Nanogels are soft, deformable networks of cross-linked polymer swollen in water. Nanogels have the unique ability to swell in response to external physiological conditions. Their stimuli-responsive nature affects degradability, drug uptake and release, which can be exploited to create tunable drug delivery systems. The ability to alter the composition and structure of nanogels imparts advantageous characteristics for targeted drug delivery applications. Antipsychotic drugs (APDs) used to treat schizophrenia, a chronic neuropsychiatric disorder, are typically hydrophobic. Prolonged dosing causes neurological and metabolic side effects due to the systemic administration of drug. Patient adherence to APD administration is low, causing complications that contribute to the substantial burden of disease. APDs would benefit from nanogel encapsulation through improved solubility and controlled release kinetics to reduce the adverse side effects associated with typical administration protocols. This thesis presents the development of hydrophobized, biodegradable poly(oligoethylene glycol methacrylate) (POEGMA)-based nanogels to deliver APDs to the brain. Both an adaptation of conventional precipitation polymerization as well as a spontaneous self-assembly technique are utilized to synthesize nanogels containing different hydrophobic domains. Incorporation of cross-linkers with different modalities of biodegradability enable stimuli-responsive degradation and drug release. The effects on nanogel swelling, biodegradability, and APD uptake and release kinetics are explored in vitro. The preclinical application of these APD-loaded nanogels is evaluated using the minimally invasive intranasal (IN) route for delivery. We show that these nanogel delivery systems have therapeutic effects in terms of significantly altering a range of rodent behaviours, including locomotion inhibition, the onset of catalepsy, and improvement in pre-pulse inhibition, over extended periods of time in relation to current administration strategies. These drug-loaded nanogel delivery systems show potential to minimize the effective therapeutic dose by enhancing APD bioavailability via IN administration, thus reducing adverse outcomes and improving potential patient adherence to APD-based therapies in clinical use. / Thesis / Doctor of Philosophy (PhD) / Nanogels are soft, deformable polymer networks swollen in water with potential for drug delivery given their easy-to-tune physicochemical properties. However, the poor water solubility of many therapeutics, including antipsychotic drugs (APDs) used to treat schizophrenia, limits drug encapsulation within nanogels. In addition, conventional synthetic techniques produce materials that degrade into poorly-defined byproducts, causing toxicity concerns. This thesis presents novel strategies to incorporate hydrophobic domains and biodegradable bonds within poly(oligo ethylene glycol methacrylate) (POEGMA) nanogels. We demonstrate how these moieties affect nanogel swelling, degradability, cytocompatability as well as the uptake and release of clinically prescribed APDs. Intranasal (IN) administration of drug-loaded nanogels is studied as a non-invasive delivery alternative to improve drug bioavailability. The proposed nanogel-based drug delivery systems can decrease drug dose, minimize adverse side effects, and improve patient adherence to therapeutic regimens relying on APDs, demonstrating their potential for clinical application.

The increasing prevalence of bacterial infections has made it necessary to find novel methods of combatting the resistance of bacteria to conventional antibiotics. Mycobacterium abscessus is an increasingly prevalent pathogen that is intrinsically drug resistant, therefore difficult to treat. The use of phytochemicals as a source of alternate antibiotics has been explored, however, the poor solubility of phytochemicals in water makes it difficult to effectively deliver them to bacterial biofilms. In this study, I investigated the efficacy of nanosponge-emulsified phytochemicals in killing M. abscessus biofilms. The nanosponge technology was used to improve the solubility and stability of the phytochemicals, allowing for improved bioavailability. Results showed that the nanosponge-emulsified phytochemicals effectively reduced the viability of M. abscessus biofilms, compared to non-emulsified phytochemicals. The findings of this study contribute to a development of new strategies for the treatment of bacterial infections and demonstrate the potential of nanosponge-emulsified phytochemicals as a promising alternative to conventional antibiotics.

