Development of Self-Assembled Amphiphilic Oligo …...Development of Self-Assembled Amphiphilic Oligo-Urethanes as Cardiovascular Drug Delivery Platforms by Maneesha Amrita Rajora

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)

ii

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

x

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


addition of LDI to NL-PEG as outlined in Scheme 4.4 with a NL-PEG:LDI feed ratio of

1:1.5.……………………………………………………………………………………………....225


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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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

xix

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

xx

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

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

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

xxiii


stream-wise addition of LDI to NL-PEG in a feed ratio of 1:1 as outlined in Scheme

4.4………………………………………………………………………………………………….210


stream-wise addition of LDI to NL-PEG in a feed ratio of 1:1.5 as outlined in Scheme

4.4………………………………………………………………………………………………….211


stream-wise addition of LDI to NL-PEG in a feed ratio of 1:2 as outlined in Scheme

4.4………………………………………………………………………………………………....211


drop-wise addition of LDI to L-PEG in a feed ratio of 1:1 as outlined in Scheme 4.5…...212


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



o and 20

o for analogue 2 films…………………………………….275



o and 20

o for analogue 2 films………………………………….....276

1

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

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

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

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.

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.

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

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.


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.

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,

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

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.


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

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.

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,

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].

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.

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].

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].

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.

18

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

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

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

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