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Progesterone-binding modified hyperbranched polyglycerols : synthesis, characterization and biological… Alizadeh Noghani, Mahsa 2016

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PROGESTERONE-BINDING MODIFIED HYPERBRANCHED POLYGLYCEROLS: SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL ASSESSMENT by  Mahsa Alizadeh Noghani  B.Sc., University of Tehran, 2005 M.Sc., University of Tehran, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2016  © Mahsa Alizadeh Noghani, 2016  ii   Abstract  Traumatic brain injury (TBI) has been proven as an established risk factor of Alzheimer’s disease (AD). Historically, progesterone (Pro) has been found to promote recovery from moderate TBI. However, the utility of this drug as a TBI treatment is severely hampered by its near total insolubility in water due to its hydrophobicity, which contributes to an inability to rapidly administer the drug after injury.  The present work describes the synthesis, characterization, development and in vitro evaluation of nanoparticulate formulations of Pro for treatment of TBI. The nanoparticles developed for Pro consist of a library of hyperbranched polyglycerols (HPGs), which were hydrophobically modified with alkyl chains (C6, 8, 10, 12, 14, 18) to enable loading the hydrophobic drug, and were further modified with MPEG chains to increase the solubility and stability of the formulations. Hydrophobically derivatized HPGs (HPG-Cn-MPEG), also known as dHPG(Cn), were characterized by GPC and NMR methods. Pro encapsulation by and release from the drug-binding pocket was determined through a reverse-phase UPLC method. Combination of binding, release and kinetic studies of the dHPG(Cn)/Pro library presented a relatively high number of drug molecules encapsulated, slow release and stable formulations.  In vitro assays, including blood biocompatibility, cytotoxicity and cellular uptake, were performed on dHPG(Cn)/Pro. Blood biocompatibility studies demonstrated that the polymer-drug formulations do not cause significant changes in blood coagulation time (APTT assay), nor have they significant effects on red blood cell aggregation, lysis or platelet aggregation. There was no platelet activation observed in this study. Study of viability of human cortical microvascular endothelial cells and human astrocytoma cells in the presence of dHPG(Cn)/Pro demonstrated no  iii  toxicity. Studies on the same cells presented significant uptake with relatively even distribution of the formulation inside the cells. Further investigations indicated no degradation pathway for dHPG(Cn) over short periods of time (~ 8 h). Overall, the in vitro studies suggest that dHPG(Cn) are compatible and harmless to cells, suitable for carrying hydrophobic drugs and molecules, such as Pro, to the target tissues.   iv  Preface  The dissertation and goals of this thesis were discussed and agreed upon between the author and research supervisor. All work described in Chapter 2 and Chapter 3 was performed solely by the author. A version of Chapter 2 and 3 has been published. Alizadeh Noghani, M. Brooks, DE. Progesterone binding nano-carriers based on hydrophobically modified hyperbranched polyglycerols, Nanoscale, 2016, 8, 5189-5199. The first draft of the manuscript and all work described therein were completed by the principal author. The author was the primary individual responsible for the design of the studies, sample preparations and analyzing data in Chapter 4. Benjamin Lai was responsible for the conducting of all the experiments in Chapter 4 except Scanning Electron Microscopy (SEM). Manu Thomas Kalathottukaren was responsible for conducting the SEM experiment. The author was the primary individual responsible for the design of the studies, sample preparations and analyzing data in Chapter 5. The author and Dr. Jerome Robert were responsible for conducting the uptake study and cell cytotoxicity experiments. Dr. Jerome Robert was responsible for conducting confocal microscopy.  v   Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiv List of Symbols ....................................................................................................................... xxviii Glossary .................................................................................................................................... xxxi Acknowledgements ................................................................................................................. xxxii Dedication ............................................................................................................................... xxxiv Chapter 1: Introduction ................................................................................................................1 1.1 Blood Brain Barrier......................................................................................................... 1 1.2 Traumatic Brain Injury ................................................................................................... 3 1.2.1 Brain Injury, Complexity and Treatments .................................................................. 3 1.2.2 Progesterone as a Good Candidate for TBI Pharmacotherapy ................................... 4 1.2.3 Clinical Studies of Pro in TBI ..................................................................................... 5 1.3 Human Blood Components ............................................................................................. 6 1.3.1 Red Blood Cells (Erythrocytes) .................................................................................. 6 1.3.1.1 Red Blood Cell Aggregation ............................................................................... 7 1.3.1.2 Red Blood Cell Lysis (Hemolysis) ..................................................................... 7 1.3.2 White Blood Cells (Leukocytes) ................................................................................. 7 1.3.3 Platelets ....................................................................................................................... 8  vi  1.4 Blood Coagulation .......................................................................................................... 8 1.5 Biocompatibility ............................................................................................................. 9 1.6 Nanoparticles ................................................................................................................ 10 1.6.1 Polymer-Based Nanodevices .................................................................................... 10 1.6.2 Dendrimers and Hyperbranched Polymers ............................................................... 12 1.7 Hyperbranched Polyglycerol (HPG)-Based Nanomedicine ......................................... 14 1.7.1 Biocompatibility and Applications ........................................................................... 17 1.7.1.1 Biocompatibility of Poly(ethylene glycol) and HPGs ...................................... 17 1.7.1.2 Applications of HPGs ....................................................................................... 20 1.8 Hydrophobically Derivatized Hyperbranched Polyglycerols dHPG(Cn) ..................... 21 1.9 Biodegradable Hyperbranched Polyglycerols (BHPGs) ............................................... 24 1.10 Thesis Goals .................................................................................................................. 28 1.11 Specific Aims ................................................................................................................ 29 1.11.1 Development of dHPG(Cn)/Pro Formulation........................................................ 29 1.11.1.1 Synthesis of dHPG(Cn) ................................................................................. 29 1.11.1.2 In vitro Study of Binding and Release of Pro and dHPG(Cn)....................... 29 1.11.2 Efficacy of dHPG(Cn)/Pro Formulation ................................................................ 30 1.11.2.1 Blood Compatibility...................................................................................... 30 1.11.2.2 Cellular Uptake and Cytotoxicity ................................................................. 30 Chapter 2: Synthesis and Characterization of Hydrophobically Modified Hyperbranched Polyglycerols ..................................................................................................31 2.1 Synopsis ........................................................................................................................ 31 2.2 Materials and Methods .................................................................................................. 31  vii  2.2.1 Chemicals .................................................................................................................. 31 2.2.2 Synthesis and Characterization of 1,2-epoxyoctadecane .......................................... 32 2.2.3 Synthesis and Characterization of MPEG-epoxide ................................................... 33 2.2.4 Synthesis of dHPG(Cn) ............................................................................................. 36 2.2.5 Characterization of dHPG(Cn) .................................................................................. 37 2.3 Results ........................................................................................................................... 38 2.3.1 Characterization of dHPG(Cn) .................................................................................. 38 2.4 Discussion ..................................................................................................................... 45 2.5 Summary ....................................................................................................................... 47 Chapter 3: Studies of Loading and Release Kinetics of Progesterone Bound to Hydrophobically Derivatized Hyperbranched Polyglycerols (dHPG(Cn)/Pro) .......................48 3.1 Synopsis ........................................................................................................................ 48 3.2 Materials and Methods .................................................................................................. 48 3.2.1 Chemicals .................................................................................................................. 48 3.2.2 Estimates of Water Bound to dHPG(Cn) .................................................................. 49 3.2.3 Drug Incorporation into dHPG(Cn) ........................................................................... 50 3.2.4 Drug Quantification Study ........................................................................................ 51 3.2.5 Temperature Effect on Pro Binding .......................................................................... 53 3.2.6 Does Pro Cause dHPG(Cn) to Aggregate? ................................................................ 54 3.2.7 Drug Release from dHPG(Cn) .................................................................................. 54 3.3 Results ........................................................................................................................... 55 3.3.1.1 Water Binding by dHPG(Cn) ............................................................................ 55 3.3.2 Drug Binding into dHPG(Cn).................................................................................... 56  viii  3.3.3 Temperature Effect on Pro Binding .......................................................................... 59 3.3.4 Effect of Pro on dHPG(Cn) Size ............................................................................... 61 3.3.5 Drug Release from dHPG(Cn) .................................................................................. 62 3.3.5.1 Release in PBS .................................................................................................. 62 3.3.5.2 Release in Platelet Poor Plasma ........................................................................ 64 3.3.6 Release Kinetics ........................................................................................................ 65 3.3.6.1 Kinetics in PBS ................................................................................................. 65 3.3.6.2 Kinetics in Platelet Poor Plasma ....................................................................... 70 3.4 Discussion ..................................................................................................................... 72 3.4.1 Correlation of Binding and Release Behaviour with Material Properties ................ 72 3.4.1.1 Pro Loading Capacity and Effect on the Size ................................................... 72 3.4.2 Pro Release Kinetics ................................................................................................. 74 3.4.3 DSC Determination of Structured Water .................................................................. 76 3.5 Summary ....................................................................................................................... 79 Chapter 4: In vitro Blood Compatibility Study of dHPG(Cn)/Pro Formulations ..................81 4.1 Synopsis ........................................................................................................................ 81 4.2 Materials and Methods .................................................................................................. 81 4.2.1 Blood Collection ....................................................................................................... 81 4.2.2 Plasma Clotting Assays Analysis: APTT ................................................................. 81 4.2.3 Red Blood Cell Aggregation Analysis ...................................................................... 82 4.2.4 Red Blood Lysis Analysis: Drabkin’s Method ......................................................... 83 4.2.5 Platelet Activation Analysis: Flow Cytometry ......................................................... 83 4.2.6 Platelet Aggregation Analysis ................................................................................... 84  ix  4.2.7 Thromboelastography (TEG) Analysis ..................................................................... 84 4.2.8 Scanning Electron Microscopy (SEM) ..................................................................... 85 4.3 Results ........................................................................................................................... 86 4.3.1 APTT Analysis.......................................................................................................... 86 4.3.2 Red Blood Cell Aggregation ..................................................................................... 88 4.3.3 Red Blood Cell Lysis ................................................................................................ 90 4.3.4 Platelet Activation ..................................................................................................... 92 4.3.5 Platelet Aggregation.................................................................................................. 93 4.3.6 Thromboelastography (TEG) Analysis ..................................................................... 96 4.3.6.1 Effect on Whole Blood ..................................................................................... 96 4.3.6.2 Effect on Platelet Poor Plasma (PPP) ............................................................. 100 4.3.6.3 Effect on Platelet Rich Plasma (PRP) ............................................................. 102 4.3.7 Scanning Electron Microscopy (SEM) ................................................................... 105 4.4 Discussion ................................................................................................................... 107 4.5 Summary ..................................................................................................................... 111 Chapter 5: In vitro Cellular Cytotoxicity and Uptake Study of dHPG(Cn)/Pro Formulation ................................................................................................................................112 5.1 Synopsis ...................................................................................................................... 112 5.2 Materials and Methods ................................................................................................ 113 5.2.1 Chemicals ................................................................................................................ 113 5.2.2 Cell Lines ................................................................................................................ 113 5.2.3 Fluorescent Labeling of dHPG(Cn) ......................................................................... 114 5.2.4 Cellular Uptake Study ............................................................................................. 115  x  5.2.5 Cellular Uptake Study with Anti-LAMP1 Antibody – Lysosome Marker ............. 116 5.2.6 Cellular Uptake Study with Anti-Progesterone Receptor Antibody ....................... 116 5.2.7 Cellular Viability Study .......................................................................................... 117 5.3 Results ......................................................................................................................... 117 5.3.1 Cellular Uptake ....................................................................................................... 117 5.3.2 Cellular Uptake with Anti-Lysosome Antibody ..................................................... 122 5.3.3 Cellular Viability .................................................................................................... 124 5.3.4 Cellular Uptake with Anti-Progesterone Receptor Antibody ................................. 127 5.4 Discussion ................................................................................................................... 133 5.5 Summary ..................................................................................................................... 136 Chapter 6: Conclusions and Future Work ..............................................................................137 6.1 Thesis Summary.......................................................................................................... 137 6.2 Future Work ................................................................................................................ 141 6.2.1 Animal Study and Biodistribution Experiment....................................................... 141 6.2.2 Binding and Release Study of Radiolabeled Pro into and from dHPG(Cn) ............ 141 6.2.3 Encapsulation of Water-Soluble Analogue of Pro by dHPG(Cn) ........................... 142 Bibliography ...............................................................................................................................144 Appendices ..................................................................................................................................158 Appendix A ............................................................................................................................. 158 A.1 1H NMR Spectra (300 MHz, CDCl3) and GPC Chromatogram of dHPG(Cn) (n = 6, 10, 14, 18) ........................................................................................................................... 158 A.2 13C NMR (400 MHz, methanol-d4) ........................................................................ 163 Appendix B ............................................................................................................................. 164  xi  B.1 Binding Profiles of Pro into dHPG(Cn) .................................................................. 164 Appendix C ............................................................................................................................. 167 C.1 Effect of Pro Binding on dHPG(C10) Size .............................................................. 167 Appendix D ............................................................................................................................. 168 D.1 In vitro Release Profiles of Pro from dHPG(Cn) .................................................... 168 Appendix E ............................................................................................................................. 172 E.1 Kinetic Profiles of Pro Released from dHPG(Cn) in PBS and Plasma ................... 172 Appendix F.............................................................................................................................. 179 F.1 t-Test Analysis on APTT Results ........................................................................... 179 F.2 Red Blood Cell Aggregation after Incubation with dHPG(Cn) at 1 mg/mL ........... 180 F.3 Red Blood Cell Aggregation after Incubation with dHPG(Cn) and dHPG(Cn)/Pro at 10 mg/mL ............................................................................................................................ 180 F.4 Platelet Aggregation after Incubation with dHPG(C8) and dHPG(C12) at 1 and 10 mg/mL ........................................................................................................................... 182 F.5 SEM Imaging of Dynamic Clots............................................................................. 184 Appendix G ............................................................................................................................. 185 G.1 1H NMR (300 MHz, CDCl3): dHPG(C8) Modified with EPP ................................ 185 G.2 1H NMR (300 MHz, D2O): dHPG(C8) Modified with Primary Amine .................. 186 Appendix H ............................................................................................................................. 187 H.1 dHPG(C8) Uptake by HCMEC/D3 Visualized by Anti-Lysosome Antibody ........ 187   xii  List of Tables  Table 1.1 Circulation half-life and organ accumulation of radio-labelled HPGs and their derivatives in mice ........................................................................................................................ 19 Table 2.1 Characteristics of dHPG(Cn) (n = 6, 8, 10, 12, 14, 18); a: Mole fractions of alkyl and MPEG monomers were calculated from proton NMR; number of hydrophobic carbons in R chain (n-2) per polymer molecule was calculated from multiplying the number of alkyl chain per polymer molecule by the number of carbons in R chain (n-2); b: Hydrodynamic radii were calculated from dynamic light scattering (QELS), GPC; yield% calculation was based on starting polymer weight ............................................................................................................................. 42 Table 3.1 Summary of DSC results: number of moles of water affected per mole of dHPG(Cn) at the two polymer concentrations shown ......................................................................................... 56 Table 3.2 Binding characteristics of dHPG(Cn) ............................................................................ 59 Table 3.3 Effect of loaded Pro on dHPG(Cn) size ........................................................................ 61 Table 3.4  Pro release rate constants from dHPG(Cn) .................................................................. 70 Table 3.5  Pro release rate constants from dHPG(C8) in plasma vs. PBS .................................... 71 Table 3.6 a: Number of alkyl carbon atoms per dHPG(Cn) molecule; b: Number of n-alkyl carbons external to oxygen per dHPG(Cn) molecule; c: Volume of n-alkyl carbons external to oxygen per dHPG(Cn) molecule; d: Volume of dHPG(Cn) molecule calculated from hydrodynamic radius; e: Volume of dHPG(Cn) molecule associated with HPG; f: Maximum ratio of Pro bound to dHPG(Cn) by loading protocol, moles per mole. P < 0.05 values are shown in bold font ........................................................................................................................ 75  xiii  Table 3.7 a: dHPG(Cn) at concentration of 10%; b: dHPG(Cn) at concentration of 5 % - Statistically significant results are shown in bold ......................................................................... 77 Table F.1 t-Test analysis on APTT results after incubation with dHPG(Cn), with and without the presence of Pro, at 10 mg/mL of polymer concentration; highlighted p-values demonstrate significant difference .................................................................................................................. 179 Table F.2 t-Test analysis on APTT results after incubation with dHPG(Cn) at 1 mg/mL; highlighted p-values demonstrate significant difference .................................................................................. 179   xiv  List of Figures  Figure 1.1 Central nervous system (CNS) ...................................................................................... 1 Figure 1.2 Blood brain barrier (BBB) structure .............................................................................. 2 Figure 1.3 Chemical structure of progesterone (Pro) ...................................................................... 5 Figure 1.4 RBC aggregation (on the left), in which cells lose their normal rouleaux shape (on the right); these images are taken from the incubation of derivatized hyperbranched polyglycerols with RBCs at higher (on the left) and lower (on the right) concentrations which are discussed in Chapter 4 ......................................................................................................................................... 7 Figure 1.5 Coagulation cascade: intrinsic and extrinsic pathways ................................................. 9 Figure 1.6 General structure of polymer micelle .......................................................................... 12 Figure 1.7 Example of a dendritic structure;   refers to the core and   refers to the branching points ............................................................................................................................................. 13 Figure 1.8 Example of a hyperbranched structure;   refers to the core and   refers to the branching points ............................................................................................................................ 14 Figure 1.9 Structure of HPG ......................................................................................................... 16 Figure 1.10 Structure of dHPG(C18); R refers to the 1,2-epoxyoctadecane monomer after epoxide ring is opened up through the anionic ring-opening reaction ....................................................... 22 Figure 1.11 Dextran ...................................................................................................................... 23 Figure 1.12. Hydroxyethyl starch ................................................................................................. 23 Figure 1.13 Structure of BHPG with cleavable linkage ............................................................... 26 Figure 1.14 Biodegradable HPG with randomly distributed ketal group (RBHPG) .................... 27  xv  Figure 2.1 Synthesis scheme of 1,2-epoxyoctadecane .................................................................. 32 Figure 2.2 1H NMR spectrum of 1,2-epoxyoctadecane in CDCl3 ................................................ 33 Figure 2.3 Synthesis scheme of MPEG-epoxide .......................................................................... 34 Figure 2.4 1H NMR spectrum of MPEG-epoxide in CDCl3 ......................................................... 35 Figure 2.5 Expanded 1H NMR of MPEG-epoxide in CDCl3 ....................................................... 35 Figure 2.6 Synthesis scheme of dHPG(Cn) (n = 6, 8, 10, 12, 14, 18); R refers to the hydrophobic chain of the alkyl monomer and x refers to the CH2 repeating units of the R chain .................... 37 Figure 2.7 1H NMR spectrum of dHPG(C8) in CDCl3 ................................................................. 40 Figure 2.8 1H NMR spectrum of dHPG(C12) in CDCl3 ................................................................ 40 Figure 2.9 GPC chromatogram of dHPG(C8); red line belongs to multi-angle light scattering detector and blue line belongs to refractive index detector .......................................................... 41 Figure 2.10 GPC chromatogram of dHPG(C12) ............................................................................ 42 Figure 2.11. Inverse-gated 13C NMR spectrum of dHPG(C8) in methanol-d4 with relaxation delay of 10 sec proved the branched structure of dHPG(Cn) .................................................................. 44 Figure 3.1 UPLC chromatogram represents the identification of Pro at the wavelength 240 nm and 1.9 min retention time ............................................................................................................ 52 Figure 3.2 UPLC calibration curve for Pro ................................................................................... 52 Figure 3.3 UPLC standards from 1 to 100 µg/mL. The retention time is 1.9 ± 0.1 and Pro was detected at the wavelength of 240 nm ........................................................................................... 53 Figure 3.4 Pro binding profile into dHPG(Cn) .............................................................................. 58 Figure 3.5 Temperature effect on drug binding behaviour in dHPG(C8)/Pro formulation; significant difference exists between the straight lines of the two temperatures, based on ANCOVA (p < 0.02) .................................................................................................................... 60  xvi  Figure 3.6 Temperature effect on drug binding behaviour in dHPG(C12)/Pro formulation; significant difference exists between the straight lines of the two temperatures, based on ANCOVA (p < 0.04) .................................................................................................................... 