4ºC, (3) 1hr incubation
at 37 ºC. C) MDA-MB
435/MDR1 (1) untreated cells, (2) 1 hr incubation at 37 ºC, (3)
4 hrs incubation at 37
ºC, (4) 24 hrs incubation at 37 ºC. D) MDA-MB435/MDR1 (1)
untreated cells, (2) 1 hr
incubation at 4ºC, (3) 1 hr incubation at 37 ºC..
......................................................... 100
Figure 3.8 Determination of the response of MDA-MB435/LCC6 cell
types to free doxorubicin
and doxorubicin loaded nanoparticles by MTT assay. (A-B) Cell
viability of MDA-
MB435/LCC6/WT (n=3) cells after exposure to increasing
concentrations of blank
nanoparticles (blank NPs), free doxorubicin and doxorubicin
loaded nanoparticles
(Dox-NPs) for 24 hrs (A) and 48 hours (B). (C-D) Cell viability
of
MDA435/LCC6/MDR1 (n=3) cells after exposure to increasing
concentrations of
blank NPs, free doxorubicin and Dox-NPs for 24 hrs (C) and 48
hrs (D). Cells with no
-
xvi
treatment and incubated with blank nanoparticles were used as
control for free drug
and drug loaded nanoparticle respectively. Cell viability is
expressed as the percent of
control for each treatment group.
................................................................................
102
Figure 4.1 A) Schematic diagram of PF-NPs and SA-NPs and the
reaction scheme for
conjugation of the NIR dye and loading of Dox. B) Chemical
structure of the PMAA-
PS 80-g-St polymer.
....................................................................................................
119
Figure 4.2 Size distribution and shapes of A) SA-NPs and B)
PF-NPs, as determined by dynamic
light scattering (DLS) and transmission electron microscopy
(TEM), respectively. . 120
Figure 4.3 Drug release kinetics from the SA-NPs (LC=21.1%) and
PF-NPs (LC=49.7%). Drug
release was measured in 0.15 M Tris/NaCl buffer, pH=7.4, at 37
ºC. The release of free
doxorubicin from the dialysis bag was used as control.
............................................. 121
Figure 4.4 A) Whole animal real time biodistribution and tumor
targeting of SA-NPs and PF-
NPs in mice bearing an orthotopic breast tumor model.
Nanoparticle-associated
fluorescence was determined prior to intravenous injection
(baseline), and then at
various hours following nanoparticles injection up to 14 days.
B) Time-dependent
excretion profiles of SA-NPs and PF-NPs from the whole body
(left) and tumor (right).
The fluorescence intensity for the region of interest was
recorded as average radiant
efficiency..
..................................................................................................................
124
Figure 4.5 Quantitative results of tissue distribution and tumor
accumulation for SA-NPs and PF-
NPs. Ratio of the relative fluorescence intensity in major
organs, tumor, and blood as a
function of time after intravenous injection of nanoparticles,
compared to normal
major organs and tumors not injected with NIR dye
conjugated-nanoparticles.. ....... 127
Figure 4.6 Microscopic distribution of the nanoparticles within
the tumor. Fluorescent signal
observed in tumor tissue treated with A) vehicle only; B)
FITC-labeled SA-NPs; C)
FITC-labeled PF-NPs. Tumors arising from orthotopically
implanted EMT6/WT cells
were allowed to grow for 8 days prior to injection of
nanoparticles. Four hours
following nanoparticle introduction, animals were sacrificed and
the distribution of
particles assessed within both core and peripheral regions.
....................................... 129
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xvii
Figure 4.7 Anti-tumour activity of starch based nanoparticles in
EMT6/WT tumor bearing mice.
Tumor cells were implanted orthotopicly on day zero. Mice were
treated with 5%
dextrose (n=2×4), free Dox (n=8), PF-NPs (n=2×4), and SA-NPs
(n=2×3) at a dose of
2×10 mg/kg equivalent to Dox on day 8 and 15. A) Tumor volume up
to day 62. Each
curve represents one animal. B) Kaplan Meier survival curves for
5% dextrose, free
Dox, PF-NPs, and SA-NPs. The trend in survival curves were
significantly different
(p=0.0033, Mantel
Cox)..............................................................................................
131
Figure 4.8 Time profiles of body weight of tumor-bearing mice
treated with 5% dextrose
(n=2×4), free Dox (n=2×4) , PF-NPs (n=2×4), and SA-NPs (n=2×3)
at a dose of 2×10
mg/kg equivalent to Dox. Balb/c mice were inoculated with
EMT6/WT tumor in the
mammary fat pad and received treatment on day 8 and 15 post
inoculation. Each curve
represents one animal.
.................................................................................................
132
Figure 4.9 Histological sections of lung, kidney, liver, and
heart of tumor-bearing mice treated
with 5% dextrose, free Dox, PF-NPs, and SA-NPs at a dose of 2×10
mg/kg equivalent
to Dox. The samples were not collected at the same time after
treatment, but rather
collected after euthanasia of the animals as necessitated by
tumor size end point.
Sections were stained with H&E and observed under a light
microscope. ................ 134
Figure 5.1 A) Reaction of starch with DTPA-bisanhydride to form
St-DTPA by direct acylation
of starch hydroxyl groups. DTPA-bisanhydride can react with one
starch molecule to
form St-DTPA or two starch molecules to form ST-DTPA-St. B)
Possible chemical
structures of PolyGd.
..................................................................................................
150
Figure 5.2 FTIR spectra of A) starch, B) DTPA, C) St-DTPA..
................................................ 152
Figure 5.3 1H NMR spectra of A) DTPA, B) starch, C) St-DTPA.
