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Development of Self-Assembled Amphiphilic Oligo-Urethanes as Cardiovascular Drug Delivery Platforms
by
Maneesha Amrita Rajora
A thesis submitted in conformity with the requirements for the degree of Master of Applied Science
Institute of Biomaterials and Biomedical Engineering
University of Toronto
© by Maneesha Amrita Rajora (2013)
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Development of Self-Assembled Amphiphilic Oligo-Urethanes as
Cardiovascular Drug Delivery Platforms
Maneesha Amrita Rajora
Master of Applied Science
Institute of Biomaterials and Biomedical Engineering
University of Toronto
Abstract
Current drug-coated balloon (DCB) technologies, used to prevent percutaneous coronary
intervention-related restenosis via antiproliferative agent delivery to arterial lesions, are
associated with systemic drug loss during catheter tracking and inefficient drug delivery to
target tissues. This thesis aimed to study and synthesize novel amphilphilic oligo-urethanes
(AOUs) as DCB drug carriers comprised of polyol, lysine diisocyanate and perfluoro-
alcohol (PFA) segments to enhance drug release, binding and shielding against premature
release respectively. AOU syntheses employing di-hydroxyl polyols were found to be
susceptible to pre-polymer intramolecular cyclization, which prevented PFA conjugation.
Such undesired cyclization reactions were circumvented in this thesis via the use of a
mono-functional polyol to yield AOU analogues that were water soluble (≥430 mg/mL
solubility) and relatively phase mixed. Fluorocarbon groups migrated to the surfaces of
analogue films, yielding water contact angle values ≤51o. Preliminary analogue-cell
compatibility studies and capillary electrophoresis-mediated drug-AOU dissociation
assessments were conducted and are reported.
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Acknowledgements
I would like to take this opportunity to extend my deep gratitude to my graduate research
supervisor, Dr. J. Paul Santerre. His constant support, invaluable mentorship,
encouragement and optimism have played a pivotal role in the completion of this thesis. I
would like to thank Dr. Roseita Esfand (Interface Biologics Inc.), our industrial collaborator,
for her time, insight and guidance throughout this project. I would also like to extend my
gratitude to my committee members, Dr. Julie Audet (Institute of Biomaterials and
Biomedical Engineering, University of Toronto) and Dr. Milica Radisic (Institute of
Biomaterials and Biomedical Engineering, University of Toronto) for their constructive
feedback and support during this thesis.
This thesis would be incomplete without the assistance of several individuals. Namely,
thank you to Dr. Soroor Sharifpoor for conducting cytotoxicity studies and for taking the
time to answer my many questions. Thank you to Mr. Frank Gibbs (Brockhouse Institute for
Materials Research, McMaster University) for DSC and TGA studies, Dr. Rana Sodhi
(Surface Interface Ontario, University of Toronto) for XPS studies, Dr. Jian Wang (Faculty
of Dentistry, University of Toronto) for SEM imaging, Ms. Marilyn Fernandes and Dr. Adam
Daley for GPC and Dr. Kirk Green (Department of Chemistry, McMaster University) for
MALDI-MS experiments. I am also grateful to the Audet lab (Institute of Biomaterials and
Biomedical Engineering, University of Toronto) and Kumacheva lab (Department of
Chemistry, University of Toronto) for granting me access to their equipment for CE studies
and contact angle measurements respectively. I would also like to extend my gratitude to
Dr. Meilin Yang (Faculty of Dentistry, University of Toronto), Ms. Sylvia Tjahyadi (Interface
Biologics Inc.) and Ms. Bernadette Ilagan (Interface Biologics Inc.) for their time and
technical expertise.
Thank you to all of my colleagues in the Santerre lab group who have truly helped in
making this graduate experience enjoyable and memorable. Thank you to Yasaman
Delaviz, Dr. Maria López-Donaire, Kyle Battiston, Maher Bourbia, Kate Brockman, Jane
Cheung, Dr. Soroor Sharifpoor and Meghan Wright for their camaraderie, understanding
and support throughout the highs and lows of the last two years.
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I am greatly appreciative of my family members, whose constant love, patience and
encouragement has always been a pillar of strength for me. Thank you especially to my
mother for her selfless support and understanding.
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Table of Contents
Abstract……………………………………………………..…………………………………….….ii
Acknowledgements………………………………………………………….……………………..iii
List of Abbreviations…………………….……………………………………………………….....ix
List of Tables……………………………………………………………………..………….……..xii
List of Schemes……………………………………………………………….……………………xv
List of Figures………………………………………..……………………………………………xvii
Chapter 1: Introduction……………………………………..…………………………………....1
1.1 Percutaneous coronary interventions and restenosis.……………………………..………1
1.2 Amphiphilic oligo-urethanes……………………...……….………………..……………....…4
1.3 Research objectives and hypotheses………………………………….………………….....6
1.3.1 Central research objective………………………………………………………….6
1.3.2 Central hypothesis…………………………………………………………………..6
1.3.3 Objective 1…………………………………………………………………………...6
1.3.4 Objective 2…………………………………………………………………………...7
1.3.5 Objective 3……………………………………………...………..………….……..10
Chapter 2: Review of Literature …………………………………………………………........12
2.1 Restenosis……………………………………………………………………………………..12
2.1.1 Restenosis: The thrombotic phase………...………….…..……………………..12
2.1.2 Restenosis: The neointimal progression phase………...………………………14
2.2 Drug-coated balloon technologies to prevent restenosis…………………………..……..15
2.2.1 Pharmaceutical agents…………………………………………………………….15
2.2.2 Current DCB carriers under clinical and pre-clinical evaluation……....……....21
2.3 Tailoring DCB carrier chemistry……………………………………………………………..27
2.3.1 Application of fluorinated polyurethane chemistries……………....…………....27
2.3.2 Designing drug-compatible carriers……………………………….…………......29
2.4 Polymer synthesis………………………………………………………………………….....32
2.4.1 Step-growth polymerization……………….……………………………………....32
2.4.2 Atom transfer radical polymerization………………………………………..……35
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2.5 Assessing drug release………………………………………………………………………37
2.5.1 Direct spectrophotometry……………………………………….……………….37
2.5.2 High-performance liquid chromatography…….…………....………………….39
2.5.3 Capillary electrophoresis……………………….………………………………..40
Chapter 3: Materials and Methods…………………………………………..………….........44
3.1 Materials…………………………………………………………………...…………………..44
3.2 Amphiphilic oligo-urethane syntheses and purifications……......………………...……...45
3.2.1 Determination of polyol hydroxyl content…………………………………………....45
3.2.2 Monitoring reaction kinetics: Isocyanate titrations…………….………………..….46
3.2.3 NL-PEG:LDI and L-PEG:LDI intramolecular pre-polymer cyclization studies.....47
3.2.4 Analogue 1 synthesis and purification………………………...…………………….50
3.2.5 Analogue 2 synthesis and purification………………………...……...………….....53
3.2.6 Macroinitiator 2’ synthesis and purification……………………...………...…….....55
3.2.7 Analogue 3 synthesis via atom transfer radical polymerization……..………..….56
3.2.8 Analogue 4 synthesis via atom transfer radical polymerization…………....…….57
3.2.9 Tin removal from analogue 1 and analogue 2……………….……..…….………...58
3.3 Bulk characterization of materials…………………………………………………………...59
3.3.1 Nuclear magnetic resonance spectroscopy……………………...………………...59
3.3.2 Gel permeation chromatography…………………..……………….………….…....59
3.3.3 Fluorine and bromine analysis………………………..……………………………..59
3.3.4 Matrix-assisted laser desorptive ionization mass spectrometry…………...…….59
3.3.5 Inductively coupled plasma atomic emission spectrometry……..……….……....60
3.3.6 AOU bulk water solubility ..………………..……….……….……………..…………61
3.3.7 Thermogravimetric analysis……………………….….……………………..……….62
3.3.8 Differential scanning calorimetry………………….…….…………………..……….62
3.4 Surface characterization of AOU films…………………………………….………………..62
3.4.1 Preparation of films………….……………………………………….………………62
3.4.2 Scanning electron microscopy…………………….………………….…………….63
3.4.3 Contact angle measurements…………….………………………….……………..63
3.4.4 X-ray photoelectron spectroscopy...……….……………………….……………...64
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3.5 Cytotoxicity studies…………………………………………………………………...……....64
3.5.1 Cell culture ……………………………………………………………………..…....64
3.5.2 Cytotoxicity assays …...………………………………………………………..……65
3.6 Capillary electrophoresis………………………………………………...…………………...66
3.6.1 Instrumentation…………………..……………………………...…………………...66
3.6.2 Run buffer optimization……...………………………………….….………………..67
3.6.3 NECEEM experiments………...…………………………….…….………………...67
3.7 Statistical analysis ……………………………………………...…………………………….68
Chapter 4: Intramolecular Cyclization during Amphiphilic Oligo-Urethane Syntheses:
Results and Discussion…………………………………….………………………………..…69
4.1 Cyclization within polyurethane pre-polymer reactions………………..………………….69
4.2 Cyclized species generation……………………………………………..……...…………..70
4.3 Variation of NL-PEG:lysine diisocyanate molar feed ratios…………....………………...79
4.4 Cyclization within AOU syntheses using linear polyol…………...……...…………….….91
4.5 Summary………………………………………………………………....……………………99
