In order for a drug, or any material used for the purpose of eliciting a change in an organisms’ physical or chemical state, to be effective it must reach the intended target intact and for a sustained rate over time. Drug delivery systems encapsulate a drug to protect it from degradation, prevent side reactions, increase solubility, improve accumulation rates at target sites, and release drugs at a controlled rate. Controlled and sustained release of drugs is achieved by degradation of the carrier triggered by breaking dynamic chemical bonds caused by changes in the chemical environment such as pH or redox conditions. Slow, first order kinetic release of drugs increase therapeutic efficacy while also reducing side effects and other cytotoxicity issues. Up and coming drug delivery systems include hydrogels and nanocarriers such as vesicles. Hydrogel drug delivery systems are unique three-dimensional networks of crosslinked hydrophilic polymers that contain anywhere from 50-90 wt% of water. Drugs can be loaded via encapsulation during the gelation process or may be covalently bound to the polymer backbone before gelation. Amphiphilic molecules or polymers that self-assemble in aqueous solutions to form supramolecular nanostructures, such as vesicles, can encapsulate hydrophilic drugs in the aqueous interior or hydrophobic drugs in the lipophilic bilayer membrane. This study seeks to embed vesicles into a hydrogel to create a hybrid drug delivery system which may be applied as a coating to medical devices to prevent bacterial adhesion and growth, injected directly to a target site, or as an additive for wound dressings. This hybrid system mitigates burst release from the hydrogel, as well as stabilizes the vesicles to afford a longer shelf life. Vesicles are prepared from a novel supramolecular amphiphile composed of thio-alkyl modified��-cyclodextrin as a macrocyclic host, and an adamantyl-dithiopropionic acid modified poly(ethylene glycol) as a linear guest. This host-guest system forms inclusion complexes that self-assemble to bilayered vesicles, which may encapsulate a payload, in aqueous solutions. These vesicles serve as three-dimensional multivalent junctions to form a hydrogel, which may encapsulate a second payload, through a dynamic disulfide exchange crosslinking reaction. This novel drug delivery system will be capable of dual and selective release of two different encapsulated payloads. A pH sensitive acid labile bond embedded in the crosslinker will cleave under acidic conditions to release the payload enclosed in the hydrogel matrix, while a disulfide bond embedded in the supramolecular amphiphile of the free vesicle can be cleaved in the presence of naturally occurring antioxidant glutathione, GSH, to release the second payload. It has been discovered that vesicles efficaciously form, can encapsulate a payload, and are stable for several weeks, up to a month. Vesicle stability is examined in the presence of both intracellular and extracellular concentrations of GSH, and it is found that vesicles are more stable in extracellular concentrations of GSH. Crosslinking of vesicles is attempted at several molecular weights of linear thiol terminated poly(ethylene glycol) crosslinker, concentrations ratios of crosslinker: vesicle, pHs, and temperatures. It can be concluded that the crosslinking density with the linear crosslinker is not high enough to form a hydrogel. Future studies will include 4-arm crosslinkers which are predicted to increase the number of crosslinking points and hence the crosslinking density.

This dissertation aims to both deepen and broaden our understanding of copolymers with pendent benzophenone (BP) in relation to both established applications and novel directions in materials science. Photo-reaction of these BP copolymers is explored in attempts to achieve three distinct goals: (1) robust and efficiently photo-crosslinkable solid polymer films, (2) photo-reacted polymer blends with disordered bicontinuous nanostructures, and (3) photo-patterned hydrogel materials with environmental UV stability. We begin by investigating the fundamental gelation behavior of solid polymer films, finding BP copolymers to be particularly effective crosslinkable materials. Gelation efficiency can be tuned according to comonomer chemistry, as BP hydrogen abstraction on the main polymer chain increases chain scission, reducing crosslinking efficiency. This knowledge is then applied in Chapter 3, wherein we discuss two potential methods for preparing nanostructured polymer blends from these copolymers, namely spinodal decomposition of a photo-crosslinked polymer blend and solution-state photografting to create interfacially active species. While each technique shows promise, the ultimate goal of a disordered bicontinuous morphology will require further tuning of materials systems and protocols. Finally, chemical deactivation of BP photo-crosslinker in copolymers for use as photo-patternable and environmentally stable hydrogel materials is investigated. Reduction of BP by sodium borohydride proves a feasible route toward deactivating residual photo-crosslinker in patterned hydrogel films. These results confirm the utility of copolymers with pendent benzophenone photo-crosslinkers as useful tools for complex material systems.

