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The primary objective of this study was to identify, develop and characterise a novel bioactive surface capable of binding hepatocytes and enabling the retention of hepatocyte-specific cell function during in-vitro culture. The materials were designed to exploit a unique characteristic of hepatocyte biology, with β-galactose moieties displayed to allow cellular adhesion via the specific asialoglycoprotein receptors (ASGP-R) found on hepatocytes. Hydrogels were created by modifying a commercially available block co-polymer of polyethylene glycol (PEG) and acrylamide, (PEGA) with galactose moieties contained within lactobionic acid (LA), producing a unique bioactive sugar-based gel. A control sugar, D-glucuronic acid (GA), was used as a non-ASGP-R binding control. Monomers used were mono- and bis-acryloamido PEG (Mw=1900 gmol-1), and dimethylacrylamide. The pendant PEGA amine groups were used as ligands to bind to the sugars. The resultant gels were characterised using Fourier Transform Infrared Spectroscopy (FT-IR), protein adsorption, Fmoc-Phe and dansyl chloride labelling. The biocompatibility of the gel surfaces was evaluated using a hepatocyte cell line and the degree of attachment, proliferation, and morphology was characterised using light microscopy, live/dead assays, DNA assays, immunochemical staining, flow cytometry and reverse-transcription polymerase chain reaction (RT-PCR).FT-IR analysis of LA revealed a distinctive band at approximately 1740cm-1 corresponding to carbonyl stretching (C=O) of carboxylic acid. This unique peak disappeared as the galactose moieties within the LA were incorporated into the PEGA gel. A similar trend was also observed with the control GA sugar within the PEGA gel, confirming that the sugars had been integrated into the material. Protein adsorption assays confirmed the non-fouling nature of PEGA. Cell culture experiments showed that hepatocytes attached preferentially to the sugar surfaces, with few cells seen on the PEGA surfaces. It was observed that cells on the PEGA with LA surface were more metabolically active, than the controls and proliferated to a monolayer by day 7 in culture. Immunocytochemical staining of the cells for actin, vinculin and phosphorylated focal adhesion kinase illustrated differences in cell morphology between cells grown on different surfaces. It was determined that the sugar PEGA surfaces maintained some characteristics of hepatocyte functionality e.g. urea synthesis over the course of 7 days. To improve the reproducibility of the surfaces generated, a preliminary investigation of two-dimensional PEG monolayer surfaces as a well defined platform for surface reactions was conducted. These were chemically functionalised in a stepwise manner with the sugars. The number of coupling steps and the choice of solvent were shown to affect the efficiency of the reaction. Further more, the need for careful sample preparation was highlighted as contamination could potentially inhibit the interpretation of the surface chemistry.The overall conclusion of this work is that saccharides within non-fouling surfaces composed of thin layers of PEG-acrylamide hydrogels are able to support hepatocyte attachment and the retention of cell type specific functions in culture. However, this preliminary work has shown that much further research is necessary to elucidate the role that the surface chemistry plays in the attachment of hepatocytes.

No / Peptides that self-assemble into nanostructures are of tremendous interest for biological, medical, photonic and nanotechnological applications. The enormous sequence space that is available from 20 amino acids probably harbours many interesting candidates, but it is currently not possible to predict supramolecular behaviour from sequence alone. Here, we demonstrate computational tools to screen for the aqueous self-assembly propensity in all of the 8,000 possible tripeptides and evaluate these by comparison with known examples. We applied filters to select for candidates that simultaneously optimize the apparently contradicting requirements of aggregation propensity and hydrophilicity, which resulted in a set of design rules for self-assembling sequences. A number of peptides were subsequently synthesized and characterized, including the first reported tripeptides that are able to form a hydrogel at neutral pH. These tools, which enable the peptide sequence space to be searched for supramolecular properties, enable minimalistic peptide nanotechnology to deliver on its promise.