Chapter 5: Synthesis and Characterization of Novel Amphiphilic Oligo-Urethanes:
Results and Discussion…………………………………….………………..………………..101
5.1 Novel amphiphilic oligo-urethane design criteria…………………..……….……………101
5.2 Analogue 1 synthesis………………………………...……………………….…………….101
5.2.1 Equimolar drop-wise approach…………………………………….……………103
5.2.2 Excess LDI drop-wise approach optimization…….…………...………………106
5.2.3 Reproduction of the optimized analogue 1 synthesis………………………...111
5.3 Synthesis of Analogue 2……………………………………………………………………113
5.4 Atom transfer radical polymerization syntheses…………………………………………122
5.4.1 Synthesis of the macroinitiator 2’…………………………...…………………..122
5.4.2 Synthesis of analogue 3………………………………………………………….125
5.4.3 Synthesis of analogue 4………………………………………………………….128
5.5 Residual catalyst concentrations…………………………………………………………..132
5.6 Hydrophilicity of the analogues…………………………………………………………….133
5.7 X-ray photoelectron spectroscopy…………………………………………………………137
5.8 Thermal properties of the analogues……………………………………………………...142
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5.9 Cytotoxicity studies………………………………………………………………………….146
5.10 Summary …………………………………………………………………………………...150
Chapter 6: Capillary Electrophoresis: Results and Discussion……..…………………153
6.1 Selection of a suitable run buffer……………………………………….………………….153
6.2 Non-equilibrium capillary electrophoresis of equilibrium mixtures experiments……..157
6.3 Summary…………………………………………………………………………………..…162
Chapter 7: Conclusions…………………………………………………………………..…...164
Chapter 8: Recommendations………………………………………………..……………...166
Chapter 9: References………………………………………………...………..……………..171
Appendix A: List of Reagents and Suppliers……………………...…………..………….203
Appendix B: Calibration Curves…………………………………………………..…………207
Appendix C: Gel-Permeation Chromatography Spectra………………………..……….210
Appendix D: Matrix-Assisted Laser Desorptive Ionization Mass Spectrometry Peak
Lists……………………………………………………………………………………………….213
Appendix E: High Resolution X-ray Photoelectron Spectroscopy Curve Fits….......274
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List of Abbreviations
ACE Affinity capillary electrophoresis
AlB Allylbenzene
AOU Amphiphilic oligo-urethane
ATRP Atom transfer radical polymerization (ATRP
bFGF Basic fibroblast growth factor
BHAc (2aR,4S,4aS,6R,9S,11S,12S,12bS)-9-(((2R,3S)-3-benzamido-2-
hydroxy-3-phenylpropanoyl)oxy)-12-(benzoyloxy)-4,11-dihydroxy-
4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-
dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxete-
6,12b-diyl diacetate
BIBB α-bromoisobutyryl bromide
BMS Bare metal stent
BPY 2,2’-Dipyridyl
BR Binary restenosis
CA Citric acid
CE Capillary electrophoresis
DES Drug-eluting stent
DCB Drug-coated balloon
DBA Dibutylamine
DBTDL Dibutyltin dilaurate
DMAc Dimethylacetamide
DMEM Dulbecco’s Modified Eagle Medium
DMAP 4-(Dimethylamino)pyridine
DNA Deoxyribonucleic acid
DSC Differential scanning calorimetry
EDC 1-Ethyl-3-(3-dimethylamino-propyl)carbodiimide·HCl
EDTA Ethylenediaminetetraacetic acid
EOF Electro-osmotic flow
ERK Extracellular signal related kinase
ESI Electrospray ionization
19F Fluorine-19
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GPC Gel-permeation chromatography
1H Proton
HCl Hydrochloric acid
HD Hummel-Dreyer
HDI Hexamethylene diisocyanate
HPLC High-performance liquid chromatography
GP Glycoprotein
ICP-AES Inductively coupled plasma atomic emission spectroscopy
IPA Isopropylalcohol
IL Interleukin
Kb Binding constant
KD Dissociation constant
LDI Lysine diisocyanate
LLL Late lumen loss
L-PEG Linear poly(ethylene glycol)
MACE Major adverse cardiac event
MALDI Matrix-assisted laser desorption/ionization
MCP-1 Monocyte chemoattractant protein-1
MEM Minimal essential media
Mn Number average molecular weight
Mw Weight average molecular weight
MS Mass spectrometry
NaOH Sodium hydroxide
NECEEM Non-equilibrium capillary electrophoresis of equilibrium mixtures
NH Neointimal hyperplasia
NMR Nuclear magnetic resonance
NL-PEG Non-linear PEG
nRIU Nano refractive index units
ns No significance
PAB Polyallylbenzene
PACCOCATH Treatment of In-Stent Restenosis by Paclitaxel-Coated Balloon
Catheters
PBS Phosphate buffered saline
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PCI Percutaneous Coronary Intervention
PDGF Platelet-derived growth factor
PEG Poly(ethylene glycol)
PEPCAD Paclitaxel-Eluting PTCA-Balloon Catheter in Coronary Artery Disease
PFA α-Fluoro-ω-(2-hydroxyethyl)poly(difluoromethylene)
PMDETA N,N,N′,N′,N′′-pentamethyldiethylenetriamine
PTMO Polytetramethylene oxide
PVP Polyvinylpyrrolidone
RPEG Poly(ethylene glycol) methyl ether
SEM Scanning electron microscopy
SMM Surface-modifying macromolecule
SPE Solid phase extraction
TCB 1,2,4-Trichlorobenzene
TGF-β Transforming growth factor-β
TLR Target lesion revascularization
TMS Trimethylsilane
Tris 2-Amino-2-hydroxymethyl-propane-1,3-diol
UV-Vis Ultraviolet-visible
VP Vinyl pyrrolidone
VSMC Vascular smooth muscle cell
vWF von Willebrand factor
WST Water soluble tetrazolium
XPS X-ray photoelectron microscopy
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List of Tables
Table 1.1 Proposed NL-PEG AOU analogues and the rationale behind their design……...8
Table 2.1 Examples of therapeutic agents used in DCBs and their respective modes of
action………………………………………………………………………………………………..16
Table 2.2 Examples of DCB carrier materials evaluated at pre-clinical stages and
associated with various pharmaceutical agents……………………….……………………....18
Table 2.3 Commercially developed DCB coating formulations…………………..………….22
Table 2.4 Clinical outcomes of commercially available DCBs with relevant p values….....23
Table 3.1 Preparation of materials prior to use ………………………..……………………...44
Table 3.2 Concentrations of analogues in the sample solutions tested for tin and copper
using ICP-AES……………………………………………………………………………………..61
Table 4.1 Summary of the results of characterization for the 1:1 drop-wise reaction
outlined in Scheme 4.1 and of the resulting purified oligomer……………………….……...73
Table 4.2 1H-NMR peak chemical shifts and integrations associated with the spectra of the
1:1, 1:1.5 and 1:2 stream-wise syntheses purified products shown in Figure 4.6…….…...82
Table 4.3 The apparent Mw, Mn and polydispersities (Mw/Mn) associated with the GPC
spectra, illustrated in Figure 4.9, of the quenched pre-polymer aliquots and purified
materials attained from the NL-PEG:LDI:PFA syntheses outlined in Scheme 4.4…….......84
Table 4.4 NL-PEG stream-wise reaction product MALDI-MS peak cluster assignments...88
Table 4.5 Characterization of the extent of cyclization during the NL-PEG stream-wise
reactions outlined in Scheme 4.4………………………………………………………………..90
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Table 4.6 The apparent Mw, Mn and polydispersity (Mw/Mn) associated with the peak
labelled with an asterisk in the GPC spectra, illustrated in Figure 4.13, of the quenched pre-
polymer aliquots and purified materials attained from the L-PEG:LDI:PFA syntheses
outlined in Scheme 4.5…………………………………………………………………….……...93
Table 4.7 Assignment of peak clusters in the MALDI-MS spectra, presented in Figure 4.18,
of the products of the drop-wise and stream-wise L-PEG:LDI:PFA syntheses conducted
according to Scheme 4.5………………………………………………………………………....98
Table 5.1 Summary of the characterization of the equimolar drop-wise synthesis………104
Table 5.2 Tabulation of the pre-polymer molecular weights corresponding to the GPC
spectra presented in Figure 5.4 attained for methanol-quenched aliquots collected from the
excess LDI drop-wise RPEG:LDI pre-polymer reactions outlined in Scheme 5.2……..…107
Table 5.3 Characterization of pre-polymers and fluoro-oligomers attained via Scheme
5.3……………………………………………………………………………………………........109
Table 5.4 Tin and copper contents within analogues 1, 2 and 3 as measured by ICP-
AES………………………………………………………………………………………………..133
Table 5.5 Analogue bulk water solubilities and air-water contact angles associated with
analogue films…………………………………………………………………………………….135
Table 5.6 Low resolution XPS analysis of the atomic composition at the surface of
analogue films using take-off angles of 20o, 40
o and 60
0……………………………………138
Table 5.7 High resolution XPS analysis of analogue film surfaces using take-off angles of
20o, 40
o and 60
0………………………………………………………………………………….140
Table 5.8 Temperatures associated with thermal degradation and thermal transitions within
analogues 1, 2 and 3………………………………………………………………………........143
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Table 6.1 Aqueous components of the run buffers used to generate experimental
conditions to monitor differences in migration times between BHAc and AOUs..………...154
Table A.1 List of materials and reagents and their associated supplier and catalogue
number…………………………………………………………………………………………….203
Table D.1 Peak list of the MALDI-MS spectrum of non-linear poly(ethylene glycol) starting
material and associated peak identification ………………………………………………….213
Table D.2 MALDI-MS peak list associated with the product attained from the drop-wise
addition of LDI to NL-PEG as outlined in Scheme 4.1..………………………….……….....218
Table D.3 MALDI-MS peak list associated with the product attained from the stream-wise
addition of LDI to NL-PEG as outlined in Scheme 4.4 with a NL-PEG:LDI feed ratio of 1:1.