Bio-interface topography strongly affects the nature and efficiency of interactions with living cells and biological molecules, making hydrogels decorated with micro and nanostructures an attractive choice for a wide range of biomedical applications. Despite the distinct advantages of protein hydrogels, literature in the field has disproportionately focused on synthetic polymers to the point that most methods are inherently incompatible with proteins and heat-sensitive molecules. We report the development of multiple biomolecule-friendly technologies to construct microstructured protein and bacteriophage (bacterial virus) hydrogels. Firstly, ordered and sphericity-controllable microbumps were obtained on the surface of protein hydrogels using polystyrene microporous templates. Addition of protein nanogels resulted in the hierarchical nano-on-micro morphology on the microbumps, exhibiting bacterial repellency 100 times stronger than a flat hydrogel surface. The developed microstructures are therefore especially suitable for antifouling applications. The microstructures created on protein hydrogels paved the way for applying honeycomb template on proteinous bacterial viruses. We developed a high-throughput method to manufacture isolated, homogenous, pure and hybrid phage microgels. The crosslinked phages in each phage-exclusive microgel self-organized and exhibited a highly-aligned nanofibrous texture. Sprays of hybrid microgels loaded with potent virulent phage effectively reduced heavy loads of multidrug resistant Escherichia coli O157:H7 on food products by 6 logs. / Thesis / Doctor of Philosophy (PhD) / Bacteriophages (bacterial viruses), also known as phages, are natural bacteria predators. These viruses act as direct missiles, each phage targeting limited groups of bacteria. In addition, phages are an endless resource for self-propagating nanoparticles that can be used as building blocks for new material. I developed a platform for manufacturing a large quantity of microscale beads made of millions of phages. These micro-beads can be sprayed on fresh produce and meat to remove bacterial contamination (with the added benefit of not affecting taste or smell). I also printed phages on substrates, like an ink. The printed phage ink evolved into a patented technology for designing phage coatings on surfaces with very high surface area, like the small structures on our fingers. This phage coating was successfully used to test the existence of bacteria in liquids.

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Chronic wounds have a detrimental impact on patient quality of life, a significant economic cost, and often lead to severe outcomes such as amputation, sepsis or death. The elaborate interplay between bacteria, cutaneous cells, immune cells, growth factors, and proteases in chronic wounds has complicated the development of new therapies that could improve outcomes for chronic wound patients. Existing in vitro models of chronic wounds do not appreciably mimic the complexity of the wound environment. In this work, tissue-engineered skin was developed with the goal of creating an in vitro platform appropriate for testing potential clinical therapies for chronic wounds. The Lumen-X, a digital light processing bioprinter, was used to create tissue-engineered skin from a 7.5% (w/v) gelatin methacryloyl hydrogel laden with primary dermal fibroblasts. This dermal layer was developed with an emphasis on providing a favourable microenvironment for the fibroblasts in order to mimic their in vivo phenotype. An epidermal layer of human keratinocytes was formed on the hydrogel surface and stratified through culture at the air-liquid-interface. The maturation of the epidermis was thoroughly characterized with histology, immunohistochemistry, and trans-epithelial electrical resistance analyses which showed a degree of maturation suitable for wound healing studies. To verify the suitability of this tissue-engineered skin for studying healing in vitro, sharp tweezers were used to create physical wounds in the epidermis which were then infected with Pseudomonas aeruginosa. Reepithelialisation, the production of the pro- inflammatory cytokine TNF-α, and the presence of bacteria were monitored over time, showing healing in wounds without infection and those treated with antibiotics, and potential biofilm formation in infected wounds. The tissue-engineered skin developed here is suitable for use as an in vitro model of the infected chronic wound environment. Future work includes developing better methods for creating the physical wound and characterizing the bacterial biofilm in order to improve the reproducibility and clarity of results. Such a model will then be well-poised to begin testing potential chronic wound therapies in vitro. / Graduate / 2023-07-26