Major peaks for DTPA and
starch have been assigned on the molecular schemes..
............................................... 153
Figure 5.4 Normalized differential heat (NDH) curves titrating 2
mM Gd3+
into 0.1 mg/ml
aqueous buffer solution of St or St-DTPA at 25 ºC and pH 5.6.
Titration of Gd3+
into
starch did not produce any heat while titration into St-DTPA
produced a large
endothermic heat indicating binding of Gd3+
to St-DTPA. Assuming one Gd3+
ion
-
xviii
binds to one DTPA molecule, the amount of covalently bound DTPA
to starch is
calculated at the titration end point (inflection point)
indicated by an arrow. ............ 155
Figure 5.5 The toxicity of saline, blank polymer, PolyGd, and
free Gd3+
to rat hepatocytes in
culture exposed for 30 min, 60 min, 120 min, or 240 min. “%live”
represents the
percent of hepatocytes excluding trypan blue..
........................................................... 156
Figure 5.6 Coronal T1-weighted (3D-FLASH, TE/TR 3/25 msec, flip
angle 20º) whole body
images of Balb/c mice injected with Omniscan® (0.1 mmol/kg
Gd3+
) and PolyGd
(0.025 mmol/kg Gd3+
). At one fourth the dose of Omniscan®, the PolyGd produces
a
much higher contrast over an extended period of time in the
cardiovascular system. 160
Figure 5.7 Quantitative MRI of whole-body distribution: A) R1
maps of Balb/c mice injected
with PolyGd (0.025 mmol/kg Gd+3
). B) Change in relaxation rates, ∆R1, of left
ventricular blood, liver, bladder, and kidneys for Omniscan®
(0.1 mmol/Kg Gd3+
) and
PolyGd (0.025 mmol/Kg Gd3+
) overtime relative to baseline. The Gd3+
loaded polymer
causes a much higher increase in blood relaxation rate for an
extended period of time
compared to Omniscan®..
..........................................................................................
162
Figure 5.8 MR angiography: A) MIP angiogram displaying contrast
enhancement of (1) whole
body and (2) neck and head regions, obtained prior to and at 15
minutes following
PolyGd injection at 0.025 mmol/kg Gd3+
. B) Kinetics of vascular signal to noise (S/N)
ratio and contrast to-noise (C/N) ratio measured from the
inferior vena cava in whole-
body angiograms..
.......................................................................................................
164
Figure 5.9 Tumor distribution of PolyGd (0.025 mmol/kg Gd3+
): A) T1-weighted images (1) and
the corresponding R1 maps (2). B) Time course of ∆R1 in tumor
periphery and tumor
core, displaying elevated tumor R1 even 48 hours after contrast
agent injection.. ..... 166
Figure 5.10 Biodistribution, elimination and tumor accumulation
of the PolyGd (0.025 mmol/kg
Gd3+
) in tumor bearing Balb/c mice. The Gd3+
content was determined using ICP-
AES..
...........................................................................................................................
168
Figure 6.1 A) 1H NMR spectra of 1) PS 80, 2) PMAA-PS 80-g-St in
0.1M NaOD. B) polymer
composition and physical properties of the PMAA-PS 80-g-St
nanoparticles. For size
-
xix
measurements, the particles were disperse in PBS pH of 7.4 and
ionic strength of 150
mM. For ξ-potential measurements PBS buffers of 7.4 and ionic
strength of 10 mM
was used
......................................................................................................................
180
Figure 6.2 A) Schematic diagram of self-assembly of PMAA-PS
80-g-St terpolymer into
nanoparticles upon complexation with Gd3+
and conjugation of the fluorescent
moieties. B) TEM images of the self-assembled nanoparticles in
water. ................... 182
Figure 6.3 Negative TOF-SIMS spectra of PS 80, PMAA-g-St, and
PMAA-PS 80-g-St, in the
m/z range of 0 to 300 atomic mass units.
...................................................................
183
Figure 6.4 Quantitative MRI of brain distribution: A) R1 maps of
Balb/c mice (n=3) injected with
Gd3+
loaded PMAA-PS 80-g-St nanoparticles (0.05 mmol/kg Gd+3
). B) Longitudinal
relaxation rates (R1) of sagittal sinus, ventricles, cortex, and
sub-cortex for Gd3+
loaded
PMAA-PS 80-g-St-DTPA polymer overtime..
........................................................... 185
Figure 6.5 Qualitative and quantitative results of brain
distribution and accumulation for PMAA-
PS 80-g-St nanoparticles. A) Ex-vivo near infrared fluorescence
images of the whole
brain. Ratio of the relative fluorescence intensity in brain as
a function of time after
intravenous injection of nanoparticles compared to normal brain
not injected with
nanoparticles. B) Fluorescence microscopy image of perfused mice
brains 45 minutes
following iv administration of saline, PMAA-g-St, and PMAA-PS
80-g-St. The
particles can be detected in the perivascular regions of the
brain capillaries for samples
treated with PMAA-PS 80-g-St nanoparticles.
........................................................... 188
Figure 7.1 SEM images of (A) surface (left-bottom corner) and
cross section (right-top corner),
(B) surface, (C) cross section of cross-linked starch-xanthan
gum film containing 10%
xanthan gum and 5% STMP.
......................................................................................
199
Figure 7.2 FTIR spectra of (A) pure starch, (B) starch reacted
with 20% STMP, (C) pure xanthan
gum, (D) xanthan gum reacted with 20% STMP, (E) physical mixture
of starch and
xanthan gum, (F) physical mixture of starch, xanthan gum, and
20% STMP, and (G)
mixture of starch and xanthan gum reacted with 20% STMP
.................................... 201
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xx
Figure 7.3 Schematic representation of cross-linking reaction of
starch and xanthan gum with
sodium trimethaphosphate (STMP)
............................................................................
