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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
This thesis is aimed at developing an antibacterial and biocompatible coating for platelet storage bags. Platelets are blood cells that play an essential role in stopping bleeding. Stored platelets for transfusion have a limited shelf life due to the loss of platelet quality upon storage and the risk of bacterial growth in the storage bags. The hydrophobic surface of plasticized polyvinyl chloride platelet storage bags is conducive to bacterial adhesion, biofilm formation and platelet adhesion. In Chapter 1 platelet transfusion and the associated challenges as well as biomaterials’ surface characteristics and biocompatibility are introduced. In Chapter 2 some hydrophilic polymer brushes are developed on platelet bag surfaces and characterized. Polymer coatings made of poly(N,N-dimethylacrylamide) (PDMA) and a copolymer of DMA and N-(3-aminopropyl)methacrylamide hydrochloride (APMA) (DMA/APMA: 5/1) could inhibit bacterial adhesion on platelet bag surfaces under growth conditions by 95 % and 70 % respectively. These coated surfaces also showed decreased platelet adhesion. To add bactericidal activity to the coating, antimicrobial peptides (AMPs) are then conjugated to polymer brushes and characterized in Chapter 3. These coated substrates increased the level of platelet adhesion and activation, however. Tuning the amount of peptide on bag surfaces and reproducing coated surface characteristics (with or without peptides) were both challenging. Taking another approach in Chapter 4, mussel-inspired chemistry is used to coat platelet bags as well as gold and silicon wafers with the antifouling polymeric system. The optimum polymer coating could resist fibrinogen adsorption, bacterial and platelet adhesion. An AMP-containing bactericidal coating was also synthesized and coated on silicon wafers using this approach. AMPs were mapped on the modified surfaces and they showed bactericidal activity in vitro. A significance of the applied methods in this thesis is their compatibility with commercial biomaterials containing leachable plasticizers. The chemical composition and morphology of some platelet bags used in Canada are studied in Chapter 5. Evaluation of bacterial and platelet adhesion on their surfaces suggests fabricating bags with only one textured surface inside and positioning it above a non-textured surface. Potential commercial application of the coatings developed here has been a perspective of this project.
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 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.
Master's Student Supervision (2010 - 2018)
As a classical representative of the hyperbranched polymer family, hyperbranched polyglycerol has been applied in a variety of diagnostics and therapies. After functionalizing the end groups with zwitterionic sulfabetaines (SB) and cationic quaternary amines (QA), the polymer exhibited superior hemagglutination capacity at a concentration ≥ 1 mg/mL and erythrocyte lysis was not observed. The goal in the present work is to use the monomers above to maximize the polymer adhesion to cells while eliminating the membrane damage caused by multivalent polycation exposure. The desired stickiest structure is a novel attractive bioadhesive material which may serve as a future topical hemostatic agent for bleeding wounds, overcoming the limitations of existing products in the market. To this end an optimization was performed on the synthesis scheme to achieve a library of HPG based polymer conjugates with a constant amount of one derivative (SB/QA) and a varied number of the other derivative (QA/SB). The efficacy of sulfabetaine (SB) and quaternary amine (QA) was quantified and different hemagglutination behaviors were displayed. Hemolysis, cytotoxicity and inhibition effects as well as the hydration properties of the polymer conjugates were also evaluated. In the blood aggregation analysis, HPG-SB₃₀-QA₃₀-OH₄₀ and HPG-SB₃₀-QA₄₀-OH₃₀ exhibited strong red blood cells aggregation effects even at a concentration as low as 1mg/mL. Thirty percent of SB also reduced the hemolytic activity to an undetectable level and retained high cell viability in the presence of 30% QA. The bound water of each repeat monomer unit was quantified by differential scanning calorimetry and explained the fouling resistance behavior of the cells. The addition of linear polyglycerol sulfate into the HPG-SB-QA-OH system effectively suppressed the red blood cell aggregation, which indicated the possibility of reversibly applying the materials for hemostasis. Overall, the in vitro studies suggest that the stickiest structure of the designed functionalized HPG is HPG-SB₃₀-QA₃₀-OH₄₀, which is biocompatible and harmless to cells.