60 Figure 3.7 Pro release from dHPG(Cn) in PBS ............................................................................. 64 Figure 3.8 Pro release profile from dHPG(C8) in PBS and plasma .............................................. 65 Figure 3.9 Pro’s absolute concentration [C] with change in time – dHPG(C8)/Pro ..................... 66 Figure 3.10 Natural logarithmic behaviour of Pro’s absolute concentration [C] with time in PBS – dHPG(C8)/Pro ............................................................................................................................ 67 Figure 3.11 Semi-log plot to determine initial rapid release kinetics for dHPG(C8)/Pro in PBS; R2 = 0.99 and p < 0.01 .................................................................................................................. 68 Figure 3.12 Semi-log plot to determine secondary slow release kinetics for dHPG(C8)/Pro in PBS; R2 = 0.97 and p < 0.01 ......................................................................................................... 68 Figure 3.13 Free Pro release rate in PBS; k1(s-1) calculated from the slope (R2 = 0.95 and p < 0.01) .............................................................................................................................................. 69 Figure 3.14 Semi-log plots of the initial phase of release of Pro from the dialysis cassettes for dHPG(C8)/Pro in plasma (R2 = 0.96) and for dissolved Pro alone in PBS (R2 = 0.95) and plasma (R2 = 0.95). [C1] is the Pro concentration vs. time in the rapid release phase. [C10] is the initial Pro concentration, calculated from the intercept of ln [C1] vs. time. ............................................ 71 Figure 3.15 Correlation between the maximum binding capacity of dHPG(Cn) polymeric systems for binding Pro and the their total mass of alkyl carbon external to the oxygen (R2 = 0.77 and p < 0.025) ............................................................................................................................................ 73 Figure 3.16 Dependence of k1 on Vp-Va; R2 = 0.89 and p < 0.02 ................................................. 76  xvii  Figure 3.17 Correlation between k1 values and mole of structured water per mole of dHPG(Cn) at 10% polymer concentration; R2 = 0.79 and p < 0.05 .................................................................... 78 Figure 3.18 Correlation between k1 values and mole of structured water per mole of dHPG(Cn) at 5% polymer concentration; R2 = 0.94 and p < 0.01 ...................................................................... 78 Figure 4.1 Effect of dHPG(Cn) on coagulation time at 1 mg/mL ................................................. 87 Figure 4.2 Effect of dHPG(Cn) and dHPG(Cn)/Pro formulations on coagulation time; polymer concentrations were 10 mg/mL and 125 µg/mL of Pro was loaded ............................................. 87 Figure 4.3 Effect of dHPG(C8) at 1 mg/mL on red blood cell aggregation; a) PBS control buffer; b) HPG-PEG control polymer at 1 mg/mL; c) dHPG(C8) at 1 mg/mL ........................................ 88 Figure 4.4 Effect of dHPG(C8) at 10 mg/mL, with or without Pro, on red blood cell aggregation; a) PBS control buffer; b) Pro control at 5 µg/mL; c) dHPG(C8) at 10 mg/mL; d) dHPG(C8)/Pro, where polymer concentration was 10 mg/mL loaded with 125 µg/mL Pro; e) Positive control for RBC aggregation from the similar structure (HPG-C8/10-MPEG) at 10 mg/mL, where C8/10 monomer is octyl/decyl glycidol ether .......................................................................................... 90 Figure 4.5 RBC lysis percentage was measured after incubation with dHPG(Cn) at 1 mg/mL ... 91 Figure 4.6 RBC lysis percentage was measured after incubation with dHPG(Cn) and dHPG(Cn)/Pro at 10 mg/mL of polymer and 125 µg/mL of loaded drug ..................................... 91 Figure 4.7 Platelet activation estimated from CD62 expression after incubation with dHPG(Cn) at 1 mg/mL ........................................................................................................................................ 92 Figure 4.8 Platelet activation estimated from CD62 expression after incubation with dHPG(Cn) and dHPG(Cn)/Pro at 10 mg/mL and 125 µg/mL of bound drug ................................................. 93 Figure 4.9 PBS control buffer; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right .................................................................................... 94  xviii  Figure 4.10 Pro control at 5 µg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right .................................................................................... 94 Figure 4.11 dHPG(C8) at 0.1 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right ............................................................................ 95 Figure 4.12 dHPG(C12) at 0.1 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right ............................................................................ 96 Figure 4.13 TEG profile of dHPG(C8)/Pro formulation in whole blood – PBS was used as control....................................................................................................................................................... 97 Figure 4.14 TEG profile of dHPG(C12)/Pro formulation in whole blood – PBS was used as control ........................................................................................................................................... 97 Figure 4.15 TEG profile of dHPG(C14)/Pro formulation in whole blood – PBS was used as control ........................................................................................................................................... 98 Figure 4.16 TEG profile of dHPG(C18)/Pro formulation in whole blood – PBS was used as control ........................................................................................................................................... 98 Figure 4.17 Effect of polymer concentration of dHPG(Cn) [n = 8, 10, 12, 14, 18] on blood clot characterization at 1 mg/mL – PBS and HPG-PEG at 1 mg/mL were controls ........................... 99 Figure 4.18 Effect of polymer concentration of dHPG(Cn) [n = 8, 10, 12, 14, 18] on blood clot characterization at 0.1 mg/mL – PBS and HPG-PEG at 0.1 mg/mL were controls ................... 100 Figure 4.19 Effect of polymer concentration of dHPG(Cn) [n = 8, 12] on clot characterization in PPP at 10 mg/mL – PBS and HPG-PEG at 10 mg/mL were controls ........................................ 101 Figure 4.20 Effect of polymer concentration of dHPG(Cn) [n = 8, 12] on clot characterization in PPP at 1 mg/mL – PBS was used as control ............................................................................... 101  xix  Figure 4.21 Effect of polymer concentration of dHPG(Cn) [n = 8, 12] on clot characterization in PRP at 10 mg/mL – PBS and HPG-PEG at 10 mg/mL were controls ........................................ 102 Figure 4.22 Effect of polymer concentration of dHPG(Cn) [n = 8, 12] on clot characterization in PRP at 1 mg/mL – PBS and HPG-PEG at 1 mg/mL were controls ............................................ 103 Figure 4.23 Effect of polymer concentration of dHPG(C8) on clot characterization in PRP at 1 and 0.1 mg/mL – PBS and HPG-PEG at 1 mg/mL were controls .............................................. 104 Figure 4.24 Concentration effect of dHPG(C8) on clot characterization in PRP from 0.1 to 0.01 mg/mL – PBS was used as control ............................................................................................. 104 Figure 4.25 Effect of polymer concentration of dHPG(C12) on clot characterization in PRP at 1 and 0.1 mg/mL – PBS was used as control ................................................................................. 105 Figure 4.26 SEM imaging of static clots; a) HBS control buffer; b) HPG-PEG at 1 mg/mL; c) dHPG(C8) at 1 mg/mL; d) dHPG(C8) at 0.1 mg/mL at different zooming ...................... 107 Figure 4.27 dHPG(Cn) TEG profile in whole blood; clot maximum strength values were measured and compared at 1 mg/mL and 0.1 mg/mL of polymer. * demonstrates the significant difference between the values ..................................................................................................... 108 Figure 4.28 dHPG(Cn) TEG profile in whole blood; clot initial formation time (R) values were measured and compared at 1 mg/mL and 0.1 mg/mL of polymer. No significant difference was observed among the polymers and controls ................................................................................ 109 Figure 5.1 Synthetic scheme for the fluorescent labelling of dHPG(C8) with Alexa 488 NHS ester ............................................................................................................................................. 115 Figure 5.2 Dose dependent uptake of dHPG(C8) by HCMEC/D3 (human cortical microvascular endothelial cells); cells were incubated with different doses of dHPG(C8) from 0 to 1 mg/mL for 1 h at 37 °C ; Alexa 488 represents labelled polymer, Alexa 633 represents stained membrane with  xx  WGA and Dapi represents stained nucleus; for all concentrations, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities ..................................................................................................................................... 118 Figure 5.3 Time dependent uptake of dHPG(C8) by HCMEC/D3 cells; cells were incubated with 0.01 mg/mL of dHPG(C8) from 0 to 24 h; increase in the green fluorescent intensity in cell’s cytoplasm demonstrated increase in the uptake of the polymer by cells; Alexa 488 represents labelled polymer, Alexa 633 represents stained membrane with WGA and Dapi represents stained nucleus; for all time points, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities .................................................. 119 Figure 5.4 Dose dependent uptake of dHPG(C8) by CCF (human astrocytoma cells); cells were incubated with different doses of dHPG(C8) from 0 to 1 mg/mL after 1 h at 37 °C; Alexa 488 represents labelled polymer, Alexa 633 represents stained membrane with WGA and Dapi represents stained nucleus; for all concentrations, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities 120 Figure 5.5 Time dependent uptake of dHPG(C8) by CFF cells; cells were incubated with 0.01 mg/mL of dHPG(C8) at different time points from 0 to 24 h; increase in the green fluorescent intensity in cell’s cytoplasm demonstrated increase in the uptake of the polymer into the cytosol; Alexa 488 represents labelled polymer, Alexa 633 represents stained membrane with WGA and Dapi represents stained nucleus; for all time points, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities.......................................... 121 Figure 5.6 Uptake of dHPG(C8) by CCF cells visualized by anti-lysosome antibody (LAMP1) at 37 °C; Alexa 488 represents labelled polymer, Dapi represents stained nucleus and LAMP1 represents anti-lysosome antibody; for all time points, each channel is shown in black and white  xxi  individually, while the merge of all is shown in colour for better monitoring of the intensities; yellow signal represents the convergence of the polymer and lysosome. a) Media control – b) after 2 h; cells were incubated with dHPG(C8) (on the left) and dHPG(C8)/Pro (on the right) – c) after 4 h; cells were incubated with dHPG(C8) (on the left) and dHPG(C8)/Pro (on the right) – d) after 8 h; cells were incubated with dHPG(C8) (on the left) and dHPG(C8)/Pro (on the right). In all cases polymer and Pro concentrations were 0.01 mg/mL and 0.125 µg/mL, respectively ........... 123 Figure 5.7 Dose dependence study; percent viability of HCMEC/D3 cells compared to no treatment after incubation with dHPG(C8)/Pro at 5, 7.5 and 12.5 µg of the drug loaded on 1 mg/mL of the polymer. Different concentrations of polymer loaded with the drug were compared with the polymer alone and media only; significant differences between groups and the media control (NONE) using ANOVA are demonstrated by *. DMSO was used as negative control. 125 Figure 5.8 Time dependence study; percent viability of HCMEC/D3 cells compared to no treatment after incubation with dHPG(C8)/Pro at 5, 7.5 and 12.5 µg of the drug loaded on 1 mg/mL of the polymer in 1, 24 and 48 h; significant differences between groups and the media control (NONE) using ANOVA are demonstrated by *. Free Pro as control was incubated with cells at the concentration of 12.5 µg/mL. DMSO was used as negative control ........................ 125 Figure 5.9 Dose dependence study; percent viability of CCF cells compared to no treatment after incubation with dHPG(C8)/Pro at 5, 7.5 and 12.5 µg of the drug loaded on 1 mg/mL of the polymer. Different concentrations of polymer loaded with the drug were compared with the polymer alone and media only; significant differences between groups and the media control (NONE) using ANOVA are demonstrated by *. DMSO was used as negative control ............. 126 Figure 5.10  Time dependence study; percent viability of CCF cells compared to no treatment after incubation with dHPG(C8)/Pro at 5, 7.5 and 12.5 µg of the drug loaded on 1 mg/mL of the  xxii  polymer in 1, 24 and 48 h; significant differences between groups and the media control (NONE) using ANOVA are demonstrated by *. Free Pro as control was incubated with cells at the concentration of 12.5 µg/mL. DMSO was used as negative control .......................................... 126 Figure 5.11 Pro uptake visualized by anti-progesterone receptor antibody (PR). a) Behaviour of CCF cells incubated with PR in the presence or absence of Pro – b) Behaviour of HCMEC/D3 cells incubated with PR in the presence or absence of Pro. Controls with no treatment (on the left), cells stained with PR (in the middle) and cells stained with PR and incubated with Pro at 60 µg/mL (on the right); blue represents Dapi staining of nuclei. Spread red signals inside the cells demonstrated non-specific binding of the antibody. The microscope lens magnification used for these imaging was 20X ............................................................................................................... 128 Figure 5.12 MCF7 (Michigan Cancer Foundation-7) cells stained with PR; in the presence of free Pro at 60 µg/mL (on the left), dHPG(C8) (in the middle) and dHPG(C8)/Pro at 0.01 mg/mL of polymer and 0.125 µg/mL of Pro (on the right); blue represents Dapi staining of nuclei; presence of red signals inside the nucleus proved the specific interaction between the antibody and the receptor and positive response of the cells to the PR staining .................................................... 129 Figure 5.13 dHPG(C8) uptake by MCF7 cells stained with WGA (Alexa 633); Alexa 488 represents labelled polymer, Dapi represents stained nucleus and Alexa 633 represents cell membranes stained with WGA; for all samples, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities. a) Media only as negative control – b) Cells were incubated with 1 mg/mL of dHPG(C8) (on the left) and dHPG(C8)/Pro (on the right), where Pro concentration was 12.5 µg/mL .................... 130 Figure 5.14 Uptake of dHPG(C8), with or without Pro, by MCF7 cells after 4 h at 37 °C; Alexa 488 represents labelled polymer, Dapi represents stained nucleus and PR represents cells stained with  xxiii  anti-progesterone receptor antibody; for all samples, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities. a) Media only as negative control – b) Cells incubated with PR as control (on the left) and incubated with Pro and PR (on the right) – c) Cells incubated with dHPG(C8) at 1 mg/mL and PR (on the left) and cells incubated with dHPG(C8)/Pro (at 12.5 µg/mL of drug) and PR (on the right) ................. 131 Figure 5.15 Uptake of dHPG(C8), with or without Pro, after 6 h at 37 °C; Alexa 488 represents labelled polymer, Dapi represents stained nucleus and PR represents cells stained with anti-progesterone receptor antibody; for all samples, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities. a) Cells incubated with PR as control (on the left) and incubated with Pro and PR (on the right) – b) Cells incubated with dHPG(C8) and PR (on the left) and cells incubated with dHPG(C8)/Pro and PR (on the right).......................................................................................................................... 132 Figure 6.1 General illustration of the Pro bound into dHPG(Cn) formulation and release properties..................................................................................................................................... 139 Figure 6.2 C20 presents the more electrophilic site of Pro for modification ............................... 142 Figure 6.3 Oxime linkage on Pro ................................................................................................ 143 Figure A.1 1H NMR spectrum of dHPG(C6) in CDCl3 .............................................................. 159 Figure A.2 GPC chromatogram of dHPG(C6) ............................................................................ 159 Figure A.3 1H NMR spectrum of dHPG(C10) in CDCl3 ............................................................. 160 Figure A.4 GPC chromatogram of dHPG(C10) ........................................................................... 160 Figure A.5 1H NMR spectrum of dHPG(C14) in CDCl3 ............................................................. 161 Figure A.6 GPC chromatogram of dHPG(C14) ........................................................................... 161 Figure A.7 1H NMR spectrum of dHPG(C18) in CDCl3 ............................................................. 162  xxiv  Figure A.8 GPC chromatogram of dHPG(C18) ........................................................................... 162 Figure A.9 Inverse-gated 13C NMR of dHPG(C10) in methanol-d4 proves the branched structure of dHPG(Cn)................................................................................................................................ 163 Figure B.1 Binding profile of Pro loaded on dHPG(C6) ............................................................. 164 Figure B.2 Binding profile of Pro loaded on dHPG(C8) ............................................................. 164 Figure B.3 Binding profile of Pro loaded on dHPG(C10) ........................................................... 165 Figure B.4 Binding profile of Pro loaded on dHPG(C12) ........................................................... 165 Figure B.5 Binding profile of Pro loaded on dHPG(C14) ........................................................... 166 Figure B.6 Binding profile of Pro loaded on dHPG(C18) ........................................................... 166 Figure C.1 DLS size determination of dHPG(C10) at 2 mg/mL (on the left) and dHPG(C10)/Pro at 2 mg/mL of polymer and 25 µg/mL of Pro (on the right) ...................................................... 167 Figure D.1 dHPG(C8)/Pro release profile in PBS at 37 °C and pH 7.4 ....................................... 168 Figure D.2 dHPG(C10)/Pro release profile in PBS at 37 °C and pH 7.4 ..................................... 168 Figure D.3 dHPG(C12)/Pro release profile in PBS at 37 °C and pH 7.4 ..................................... 169 Figure D.4 dHPG(C14)/Pro release profile in PBS at 37 °C and pH 7.4 ..................................... 169 Figure D.5 dHPG(C18)/Pro release profile in PBS at 37 °C and pH 7.4 ..................................... 170 Figure D.6 Free Pro release profile in PBS at 37 °C and pH 7.4 ................................................ 170 Figure D.7 HPG/Pro release profile in PBS at 37 °C and pH 7.4 ............................................... 171 Figure D.8 HPG-PEG/Pro release profile in PBS at 37 °C and pH 7.4 ...................................... 171 Figure E.1 dHPG(C10)/Pro; rapid release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.97 and p < 0.01) ....................................................................................................................... 172 Figure E.2 dHPG(C10)/Pro; slow release rate in PBS – k2(s-1) calculated from the slope (R2 = 0.99 and p < 0.01) ....................................................................................................................... 172  xxv  Figure E.3 dHPG(C12)/Pro; rapid release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.97 and p < 0.01) ....................................................................................................................... 173 Figure E.4  dHPG(C12)/Pro; slow release rate in PBS – k2(s-1) calculated from the slope (R2 = 0.86 and p < 0.01) ................................................................................................................................ 173 Figure E.5 dHPG(C14)/Pro; rapid release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.96 and p < 0.01) ....................................................................................................................... 174 Figure E.6 dHPG(C14)/Pro; slow release rate in PBS – k2(s-1) calculated from the slope (R2 = 0.99 and p < 0.01) ....................................................................................................................... 174 Figure E.7 dHPG(C18)/Pro; rapid release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.99 and p < 0.01) ....................................................................................................................... 175 Figure E.8 dHPG(C18)/Pro; slow release rate in PBS – k2(s-1) calculated from the slope (R2 = 0.95 and p < 0.01) ...................................................................................................................... 175 Figure E.9 HPG/Pro; release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.98 and p < 0.01) ............................................................................................................................................ 176 Figure E.10 HPG-PEG/Pro; release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.98 and p < 0.01) ...................................................................................................................................... 176 Figure E.11 Free Pro; rapid release rate in plasma – k1(s-1) calculated from the slope (R2 = 0.95 and p < 0.01) ...................................................................................................................... 177 Figure E.12 Free Pro; slow release rate in plasma – k2(s-1) calculated from the slope (R2 = 0.89 and p < 0.02) ............................................................................................................................... 177 Figure E.13 dHPG(C8)/Pro; rapid release rate in plasma – k1(s-1) calculated from the slope (R2 = 0.96 and p < 0.01) .................................................................................................................... 178  xxvi  Figure E.14 dHPG(C8)/Pro; slow release rate in plasma – k2(s-1) calculated from the slope (R2 = 0.98 and p < 0.01) ............................................................................................................ 178 Figure F.1 Red blood cell aggregation in the presence of dHPG(Cn); a) dHPG(C12) at 1 mg/mL; b) dHPG(C14) at 1 mg/mL........................................................................................................... 180 Figure F.2 Red blood cell aggregation in the presence of dHPG(Cn), with or without Pro; a) dHPG(C12) at 10 mg/mL; b) dHPG(C12)/Pro, where polymer concentration was 10 mg/mL loaded with 125 µg/mL Pro; c) dHPG(C14)/Pro, where polymer concentration was 10 mg/mL loaded with 125 µg/mL Pro; d) dHPG(C18)/Pro, where polymer concentration was 10 mg/mL loaded with 125 µg/mL Pro ........................................................................................................ 181 Figure F.3 dHPG(C8) at 10 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right .................................................................................. 182 Figure F.4 dHPG(C8) at 1 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right .................................................................................. 182 Figure F.5 dHPG(C12) at 10 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right .................................................................................. 183 Figure F.6 dHPG(C12) at 1 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right .................................................................................. 183 Figure F.7 SEM images of PRP dynamic clots formed in the presence of dHPG(C8); a) HBS control buffer; b) dHPG(C8) at 0.1 mg/mL; c) dHPG(C8) at 1 mg/mL .................................... 184 Figure G.1 1H NMR spectrum of dHPG(C8) modified with EPP in CDCl3 – Peaks at 7.59 ppm and 7.69 ppm belong to the aromatic ring of EPP confirm the modification ............................. 185 Figure G.2 1H NMR spectrum of dHPG(C8) modified with amine groups in D2O confirms the absence of EPP ............................................................................................................................ 186  xxvii  Figure H.1 Uptake of dHPG(C8) by HCMEC/D3 cells visualized by anti-lysosome antibody (LAMP1) at 37 °C; Alexa 488 represents labelled polymer, Dapi represents stained nucleus and LAMP1 represents anti-lysosome antibody; for all time points, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities; yellow signal represents the convergence of the polymer and lysosome. a) Media control – b) after 2 h; cells were incubated with dHPG(C8) (on the left) and dHPG(C8)/Pro (on the right) – Polymer and Pro concentrations were 0.01 mg/mL and 0.125 µg/mL, respectively ..... 187   xxviii  List of Symbols  ACN   Acetonitrile ADP   Adenosine diphosphate ANCOVA  Analysis of Covariance APTT   Activated partial thrombin time BBB   Blood brain barrier BHPG   Biodegradable hyperbranched polyglycerol CCF   Human astrocytoma cells CDCl3   Deuterated chloroform CHO   Chinese Hamster Ovary CNS   Central nervous system Da   Dalton DCM   Dichloromethane dHPG(Cn)  Hydrophobically derivatized hyperbranched polyglycerol dHPG(Cn)/Pro  Progesterone loaded hydrophobic hyperbranched polyglycerol DMF   Dimethylformamide DSC   Differential scanning calorimetry GPC   Gel permeation chromatography h   hour HCMEC  Human cortical microvascular endothelial cell HBS   HEPES buffered saline HPG   Hyperbranched polyglycerol  xxix  kDa   Kilo Dalton Ln   Natural logarithmic MALLS  Multi-angle light scattering MCF7   Michigan Cancer Foundation-7  Methanol-d4  Deuterated methanol min   Minute MPEG   Methoxy poly(ethylene glycol) Mn   Number average molecular weight Mw   Weight average molecular weight MWCO  Molecular weight cut off NaNO3   Sodium nitrate PBS     Phosphate buffered saline PDI   Polydispersity (Mw/Mn) PEG   Poly(ethylene glycol) PPP   Platelet poor plasma Pro   Progesterone PRP   Platelet rich plasma P-value  Probability R2   Coefficient of determination RBC   Red blood cell aggregation Rh   Hydrodynamic radius SEM   Scanning electron microscopy t1/2   Plasma half-life  xxx  TBI   Traumatic brain injury TEG   Thromboelastography TMP   Trimethylolpropane UPLC   Ultra performance liquid chromatography UV   Ultraviolet    xxxi   Glossary  ANCOVA: Analysis of Covariance (ANCOVA) is a general linear model which combines analysis of variance (ANOVA) and regression; it evaluates similarity of two regression models with a statistical significance level. ANOVA:  Analysis of variance (ANOVA) is a statistical method used to compare the means of two or more groups. Probability: A number between 0 and 1 describing the chance that a stated event will occur. R2: A number that indicates how well data fit a statistical model; R2 = 1 implies a perfect fit. Regression analysis: A statistical process for estimating the relationships among variables. Student t-test: Analysis tool test for equality of the population means.    xxxii  Acknowledgements  This dissertation would not have been possible without the support of many individuals. I would like to extend my appreciation to my supervisor, Professor Don Brooks for providing me the opportunity to work in his laboratory with an inspiring lab environment which allowed me to learn and grow, and for his continuous consideration, encouragement and support along the way. I am also very grateful to my co-supervisor, Dr. Jayachandran Kizhakkedathu for providing guidance and directions throughout this study. Special thanks go to Dr. Rajesh Shenoi for being my mentor and a great polymer chemistry teacher. I am grateful to Irina Chafeeva for her great help in teaching me the basics of HPG synthesis, GPC experiments and analysis. I also thank Dr. Johan Janzen for his excellent technical assistance.  I would like to thank Dr. Lawrence Amankwa and Dr. Clement Mugabe at the Centre for Drug Research and Development (CDRD) at UBC for the outstanding UPLC method they have developed and providing me the opportunity to work in their laboratory and learn the UPLC and DSC techniques as well as for ongoing discussions.  Special thanks go to Dr. Cheryl Wellington and her research group at Centre for Brain Health (CBH) at UBC, especially to Dr. Jerome Robert for his great help on the brain studies and discussions.  I would also like to thank the UBC NMR lab staff, particularly Dr. Maria Ezhova for her expert assistance. I am thankful to my colleague and friend at Centre for Blood Research (CBR) at UBC, Benjamin Lai, who has been a great help in directing blood experiments and sharing the knowledge. To my past and present lab mates at CBR at UBC; Manu Thomas Kalathottukaren,  xxxiii  Narges Hadjesfandiari, Jasmine Hamilton, and our postdoctoral fellows; Dr. Srinivas Abbina, Dr. Nima Khademmohtaram, Dr. Anil Parambath and Dr. David Yang. I thank you all for your friendship, support, encouragement and positive atmosphere throughout this work.  Most of all, special thanks are owed to the most caring and dedicated people in my life; my husband, for his timely and constant encouragement, enthusiasm, and unwavering support and love, and to my parents for their unconditional love and unrelenting spiritual supports throughout all these years.  