……………………………………………………………………………………………………..221
Table D.4 MALDI-MS peak list associated with the product attained from the stream-wise
addition of LDI to NL-PEG as outlined in Scheme 4.4 with a NL-PEG:LDI feed ratio of
1:1.5.……………………………………………………………………………………………....225
Table D.5 MALDI-MS peak list associated with the product attained from the stream-wise
addition of LDI to NL-PEG as outlined in Scheme 4.4 with a NL-PEG:LDI feed ratio of 1:2.
………………………………………………………………………………………………...…...228
Table D.6 Summary of possible identifications for peak clusters (as presented in Tables
D.3, D.4 and D.5) present in the MALDI-MS spectra of the stream-wise 1:1, 1:1.5 and 1:2
NL-PEG reactions outlined in Scheme 4.4……………………………………………….......232
Table D.7 Peak list of the MALDI-MS spectrum of linear poly(ethylene glycol) starting
material and the associated peak identifications.……………………….……………………235
Table D.8 MALDI-MS peak list associated with the product attained from the drop-wise L-
PEG:LDI:PFA synthesis outlined in Scheme 4.5 after dialysis…………………………..…240
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Table D.9 MALDI-MS peak list associated with the product attained from the stream-wise
L-PEG:LDI:PFA synthesis outlined in Scheme 4.5 after dialysis.….……………………….244
Table D.10 Summary of possible identifications for peak clusters (as presented in Tables
D.8 and D.9) present in the MALDI-MS spectra of the drop-wise and stream-wise L-
PEG:LDI:PFA syntheses outlined in Scheme 4.5…………..………………………………..248
Table D.11 Peak list of the MALDI-MS spectrum of RPEG starting material and associated
peak identities….…………….............................................................................................249
Table D.12 MALDI-MS peak list associated with analogue 1 and possible identifications for
all m/z values……………………………………………………………………………………..253
Table D.13 MALDI-MS peak list associated with analogue 2 and the associated possible
identifications for each m/z value………………………………………………………………259
List of Schemes
Scheme 2.1 Building blocks and synthesis of polyurethanes………………………………..28
Scheme 2.2 Overview of a general step-growth polymerization reaction...………………...33
Scheme 2.3 Resonance forms of isocayante groups and a simplified depiction of the
nucleophilic attack of an alcohol group at an isocyanate carbon centre yielding a urethane
bond………………………………………………………………………………………………...33
Scheme 2.4 Undesired side reactions during polyurethane synthesis which yield ureas,
biurets, alophanates and dimerized products…………………………………………………..34
Scheme 2.5 Cyclization during polyurethane pre-polymer syntheses via the conjugation of
terminal isocyanate and alchohol groups, and the conjugation of a terminal isocyanate and
main chain secondary amine………………………………………………………………….....35
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Scheme 2.6 Schematic of an ATRP reaction……………………………………………….....36
Scheme 3.1 The reaction of polyol with excess acetic anhydride, which was followed by
quenching of the reaction with water to generate acetic acid that was then titrated in order
to determine polyol molecular weight………………………………………………………..….45
Scheme 3.2 Reaction of the pre-polymer residual isocyanates with excess DBA………...46
Scheme 4.1 Synthetic scheme outlining the drop-wise addition of LDI to NL-PEG in a
stoichiometric ratio of 1:1………………………………………………………………………....71
Scheme 4.2 Synthesis of NL-PEG as outlined by Fock and Möhring and Liu et al…….....75
Scheme 4.3 Illustration of side reactions generating monohydroxylated polyols through the
alkoxide-initiated proton abstraction and subsequent ring-opening polymerization of
ethylene oxide or the cyclic ketal (i)……………………………………………………….…….76
Scheme 4.4 NL-PEG:LDI:PFA stream-wise syntheses conducted with varying NL-PEG:LDI
molar reaction feed ratios ……………………………………………………………………......79
Scheme 4.5 Synthetic scheme depicting AOU syntheses using L-PEG and LDI in a 1:1
molar ratio…………………………………………………………………………………………..91
Scheme 5.1 The equimolar drop-wise approach towards analogue 1 synthesis………..103
Scheme 5.2 RPEG:LDI pre-polymer syntheses conducted with a two-fold excess of LDI
and the drop-wise addition of RPEG to LDI over the course of one hour ….…………….106
Scheme 5.3 Optimization of the synthesis of analogue 1 using a two-fold excess of LDI
and the drop-wise addition of RPEG over one hour via three conditions....………….......108
Scheme 5.4 The hydrolysis of the ester group in analogue 1 to generate the carboxylic
acid 1’…………………………………………………………………………………………......114
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Scheme 5.5 Illustration of the EDC-DMAP approach, which was attempted for the
synthesis of analogue 2 from the carboxylic acid 1’………………………………………….117
Scheme 5.6 The synthesis of analogue 2 via the direct coupling of Tris to analogue 1..118
Scheme 5.7 Synthetic scheme illustrating the conjugation of BIBB to analogue 2……...124
Scheme 5.8 The ATRP reaction conducted to generate analogue 3 through the grafting of
VP to the macroinitiator 2’……………………………………………………………………....125
Scheme 5.9 Example of radical disproportionation during the synthesis of analogue 3,
which would yield non-“living” products with terminated VP chains……………………......128
Scheme 5.10 The ATRP reaction conducted to generate analogue 4 through the grafting of
AlB to the macroinitiator 2’……………………………………………………………………...129
Scheme 5.11 Examples of mechanisms by which radicals generated from 2’ could
undergo termination…………………………………………………………………………......131
List of Figures
Figure 1.1 Proposed AOU model post synthesis and self-assembly as a film……………...5
Figure 2.1 Summary of the cascade of events which are initiated by vascular injury from
PCI balloon inflation and stent implantation, and which lead to restenosis of the treated
blood vessel……………………………………………………………………………………......13
Figure 2.2 Structure of paclitaxel with labelled carbon atoms……………………………….30
Figure 2.3 The components of a typical CE system……………………………………………...41
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Figure 2.4 Example of a typical electropherogram obtained from a NECEEM
experiment................................................................................................................... ........42
Figure 4.1 Illustration of NL-PEG starting material and a 1:1 NL-PEG:LDI cyclized product
that would result from intramolecular cyclization during NL-PEG:LDI pre-polymer
reactions.................................................................................................................... ..........70
Figure 4.2 1H-NMR spectrum (in deuterated chloroform) of the purified product obtained
from the drop-wise addition of LDI to NL-PEG in a stoichiometric feed ratio of 1:1, followed
by PFA end-capping of non-cyclized species as per Scheme 4.1…………………………...72
Figure 4.3 19
F-NMR spectrum (in deuterated chloroform) of the purified material obtained
from the drop-wise addition of LDI to NL-PEG in a stoichiometric feed ratio of 1:1, followed
by PFA end-capping of non-cyclized species as per Scheme 4.1…..……………………....72
Figure 4.4 GPC spectra of the pre-polymer (pre-PFA addition) and purified product (post-
PFA addition) attained through the drop-wise addition of LDI to NL-PEG in a 1:1
stoichiometric feed ratio as outlined by Scheme 4.1…………………………………………..73
Figure 4.5 MALDI-MS spectra of NL-PEG starting material and the purified 1:1 drop-wise
product synthesized as per Scheme 4.1.…….....................................................................78
Figure 4.6 1H-NMR (in deuterated chloroform) spectra of the purified products attained
from the 1:1, 1:1.5 and 1:2 stream-wise syntheses……………………………………....…...81
Figure 4.7 19
F-NMR (in deuterated chloroform) of PFA starting material and purified
stream-wise reaction products…………………..…………………………………………….....82
Figure 4.8 Pre-polymer isocyanate conversions prior to PFA addition and purified product
fluorine content for the stream-wise syntheses outlined in Scheme 4.4…………………….83
Figure 4.9 GPC spectra of the pre-polymers and purified materials obtained through the
stream-wise addition of LDI to NL-PEG as outlined in Scheme 4.4…………………..….….85
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Figure 4.10 MALDI-MS spectra of the purified reaction products associated with the
stream-wise addition of LDI to NL-PEG as outlined in Scheme 4.4……………………...….86
Figure 4.11 Peak clusters within m/z ranges of 1100-1225, 2325-2400 and 3300-3500
observed in the MALDI-MS spectra of purified products attained from the 1:1, 1:1.5 and 1:2
NL-PEG stream-wise reactions…………………………………………………………………..87
Figure 4.12 Pre-polymer isocyanate conversion directly prior to the addition of PFA to the
L-PEG:LDI pre-polymers, and fluorine contents of the purified materials synthesized
according to Scheme 4.5 via drop-wise and stream-wise addition approaches…………...92