Recent progress in nanotechnology has attracted interest to a biomedical application of the carbon nanoparticle C60 fullerene (C60) due to its unique structure and versatile biological activity. In the current study the dual functionality of C60 as a photosensitizer and a drug nanocarrier was exploited to improve the efficiency of chemotherapeutic drugs towards human leukemic cells. Pristine C60 demonstrated time-dependent accumulation with predominant mitochondrial localization in leukemic cells. C60’s effects on leukemic cells irradiated with high power single chip LEDs of different wavelengths were assessed to find out the most effective photoexcitation conditions. A C60-based noncovalent nanosized system as a carrier for an optimized drug delivery to the cells was evaluated in accordance to its physicochemical properties and toxic effects. Finally, nanomolar amounts of C60-drug nanocomplexes in 1:1 and 2:1 molar ratios were explored to improve the efficiency of cell treatment, complementing it with photodynamic approach. A proposed treatment strategy was developed for C60 nanocomplexes with the common chemotherapeutic drug Doxorubicin, whose intracellular accumulation and localization, cytotoxicity and mechanism of action were investigated. The developed strategy was revealed to be transferable to an alternative potent anticancer drug – the herbal alkaloid Berberine. Hereafter, a strong synergy of treatments arising from the combination of C60-mediated drug delivery and C60 photoexcitation was revealed. Presented data indicate that a combination of chemo- and photodynamic treatments with C60-drug nanoformulations could provide a promising synergetic approach for cancer treatment. / Kürzliche Fortschritte in der Nanotechnologie haben Interesse an einer biomedizinischen Anwendung des Kohlenstoffnanopartikels C60 Fulleren (C60) aufgrund seiner einzigartigen Struktur und breiten biologischen Aktivität geweckt. In der aktuellen Studie wurde die doppelte Funktionalität von C60 als Photosensibilisator und als Wirkstoff-Nanoträger genutzt, um die Wirkung von Chemotherapeutika auf menschliche Leukämiezellen zu verbessern. C60 alleine zeigte in den Zellen eine zeitabhängige Akkumulation mit vorherrschender mitochondrialer Lokalisation. Die Wirkung von C60 auf Leukämiezellen, die mit unterschiedlicher Wellenlänge bestrahlt wurden, wurde bewertet, um die effektivsten Photoanregungsbedingungen zu finden. Die physikochemischen Eigenschaften und toxischen Wirkungen von C60 auf die Leukämiezellen wurden nach nicht kovalenter Bindung von Arzneistoffen bewertet. Schließlich wurden nanomolare Mengen von C60-Wirkstoff-Nanokomplexen in Molverhältnissen von 1:1 und 2:1 untersucht, um die Effizienz der Behandlung von Zellen zu verbessern und sie durch photodynamischen Ansatz zu ergänzen. Mit dem gängigen Chemotherapeutikum Doxorubicin wurde eine Behandlungsstrategie entwickelt und dessen intrazelluläre Akkumulation und Lokalisation, Zytotoxizität und Wirkmechanismus untersucht wurden. Es wurde gezeigt, dass die entwickelte Strategie auch auf ein alternatives Krebsmedikament übertragbar ist – das pflanzliche Alkaloid Berberin. Die erhaltenen Daten deuten darauf hin, dass eine Kombination von chemo- und photodynamischen Behandlungen mit C60-Nanokomplexen einen vielversprechenden synergetischen Ansatz für die Krebsbehandlung bieten könnte.

Over the past few decades, tremendous efforts have been made to develop nanomaterials for biotechnological applications such as therapeutics. Understanding and engineering interfaces between biomacromolecules and nanomaterials is a key to the creation of successful therapeutic systems. My research has been oriented toward developing therapeutic systems using gold nanoparticles (AuNPs) incorporating material science, organic synthesis, and biology. For this purpose, mixed monolayer protected AuNPs (~2 nm core size) with various functional groups have been employed for triggering therapeutic effects. Several strategies have been accomplished using anticancer drugs that non-covalently and covalently incorporate onto AuNPs as a drug delivery carrier. Alternatively, AuNPs were developed by regulating host-guest complexation processes inside the cell, allowing control of the therapeutic effect of the AuNP. In addition, by using host-guest chemical events on the AuNPs, exocytosis of the AuNPs was controlled, enabling their prolonged retention inside of the cells, providing new strategies for improving conventional drug delivery systems. Therefore, engineering of the AuNP surface can afford new pathways for designing and improving therapeutics.