xxxiv  Dedication  To my loving husband, Masoud, for being the biggest inspiration and support, and the best company during the PhD program and ever in my life.    To my devoted parents, Mehdi and Akram, for every sacrifice they have made for me and convincing me that I could achieve anything.  To my brothers, Siamak and Kyoomars, for being my best friends forever and never leaving my side. 1  Chapter 1: Introduction  1.1 Blood Brain Barrier The central nervous system (CNS) consists of the brain and spinal cord (Figure 1.1), and is separated from the circulation by the blood brain barrier (BBB). The BBB is recognized as the main element for precisely controlling the environment of neurons in the CNS for their optimal function. It firmly controls the exchanges between blood and brain compartments, providing protection to the brain against toxic compounds and pathogens [1], [2]. The BBB is known as one of the most challenging obstacles for the treatment of CNS disease [3].   Figure 1.1 Central nervous system (CNS)  The BBB cells consist of endothelial tight junctions, prohibiting paracellular transport of macromolecules [4], and physically restricting the exchange of solutes between the blood and the brain (Figure 1.2). Protecting the brain from its extracellular environment, continuously providing BrainSpinal cordCNS 2  nutrients for the brain by a specific transport system, maintenance and regulation of the CNS and the neuronal microenvironment are reported as main responsibilities of the BBB [2], [5]. Therefore, application of neuropharmaceuticals to deliver drugs to the CNS is highly restricted due to the brain barrier cells which limit molecules larger than 500Da from accessing the CNS [6].      Figure 1.2 Blood brain barrier (BBB) structure  Zebrafish, which have a BBB functionally homologous to humans’, were studied as a novel in vivo model for understanding the functionality and structure of the BBB as it possesses many appropriate characteristics for study of drug delivery to the human BBB [7].   Brain endothelial cell (BBB)Tight junctionsNeuronsAstrocytomaRed blood cellCapillary 3  1.2 Traumatic Brain Injury 1.2.1 Brain Injury, Complexity and Treatments Traumatic Brain Injury (TBI) is a significant public health problem and by far the most common cause of morbidity, mortality and disability in North American adults under the age of 40 [8]. It is also known as a major problem in the elderly, among whom falls lead to brain injury. The most common causes are attributed to motor vehicle crashes, falls and violence [9]. Pediatric TBI can also lead to development of injury and life-long disability among children [10]. For over 10 years, it has been reported that many physiological processes, including production of an oxidative stress reaction in heart, kidney, lung, liver and other body tissues can take effect in the first 24 h after a closed head injury [11]. TBI has been established as a risk factor for Alzheimer’s disease (AD) through linkage with pathologically confirmed AD in several individual case reports [12], [13]. Systemic toxic events in the brain are a known cause of TBI [14]. Therefore, TBI, a very complex disease, presents significant short and long-term challenges to worldwide health.  The brain injury cascade initiates rapidly as a result of forces that produce tissue distortion at the moment of injury, which can cause long-lasting physical and psychological dysfunction in both mild and severe cases [15], [16]. These deformations lead to a primary injury, which is considered irreversible and directly affects blood vessels, axons, neurons and glia. Following that, secondary processes cause complex inflammatory, cellular, neurochemical and metabolic modifications [17], [18]. Also, other reports confirm that the consequences of head injury could affect heart, kidney, lung, liver and other body tissues in the first 24 h after injury [11].  In preclinical studies of TBI, over 30 therapeutic candidates failed to improve the clinical outcomes [19]. However, several therapeutic agents have been used in an animal TBI model and some improvements have been made; Cyclosporine (an immunosuppressive drug in clinical use)  4  was used due to its neuroprotective properties. Although the drug could not reduce the mortality rate, it improved the functional outcomes [20]. A modified form of erythropoietin, Darbepoetin alfa (Aranesp), demonstrated a decrease in oxidative stress in injured brain tissue [21], [22].  Glyburide also demonstrated a beneficial impact in reducing progressive hemorrhagic (abnormal bleeding), lesion volume and neurobehavioral dysfunction [23].  1.2.2 Progesterone as a Good Candidate for TBI Pharmacotherapy While there was no established pharmaceutical agent among current agents in clinical trials that could enhance functional outcomes following TBI, the neurosteroid progesterone (Pro), first recognized by Baulieu and his colleagues, appeared to hold considerable therapeutic promise [24], [25]. Pro (Figure 1.3), a naturally occurring steroid hormone, has been considered for a long time only as a female reproductive hormone. However, studies show the beneficial role of Pro in other treatments such as ischemic stroke [26], [27] and axonal degenerative disorder [28]. Additionally, reports confirm Pro as a post-injury treatment with acute and traumatic injuries of brain and spinal cord [29]. Hormonal Pro, produced by the adrenal glands, exists in both male and female brains and plays a key role in sustaining the level of myelin in some types of neurodegenerative disorders [30]. This hormone is synthesized by oligodendrocytes and some neurons in approximately equal amount in the brains of male and female [31]. Pro may retain its neuroprotective properties by increasing the viability of neurons in the brain and spinal cord as well as promoting the formation of new myelin sheaths. Also, Pro is demonstrated to be permeable to the blood-brain barrier [32]. Treatment with Pro improves behavioural recovery and reduces inflammation, apoptosis, cerebral edema, neuronal cell death, and improves functional recovery when given after TBI [33].   5   Figure 1.3 Chemical structure of progesterone (Pro)  Evidence confirms a gender difference in vulnerability to TBI in humans in terms of risk and consequence; better predicted functional outcome (p < 0.015) was observed in women compared to men of the same age with similar severity, suggesting that Pro, acting as a neuroprotective agent, may explain the results [34]. The neuroprotection potential of Pro was initially exposed by the observation that female rats recover better from TBI than male rates in terms of functional impairments, and the recovery in females with the higher level of internal Pro was better at the time of injury [35].  1.2.3 Clinical Studies of Pro in TBI Based on studies of the neuroprotective effects of Pro, the first randomized clinical trial, ProTECT I (Progesterone for Traumatic Brain injury, Experimental Clinical Treatment), was designed in Atlanta, Georgia, including 100 trauma patients with moderate to acute TBI [36]. The outcome reported that Pro was well tolerated in terms of safety and potential benefits of its administration after the head injury. Followed after randomly receiving Pro intravenously, patients showed lower 30-day mortality rate than controls in severe cases. However, patients with moderate, rather than acute, TBI presented better neurologic results. Prolonged infusion of high  6  doses of Pro presented no harm in this trial [36]. Therefore, based on several experimental studies, which provided supporting evidence, Pro is a potent neurosteroid, which reduces lesion volume, cerebral edema, inflammation, and provides support to damaged neurons, when administered immediately [37].   Despite its efficiency, the major challenge to widespread use of Pro for TBI is its aqueous insolubility due to its hydrophobicity, which contributes to an inability to rapidly administer the drug after injury and store and transport stable Pro formulations [36], [37]. Plasma half-life (t1/2) of Pro is reported as short as 25 min [38]. To overcome this issue, synthesis of water-soluble analogues of Pro has been reported with sustainable efficacy in an  animal model of TBI, resulting in improved solubility and pharmacokinetic properties of the drug [37], but their efficacy in clinical studies is not known yet.  1.3 Human Blood Components  Plasma is the liquid phase of blood, in which blood cells are suspended. In addition, it contains a huge number of proteins, glucose, mineral ions and many other components, including hormones.   1.3.1 Red Blood Cells (Erythrocytes) Red blood cells (RBCs) are the most common type of blood cells. Their cytoplasm contains hemoglobin; its main role is to transport oxygen to the body tissues through the circulation. RBCs contain no nucleus and have flexible membranes which helps them to pass through smaller spaces than their diameter [39].     7  1.3.1.1 Red Blood Cell Aggregation RBCs naturally aggregate in plasma, forming linear aggregates known as rouleaux. However, when RBCs form stronger aggregation, which is hard to breakdown, they increase blood viscosity (Figure 1.4) [40].     Figure 1.4 RBC aggregation (on the left), in which cells lose their normal rouleaux shape (on the right); these images are taken from the incubation of derivatized hyperbranched polyglycerols with RBCs at higher (on the left) and lower (on the right) concentrations which are discussed in Chapter 4  1.3.1.2 Red Blood Cell Lysis (Hemolysis) When the membrane of a RBC breaks, it is called hemolysis. Loss of RBC deformability, phagocytosis by macrophages, or disruption of membrane integrity are some of the possible mechanism of hemolysis [39].  1.3.2 White Blood Cells (Leukocytes) These cells are the blood immune cells and are responsible for the body’s defence against infections, diseases and foreign attackers, and are located in the blood and lymphatic system [39].  8  1.3.3 Platelets Platelets are the smallest cells (1-3 microns) in the blood, one fourth of the size of RBCs. Their role is to plug holes in blood vessels. When an injury happens in a blood vessel, platelets are activated and adhere to the injured site, forming a mass to prevent bleeding [39].  1.4 Blood Coagulation When injury occurs, a blood clot must form quickly in order to stop bleeding (thrombosis). However, the formed clot should be only limited to the injured site. It is essential that clot formation and clot lysis should be in balance to stop bleeding, while not interfering with blood flow.  Coagulation is the process that changes the blood from liquid to gel. This process involves a series of enzymatic reactions. The coagulation cascade has two initial pathways and both will lead to fibrin formation, which is a protein that forms from cross-linked fibrinogen in and around platelet plugs in the coagulation process and strengthens the platelet plug. In both pathways, prothrombin (coagulation factor II) cleaves to form thrombin, which converts soluble fibrinogen into insoluble fibrin (Figure 1.5) [41], [42].    9    Figure 1.5 Coagulation cascade: intrinsic and extrinsic pathways   1.5 Biocompatibility Biomaterials are materials that have the ability to exist in contact with human body tissues without causing undesirable harm or damage. Their behaviours are usually investigated under the subject of biocompatibility. Biocompatibility traditionally refers to implantable devices or materials that are aimed to remain in a body for a long time, but has broader implications for medically oriented techniques such as tissue engineering, drug delivery and gene transfection systems.  When a new compound is placed in the body, several reactions may occur, such as cellular toxicity, neutrophil activation, platelet activation, activation of the clotting cascade, red blood cell  10  aggregation, hemolysis or even tumor formation. Based on that, the biocompatibility characteristics of any synthetic materials or devices which are designed for any application in the body should be examined in vitro before attempting an in vivo study [43], [44].  1.6 Nanoparticles 1.6.1 Polymer-Based Nanodevices Due to the limitations of specific targeting agents and therapeutics designed to react inside cells, developing a smart delivery system for transporting drugs to target cells remains challenging [45]. However, nanotechnology has created a growing list of nanoparticles aimed at regulating processes in living cells. Nanoparticles are materials or devices with nanoscale diameters between 5 and 1000 nm, such as natural or synthetic polymers, lipids or phospholipids, liposomes, and carbon nanotubes. These materials are assembled from different biodegradable ingredients with different physical and chemical properties [46]. Such flexibility in design enables them to serve in specific intracellular applications such as drug delivery, in which drugs may be encapsulated, absorbed or dispersed [47]–[49]. Over the years, research has indicated that controlling size, shape and chemical targeting of nanoparticles can significantly improve specific in vivo delivery to pathological sites. For instance, a glycol chitosan nanoparticle was successful in tumor-targeted delivery during many in vivo experiments [50]. These delivery systems can be injected directly into the systemic circulation due to their nanometre scale without blocking blood vessels [51], [52]. Nanoparticles can serve as drug delivery vehicles, offering several advantages over conventional delivery models, by maintaining, through controlled release kinetics, the optimum  11  therapeutic concentration of drug in the blood or cells, which increases patient satisfaction as a result of reduction in frequency of dosing [53], [54].  Chemical modification of nanoparticles with different biomolecules, such as particular proteins or antibodies, enables them to specifically target receptors in disease cells. Nanoparticles’ relative stability is required so they can carry their payload through blood and across the cell membrane. However, they should be able to break down and release their cargo without causing toxicity [55].  As previously reviewed, polymer micelles ranging from 10 to 100 nm in diameter are formed when amphiphilic block copolymers are dissolved in water at or above their critical micelle concentration (CMC). The hydrophobic core of these systems enables them to encapsulate hydrophobic molecules, such as drugs and hormones, through hydrophobic interactions, while the hydrophilic shell keeps them soluble in water (Figure 1.6). The outer shell provides a protective interface between the micelle core and external medium. The surface of the carriers can be designed to try to avoid recognition by host defense systems, leading to longer circulation half-lives [56]–[59]. The ease of chemical modification of these copolymers allows for optimization of drug loading, release and surface modifications of target moieties. However, the tumor microenvironment can cause limitations for effective transport of drugs to the tumor site via in vivo block copolymer mediated delivery, including stability in the blood compartment and prolonged circulation, penetration into deep layers of tumor tissue and tumor bioavailability of the drug within cancer cells [60], [61].     12    Figure 1.6 General structure of polymer micelle  Nanocarriers represent engineering of particles smaller than 100 nm and in general can better deliver drugs to protected sites within the body due to their greater access compared to larger scale systems. Therefore, nanotechnology promises to link the gaps between the structure and the functions of biomolecules and larger scale materials by improving the problems of short half-life and poor bioavailability of unprotected biotech drugs. However, several limitations are reported such as difficulties in handling and storage of nanocarriers, mainly because of their aggregation tendency [45].   1.6.2 Dendrimers and Hyperbranched Polymers Dendrimers and hyperbranched polymers are macromolecules that are characterized by a highly branched structure. Their three-dimensional, highly-branched, multi-functional, and special chemical and physical properties make them strong candidates for many biological applications. Dendrimers are large, complex molecules with a regular and perfectly branched structures, and A-B block copolymerHydrophobic blockHydrophilic blockHydrophobic molecule 13  originated by Fritz Vogtle in 1978 [62] and Didier Astruc in 1979 [63]. These molecules consist of two types of structural units, terminal and dendritic. They are monodisperse and demonstrate a defined end-group multiplicity and functionality (Figure 1.7), but they are prepared in a time-consuming, stepwise synthetic method, which limits their practical use. Hyperbranched polymers, on the other hand, have three types of structural units, linear, terminal and dendritic. Linear and dendritic units are randomly distributed, resulting in an irregular structure (Figure 1.8). Dendrimers and hyperbranched polymers are used for design of homogeneous or amphiphilic dendritic polymers as they can carry numerous functional groups [64], [65] .    Figure 1.7 Example of a dendritic structure;   refers to the core and   refers to the branching points   14   Figure 1.8 Example of a hyperbranched structure;   refers to the core and   refers to the branching points  Hyperbranched polymers are highly but imperfectly branched macromolecules with three-dimensional dendritic architecture. Their unique physical and chemical properties, make them potential candidates in many fields from drug-delivery to coatings [66].  Compared to dendrimers, hyperbranched polymers can be prepared in a convenient single pot through a random polymerization of multifunctional monomers of the ABm-type. Therefore, this type of polymer can be prepared in a less expensive way, which makes them potential alternatives to dendrimers when an exact branched structure is not necessary [67], [68].  1.7 Hyperbranched Polyglycerol (HPG)-Based Nanomedicine Recently branched (dendritic) polymers with a covalently linked hydrophobic core and hydrophilic shell, resembling micellar characteristics in a single molecule, have been developed to overcome the instability of conventional polymer micelles in the blood stream. As a result, they  15  have been designated by some as “unimolecular micelles” with no CMC yet have an amphiphilic structure [69]. Their natural stability to various environmental effects such as dilution, shear force, and pH combined with their binding capacity makes these formulations excellent drug delivery candidates [70], [71]. Moreover, they are smaller (with hydrodynamic radius (Rh) less than 10 nm), and have correspondingly low intrinsic viscosities; they are also denser and diffuse faster than conventional polymer micelles [72], [73]. The outer surface of these molecules can be derivatized with suitable functionalities for enhanced solubility and multivalent interactions for enhanced drug delivery [74], [75].   Slow monomer addition has become the successful way to overcome the large polydispersity of hyperbranched polymers, resulting in a controlled polymerization. Degree of branching (DB) of these polymers is controlled by the nature and functionality of internal constructing blocks and the chain ends [72], [76]. Glycidol, a highly reactive hydroxyl epoxide, represents a cyclic AB2-type monomer, which can be polymerized to hyperbranched polyethers through a ring-opening multibranching step [76]; the first attempt to polymerize it was by Sandler and Berg [77]. The first branched structure of polymerization of glycidol was characterized by Vandenberg et al [78], but low polydispersities (PDIs) were not achieved until 1999 when controlled synthesis of hyperbranched polyglycerols (HPG), through anionic ring-opening multibranching polymerization of glycidol with low PDI and predetermined molecular weight was reported by Sunder et al [76]. In this method, anionic polymerization of glycidol occurred through partial deprotonation of an alkoxide initiator (trimethylolpropane (TMP)) and slow monomer addition. Both intra and intermolecular transfer steps of hydrogen transfer after the ring-opening step results in a branched structure.    16    Figure 1.9 Structure of HPG  Synthesis and characterization of very high molecular weight HPG (Figure 1.9) with low polydispersity and hydrodynamic radius of less than 10 nm, in the presence and absence of emulsifying agents, has been reported by the group at the Centre for Blood Research (CBR) at the University of British Columbia [79]. These polymers potentially can be used as components in adhesives, advanced coatings as well as hydrogels and composites. Furthermore, they can have applications in drug delivery and nanotechnology.    17  1.7.1 Biocompatibility and Applications  1.7.1.1 Biocompatibility of Poly(ethylene glycol) and HPGs Poly(ethylene glycol) (PEG) is reported to have broad application as a covalent modifier of biological macromolecules as well as a suitable carrier for low molecular weight drugs as it is found to enhance biocompatibility. This polymer is non-toxic, very weakly immunogenic, and highly soluble in water. The application of conjugation of drugs and proteins with PEG has been reported repeatedly [80]–[83]. Using higher molecular weight PEGs (40,000 Da) resulted in long plasma circulation half-life of about 10 h in mice, which led the system to be a good clinical candidate. These polymers dissolve in both water and organic solvents. Being non-toxic, with easy elimination pathways if small enough to pass out via the kidney, makes them strong candidates in pharmaceutical applications [84].  Conjugation of proteins with PEG reduces the recognition of protein by the host defense system, inhibiting them from rapid clearance [85]. Nonspecific protein adsorption to surfaces such as catheters, implants or artificial organs which are exposed to the biological environment, may cause irritation, chronic infections or allow recognition as a foreign material. To prevent such unfavorable protein adsorption, coating the surface with linear, hydrophilic and water-soluble PEG has been reported [86]–[88]. It is also well understood that coating or grafting of PEG chains onto the surfaces reduces platelet adhesion, as well as providing longer circulation half-lives, resulting in strong therapeutic promise. Reports suggesting that dendritic PEG provides highly protein resistant surfaces through their hydrophilicity and hyperbranched structure have also appeared [89].  High molecular weight HPGs, carrying a large number of reactive sites per molecule, are also shown to be powerful candidates for various applications in nanomedicine, suggesting roles  18  for these materials as transporters or protein substitutes in nanobiotechnology [90]. These polymers are compact and have a spherical conformation in water with no sign of aggregation [79]. The circulation half-life in mice depends on the molecular weight of the polymer, reaching almost 60 h for 540,000 Da and can be controlled by manipulating the molecular weight [91]. Unlike many other polymers and carriers used in nanomedicine, HPGs have shown very limited organ accumulation after intravenous injection (Table 1.1) [91], [92]. They are highly biocompatible, water-soluble, inexpensive and easy to synthesize with good polymerization control. Derivatization of HPG with active groups can affect their properties, requiring some additional modification, typically by adding PEG protection. Modification of derivatized HPGs with PEG chains enhances aqueous solubility, stabilizes against any aggregation that derivatization might produce and protects against host defence that might be activated by the derivatized material. PEG decoration enhances the circulation half-life and helps the system serve as a delivery vehicle in a more effective manner [53].      19  Sample Mn (Da) (Mw/Mn) Blood Compatibility Hydrodynamic Radius (Rh) (nm) Plasma Half Life (h) @ Organ Accumulation  (% in dose) Liver Spleen 1 h 48 h 1h 48 h 1HPG 106,000  (2.9) Excellent  (up to 100 mg/mL) 4.8 32.4 ± 2.7 5.5 9.6 4.1 7.7 1HPG 540,000  (1.1) Excellent (up to 100 mg/mL) 6.8 57.5 ± 9.1 6.4 10.2 5.1 10.2 2dHPG 40,000  (1.6) Excellent  (up to 100 mg/mL) 6.5 24.9 ± 6.2 3.1 3.2 1.8 2.6 3dHPG 83,000  (1.8) Excellent  (up to 100 mg/mL) 7.7 31.0 ± 2.9 2.9 3.5 2.4 3.2  Table 1.1 Circulation half-life and organ accumulation of radio-labelled HPGs and their derivatives in mice 1. Unmodified HPGs, lower and higher molecular weights - Total injected dose for unmodified HPGs was 1000 mg/kg [91].  2. dHPG(C18) is a hydrophobically modified drug-binding HPG; composition: 78 mole% Polyglycerol, 20.4 mole% MPEG and 1.6 mole% C18 chains per HPG molecule. Total injected dose was 500 mg/kg [92].  3. dHPG(C18) is a hydrophobically modified drug-binding HPG; composition: 69 mole% Polyglycerol, 29.4 mole% MPEG and 1.6 mole% C18 chains per HPG molecule [92].  Total injected dose was 500 mg/kg. # Mn - number average molecular weight; Mw/Mn - polydispersity; @ - % of injected treated HPGs    20  1.7.1.2 Applications of HPGs Due to the structural similarity between HPGs and PEG, HPGs are also expected to be biocompatible. High biocompatibility of HPGs and their ability to serve as potential candidates for conjugation of drugs and bioactive molecules such as peptides and carbohydrates were reported in several cases [93]–[97]. Studies from our group showed that high molecular weight HPGs, (up to 700,000 Da) are blood compatible with insignificant effects on red blood cell aggregation, platelet activation, coagulation time and cytotoxicity [90]. HPGs are also reported to be highly biocompatible in vivo; they are hydrophilic, non-immunogenic and non-toxic with no evident animal toxicity. Their compact structure removes many disadvantages associated with the exposure of high molecular weight linear polymers to blood and cells [79], [91], [92], [98].  Covalent linking of biologically compatible and hydrophilic macromolecules (e.g. PEG) to a cell membrane helps to minimize immunogenicity, enzymatic degradation and improves the compatibility of the cell in vivo with no harm to the tissues [43], [99]–[103]. Based on that observation, modification of some of the functional groups of HPGs in order to covalently bind them to red blood cells (RBC) has been studied. Results demonstrated good cell viability and low toxicity [104]. Furthermore, in vivo studies demonstrated that covalently binding desferrioxamine (DFO) – the iron chelator used for treatment of “iron overload” disorder – to modified HPG improved DFO’s toxicity and short plasma half-life. HPG-DFO conjugation was successful in removing the excess of iron with no detectable cellular toxicity. The results were supported by excellent outcomes of blood compatibility analysis [105]–[107]. Synthesis and application of HPGs modified with anionic groups as heparin analogues and as anti-inflammatory agents have been reported [108]–[110]. Cationically derivatized HPGs have  21  also been reported to bind heparin-based anticoagulant drugs – used widely in surgery, dialysis and vascular treatments – as a method to reverse anticoagulation where necessary to improve safe use and bleeding risks associated with these drugs. The safety and efficacy of this polymeric therapeutic was supported by in vitro and in vivo results [111]. Application of HPG functionalized with cationic groups in binding nucleic acids to serve as potential delivery system has been reported as well [112], [113].   1.8 Hydrophobically Derivatized Hyperbranched Polyglycerols dHPG(Cn) Synthesis and application of HPGs, modified with aliphatic hydrophobic chains (presumably in the core) and methoxy poly(ethylene glycol) (MPEG) chains (presumably as a shell), have been reported by our group. We have synthesized HPG with a hydrophobic character by including some branches terminated with alkyl groups through a single pot synthetic strategy; the hydrophobic fraction provides a suitable region for binding hydrophobic molecules. The polymer as a whole is protected by MPEG chains (Figure 1.10). Combination of these modifications enables the polymeric system to encapsulate hydrophobic drugs and molecules, and enhance the solubility of the drug, stability of the formulation, and circulation half-life. The binding capacity of these polymers for encapsulation of hydrophobic species can be tuned by the hydrophilic to hydrophobic ratio. Hyperbranched polymers modified with C18 alkyl chains and MPEG 350 chains (dHPG(C18)), demonstrated only a small effect on the plasma viscosity.  The dHPG(Cn) could effectively bind fatty acid and pyrene; they showed no sign of activation of the complement system, platelets or coagulation. Also no blood cell aggregation was reported after incubation with dHPG(Cn), although HPG-C18-PEG does absorb to RBCs [114]. Circulation half-life of these polymers in mice varied between 25 to 34 h, which was controlled  22  by the molecular weight as well as the PEG content of the polymer. Administration of derivatized HPG polymers through intravenous injection in mice produced no sign of toxicity and organ abnormality. Studies demonstrated that coagulation effects of dHPG(Cn) depended on their molecular weight as well as the rate of in vivo degradation [53].      Figure 1.10 Structure of dHPG(C18); R refers to the 1,2-epoxyoctadecane monomer after epoxide ring is opened up through the anionic ring-opening reaction  Development of a second-generation synthetic substitute for human serum albumin (HSA), the most abundant plasma protein [115], based on hydrophobically derivatized material has been reported elsewhere [92]. This material holds advantages over the current linear plasma expanders used clinically (such as dextran, hydroxyethyl starch and collagen), as these expanders have shown  23  significant failures in increasing plasma viscosity, resulting in red cell aggregation (Figure 1.11 and Figure 1.12). dHPG(Cn) have demonstrated low intrinsic viscosity because of their highly branched structure. As a result, they only slightly increase plasma viscosity. Another advantage of these polymers over current plasma expanders is that they can be administered repeatedly without causing changes in plasma half-lives [116]–[119]. Moreover, they are unique in having capacity to bind and transport fatty acids, one of the major functions of HSA.   