202
Figure 7.4 A) 31
P NMR spectra of (I) pure starch, (II) starch reacted with 5%
STMP, (III) pure
xanthan gum, (IV) xanthan gum reacted with 5% STMP. B) 31
P NMR spectra of (I)
starch-xanthan gum without STMP, (II) STMP, (III) physical
mixture of starch,
xanthan gum and STMP, (IV) starch and 5% xanthan gum reacted
with 5% STMP. C)
31P NMR spectra of (I) starch and 10% xanthan gum reacted with
2% STMP, (II)
starch and 10% xanthan gum reacted with 5% STMP, (III) starch
and 10% xanthan
gum reacted with 20% STMP.
....................................................................................
205
Figure 7.5 Swelling kinetics of cross-linked starch-xanthan gum
films containing 5% xanthan
gum and 2% or 10% STMP in 0.15M phosphate buffer of pH=7.4
........................... 207
Figure 7.6 Equilibrium swelling behavior of modified starch
xanthan gum films of various
compositions with respect to change in (A) xanthan gum
concentration in DDIW (B)
pH with constant ionic strength of 0.15M.
.................................................................
208
Figure 7.7 Relative diffusion coefficients of molecular probes
as a function of molecular radius
across modified starch-xanthan gum films with various STMP
levels. ..................... 211
Figure 7.8 A plot of Y/Q-1 as a function of the hydration factor
(Q-1)-1
for starch-xanthan gum
hydrogels of various compositions. The slope of the line is
equal to the scale factor, Y.
....................................................................................................................................
212
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xxi
List of Abbreviations
1H-NMR hydrogen nuclear magnetic resonance spectroscopy
31P-NMR phosphorous nuclear magnetic resonance spectroscopy
AcAn acetic anhydride
ALT alanine aminotransferase
α-MEM alpha-modified minimal essential medium
ANOVA analysis of variance
ATP adenosine-5’-triphosphate
AUC area under the curve
BBB blood brain barrier
CCAC Canada council on animal care
CK creatine kinase
CO2 carbon dioxide
D2O deuterated water
DAPI 4’,6-diamino-2-phenylindole
DDIW distilled deionized water
DLS dynamic light scattering
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
Dox doxorubicin
DTPA diethylenetriaminepenta acetic acid
DTPA-bis-An diethylenetriaminepenta acetic acid bisanhydride
EDC N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride
EE encapsulation efficiency
-
xxii
EPR enhanced permeability and retention effect
EtOH ethanol
FA fluoresceinamine isomer I
FBS fetal bovine serum
FDA food and drug administration
FITC fluorescein isothiocyanate
FTIR Fourier transform infrared spectroscopy
Gd gadolinium
GD growth tumor delay
GSH glutathione
H&E hematoxylin and eosin
HCl hydrochloric acid
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HF 750 HiLyte Fluor TM
750 hydrazide
IC50 inhibitory concentration for 50% effect
ITC isothermal titration calorimetry
Kel elimination rate constant
KPS potassium persulfate
LDH lactate dehydrogenase
λem emission wavelength
λex excitation wavelength
LC loading content
LRP lung resistance protein
LV left ventricle
MAA methacrylic acid
MBA N,N′-Methylenebisacrylamide
-
xxiii
MDR multidrug resistance
MPS mononuclear phagocytic system
MRI magnetic resonance imaging
MRP1 multidrug resistance protein 1
MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide
NADPH nicotinamide adenine dinucleotide phosphate
NaOH sodium hydroxide
NHS N-Hydroxysuccinimide
NIR near-infrared
NPs nanoparticles
PBS phosphate buffered saline
PEG polyethylene glycol
PET positron emission tomography
PF-NPs preformed nanoparticles
Pgp P-glycoprotein
PMAA polymethacrylic acid
PMAA-PS 80-g-St polymethacylic acid grafted starch
PS 80 polysorbate 80
Py pyridine
Ri relaxation rate
ri relaxivity
ROS reactive oxygen species
S/V surface area to volume ratio
SA-NPs self-assembled nanoparticles
SD standard deviation
SDS sodium dodecyl sulphate
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xxiv
SEM standard error of the mean
St starch
STMP sodium trimethaphosphate
STS sodium thiosulfate
t1/2 half life
TEM transmission electron microscopy
v/v volume by volume
w/v weight by volume
XG xanthan gum
XRPD x-ray powder diffraction
ξ zeta potential
-
1
Chapter 1. Introduction
-
2
1.1 Breast Cancer
1.1.1 Epidemiology
Cancer is a serious and prevalent health problem that affects us
all. There were 12,661,000 new
cases of cancer in 2008, and 7,564,000 people had died from
previous cancer diseases [1]. In
2011, 177,800 Canadians were newly diagnosed with cancer, and
cancer related deaths
accounted for 29.3% of total deaths in Canada [2].
Breast cancer is the most prevalent form of cancer among women.
There were 1,384,000 new
cases in 2008, making up 23% of all women’s cancer [1]. With
458,000 deaths worldwide, breast
cancer is considered the most frequent cause of cancer related
death in women. According to
Canadian Cancer Society, there were 23,600 new cases of breast
cancer in 2011, making it the
most common cancer in Canadian women. [2]. Although the
mortality rates are on a gradual
decline in most developed countries since 1990, mainly due to
better screening techniques and
more effective treatment strategies, the disease claimed the
lives of 5,100 Canadian women,
comprising 14.8% of total cancer deaths, second only to lung
cancer [2].