Chemically cross-linked hydrogels (chemical gels) offer a number of enhanced properties over their physical counterparts, particularly in biomedical applications such as drug delivery, tissue engineering, and cell encapsulation. Conventional chemical gels are generally too elastic to be introduced into the body without requiring surgical implantation, making them challenging to use in a clinical context. In response, this thesis is focused on developing injectable analogues of conventional hydrogel-based biomaterials as well as advanced, engineered injectable hydrogels, enabling the facile use of these hydrogels in biomedical applications. Cross-linking is achieved using hydrazone chemistry, in which one precursor is functionalized with aldehyde groups and the other is functionalized with hydrazide groups. Following coextrusion of the reactive precursors, a stable hydrogel network spontaneously forms within seconds. By employing this chemistry as a standard in all of this work, a number of injectable hydrogel systems with well-defined properties (including swelling, drug loading and release, optical properties, gel formation and degradation kinetics, response to the temperature of the surrounding environment, and tissue response) have been generated that can be tuned by rationally varying the charge content in the precursor polymers, the number of cross-linking functional groups used, the reactivity of the electrophilic cross-linking units, and the length and number of hydrophobic affinity domains present within the gels. This work therefore presents a series of independent methods for customizing hydrogels so that they may be adapted to a number of different biomedical applications. / Dissertation / Doctor of Philosophy (PhD)

The in vivo 3D extracellular matrix provides a temporal regulatory environment of chemical cues. Understanding this dynamic environment will be crucial for efficient drug screening, diseases mechanism elucidation, and tissue engineering. Therefore, in vitro 3D cell culture systems with reversible chemical environments are required. To this end, we developed a non-cytotoxic agarose-desthiobiotin hydrogel to sequester streptavidin biomolecule conjugates (KD 10-11 M), which can then be displaced by the addition of biotin (KD 10-15 M). Streptavidin biomolecule conjugates were simultaneously and sequentially immobilized by changing media components. The time required for biochemical environment exchange was minimized by increasing the surface area to volume ratios and pore size of the hydrogels. We temporally controlled the cell adhesive properties of hydrogels with RGD modified streptavidin to influence endothelial cell tube formation. / Thesis / Master of Science (MSc)