Figure 1.11 Dextran   Figure 1.12. Hydroxyethyl starch  HPGs derivatized with hydrophobic groups (HPG-C8/10-MPEG) with a hydrodynamic radius of less than 10 nm have also been reported as good candidates as drug delivery systems for hydrophobic anticancer drugs, such as paclitaxel and docetaxel. These nanoparticle formulations were non-toxic and rapidly taken up into cells, and also provided continuous controlled release of   24  the drugs [120], [121]. Furthermore, the surface modification of HPG-C8/10-MPEG with amine groups resulted in a charged nanoparticle, with developed in vitro and in vivo properties as a treatment for bladder cancer. Continuous controlled release of docetaxel was also observed, compared to dHPG(Cn) with no amine modifications [122]. These HPGs, even with loaded drugs, form nanoparticles less than 10 nm in size, resulting in significantly smaller nanoparticles than other polymeric ones, including micelles, nanocapsules and polymersomes [47]. They are stable nanoparticulate formulations, which can stabilize and increase the solubility of hydrophobic molecules and drugs. This depends on the alkyl content, assumed to associate into the hydrophobic pocket in the polymers, which can be easily manipulated by adjusting the polymer composition during synthesis [53], [121], [123].  1.9 Biodegradable Hyperbranched Polyglycerols (BHPGs) Development of biodegradable polymers and their different applications in food science, drug delivery, medical devices and tissue engineering has become a huge area of interest. Therefore, designing a polymeric system with capability of degrading in a controlled manner in a specific environment is desired. Studies have shown decent degradation properties for linear polymers, in which removing the stabilizing end-cap through a single-bond cleavage initiates the cascade of gradual degradation. These systems were further investigated for providing release of multiple drugs at the same time, which were sensitive to reducing conditions or enzymes [124].  Binding drugs and conjugation of carbohydrates and proteins to the high molecular weight HPGs demonstrated advantages such as better bioavailability due to the longer circulation half-life for the drugs  as well as capability of multivalent interactions with protein and cells [125], [126]. However, the accumulation of higher molecular weight HPGs in the organs compared to lower  25  molecular weights, makes essential the need for designing biodegradable HPGs, which helps the clearance of degraded fragments from the kidney. Therefore, synthesis and characterization of water-soluble biodegradable HPGs (BHPGs) containing different numbers of acid degradable dimethyl and cyclohexyl  ketal linkages have been reported by our group for the first time [127]. This polymer is synthesized through ring-opening polymerization of glycidol, using initiators containing a ketal linkage which is cleavable in an acidic environment and vary in number of ketal groups (Figure 1.13). Narrow polydispersity proved controlled polymerization. This delivery vehicle can have applications in transporting antitumor agents, because of the acidic environment of tumors, as well as proteins and nucleic acid due to the capability of the polymer to go through endosomal degradation, releasing the therapeutic agent to the target cell, as reported previously in several cases of degradable polymers [128]–[131]. BHPG was reported to be stable at physiological pH and showed controlled degradation in acidic pH, followed by excellent blood compatibility. Through changing the ketal group structure of the initiator, the degradation of BHPGs can be tuned which makes these polymers potential candidates for drug delivery applications [127].     26   Figure 1.13 Structure of BHPG with cleavable linkage   Followed by that, synthesis and application of water-soluble biodegradable hyperbranched polyglycerols with randomly distributed ketal groups (RBHPGs) has been reported (Figure 1.14) [132], suggesting that the degradation of ketal groups into easily excretable, non-toxic low molecular weight fragments ought to minimize organ accumulation. This polymer is synthesized through copolymerization of glycidol and ketal-containing epoxide monomers. An in vitro degradation study demonstrated that the ketal groups were relatively stable in neutral pH. However, in acidic pH hydrolysis of the ketal group resulted in non-toxic low molecular weight products. In vivo studies also demonstrated a significantly lower plasma half-life of this new formulation, as well as much less tissue accumulation and faster clearance, compared to the non-degradable HPG [132]. Biodegradable HPG shortens the plasma half-life from 39.2 h for non-Acid cleavable linkage 27  degradable HPG to 2.7 h at the similar molecular weight. Therefore, these characteristics of RBHPGs make them attractive in several biomedical applications such as drug delivery and tissue engineering.    Figure 1.14 Biodegradable HPG with randomly distributed ketal group (RBHPG)   Degradable linkage 28  1.10 Thesis Goals Delivery of hydrophobic drugs and hormones to target cells is one of the major research challenges in pharmaceutical science due to their high insolubility in aqueous environments. Nanocarriers with flexible structures can encapsulate hydrophobic drugs and molecules and transport them to therapeutic sites. Regulation of physiochemical properties of nanocarriers, such as surface charge, particle size and lipophilicity play an important role in their functionalities. Polymer-based therapeutics can be designed to enhance drug solubility, which offers a wide range of opportunities to elevate the effectiveness and decrease the toxicity of drugs [133], [134].  Progesterone (Pro) is a potential neurosteroid for the treatment of traumatic brain injury (TBI), provided it is administered rapidly after the head injury. However, a major barrier for the drug to act efficiently after administration is its instability and very low solubility in the blood stream due to its hydrophobicity, preventing it from reaching the target cells. This work reports a newly designed drug delivery system for Pro, which is stable and has the potential to deliver the drug by increasing its solubility.  Hyperbranched polyglycerols (HPGs) are macromolecules with a well-defined architecture, a high degree of branching and end-group functionalities, good aqueous solubility and no toxicity. HPGs offer a broad range of applications in medicine and pharmacology [133]. In this work, a polymeric system consisting of HPG-Cn-MPEG (also known as dHPG(Cn)) with a hydrophobic alkyl fraction and hydrophilic PEG chains is synthesized and tested as a suitable delivery system for Pro. dHPG(Cn) are water-soluble and can be synthesized in a variety of molecular weight ranges depending on the use, which makes them flexible for different applications [53]. Binding capacity for encapsulation of hydrophobic drugs and molecules can be manipulated by changing the hydrophilic to hydrophobic ratio of the polymer; the hydrophobic  29  fraction should increase the binding affinity of the drug to the polymeric system, while the hydrophilic PEG fraction ought to keep the system soluble in an aqueous environment and provide stability for the dHPG(Cn)/Pro formulation. These polymers are blood compatible and non-toxic, offering strong candidates for a drug-binding pocket to deliver Pro to brain cells.  1.11 Specific Aims We have formulated two specific aims to test our hypothesis in this thesis proposal; development of dHPG(Cn)/Pro system and efficacy of the formulation.  1.11.1  Development of dHPG(Cn)/Pro Formulation  Intelligently designed Pro binding pocket within the dHPG(Cn) enables controlled binding and release, and stabilizes the Pro formulation in aqueous systems (Chapters 2 and 3).   1.11.1.1 Synthesis of dHPG(Cn)  The development of a library of high molecular weight dHPG(Cn) polymers is carried out, where the hydrophobic alkyl chain vary from 6 to 18 carbons, while the composition of  the PEG as well as the molecular weights of the polymer remain similar in all cases. This helps to demonstrate the effect of the different chain lengths in the binding affinity and stability of Pro into and release from the drug-binding pocket. The results of this study are described in Chapter 2.  1.11.1.2 In vitro Study of Binding and Release of Pro and dHPG(Cn) The capability of the system for loading drug and maintaining stability is measured. This helps us to quantify the maximum molar ratio of the drug to the polymer in order to find the limit  30  for in vitro applications and biocompatibility assays. Additionally, the release rate of the drug from the formulation is determined by kinetic studies and correlated with system properties. The results of this study are described in Chapter 3.  1.11.2 Efficacy of dHPG(Cn)/Pro Formulation  To support the idea, we compare Pro, loaded in a dHPG(Cn) pocket, to the free drug in various in vitro assays (Chapters 4 and 5).  1.11.2.1 Blood Compatibility The influence of the dHPG(Cn) system on blood compatibility using human blood components is measured. The results of this study are described in Chapter 4.  1.11.2.2 Cellular Uptake and Cytotoxicity The toxicity effect of dHPG(Cn)/Pro formulation on astrocytoma and blood brain barrier cell lines is investigated. This is followed by a study of the capability of these cells to uptake the formulation within a specific time frame. The results of this study are described in Chapter 5.   31  Chapter 2: Synthesis and Characterization of Hydrophobically Modified Hyperbranched Polyglycerols  2.1 Synopsis This chapter documents synthesis and characterization of hydrophobic derivatized hyperbranched polyglycerols [dHPG(Cn)]. Modification of HPG molecules with hydrophobic carbon chains increases the binding affinity for the hydrophobic molecules, whereas modification with MPEG increases the system’s solubility and stability in aqueous environments. These assemblies are intended to serve as a model for delivery of hydrophobic drugs with low water solubility.  2.2 Materials and Methods 2.2.1 Chemicals 1,2-epoxyalkanes (except 1,2-epoxyoctadecane) were purchased from TCI America Ltd. (Portland, OR), while all other chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON) and were used without any further purification. All solvents were HPLC-grade from Fisher Scientific (Ottawa, ON). Deuterated solvents for NMR spectroscopy were purchased from Cambridge Isotope Laboratories Inc. (Andover, MA). Glycidol (96%) was purified using vacuum distillation and stored over molecular sieves in a refrigerator at 4 °C. 1,2-epoxyoctadecane was synthesized by peroxidation of octadecane with m-chloroperbenzoic acid. Methoxy poly(ethylene glycol) 350 epoxide (MPEG-epoxide) was synthesized by a reaction of MPEG 350 Da, sodium hydroxide and epichlorohydrine. Cellulose acetate dialysis tubing (MWCO 10,000 g/mol)  32  was purchased from Spectrum Laboratories, Inc. (Houston, TX). Proton NMR and carbon-13 NMR spectra were recorded on Bruker Avance 300 MHz and Bruker Avance 400 MHz NMR spectrometers respectively, using deuterated solvents (CDCl3 and methanol-d4 ), with the solvent peak as a reference. Molecular weights and polydispersities of the polymers were determined by gel permeation chromatography (GPC) using a DAWN-EOS multi-angle laser light scattering (MALLS) detector; details have been reported previously [79].  2.2.2 Synthesis and Characterization of 1,2-epoxyoctadecane 1,2-epoxyoctadecane was synthesized by first dissolving 4.8 g of m-chloroperbenzoic acid in 50 mL chloroform (CHCl3). The reaction mixture was kept in an ice-bath, followed by drop-wise addition of 5 g (6.5 mL) of octadecene to the reaction mixture. The system was kept at 4 °C for two days. In order to remove the excess acid byproduct, the reaction mixture was washed with saturated sodium bicarbonate (NaHCO3) solution and then dried over sodium sulfate (Na2SO4). Drying agent was filtered and excess chloroform was then removed using a rotary evaporator (Figure 2.1). The obtained 1,2-epoxyoctadecane was dissolved in hexane and passed through a silica gel column to remove the trace of impurities. Hexane was then evaporated and the product was obtained through freeze drying.   Figure 2.1 Synthesis scheme of 1,2-epoxyoctadecane    33  1,2-epoxyoctadecane was then characterized using proton NMR spectroscopy on a Bruker Avance 300 MHz 1H NMR spectrometer (Figure 2.2). 1H NMR (300 MHz, CDCl3) δ(ppm): 0.89 (3H, t, J = 6.7 Hz, CH3 from C18 chain); 1.26-1.60 (30H, m, CH2 from C18 chain); 2.45-2.48 (1H, dd, J = 2.7, 5.0 Hz, CH2 from epoxide ring); 2.74-2.77 (1H, m, CH2 from epoxide ring); 2.88-2.94 (1H, m, CH from epoxide ring).   Figure 2.2 1H NMR spectrum of 1,2-epoxyoctadecane in CDCl3  2.2.3 Synthesis and Characterization of MPEG-epoxide Synthesis of MPEG 350 epoxide was carried out by starting the reaction between 220 g of MPEG with average molecular weight of 350 g/mol and excess (74 g) sodium hydroxide (NaOH) in an ice-bath for a few hours, followed by drop-wise addition of 106 mL epichlorohydrin within -CH3 -CH2-(from the chain)-CH-(epoxide ring)-CH2-(epoxide ring)-CH2-(epoxide ring)CDCl3 34  2 h and stirring the reaction mixture for 48 h. A 600 mL aliquot of dichloromethane (DCM) was added to the mixture and sodium chloride precipitations were filtered out using suction filtration and dried with sodium sulfate overnight. The drying agent (Na2SO4) was filtered out and DCM and excess epichlorohydrin were removed by rotary evaporator for a few hours at 50 °C (Figure 2.3). The obtained MPEG-epoxide was kept under argon flow for a few min and stored in a 4 °C refrigerator.    Figure 2.3 Synthesis scheme of MPEG-epoxide   The purity of the MPEG-epoxide was determined by proton NMR spectroscopy run on a Bruker Avance 400 MHz 1H NMR spectrometer using deuterated chloroform (Figure 2.4 and Figure 2.5). 1H NMR (400 MHz, CDCl3) δ(ppm): 2.60-2.63 (1H, dd, J = 2.7, 4.8 Hz, CH2 from epoxide ring (Ha or Hb)); 2.79-2.81 (1H, m, CH2 from epoxide ring (Ha or Hb)); 3.15-3.19 (1H, m, CH from epoxide ring (Hc));  3.38 (3H, s, OCH3 (Hg)); 3.41-3.46 (1H, dd, J = 6.1, 11.8 Hz, CH2 proximity of epoxide ring (Hd or He)); 3.54-3.72 (avg. 28H, m, CH2 (Hf)); 3.77-3.81 (1H, dd, J = 3.0, 11.6 Hz, CH2 proximity of epoxide ring (Hd or He)).  35   Figure 2.4 1H NMR spectrum of MPEG-epoxide in CDCl3   Figure 2.5 Expanded 1H NMR of MPEG-epoxide in CDCl3 Ha or HbHfHcHd or HeHd or He Ha or HbHg 36  2.2.4 Synthesis of dHPG(Cn) The polymerizations of Cn alkyl (n = 6, 8, 10, 12, 14, 18) core modified HPGs were carried out according to protocols developed earlier by our group [53]. Polymerization was carried out in a three-neck round-bottom flask, equipped with a mechanical stirrer and under argon atmosphere, through a simple single-pot synthetic procedure based on anionic ring-opening polymerization of epoxide. The initiator 1,1,1-tris(hydroxylmethyl)propane (TMP; 120 mg) was added to the three-neck round-bottom flask, followed by addition of 0.1 mL potassium methylate solution in methanol (25% w/v). The mixture was stirred at 60 °C using a magnetic stir bar for 30 min and the excess methanol was removed under the vacuum afterwards for several hours. Eight mL of glycidol and 5 mL of Cn alkyl epoxide mixture were injected to the initiator using a syringe pump drop-wise in 15 h at a rate of 0.9 mL/h. The reaction was carried out under argon at 95-100 °C overnight. The stirring rate was fixed at 68 rpm using a digital overhead stirring system (BDC2002). Potassium hydride (KH; 0.1 mL) was then added to the flask. The mixture was stirred for 1 h, after which about 20 mL of MPEG 350 epoxide was added drop-wise in 15 h as a final step in the “one pot” synthesis using a syringe pump at a rate of 1.2 mL/h. The stirring rate was then increased to 90 rpm and the reaction was continually carried out at 95-100 °C overnight.  After completion of monomer addition, the mixture was stirred for an additional 6 h. The product was then dissolved in methanol and neutralized by passing three times through a cation exchange column (Amberlite IRC-150, Rohm and Haas Co., Philadelphia, PA) (Figure 2.6). Unreacted alkyl epoxides were removed from the methanolic polymer solution by extraction with hexane. Purified polymers were obtained by precipitating in diethyl ether twice, followed by extraction with hexane for a couple of times to remove the unreacted alkyl monomer. Methanol was removed under vacuum and an aqueous solution of the polymer was then dialyzed for three  37  days against water, using cellulose acetate dialysis tubing (MWCO 10,000 g/mol), with three water changes per day. Dry polymer was then obtained by freeze drying.   Figure 2.6 Synthesis scheme of dHPG(Cn) (n = 6, 8, 10, 12, 14, 18); R refers to the hydrophobic chain of the alkyl monomer and x refers to the CH2 repeating units of the R chain   2.2.5 Characterization of dHPG(Cn) The compositions of MPEG and alkyl chains in dHPG(Cn) were estimated on a Bruker Avance 300MHz 1H NMR spectrometer experiments using deuterated solvents (Cambridge Isotope Laboratories, 99.8% D). Chemical shifts were referenced to the residual solvent peak. A peak corresponding to OCH3 of MPEG and the absence of any epoxide groups from MPEG-epoxide and epoxyalkanes represented the absence of contamination of polymer by unreacted  38  monomers. A quantitative estimation of OH/glycidol groups per molecule and the composition was obtained in CDCl3. Molecular weights and polydispersities of polymeric systems were calculated by GPC on a Waters 2690 separation module with multi-angle laser light scattering detector (MALLS) from Wyatt Technology Inc. As previously reported by our group [79], this GPC system provides refractive index and 90° for dynamic light-scattering applications. An aqueous 0.1 N NaNO3 solution was used as the mobile phase, at a flow rate of 0.8 mL/min. An Ultrahydrogel 120 column with a bead size of 6 µm (elution range 150 – 5 x 103 Da) and an Ultrahydrogel linear column with a bead size of 6-13 µm (elution range 103 – 5 x 106 Da) from Waters were used. The dn/dc value for HPG polymers was calculated to be 0.120 in aqueous 0.1 N NaNO3 solutions and was used for molecular weight determinations.   2.3 Results 2.3.1 Characterization of dHPG(Cn) A series of proton NMR spectroscopy runs was conducted to characterize the structure of the polymers and to calculate the mole percentage of hydrophobic and hydrophilic fractions.  The mole fractions of glycidol, MPEG and alkyl monomers per polymer were calculated from the 1H NMR spectra; the number of OH groups per molecule can be estimated assuming on average ~one OH group per glycidol monomer from published 13C NMR data [79]. The number of alkyl chains and OH/glycidol groups per molecule were calculated using: 𝑛𝑖 =𝑀𝑛𝑓𝑖∑ 𝑓𝑖𝑀𝑖      [1] where:  39  ni = number of moles of monomer i in polymer molecule; i refers to glycidol, alkyl or MPEG fi = mole fraction of monomer i in polymer molecule Mi = molecular weight of monomer i incorporated in polymer molecule Mn = number average molecular weight of polymer  The sum is taken over all monomer species in the molecule. Total number of alkyl carbon in each system was calculated by multiplying of number of alkyl chains by the number of carbons in the alkyl epoxide monomer, n. In order to correlate the effects of hydrophobic modification on polymer behaviour, the number of purely hydrophobic carbons per chain (R) was used for calculations which is (n-2) carbons, without considering the two carbons originating from the epoxide ring of the epoxyalkane monomer separated by the epoxide oxygen from the linear hydrophobic chain. Proton NMR spectra of two dHPG(Cn) (n = 8 and 12 as examples of HPGs containing alkyl monomers with a short and a long chain) are given below (Figure 2.7 and Figure 2.8) and those for other dHPG(Cn) (n = 6, 10, 14, 18) are included in Appendix A.1. 1H NMR (300 MHz, CDCl3) δ(ppm): 0.90 (3H, t, J = 6.0 Hz, CH3 from C8 monomer); 1.25-1.50 (10H, m, CH2 from C8 monomer); 3.50-3.95 (CH and CH2 from HPG core); 3.39 (3H, s, OCH3  from MPEG). 1H NMR (300 MHz, CDCl3) δ(ppm): 0.88 (3H, t, J = 6.0 Hz, CH3 from C12 monomer); 1.20-1.50 (18H, m, CH2 from C12 monomer); 3.50-3.95 (CH and CH2 from HPG core); 3.38 (3H, s, OCH3  from MPEG).   40   Figure 2.7 1H NMR spectrum of dHPG(C8) in CDCl3   Figure 2.8 1H NMR spectrum of dHPG(C12) in CDCl3 -CH3 (from C8 chains)-CH2-(from C8 chains)-CH2-(from HPG and PEG)CDCl3-OCH3-CH3 (from C12 chains)-CH2-(from C12 chains)-CH2-(from HPG and PEG)CDCl3-OCH3 41  Through GPC analysis, the molecular weight, polydispersity and the hydrodynamic radius were calculated for each product. GPC chromatograms for dHPG(Cn) (n = 8 and 12) are given below (Figure 2.9 and Figure 2.10) and those for other dHPG(Cn) (n = 6, 10, 14, 18) are included in Appendix A.1. For all dHPG(Cn) preparations, the targeted molecular weight was around 100,000 g/mol. In order to determine the effects of different chain lengths and hydrophobic properties of the polymeric systems on drug binding and releasing, the contributions of MPEG composition were maintained almost the same in all systems. Characteristics of all dHPG(Cn) are summarized in Table 2.1.   Figure 2.9 GPC chromatogram of dHPG(C8); red line belongs to multi-angle light scattering detector and blue line belongs to refractive index detector   Define PeaksLS dRI     volume (mL)0.0 10.0 20.0 30.0Relative Scale0.00.51.01 1 42   Figure 2.10 GPC chromatogram of dHPG(C12)  dHPG(Cn) Cn mol%a MPEG mol%a Mn  × 10-3 (g/mol) Mw / Mn No. of alkyl chain/ polymer molecule No. of hydrophobic carbon (R)/ polymer molecule Rh avgb (nm) Yield% dHPG(C6) 30 29 68 1.98 115 460 6.8 (± 0.24%) 61 dHPG(C8) 28 36 99 1.26 137 822 4.4 (± 0.41%) 74 dHPG(C10) 15 31 110 1.74 89 712 5.5 (± 0.27%) 68 dHPG(C12) 12 30 168 1.82 111 1110 7.8 (± 0.03%) 76 dHPG(C14) 9 34 125 1.92 60 720 6.7 (± 0.24%) 71 dHPG(C18) 3 30 113 1.47 17 272 7.5 (± 0.24%) 69  Table 2.1 Characteristics of dHPG(Cn) (n = 6, 8, 10, 12, 14, 18); a: Mole fractions of alkyl and MPEG monomers were calculated from proton NMR; number of hydrophobic carbons in R chain (n-2) per polymer molecule was calculated from multiplying the number of alkyl chain per polymer molecule by the number of carbons in R chain (n-2); b: Hydrodynamic radii were calculated from dynamic light scattering (QELS), GPC; yield% calculation was based on starting polymer weight Define PeaksLS dRI     volume (mL)0.0 10.0 20.0 30.0Relative Scale0.00.51.01 1 43  Inverse-gated 13C NMR spectroscopy proved the branched structure of HPGs conjugated with alkyl chains and MPEG chains. The 13C NMR spectrum of dHPG(C8) in methanol-d4 is shown below (Figure 2.11). Calculating degree of branching (DB), as reported previously for HPGs between 0.5-0.6 [76], confirmed the branched structure of dHPG(C8) with the value of 0.61 through the below formulation: 𝐷𝐵 =2𝐷2𝐷+ 𝐿13+𝐿14      [2] where: D is dendritic, T is terminal, L13 is linear (primary hydroxyl), and L14 is linear (secondary hydroxyl). 13C NMR (400 MHz, methanol-d4) δ(ppm): 14.72 (CH3 from alkyl on C8 monomer); 23.87, 26.81, 30.74, 33.18 and 34.67 (CH2 from alkyl on C8 monomer); 59.27 (OCH3 from MPEG); 62.99-81.53 (CH and CH2 from HPG core).  44   Figure 2.11. Inverse-gated 13C NMR spectrum of dHPG(C8) in methanol-d4 with relaxation delay of 10 sec proved the branched structure of dHPG(Cn)  45  To confirm the consistency among the structure of dHPG(Cn), the same study was done on dHPG(C10) and DB was calculated as 0.59 (Appendix A.2).  2.4 Discussion dHPG(Cn) synthesis was carried out in a simple one-pot procedure, based on anionic ring-opening multibranching polymerization of epoxides. The initiator (TMP) was first activated and partially deprotonated by potassium methylate and then reacted with glycidol and 1,2-epoxyalkane (Cn), ranging from shortest chain (C6) to the longest chain (C18), to create HPG-Cn-OH with a hydrophobic core. This modification should have provided suitable area to bind the hydrophobic drug. Due to the reaction of the alcohol initiator with a suitable deprotonating agent, only 10% of the hydroxyl groups were converted into the alkoxide. This step enabled control of the concentration of active sites (alkoxides) in the polymerization, leading to simultaneous growth of all chain ends and consequently control of molecular weight with considerable narrowing of the polydispersity, as a result of this control. In the subsequent propagation step, the alkoxide initiator reacted with the epoxide ring on its unsubstituted end and thus produced a secondary alkoxide of all chain ends. Both inter and intramolecular transfer steps after the ring-opening reaction can lead to the formation of a primary alkoxide as active site, which further propagates, resulting in branched structures. MPEG 350 epoxide was added in the terminal phase of the reaction to form HPGs with a more hydrophilic shell (presumably on the surface), designated as HPG-Cn-MPEG (dHPG(Cn)) systems, compared to HPG-Cn-OH. Decoration with MPEG chains are intended to increase the aqueous solubility of hydrophobically modified HPG. Although synthesis of dHPG(Cn) is described above as several simple and manageable steps, controlling the molecular weights and polydispersities pose significant challenges in this  46  type of polymerization. Dryness and purity of the monomers have an important role in their incorporation into the polymerization as well as their effects on narrowing polydispersities. This was the most challenging in the case of the MPEG-epoxide monomer. This monomer is highly hydrophilic and has a high tendency to absorb water. Therefore, a proper drying step and removing the traces of unreacted epichlorohydrin highly contributes to the polymerization and polydispersity reduction. Since moisture causes the reaction to quench, keeping all glassware dry by using multiple flaming and vacuum is essential. Constant temperature and homogenous stirring with constant speed throughout the procedure also significantly contribute to controlling the rate. As previously mentioned, the concentration of the active chain ends should be controlled to allow the polymer to grow from all sites at the same time. To do this, deprotonation of the initiator, along with slow monomer addition over time, lead to complete incorporation of the alkoxide initiator into the hyperbranched macromolecule. A high ratio of monomer to initiator provides higher chance of cyclization by intramolecular ring-opening polymerization of the monomer, broadens the polydispersity and lowers the achievable molecular weight. Therefore, careful control of polymerization conditions provide suppression of cyclization reaction. Physicochemical characterization of dHPG(Cn) was performed by proton and carbon-13 NMR spectroscopy to prove the absence of any contamination of polymers by unreacted monomers. Inverse-gated 13C NMR proved the hyperbranched structure of a typical example of these polymers and demonstrated that alkylation does not affect the branching of HPG. Proton NMR spectra demonstrated almost the same contributions of hydrophilic fractions (MPEG chains) in all polymeric systems, whereas the length and incorporation of alkyl chains in the hydrophobic species varied in each case. To create a water-soluble polymeric system without any sign of aggregation, keeping the balance of hydrophilic and hydrophobic fractions had an important role.  47  In order to prove this idea, measuring the hydrodynamic size of different concentrations of an example of these polymers in aqueous environment is discussed in Chapter 3.  As the length of hydrophobic chain increased from 6 to 18, the contribution of the hydrophobic fraction was decreased, which was most extreme in the dHPG(C18) system. This could be explained if the increase in chain length resulted in steric hindrance and inhibition of reactions with more groups. Through GPC-MALLs, the molecular weights, polydispersities and hydrodynamic radii of the dHPGs were measured. The properties of aqueous 0.1 N NaNO3 solutions of all systems were consistent with expectations based on highly branched, non-aggregated structures.   2.5 Summary In this chapter, synthesis and characterization of hydrophobically modified HPGs dHPG(Cn) based on ring-opening polymerization of epoxides through a single-pot synthetic procedure are reported. Strong advantages of dHPG(Cn) for application as delivery systems are the absence of intermediate purification steps and flexibility to optimize hydrophobicity. These materials with hydrodynamic radii less than 10 nm that do not aggregate at high concentrations are characteristically compact and highly water-soluble, which can make them strong candidates for delivery vehicles for hydrophobic drugs. Also, there is a strong potential for these molecules to increase their binding capacity for hydrophobic species by manipulating the hydrophilic to hydrophobic ratio. Based on the structural flexibility of dHPG(Cn), these polymers show a noticeable ability to encapsulate a hydrophobic drug, such as Pro, and improve its solubility and stability, as shown in Chapter 3.    48  Chapter 3: Studies of Loading and Release Kinetics of Progesterone Bound to Hydrophobically Derivatized Hyperbranched Polyglycerols (dHPG(Cn)/Pro)   3.1 Synopsis Steroid gender hormones are prospective neuroprotective applicants following central nervous system damage. Experimental studies demonstrated that Pro, a neutral hydrophobic compound, is a potential neurosteroid that promotes recovery from moderate Traumatic Brain Injury (TBI). However, the applicability of Pro as a TBI treatment is severely impeded by its poor water solubility due to its hydrophobicity, which presents a major challenge during formulation. Studies in this chapter demonstrate that development of Pro encapsulated by hydrophobically modified hyperbranched polyglycerol (HPG)-based biocompatible nanocarriers (dHPG(Cn)/Pro), enhances solubility and stability of the hormone.   