1.1.2 Breast Cancer Cells and Tumor Microenvironment
Breast cancer, as in other solid cancers, is fundamentally
characterized by continuous and
uncontrolled growth. Many researchers share the view that
tumorigenesis proceeds via a process
analogous to Darwinian evolution, in which a succession of
genetic changes, each conferring one
or another type of growth advantage, leads to the progressive
conversion of normal human cells
into cancer cells. Hanahan and Weinberg have proposed that six
essential alterations in cell
physiology enabled by the genetic instability result in
malignant growth [3, 4]. These include
continuous proliferation independent of cells’ microenvironment,
acquired resistance to anti-
proliferative signals, evasion of apoptosis, ability to
replicate without limits, ability to induce
-
3
angiogenesis, and the ability of some cancer cells to undergo
metastasis. These acquired
physiological alterations generally apply to solid tumors,
including breast cancer.
Breast tumors are not merely an accumulation of neoplastic
cells, but rather a complex tissue
with blood vessels, stromal cells, infiltrating immune-competent
cells, and a differentiated
extracellular matrix [3-5]. All these cell types interact with
each other to build a unique tumor
microenvironment. This heterogeneous mass grows until it reaches
an approximate volume of 2
mm3, beyond which the diffusion of nutrients and oxygen cannot
take place and areas of hypoxia
and acidosis develop [6, 7]. As a result, the tumor contains
interspersed regions of well
oxygenated (pO2 > 2.5 mmHg) and poorly oxygenated (pO2 ≤ 2.5
mmHg), or hypoxic, tissue
heterogeneously distributed throughout the tumor mass. Cancer
cells of a breast tumor can adapt
to thrive in areas of low oxygen concentrations that would
otherwise induce normal cell death [8,
9]. Normal cells fulfill 90% of their energy requirements
through the Krebs cycle which uses
pyruvate formed from glycolysis in a series of reactions that
donate electrons via NADH and
FADH2 to the respiratory chain complexes in mitochondria [8].
This high efficiency glucose
metabolism requires oxygen. With limited oxygen, such as with
muscles that have undergone
prolonged exercise, pyruvate is not used in the Kreb’s cycle and
is converted into lactic acid by
lactate dehydrogenase (LDH) in a process termed anaerobic
glycolysis. This process can also
produce cellular energy but with poor efficiency. Warburg was
the first one to report that even in
the presence of oxygen, 50% of tumor ATP is produced through
glycolytic catabolism [8]. This
switch from high efficiency aerobic oxidative phosphorylation to
low efficiency anaerobic
glycolysis for cellular chemical energy production in tumors is
known as the Warburg effect [8,
9]. Through reduction of the oxygen consumption, the Warburg
effect enhances cancer cell
survival in hypoxic tumor microenvironment by decreasing the
oxygen-starved fraction of the
tumor distal from blood vessels, and by reducing the production
of the reactive oxygen species
-
4
(ROS) that are the by-product of electron transport in the
mitochondria during the Kreb cycle
[10]. Due to cytotoxic nature of the ROS, reduction in their
production by the Warburg effect
provides cancer cells with a survival advantage in hypoxic tumor
tissues [11].
Tumor growth beyond 2 mm3 is angiogenesis dependent.
Angiogenesis is defined as the process
of formation of the new blood vessels. The hypoxic nature of the
tumor environment activates a
series of hypoxia-sensitive transcription factors in cancer
cells, stromal fibroblasts, and tumor
associated macrophages. These cells all work in harmony to
generate new tumor blood vessels
[12-14]. Formation of new vascular network by angiogenesis
supply the tumor with oxygen and
nutrients required to support its continued growth. However,
tumor vasculature is significantly
different in terms of its structure and physiology from healthy
blood vessels. Many tumors reveal
chaotic networks of tortuous and distended veins, venules and
venous capillaries along with
intertwining capillaries branching from arterioles and veins
[15, 16]. Irregularities of vascular
wall structures in tumors have also been described with walls
composed of a mosaic of cancer
and endothelial cells which leads to widened interendothelial
junctions and numerous endothelial
fenestrations with the net effect of leaky blood vessels unique
to tumor neovasculature [17].
These blood vessels are highly permeable to the extravasation of
therapeutic macromolecules
and small colloidal particles [18, 19].
The resulting intratumoral circulation is characterized by
tortuous microvessels lacking the
normal hierarchical arrangement of arterioles, capillaries and
venules. Within this altered
microenvironment, blood flow is sluggish with unstable rheology,
anomalous and generally
stagnant [15, 16]. As a result, these characteristics lead to
heterogeneous perfusion with hypoxia
and acidity in low-flow regions. In fact, hypoxia is a
pathophysiological property of breast
tumors, with up to 60% of the tumor existing in a hypoxic state
[7].
-
5
Accumulation of lactic acid in the tumor microenvironment
coupled with insufficient blood
supply and poor lymphatic drainage results in acidic pH states
in the solid tumor
microenvironment. Although there is a distribution, in vivo pH
measurements, made by needle
type microelectrodes on human patients having various solid
tumors (adenocarcinoma, squamous
cell carcinoma, soft tissue sarcoma, and malignant melanoma) in
readily accessible areas (limbs,
neck, or chest wall), show the mean pH value to be 6.9 with
values as low as 5.7 being reported
for some tumors [20, 21]. Increasingly, it has been proposed by
many researchers that the mildly
acidic tumor environment can be exploited to achieve high local
drug concentrations and to
minimize overall systemic exposure [22, 23] . The use of pH
responsive carrier systems in the
delivery of chemotherapeutics will be discussed in section
1.1.5.3.