Molecular recognition is an important tool for developing tunable controlled release systems and fabricating biosensors with increased selectivity and sensitivity. The development of polymer-based materials that exploit molecular recognition events such as host-guest complexation, enzyme-substrate and enzyme-inhibitor interactions and nucleic acid hybridization was pursued in this thesis. Using polymers as an anchor for molecular recognition can enhance the affinity, selectivity, and the capacity for immobilization of recognition units, enabling the practical use of affinity-based systems in real applications. To introduce the potential for immobilization while preserving or enhancing the affinity of small molecule recognition units, the affinity of derivatized cyclodextrins for the hydrophobic drug, dexamethasone, was investigated. Cyclodextrins (CDs) are molecules that possess a hydrophilic exterior and a hydrophobic cavity capable of accommodating a wide range of small molecule guests. Analysis of the solubilization capacities, thermodynamic parameters and aggregative potentials of carboxymethyl and hydrazide derivatives of CDs established the dextran-conjugated βCD derivative as an ideal carrier of hydrophobic drugs and the hydrazide βCD derivative as an optimal solubilizer of lipophilic pharmaceuticals, both alone and when incorporated in a polymer-based drug delivery vehicle. To enable non-covalent immobilization and stabilization of biomacromolecular recognition units, a printed layer hydrogel was investigated as a selective diffusion barrier for analyte sensing and enzyme inhibitor recognition. A printable hydrogel platform was developed from an established injectable system composed of aldehyde- and hydrazide-functionalized poly(oligoethylene glycol methacrylate) polymers. The printed layer hydrogel effectively immobilized a wide range of enzymes and protected enzyme activity against time-dependent and protease-induced denaturation, while facilitating the diffusion of small molecules. Furthermore, to demonstrate the potential of the printed film hydrogel immobilization layer to enhance the selectivity of the target, the printable hydrogel platform was used to develop a microarray-based assay for the screening of inhibitors of the model enzyme, β-lactamase. The assay was able to accurately quantify dose-response relationships of a series of established inhibitors, while reducing the required reagent volumes in traditional drug screening campaigns by 95%. Most significantly, the assay demonstrated an ability to discriminate true inhibitors of β-lactamase from a class of non-specific inhibitors called promiscuous aggregating inhibitors. Finally, to enable non-covalent immobilization of DNA recognition units, the printable hydrogel-based microarray was tested for its ability to immobilize DNA recognition sites and promote the detection of DNA hybridization events. A long, concatameric DNA molecule was generated through rolling circle amplification and was used as a sensing material for the detection of a small, fluorophore labeled oligonucleotide. The printable hydrogel was able to effectively entrap the rolling circle amplification product. Properties of the printable hydrogel were investigated for their ability to support the detection of DNA hybridization events. / Thesis / Doctor of Philosophy (PhD) / This thesis describes the development of polymer-based materials that exploit molecular recognition events for drug delivery and biosensing applications. First, cyclodextrins (CDs) are molecules that are capable of binding a wide range of small molecules. A comprehensive analysis of the complexation properties of CD derivatives revealed critical insight regarding their application in polymer-based drug delivery vehicles. Second, a printable hydrogel platform was developed to support the immobilization and activity of biomolecules and establish a biosensing interface that facilitates the diffusion of small molecules but not molecular aggregates. A microarray-based assay was developed by employing the printed hydrogel interface for the screening of inhibitors of the model enzyme, β-lactamase, and the detection of DNA hybridization events.

Hydrogels are defined as swellable polymer networks with the mechanical, interfacial, and physical properties similar to native tissues in the body. Nanocomposite hydrogels, defined as hydrogels that either entirely consist of or have embedded nanoparticle phases, have been shown to further expand the range of properties achievable with hydrogels and be suitable in many applications such as building tissue scaffolds. In particular, nanocomposite phases that can be eroded offer interesting potential to construct nanoscale voids that can be made in the gel that may be highly beneficial for applications in drug delivery, bioseparations, and tissue engineering. In this thesis, two methods of incorporating starch nanoparticles (SNPs) into a ultraviolet (UV)-cured poly(ethylene glycol methacrylate) (POEGMA) matrix are described. In the first method, the SNPs were physically entrapped during the curing of the gels. An investigation of the effect of fabrication parameters such as monomer ratios, crosslinker amounts, and entrapped SNP concentration on swelling and shear storage modulus (G’) was performed. Enzymatic degradation of the nanophase was also observed to be possible upon amylase treatment, and the resulting internal morphology was confirmed to have increased internal porosity based on a methylene blue uptake experiment. In the second method, chemically functionalized SNPs were used as the exclusive crosslinker to create the POEGMA network. The swelling and mechanical performance of the SNP-crosslinked hydrogels were investigated and compared to the entrapped SNP gels. A preliminary study of the consequences of degradation of a naturally occurring crosslinker to the enzyme α-amylase was also performed. The combination of cytocompatible components and potential for internal porosity control make gels an interesting platform for tissue scaffolding and bioseparation applications. / Thesis / Master of Applied Science (MASc)