Figure 4.13 GPC spectra of the quenched pre-polymer aliquots and purified materials
attained from the L-PEG:LDI:PFA syntheses outlined in Scheme 4.5………………….…...93
Figure 4.14 GPC spectra of the purified products attained from the L-PEG:LDI:PFA
reactions outlined in Scheme 4.5 post dialysis…………………………………………………94
Figure 4.15 1H-NMR spectra of the purified products post dialysis attained from the drop-
wise and stream-wise L-PEG:LDI:PFA reactions outlined in Scheme 4.5…………….…....95
Figure 4.16 19
F-NMR spectra of the purified products attained from the drop-wise and
stream-wise L-PEG:LDI:PFA reactions outlined in Scheme 4.5 after dialysis……………..96
Figure 4.17 MALDI-MS spectra of the purified reaction products associated with the drop-
wise and stream-wise L-PEG:LDI:PFA syntheses outlined in Scheme 4.5. ………….…....96
Figure 4.18 MALDI-MS spectra of the dialyzed L-PEG:LDI:PFA drop-wise and stream-wise
reaction products expanded between m/z values of 1200-1300, 2200-3000 and 3400-
3500………………………………………………………………………………….....................97
Figure 5.1 Illustration of analogue 1 …………………………...……………………………..102
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Figure 5.2 19
F-NMR and 1H-NMR spectra (in deuterated chloroform) of the purified fluoro-
oligomer attained via the equimolar drop-wise synthesis outlined in Scheme 5.1………105
Figure 5.3 GPC spectra of the pre-polymer and purified oligomer associated with the
equimolar drop-wise synthesis outlined in in Scheme 5.1…………………………………..105
Figure 5.4 GPC spectra of pre-polymer aliquots collected from the reaction of LDI and
RPEG as described in Scheme 5.2………….………………………………………………..106
Figure 5.5 Isocyanate consumption during the RPEG:LDI pre-polymer reactions conducted
as per Scheme 5.2……………………………………………..………………………………..107
Figure 5.6 Comparison of the GPC spectra of pre-polymer aliquots and purified fluoro-
oligomers attained via the conditions specified in Scheme 5.3……………..….................110
Figure 5.7 1H-NMR spectra of the purified fluoro-oligomers obtained from the excess LDI
drop-wise syntheses outlined in Scheme 5.3………………………………………………...110
Figure 5.8 GPC spectra of the pre-polymers and fluoro-oligomers obtained via the
repeated synthesis of analogue 1……………………………………………………………...112
Figure 5.9 Pre-polymer isocyanate conversion directly prior to PFA addition as outlined in
Scheme 5.3c and the fluorine content of the resulting purified fluoro-oligomers………..112
Figure 5.10 MALDI-MS spectrum of the purified fluoro-oligomer attained via the optimized
excess LDI drop-wise condition outlined in Scheme 5.3c…………………………………..113
Figure 5.11 Illustration of analogue 2………………………………………………………....114
Figure 5.12 1H-NMR spectra (in deuterated chloroform) of the products of the hydrolysis of
the analogue 1 ester group using hydrolysis times of 6, 9 and 25 hours…………………115
Figure 5.13 GPC spectrum of the hydrolysis product 1’……………………………………116
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xxi
Figure 5.14 1H-NMR spectra (in deuterated methanol) of the aliquots obtained from the
reaction mixture of 1’ and Tris as outlined by Scheme 5.5……………………………….....117
Figure 5.15 1H-NMR spectra (in deuterated methanol) of reaction mixture aliquots and the
final product associated with the direct coupling of Tris to analogue 1 as illustrated in
Scheme 5.6……………………………………………………………………………………….119
Figure 5.16 19
F-NMR spectrum (in deuterated chloroform) of analogue 2………………..121
Figure 5.17 Comparison of the GPC spectra of analogues 1 and 2……………………....122
Figure 5.18 MALDI-MS spectrum of analogue 2………………………………………….....122
Figure 5.19 The chemical structures of analogues 3 and 4……………………………..….123
Figure 5.20 1H-NMR and
19F-NMR spectra of the macroinitiator 2’ in deuterated
methanol………………………………………………………………………………………..…124
Figure 5.21 GPC spectrum of the macroinitiator 2’………………………………………….125
Figure 5.22 1H-NMR and
19F-NMR spectra (in deuterated methanol) of analogue 3, which
was synthesized according to Scheme 5.8…………………………………………………...126
Figure 5.23 GPC spectrum of analogue 3 and its comparison to the GPC spectrum of the
macroinitator 2’…………………………………………………………………………………..128
Figure 5.24 1H-NMR spectrum (in deuterated methanol) of the reaction product attained
from the ATRP reaction attempted towards the synthesis of analogue 4 as outlined in
Scheme 5.10……………………………………………………………………………………...131
Figure 5.25 Overlay of the GPC spectra of 2’ and the purified reaction product of the ATRP
reaction, outlined in Scheme 5.10, attempted towards the synthesis of analogue 4….....132
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xxii
Figure 5.26 SEM images of films, cast onto nylon coupons, composed of analogues 1, 2
and 3, 1:2:2 NL-PEG:LDI:PFA reaction product and AOU-2 ….………………….....……..134
Figure 5.27 Illustration of contact angles measured for liquid-air-solid interfaces…........136
Figure 5.28 TGA plots and DSC thermograms of RPEG, analogue 1, analogue 2 and
analogue 3 obtained from the second heat runs.…………………………………………….145
Figure 5.29 Cyotoxicity of analogues 1, 2 and 3 on A-10 VSMCs at doses of 0.1, 1 and 10
mg/mL in growth medium as assessed by Hoescht DNA and WST-1 assays……..…….148
Figure 6.1 Electropherograms of BHAc, AOU-1 and AOU-2 using a variety of run buffer
systems…………………………………………………………………………………………...156
Figure 6.2 Electropherograms obtained from the AOU-1/BHAc NECEEM
experiments……………………………………………………………………………………....158
Figure 6.3 Electropherograms obtained from the AOU-2/BHAc NECEEM
experiments……………………………………………………………………………………….159
Figure B.1 Inductively coupled plasma atomic emission spectroscopy calibration curves for
the tin and copper standards…………………………………………………………………...207
Figure B.2 Water soluble tetrazolium assay cell seeding and incubation duration
calibration…………………………………………………………………………………………208
Figure B.3 Intensity of fluorescence associated with varying masses of DNA
standards...........................................................................................................................209
Figure C.1 GPC spectra of the pre-polymer and purified oligomer obtained through the
drop-wise addition of LDI to NL-PEG in a feed ratio of 1:1 as outlined in Scheme 4.1….210
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xxiii
Figure C.2 GPC spectra of the pre-polymer and purified oligomer obtained through the
stream-wise addition of LDI to NL-PEG in a feed ratio of 1:1 as outlined in Scheme
4.4………………………………………………………………………………………………….210
Figure C.3 GPC spectra of the pre-polymer and purified oligomer obtained through the
stream-wise addition of LDI to NL-PEG in a feed ratio of 1:1.5 as outlined in Scheme
4.4………………………………………………………………………………………………….211
Figure C.4 GPC spectra of the pre-polymer and purified oligomer obtained through the
stream-wise addition of LDI to NL-PEG in a feed ratio of 1:2 as outlined in Scheme
4.4………………………………………………………………………………………………....211
Figure C.5 GPC spectra of the pre-polymer and purified oligomer obtained through the
drop-wise addition of LDI to L-PEG in a feed ratio of 1:1 as outlined in Scheme 4.5…...212
Figure C.6 GPC spectra of the pre-polymer and purified oligomer obtained through the
stream-wise addition of LDI to L-PEG in a feed ratio of 1:1 as outlined in Scheme 4.5...212
Figure D.1 Example of high resolution XPS curve fitting conducted for spectra attained at
take-off angles of 60o, 40
o and 20
o for analogue 1 films…………………………………….274
Figure D.2 Example of high resolution XPS curve fitting conducted for spectra attained at
take-off angles of 60o, 40
o and 20
o for analogue 2 films…………………………………….275
Figure D.3 Example of high resolution XPS curve fitting conducted for spectra attained at
take-off angles of 60o, 40
o and 20
o for analogue 2 films………………………………….....276
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Chapter 1:
Introduction
1.1 Percutaneous coronary interventions and restenosis
Cardiovascular disease, the root of one third of all Canadian mortalities, often stems from
atherosclerosis, in which the accumulation of cholesterol and fatty deposits in arteries
leads to arterial hardening and the formation of plaques [1, 2]. The restoration of blood flow
to such vessels, termed as revascularization, can be conducted via percutaneous coronary
interventions (PCIs). This minimally invasive procedure, conducted on 622, 000 patients in
2007 within the United States alone [3], involves the mechanical opening of an occluded
blood vessel via a balloon, which is guided to the occlusion site by a guide wire within a
catheter system that is introduced percutaneously to the body [4]. PCIs, as minimally
invasive alternatives to surgery, are used more prominently than traditional invasive
treatment options such as coronary artery bypass surgery in Canada [5] and have
undergone much development since their inception.