Recent technologies in micro-devices for investigation of functional biology in a controlled microenvironment are continually growing and evolving. In particular, electric-field mediated microfluidic platforms are evolving technologies that have significant applications in drug delivery and cell migration investigations. Although drug delivery has had several successes, in some areas, it continues to be a challenge; in recent years, the positive impact of electric fields is being explored. The primary objectives of the dissertation are to design, fabricate, and employ two novel microfluidic platforms for drug delivery and cell migration in the presence of electric fields. Description of iontophoretic carboplatin delivery into the MDA-MB-231 triple-negative breast cancer cells and investigation of neutrophil electro taxis are two main aims of the dissertation. Transdermal drug delivery systems such as iontophoresis are useful tools for delivering chemotherapeutics for tumor treatment not only because of their non-invasiveness but also due to their lower systematic toxicity compared to other drug delivery systems. While iontophoresis animal models are commonly being used for the development of new cancer therapies, there are some obstacles for precise control of the tumor microenvironment's chemoresistance and scaffold in the animal models. We employed experimental and computational approaches, the iontophoresis-on-chip and the fraction of tumor killed mathematical model, for predicting the outcome of iontophoresis treatment in a controlled microenvironment. Also, precise control over the cell electromigration is a challenging investigation which we will address in the second aim of the dissertation. Here, we developed a microfluidic platform to study the consequences of DC electric fields on neutrophil electromigration (electrotaxis), which has an application of directing neutrophils away from healthy tissue by suppressing the migration of neutrophils toward pro-inflammatory chemoattractant. / Doctor of Philosophy / Recent technologies in the micro-scale medical devices for diagnosis and treatment purposes are continually growing and evolving. Microfluidic platforms are reproducible devices with the dimensions from tens to hundreds of micrometers for manipulating and controlling fluids. In particular, electric-field mediated microfluidic platforms, are developing technologies that have significant applications in drug delivery and biological cell directional movement investigations. Although drug delivery has had several successes, in some areas, it continues to be a challenge. In recent years, the positive impact of electric fields is a significant advancement in drug delivery techniques. Transdermal drug delivery systems such as iontophoresis are useful tools for delivering chemo drugs for tumor treatment not only because of their sensitivity but also to their lower systematic toxicity compared to injection or oral drug delivery. While iontophoresis animal models are conventional for the development of new cancer therapies, there are some obstacles to precise control of the tumor scaffold in the animal models. We also developed a novel microfluidic platform to study the consequences of DC electric fields on white blood cells' (WBC) directional movement, which has an application of directing WBC away from healthy tissue by suppressing the damage of WBC accumulation in healthy organs.

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Hypoxia is a characteristic pathophysiological feature of many solid tumors, which contributes significantly to resistance to chemotherapy and radiotherapy. It also induces numerous intracellular signaling pathways, which in turn trigger the upregulation of various key proteins promoting tumor cell survival, progression and metastasis. In this context, novel therapeutic approaches are urgently needed to facilitate the early detection and improve the treatment of hypoxic tumors. Focusing on the hypoxic tumor microenvironment, one can recognize that the membrane bound carbonic anhydrase IX (CA IX) isozyme represents a potential biomarker and a compelling therapeutic target for better diagnosis and management of hypoxic tumors. CA IX is significantly overexpressed under hypoxic conditions as compared to normal tissues and it assists tumor cell to maintain neutral intracellular pH values. Building on this hypothesis, we are focusing our efforts in this thesis towards the development and the optimization of drug delivery nanosystems capable of selectively targeting CA IX that is overexpressed in the hypoxic tumor niche, which in turn will enhance the early detection of hypoxic tumors as well as improve the accumulation of chemotherapeutic drugs in hypoxic cancer cells. This strategy is expected to overcome the chemoresistance associated with tumor hypoxia and minimize the systemic side effects associated with chemotherapeutic drugs administration. In chapter 2, we focused our efforts towards the development of the in vitro biological models for testing our nanoparticles. This process was achieved through screening a series of cancer cell lines for the expression of our target epitope under hypoxic conditions. We induced hypoxia either chemically, using cobalt chloride, or physicochemically, using a hypoxia chamber purged with hypoxia gas mixture containing 1% O2. Screening for CA IX overexpression under hypoxic conditions was done both in 2D monolayer cells and 3D tumor spheroids, which become naturally hypoxic due to their 3D growth. Western blot analysis was used to confirm the expression of our target protein and we have identified three cell lines with a high level of expression of CA IX under hypoxic conditions, namely HT-29 colorectal cancer, SKOV-3 ovarian cancer and MDA-MB-231 breast cancer cell lines. In chapter 3, we optimized a theranostic liposomal delivery system through the use of a combination of zwitterionic amphiphilies of different packing parameters to encapsulate