This thesis describes the investigation of the properties of cellulose nanocrystals (CNCs) in water and at interfaces in the presence of different water-soluble polymers and surfactants. The potential of producing hydrogels, emulsions, and foams using both CNCs and surfactants and polymers is extensively explored herein. Interactions between CNCs and polymers were studied by measuring adsorption of polymers on CNC-coated surface in quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) instruments. Hydroxyethyl cellulose, hydroxypropyl guar, and locust bean gum adsorbed onto CNC-coated surfaces, whereas dextran did not adsorb. Gelation of CNC dilute dispersions was found for the samples added with adsorbing polymers, whereas the introduction of non-adsorbing polymers showed no such change of rheological behaviors of CNC dilute dispersions. The further addition of negative surfactant SDS or non-ionic surfactant Triton X-100 disrupted the gels whereas cationic surfactant CTAB did not. These behaviors illustrate the complexities associated with including CNC dispersions in formulated water-based products where polymers and surfactants are commonly used as well. The adsorption of cationic surfactants on CNC particle surfaces and the associated change of CNC hydrophobicity were investigated. Surfactant-modified CNCs were then employed as emulsifying agents to determine the effects of stabilizing oil-water interface with CNCs after surfactant addition. Emulsion stability was substantially enhanced with the introduction of surfactants. Based on the chemistry of cationic surfactants, and the extent CNC surface hydrophobicity increases after surfactant binding, either oil-in-water or water-in-oil emulsions were successfully produced. This in situ surfactant adsorption method thus offers a simple way of modifying surface hydrophobicity of CNCs and allows fine tuning of CNC-based emulsion properties. Adsorbing polymers were used together with CNCs to prepare stable emulsions. The introduction of polymers facilitated the production of emulsion droplets with enhanced stability and smaller diameters. Both polymer-coated CNCs and the extra polymers partitioned at the interface and worked as the emulsifiers in a synergistic manner, leading to a reduction in CNC coverage on the emulsion droplet surfaces. Furthermore, reversible thermogelation of the emulsion was obtained when thermosensitive polymers were added. No noticeable emulsion coalescence occurred after multiple cycles of heating and cooling treatments of the emulsion gels. Freeze-drying and air-drying of these emulsion gels produced oil powders containing oil content as high as 94 wt. %. Finally, highly stable wet foams were successfully produced using CNCs and the water-soluble polymer, methyl cellulose. The effect of CNC and methyl cellulose concentration on the stability of air-water interfaces was elucidated. Both foamability and foam stability were greatly improved by adding CNCs to methyl cellulose solutions. The CNC particles helped to retain fluid in the films and plateau borders between bubbles, increasing bulk viscosity, and impeding water drainage. We also demonstrated that adding various monomers to CNCs- methyl cellulose wet foams did not lead to noticeable foam breaking. The successful production of macroporous structures with tailored chemistry and properties was achieved by subsequent polymerization of the monomers added to the foam. / Thesis / Doctor of Philosophy (PhD)

This thesis describes the investigation of the properties of cellulose nanocrystals (CNCs) in water and at interfaces in the presence of different water-soluble polymers and surfactants. The potential of producing hydrogels, emulsions, and foams using both CNCs and surfactants and polymers is extensively explored herein. Interactions between CNCs and polymers were studied by measuring adsorption of polymers on CNC-coated surface in quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) instruments. Hydroxyethyl cellulose, hydroxypropyl guar, and locust bean gum adsorbed onto CNC-coated surfaces, whereas dextran did not adsorb. Gelation of CNC dilute dispersions was found for the samples added with adsorbing polymers, whereas the introduction of non-adsorbing polymers showed no such change of rheological behaviors of CNC dilute dispersions. The further addition of negative surfactant SDS or non-ionic surfactant Triton X-100 disrupted the gels whereas cationic surfactant CTAB did not. These behaviors illustrate the complexities associated with including CNC dispersions in formulated water-based products where polymers and surfactants are commonly used as well. The adsorption of cationic surfactants on CNC particle surfaces and the associated change of CNC hydrophobicity were investigated. Surfactant-modified CNCs were then employed as emulsifying agents to determine the effects of stabilizing oil-water interface with CNCs after surfactant addition. Emulsion stability was substantially enhanced with the introduction of surfactants. Based on the chemistry of cationic surfactants, and the extent CNC surface hydrophobicity increases after surfactant binding, either oil-in-water or water-in-oil emulsions were successfully produced. This in situ surfactant adsorption method thus offers a simple way of modifying surface hydrophobicity of CNCs and allows fine tuning of CNC-based emulsion properties. Adsorbing polymers were used together with CNCs to prepare stable emulsions. The introduction of polymers facilitated the production of emulsion droplets with enhanced stability and smaller diameters. Both polymer-coated CNCs and the extra polymers partitioned at the interface and worked as the emulsifiers in a synergistic manner, leading to a reduction in CNC coverage on the emulsion droplet surfaces. Furthermore, reversible thermogelation of the emulsion was obtained when thermosensitive polymers were added. No noticeable emulsion coalescence occurred after multiple cycles of heating and cooling treatments of the emulsion gels. Freeze-drying and air-drying of these emulsion gels produced oil powders containing oil content as high as 94 wt. %. Finally, highly stable wet foams were successfully produced using CNCs and the water-soluble polymer, methyl cellulose. The effect of CNC and methyl cellulose concentration on the stability of air-water interfaces was elucidated. Both foamability and foam stability were greatly improved by adding CNCs to methyl cellulose solutions. The CNC particles helped to retain fluid in the films and plateau borders between bubbles, increasing bulk viscosity, and impeding water drainage. We also demonstrated that adding various monomers to CNCs- methyl cellulose wet foams did not lead to noticeable foam breaking. The successful production of macroporous structures with tailored chemistry and properties was achieved by subsequent polymerization of the monomers added to the foam. / Thesis / Doctor of Philosophy (PhD)