1.1.3 Doxorubicin: a Potent Drug for Breast Cancer
Chemotherapy
Doxorubicin (Dox), Figure 1.1, is a chemotherapeutic agent which
belongs to the anthracycline
antibiotics family. The drug has been widely adopted as a first
line chemotherapy agent, most
often in combination with other agents, for the treatment of
various types of cancer including
hematological malignancies, carcinomas, and sarcomas [24, 25].
Its widespread use in the
treatment of breast cancer has been recognized as one of the
reasons for the decreased mortality
rates of the disease [26]. A number of mechanisms have been
proposed to account for the broad-
spectrum anticancer activity of Dox. These include: (1) DNA
intercalation by its planar three-
ring structure, (2) poisoning topoisomerase II enhancing DNA
strand breaks, (3) generation of
free radicals, (4) possible disruption of the cell membrane
functionality [27-29]. The cell nucleus
is the main cellular target of Dox even though involvement of
mitochondria in Dox cytotoxicity
such as mitochondria-mediated apoptosis has also been observed
[30]. It is likely that multiple
pathways are used by Dox to inflict damage to cancer cells.
-
6
Dox is commonly administered intravenously in the form of
commercially available injections
Adriamycin® and Rubex® for maintaining the therapeutic levels in
blood. In addition, two
PEGylated liposomal formulations of Dox, Doxil® and Caelyx®, are
also available.
Dox administration in breast cancer chemotherapy is often
limited by serious systemic side
effects such as myelosuppression and congestive heart failure
[31-33]. The Dox induced
cardiomyopathy appears to be cumulative limiting the maximum
lifetime dose of the drug in
humans to 450 mg/m2 [33]. The detailed mechanism of
cardiotoxicity is not fully understood;
however, it is believed that Ca2+
activated ATPase, cAMP, and lipid peroxidation are involved,
as well as the ability of Dox to produce free radicals.
Myocardium cells seem to have an
enhanced sensitivity to these effects leading to cardiomyopathy,
and decreased cardiac
ventricular ejection fraction [29, 31, 32, 34].
Although Dox is highly effective in a variety of non-resistant
breast cancer cell lines, its efficacy
is significantly reduced in multi-drug resistant (MDR) sub-cell
lines such as murine
EMT6/AR1.0 and human MDA435/LCC6/MDR1. Hence Dox is an excellent
compound for
evaluation of novel MDR-reversal approaches.
Figure 1.1 Chemical structure of Doxorubicin
-
7
1.1.4 Barriers to Cancer Chemotherapy
1.1.4.1 Drug Resistance
Drug resistance is one of the major obstacles in successful and
effective treatment of breast
cancer. The underlying causes of drug resistance are complex and
multi-factorial providing the
cancer cells with many ways to survive cancer chemotherapy. In
general, the mechanisms of
drug resistance can be classified into non-cellular resistance
and cellular resistance. These two
mechanisms are described in more detail below.
Non-cellular resistance: Poor efficacy in classical chemotherapy
often is associated with the
unique anatomical or physiological features of the solid tumors.
It is a well-known fact that
multi-cellular spheroids of tumor cells are more resistant to
anticancer agents than the
corresponding monolayer cultures [35]. Solid tumors are also
found to be less sensitive to
chemotherapy than malignancies consisting of individual cells,
e.g. leukemia. As discussed in the
previous section, the tumor environment and blood vessel
architecture is unique and
characterized by hypoxic regions, acidic microenvironment,
sluggish, and inhomogeneous blood
circulation. Most blood vessels inside tumor are highly
disorganized as they take tortuous turns
and many of these twisted blood vessels near the center of
tumor, are crushed due to the irregular
growth of tumor in a confined space. Some sites in the tumor are
thus far away from the blood
supply making it difficult for drug molecules, especially those
with larger molecular weight and
lipophilicity, to reach these sites [36]. In addition, due to
the elevated vascular permeability of
tumor vessels along with the absence of a functional lymphatic
network, in this confined space,
interstitial fluid is collected more abundantly than in normal
tissue, creating another impediment
to drug distribution inside tumor mass. The accumulation of this
fluid leads to elevated
interstitial fluid pressure that decreases from the core toward
the periphery of the tumor [37]. The
-
8
altered composition of extracellular fluid, associated with
increased interstitial pressure and
sluggish blood flow hinders drug distribution and therefore the
efficacy of chemotherapy.
As discussed previously poor blood supply and excessive growth
leads to development of
hypoxic regions in tumors. Hypoxic cells may become quiescent
and resistant to cytotoxic agents
that target proliferating cells [38]. The reduction in oxygen
levels also compromises the anti-
cancer efficacy of some drugs such as Dox which is known to be
much more effective in
oxygenated regions of the tumor by generating ROS species
through the redox cycling of its
quinine nucleus [28]. Hypoxia, especially chronic hypoxia, can
promote metabolic changes and
genomic instability of cancer cells, further enabling the
acquisition of random mutations and
driving the malignancy of the tumor [38, 39]. Furthermore, the
acidic environment of the tumor
due to production of lactic acid may potentially deactivate weak
base drugs such as doxorubicin
[40].
In summary, non-cellular resistance is one of the major causes
of poor efficacy of conventional
chemotherapeutic agents in solid tumors as only a limited
fraction of the systemically
administered anticancer drug can reach and penetrate the target
tumor site and remain active.
Cellular resistance: The cancer cells may still survive despite
the significant levels of drug in
the tumor. Multidrug resistance (MDR) is a major barrier to
effective treatment of cancer. This is
due to the ability of cancer cells to effectively neutralize the
cytotoxicity of classical agents such
as Dox. MDR can be acquired following failed rounds of drug
therapy, or it can be innate, pre-
programmed into the genetic code of the cancer cells [40, 41,
42]. In addition, the drug resistance
in MDR cells is rarely specific to one drug and generally
develops as cross-resistance to a range
of structurally and functionally unrelated compounds [43].