3.2 Materials and Methods 3.2.1 Chemicals dHPG(Cn) polymeric systems were synthesized as explained in the previous chapter. All solvents were HPLC grade from Fisher Scientific (Ottawa, ON). GD/X 13 mm Whatman Syringe Filters were purchased from General Lab Supply Inc. (Houston, TX, US). UPLC vials were purchased from Canadian Life Science (Peterborough, ON). Pro powder was obtained from Steraloids Inc. (Newport, RI, USA). Dialysis membrane tubing was purchased from Spectrum Laboratories (Rancho Dominguez, CA), and dialysis cassettes were purchased from Thermo Scientific (Rockford, IL). Platelet-poor plasma (PPP) was collected by centrifuging the citrated  49  whole blood at 826 × g for 20 min, in an Allegra X-22R Centrifuge (Beckman Coulter, Canada); Blood was drawn from healthy un-medicated consenting donors at the Centre for Blood Research at UBC (UBC Ethics approval no: H07-02067). The chromatography equipment used consists of an Ultra Performance Liquid Chromatograph (Waters® Acquity™ UPLC) with tandem Waters Acquity PDA detectors. A symmetric C18 column (Acquity BEH C18, Waters) was used for measuring the concentration of Pro bound into and released from dHPG(Cn) using the gradient mobile phase of water and acetonitrile (ACN), containing 0.1% formic acid.  3.2.2 Estimates of Water Bound to dHPG(Cn)  Since water bound by dHPG(Cn) could influence their drug binding and release, the amount of non-freezing water bound to or structurally altered by the polymers was measured using Q2100 differential scanning calorimetry (DSC) (TA instrument, New Castle, DE, USA). The method has been described previously [135], [136]. As temperature is ramped up from -20 °C, the instrument evaluates the change in heat absorbed around 0 °C, when bulk water melts in the presence of polymer. Briefly, about 20 µl of 5% and 10% w/w (polymer-water) solution was cooled down to -20 °C, followed by heating up to -5 °C at the rate of 2 °C /min. The sample heating was continued from -5 °C to +5 °C at the rate of 0.2 °C/min and at 2 °C/min to +20 °C thereafter. The heat flow (J/g) was recorded as a function of temperature and the enthalpy of fusion of water was calculated from the integration of the area under the peak and compared to pure water as control. The moles of water bound or altered per mole of dHPG(Cn), N, was then calculated from the following equation:  50  𝑁 =((∆𝐻0(1−𝐶𝑝)−∆𝐻𝑝)𝑀𝑛)(∆𝐻018.02𝐶𝑝)     [3] where ΔH0 is the fusion enthalpy of pure water, ΔHp is the fusion enthalpy of free water in the polymer solution, Mn is the molecular weight of the polymer, and Cp is the mass fraction of dHPG(Cn) in the solution.  3.2.3 Drug Incorporation into dHPG(Cn) For each dHPG(Cn)-drug preparation, the binding capacity of Pro and the stability (half-life) of the mixture have been studied. Different concentrations of Pro and a constant concentration of dHPG(Cn) were dissolved in 1 mL ACN solution in a 4 mL vial, dried in an oven at 60 °C for 60 min and flashed with a nitrogen stream for few min to eliminate any traces of the organic solvent. The resulting dHPG(Cn)/Pro mixture was hydrated with 10 mM phosphate buffered saline (PBS - 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4) at pH 7.4 at room temperature and vortexed for a few min. The resulting solutions were generally clear but in order to prevent formation of any white precipitate, the solutions were filtered with a 0.2 µm pore size syringe filter into new vials. All dHPG(Cn)/Pro solutions were used on the same day as they were prepared. The concentration of Pro was increased from 50 to 375 µg/mL (50, 75, 100, 125, 175, 250, 325 and 375 µg/mL), while the concentration of dHPG(Cn) remained constant at 20 mg/mL. The maximum loading capacity and stability of Pro loaded into dHPG(Cn) were determined by gradient reverse-phase UPLC. Similarly, the stability of Pro loaded into dHPG(Cn) was evaluated within 24 h.    51  3.2.4 Drug Quantification Study The amount of Pro incorporated in dHPG(Cn) was determined by a gradient reverse-phase UPLC method, as established previously for similar hydrophobic drugs, such as paclitaxel and docetaxel [65], [120], [121], [137]. Regarding the precision and linearity, the UPLC method was validated first by standard protocols and all samples were diluted to the concentrations within the linear range of calibration. Drug content analysis was performed using a symmetric C18 column (Acquity BEH C18, Waters - 1.7 µm, 2.1×50 mm) with two mobile phases containing 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in ACN (mobile phase B) at a flow rate of 0.3 mL/min. Composition of mobile phases was set from the initial to 1.50 min at 70% A and 30% B, followed by changing from 1.50 - 2.10 min at 5% A and 95% B, and back to 70% A and 30% B from 2.10 min to the end. Sample injection volumes were 20 µl and Pro detection was performed using ultraviolet detection at a wavelength of 240 nm. The total run time was set to 4.5 min and the Pro retention time was 1.9 ± 0.1 min (Figure 3.1).    52   Figure 3.1 UPLC chromatogram represents the identification of Pro at the wavelength 240 nm and 1.9 min retention time  The limit of quantification for Pro was 1 µg/mL with a linear dynamic range of 1-100 µg/mL (Figure 3.2 and Figure 3.3).   Figure 3.2 UPLC calibration curve for Pro  Progesterone - 1.892AU0.000.200.400.600.801.001.201.401.60Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50Area01x1062x1063x106Amount0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 53    Figure 3.3 UPLC standards from 1 to 100 µg/mL. The retention time is 1.9 ± 0.1 and Pro was detected at the wavelength of 240 nm  For this, 500 µl of 1 mg/mL solution of Pro in ACN was dissolved with 500 µl of ACN/PBS (50:50 v/v). Different volumes of this stock solution were dissolved with different volumes of ACN to prepare 1, 5, 15, 25, 50, 75 and 100 µg/mL solutions to be used as standards for the calibration curve.  3.2.5 Temperature Effect on Pro Binding In order to evaluate the effect of temperature variation on the binding capacity of dHPG(Cn) for drug, samples containing Pro loaded on dHPG(C8) and dHPG(C12) were hydrated with PBS at 37 °C and the amount of Pro bound into these two formulations were determined and compared to that bound at room temperature (22 °C). Analysis of covariance (ANCOVA) was then used to compare the results.   AU0.200.400.600.801.001.201.40Minutes1.64 1.66 1.68 1.70 1.72 1.74 1.76 1.78 1.80 1.82 1.84 1.86 1.88 1.90 1.92 1.94 1.96 1.98 2.00 2.02 2.04 2.06 2.08 2.10 2.12 2.14 2.16 2.18 54  3.2.6 Does Pro Cause dHPG(Cn) to Aggregate? In order to investigate whether the loaded hydrophobic drug causes any aggregation of the polymeric system, we measured the size (Rh value) for the polymer, before and after loading drug at different concentrations. Samples were prepared in 0.1 M NaNO3 and directly injected into the flow cell in a light scattering batch experiment using the MALLS detector. To make the stock solution, 250 µg/mL Pro was loaded on 20 mg/mL dHPG(C10) in ACN, followed by evaporation of solvent and hydration of the system with 1 mL of NaNO3.  From that, different solutions at the polymer concentration range from 0.05 to 2 mg/mL were prepared. The Rh values of the dHPG(Cn)/Pro were measured using LS and QELS detectors and compared to the same concentration of polymer alone.  3.2.7 Drug Release from dHPG(Cn) Each dHPG(Cn)/Pro solution (2 mL of 20 mg/mL) at the drug concentration of 250 µg/mL was transferred into a dialysis cassette (molecular weight cut off 3500) in a bottle containing 1 L of 10 mM PBS (pH 7.4) at 37 °C with slight agitation. The total volume of the release medium was chosen such that when Pro was completely released, its concentration was below its solubility in water, which is 10 µg/mL at 37 °C [138]. Decrease in the drug concentration in the dialysis cassette was monitored over a week. At different time points, the concentration of Pro was measured on 75 µl from the cassette diluted into 225 µl of ACN and assayed by UPLC, as described above. To observe the effect of alkylation of dHPG(Cn) on release behaviour of the drug, control experiments were done using HPG and HPG-PEG, for which Pro was loaded at 125 and 100 µg/mL respectively. To investigate the effect of the presence of dHPG(Cn) in the formulation on the drug release profile, unbound drug clearance from the dialysis cassette was also measured as control by  55  adding 2 mL PBS containing 5 µg of free drug to the cassette (to maintain solubility), followed by dialyzing against PBS and analyzing the contents at different time points, as for the dHPG(Cn).   Release kinetics of the drug from one dHPG(Cn) were also studied in human anticoagulated human platelet-poor plasma (PPP) to observe the effect of plasma proteins’ interactions with Pro on the release profile. In this study, dHPG(C8)/Pro solutions (2 mL of 20 mg/mL) in PPP at the drug concentration of 250 µg/mL were transferred into dialysis cassettes (molecular weight cut off 3500) in a bottle containing 1 L of 10 mM PBS (pH 7.4) at 37 °C with slight agitation. At different time points, the concentration of Pro was measured by taking the sample from the dialysis bag as above, diluting 4 times with ACN, followed by centrifugation for removal of precipitated proteins to separate the top ACN layer and assayed by UPLC, as described above.  3.3 Results 3.3.1.1 Water Binding by dHPG(Cn) Previous DSC measurements have shown that HPG interacts with water in a substantial way and that in the presence of HPG a fraction of the water in a solution does not participate in freeze-thaw heat exchange around T = 0 °C [136], [139]. The solutions behave as if a significant amount of water was bound or otherwise structured in a way distinct from pure water when HPG is present. If this bound or structured non-freezing water had different solution properties for Pro an effect on the release kinetics of Pro from dHPG(Cn) might occur. To test this idea we performed DSC measurements on the series of dHPG(Cn) species under investigation, as described. The results are shown in (Table 3.1) where the reduction in heat of fusion measured as a function of dHPG(Cn) structure and concentration is interpreted in terms of number of moles of water affected  56  per mole of polymer. HPG-PEG with the molecular weight of 93 kDa and 27% of MPEG fraction was run as control.   dHPG(Cn) Mole water per mole dHPG(Cn) (10 %) × 10-3 Mole water per mole dHPG(Cn) (5 %) × 10-3 dHPG(C8) 6.3 5.2 dHPG(C10) 6.9 8.4 dHPG(C12) 8.2 11.4 dHPG(C14) 9.4 10.6 dHPG(C18) 12.6 15.4 HPG-PEG 13.9 17.9  Table 3.1 Summary of DSC results: number of moles of water affected per mole of dHPG(Cn) at the two polymer concentrations shown  3.3.2 Drug Binding into dHPG(Cn) The dHPG(Cn) systems are nanoparticles of less than 10 nm in diameter as verified in the previous chapter. Uncomplexed Pro is a hydrophobic drug with a low aqueous solubility of < 7.8 µg/mL at room temperature (22 °C) [37], 10.4 µg/mL at 37 °C at neutral pH [138]; 1 mg/mL of dHPG(C8, 10, 12, 14) increased the aqueous solubility of Pro to ~ 20 µg/mL per milligram of polymer with different molar ratios. However, the maximum number of loaded drug molecules decreased in dHPG(Cn) polymeric systems containing C6 and C18 chains. This could be explained by short chains and less hydrophobic properties of dHPG(C6), insufficient to bind a higher amount of the hydrophobic drug, or long chains in dHPG(C18) resulting in steric hindrance and decrease in  57  number of bound drug molecules. The maximum molar ratio of drug to polymer was reached at highest loading for the dHPG(C12)/Pro formulation (Figure 3.4). Then, number of bound drug molecules per polymer molecule dropped as more Pro was loaded into the polymer. The formulations became destabilized, resulting in aggregation of unbound hydrophobic drug molecules and drug precipitation in the aqueous system, producing visible cloudiness.   dHPG(Cn) polymers containing C6 chains demonstrated the least amount of bound Pro among all the polymer-drug formulations. This could be explained by C6 being the shortest length of the chain, resulting in the least hydrophobic interaction between each alkyl group and Pro. Maximum binding capacity was obtained at 175 µg/mL of the loaded drug into the polymeric system, and was reached at 161 µg/mL of the bound drug. From this value, the maximum molar ratio of drug to polymer was calculated as 1.7.   As the length of hydrophobic chain in the polymer core increased in dHPG(C8), the maximum number of bound Pro molecules was reached at higher loaded Pro values, compared to dHPG(C6). This copolymer bound the highest mass of Pro per gram of polymer, compared to the other formulations, with 320 µg/mL of the drug bound when Pro was loaded at 325 µg/mL, producing a maximum molar ratio of drug to polymer of 5.1.  Maximum binding capacity of Pro loaded into dHPG(C10) was 278 µg/mL when 325 µg/mL Pro was loaded into the polymer. The maximum ratio of bound drug was then calculated as 4.8 molecules per polymer molecule. dHPG(C12) presented the highest number of drug molecules bound per polymer molecule, compared to all other polymeric systems. Maximum binding capacity obtained was 302 µg/mL after loading 325 µg/mL of Pro. Molar ratio of drug to polymer at this point was then calculated as 8.1 moles Pro per mole of polymer.    58  Maximum binding capacity for Pro loaded into dHPG(C14) reached was 325 µg/mL, and was 303 µg/mL of the bound drug. The molar ratio of drug to polymer at this point was then calculated as 6.0 moles of Pro per mole of HPG.   The number of Pro molecules loaded into the dHPG(C18) system dropped down to a mole ratio of 3.5 (drug to polymer). This could be explained by steric hindrance of C18 long chains, which could avoid presenting stronger hydrophobic interaction between the alkyl chains and Pro molecules. This could also be related to the lowest incorporation of C18 monomer into the HPG structure, compared to other alkyl monomers, including C8, 10, 12. 14. Maximum binding capacity of drug loaded into dHPG(C18) reached was 196 µg/mL after loading 250 µg/mL of Pro. Individual binding plots for each dHPG(Cn) are presented in Appendix B. Results from binding study are summarized in Table 3.2.   Figure 3.4 Pro binding profile into dHPG(Cn)  0501001502002503003504000 50 100 150 200 250 300 350 400Bound Pro (µg/ml)Loaded Pro (µg/ml)dHPG(C₆)/ProdHPG(C₈)/ProdHPG(C₁₀)/ProdHPG(C₁₂)/ProdHPG(C₁₄)/ProdHPG(C₁₈)/Pro 59  Sample No. of hydrophobic carbons per polymer molecule (n-2) Maximum molar ratio (drug/polymer) dHPG(C6)/Pro 460 1.7 dHPG(C8)/Pro 822 5.1 dHPG(C10)/Pro 712 4.8 dHPG(C12)/Pro 1110 8.1 dHPG(C14)/Pro 720 6.0 dHPG(C18)/Pro 272 3.5  Table 3.2 Binding characteristics of dHPG(Cn)  3.3.3 Temperature Effect on Pro Binding Results from the Pro loading study into dHPG(C8) and dHPG(C12) at 37 °C demonstrated the binding capacity increased slightly compared to the ones at room temperature. This rise was observed more at higher concentrations of Pro loaded. The maximum molar ratio of Pro to dHPG(C8) was increased slightly from 5.09 at room temperature (22 °C) to 5.13 at 37 °C (Figure 3.5). Similar behaviour was observed for the dHPG(C12)/Pro formulation. The maximum molar ratio of bound drug per polymer was increased from 8.1 to 8.73 by the increase in temperature (Figure 3.6). For both formulations, ANCOVA demonstrated significant difference in the amount of bound Pro for the two temperatures.   60   Figure 3.5 Temperature effect on drug binding behaviour in dHPG(C8)/Pro formulation; significant difference exists between the straight lines of the two temperatures, based on ANCOVA (p < 0.02)   Figure 3.6 Temperature effect on drug binding behaviour in dHPG(C12)/Pro formulation; significant difference exists between the straight lines of the two temperatures, based on ANCOVA (p < 0.04)  0501001502002503003504000 50 100 150 200 250 300 350 400Bound Pro (µg/ml) Loaded Pro (µg/ml) Room temp.  37 deg C0501001502002503003504000 50 100 150 200 250 300 350 400Bound Pro (µg/ml) Loaded Pro (µg/ml) Room temp.  37 deg C 61  3.3.4 Effect of Pro on dHPG(Cn) Size Results from measuring the Rh value demonstrated that loading Pro on dHPG(Cn) does not cause aggregation in the system and no change in the size of the dominant population was observed at different concentrations compared to the polymer alone at the same concentrations as control. This will confirm the non-micelle structure of dHPG(Cn) (Table 3.3).   Sample Concentration (mg/mL) Rh (nm) dHPG(C10) 0.05 5.6 (±1.0 %) dHPG(C10)/Pro 0.05 5.4 (±0.9 %) dHPG(C10) 1 5.6 (±0.8 %) dHPG(C10)/Pro 1 5.6 (±0.7 %) dHPG(C10) 1.5 5.6 (±0.7 %) dHPG(C10)/Pro 1.5 5.5 (±0.6 %) dHPG(C10) 2 5.3 (±0.8 %) dHPG(C10)/Pro 2 5.3 (±1.0 %)  Table 3.3 Effect of loaded Pro on dHPG(Cn) size  On the other hand, the intensity distribution from DLS measurement revealed the presence of a small population at a larger hydrodynamic radius range (~ 400 nm) at low shear rates in the solution of the polymer alone. However, when the polymer was loaded with Pro, a decrease in the peak’s intensity as well as a slight shift to a smaller hydrodynamic radius range (~ 250 nm) was observed (Appendix C). This result is aligned with what was observed previously for dHPG(C18),  62  where in the presence of the hydrophobic ligand (pyrene), the intensity of the second peak at a larger size was reduced and almost disappeared at different concentrations of the polymer, compared to the polymer alone. The presence of the second peak with a larger size could be due to a small range of intramolecular interactions among alkyl chains. It has been suggested that loading the hydrophobic ligand could prevent the alkyl chains from forming aggregates [54]. This study was furthered clarified by examining the effect of the larger size fraction on the pyrene’s binding; results demonstrated linear correlation between the binding of pyrene and the polymer’s concentration, conditions that did not produce aggregation. This proved that the presence of small aggregates did not affect the pyrene’s binding to the polymer, however [53]. In the case of dHPG(C10), the presence of the larger size population demonstrated no sign of further aggregation as a result of binding with Pro. This suggested that Pro binding to the polymer was not affected by the presence of the second population.  3.3.5  Drug Release from dHPG(Cn) 3.3.5.1 Release in PBS Results of the binding study and comparison of the maximum molar ratios demonstrated that the polymeric systems varied by a fraction of about 5 in their capacity to bind Pro. dHPG(C6) bound the lowest molar ratio of drug to polymer (1.7), whereas the highest ratio (8.1) was observed for the dHPG(C12) system. To evaluate dHPG(Cn)’s potential as a drug carrier and its ability to hold the drug, the release rates of Pro from the polymeric systems were measured at 37 °C in PBS through a dialysis method. For this study, dHPG(Cn) with higher numbers of bound drug molecules were selected such as dHPG(C8, 10, 12, 14). Selecting the drug concentration for the release was an important factor, since the drug had to be stable at that concentration for more than 24 h. Therefore,  63  a concentration (250 µg/mL) just lower than the drug’s maximum loaded concentration for Pro, obtained from the binding study, was selected. dHPG(C18) with a low amount of bound drug was also a point of interest for the release study due to its having the longest hydrophobic chains. However, in this case, 175 µg/mL of Pro was selected to load into the polymer. The release behaviour of Pro from dHPG(Cn) were also compared to free Pro; HPG with the molecular weight of 98 kDa and HPG-PEG with the molecular weight of 93 kDa and 27% of MPEG fraction were run as controls. The percent released from the polymer-drug formulations and the free drug was then plotted versus time, and the release properties were compared (Figure 3.7).  Comparing the release profiles of all dHPG(Cn)/Pro formulations, the slowest release belonged to dHPG(Cn)/Pro containing C8 alkyl chain, in which about 60% of the drug was released in the first 8 h and about 98% was released within 5 days. In the most rapid system, in comparison, dHPG(C18) released ≈ 80% in the first 8 h.  Overall, two different behaviours were observed in the release study; a rapid-release phase in the first 8-10 h followed by a slow-release phase within a week. Free Pro at 5 µg/mL was released from the cassette by 99% within 4 h. This demonstrated the important effect of dHPG(Cn) in stabilizing drug formulations, resulted in a significant decrease in the release profile compared to the free drug. Individual release profiles of Pro from each dHPG(Cn) are presented in Appendix D.     64     Figure 3.7 Pro release from dHPG(Cn) in PBS  3.3.5.2 Release in Platelet Poor Plasma Pro release behaviour from dHPG(C8) polymeric system in plasma was also studied and compared with the results from release of the same system in PBS. This study helps to monitor only the effect of plasma proteins’ presence on release properties of the drug. Pro release behaviour 0%20%40%60%80%100%0 1 2 3 4 5 6 7 8ReleaseTime (h)dHPG(C₈)/Pro dHPG(C₁₀)/ProdHPG(C₁₂)/Pro dHPG(C₁₄)/ProdHPG(C₁₈)/Pro HPG-PEG/ProHPG/Pro Free Pro0%20%40%60%80%100%0 10 20 30 40 50 60 70 80 90 100 110 120ReleaseTime (h)dHPG(C₈)/ProdHPG(C₁₀)/ProdHPG(C₁₂)/ProdHPG(C₁₄)/ProdHPG(C₁₈)/ProHPG-PEG/ProHPG/ProFree Pro 65  indicated a significant decline in plasma, presumably due to an additional affinity of the hydrophobic drug to interact with the plasma proteins, which were retained in the dialysis cassette (Figure 3.8). The outcomes were compared with the free Pro in plasma and PBS. Results demonstrated much faster release for free Pro in PBS, compared to all. Also, Pro release from dHPG(C8) was observed the slowest in plasma, followed by in PBS.    Figure 3.8 Pro release profile from dHPG(C8) in PBS and plasma  3.3.6 Release Kinetics 3.3.6.1 Kinetics in PBS To compare the release rate for each drug-polymer formulation, the rate of drug diffusion from the dialysis bag to the media was studied by monitoring the decrease in absolute concentration of the drug inside the cassette with time (Figure 3.9). As illustrated in Figure 3.7 and 0%20%40%60%80%100%0 10 20 30 40 50 60 70 80 90 100 110 120ReleaseTime (h)dHPG(C₈)/Pro in PlasmadHPG(C₈)/Pro in PBSFree Pro in PlasmaFree Pro in PBS 66  Figure 3.8, the release profiles of Pro from dHPG(Cn) occurred in two phases, a rapid phase over the first 8-10 h, followed by slower release, which continued for several days.     Figure 3.9 Pro’s absolute concentration [C] with change in time – dHPG(C8)/Pro  Commonly, it was found the natural log (ln) of drug concentration versus time plot was not a straight line, such as expected for a simple first order drug elimination [140], where the drug concentration would fall mono-exponentially with time (Figure 3.10). We observed an initial early linear section, followed by deviation to a second log-linear phase. The initial phase was a more rapid drop in the drug concentration before settling into the longer term log-linear fall over several days. This suggested that the system was not behaving as a single compartment and that the drug was distributed between two or more compartments. Rate constants of the two release phases were determined by calculating the slopes of both rapid and slow-release phases from the natural log of absolute concentration in time.  0501001502002500 20 40 60 80 100 120[C]Time (h) 67    Figure 3.10 Natural logarithmic behaviour of Pro’s absolute concentration [C] with time in PBS – dHPG(C8)/Pro  By calculating the ln ratio of each concentration [C1] for the first 8 h relative to the concentration at time zero [C10] in Figure 3.10 (extracted from the trend line of rapid-release data), and plotting these values versus time, the rate constant for the rapid-release phase [k1] was obtained (Figure 3.11). This was followed by plotting the ln ratio of absolute concentrations for the times 24 to 120 h [C2] relative to the extrapolated estimate of the concentration at time zero [C20] in Figure 3.10 (extracted from the trend line of slow-release data) versus time, from which the rate constant for the slow-release phase [k2] was then calculated from the slope (Figure 3.12).  0123450 10 20 30 40 50 60 70 80 90 100 110 120ln [C]Time (h)ln[C10]ln[C20] 68   Figure 3.11 Semi-log plot to determine initial rapid release kinetics for dHPG(C8)/Pro in PBS; R2 = 0.99 and p < 0.01   Figure 3.12 Semi-log plot to determine secondary slow release kinetics for dHPG(C8)/Pro in PBS; R2 = 0.97 and p < 0.01  -1.2-1.0-0.8-0.6-0.4-0.20.10 1 2 3 4 5 6 7 8ln[C1/C10] Time (h)-3.5-3.0-2.5-2.0-1.5-1.0-0.50.00 20 40 60 80 100 120 140ln[C2/C20]Time (h) 69  The same analysis was done for dHPG(C10, 12, 14, 18)/Pro as well and the rate constants for both rapid release and slow release phases for different formulations were calculated. Release rate constants were also compared with HPG/Pro and HPG-PEG/Pro systems as controls. Comparing all the kinetic profiles mentioned above with the free Pro profile (Figure 3.13), the absolute concentration over time changed much faster and the drug leaves the cassette in the first 4 h in the free Pro, HPG/Pro and HPG-PEG/Pro cases. The release rate constants were calculated from one phase in these cases therefore. The two phases of release into PBS with two rate constants are characterized as described above and their values are recorded in Table 3.4. Individual kinetic profiles for each dHPG(Cn)/Pro formulation are presented in Appendix E.     Figure 3.13 Free Pro release rate in PBS; k1(s-1) calculated from the slope (R2 = 0.95 and p < 0.01)  -5.0-4.0-3.0-2.0-1.00.00 0.5 1 1.5 2 2.5 3 3.5 4 4.5ln [C/C0]Time (h) 70   Sample k1(s-1) × 106 Rapid-release phase rate constant in PBS k2(s-1) × 106 Slow-release phase rate constant in PBS dHPG(C8)/Pro 33.4 ± 1.1 0.6 ± 0.5 dHPG(C10)/Pro 41.1 ± 3.1 0.5 ± 0.3 dHPG(C12)/Pro 58.7 ± 4.6 0.3 ± 0.7 dHPG(C14)/Pro 55.4 ± 5.0 0.4 ± 0.2 dHPG(C18)/Pro 66.5 ± 2.1 0.4 ± 0.5 HPG/Pro 326 ± 26.8 N/A HPG-PEG/Pro 274 ± 24.8 N/A Free Pro 276 ± 32.8 N/A  Table 3.4  Pro release rate constants from dHPG(Cn)  3.3.6.2 Kinetics in Platelet Poor Plasma The drug release rate from dHPG(C8)/Pro formulation dissolved in plasma was also studied and compared with free Pro. The release of Pro from dHPG(C8) into plasma was again found to occur in two phases, the more rapid one of which was slower from the equivalent phase of release into PBS. The free Pro release from the cassette also exhibited two phases, unlike the case of free drug in PBS, but the rapid phase rate constant was only about 1/6 of that in PBS. Both of these results are consistent with Pro interacting with a relatively high affinity with components in plasma (Figure 3.14). The kinetic constants for these systems are summarized in Table 3.5.   71   Figure 3.14 Semi-log plots of the initial phase of release of Pro from the dialysis cassettes for dHPG(C8)/Pro in plasma (R2 = 0.96) and for dissolved Pro alone in PBS (R2 = 0.95) and plasma (R2 = 0.95). [C1] is the Pro concentration vs. time in the rapid release phase. [C10] is the initial Pro concentration, calculated from the intercept of ln [C1] vs. time.  Sample k1(s-1) × 106 in plasma k2(s-1) × 106 in plasma k1(s-1) × 106 in PBS k2(s-1) × 106 in PBS dHPG(C8)/Pro 14.3 ± 1.1 0.6 ± 0.4 33.4 ± 1.1 0.6 ± 0.5 Free Pro 48.5 ± 4.4 0.4 ± 0.8 276 ± 32.8 N/A  Table 3.5  Pro release rate constants from dHPG(C8) in plasma vs. PBS  -5-4-3-2-100 1 2 3 4 5 6 7 8 9ln[C1/C10]Time (h)dHPG(C₈)/Pro in PlasmadHPG(C₈)/Pro in PBSFree Pro in PlasmaFree Pro in PBS 72  3.4 Discussion 3.4.1 Correlation of Binding and Release Behaviour with Material Properties 3.4.1.1 Pro Loading Capacity and Effect on the Size In this chapter, the physicochemical properties of Pro loaded into dHPG(Cn) are evaluated. The presence of Cn alkyl chains in HPGs significantly enhances loading of hydrophobic drugs, like Pro. The changes in the material properties through the dHPG(Cn)/Pro formulations allowed an analysis using linear correlations of structure-function properties which are presented below.  Comparing all Pro binding profiles into dHPG(Cn) (Figure 3.4) and results from Table 3.2, the best correlation between loading capacity and a structural property was the dependence on the sum per dHPG(Cn) molecule of the number of carbon atoms in the alkyl units external to the oxygen contributed from the epoxide group, two less than the number of carbons in the alkyl monomers (n). The C12 dHPG(Cn) had the highest value for this parameter and the highest loading capacity. The correlation implies the hydrophobic group length was a strong determinant of loading capacity (Figure 3.15). The loading capacity also increased slightly with a slight rise in temperature. This could be explained by an increase in hydrophobic interactions between the drug and the polymer at the higher temperature; hydrophobic interactions are known to increase with increasing temperature in some cases [86].   73   Figure 3.15 Correlation between the maximum binding capacity of dHPG(Cn) polymeric systems for binding Pro and the their total mass of alkyl carbon external to the oxygen (R2 = 0.77 and p < 0.025)   Results from the effect of Pro encapsulation on the size of dHPG(C10) demonstrated the independency of the size from the concentration of the polymer in both the presence and absence of the hydrophobic drug. This is aligned with previous observations for a dHPG(C18) formulation, in which the drug’s solubility was changed with the polymer concentration at the same rate, resulting in no dependency between binding capacity of the hydrophobic molecule and the polymer concentration [53]. In addition, other studies have demonstrated the noticeably greater size of other polymeric micelles compared to HPGs. For example, the hydrodynamic radii of the formed micelle from polylactides grafting PEG and alkyl chains, with the molecular weight of about 100,000 Da and above, were in the range of 60 nm. Other examples, such as micelle formation from amphiphilic block copolymers of poly(ethylene oxide) derivatives in water, confirmed the large hydrodynamic radii, compared to their molecular weights [141], [142]. Given the size of these  - 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.00 200 400 600 800 1000 1200Maximum bound molar ratio (Pro/polymer)Number of alkyl carbons (n-2)/moleculedHPG(C18)dHPG(C12)dHPG(C8)dHPG(C14)dHPG(C10)dHPG(C6) 74  micellar structures and dHPG(Cn), the small hydrodynamic radii for dHPG(Cn) compared to their high molecular weights implies non-micellar structure for these polymers.  