The first PCIs, conducted in 1977 by Andreas Grüntzig, generated great enthusiasm in the
medical community [4, 6]. However, long-term angiographic follow up of treated patients
yielded unfavourable results, as restenosis, the re-closure of treated vessels, was found to
occur in 30 to 60% of treated vessels [7, 8]. Such restenosis, in addition to the elastic recoil
demonstrated by treated vessel walls due to balloon inflation, led to the use of stenting, in
which a wire mesh tube is expanded via a catheter balloon into the diseased artery to
prevent lumen closure [6, 9]. Though the use of such bare metal stents (BMSs) addressed
the issue of elastic recoil incurred by balloon inflation in PCI-treated arteries, restenosis
continued to occur in up to 32% of stented vessels [10]. Despite advances in stent
technologies, restenosis continues to plague patients receiving BMS PCIs, who ultimately
require repeat interventions that place an additional $3,000 per patient burden on the
health care system [11].
BMS-PCI-associated restenosis was demonstrated to be a result of neointimal hyperplasia
from vascular smooth muscle cell (VSMC) proliferation and migration [12, 13]. Balloon
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2
expansion and stent implantation cause endothelial denudation and vascular injury [14,
15]. Such injury leads to early VSMC proliferation as a result of platelet activation and
adhesion [16]. Mitogenic agents and growth factors are secreted and activated during this
inflammatory response, leading to medial VSMC proliferation and migration into the intima,
thus yielding a stenosed vessel [14, 15]. A detailed review of the mechanisms governing
neointimal hyperplasia is provided in Section 2.1. This development of restenosis,
attributed to BMS implantation, is clinically detrimental, leading to myocardial infarctions or
unstable anginas in over 35% of in-stent restenosis cases [17].
To circumvent the occurrence of such neointimal hyperplasia, the systemic administration
of anti-proliferative agents was attempted. However, as reviewed by Rajagopal et al., the
administration of a variety of pharmaceutical agents, such as heparin, angiotensin
converting enzyme inhibitors and cilostazol, yielded no attenuation in restenosis incidence,
perhaps as a result of delivering therapeutically insufficient concentrations of the
pharmaceutical agents to the target tissues [6, 18]. The need for localized drug delivery
was first addressed by the utilization of drug-eluting stents (DESs), in which anti-
proliferative agents such as sirolimus (CYPHER stent) and paclitaxel (Taxus Liberté stent)
are embedded and released from a polymer matrix located on stents, directly to diseased
tissues in a controlled manner [19-21] in order to inhibit VSMC proliferation and
subsequent migration into the intima. Initial short-term clinical trials demonstrated the
efficacy of both paclitaxel and sirolimus-eluting DESs in preventing restenosis over BMSs,
promoting their wide use during PCIs in the early part of the last decade [20-22].
Though DESs became the gold-standard PCI approach, the technology was associated
with higher stent thrombosis risk over BMS technologies, leading also to higher mortality
and myocardial infarction rates [23-25]. The prolonged presence of pro-thrombotic polymer
materials and, primarily, the incomplete re-endothelialization of treated tissues, promoted
by the extended use of anti-proliferative agents, are implicated in the development of such
DES-related thrombi [24, 26-28]. Furthermore, a study directly comparing DES and BMS
clinical outcomes contested the long term efficacy of DESs over BMSs at preventing
restenosis and repeat revascularization [25]. It was found that late stent thrombosis
associated with DESs resulted in increased mortality rates, an increase of myocardial
infarctions by 38% over BMSs, and required repeat revascularization procedures in 28% of
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3
the target lesions [25]. These unfavourable results warranted the need for a transient PCI
technique able to address DES-associated thrombosis and prevent PCI-associated
neointimal hyperplasia.
In order to address the limitations of DES technologies, the use of drug-coated balloons
(DCBs) is under review. The use of DCBs to address in-stent restenosis is presented as
advantageous over options such as the systemic administration of antiplatelet therapies
(which leads to a high risk of bleeding) and stent-in-stent interventions, 20% of which lead
to restenosis [9, 26, 29]. Alternatively, the use of DCBs allows for the targeted and
homogeneous delivery of anti-proliferative agents to address in-stent restenosis (DESs are
incapable of homogeneous drug transfer as drug concentrations are higher at the stent
struts than at the margin). As a transient technique, it ultimately minimizes material-related
thrombotic events resulting from residual polymeric drug carrier matrices and the extended
delivery of anti-proliferative agents [9, 30]. Furthermore, the transient nature of DCBs
overcomes the limitation of delayed re-endothelialization of injured arteries associated with
stent technologies. The versatility of DCBs is advantageous as the technology can be used
in combination with BMSs, occluded DES-stented vessels and solitarily for the treatment of
bifurcation lesions, below the knee interventions, and small and tortuous vessel occlusions
in which stent fracture and ineffective stenting are concerns [9, 30, 31].
The efficacy of DCBs in preventing post-PCI restenosis is contingent upon the use of a
drug carrier that appropriately delivers anti-proliferative agents to target vascular tissue.
Such a carrier must retain drug during catheter transit to the occluded vessel, with
minimum systemic drug loss, and subsequently rapidly release drug to the targeted tissue
upon balloon inflation. While direct coating strategies of the anti-proliferative agents
paclitaxel and sirolimus onto angioplasty balloons largely prevented premature drug loss
during catheter transit, such a coating strategy also yielded low drug transfer to tissue upon
balloon inflation [32]. Contrarily, the more prominently explored hydrophilic carriers, from
which embedded drug is released, yielded higher drug transfer to tissue upon balloon
deployment than direct coating strategies, but also resulted in higher drug loss during
transit [32]. Such premature drug loss associated with current DCB technologies yields
inefficient drug transfer, where some carrier formulations are associated with up to 90%
drug loss pre-DCB deployment with merely 6% of the initial dose reaching the targeted
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4
tissues [30]. Such limitations must be addressed through the development of non-
thrombogenic, bioresorbable DCB drug carrier systems, specifically designed to both
prevent premature drug loss during catheter transit, and ensure efficient and targeted
delivery of drug to occlusion sites to prevent PCI-induced restenosis. Given the prevalent
use of PCIs for the treatment of atherosclerosis in North America, and the need for an
effective PCI approach to prevent restenosis and repeat revascularization, such a carrier
could have great potential impact in the field of interventional cardiology.
1.2 Amphiphilic oligo-urethanes
In order to address the limitations of inefficient drug transfer to target tissue associated with
current DCB technologies, an amphiphilic oligo-urethane (AOU) model has been conceived
for application as DCB drug carrier molecules. This model is comprised of a bioresorbable
oligo-urethane generated from the addition of a polyol core, diisocyanate and a fluoro-
alcohol, as shown in Figure 1.1. The polyol core is proposed to establish drug release
kinetics, the diisocyanate components to bind drug during transit, and the fluorinated
segments to shield the drug from premature systemic release and to enhance the blood-
compatibility of the material. Such shielding potential is based on studies conducted in the
Santerre group, in which terminal fluorocarbon groups, added as surface modifying
macromolecules (SMMs) to polyurethane materials, were demonstrated by X-ray
photoelectron microscopy (XPS) to migrate to the surface of such materials when cast as
films [33-36]. As such, the surface of these films were hydrophobic in nature and were
found to yield water-contact angles similar to, and in several instances exceeding, those of
Teflon® [33-36]. AOUs, which are composed of similar chemical components, are thus
proposed to self-assemble to yield a fluorinated DCB surface which shields against drug
dissolution during catheter tracking. Furthermore, SMMs were shown to significantly
decrease platelet and fibrinogen activation, which are indicators of thrombosis, relative to
bare polyurethane materials, reduce polyurethane hydrolytic degradation, and yield
fibrinogen adsorption in a manner which minimized protein denaturation leading to blood
coagulation [37-41]. Thus, the inclusion of such fluorinated segments in the AOU model
provides the potential to generate a drug carrier that is non-thrombogenic, and able to
shield the carrier and drug from dissolution and environmental breakdown by blood
components during catheter transit.