Hydrophobic, amphiphathic drugs
such as vinca alkaloids (vincristine, vinblastine), taxanes
(paclitaxel, docetaxel),
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9
epipodophyllotoxins (etoposide, teniposide), anthracyclines
(doxorubicin, daunorubicin) are
more frequently associated with MDR phenomena. MDR is often
attributed to cell membrane
drug transport proteins, metabolic pathways, and intracellular
targets common to cancer
chemotherapeutics [41, 43, 44].
Over-expression of ATP Binding Cassette (ABC) efflux pumps is
one of the major mechanisms
of the MDR. P-glycoprotein (P-gp) and Multidrug Resistance
Protein 1 (MRP1) are two main
ABC efflux pumps that are over-expressed in drug resistant
breast cancers [45, 46]. Like all
ABC family members, both P-gp and MRP1 utilize the energy
released from ATP hydrolysis to
transport drug molecules in the cell membrane to the outside.
P-gp and MRP1 works in concert
to effectively decrease the intracellular concentration of the
drugs before they become active and
reach their cellular target [41]. P-gp preferentially transports
neutral or mildly cationic
compounds while MRP1 is more effective against lipophilic anions
(e.g. glucuronate or
glutathione conjugates) [45]. The expression of both pumps is
controlled by hypoxia-inducible
transcription factors, and is responsive to changes in
intracellular redox status and ROS
production [47, 48]. Hence, the hypoxic nature of the tumor
microenvironment as well as the
redox cycling associated with certain cytotoxic drugs such as
Dox can upregulate the expression
of these plasma membrane efflux pumps induce acquired MDR. It
has been suggested that
certain non-ionic surfactants (e.g. polysorbate 80), and block
co-polymers (e.g. PluoronicTM P85)
may inhibit the P-gp efflux pumps [49]. In addition to P-gp and
MRP1, breast cancer resistance
(BCRP) has also been reported in some resistant breast cancer
cells [45]. More than one of the
mentioned membrane-associated drug transports may be present in
the same cancer cell and
render the cell even more resistant to chemotherapy.
-
10
Alternatively, cancer cells may become resistant by
sequestration of drugs in cytoplasmic
vesicles. Using fluorescence microscopy and taking advantage of
the fluorosence properties of
Dox, Beyer et al. have demonstrated that the subcellular
distribution of the drug following cell
uptake was significantly different between chemosensitive and
MDR cell lines with higher
accumulation of the drug in the cytosolic vesicles being
observed in MDR cells [50]. By
preventing the drug from interacting with its cellular targets,
MDR cancer cells can effectively
neutralize the chemotherapeutic agents.
MDR can also be the result of cell ability to evade apoptosis.
Apoptosis is the process of
programmed cell death or cell suicide that can be elicited by a
number of stimuli such as DNA
damage caused by certain anticancer agents (e.g doxorubicin and
cisplatin) [51]. The apoptotic
pathway involves highly organized and specific signal
transduction that is negatively or
positively regulated by anti-apoptotic factors and pro-apoptotic
factors. Pakunlu et al.
demonstrated that Bc12, an anti-apoptotic factor, is upregulated
in Dox resistant tumor cells [52].
By avoiding the occurrence of apoptosis, cancer cells become
less sensitive to certain
chemotherapeutic agents.
Some drug-resistance cancer cells were also found to have more
active drug detoxification
systems. Glutathione (GSH) and glutathione-S-transferase act to
sequester a number of
anticancer agents and are overexpressed in MDR cells [42]. An
increase in intracellular GSH
facilitates the removal of the drug-GSH conjugates from the cell
via MRP1 which is also
upregulated in MDR cells [48].
Cancer cells may also protect themselves from the action of
cytotoxic drugs by alteration in drug
targets themselves. By altering the conformation of the drug
target or masking the drug docking
site through post-translational modification, the efficiency
with which the target is modified or
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11
its function disrupted by the drug is decreased, rendering the
cell resistant to the action of the
drug [41]. For example, altered tubulin structure may compromise
the effectiveness of anti-
microtubule agents [53].
Many anticancer drugs exert their action by inflecting damage to
DNA. The resistant cells often
modify their DNA repair mechanisms through upregulation of the
DNA repair proteins [44].
Increased repair of Dox-DNA adducts would decrease the rate of
induced DNA lesions and
promote the restitution of DNA structure, overcoming Dox
genotoxicity. In addition, DNA
damage signalling pathways responsible for relaying the
information regarding genotoxicity to
effectors of apoptosis are shut down, rendering the cells
resistant to the cytotoxic drugs [41, 44].
As discussed, clinical drug resistance is a multi-factorial
phenomenon, and the development of
MDR phenotype in cancer patients may involve any combination of
the several physiological
alterations outlined above (Figure 1.2). While MDR may be
acquired through different
mechanisms in different patients, the development of multidrug
resistance is almost universal.
Figure 1.2 Factors leading to MDR in cancer. One of the leading
causes of treatment failure in
cancer is the onset of resistant disease. MDR can be divided
into two broad categories: Cellular
resistance and non-cellular resistance.