Enhancement of hemostasis at the site of wound is a very attractive method to limitbleeding and to reduce the need for blood transfusion support. However, many commerciallyavailable bioadhesives and hemostatic agents fail to fulfill the design requirements of efficacy,safety and cost. There is a need to develop novel bioadhesive and hemostatic agents that wouldovercome these limitations.A library of hyperbranched polyglycerol (HPG) based macromolecular structuresfunctionalized with different mole fractions of zwitterionic sulfabetaine and cationic quaternaryammonium ligands were synthesized and characterized. A post-polymerization method wasemployed that utilized double bond moieties on the HPG backbone for the coupling of thiolcappedfunctional groups via UV initiated thiol-ene “click” chemistry. The proportions ofdifferent ligands were precisely controlled by varying the monomer concentration during theirradiation process.The effect of the polymer on hemostasis has been investigated using whole blood. It wasfound that polymer with 40% or more positive charged groups caused hemagglutination withoutcausing red blood cell lysis. The quaternary ammonium groups can interact with the negativecharged sites on the membranes of erythrocytes, which improves the bioadhesiveness. Thezwitterionic sulfabetaine can provide a hydration layer to partially mask the adverse effects thatare likely to be caused by cationic moieties on the integrity of cell membrane. The conjugate wasalso found to be able to enhance platelet aggregation and activation in a concentration andpositive charge density dependent manner, which would contribute to the initiation ofhemostasis. The polymer-induced hemostasis is obtained by a process independent of the normaliiiblood coagulation cascade but dependent on red blood cell agglutination, where the polymerspromote hemostasis by linking erythrocytes together to form a lattice to entrap the cells.
Use of synthetic materials in medical applications is one of the most common practices in modern medicine. Yet occurrence of surface-induced thrombus formation on these materials, especially those associated with cardiovascular applications, generates a need for surface modifications. Limiting thrombus formation on a biomaterial surface represents the ultimate success for blood contacting devices. One interesting approach is to enhance fibrinolysis before the blood clot becomes stabilized. Herein, two synthetic polymers, poly-N- [(2, 2-dimethyl-1, 2-dioxolane) methyl] acrylamide (PDMDOMA) and poly- (N-isopropylacrylamide) (PNIPAm), were tested for this particular antithrombotic property. Surface-grafted PNIPAm samples, brush-PNIPAm and star-PNIPAm, were also tested for the biological activity.We evaluated the influence of these synthetic polymers on blood hemostasis by studying the fibrin polymerization process, the three-dimensional clot structure, and the mechanical properties of blood clot such as its clot strength, clot elasticity and clot fibrinolysis. Both linear PDMDOMA and PNIPAm altered the normal fibrin polymerization by changing the rate of protofibril aggregation and resulting in a 5-fold increase in the overall turbidity. Fibrin clots formed in presence of these synthetic polymers exhibited thinner fibers with less branching and resulted in a more porous and heterogeneous clot structure in scanning electron micrographs. The structural changes in these clots led to significant difference to their mechanical properties. Lower clot strength and clot elasticity were recorded from the thromboelastography study. More interestingly, enhanced clot lysis was measured by thromboelastography when whole blood was clotted in presence of PDMDOMA or PNIPAm. Further evidence of the altered clot structure and clot cross-linking was obtained from the significant decrease in D-dimer levels measured from degraded plasma clot. Similar results were obtained when star-form of PNIPAm was used but not for brush-form PNIPAm.The antithrombotic activity of soluble PDMDOMA and PNIPAm could potentially lead to the development of novel antithrombotic agents that could enhance endogenous fibrinolytic activity by modulating the fibrin clot structure. In the exploratory analysis of surface grafted PNIPAm (brush-PINPAm), brush-PNIPAm showed that the biological activity of attached chains is quite different from soluble polymers and several parameters need to be optimized to generate an antithrombotic coating for biomaterials.