3.4.2 Pro Release Kinetics It has been previously demonstrated that more hydrophilic drugs are released more rapidly from the polymer matrix as water penetrates the polymer matrix [120]. All the release profiles demonstrated significant effects of dHPG(Cn) in stabilizing the formulation, compared to the free drug (Figure 3.7 and Table 3.4). Pro release occurred in two phases, each of which could be characterized by a first order kinetic constant. The initial phase was the most rapid and its kinetic constant, k1, was roughly 5 to 20 times greater than k2 characterizing the second phase.  The rates of release demonstrated the formulation of Pro reversibly bound to dHPG(Cn) caused enhanced solubility and greater stability which should result in longer circulation half-lives for the drug. This can be explained by stronger and prolonged binding of Pro due to alkyl content of the dHPG(Cn) through hydrophobic interactions, while PEG chains solubilized the system in the aqueous environment.  In the case of release in plasma, plasma proteins may have bound much of the free drug and slowed the whole process down as observed. Comparing the dHPG(Cn)/Pro systems and free Pro, either in PBS or plasma, indicated that the more hydrophobic the carrier, the stronger the hydrophobic interaction with the drug and the longer the retention of the drug as a combination (Table 3.5). Regression analysis data (Table 3.6) showed some significant dependences: both k1 and k2 are significantly correlated (p < 0.025) with the hydrated polymer volume (Vp), which was calculated from the hydrodynamic radius, and with the volume of the polymer with the alkyl  75  contribution subtracted (Vp – Va(n-2)). The strongest correlation (p < 0.03) for the Pro loading capacity of dHPG(Cn) was found with two less than the number of carbons in the alkyl monomers (n-2), where n is the total number of carbon atoms in the alkyl epoxide monomer. This is considered to be a measure of the hydrophobicity of each molecular species.    N=5  No. C(n)a No. C(n-2)b Va(n-2)c Vpd Vp-Va(n-2)e R2 p R2 p R2 p R2 p R2 p Max Pro per dHPG(Cn)f 0.559 0.087 0.743 0.027 0.710 0.073 0.119 0.57 0.111 0.58 k1 0.211 0.436 0.119 0.570 0.220 0.43 0.880 0.018 0.889 0.016 k2 0.033 0.770 0.003 0.934 0.038 0.75 0.863 0.022 0.863 0.022  Table 3.6 a: Number of alkyl carbon atoms per dHPG(Cn) molecule; b: Number of n-alkyl carbons external to oxygen per dHPG(Cn) molecule; c: Volume of n-alkyl carbons external to oxygen per dHPG(Cn) molecule; d: Volume of dHPG(Cn) molecule calculated from hydrodynamic radius; e: Volume of dHPG(Cn) molecule associated with HPG; f: Maximum ratio of Pro bound to dHPG(Cn) by loading protocol, moles per mole. P < 0.05 values are shown in bold font   76   Figure 3.16 Dependence of k1 on Vp-Va; R2 = 0.89 and p < 0.02  3.4.3 DSC Determination of Structured Water Since HPG is known to cause some water to structure or bind at the temperature used and not freeze near 0 °C [135], if this bound or structured non-freezing water had different solution properties for Pro an effect on the release kinetics of Pro from dHPG(Cn) might occur. To test this idea, we performed DSC measurements on the series of dHPG(Cn) species as described. The results are shown in Table 3.1, where the reduction in heat of fusion measured as a function of dHPG(Cn) structure and concentration is interpreted in terms of number of moles of water affected per mole of polymer. Regression analysis demonstrated strong correlation between the rapid release rate constant and the total number of water molecules bound to dHPG(Cn) both in 5% and 10% of polymer concentration. 010203040506070800 200 400 600 800 1000 1200 1400 1600 1800 2000k1(s-1) ×106Vp-VadHPG(C8)dHPG(C10)dHPG(C14)dHPG(C12)dHPG(C18) 77   N=5  Water/dHPG(Cn)a 10 % Water/dHPG(Cn)b 5 % R2 p R2 p k1 0.793 0.043 0.940 0.006 k2 0.392 0.258 0.720 0.069  Table 3.7 a: dHPG(Cn) at concentration of 10%; b: dHPG(Cn) at concentration of 5 % - Statistically significant results are shown in bold   The results of the DSC measurements in Table 3.1 were examined for correlation with the kinetic constants of Pro release from the dHPG(Cn) species. Regression analysis of dependence of k1 and k2 on the amount of bound water are presented in Table 3.7. It is seen the most significant correlation is between k1 and the number of moles of water affected per mole of dHPG(Cn) at the lower polymer concentration; detailed plots are shown in Figure 3.17 for polymer at 10% concentration and Figure 3.18 for polymer at 5% concentration. However, the k2 values did not correlate significantly with the water-related parameters. Since the release kinetics took place from 2% polymer solutions the 5% water binding data is likely the most relevant.    78   Figure 3.17 Correlation between k1 values and mole of structured water per mole of dHPG(Cn) at 10% polymer concentration; R2 = 0.79 and p < 0.05   Figure 3.18 Correlation between k1 values and mole of structured water per mole of dHPG(Cn) at 5% polymer concentration; R2 = 0.94 and p < 0.01  010203040506070800 2000 4000 6000 8000 10000 12000 14000k1(s-1)×10 6Mole of water/Mole of polymerdHPG(C18)dHPG(C14)dHPG(C12)dHPG(C8)dHPG(C10)010203040506070800 2000 4000 6000 8000 10000 12000 14000 16000 18000k1(s-1)×106Mole of water/mole of polymerdHPG(C18)dHPG(C14)dHPG(C12)dHPG(C8)dHPG(C10) 79  3.5 Summary Studies in this chapter conclude that dHPG(Cn) stabilizes the Pro formulation and increases the solubility of the drug. Encapsulation of Pro by dHPG(Cn) depends on the number of carbon atoms of the hydrophobic chain of alkyl monomer, incorporated in the polymer structure; the more carbon mass contained within the HPG, the more available binding sites and the higher the amount of bound Pro. Release of Pro from dHPG(Cn) formulations depends on the hydrodynamic size of the polymer, and more particularly on the amount of water that penetrates the polymer structure. Kinetic studies in both PBS and plasma confirm two rates of drug release from the drug-binding pocket; rapid and slow release phases. The release constant of the rapid phase (k1) depends on the hydrodynamic size of the polymer; the smaller the polymer size, the better the distribution of hydrophobic fractions and the higher the chances of hydrophobic interactions with the drug, reducing the release rate. This result is also aligned with the number of structured water molecules per polymer molecule, where the polymer with the smaller size binds the least number of water molecules.  The remarkably strong correlation of the amount of bound water and the release rate constant (k1) show the unique property of the designed systems with the relatively similar molecular weight distribution and alkyl content for encapsulation of Pro. This unique observation could provide more details to be able to simulate the formulation’s behaviour in the bio applications. Also, these results strongly suggest considering the hydration properties of the transport systems in designing drug delivery vehicles for encapsulation of hydrophobic molecules and hormones. However, the release rate of the slow phase (k2) correlates with the size of the polymer, but does not significantly correlate with the amount of bound water to the polymer. The  80  values of k2 are small, however, and could reflect some infrequent property of a small fraction of each preparation that is difficult to recognize.    81  Chapter 4:  In vitro Blood Compatibility Study of dHPG(Cn)/Pro Formulations   4.1 Synopsis In this chapter, biocompatibility testing of dHPG(Cn)/Pro formulations conducted in vitro is reported. The in vitro studies include hemocompatibility testing of the influence of dHPG(Cn) with and without drug loaded on coagulation (Activated Partial Thromboplastin Time (APTT) and Thromboelastrograph parameters (TEG)), platelet activation (CD62 expression), platelet aggregation, red blood cell aggregation as well as red blood cell lysis.   4.2 Materials and Methods 4.2.1 Blood Collection All blood collection was performed on consenting donors at the University of British Columbia, Centre for Blood Research’s blood collection suite. Blood was drawn from healthy un-medicated consenting donors under UBC Ethics approval no. H07-02067. Platelet rich plasma (PRP) was prepared by centrifuging the citrated whole blood at 74 × g for 10 min and platelet poor plasma (PPP) was collected by centrifuging the citrated whole blood at 826 × g for 20 min, in an Allegra X-22R Centrifuge (Beckman Coulter, Canada). Serum was obtained by centrifuging the serum tube at 826 × g for 20 min, after the whole blood had been allowed to clot for thirty min.  4.2.2 Plasma Clotting Assays Analysis: APTT  The effect of dHPG(Cn) and dHPG(Cn)/Pro formulations on the intrinsic pathway of coagulation was determined by APTT analysis. Activated Partial Thromboplastin Time were measured and repeated in triplicate on a coagulation analyzer using mechanical endpoint  82  determination; STart®4 coagulometer (Diagnostica Stago, France). Sodium citrate anticoagulated PPP was used for APTT analysis. Thromboplastin reagent actin FSL was used for this study. The effect of polymers on the coagulation cascade was examined by mixing PPP with the polymer solution (9:1 v/v to prepare 1 mg/mL or 10 mg/mL final concentration) at 37 °C. Control experiments were performed with PBS added to PPP. The average values of the clotting time were reported from at least three different donors. One hundred and eighty microliters of PPP with 20 µl of stock polymer solution was mixed (final concentrations were 1 mg/mL and 10 mg/mL) at room temperature. The same volume (200 µl) of the actin FSL was added. Then, 100 µl of the polymer-PPP-reagent mixture was added into the cuvette-strip and warmed up at 37 °C for 180 s. Fifty microliters of pre-warmed 0.025 M CaCl2 was then added to the cuvette and clot formation, assessed optically, was timed and recorded. Mean values of three technical replicates with standard deviation from three different donors were reported.  4.2.3 Red Blood Cell Aggregation Analysis Red blood cell (RBC) aggregation and morphology were measured in citrated anticoagulated blood. Ninety microliters of citrated whole blood was incubated with 10 µl of polymer stock solutions (final concentrations were 1 mg/mL and 10 mg/mL) for 1 h at 37 °C. PBS was used as normal control for the study. After incubation, the polymer-whole blood mixture was centrifuged at 5877 × g for 3 min. Two microliters of the cell suspension was placed on a clean glass slide and 6 µl of PPP was added to the top of the blood drop. The blood-plasma drop was mixed and microscopic images were taken with the Axioskop2 plus microscope (Zeiss). All images were taken at 40X magnification.  83  4.2.4 Red Blood Lysis Analysis: Drabkin’s Method For the hemolysis study, 30 µl of each of the stock polymer concentrations (to prepare 1 mg/mL or 10 mg/mL final concentration) was mixed with 270 µl of 10% hematocrit whole blood for 1 h at 37 °C. The complete lysis of red blood cells by H2O acted as the positive control (100% lysis) and the cell suspension incubated with PBS solution acted as the negative control for the study. The percent of red blood cell lysis was measured using the Drabkin’s method [143]. Twenty microliters of the whole blood/polymer solution was added to 1 mL of Drabkin’s solution. After centrifugation, the supernatant of the whole blood/polymer solution was also subjected to 1 mL of Drabkin’s solution. The difference in optical density (OD) was measured at 540 nm. The percent of red blood cell lysis in the sample is the OD of supernatant divided the OD of sample.  4.2.5 Platelet Activation Analysis: Flow Cytometry The level of platelet activation in PRP upon incubation with dHPG(Cn)/Pro formulations was quantified by flow cytometry. Thrombin and fibrinogen from human plasma, calcium chloride and antibody CD62-PE were purchased from Sigma-Aldrich Canada Ltd. Ninety microliters of PRP was incubated at 37 °C with 10 µl of stock polymer samples (final concentration was 1 mg/mL or 10 mg/mL). After 1 h, aliquots of the incubation mixtures were removed for assessment of the platelet activation state. Five microliters of post-incubation platelet/polymer mixture, diluted in 45 µl HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, was incubated for 20 min in the dark with 5 µl of fluorescent monoclonal anti-CD62-PE (Immunotech laboratories Inc., Monrovia, CA). The incubations were then stopped with 0.3 mL of PBS solution. The level of platelet activation was analyzed in a BD FACSCanto II flow cytometer (Becton Dickinson) by gating platelet specific events based on their light scattering profile. Activation of platelets was  84  expressed as the percentage of platelet activation marker CD62-PE fluorescence detected in the 10,000 total events counted. Duplicate measurements were done, the mean of which was reported. Controls were done for the flow cytometric analysis. One NIHU/mL of human thrombin (Sigma) was used as a positive control, and PE conjugated goat anti-mouse IgG polyclonal antibodies (Immunotech) were used as the non-specific binding control.  4.2.6 Platelet Aggregation Analysis Four hundred and fifty microliters of citrated PRP was pre-warmed in the whole blood Lumi-aggregometer (Chrono-Log Corporation) for 15 min at 37 °C. Fifty microliters of polymer stock solutions (for 1mg/mL or 10 mg/mL final concentration) was later added to PRP and the level of platelet aggregation was monitored for 15 min in the aggregometer. Platelet aggregation is recorded as percent of light transmittance in PRP compared to PPP sample. Fifty microliters of ADP (final concentration of 5 µM) and PBS were added to PRP and serve as the positive and negative controls of the analysis respectively.  4.2.7 Thromboelastography (TEG) Analysis  The effect of different dHPG(Cn), with and without Pro loaded, was examined using the TEG technique, which is the most comprehensive in vitro assaying technique for blood coagulation [144], [145]. TEG measures the physical properties of the fibrin clot formed and produces values for two main parameters that relate to the kinetics and strength of clot formation: R, the time from the start of a run when Ca2+ is added to a final concentration of polymer stock solution (10 mg/mL, 1 mg/mL or 0.1 mg/mL) until the first signs of detectable clot formation; and maximum amplitude (MA), a measurement of the maximum strength or stiffness of the formed clot. Whole blood from  85  healthy, consenting donors was collected into 3.8% sodium citrated blood collection tubes, with a blood to anticoagulant ratio of 9:1. Citrated human whole blood mixed with the polymers were subjected to a coagulation clotting study using a Thromboelastography hemostasis system 5000 (Haemscope Corporation); TEG cup and pin were purchased from Haemscope Inc. (Niles, IL). In each experiment, 40 µl of polymer stock solutions was incubated with 360 l of citrated whole blood. For all experiments, blood samples were used within 5 min of blood collection. Three hundred and forty microliters of the polymer-whole blood sample suspension were transferred to TEG cups; the coagulation analysis began when the anti-coagulated whole blood was re-calcified with 20 l of 0.20 M CaCl2 solution. PBS solution mixed with whole blood served as the normal control. The effects of different dHPG(Cn) on clotting of whole blood, PRP and PPP using TEG experiments were determined at 37 °C and ended after 2 h.  4.2.8 Scanning Electron Microscopy (SEM) For this study, plasma clots of 200 µl were formed in sterile round-bottomed 5 mL polypropylene tubes (BD Falcon) by recalcifying human PRP with 20 mM CaCl2 in the absence or presence of  dHPG(C8) in 20 mM HEPES buffered saline (pH 7.4 and 150 mM NaCl). Platelets in PRP were adjusted to 2.5×108 cells/mL using PPP. After 1 h at 37 °C, clots were immediately fixed using Karnovsky fixative (2.5% glutaraldehyde and 4% formaldehyde)  and repeatedly washed with 0.1 M sodium cacodylate buffer at pH 7.4 followed by post-fixation with 1% v/v osmium tetroxide. The samples were washed three times with distilled water and then dehydrated with a gradient ethanol series (20-95 % v/v). Clots were then critical point dried with CO2 in a Tousimis Autosamdri 815B critical point dryer, mounted onto stubs, and gold sputter-coated for  86  SEM examination using a Hitachi S-4700 field emission scanning electron microscope at 2,500 X and 5,000 X magnifications. Images of two different areas of each clot were captured.  4.3 Results 4.3.1 APTT Analysis    Coagulation is the process by which blood forms a clot, resulting in prevention of blood loss from a damaged vessel. The APTT assay was used to evaluate the common coagulation pathway and the results were expressed in seconds required for a fibrin clot to form in plasma after actin (APTT reagent) and calcium chloride were added to the sample. The results demonstrated a slight increase of the coagulation time after treatment by dHPG(Cn) with and without Pro, compared to controls (Figure 4.1 and Figure 4.2). dHPG(Cn) at 1 mg/mL increased the coagulation time less than 15% at the most and dHPG(Cn) at 10 mg/mL increased the coagulation time less than 30% at the most. Details of the probability values from t-Test among the groups are presented in Appendix F.1.     87   Figure 4.1 Effect of dHPG(Cn) on coagulation time at 1 mg/mL Figure 4.2 Effect of dHPG(Cn) and dHPG(Cn)/Pro formulations on coagulation time; polymer concentrations were 10 mg/mL and 125 µg/mL of Pro was loaded 33.432.430.2 30.7 30.129.2 29.2010203040dHPG(C₈) dHPG(C₁₀) dHPG(C₁₂) dHPG(C₁₄) dHPG(C₁₈) Free Pro PBS BufferTime (s)Samples and controls40.738.235.636.735.240.738.235.037.034.931.7 31.601020304050Time (s)Samples and controls 88  4.3.2 Red Blood Cell Aggregation The response of RBCs to dHPG(Cn)/Pro formulations when added to the whole blood in vitro at different concentrations was evaluated by microscope examination. At incubation with dHPG(Cn) at the concentration of 1 mg/mL, no enhancement of RBC aggregation was observed (Figure 4.3). RBCs were naturally aggregated and formed rouleaux.   a)   b)   c)   Figure 4.3 Effect of dHPG(C8) at 1 mg/mL on red blood cell aggregation; a) PBS control buffer; b) HPG-PEG control polymer at 1 mg/mL; c) dHPG(C8) at 1 mg/mL    89  Also, when the concentration of the polymer was increased 10 times, there was no abnormal aggregation of red blood cells observed compared to the free Pro and PBS control, neither by incubating with dHPG(Cn), nor with dHPG(Cn)/Pro at the concentration of 125 µg/mL of the drug (Figure 4.4). More results from this study are presented in Appendices F.2 and F.3.  a)   b)   c)      90  d)  e)  Figure 4.4 Effect of dHPG(C8) at 10 mg/mL, with or without Pro, on red blood cell aggregation; a) PBS control buffer; b) Pro control at 5 µg/mL; c) dHPG(C8) at 10 mg/mL; d) dHPG(C8)/Pro, where polymer concentration was 10 mg/mL loaded with 125 µg/mL Pro; e) Positive control for RBC aggregation from the similar structure (HPG-C8/10-MPEG) at 10 mg/mL, where C8/10 monomer is octyl/decyl glycidol ether  4.3.3 Red Blood Cell Lysis Lysis of RBCs was tested after incubation of whole blood with dHPGs at 1 mg/mL of dHPGs and 10 mg/mL of dHPG(Cn) and dHPG(Cn)/Pro formulations for 1 h at 37 °C. The percentage of lysis was determined through measuring the optical density of hemoglobin at 540 nm, using Drabkin’s reagent, which reacts with hemoglobin released into the media. No hemolysis was observed for any formulations, while the positive control (H2O) was associated with about 80% lysis (Figure 4.5 and Figure 4.6).   91  Figure 4.5 RBC lysis percentage was measured after incubation with dHPG(Cn) at 1 mg/mL    Figure 4.6 RBC lysis percentage was measured after incubation with dHPG(Cn) and dHPG(Cn)/Pro at 10 mg/mL of polymer and 125 µg/mL of loaded drug 2.22 2.42 2.29 2.30 2.52 2.49 2.2878.70102030405060708090100dHPG(C₈) dHPG(C₁₀)dHPG(C₁₂)dHPG(C₁₄)dHPG(C₁₈) HPG-PEG PBS H₂O % RBC lysisSamples and controls2.60 2.68 2.55 2.43 2.37 2.45 2.42 2.33 2.63 2.38 3.02 2.7083.40102030405060708090100% RBC lysisSamples and controls 92  4.3.4  Platelet Activation The influence of dHPG(Cn) formulation on blood clot initiation (aggregation of platelets) was studied by examining their effects on platelets. The expression of platelet surface receptor glycoproteins, indicative of activated platelets after incubation at 37 °C with dHPG(Cn) at 1 mg/mL and 10 mg/mL and dHPG(Cn)/Pro formulation (loaded with 125 µg/mL Pro) were studied and compared to the PBS buffer (as a negative control), thrombin (as a positive control) and free Pro at 5 µg/mL. The results demonstrated no platelet activation in any case, while the positive control (Thrombin) was associated with about 100% activated platelets (Figure 4.7 and Figure 4.8).   Figure 4.7 Platelet activation estimated from CD62 expression after incubation with dHPG(Cn) at 1 mg/mL   10.28 10.04 9.83 10.40 10.18 9.51 10.7797.60102030405060708090100dHPG(C₈) dHPG(C₁₀)dHPG(C₁₂)dHPG(C₁₄)dHPG(C₁₈) HPG-PEG PBS Thrombin% CD62-PESamples and controls 93   Figure 4.8 Platelet activation estimated from CD62 expression after incubation with dHPG(Cn) and dHPG(Cn)/Pro at 10 mg/mL and 125 µg/mL of bound drug   4.3.5 Platelet Aggregation Incubation of PRP with dHPG(C8) and dHPG(C12) at different concentrations (0.1 mg/mL, 1 mg/mL and 10 mg/mL) with PRP at 37 °C and their effect on platelet aggregation was examined after 15 min. No platelet aggregation was caused by control buffer, free Pro (5 µg/mL) or polymer samples at the first 15 min. By adding ADP (platelet aggregation reagent) to the control samples for another 15 min, full aggregation response to ADP was observed, so no inhibition took place (Figure 4.9 and Figure 4.10).   2.95 2.50 2.40 4.28 3.83 3.55 3.90 4.256.30 6.48 5.10 2.4595.500102030405060708090100% CD62-PESamples and controls 94     Figure 4.9 PBS control buffer; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right         Figure 4.10 Pro control at 5 µg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right       95  The dHPG(Cn) solutions, however, at high concentration (1 and 10 mg/mL) made a slight change to this behaviour in that the curves flattened and were constant at a little less than 100% transmission (Appendix F.4), which was hard to interpret, but almost full aggregation by ADP was observed by reducing the concentration to 0.1 mg/mL (Figure 4.11 and Figure 4.12). Polymers of this general structure are known to absorb to at least RBC surface [114], so such inhibition is possible in principle.      Figure 4.11 dHPG(C8) at 0.1 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right     Less influence of the polymer on the aggregation pathway was observed for dHPG(C12) even at higher concentrations (Appendix F.4), compared to the dHPG(C8). This could have happened as a result of the difference in hydrophobicity of these polymers or different distribution of the hydrophobic chain in the polymer structure.     96      Figure 4.12 dHPG(C12) at 0.1 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right     4.3.6 Thromboelastography (TEG) Analysis The healthy body response to prevent bleeding from injuries is formation of the blood clot (thrombus). Therefore, it is important that any external material contacting the circulation does not change the process of thrombus formation. In this study the effect of dHPG(Cn) formulation on the blood clot formation pathway was examined.   4.3.6.1 Effect on Whole Blood dHPG(Cn) at the concentration of 10 mg/mL, with and without 125 µg/mL of Pro loaded, and dHPG(Cn) at 1 mg/mL were incubated with whole blood and their effect on clot formation was observed and compared to free Pro at 5 µg/mL and PBS control buffer (Figure 4.13, Figure 4.14, Figure 4.15 and Figure 4.16). Time zero (T = 0) is the time of Ca2+ addition to the samples for all TEG profiles. Blood samples were taken from different donors at different times.  97   Figure 4.13 TEG profile of dHPG(C8)/Pro formulation in whole blood – PBS was used as control   Figure 4.14 TEG profile of dHPG(C12)/Pro formulation in whole blood – PBS was used as control    R TimeT = 0 T = 2 hdHPG(C8) (1mg/ml)dHPG(C8)/ProdHPG(C8) (10 mg/ml)PBS (control) Free ProOverall StrengthR TimeT = 0 T = 2 hFree ProPBS (control)dHPG(C12) (1mg/ml)dHPG(C12)/ProdHPG(C12) (10 mg/ml)Overall Strength 98   Figure 4.15 TEG profile of dHPG(C14)/Pro formulation in whole blood – PBS was used as control   Figure 4.16 TEG profile of dHPG(C18)/Pro formulation in whole blood – PBS was used as control  Comparing all TEG profiles above, free Pro did not show any large effects on the characterization of the blood clot compared to control buffer. However, dHPG(Cn) in all cases from C8 to C18 at high concentration (10 mg/mL) caused significant effects on the signal representing the strength of the clot and apparently caused the clots to collapse, while no significant effect on the clot initial formation time (R time) was observed. Loading systems with Pro slightly R TimeT = 0 T = 2 hFree ProdHPG(C14) ( 1mg/ml)dHPG(C14)/ProdHPG(C14) (10 mg/ml)PBS (control)Overall StrengthR TimeT = 0 T = 2 hdHPG(C18) (1mg/ml)PBS (control)Free ProdHPG(C18)/ProdHPG(C18) (10 mg/ml)Overall Strength 99  improved clot’s overall strength, which was most noticeable with the dHPG(C18)/Pro formulation. However, when the concentration of the polymers was decreased to 1 mg/mL, a smaller reduction was observed on the overall strength of the formed clot in all cases with no reduction observed for the dHPG(C18) polymeric system. Based on the usual interpretation of TEG data, high polymer concentration affected the blood clot strength parameter significantly. The effects of all dHPG(Cn) at 1 mg/mL (Figure 4.17) and 0.1 mg/mL (Figure 4.18) were also measured and compared with control buffer (PBS) and control polymer (HPG-PEG with no alkylation) in order to find the threshold concentration of polymers needed to give parameters typical of a normal clot.   Figure 4.17 Effect of polymer concentration of dHPG(Cn) [n = 8, 10, 12, 14, 18] on blood clot characterization at 1 mg/mL – PBS and HPG-PEG at 1 mg/mL were controls  dHPG(C8)HPG-PEG (Control)dHPG(C18)dHPG(C14)dHPG(C12)dHPG(C10)PBS (Control)Overall StrengthR TimeT = 0 T = 2 h 100   Figure 4.18 Effect of polymer concentration of dHPG(Cn) [n = 8, 10, 12, 14, 18] on blood clot characterization at 0.1 mg/mL – PBS and HPG-PEG at 0.1 mg/mL were controls  The TEG profile in whole blood incubated with polymers at 1 mg/mL and 0.1 mg/mL, verified a significant effect of concentration on the clot overall strength signal in all systems. dHPG(C8) and dHPG(C10) at the higher concentration caused more significant reduction in this parameter, while at the lower concentration, most of the systems acted like control buffer and HPG-PEG control, except for dHPG(C8).  4.3.6.2 Effect on Platelet Poor Plasma (PPP) Results from the whole blood in the previous experiment demonstrated that dHPG(Cn) affects the signal associated with blood clot’s overall strength. However, by reducing the concentration from 10 mg/mL to 0.1 mg/mL, the presence of most of the polymeric systems in blood did not affect the clot characteristics. To better understand this effect, PPP also was incubated with dHPG(Cn) at 10 mg/mL and 1 mg/mL to examine the plasma components’ contribution free of cells. For this study, dHPG(C8) and dHPG(C12) were selected. PBS and HPG-PEG (with no alkylation) were also used as controls (Figure 4.19 and Figure 4.20). Time zero (T dHPG(C10)dHPG(C12)dHPG(C14)dHPG(C18)dHPG(C8)HPG-PEGPBS (control)R TimeT = 0 T = 2 hOverall Strength 101  = 0) is the time of Ca2+ addition to the samples for all TEG profiles. Blood samples were taken from different donors at different times.   Figure 4.19 Effect of polymer concentration of dHPG(Cn) [n = 8, 12] on clot characterization in PPP at 10 mg/mL – PBS and HPG-PEG at 10 mg/mL were controls   Figure 4.20 Effect of polymer concentration of dHPG(Cn) [n = 8, 12] on clot characterization in PPP at 1 mg/mL – PBS was used as control  T = 0 T = 2 hR TimeHPG-PEGdHPG(C12)dHPG(C8) PBS (control)Overall StrengthT = 0 T = 2 hR TimedHPG(C8)PBS (control)dHPG(C12)Overall Strength 102  Compared to the whole blood results, polymers at the same or similar concentration exhibited much less effect on the clot strength. This was even more noticeable at the high concentration (10 mg/mL) of the polymers (Figure 4.19).   4.3.6.3 Effect on Platelet Rich Plasma (PRP) The same experiments were done by incubation of PRP with dHPG(C8) and dHPG(C12) to observe the effect of polymers on platelets in plasma. The experiments were run with 10 mg/mL and 1 mg/mL of polymers (Figure 4.21 and Figure 4.22). Time zero (T = 0) is the time of Ca2+ addition to the samples for all TEG profiles. Blood samples were taken from different donors at different times.   Figure 4.21 Effect of polymer concentration of dHPG(Cn) [n = 8, 12] on clot characterization in PRP at 10 mg/mL – PBS and HPG-PEG at 10 mg/mL were controls   T = 0 T = 2 hR TimeHPG-PEGdHPG(C12) dHPG(C8)PBS (control)Overall Strength 103   Figure 4.22 Effect of polymer concentration of dHPG(Cn) [n = 8, 12] on clot characterization in PRP at 1 mg/mL – PBS and HPG-PEG at 1 mg/mL were controls   Compared to the whole blood and PRP results, the effects of polymers at the same concentrations followed the same behaviour seen in whole blood, in which the polymers affected the clot strength signal significantly, especially at the higher concentration in both cases (Figure 4.21). Concentration effect of the polymers on the clot properties was the same as their effect on the platelet aggregation at the higher concentration.  