Factors leading to multi-drug resistance
Cellular resistance
Drug efflux pumps
up-regulation
Intracellular Sequestration
Drug detoxification
enzymes
up-regulation
Increased DNA repair
capacity
Anti-apoptotic proteins
up-regulation
Drug targets down-
regulation
Non-cellular resistance
Tumor Hypoxia
and acidity
Sluggish blood flow
Low intratumoral
drug concentration
Elevated interstitial
fluid pressure
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12
1.1.4.2 Systemic Side Effects
Chemotherapeutics are often non-specific to cancer cells. A
systemically administered cytotoxic
drug causes toxicity in both normal and cancer cells. As a
result, a major limitation of cancer
chemotherapy is the systemic toxicity of the agents which
ultimately influences their therapeutic
efficacy. While most of the classical chemotherapeutics such as
Dox are more toxic towards
highly proliferating cells, these drugs will still induce
toxicity in other cell populations due to
their ability to produce ROS through redox cycling reactions
[28]. In addition to cancer cells,
other cells in the body such as the cells of gastrointestinal
tract, hair follicles, and bone marrow
are continually proliferating in an adult, reducing the
selectivity of classical chemotherapy.
Hence, some side effects such as nausea, vomiting, alopecia, and
myelosuppression are common
for all cytotoxic drugs. While other side effects are more drug
specific. For example, Dox
administration is associated with irreversible cardiotoxicity
that is attributed to its major phase I
metabolite doxorubicinol, limiting the maximum allowable
lifetime dose of the drug [54, 55]. It
has been suggested that Dox induces cardiomyopathy and
congestive heart failure by interfering
with the sarcoplasmic reticulum. Co-administration of the
cardio-protectants or the use of
anthracycline derivatives with lower demonstrated cardiotoxicity
have been attempted by
different researchers [56-58]. However, these approaches have
often led to reduced anti-cancer
efficacy, preventing effective anti-cancer outcomes.
Because anticancer drugs often show a steep dose-response curve,
it is has been suggested by
some clinicians that aggressive chemotherapy is associated with
greater therapeutic efficacy [59],
but high doses of chemotherapeutics are generally associated
with high levels of acute and
chronic toxicity. In order to improve therapeutic efficacy, the
drug associated systemic toxicity
needs to be reduced. This may be achieved through limiting the
drug exposure to the healthy
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cells while increasing the drug concentration in the cancer
cells. This rational forms the basis for
the use of nanotechnology as drug delivery vector for cancer
chemotherapy.
1.1.5 Nanoparticulate Systems in Cancer Therapy
Despite outstanding progress in the field of cancer biology and
understanding the fundamentals
of the disease, challenges remain in our ability to translate
our understanding of fundamental
cancer biology to the clinic. Classical chemotherapies suffer
many challenges which include
dose limiting systemic toxicity, hypoxia, relatively low
intra-tumoral drug levels, and MDR
phenotypes of cancer cells, often limiting their therapeutic
potentials. Over the past two decades,
nanotechnology-based approaches have emerged as an exciting
field with promises to remedy
these limitations. First, a well-designed nanoparticulate system
with optimum size and
circulation half-life would facilitate controlled and tumor
specific drug accumulation and release,
thus reducing the systemic toxicity that is often associated
with classical chemotherapeutics and
increasing the possibility of more aggressive chemotherapy and
possibly better clinical outcome
[60]. Second, the encapsulated drug is protected from the harsh
environments of the body, drug
metabolizing enzymes, and extensive binding to serum proteins
while in the circulation leading
to improved therapeutic efficacy [61]. Third, due to leaky
nature of the tumor vasculature and its
poor lymphatic drainage, drug carriers with optimum size (50-300
nm) and prolonged blood
circulation will preferentially accumulate in the tumor, a
phenomenon known as Enhanced
Permeation and Retention effect (EPR) (Figure 1.3) [18, 19].
Finally, the sub-micron size of the
drug carriers affords the ability to enter deep within the cell
and overcome the MDR efflux pump
mechanisms which are currently one of the major causes of
treatment failure in the clinic (Figure
1.4) [60, 62-67]. By designing an optimum nanoparticulate drug
delivery system, the right drug
can be delivered to the right site in the body at the right
time.
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14
Figure 1.3 Passive targeting of the tumor by EPR. The EPR effect
is the balance of enhanced
tumor permeability with poor tumor interstitial fluid drainage,
resulting in the selective uptake
and retention of nanoparticles in the tumor tissue [18, 19].
Figure 1.4 Drug loaded nanoparticles can overcome MDR cancer
cells. Endocytosis of the drug
loaded nanoparticles in membrane bound vesicles protects the
drug from the action of the
membrane efflux pumps. The nanoparticles release the drug deep
inside the cell and the drug can
gain access to its cellular target site (e.g. DNA)
Enhanced Permeability & Retention Effect (EPR)
1. Tumor-associated leaky vasculature2. Poor lymphatic
drainage
Tumor
Neovasculature
Loose
Interendothelial
Junctions
(Fenestrations)
Blood
Flow
Lymph
Flow Tumor
~ < 300 nm
Blood
pH =7.4
Lysosomal
pH < 6
Tumor
pH < 7Drug
Dox efflux by
P-gp pumps
Leaky tumor
vasculatureActive uptake of NPs
by tumor cells
Dox
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1.1.5.1 Requirements of an Ideal Nanoparticulate Drug Delivery
System for Cancer Chemotherapy
Ideally, a drug carrier should have as many of the following
properties as possible: (1) good
biocompatibility profile, (2) biodegradable with non-toxic
degradation products, (3) convenient,
cost-effective, and reproducible preparation, (4) ability to
efficiently load the drug at high
contents, (5) controlled and tumor specific drug release
kinetics, (6) optimum size and
circulation half-life, (7) passive and/or active tumor targeting
capabilities, and (8)
“upgradability”, i.e., allowing further surface
modifications.