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The development of structured macroporous hydrogels are of great interest in many industries due to their high permeabilities, large surface areas and large pore volumes. In drug delivery and wound healing applications, these macropores may theoretically be utilized as large drug reservoirs to deliver anti-inflammatory drugs to a wound site, while simultaneously absorbing exudate and maintaining a hydrated environment in which the wound may heal. However, current methods of generating macroporous structured hydrogels are low-throughput, expensive, and require the use of organic solvents, salts, and other additives that are difficult to remove from the crosslinked hydrogel scaffold. In contrast, the Pressurized Gas eXpanded liquid (PGX) processing technology, patented by the University of Alberta and licensed for all industrial applications by Ceapro Inc., has been shown to generate purified and exfoliated biopolymer scaffolds in a less expensive and more efficient way. Herein, the tunability of the PGX processing method was investigated in depth, varying solvent/anti-solvent ratios, nozzle mixing volume, polymer molecular weight, and polymer concentration to examine the resulting effects on produced polymer morphologies. PGX-processed chitosan and alginate scaffolds were stabilized as bulk hydrogels through post-processing crosslinking methods using anti-solvents, solid-state chemistries, and/or rapid gelation kinetics. The mechanical strength, swelling/degradation kinetics, affinity for protein uptake, and cytotoxicity of these stabilized scaffolds were subsequently examined and compared to hydrogels produced without the use of PGX processing. Furthermore, in situ crosslinking methods were explored, in which alginate and poly(oligoethylene glycol methacrylate) polymers were shown to form stable aerogels during the standard PGX processing method. Finally, the PGX apparatus was reconfigured to enable the impregnation of a model hydrophobic drug into pre-processed polymer scaffolds via circulation of supercritical CO2. The total loading was calculated and the release kinetics from loaded-scaffolds examined. In conclusion, this work outlines a novel method of creating structured macroporous hydrogels from PGX processed biopolymers with the potential to provide improved drug loadings and sustained release profiles. It is expected that this work will provide a basis for a great deal of research into the further stabilization of scaffolds for use in other applications, the investigation of a larger range of bioactive molecules for impregnation and release, and the exploration of PGX hydrogel scaffolds for in vivo wound healing. / Thesis / Master of Applied Science (MASc)

The advent of highly sophisticated drugs designed to interfere with specific cellular functions has created the demand for “intelligent” carriers that can efficiently deliver therapeutic agents in response to a pathophysiogical condition. Nanoscale intelligent systems can maximize the efficacy of therapeutic treatments in numerous ways because they have the ability to rapidly detect and response to disease states directly at the site and sparing physiologically healthy cells and tissues, thereby improving a patient’s quality of life. Nanoparticle fabrication has primarily relied on emulsions, self-assembly and micelles based methods which inherently generate polydisperse spherical particles with little control over particle geometry. Despite significant progress in such drug delivery systems, critical limitations remain in synthesizing nanocarriers with highly controllable architecture (size, shape or aspect ratio) that can, at the same time, impart response-sensitive release mechanisms. These parameters are essential for controlling the in-vivo transport, bio-distribution, and drug release mechanisms. The objective of my dissertation is to employ the nanofabrication technique Step and Flash Imprint Lithography (S-FIL) to synthesize stimuli-responsive nanocarriers of precise architectures and composition. Applying S-FIL technology, fabrication of nanocarriers of a variety of shapes and sizes (down to 36nm length scale) that are also environmentally responsive by incorporating enzymatically-degradable peptides into the nanocarrier hydrogel matrix, to provide triggered release of encapsulated therapeutic agents in response to specific pathophysiological conditions, has been accomplished. Besides disease-responsive release, the two key properties of an effective nanocarrier are (a) efficient targeting to specific tissues and cells and (b) avoiding rapid clearance and remaining in circulation in the blood stream for a significant amount of time to increase particle uptake in target tissues. These two properties are expected to be dependent on the shape and size of the carriers. Using various shape and size S-FIL fabricated nanoparticles, the effects of particle geometry on intracellular uptake has also been evaluated. In this dissertation, I will present the extensive work that has been done in the fabrication and optimization of the S-FIL nanocarriers, evaluation of the nanocarrier’s in vitro properties, and evaluation of the effects of nanocarrier geometry on intracellular uptake. / text

No description available.