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5
Figure 1.1 Proposed AOU model post synthesis and self-assembly as a film. This model
comprises of a polyol, diisocyanate, and fluoro-alcohol reacted to generate the oligo-
urethane, which then self-assembles to yield fluorine groups migrated to the film surface.
Drug is proposed to bind to the diisocyanate domains of the model.
Initial hydrophobic anti-proliferative drug release studies conducted on a variety of AOU
formulations have identified two oligomers of interest: the hydrophilic AOU-1 and
hydrophobic AOU-2. AOUs hold the potential to shield drug during catheter transit via the
fluorinated surface, which, when disrupted upon balloon inflation, could allow for carrier
hydration and rapid release of a hydrophobic drug from the hydrophilic carrier. When
synthesized in a reproducible manner, which successfully incorporates all components of
the model, such AOUs may address limitations incurred by current DCB technologies.
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6
1.3 Research objectives and hypotheses
1.3.1 Central research objective
This project aims to exploit the blood-compatibility and surface migratory properties of
fluorinated polyurethane-SMM systems to develop both a novel class of fluorinated
polyurethane materials in a unique AOU model that could, in future studies, be examined
for their release of anti-proliferative drugs, and to establish a method to evaluate drug
release from films composed of such AOUs. Such AOUs were designed to serve as DCB
drug carriers. They are comprised of a poly(ethylene glycol) (PEG) core to enhance drug
release kinetics, lysine diisocyanate (LDI) to establish chemical functionalities for increased
drug binding and loading, and the perfluoro-alcohol α-fluoro-ω-(2-
hydroxyethyl)poly(difluoromethylene) (PFA) to enhance the blood-compatibility of the
coating and to shield against premature drug release during catheter transit.
1.3.2 Central hypothesis
It is hypothesized that the incorporation of a water-soluble core, hydrogen-bonding
moieties and fluorinated segments in an AOU model will yield a potential drug delivery
platform in formulations which are synthetically feasible and reproducible, water soluble
and yield high threshold concentration values in cytotoxicity.
1.3.3 Objective 1 (Chapter 4)
To study the step-growth polymerization reaction used to generate non-linear (NL)
PEG:LDI:PFA AOU systems, with the aim of characterizing the extent to which side
reactions, such as pre-polymer cyclization, occur within NL-PEG AOU syntheses.
Rationale
AOU syntheses using α-[2,2-bis(hydroxymethyl)butyl]-ω-methoxy poly(oxy-1,2-ethanediyl)
(NL-PEG) may be prone to intramolecular cyclization due to the proximity of the hydroxyl
functionalities of NL-PEG. Cyclization impedes PFA conjugation to NL-PEG:LDI pre-
polymer systems due to the consumption of isocyanate groups via intramolecular
conjugation of reactive end groups. Cyclization reactions would thus yield oligomers with
low fluorine contents. As the fluorinated segments of the AOU model are proposed to play
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7
a key role in the prevention of drug loss during DCB catheter tracking and in enhancing
AOU blood-compatibility, the study of such cyclization reactions is vital towards the
successful synthesis of oligomers consistent with the AOU model.
Sub-Hypothesis
Cyclization will manifest itself in NL-PEG:LDI:PFA AOU syntheses conducted with varying
stoichiometric ratios of NL-PEG and LDI. It is hypothesized that the use of NL-PEG, in
which the di-hydroxyl functionalities are in close proximity at one end of the PEG chain, will
promote intramolecular cyclization in the AOU syntheses to a greater extent than when
linear (L)-PEG of an equivalent molecular weight, bearing di-hydroxyl functionalities
separated by the PEG chain, is used as the polyol core in AOU syntheses.
Approach
AOU syntheses were conducted with varying polyol:LDI stoichiometric ratios.
The kinetics of the reactions were monitored via isocyanate titrations of reaction
mixture aliquots.
Gel-permeation chromatography (GPC) and matrix-assisted laser desorptive
ionization mass spectrometry (MALDI-MS) were used to assess oligomer molecular
weights and the presence and extent of cyclization reactions within AOUs
syntheses.
Elemental analysis of purified oligomers was used to quantitate fluorine content
associated with PFA conjugation.
Proton (1H) and fluorine-19 (
19F) nuclear magnetic resonance (NMR) spectroscopy
was conducted towards oligomer chemical structure validation.
1.3.4 Objective 2 (Chapter 5)
To synthesize, purify and characterize (chemically, physically and biologically) novel fluoro-
oligomer drug carriers as alternatives to NL-PEG:LDI:PFA AOUs, using mono-functional
RPEG as the AOU polyol core, LDI as the diisocayante and PFA as the fluoro-alcohol. The
four proposed analogues and specific corresponding design rationales are provided in
Table 1.1.
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8
Rationale
The use of a mono-functional polyol segment prevents the occurrence of intramolecular
cyclization during urethane bond formation, which is a challenge encountered during AOU
syntheses employing polyols bearing di-hydroxyl functionalities. The RPEG-containing
oligomers still remain consistent with the AOU model as they incorporate polyol,
diisocyanate and fluorocarbon segments. As indicated in Table 1.1, these analogues
contain moieties able to engage in non-covalent interactions with anti-proliferative drugs,
potentially allowing for good drug loading and binding during catheter transit.
Table 1.1 Proposed NL-PEG AOU analogues and the rationale behind their design.
Analogue Structure Rationale
1
Use of RPEG prevents extended step-
growth polymerization and, thus too, any
formation of cyclized, non-fluorinated
oligo-urethane species within the AOU
reaction mixture.
Use of LDI as the diisocyanate is
rationalized as follows:
a) lysine would be generated as a non-
toxic degradation product
b) the ester group may engage in
hydrogen bonding with drug and can
also serve as a point for further
chemical functionalization.
2
The conjugation of 1 to 2-amino-2-
hydroxymethyl-propane-1,3-diol (Tris)
increases the number of moieties
available to engage in hydrogen bonding
to drug and water in order to
respectively increase drug binding and
AOU hydrophilicity.
Tris incorporation introduces a site,
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9
specifically the triol group, for covalent
conjugation of additional drug-binding
moieties.
3
Grafting of poly(vinyl pyrrolidone) (PVP)
units to 2 increases the availability of
hydrogen bond acceptors for enhanced
drug binding and AOU hydrophilicity.
PVP grafting has been found to increase
the blood-compatibility of blood-
contacting polymeric systems [42, 43].
The number of vinyl pyrrolidone unit
additions may be controlled via atom
transfer radical polymerization (ATRP)
chemistry to tailor drug loading and
release from AOU films.
4
Grafting of poly(allylbenzene) units to 2
introduces aromatic moieties which can
engage in pi-pi stacking interactions with
drugs to promote drug binding and
loading.
The number of allylbenzene group
additions may be controlled via ATRP
chemistry to tailor drug release.
Sub-Hypothesis
With the optimization of synthesis and purification methodologies, it is hypothesized that
AOUs 1 through 4 will be successfully and reproducibly generated. These analogues are
hypothesized to be water soluble, with analogues 2 and 3 exhibiting higher water
solubilities than analogues 1 and 4. Furthermore, when cast as films, it is anticipated that
upon self-assembly, the fluorinated segments of the analogues will migrate to the surface
of the films. Upon successful removal of metal catalysts used during AOU syntheses from
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10
reaction products, it is hypothesized that the analogues will yield high cytotoxicity threshold
concentrations in vitro when compared to positive cytotoxic controls.
.Approach
NMR spectroscopy, GPC, MALDI-MS, elemental analysis and pre-polymer
isocyanate titrations were used to evaluate the outcome and reproducibility of
analogue syntheses.
Water contact angle measurements and bulk solubility testing were conducted to
determine AOU film surface and bulk hydrophilicity respectively.
Surface characterization of AOU films was conducted with high and low resolution
XPS and scanning electron microscopy.
Differential scanning calorimetry and thermogravimetric analysis were used to
characterize the bulk thermal properties of the AOUs.
Inductively coupled plasma atomic emission spectroscopy was used to confirm the
removal of metal catalysts during the purification of analogues 1 through 4.
Cytotoxicity evaluation of the AOUs was conducted via water-soluble tetrazolium
cell viability assays and deoxyribonucleic acid quantification using rat aortic VSMCs.
1.3.5 Objective 3 (Chapter 6)
To develop a capillary electrophoresis (CE) method for the separation of AOUs and the
antiproliferative agent BHAc, and to apply such a method towards the evaluation of
AOU:BHAc dissociation constants using AOU-1 and AOU-2 as model materials via the
“non-equilibrium capillary electrophoresis of equilibrium mixtures” (NECEEM) technique.