Based on the PRP and whole blood results, dHPG(C8) presented the greatest effect on the clot strength signal. In order to find out the threshold concentration of this system for forming a normal clot, PRP was incubated with different concentrations of the polymer from 0.1 mg/mL to 0.01 mg/mL (Figure 4.23 and Figure 4.24). T = 0T = 2 hR TimePBS (control)dHPG(C8) dHPG(C12)HPG-PEGOverall Strength 104   Figure 4.23 Effect of polymer concentration of dHPG(C8) on clot characterization in PRP at 1 and 0.1 mg/mL – PBS and HPG-PEG at 1 mg/mL were controls     Figure 4.24 Concentration effect of dHPG(C8) on clot characterization in PRP from 0.1 to 0.01 mg/mL – PBS was used as control   In the case of dHPG(C12) in PRP, by reducing the concentration from 1 mg/mL to 0.1 mg/mL, normal clot was indicated (Figure 4.25). T = 0 T = 2 hR TimeHPG-PEG dHPG(C8) (0.1 mg/ml)dHPG(C8) (1 mg/ml) PBS (control)Overall StrengthT = 0 T = 2 hR TimedHPG(C8) (0.01 mg/ml)dHPG(C8) (0.05 mg/ml)dHPG(C8) (0.1 mg/ml)PBS (control)Overall Strength 105   Figure 4.25 Effect of polymer concentration of dHPG(C12) on clot characterization in PRP at 1 and 0.1 mg/mL – PBS was used as control   4.3.7   Scanning Electron Microscopy (SEM) Based on the results of TEG in whole blood and PRP, the signals representing the clot’s overall strength was significantly reduced by dHPG(C8) polymeric systems, the more so the higher the concentration. To try to confirm this interpretation. SEM images were made from similar systems of PRP allowed to clot under stagnant conditions for 1 h. The results were again compared to HPG-PEG at the same concentrations and HBS buffer as controls (Figure 4.26). Images were taken from static clots. SEM images from the dynamic clots (separated from the TEG samples) are presented in Appendix F.5.   T = 0 T = 2 hR TimePBS (control)dHPG(C12) (1 mg/ml)dHPG(C12) (0.1 mg/ml)Overall Strength 106  a)   b)   c)    107  d)     Figure 4.26 SEM imaging of static clots; a) HBS control buffer; b) HPG-PEG at 1 mg/mL; c) dHPG(C8) at 1 mg/mL; d) dHPG(C8) at 0.1 mg/mL at different zooming  4.4 Discussion Results from APTT demonstrated moderate increase of the coagulation time after treatment with dHPG(Cn) at their highest concentration. However, this effect was decreased by reducing the polymer concentration. Presence of Pro in all cases did not cause any effect. Results from RBC aggregation, RBC lysis and platelet activation demonstrated that dHPG(Cn) even at high concentration with or without drug loaded do not cause any destructive influence. However, the platelet aggregation study presents a slight change in the behaviour after incubation with dHPG(C8), which is observed as a concentration dependent effect. The same behaviour is noticed  108  the most on the maximum strength of the clot after incubation treatment with dHPG(C8). More significant interactions could result from the smallest volume of dHPG(C8) and more uniform distribution of the hydrophobic chains in the polymeric structure, compared to the other dHPG(Cn). Results from the TEG study demonstrated concentration dependent effect of the dHPG(Cn) on the strength of the blood clot, of which dHPG(C8) significantly influenced the clot maximum strength at the highest of studied concentrations (Figure 4.27). However, the clot initial formation time (R time) was not affected in any cases at any concentrations (Figure 4.28).    Figure 4.27 dHPG(Cn) TEG profile in whole blood; clot maximum strength values were measured and compared at 1 mg/mL and 0.1 mg/mL of polymer. * demonstrates the significant difference between the values  010203040506070PBS HPG-PEG dHPG(C₈) dHPG(C₁₀) dHPG(C₁₂) dHPG(C₁₄) dHPG(C₁₈)Maximum amplitude (mm)Polymers and controls0.1 mg/ml 1 mg/ml 109   Figure 4.28 dHPG(Cn) TEG profile in whole blood; clot initial formation time (R) values were measured and compared at 1 mg/mL and 0.1 mg/mL of polymer. No significant difference was observed among the polymers and controls  TEG data suggests that under the conditions of the TEG analysis the dHPG(Cn), with or without Pro present reduces the strength of the clot to varying degrees, depending on concentration and composition of the polymer species and whether or not RBCs or platelets were present in the test samples. SEM images of some of the same compositions in PRP after 1 h of clotting in a static tube did not show any obvious flaws in the fibrin or platelet aggregates, however, even with the dHPG(C8) species at 1 mg/mL which caused a significant reduction in apparent clot strength in the TEG assay (Figure 4.13 and Figure 4.17). To see if the shearing of the sample could have affected the clot image, samples were taken from the TEG cup after shearing for 1 h and processed for SEM. The images for the HPG-PEG controls (Figure 4.26(b)) showed a somewhat tighter mesh in the fibrin mat than static controls but otherwise there were no obvious differences. A 024681012141618PBS HPG-PEG dHPG(C₈) dHPG(C₁₀) dHPG(C₁₂) dHPG(C₁₄) dHPG(C₁₈)R Time (min)Polymers and controls0.1 mg/ml 1 mg/ml 110  tighter fibrin mesh compared to static samples was also seen when either 1 mg/mL (Figure 4.26(c)) or 0.1 mg/mL (Figure 4.26(d)) dHPG(C8) was present, but otherwise platelet aggregates associated with the fibers were present at both concentrations and no obvious abnormalities in the clot infrastructure was evident.   The above comparison suggests that the TEG signals may not be reflecting only the dynamic mechanical properties of the clot since clotting certainly is taking place in an apparently normal way in so far as the SEM images represent them. Some clear anomalies are evident, for instance in Figure 4.13 and Figure 4.17 after 1 h the strength parameter (amplitude of the curve compared to zero) is essentially zero yet the SEMs clearly show clotting is present. This could be explained by the following mechanism.  The TEG apparatus consists of a cylindrical cup into which the sample is pipetted, calcium added, then an inner element with a smaller radius is lowered into the blood sample. The outer cup then oscillates at a low frequency (~1 Hz) and the oscillatory force transmitted by the elastic properties of the sample are recorded by electronics linked to the inner element. In order for a significant signal to appear at the surface of the inner element the sample must interact strongly with both the surface of the cup, so that the oscillation can be transmitted to the sample, and with the surface of the inner element to transmit the elastic strength of the sample to the measuring circuitry. The system is optimized for the low elastic properties of clotting blood and requires close association of the clot to the surfaces involved. If the clotting material in the gap does associate with the two surfaces, normally the signal will be low or zero, even though the clot itself could exhibit normal elastic properties. This is not known to occur in clinical conditions that produce variations in clot properties but the present samples show inconsistencies only when dHPG(Cn) are included. If these polymers were to bind to the TEG cup and inner element surfaces and  111  prevented the clot from associating with the surfaces the kind of anomalies seen in the present work could occur. As the disposable cups are made from an acrylic-based multipolymer compound, the binding of hydrophobic polymer to the inner wall of the cups could be possible. There is data from our group that shows that alkylated HPGs can adsorb to red cells much more strongly than HPG itself so it is possible that a similar difference could exist for the materials used in the TEG apparatus [114]. This possibility has not been examined directly yet. It seems likely that the detailed conclusions on effects of dHPG(Cn) on clot strength based on the TEG results may not be accurate, however. The reproducibility of TEG results did vary depending on the donor and different interactions of the material with the wall of the TEG cups.  4.5 Summary This chapter presents blood biocompatibility properties of dHPG(Cn) in vitro, with or without Pro loaded. Results prove that free and bound drug do not affect any blood characteristics by themselves. In most cases, dHPG(Cn) are blood compatible and harmless materials in the presence and absence of Pro. However, the observed differences among various formulations could be correlated with their hydrophobicity, given the volume of the polymers and distribution of the hydrophobic volume in the polymeric structures differ from one structure to another. The more uniform distribution of the hydrophobic chains could result in better interactions with hydrophobic materials. However, there is not enough evidence to support this and more detailed experiments should be done on the effect of these polymers on the blood coagulation properties.     112  Chapter 5: In vitro Cellular Cytotoxicity and Uptake Study of dHPG(Cn)/Pro Formulation  5.1 Synopsis Polymers have been often used to improve the therapeutic efficiency, drug-delivery and the pharmacokinetic properties of drugs [79], [90]–[92], [104], [111], [135] and [146]. The blood brain barrier (BBB) is recognized as the main element for precisely controlling the environment of neurons of the central nervous system for their optimal function, and limits the delivery of therapeutics into the CNS [147]. After many years of extensive research on the use of Pro in TBI, it is now clear that Pro is a neurosteroid which can affect multiple mechanisms involved in neuroprotection and repair, following numerous types of brain injury. Pro has been tested in different injury models in humans, including TBI, stroke, spinal cord injury and neurodegenerative disorders [29], [148], [149]. Therefore, the proper delivery system, which can carry the drug to the target cells and transport through the BBB, has an important role in the treatment.  This chapter includes cellular uptake studies of fluorescently-labelled dHPG(Cn) by human cortical microvascular endothelial cells (HCMEC/D3) and human astrocytoma cells (CCF-STTG1), as two representatives of brain cells. The viabilities of these cells after treatment with the polymeric formulations, loaded with and without Pro, are also described. To get more information regarding the endocytosis process of the polymer, incubation of these cells with anti-lysosome antibody is also examined. In addition, to monitor the presence of Pro, Michigan Cancer Foundation-7 (MCF7) cells loaded with the polymer inside and stained with anti-progesterone receptor antibody are used as positive control cells. Confocal microscopy is used for the imaging  113  process. However, this techniques is not used for a quantitative study, but rather used to monitor the presence of fluorescently-labelled materials inside the cells.  5.2 Materials and Methods 5.2.1 Chemicals All solvents were HPLC grade from Fischer Scientific (Ottawa, ON). N-(2,3-epoxypropyl)-phthalimide (EPP), sodium hydride (NaH) and sodium borohydride (NaBH4) were purchased from Sigma-Aldrich (Oakville, ON). Alexa Fluor® 488 NHS ester (succinimidyl ester), Wheat Germ Agglutinin (WGA), Alexa Fluor® 633 conjugate and ProLong® Gold Antifade Reagent with DAPI were obtained from Molecular Probes (Burlington, ON). Anti-LAMP1 antibody - Lysosome marker and anti-progesterone receptor antibody [YR85] were purchased from Abcam (Toronto, ON).  5.2.2 Cell Lines Immortalized human cortical microvascular endothelial cells (HCMEC/D3) were purchased from Fisher Scientific (Ottawa, ON). Human astrocytoma cells (CCF-STTG1, ATCC) were purchased from ATCC (Manassas, VA). Michigan Cancer Foundation-7 (MCF7) cells were purchased from ATCC (Manassas, VA). Cells were cultivated as previously described [147], [150]. Briefly, HCMEC/D3 were cultivated in EGM2 medium (supplemented with VEGF, hrIGF-1, hEGF, gentamycin, amphotericin-B, hydrocortisone, ascorbic acid, heparin, and 2% FBS; LONZA Inc.) and CCF-STTG1 in DMEM (supplemented with L-glutamine, pen/strep and 10% FBS, LifeCell technology).  MCF7 were cultivated in DMEM/F-12 (supplemented with 10% FBS and pen/strep).  114  5.2.3 Fluorescent Labeling of dHPG(Cn) Modification of HPGs with amines has been reported previously [122], [151]. dHPG(C8) was selected for the brain cell uptake and viability study based on the results from previous chapters. Two to five percent of free hydroxyl groups of dHPG were targeted to be modified into primary amine groups, followed by reacting with Alexa 488 (NHS ester). For this, 500 mg of dHPG(C8) was dissolved in 15 mL of anhydrous dimethylformamide (DMF). Eight milligrams of NaH (0.33 mmol, 4 equiv) was then added to the solution and stirred at room temperature for 2 h to form a clear solution. The temperature was then increased to 65-70 °C, and 67 mg (0.33 mmol) of EPP was slowly added to the solution and the reaction mixture was stirred for 24 h. The polymer mixture was then precipitated two times in diethyl ether and the modified dHPG was obtained by centrifuging at 574 × g and at 15 °C for 15 min. Conversion of the hydroxyl group to the N-phthalimide was confirmed by proton NMR (Appendix G.1). Cleavage of the N-phthalimide function was achieved by slow addition of an excess amount of NaBH4 to the aqueous solution of dHPG(C8) N-phthalimide at room temperature and the solution was then stirred at 50 °C for 15 h. Acetic acid (300 µl) was added drop-wise to the solution to reach a pH of about 5 and the residual acetic acid and unreacted materials were cleared out by dialysis of the polymer solution against water for two days using a dialysis membrane of 1000 g/mol MWCO. The modified polymer with primary amine groups was obtained after freeze drying and the absence of peaks from N-phthalimide function was confirmed by proton NMR (Appendix G.2). The amine-functionalized polymer was dissolved in 15 mL of anhydrous DMF and 1 mg of Alexa 488 NHS ester was added to the solution and stirred for several hours. The purified fluorescent polymer was obtained after dialysis against water for 3 days using 3500 g/mol MWCO dialysis membrane, followed by freeze drying (Figure 5.1). Pro was encapsulated by dHPG(C8) as explained in Chapter 3.   115   Figure 5.1 Synthetic scheme for the fluorescent labelling of dHPG(C8) with Alexa 488 NHS ester  5.2.4 Cellular Uptake Study HCMEC/D3 and CCF-STTG1 cells were seeded on glass cover-slides two days prior to the assay. Then cells in growth media were incubated with 0.001 to 1 to mg/mL of labelled dHPG(C8) compound for 1 h or with 0.01 mg/mL for 15 min, 1, 2, 4, 8, 24 and 48 h 37 °C. After incubation, cells were washed three times in PBS and fixed with 3.75 % paraformaldehyde (PFA) at room temperature for 30 min. The fixation was stopped by washing with 0.5 mM Tris-HCl at pH 7.4 solution for 10 min. After washing with PBS twice, cells were incubated with 5 mg/mL of WGA Alexa 633 for 15 min at room temperature in order to label cells’ membranes. Later, following three additional PBS washes, cells were mounted in embedding solution containing DAPI to visualize the nuclei. The cells were visually inspected using an SP5 inverted confocal microscope (Leica), connected to the HyD detector with the scan speed of 400 Hz. An average of 3 scans were taken for each image. The microscope lens magnification used was 63X. All uptake experiments in this chapter were repeated 3 times on average. R=Alexa  116  5.2.5 Cellular Uptake Study with Anti-LAMP1 Antibody – Lysosome Marker HCMEC/D3 or CCF cells were seeded on glass cover-slides two days prior to the assay. Then cells in growth media were incubated with 0.01 mg/mL of fluorescently-labelled dHPG(C8) compound for 2, 4 or 6h 37 °C. After incubation, cells were washed three times in PBS and fixed with 3.75 % paraformaldehyde (PFA) at room temperature for 30 min. The fixation was stopped by washing with 0.5 mM Tris-HCl at pH 7.4 solution; after three additional PBS washes, cells were permeabilized with 0.2% Triton X-100 (average MW of 625 g/mol) in PBS buffer (at the concentration of 0.05% (v/v)) for 5 min. After three additional washes with PBS, cells were incubated with 1:400 anti-LAMP1 antibody (Abcam) overnight at 4 °C [152]. After three additional PBS washes, cells were incubated with Alexa Fluor 594 secondary labelled antibody for 45 min at room temperature. Later, cells were washed three more times with PBS and mounted in embedding solution containing DAPI to visualize the nuclei. The cells were visually inspected using an SP5 inverted confocal microscope (Leica).  5.2.6 Cellular Uptake Study with Anti-Progesterone Receptor Antibody MCF7 cells were seeded on cover-slides two days prior to the assay. Cells in growth media were then incubated with 0.01 mg/mL of fluorescently-labelled dHPG(C8) compound for 2 and 4 h or with 0.1 to 1 mg/mL for 2, 4 and 24 h 37 °C. Pro at a concentration of 60 µg/mL was also incubated with cells as a control. After incubation, cells were washed three times in PBS and fixed with 3.75 % paraformaldehyde (PFA) at room temperature for 30 min. The fixation was stopped by washing with 0.5 mM Tris-HCl at pH 7.4 solution, after three additional PBS washes, cells were permeabilized with 0.2% Triton X-100 in PBS buffer for 5 min. After three additional washes with PBS, cells were incubated with 1:300 anti-progesterone receptor antibody [YR85] (Alexa  117  Fluor® 647) overnight at 4 °C [153]. Later, cells were washed three more times with PBS and mounted in embedding solution containing DAPI to visualize the nuclei. The cells were visually inspected as above.  5.2.7 Cellular Viability Study Pro was loaded into dHPG(C8) as described in Chapter 3. HCMEC/D3 and CCF-STTG1 cells were seeded in 96 well plates two days before the assay. Cells in growth media were incubated with 0.01 to 1 mg/mL of compounds containing 0, 5, 7.5 or 12.5 g of Pro per mg/mL of polymer for 1 h or with 0.01 mg/mL for 1, 24 or 48 h 37 °C. After incubation, cell viability was tested using Cell Titer-Blue as recommended by the manufacturer (Promega). Cells were also incubated with free Pro as control at 12.5 µg/mL of drug concentration at different time points.   5.3 Results 5.3.1 Cellular Uptake Results from uptake studies with CCF and HCMEC/D3 cells showed uptake occurred and was dose dependent and time dependent. In order to find out the best dose of dHPG(C8) for the uptake study, the concentration of the polymer was increased from 0 to 1 mg/mL and the uptake was measured after 1 h of incubation of cells with polymer and compared to the media only. Selecting the proper concentration of the carrier in order to deliver the correct amount of the drug to the target has an important role, given the human plasma concentration of Pro has been reported to be 0.3 to 300 nM [154]. It has been demonstrated from the binding study in Chapter 3 that 1 mole of dHPG(C8) can carry 5 moles of Pro. Therefore, 0.01 mg/mL of dHPG(C8) (10-7 M) was selected for this study, which can carry about 500 nM of Pro (Figure 5.2).   118   Figure 5.2 Dose dependent uptake of dHPG(C8) by HCMEC/D3 (human cortical microvascular endothelial cells); cells were incubated with different doses of dHPG(C8) from 0 to 1 mg/mL for 1 h at 37 °C ; Alexa 488 represents labelled polymer, Alexa 633 represents stained membrane with WGA and Dapi represents stained nucleus; for all concentrations, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities  dHPG(C8) at 0.01 mg/mL was then incubated with HCMEC/D3 cells at different time points from 0 to 24 h and the change in fluorescent intensity was monitored using confocal microscopy (Figure 5.3). mg/mL1                  0.1               0.01         0.001            0Alexa 488 Alexa 633 Dapi Merge10 m 119    Figure 5.3 Time dependent uptake of dHPG(C8) by HCMEC/D3 cells; cells were incubated with 0.01 mg/mL of dHPG(C8) from 0 to 24 h; increase in the green fluorescent intensity in cell’s cytoplasm demonstrated increase in the uptake of the polymer by cells; Alexa 488 represents labelled polymer, Alexa 633 represents stained membrane with WGA and Dapi represents stained nucleus; for all time points, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities Alexa 488 Alexa 633 Dapi Merge10 µmTime (h)24     8             4              2            1                   0.25                  0 120  The same studies were done for CCF cells and very similar results were observed for dose and time dependence as above (Figure 5.4 and Figure 5.5).     Figure 5.4 Dose dependent uptake of dHPG(C8) by CCF (human astrocytoma cells); cells were incubated with different doses of dHPG(C8) from 0 to 1 mg/mL after 1 h at 37 °C; Alexa 488 represents labelled polymer, Alexa 633 represents stained membrane with WGA and Dapi represents stained nucleus; for all concentrations, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities Alexa 488 Alexa 633 Dapi Merge10 µmmg/mL1                  0.1               0.01         0.001            0 121    Figure 5.5 Time dependent uptake of dHPG(C8) by CFF cells; cells were incubated with 0.01 mg/mL of dHPG(C8) at different time points from 0 to 24 h; increase in the green fluorescent intensity in cell’s cytoplasm demonstrated increase in the uptake of the polymer into the cytosol; Alexa 488 represents labelled polymer, Alexa 633 represents stained membrane with WGA and Dapi represents stained nucleus; for all time points, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities Alexa 488 Alexa 633 Dapi Merge10 mTime (h)24     8             4              2            1                   0.25                  0 122  5.3.2 Cellular Uptake with Anti-Lysosome Antibody  Results from the cellular uptake above using CCF and HCMEC cells demonstrated the long lasting properties of the dHPG(C8) inside the cells for at least 24 h. In order to monitor what happens when the polymer enters the cells, cells incubated with polymer at 0.01 mg/mL were stained with anti-lysosome antibody (LAMP1) and monitored for 8 h. Results from this study showed, at most, a low endolysosomal accumulation of dHPG(C8) with or without Pro loaded (at the concentration of 0.125 µg/mL) within the examined time (Figure 5.6). Co-localization of the polymer with lysosomes produced a yellow signal in merged images.      a)           b)       DapiLAMP1 MergeAlexa 48810 µmDapi Alexa 488MergeLAMP1Dapi Alexa 488MergeLAMP110 µm10 µm 123     c)          d)     Figure 5.6 Uptake of dHPG(C8) by CCF cells visualized by anti-lysosome antibody (LAMP1) at 37 °C; Alexa 488 represents labelled polymer, Dapi represents stained nucleus and LAMP1 represents anti-lysosome antibody; for all time points, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities; yellow signal represents the convergence of the polymer and lysosome. a) Media control – b) after 2 h; cells were incubated with dHPG(C8) (on the left) and dHPG(C8)/Pro (on the right) – c) after 4 h; cells were incubated with dHPG(C8) (on the left) and dHPG(C8)/Pro (on the right) – d) after 8 h; cells were incubated with dHPG(C8) (on the left) and dHPG(C8)/Pro (on the right). In all cases polymer and Pro concentrations were 0.01 mg/mL and 0.125 µg/mL, respectively  LAMP1Dapi Alexa 488MergeDapi Alexa 488MergeLAMP110 µm 10 µmDapi Alexa 488 Dapi Alexa 488Merge MergeLAMP1LAMP110 µm 10 µm 124  The same study was done on HCMEC/D3 cells; within the first 2 h, a low accumulation of the polymer in the lysosomes was observed (Appendix H). As shown in Figure 5.6, a high amount of lysosomal fluorescence from the antibody was observed, but only a small amount was co-localized with the HPG label.  5.3.3 Cellular Viability The percentage of live cells was measured after incubation of brain endothelial cells and astrocytoma cells with dHPG(C8) and dHPG(C8)/Pro formulations. Results from dose-dependent studies of both cell lines demonstrated that not only unloaded polymer, but also different concentrations of Pro loaded on dHPG(C8) did not cause any significant changes in the number of live cells. No significant differences were observed among the formulations compared to the media only and free Pro as controls in 1, 24 or 48 h (Figure 5.7 and Figure 5.9). Also, the results from the effect of the time of incubating cells with the loaded and unloaded dHPG(C8) demonstrated that  there was no significant effect after treatment with polymer in time over 48 h, compared to the media, and more than 80% of the cells stayed alive (Figure 5.8 and Figure 5.10). Analysis of variance (ANOVA) was done to find out the whether there were any significant differences between the groups and the media.     125     Figure 5.7 Dose dependence study; percent viability of HCMEC/D3 cells compared to no treatment after incubation with dHPG(C8)/Pro at 5, 7.5 and 12.5 µg of the drug loaded on 1 mg/mL of the polymer. Different concentrations of polymer loaded with the drug were compared with the polymer alone and media only; significant differences between groups and the media control (NONE) using ANOVA are demonstrated by *. DMSO was used as negative control   Figure 5.8 Time dependence study; percent viability of HCMEC/D3 cells compared to no treatment after incubation with dHPG(C8)/Pro at 5, 7.5 and 12.5 µg of the drug loaded on 1 mg/mL of the polymer in 1, 24 and 48 h; significant differences between groups and the media control (NONE) using ANOVA are demonstrated by *. Free Pro as control was incubated with cells at the concentration of 12.5 µg/mL. DMSO was used as negative controlNONE 0.010.1 10.010.1 10.010.1 10.010.1 1DMSO050100150StimuliViability %12.5/17.5/15/10/1NONE 0.01mg/ml0.1mg/ml1mg/mlDMSONONE  1h 24h48h 1h 24h48h 1h 24h48h 1h 24h48h 1h 24h48hDMSO050100150StimuliViability %12.5/17.5/15/10/1Free Pro 126    Figure 5.9 Dose dependence study; percent viability of CCF cells compared to no treatment after incubation with dHPG(C8)/Pro at 5, 7.5 and 12.5 µg of the drug loaded on 1 mg/mL of the polymer. Different concentrations of polymer loaded with the drug were compared with the polymer alone and media only; significant differences between groups and the media control (NONE) using ANOVA are demonstrated by *. DMSO was used as negative control   Figure 5.10  Time dependence study; percent viability of CCF cells compared to no treatment after incubation with dHPG(C8)/Pro at 5, 7.5 and 12.5 µg of the drug loaded on 1 mg/mL of the polymer in 1, 24 and 48 h; significant differences between groups and the media control (NONE) using ANOVA are demonstrated by *. Free Pro as control was incubated with cells at the concentration of 12.5 µg/mL. DMSO was used as negative controlNONE 0.010.1 10.010.1 10.010.1 10.010.1 1DMSO050100150StimuliViability  %NONE 0.01mg/ml0.1mg/ml1mg/ml12.5/17.5/15/1 0/1DMSONONE  1h 24h48h 1h 24h48h 1h 24h48h 1h 24h48h 1h 24h48hDMSO050100150StimuliViability %12.5/17.5/15/1 0/1Free Pro 127  5.3.4 Cellular Uptake with Anti-Progesterone Receptor Antibody Results from the uptake of dHPG(C8), with or without Pro bound, by CCF and HCMEC/D3 cells demonstrated no difference in the uptake, whether the Pro was loaded on the labelled polymer or not. To further examine this and observe if the polymer can carry the drug into the cells, treated cells with polymer were stained with anti-progesterone receptor antibody (PR); in the presence of Pro, the hormone’s receptor translocates into the nucleus and the red signal appears inside of the nucleus as a result of staining with the receptor’s antibody, which binds to the receptor [153]. However, the antibody showed non-specific binding in both cells after treatment with or without 60 µg/mL Pro; the red signals were spread all over the cytosol (Figure 5.11). Each experiment was repeated 3 times on average.    128  a)  b)  Figure 5.11 Pro uptake visualized by anti-progesterone receptor antibody (PR). a) Behaviour of CCF cells incubated with PR in the presence or absence of Pro – b) Behaviour of HCMEC/D3 cells incubated with PR in the presence or absence of Pro. Controls with no treatment (on the left), cells stained with PR (in the middle) and cells stained with PR and incubated with Pro at 60 µg/mL (on the right); blue represents Dapi staining of nuclei. Spread red signals inside the cells demonstrated non-specific binding of the antibody. The microscope lens magnification used for these imaging was 20X   This study was continued using Michigan Cancer Foundation-7 (MCF7) cells as positive control cells, given they strongly express the Pro receptor [153]. As a result of staining cells with PR, the red signal appeared inside of the nucleus in the presence of Pro (Figure 5.12). However, no signal was observed when cells were incubated with the labelled polymer at 0.01 mg/mL, with or without Pro loaded at 0.125 µg/mL.   PR+ProPRControl 10 µmPR+ProPRControl 10 µm 129    Figure 5.12 MCF7 (Michigan Cancer Foundation-7) cells stained with PR; in the presence of free Pro at 60 µg/mL (on the left), dHPG(C8) (in the middle) and dHPG(C8)/Pro at 0.01 mg/mL of polymer and 0.125 µg/mL of Pro (on the right); blue represents Dapi staining of nuclei; presence of red signals inside the nucleus proved the specific interaction between the antibody and the receptor and positive response of the cells to the PR staining   Based on what has been reported in several studies on the uptake of nanoparticles by MCF7 cells [155]–[157], it seems likely that the polymer concentration used for this study was too low to detect. Therefore, the dHPG(C8) concentration was increased from 0.01 mg/mL to 1 mg/mL and consequently, the signals representing the polymer were observed inside the cells. To make sure this concentration caused no rupture in the cell membranes, the cells incubated with polymer, with or without Pro loaded, were stained with WGA (Alexa 633) (Figure 5.13). Results demonstrated intact cell membranes in both cases after 24 h, although there was no positive control for comparison available to demonstrate a leaky membrane. Given the polymer at 1 mg/mL did not cause damage to the membrane or nucleus of MCF7 based on what has been observed above, an uptake study using PR staining was done at the selected concentration. Polymer at 1 mg/mL, with or without 12.5 µg/mL Pro bound, was incubated with the cells at 4 h and 6 h, followed by staining with PR. The results were then compared to free Pro at 60 µg/mL and media only as controls (Figure 5.14 and Figure 5.15). Each time point was PR+Pro dHPG(C8) dHPG(C8)/Pro10 µm 130  repeated 3 times on average with the same qualitative results (Green signals from the polymer are more evident in the electronic file).            