a potent fluorescent carbonic anhydrase inhibitor (CAI), as a novel approach to facilitate the detection of colorectal cancer. Our main focus was to increase the aqueous concentration of poorly water-soluble CAI, to correlate its delivery efficiency with the lipid type and composition of the liposomal nanosystem, as well as to enhance the tissue permeability, allowing easy detection of small tumor polyps. Our optimized DMPC/DOPE liposomal formulation demonstrated an optimum size, high encapsulation efficiency of CAI, and a phase transition temperature below 37 ᴼC that allows efficient delivery of CAI and good tissue penetrability towards the hypoxic tumor cells overexpressing CA IX. In chapter 4, we optimized a CAI-targeted long circulating liposomal delivery system encapsulating doxorubicin. Our main focus was to enhance the accumulation of doxorubicin in hypoxic tumors through targeting CA IX protein overexpressed under hypoxic conditions. This strategy proved to enhance the internalization of the drug carrier into hypoxic cancer cells thus overcoming chemoresistance associated with hypoxia and also minimize the systemic side effects associated with the intravenous administration of non-targeted Doxil®-like formulations. In chapter 5, we optimized a pH sensitive gold nanoplatform functionalized with CAI based moieties to enhance the selective delivery of doxorubicin to hypoxic tumors in a controlled release manner. Our main focus was to combine the advantage of targeting CA IX overexpressed under hypoxic conditions with the intracellular triggered release of doxorubicin in the lysosomes inside the cell in order to enhance the delivery of doxorubicin inside the cancer cells and to overcome the chemoresistance associated with hypoxia. / Pharmaceutical Sciences

Microgels, colloidal networks of crosslinked water-soluble polymers with dimensions < 1 μm, have been demonstrated to be useful materials in a wide range of biomedical and environmental applications. In particular, temperature-responsive microgels based on poly(N- isopropylacrylamide) (PNIPAM) have attracted significant research interest in drug delivery applications. However, conventional precipitation-based PNIPAM microgels are functionally non-degradable, problematic for biomedical applications. To resolve this issue, a thermally- driven self-assembly approach based on hydrazide and aldehyde functionalized PNIPAM oligomers to form an acid-labile hydrazone bond was developed in the Hoare Lab to produce thermoresponsive, colloidally stable, monodisperse and degradable microgels. In this thesis, the internal structure of these self-assembled microgels was investigated using small and ultra-small angle neutron scattering and surface force experiments. Contrary to expectations based on the assembly technique, all these characterization strategies suggested that self-assembled microgels have a homogeneously cross-linked internal structure. It is anticipated that these well-defined degradable and homogeneous nanoscale gel networks offer opportunities for addressing challenges in drug delivery, biosensing, and optics by exploiting the predictable diffusive and refractive properties of the homogeneous microgel networks. In addition, the co-self-assembly of a moderately hydrophobic anti-inflammatory drug (dexamethasone) during the microgel self-assembly process was demonstrated to enable five-fold higher drug encapsulation (75-80%) relative to the conventional partition/diffusion- based drug loading processes. This result addresses a key challenge in delivering hydrophobic drugs using conventional precipitation-based microgel systems due to the inherent hydrophilicity of the crosslinked network. The potential of the self-assembly approach to fabricate multi-responsive smart microgels was demonstrated by incorporating pH-ionizable functional groups (via the copolymerization of acrylic acid and 2-dimethylaminoethylmethacrylate to introduce anionic and cationic charges respectively) into the hydrazide and aldehyde-functionalized precursor polymers prior to self-assembly. The self-assembled charged microgels showed the same pH- responsive swelling behaviours of conventional microgels, including amphoteric microgels that can be formed at any desired cationic:anionic charge density by simply mixing different ratios of cationic and anionic precursor polymers. Such microgels offer significant potential to improve the performance of microgels in applications demanding dual pH/temperature specific drug delivery. / Thesis / Master of Applied Science (MASc) / Medications can exist in many different forms. From pills to injections, existing drug delivery systems require a high frequency of drug administration and often result in low efficacy of drug once administered to the human body. Polymer-based drug delivery systems have the potential to improve this delivery. In particular, microgels, water-filled crosslinked polymer networks with a size less than one micron, offer promise as a drug delivery vehicle. The size and chemical composition of microgels can be tailored to enable their use in a wide array of drug delivery applications. In addition, microgels can be loaded with a therapeutic agent and transported in the blood stream to deliver drug at a rate and/or location tunable based on the internal structure of the microgel. “Smart” microgels have the particularly attractive ability to change their properties in response to certain environmental stimuli (i.e. temperature or pH). However, current smart microgel systems are non-degradable and would accumulate in the body, causing undesired side-effects. In this thesis, a new self-assembly approach has been used to produce degradable microgels with the potential to switch properties in response to both temperature and pH. Water-insoluble drugs can be encapsulated more efficiently with this method, and the dual-responsive behaviour is expected to improve our capacity to deliver drug at the rate and location desired in the body.