Rationale
In order to quantify the release of embedded drug from balloons coated with AOUs, a
sensitive, reproducible and efficient analytical method must be employed. Although high
performance liquid-chromatography (HPLC) is conventionally used to analyze drug release
from various delivery systems, it’s inadequacy in detecting and differentiating between
AOUs, AOU-drug complexes and free drug in a solution does not allow for the complete
characterization of drug release from an AOU-based drug delivery system. Such
information is vital in determining the amount of drug released from AOU systems, as the
relative amount of free and bound drug states may influence the bioavailability, and thus
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11
the effective delivery, of drug. Contrary to the limitations of HPLC, CE allows for the rapid
separation and quantification of free ligand, ligand-bound target and free target [44]. Thus,
if applied to AOU-BHAc systems, CE may allow for the complete characterization and
comparison of AOU drug release profiles. For a rapid drug dissociation system, which is
anticipated for the proposed AOUs, the NECEEM protocol is advantageous as it does not
rely on the assumption that equilibrium binding will be maintained during separation.
Rather, this technique involves the evaluation of the decay of bound systems starting at
equilibrium [45]. Details of this technique are further discussed in Section 2.5.
Sub-Hypothesis
Through the optimization of run buffers and run voltages, it is hypothesized that a CE
method will be established which yields differing migration times of AOU-1 and AOU-2 from
BHAc. If applied to assess dissociation constants via the NECEEM technique, it is
hypothesized that AOU-2 will yield lower AOU:BHAc complex dissociation than AOU-1 for
established AOU:BHAc formulations.
Approach
A variety of run buffers composed of aqueous buffer and tetrahydrofuran, to enable
the dissolution of BHAc, were used to determine the migration times of AOU-1,
AOU-2 and BHAc injected individually into a silica capillary-based CE system. Run
voltage variation was also explored for the run buffer yielding the greatest difference
in migration time between the AOUs and BHAc.
Solutions of AOU-1:BHAc and AOU-2:BHAc formulations within the optimized run
buffer were generated. Aliquots of such solutions were obtained at various
dissolution time points for CE analysis using the optimized run system.
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12
Chapter 2:
Review of Literature
2.1 Restenosis
Restenosis is defined as a 50% lumen closure following revascularization [46]. While
negative remodelling (a process in which the treated vessel shrinks or fails to expand in
order to accommodate thrombus and neointimal formation) contributes largely to vessel re-
narrowing post percutaneous coronary interventions (PCIs) in which stents are not used,
neointimal hyperplasia (NH) is thought to be the dominating factor in the formation of
restenotic lesions post stent-based PCIs [47-49]. NH involves a phenotypic switch of
vascular smooth muscle cells (VSMCs) from a normal contractile state to a synthetic state,
causing VSMC proliferation and migration from the media to the intima [46, 50, 51]. These
VSMCs, in combination with extracellular matrix components such as elastin, collagen and
proteoglycan, form a neointima and ultimately lumen re-closure at the site of vessel
treatment [14, 48, 51]. The cascade of events triggering NH originates from vascular injury
from balloon inflation and stent implantation [14, 46, 48, 50-52], the summary of which is
illustrated in Figure 2.1
2.1.1 Restenosis: The thrombotic phase
Neointimal formation first involves a thrombotic phase, which is triggered by vascular injury
from balloon inflation and stent implantation [47]. Endothelial denudation exposes sub-
endothelium layers which consist of adhesive proteins such as collagen, fibronectin,
vitronectin, laminin, and the von Willebrand factor (vWF) [53]. These proteins serve as
ligands to bind platelets through membrane glycoprotein (GP) receptor complexes such as
GP Ib-IX, Ia-IIa, and Ic-IIa [53]. The aggregation of platelets, which is mediated via
fibrinogen and the activated GP receptor complex IIb-IIIa [46, 53], and platelet binding to
vWF via GP IIb-IIIa and to type I collagen via GP Ia-IIa, leads to platelet activation [53, 54].
Activated platelets express adhesion molecules, such as P-selectin GP, which bind
leukocytes circulating in the blood stream [48, 55]. Leukocytes commence a rolling process
across the platelet layer and subsequently tightly bind to the platelet surface through the
interaction of the leukocyte integrin Mac-I and platelet GP receptor complex Ibα [48, 55,
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13
56]. Platelet activation also triggers the release of coagulation factors, such as factor VII,
which binds to tissue factor secreted by the recruited leukocytes [46]. This binding initiates
the coagulation cascade in which prothrombin is converted to thrombin. This, in turn,
facilitates the conversion of fibrinogen to fibrin [46, 57]. A matrix of aggregated platelets
and fibrin is thus formed at the site of vessel injury, which entraps erythrocytes leading to
thrombosis and lumen re-closure [46, 57].
Figure 2.1 Summary of the cascade of events which are initiated by vascular injury from
PCI balloon inflation and stent implantation, and which lead to restenosis of the treated
blood vessel [58].
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14
2.1.2 Restenosis: The neointimal progression phase
In addition to triggering thrombus formation, endothelial denudation, activated platelets and
leukocytes also play key roles in the post-PCI restenotic process through the initiation of
NH. Activated platelets secrete platelet-derived growth factor (PDGF) and transforming
growth factor-β (TGF-β) [46]. TGF-β triggers the synthesis of deoxyribonucleic acid (DNA)
in VSMCs and also promotes VSMC migration [46, 59]. The platelet vitronectin receptor,
αvβ3, is also implicated in TGF-β production and thrombin-induced VSMC proliferation and
migration [46, 53, 60]. Such migration is also enhanced through the binding of platelet
factor VII to leukocyte derived tissue factor [46]. PDGF contributes to NH by directly
stimulating VSMC proliferation and also by acting as a VSMC chemoattractant to initiate
VSMC migration [61, 62]. PDGF also mediates monocyte chemoattractant protein-1 (MCP-
1), which in turn regulates the chemotaxis of monocytes and macrophages to the site of
vessel injury [46]. These leukocytes produce interleukin-1 (IL-1), a cytokine which acts as a
growth factor to stimulate VSMC proliferation [63]. VSMC proliferation and migration are
also directly promoted by endothelium damage. Endothelial cells produce prostacyclin,
nitric oxide and heparan sulfate, which act to inhibit VSMC proliferation. Upon endothelial
damage, the production of these molecules decreases, which increases VSMC proliferation
[64, 65]. Furthermore, endothelial injury increases the expression of the VSMC mitogen
basic fibroblast growth factor (bFGF), thus further promoting VSMC proliferation [65, 66].
VSMC migration and proliferation during NH are consequently a result of multiple signalling
events involving platelets, endothelial cells, leukocytes and VSMCs (Figure 2.1).
Collectively, these events lead to NH through an alteration of VSMCs from a normal
contractile phenotype to a proliferative phenotype via the initiation of the G1 phase of the
cell cycle [67]. Mitogenic stimulation initiates the assembly and phosphorylation of
cyclin/cyclin dependent kinase complexes, which instigate the progression of the G1 phase
[50, 67]. As such, VSMCs exit the G0 phase of the cell cycle once stimulated and transition
through the G1, S, G2 and M phases of the cell cycle, ultimately leading to cellular division
[50]. Mitogenic stimulation of the VSMCs within the G1 to G1/S transition window also
leads to VSMC migration [50]. VSMCs thus undergo proliferation and migration to the
media, and in conjunction with VSMC-synthesized extracellular matrix, form a restenotic
lesion within the treated blood vessel.
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15
2.2 Drug-coated balloon technologies to prevent restenosis
2.2.1 Pharmaceutical agents
The advantages of employing drug-coated balloons (DCBs) during PCIs, with the aim of
preventing restenosis, are contingent upon the delivery of an appropriate pharmaceutical
agent. Several therapeutic agents with differing modes of activity were explored in order to
inhibit the cascade of events leading to restenosis, described in the previous section.
These drugs and their respective modes of action are summarized in Table 2.1. Several
studies conducted in the 1990’s, as listed in Table 2.2, evaluated the use of balloon-
delivered anti-coagulants, such as heparin and argatroban, to prevent angioplasty-
mediated restenosis. Although the local delivery of these agents demonstrated lower
platelet deposition and VSMC proliferation at the site of vessel injury when compared to
controls in animal models, the long-term and in-human efficacy of these agents at
preventing restenosis was contested [68-73]. As anti-coagulants, these pharmaceutical
agents inhibit the inflammatory and VSMC proliferation-inducing effects of thrombin.
However, as can be seen in Figure 2.1, NH leading to restenosis can progress via multiple
pathways. As such, therapeutic agents specifically targeting VSMC proliferation were
explored.
Attention thus turned towards the use of anti-proliferative agents such as paclitaxel or
drugs within the limus family. These drugs are hydrophobic and therefore diffuse easily
across target cell membranes to elicit their respective activities, which are summarized in
Table 2.1 [74-76]. Drugs of the limus family, such as sirolimus (rapamycin), zotarolimus
and everolimus, bind and inhibit the mammalian target of rapamycin to act as cytostatic
agents by arresting the cell cycle at the G1 stage [76]. On the other hand, paclitaxel binds
to β-tubulin and affects the spindle microtubule assembly dynamics via microtubule
stabilization, which leads to over-polymerization of microtubules [74, 75]. Mitosis, which
relies on appropriate microtubule assembly dynamics, is thus disrupted. Depending on
paclitaxel doses used, this can elicit a cytotoxic apoptotic effect or a cytostatic effect to
ultimately inhibit cellular proliferation [74, 75].