As mentioned previously, chemotherapeutics typically show a
steep dose-response curve. To
ensure therapeutic success sufficiently high dose intensity has
to be used. In other words,
reasonably high drug loading capacity and sufficient drug
release at the diseased site are required
or else an unreasonably large quantity of the drug carrier has
to be administered for effective
cancer treatment [68]. Cytotoxic drugs generally do not
discriminate between cancer cells and
healthy cells in the body; hence, an ideal drug delivery system
should exhibit minimal non-
specific drug release while in the circulation followed by
increased release rate upon
accumulation in the tumor. Size, blood circulation time, and
colloidal stability of the
nanoparticles are all important characteristics of the drug
carrier. Nanoparticles which are
smaller than 10 nm are rapidly cleared by the renal route
preventing adequate time for tumor
accumulation, while passive targeting by the EPR effect is
significantly reduced with particles
larger than 300 nm [69, 70]. The size range of 50-200 nm has
found to be optimal in promoting
the passive targeting of the nano-carriers to the tumor site.
However, it has to be pointed out that
the range of acceptable nanoparticle sizes for optimized
chemotherapy is highly material
dependent and will change from polymeric to inorganic to lipid
based formulations. The long
circulation of the nanoparticulate drug delivery system is
required to increase the number of
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16
exposures of tumor tissue to the drug delivery system, promoting
the passive tumor targeting by
EPR effect. In fact, it is believed that EPR effect-mediated
passive tumor uptake is optimized
with circulation times of at least 4 hours [61, 70]. Surface
grafting of some hydrophilic polymers
to the nanoparticles can prolong the circulation half-life of
nanoparticles, and will be reviewed in
the next section.
1.1.5.2 Surface Modification to Prolong Nanoparticles Blood
Circulation
Plasma proteins, including immunoglobulins and complement
proteins readily bind to foreign
matters in the blood [61, 70]. These plasma proteins, or
opsonins, effectively, tag the foreign
particles for removal from the circulation by resident tissue
macrophages of the liver, lymph, and
spleen, known as the mononuclear phagocytic system (MPS) [71].
The removal of opsonised
nanoparticles from the systemic circulation reduces the
longevity of nanoparticle circulation and
hence compromises its therapeutic efficacy. The surface of the
nanoparticles can be modified to
prevent this rapid uptake producing long circulating or
“stealth” particles which promote both
passive and/or active targeting. Vonarbourg et al. have reviewed
the general conditions for the
stealthiness of colloidal drug carriers [72]. These include
having a small size, with a neutral and
hydrophilic surface, as well as a thick, well-anchored and
flexible coating.
The use of hydrophilic polymer coatings to nanoparticulates is a
common practice to achieve a
long circulation time. The most commonly used polymer for this
purpose is poly(ethylene
glycol) (PEG) [73]. It is generally accepted that the PEGylation
of the particle surface prevents
opsonisation by enhancing the steric repulsion between
nanoparticles’ surfaces and blood
components, and by forming an inert, impenetrable polymer layer
over the surface of the
nanoparticles with the effectiveness mainly depending on the
degree of surface coverage and
molar mass [74]. Additionally, it has been suggested that the
PEG coats allow the selective
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17
adsorption of some serum proteins that are sometimes known as
dysopsonins, i.e. plasma
components which are believed to prevent opsonization. In other
words, it may be the selective
adsorption, and not the prevention of adsorption, that alters
the pharmacokinetics and fate of
nanoparticles [75, 76]. Nevertheless, it is the general
consensus that the surface PEGylation of
the nanoparticles prolongs their blood circulation.
However, PEG is not biodegradable; also, chemical attachment of
additional groups such as
targeting moieties, and/or metal chelators, to PEGylated
surfaces is difficult and involves
complicated reaction schemes mostly due to the absence of
reactive groups on the PEGylated
surfaces. This is one of the reasons why polysaccharide coatings
have been considered as an
alternative to the PEG coatings. Additionally, oligo and
polysaccharides may achieve active
targeting per se since they have specific receptors in certain
cells or tissues [77]. Moreover,
polysaccharides display well-documented biocompatibilities and
biodegradabilities, which are
the desired basic characteristics for polymers used as
biomaterials. Polysaccharides have been
suggested as biocompatible polymer coatings for nanoparticles.
For example, heparin coating of
poly(methyl methacrylate), PMMA, nanoparticles exhibited an
increased circulation half-life of
5 h compared to only a few minutes for the bare nanoparticles
[78]. Similarly, coating of
superparamagnetic iron oxide nanoparticles (SPIONs) with dextran
increased their half-life up to
4.5 h [79]. However, there have been reports of hypersensitivity
reactions upon administration of
dextran in the clinic [80]. Hydroxyethyl starch (HES) is
currently investigated at the industrial
level as a biodegradable substitute for PEG, so that HESylation
of proteins could substitute
PEGylation [81]. In one study poly(lactic-co-glycolic acid),
PLGA, nanoparticles stabilized with
HES exhibited reduced human serum albumin (HSA) and fibrinogen
(FBG) adsorption, and
uptake by phagocytic cells in vitro [82].
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In summary, the polysaccharide coating may provide steric
protection against protein adsorption
and macrophage uptake. Additionally, as polysaccharides offer
many available reactive groups,
active targeting could be obtained by grafting ligands onto the
nanoparticle surface. Considering
the very large variety of polysaccharides in nature, one could
imagine the wide array of surface-
engineered nanoparticles adapted to a given therapeutic and
monitoring purpose.
1.1.5.3 pH-responsive Nanoparticles in Cancer Chemotherapy
As discussed previously, tumor environment is acidic with
average pH of 6.9 but