a)               b)        Figure 5.13 dHPG(C8) uptake by MCF7 cells stained with WGA (Alexa 633); Alexa 488 represents labelled polymer, Dapi represents stained nucleus and Alexa 633 represents cell membranes stained with WGA; for all samples, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities. a) Media only as negative control – b) Cells were incubated with 1 mg/mL of dHPG(C8) (on the left) and dHPG(C8)/Pro (on the right), where Pro concentration was 12.5 µg/mL  DapiAlexa 633Alexa 488MergeDapiAlexa 633Alexa 488MergeDapiAlexa 633Alexa 488Merge 131    a)        b)     c)    Figure 5.14 Uptake of dHPG(C8), with or without Pro, by MCF7 cells after 4 h at 37 °C; Alexa 488 represents labelled polymer, Dapi represents stained nucleus and PR represents cells stained with anti-progesterone receptor antibody; for all samples, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities. a) Media only as negative control – b) Cells incubated with PR as control (on the left) and incubated with Pro and PR (on the right) – c) Cells incubated with dHPG(C8) at 1 mg/mL and PR (on the left) and cells incubated with dHPG(C8)/Pro (at 12.5 µg/mL of drug) and PR (on the right)   DapiPRAlexa 488Merge10 µmDapiPRAlexa 488MergeDapiPRAlexa 488Merge 10 µm10 µmDapiPRAlexa 488MergeDapiPRAlexa 488Merge10 µm10 µm 132     a)        b)    Figure 5.15 Uptake of dHPG(C8), with or without Pro, after 6 h at 37 °C; Alexa 488 represents labelled polymer, Dapi represents stained nucleus and PR represents cells stained with anti-progesterone receptor antibody; for all samples, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities. a) Cells incubated with PR as control (on the left) and incubated with Pro and PR (on the right) – b) Cells incubated with dHPG(C8) and PR (on the left) and cells incubated with dHPG(C8)/Pro and PR (on the right)  Results from Figure 5.14 and Figure 5.15 proved the translocation of the Pro receptor in the presence of free drug, as expected. At 4 h, Pro loaded on the polymer demonstrated a stronger signal than appeared with free Pro from the nucleus, which implied Pro was delivered into the cytosol by the polymer. This was observed consistently in these experiments. However, weaker DapiPRAlexa 488Merge 10 µmDapiPRAlexa 488Merge 10 µmDapiPRAlexa 488MergeDapiPRAlexa 488Merge10 µm 10 µm 133  red signals were also observed after incubation with polymer alone, so some non-specific signals were caused by the polymer. At 6 h, however, the free Pro showed more PR activity than the dHPG(C8)/Pro treatment and the PR staining was little different from the polymer alone. Comparing the merged channels of polymer/Pro and polymer alone in both figures above suggests a greater number or more intensity of red nuclei occurred when the polymer was loaded with Pro, but the results were complicated by an effect of the polymer on PR staining in the absence of Pro. Hence, there is not enough evidence to be sure if this difference proves that Pro was truly delivered into the cytosol by the polymer, perhaps with different kinetics from the free Pro effect. A more quantitative approach, perhaps utilizing radiolabelled Pro and/or polymer may be needed to provide a clearer answer.    5.4 Discussion Different approaches exist for enhancement of drug delivery to the brain, of which targeted delivery using polymeric nanoparticle carriers has been considered as one of the most attractive. Improvement of therapeutic efficacy and decrease in toxicity have been demonstrated for a variety of drugs associated with nanoparticles [158]. Intracellular targeting refers to the delivery of agents, such as drugs, proteins or DNA, to specific compartments within the cells, which could result in higher bioavailability of a therapeutic agent at its site of actions and lower side effects associated with the drug. However, designing proper nanocarriers to improve low permeability of the drug through cell membranes, poor accessibility of the drug to its targeted site of action within the cells, degradation of the drug in specific cell compartments, as well as decrease in toxicity caused by the drug have always been controversial [159]. Different ways have been proposed to enhance the adhesion to and absorption of nanoparticles into the cells, such as coating the particles with  134  appropriate bioadhesive materials. It has been found that  particle size, polydispersity and surface modification may have critical effects on their adhesion and interaction with biological cells as well as stability and endocytosis process of nanoparticles [158], [160], [161]. It has been demonstrated that the in vivo uptake efficacy of particles with the size of 100 nm is 15-250 fold greater than larger particles as they can penetrate throughout the cells’ membrane faster [159].  Endocytosis is a vital function of cells, where extracellular substances are taken up into the cells, usually by folding in of an outer layer of plasma membrane, forming a vesicle. This process controls nutrient uptake, cell adhesion and migration, receptor signaling, and drug delivery. Generally, intracellular localization of nanoparticles is completed by the endosomal pathway, followed by the lysosomal pathway, which provides a significant drop in pH [133], [134], [159].  Studies have demonstrated that fluorescently-labelled amino-modified HPG with higher molecular weight range from 40 to 870 kDa with a maximum diameter of 23 nm, accumulate much better than the lower molecular weight HPGs within different cell lines, such as human lung cancer cells, human epidermoid carcinoma cells and human umbilical vein endothelial cells (HUVEC) [133]. These results suggest that the endocytosis process for macromolecules can be suppressed by lower molecular weights. It has also been demonstrated that fluorescently-labelled HPGs, modified with choline phosphate (CP) on the surface, with the molecular weight of 65 kDa were rapidly taken up into the cytoplasm of Chinese Hamster Ovary (CHO) cells [162]. Also, a cellular uptake study of high molecular weight fluorescently-labelled poly(ketal hydroxyl ethers) on CHO cells within 24 h demonstrated the slower but significant uptake of these polymers into the cytosol [163]. In this chapter, results from the in vitro uptake studies of dHPG(C8) by astrocytoma and brain endothelial cells confirmed a strong uptake and an even distribution of the polymer at  135  different concentrations. In this study, dHPG(C8), with or without Pro loaded, did not damage cells, given the nucleus remained intact and healthy at all studied concentrations. Higher polymer concentration, in addition to longer incubation time with cells, exhibited higher intracellular distribution of the polymer. Uptake studies of dHPG(C8) stained with anti-lysosome antibody in the same cells demonstrated the ingestion of the polymer by a non-lysosomal pathway within the first 8 h. These results provide evidence that dHPG(Cn) can enter into the cytosol without showing any obvious co-localization with lysosomes at incubation time points. However, more investigation on what eventually happens to the polymer inside the cells is essential.  Results from the cell viability studies demonstrated that dHPG(C8) at the examined concentrations, with or without Pro loaded, did not show toxicity within 48 h, which confirms that dHPG(Cn) are good candidates for delivering Pro, as they are non-toxic and also show very good uptake.  To study whether Pro is delivered into the cytosol by dHPG(C8), uptake of this polymer, with and without Pro loaded, by MCF7 cells was examined using anti-progesterone receptor antibody staining at 4 and 6 h. While signals from the nucleus, both in the presence of free Pro and dHPG(C8) loaded with Pro, proved the translocation of the Pro receptor into the nucleus, polymer in the absence of the drug also caused this translocation, albeit more weakly. Comparing the signals’ intensity or the population of the stained nuclei after incubation with dHPG(C8) and dHPG(C8)/Pro provides positive evidence that Pro is delivered inside the cells by the polymer, but further, more detailed approaches beyond the scope of this thesis will be required to prove the point.   136  5.5 Summary In this chapter, results from in vitro uptake and toxicity studies of fluorescently-labelled dHPG(C8), with or without Pro loaded, are presented. These data on all the examined cell lines demonstrate non-toxic and intracellular distribution of polymer-drug formulation within the cells. Also, lack of accumulation of the formulation in the lysosomal region for the first couple of hours of incubating cells with the polymer suggests that it is a harmless and potentially useful material, which could represent a stable carrier for delivering Pro without exposure to an environment capable of degradation of many nanocarriers. However, more detailed studies are essential to find out the fate of the drug and the state of the polymer after unloading the drug. Study of the presence of Pro within the polymeric formulation in the cytoplasmic area, co-localizing with anti-progesterone receptor antibody, suggests this as a promising way to gain information on the capability of the polymer to transport Pro into the cells. However, based on the signals received from the nucleus, it is not fully understood whether the polymer was truly able to deliver the drug to the target sites. Therefore, further investigation is required in order to monitor the presence of the drug in the cells; different methods could provide more information on monitoring Pro into the cytosol, such as encapsulation of radiolabelled drug by dHPG(Cn) or conjugation of Pro to the fluorescently-labelled polymer through a degradable linkage. Further details are discussed in future work in Chapter 6.   137  Chapter 6: Conclusions and Future Work   6.1 Thesis Summary Delivery of hydrophobic drugs to therapeutic sites is one of the major research challenges in pharmaceutical science due to their poor aqueous solubility. Polymer-based nanoparticles are promising carriers for the targeted delivery of therapeutics to the intracellular sites of action, which can be designed to enhance aqueous solubility of drugs and specific cellular uptake. Several formulations, including micelles, have been studied in detail. Among current approaches, unimolecular micelles are considered as very promising carriers due to their structural stability. This work was focused on studying binding and release properties of a hydrophobic neuroptotective agent, hormone progesterone (Pro), by hydrophobically derivatized hyperbranched polygelycerols dHPG(Cn), as well as in vitro characterizations of the formulations, in order to design efficient delivery systems.  In this study, synthesis and characterization of dHPG(Cn) have been reported. These systems, which were synthesized by multibranching anionic ring-opening polymerization were demonstrated to bind hydrophobic molecules and drugs. These polymers have been produced through a single pot synthesis method with no intermediate purification steps, which makes this polymerization cost effective for bulk production. dHPG(Cn) are water-soluble and can be produced in a variety of molecular weight ranges depending on the use, which makes them flexible for different applications. These polymers show no sign of aggregation in water at high concentrations under the flow conditions of GPC. Binding capacity for encapsulation of hydrophobic drugs and molecules can be manipulated by changing the hydrophilic to hydrophobic ratio of dHPG(Cn). These polymers were synthesized using alkyl monomers (Cn) with different  138  chain lengths from 6 to 18 carbons, while the MPEG fraction of the polymer remained almost constant in all systems. The hydrophobic chains bind Pro molecules, whereas the presence of MPEG chains keeps the system soluble in aqueous solutions.  Following that, the preparation and characterization of Pro carriers in a nanoparticulate formulation based on these hydrophobically derivatized hyperbranched polyglycerols dHPG(Cn) was described. Encapsulation of Pro by dHPGs demonstrated significant correlation with the total mass of hydrophobic chains from the alkyl monomer, which was maximized in the dHPG(C12)/Pro system. Analyzing the hydrodynamic radius of polymeric systems before and after loading drug confirmed the non-micellar structure of these system. This is known as one of the main advantages of these polymers for binding hydrophobic molecules. Kinetic studies of Pro release from dHPG(Cn) provided detailed information on the behaviour of drug release from the polymeric vehicle, depending on the polymer characteristics. Pro release rates from dHPG(Cn) could be characterized by two kinetic constants, of which the major one (burst release) correlated with the volume of the hydrated polymer and more directly with the amount of water structurally affected per mole of polymer, as shown by DSC measurements. Pro release in human plasma was slower than in buffer but had the same general characteristics. These studies provide insight into the nature of binding of a hydrophobic drug into alkylated HPGs and permit a connection to be drawn between the release rates and the amount of bound or structured water associated with the polymers. This unique observation offers some guidelines for the design of Pro carriers with some control over loading and release properties (Figure 6.1).   139   Figure 6.1 General illustration of the Pro bound into dHPG(Cn) formulation and release properties  In vitro studies demonstrated that dHPG(Cn), with and without Pro loaded, are blood compatible in general; no platelet activation or red blood cell aggregation and red blood cell lysis were observed. However, detailed study of formation of a blood clot using Thromboelastography and Scanning Electron Microscopy presented some differences among polymeric formulations in their effect on blood coagulation, which could be explained by the difference in the polymers’ volume and the distributions of the hydrophobic fractions in different formulations and their affinity to bind to hydrophobic materials. This finding may play an important role in understanding the formation of a healthy clot after incubation of blood with polymer. Effect of dHPG(Cn) on the maximum strength of blood clot was concentration dependent. As a result, the maximum strength of the formed clot was significantly affected at higher concentrations of polymeric formulation, causing the TEG signal to collapse in a shorter period of time. At the same time, free Pro did not HPGProgesterone (Pro)0%20%40%60%80%100%0 20 40 60 80 100 120ReleaseTime (h)Free ProPro bound to HPG 140  show any effect on any blood characteristics. In order to conclude the precise effect of these polymeric systems on blood coagulation and the mechanisms behind it, more detailed studies of what have been observed is essential, including more SEM imaging and measuring clot’s characteristics after incubation with dHPG(Cn). Since the target of the project was to deliver Pro to the brain, the results from in vitro cytotoxicity on the brain cells and also uptake studies were reported. Results from the uptake study of two brain cell lines (Astrocytoma cell line (CCF) and blood brain barrier cell line (HCMEC/D3)) demonstrated a significant uptake of fluorescent-labelled dHPG(C8) formulation at different concentrations. Since the desired amount of Pro was reported previously to be between 30-300 nM, the concentration of the polymer which can carry this amount of Pro was chosen for the time dependent cell uptake study. Results of this study confirmed significant increase in uptake of dHPG(C8) by cells without killing cells after 24 h exposure, which could be considered a promising result for this formulation to be used as a safe delivery system in clinical applications. More detailed studies on the uptake showed no exposure to lysosomal pathway for the polymer in the first couple of hours. Additionally, further studies on the Pro’s receptor suggested the presence of Pro inside cells. The combination of all these results supported the strong possibility of delivering Pro encapsulated by dHPG(Cn) to the target cells. Additionally, cytotoxicity was studied by incubating cells with polymer at different concentration with and without loading Pro. These results confirmed that dHPG(Cn) is non-toxic to the cells at any examined concentration or time of incubation within 48 h. At the same time, free drug does not show any toxicity to the cells either.   141  The work presented in this thesis highlights binding and releasing properties of Pro through encapsulation of water-soluble, high molecular weight polyglycerols that can be synthesized in a controlled manner. These polymers are blood compatible and demonstrate significant uptake by brain cells with no sign of damage. Results from this work suggest a bright promise in using these formulations in more clinical applications in order to safely deliver Pro to the brain for treatment of traumatic brain injury.   6.2 Future Work As Pro is reported to act as a neuroprotective agent in both animal models and human, effective delivery to the brain is very important in TBI treatment models. It’s also very important to make sure that the delivery vehicle leaves the circulation system after transporting the drug to the target. Therefore, there are some points to be considered as a future work of this project.   6.2.1 Animal Study and Biodistribution Experiment Injecting labelled dHPG(Cn)/Pro formulations into mice will help measure plasma half-lives of these systems and observe their pharmacokinetic behaviour, which is very important in directing us into clinical applications. Study of accumulation of polymers in different organs such as brain, liver, kidney, and spleen will be done by radiolabeling dHPG(Cn). Results from these experiment have an important role in finding the proper dose for clinical applications.   6.2.2 Binding and Release Study of Radiolabeled Pro into and from dHPG(Cn)  To monitor the exact amount of Pro, loaded into the radiolabeled dHPG(Cn), and its transportation within the polymeric systems into the cells, usage of radiolabeled Pro (with a  142  different radiolabel marker) could provide significant improvements on precise monitoring of the drug for in vivo applications. This could be a very helpful way to reduce the risk of unmonitored precipitation of Pro from the formulations. Also, the amount of radiolabeled Pro released from dHPG(Cn) can be counted directly from the media.  Similarly, this will be helpful in some in vitro assays such as cellular uptake studies; for this, radiolabeled drug loaded into radiolabeled dHPG(Cn) would allow monitoring of the drug and the carrier separately inside the cells.   6.2.3 Encapsulation of Water-Soluble Analogue of Pro by dHPG(Cn)  Pro with two active functional groups demonstrates the potential for modification; C3 and C20 are chemically active sites of the drug, of which C20 presents the more active electrophile for nucleophilic substitution reaction (Figure 6.2).    Figure 6.2 C20 presents the more electrophilic site of Pro for modification   Reacting the more electrophilic carbonyl group with some water-soluble reagents like derivatives of hydroxyl amines (NH2OR) could be a good way to modify Pro into a more water-soluble analogue (Figure 6.3) [37]. Such material would retain much of its hydrophobic character  143  and bind to alkylated HPGs, but should be more rapidly released in aqueous media due to its enhanced water solubility. The oxime linker can undergo hydrolysis in vivo [161]. Moreover, the oxime modification causes more reduction in neuronal cell death at a lower dose, compared to free Pro [37]. Encapsulation of this analogue of Pro by dHPG(Cn) could provide a more stable delivery system by improving the binding affinity and release properties of Pro from the drug-binding pocket.   Figure 6.3 Oxime linkage on Pro     144  Bibliography [1] Abbott, N. J., “Blood-brain barrier structure and function and the challenges for CNS drug delivery”, J. Inherit. Metab. Dis. 2013, 36, 437-449. [2] Cardoso, F. L.; Brites, D.; Brito, M. A., “Looking at the blood-brain barrier: Molecular anatomy and possible investigation approaches”, Brain Res. Rev. 2010, 64, 328-363. [3] Saunders, N. R.; Ek, C. J.; Habgood, M. D.; Dziegielewska, K. 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Soc. 2012, 134, 14945-14957.     158  Appendices Appendix A   A.1 1H NMR Spectra (300 MHz, CDCl3) and GPC Chromatogram of dHPG(Cn) (n = 6, 10, 14, 18)  1H NMR (300 MHz, CDCl3) δ(ppm): 0.90 (3H, t, J = 6.0 Hz, CH3 from C6 monomer); 1.16-1.53 (6H, m, CH2 from C6 monomer); 3.30-3.95 (m, CH and CH2 from HPG core); 3.38 (3H, s, OCH3 from MPEG). 1H NMR (300 MHz, CDCl3) δ(ppm): 0.88 (3H, t, J = 6.0 Hz, CH3 from C10 monomer); 1.20-1.50 (14H, m, CH2 from C10 monomer); 3.50-3.95 (CH and CH2 from HPG core); 3.38 (3H, s, OCH3 from MPEG). 1H NMR (300 MHz, CDCl3) δ(ppm): 0.88 (3H, t, J = 6.0 Hz, CH3 from C14 monomer); 1.20-1.50 (22H, m, CH2 from C14 monomer); 3.50-3.95 (CH and CH2 from HPG core); 3.38 (3H, s, OCH3 from MPEG). 1H NMR (300 MHz, CDCl3) δ(ppm): 0.88 (3H, t, J = 6.0 Hz, CH3 from C18 monomer); 1.20-1.40 (30H, m, CH2 from C18 monomer); 3.50-3.95 (CH and CH2 from HPG core); 3.38 (3H, s, OCH3 from MPEG).       159   Figure A.1 1H NMR spectrum of dHPG(C6) in CDCl3   Figure A.2 GPC chromatogram of dHPG(C6)  Define PeaksLS dRI     time (min)0.0 10.0 20.0 30.0 40.0Relative Scale0.00.51.01 1 160    Figure A.3 1H NMR spectrum of dHPG(C10) in CDCl3   Figure A.4 GPC chromatogram of dHPG(C10)  Define PeaksLS dRI     time (min)0.0 10.0 20.0 30.0 40.0Relative Scale0.00.51.01 1 161     Figure A.5 1H NMR spectrum of dHPG(C14) in CDCl3   Figure A.6 GPC chromatogram of dHPG(C14)  Define PeaksLS dRI     time (min)0.0 10.0 20.0 30.0 40.0Relative Scale0.00.51.01 1 162    Figure A.7 1H NMR spectrum of dHPG(C18) in CDCl3   Figure A.8 GPC chromatogram of dHPG(C18)  Define PeaksLS dRI     time (min)0.0 10.0 20.0 30.0 40.0Relative Scale0.00.51.01 1 163  A.2 13C NMR (400 MHz, methanol-d4)  13C NMR (400 MHz, methanol-d4) δ(ppm): 14.72 (CH3 from alkyl on C10 monomer); 23.88-33.19 (CH2 from alkyl on C10 monomer); 59.27 (OCH3 from MPEG); 62.91-81.75 (CH and CH2 from HPG core).   Figure A.9 Inverse-gated 13C NMR of dHPG(C10) in methanol-d4 proves the branched structure of dHPG(Cn)   164  Appendix B   B.1 Binding Profiles of Pro into dHPG(Cn)   Figure B.1 Binding profile of Pro loaded on dHPG(C6)   Figure B.2 Binding profile of Pro loaded on dHPG(C8) 0501001502000 50 100 150 200 250 300Bound Pro (µg/ml) Loaded Pro (µg/ml) 0501001502002503003500 50 100 150 200 250 300 350 400Bound Pro (µg/ml) Loaded Pro (µg/ml)  165   Figure B.3 Binding profile of Pro loaded on dHPG(C10)   Figure B.4 Binding profile of Pro loaded on dHPG(C12)  0501001502002503003500 50 100 150 200 250 300 350 400Bound Pro (µg/ml) Loaded Pro (µg/ml) 0501001502002503003500 50 100 150 200 250 300 350 400Bound Pro (µg/ml) Loaded Pro (µg/ml)  166   Figure B.5 Binding profile of Pro loaded on dHPG(C14)   Figure B.6 Binding profile of Pro loaded on dHPG(C18)     0501001502002503003500 50 100 150 200 250 300 350 400Bound Pro (µg/ml) Loaded Pro (µg/ml) 0501001502002500 50 100 150 200 250 300 350Bound Pro (µg/ml) Loaded Pro (µg/ml)  167  Appendix C   C.1 Effect of Pro Binding on dHPG(C10) Size   Figure C.1 DLS size determination of dHPG(C10) at 2 mg/mL (on the left) and dHPG(C10)/Pro at 2 mg/mL of polymer and 25 µg/mL of Pro (on the right)     168  Appendix D   D.1 In vitro Release Profiles of Pro from dHPG(Cn)    Figure D.1 dHPG(C8)/Pro release profile in PBS at 37 °C and pH 7.4    Figure D.2 dHPG(C10)/Pro release profile in PBS at 37 °C and pH 7.4 0%20%40%60%80%100%0 10 20 30 40 50 60 70 80 90 100 110 120ReleaseTime (h)0%20%40%60%80%100%0 10 20 30 40 50 60 70 80 90 100 110 120ReleaseTime (h) 169    Figure D.3 dHPG(C12)/Pro release profile in PBS at 37 °C and pH 7.4    Figure D.4 dHPG(C14)/Pro release profile in PBS at 37 °C and pH 7.4 0%20%40%60%80%100%0 10 20 30 40 50 60 70 80 90 100 110 120ReleaseTime (h)0%20%40%60%80%100%0 10 20 30 40 50 60 70 80 90 100 110 120ReleaseTime (h) 170    Figure D.5 dHPG(C18)/Pro release profile in PBS at 37 °C and pH 7.4    Figure D.6 Free Pro release profile in PBS at 37 °C and pH 7.4 0%20%40%60%80%100%0 10 20 30 40 50 60 70 80 90 100 110 120ReleaseTime (h)0%20%40%60%80%100%0 1 2 3 4ReleaseTime (h) 171    Figure D.7 HPG/Pro release profile in PBS at 37 °C and pH 7.4    Figure D.8 HPG-PEG/Pro release profile in PBS at 37 °C and pH 7.4   0%20%40%60%80%100%0 1 2 3 4ReleaseTime (h)0%20%40%60%80%100%0 1 2 3 4ReleaseTime (h) 172  Appendix E   E.1 Kinetic Profiles of Pro Released from dHPG(Cn) in PBS and Plasma     Figure E.1 dHPG(C10)/Pro; rapid release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.97 and p < 0.01)    Figure E.2 dHPG(C10)/Pro; slow release rate in PBS – k2(s-1) calculated from the slope (R2 = 0.99 and p < 0.01) -2.0-1.5-1.0-0.50.00 2 4 6 8ln[C1/C10] Time (h)-3.5-3.0-2.5-2.0-1.5-1.0-0.50.00 20 40 60 80 100 120ln[C2/C20] Time (h) 173    Figure E.3 dHPG(C12)/Pro; rapid release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.97 and p < 0.01)    Figure E.4  dHPG(C12)/Pro; slow release rate in PBS – k2(s-1) calculated from the slope (R2 = 0.86 and p < 0.01)  -2.0-1.5-1.0-0.50.00 2 4 6 8ln[C1/C10] Time (h)-2.0-1.5-1.0-0.50.00 20 40 60 80 100 120 140ln[C2/C20] Time (h) 174    Figure E.5 dHPG(C14)/Pro; rapid release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.96 and p < 0.01)    Figure E.6 dHPG(C14)/Pro; slow release rate in PBS – k2(s-1) calculated from the slope (R2 = 0.99 and p < 0.01)  -2.0-1.5-1.0-0.50.00 1 2 3 4 5 6 7 8ln[C1/C10] Time (h)-2.0-1.5-1.0-0.50.00 20 40 60 80 100 120ln[C2/C20] Time (h) 175    Figure E.7 dHPG(C18)/Pro; rapid release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.99 and p < 0.01)    Figure E.8 dHPG(C18)/Pro; slow release rate in PBS – k2(s-1) calculated from the slope (R2 = 0.95 and p < 0.01)    -2.0-1.5-1.0-0.50.00 1 2 3 4 5 6 7 8ln[C1/C10] Time (h)-2.0-1.5-1.0-0.50.00 20 40 60 80 100 120ln[C2/C20] Time (h) 176    Figure E.9 HPG/Pro; release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.98 and p < 0.01)    Figure E.10 HPG-PEG/Pro; release rate in PBS – k1(s-1) calculated from the slope (R2 = 0.98 and p < 0.01)  -4.0-3.0-2.0-1.00.10 0.5 1 1.5 2 2.5 3 3.5 4ln[C1/C10]Time (h)-5.0-4.0-3.0-2.0-1.00.00 1 2 3 4 5ln[C1/C10]Time (h) 177    Figure E.11 Free Pro; rapid release rate in plasma – k1(s-1) calculated from the slope (R2 = 0.95 and p < 0.01)    Figure E.12 Free Pro; slow release rate in plasma – k2(s-1) calculated from the slope (R2 = 0.89 and p < 0.02)  -2.0-1.5-1.0-0.50.00 2 4 6 8ln [C1/C10]Time (h)-2.2-1.7-1.2-0.7-0.2 0 20 40 60 80 100 120ln [C2/C20]Time (h) 178    Figure E.13 dHPG(C8)/Pro; rapid release rate in plasma – k1(s-1) calculated from the slope (R2 = 0.96 and p < 0.01)    Figure E.14 dHPG(C8)/Pro; slow release rate in plasma – k2(s-1) calculated from the slope (R2 = 0.98 and p < 0.01)   -2.0-1.5-1.0-0.50.00 1 2 3 4 5 6 7 8ln [C1/C10]Time (h)-2.6-2.1-1.6-1.1-0.6-0.10 20 40 60 80 100 120 140ln [C2/C20]Time (h) 179  Appendix F   F.1 t-Test Analysis on APTT Results  Sample dHPG(C8)/Pro dHPG(C10)/Pro dHPG(C12)/Pro dHPG(C14)/Pro dHPG(C18)/Pro Free Pro PBS dHPG(C8) 0.485     0.000 0.022 dHPG(C10)  0.488    0.000 0.051 dHPG(C12)   0.243   0.001 0.104 dHPG(C14)    0.348  0.000 0.073 dHPG(C18)     0.387 0.005 0.105 Free Pro 0.000 0.000 0.003 0.000 0.004   PBS 0.021 0.043 0.117 0.064 0.124 0.486   Table F.1 t-Test analysis on APTT results after incubation with dHPG(Cn), with and without the presence of Pro, at 10 mg/mL of polymer concentration; highlighted p-values demonstrate significant difference  Sample Free Pro PBS dHPG(C8) 0.000 0.027 dHPG(C10) 0.000 0.025 dHPG(C12) 0.016 0.125 dHPG(C14) 0.003 0.069 dHPG(C18) 0.021 0.131 Free Pro N/A 0.485  Table F.2 t-Test analysis on APTT results after incubation with dHPG(Cn) at 1 mg/mL; highlighted p-values demonstrate significant difference   180  F.2 Red Blood Cell Aggregation after Incubation with dHPG(Cn) at 1 mg/mL    a)      b)   Figure F.1 Red blood cell aggregation in the presence of dHPG(Cn); a) dHPG(C12) at 1 mg/mL; b) dHPG(C14) at 1 mg/mL  F.3 Red Blood Cell Aggregation after Incubation with dHPG(Cn) and dHPG(Cn)/Pro at 10 mg/mL    a)      181    b)     c)     d)    Figure F.2 Red blood cell aggregation in the presence of dHPG(Cn), with or without Pro; a) dHPG(C12) at 10 mg/mL; b) dHPG(C12)/Pro, where polymer concentration was 10 mg/mL loaded with 125 µg/mL Pro; c) dHPG(C14)/Pro, where polymer concentration was 10 mg/mL loaded with 125 µg/mL Pro; d) dHPG(C18)/Pro, where polymer concentration was 10 mg/mL loaded with 125 µg/mL Pro    182  F.4 Platelet Aggregation after Incubation with dHPG(C8) and dHPG(C12) at 1 and 10 mg/mL     Figure F.3 dHPG(C8) at 10 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right     Figure F.4 dHPG(C8) at 1 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right  183     Figure F.5 dHPG(C12) at 10 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right     Figure F.6 dHPG(C12) at 1 mg/mL; before addition of ADP reagent (0-15 min) on the left; after addition of ADP (16-30 min) on the right     184  F.5 SEM Imaging of Dynamic Clots a)    b)   c)   Figure F.7 SEM images of PRP dynamic clots formed in the presence of dHPG(C8); a) HBS control buffer; b) dHPG(C8) at 0.1 mg/mL; c) dHPG(C8) at 1 mg/mL  185  Appendix G   G.1 1H NMR (300 MHz, CDCl3): dHPG(C8) Modified with EPP    Figure G.1 1H NMR spectrum of dHPG(C8) modified with EPP in CDCl3 – Peaks at 7.59 ppm and 7.69 ppm belong to the aromatic ring of EPP confirm the modification    186  G.2 1H NMR (300 MHz, D2O): dHPG(C8) Modified with Primary Amine    Figure G.2 1H NMR spectrum of dHPG(C8) modified with amine groups in D2O confirms the absence of EPP     187  Appendix H   H.1 dHPG(C8) Uptake by HCMEC/D3 Visualized by Anti-Lysosome Antibody          a)              b)        Figure H.1 Uptake of dHPG(C8) by HCMEC/D3 cells visualized by anti-lysosome antibody (LAMP1) at 37 °C; Alexa 488 represents labelled polymer, Dapi represents stained nucleus and LAMP1 represents anti-lysosome antibody; for all time points, each channel is shown in black and white individually, while the merge of all is shown in colour for better monitoring of the intensities; yellow signal represents the convergence of the polymer and lysosome. a) Media control – b) after 2 h; cells were incubated with dHPG(C8) (on the left) and dHPG(C8)/Pro (on the right) – Polymer and Pro concentrations were 0.01 mg/mL and 0.125 µg/mL, respectively DapiLAMP1 MergeAlexa 48810 µmDapiAlexa 594 MergeAlexa 48810.0 µmDapiLAMP1Alexa 488MergeDapiLAMP1Alexa 488Merge10 µm 10 µm

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