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16
Table 2.1 Examples of therapeutic agents used in DCBs and their respective modes of action.
Family Therapeutic
agent Mode of action
Anti-
coagulant
Heparin
Binds to the plasma protease inhibitor antithrombin-III to accelerate the binding and,
consequently, inhibition of the protease activity of thrombin [77]. This ultimately inhibits the anti-
inflammatory and VSMC proliferation-inducing effects of thrombin.
Argatroban Binds directly to the catalytic site of thrombin to inhibit its coagulant activity [77].
Nitric oxide
donors
Activates guanylate cyclase to increase intracellular cyclic guanosine monophosphate
concentrations, which results in increased protein kinase A and G activities [78]. These kinases
are implicated in a number of pathways which inhibit platelets and attenuate VSMC proliferation
and migration, including the inhibition of PDGF, attenuation of GP IIb-IIIa and P-selectin
expression, and arrest of the cell cycle at G1 and S phases [78].
Anti-
proliferative
Paclitaxel
Binds to β-tubulin and affects the spindle microtubule assembly dynamics via microtubule
stabilization, which leads to over-polymerization of microtubules [74, 75]. Disruption of normal
microtubule assembly also disrupts mitosis, resulting in the prevention of cell proliferation [74, 75].
Limus family
(sirolimus,
zotarolimus,
everolimus)
Bind to the FK506-binding protein 12. This complex then binds to and inhibits the mammalian
target of rapamycin, which is involved in the transition of the cell cycle from the G1 to S phase.
These agents thus act cytostatically to arrest the cell cycle at the late G1 phase [76]
C6-
Ceramide
Permeates through cell membranes to regulate mitogen-activated protein kinase cascades
involved in mitosis and to inhibit kinases, such as protein kinase B, involved in cell survival to
ultimately prevent VSMC proliferation and induce apoptosis [79].
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17
Although both paclitaxel and drugs of the limus family are hydrophobic and thus permeate
cell membranes via diffusion, paclitaxel has a significantly higher arterial tissue binding
capacity than rapamycin, allowing for its biological activity to be more strongly exerted over
the course of its residency time of one week in tissues [80, 81]. The tissue distribution of
the two anti-proliferatives also differs. Whereas paclitaxel is believed to concentrate more
highly in the media and adventia, important sites of origin for VSMC proliferation and
migration, rapamycin is distributed evenly across the arterial wall [80]. Furthermore, a study
by Axel et al. demonstrated the ability of paclitaxel, administered as a single burst at
nanomolar concentrations, to inhibit human arterial smooth muscle cell proliferation for 14
days without eliciting any significant non-specific cytotoxicity or apoptosis [82]. Such
inhibition was sustained even in the presence of exogenously added PDGF, thrombin and
bFGF [82]. This single exposure to paclitaxel was shown in a porcine model by Speck et al.
to prevent restenosis as effectively as the Cypher stent one month after PCI [83]. These
characteristics have made paclitaxel the most commonly used therapeutic in commercial
DCB systems. Table 2.3 lists several paclitaxel-based DCB systems that have been
studied in recent years.
Although several investigations have looked into alternative anti-proliferative therapeutics
to inhibit restenosis post PCI, paclitaxel remains the drug of choice for DCB inclusion. Such
therapeutics include the cell-permeable sphingolipid C6-ceramide, genestein (a tyrosine
kinase inhibitor) and DNA plasmids encoding for the apoptosis regulating protein kinase C
[79, 84-86]. Although the delivery of these agents from DCBs attenuated NH when
compared against the use of uncoated balloon controls in short-term animal model studies,
unlike paclitaxel, such activity often did not translate into the inhibition of restenosis at
periods longer than one month post PCI [79, 84-86]. Furthermore, studies evaluating the
efficacy of these anti-proliferative agents are limited in number and also are limited to in
vitro or animal model trials.
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Table 2.2 Examples of DCB carrier materials evaluated at pre-clinical stages and associated with various pharmaceutical agents.
Drug DCB carrier
example
Author/
inventor Description of DCB and outcomes
Heparin
Polyacrylic acid
cross-linked
hydrogel coated
on a polyethylene
balloon
Azrin et
al.[70]
Without sleeving, 96% systemic loss of heparin in 30 seconds
2.3% of loaded drug delivered in vivo to porcine carotid/iliac arteries
Heparin levels after 48 hours post angioplasty were below the detection limit
81% reduction in platelet deposition and 25% reduction in SMC proliferation
compared to control
High molecular
weight polyacrylic
acid cross-linked
hydrogel
Johnson
et al.[71]
Less than 0.1% of loaded nadroparin found in porcine iliac arteries at
angioplasty sites post balloon inflation for 2 minutes
Platelet deposition and SMC proliferation reduced by 18.4% and 22.4%
respectively compared to control
Argatroban
Polyacrylic acid
cross-linked
hydrogel
Imanishi
et al.[72]
24.6% of argatroban delivered to rabbit carotid artery tissue upon balloon
inflation
No detectable argatroban 24 hours post angioplasty in tissue
38% reduction in platelet deposition 2 hours after angioplasty and 30%
reduction in late restenosis 4 months post angioplasty
Anionic surface-
modified
polyethylene
Richey
et al.[73]
Anionic coating of acrylic acid or 2-(dimethylamino) ethyl methacrylate
(DMAEMA) grafted on polyethylene
Argatroban adsorbed via ionic interactions with acrylic acid or DMAEMA
grafts (70 µg/cm2 and 48 µg/cm
2 loading respectively)
5.5% of loaded drug transferred to rabbit carotid arteries
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19
Nitric oxide
donor photo-
polymerized S-
nitrosocysteine
Ultra-thin Glidex-
TM (Boston
Scientific
Corporation)
hydrogel coating
Rolland
et al.[87]
8% of drug released 2 minutes post inflation in buffer solution
Lack of VSMC proliferation in the porcine iliac artery 24 hours after
angioplasty
No pathological evidence of restenosis 3 months post angioplasty of porcine
iliac artery
Pacltaxel
Direct coating with
unspecified
excipient
Berg et
al.[88]
Paclitaxel and excipient were directly coated via organic solvent (termed
formulation d or j)
17% (formulation d) or 42% (formulation j) loss of loaded paclitaxel during
catheter tracking
8-12% of loaded paclitaxel delivered to porcine coronary arteries
0.27 mm and 0.23 mm late lumen loss 4 weeks post angioplasty for
formulations d and j respectively (16% and 19% less than control)
Iopromide contrast
agent excipient
coated using ethyl
acetate or acetone
Scheller
et al.[89]
6% of loaded drug was lost during tracking, 80% released; percent up-taken
by porcine coronary arteries was not recorded
0.49 mm late lumen loss 35 days post angioplasty (24% less than without
paclitaxel
Porous balloon
catheter
Oberhoff
et al.[90]
34.2 µg of paclitaxel in solution was injected and delivered to rabbit carotid
artery tissue through 75 µm pores
Systemic exposure to paclitaxel: plasma levels of 26.2 ng/mL directly after
balloon inflation; paclitaxel undetectable 30 minutes post angioplasty
No significant change in percent stenosis of artery pre and post treatment
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20
Limus family
agents
Poly (vinyl
pyrolidone)
excipient with
glycerol plasticizer
Stankus
et al.[91,
92]
1:1:0.4 ratio of zotarolimus: poly (vinyl pyrrolidone): glycerol yielded
maximum zotarolimus release after inflation (85% release) in vitro
30% of loaded coating/dose delivered to porcine arteries post angioplasty
Sirolimus
encapsulated
phospholipid
nanoparticles
coated onto the
balloon
Gandhi
and
Murthy
[93]
Polyethylene glycol (PEG) and Tomadol used as binder and surfactant
respectively
Only 42% of loaded sirolimus was released one minute after inflation and
64% released 45 seconds after second inflation (2 minutes after the first
inflation into phosphate buffered saline solution
C6-Ceramide
Vitamin E based
oil excipient
Schultz
[94]
Extracellular signal related kinase (ERK) levels were similar for treated
rabbits in a rabbit carotid stretch model and un-injured rabbits (inhibition of
ERK conversion to phosphorylated ERK correlates to restenosis inhibition)
50% less lumen loss compared to un-coated balloon
Lipid gel in 90:10
ethanol:
dimethylsulfoxide
(0.5% ceramide)
O’Neill
et al.
[86]
14% porcine artery stenosis 30 days post angioplasty (32% in control)
Similar inhibition of VSMC proliferation 24 hours post angioplasty as
paclitaxel and rapamycin but with no significant inhibition of endothelial cell
pro