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Assisted reproductive technology (ART) has been rapidly expanding since the birth of Louise Brown, the first test tube baby, in 1978. Although an increasingly complex array of laboratory skills and procedures have been developed for infertility treatments, the success rate of ART remains low. In an attempt to make ART safer and more efficient, international medical practice is trending towards single embryo transfers and the use of innovative, sophisticated technologies to identify promising gametes and embryos with the highest potential to generate a pregnancy. Laser technology is increasingly being used to accomplish these aims. The application of lasers for ART has been successfully employed in clinical practice for some time now and is continually the subject of investigative research in order to generate new methods to improve operations. Moreover, lasers serve as a powerful tool at the forefront of investigative research in the reproductive sciences, assisting in broadening our understanding of reproductive and developmental biology. Nevertheless, there is a paucity of literature pertaining to the safe standardisation of such laser procedures with evidence at the molecular level. The primary aim of this thesis was to optimise applications of laser technology for clinical ART and research applications in the reproductive sciences. This thesis utilised the mouse embryo model to investigate potential deleterious effects of different laser treatment applications, both by the operator and hardware manufacturer. Safe and unsafe laser operator parameters were elucidated by assessing deleterious effects to the plasma membrane integrity, blastocyst survival rate, DNA fragmentation levels, and changes in gene expression of key developmental genes. The effect of altering the laser hardware to lower the power output was evaluated and it was determined that if a lower power laser is used to deliver a set amount of energy over a longer period of time, a smaller amount of damage is incurred. Work in this thesis also established a new method in which laser technology can be used as a research tool for the reproductive sciences, by creating a novel stimuli-responsive laser-activated nanoparticle delivery system with spatial control and increased efficiency in a mammalian cell model. The field of reproductive science continues to benefit greatly from laser application clinically to improve infertility treatments, and in research, to elucidate mechanisms underlying infertility, with a hope of increasing our understanding and eventually developing new treatment options.

Glioblastoma multiforme (GBM) is resilient to the current standard of care treatment of surgical resection followed by concurrent radiotherapy and temozolomide (TMZ) chemotherapy. GBM patient responses are poor and variable, resulting in more than 90% tumor recurrence and grim survival. The high mortality of GBM is attributed to its invasive peripheral growth, partially intact blood-brain barrier (BBB), regions of hypoxia, and high cellular heterogeneity that includes brain tumor initiating cells (BTICs) and immunosuppressive cells. These features of GBM work together to restrict the delivery of drugs throughout the tumor, suppress immune recognition of tumor cells, and facilitate tumor progression. Nanoparticles are well-suited to address limitations associated with the treatment of GBM by enhancing drug delivery to the tumor and reducing side effects. The overall objective of the work in this dissertation is to develop systemically administered nanoparticles that overcome barriers to drug distribution and cellular heterogeneity in GBM to improve therapeutic responses. In murine GBM models, the effective delivery of Doxorubicin (DOX) chemotherapy, BTIC inhibitor, and immune-stimulating agonists were evaluated using two distinct mesoporous silica nanoparticles (MSNs): 1) the Fe@MSN particle and 2) the immuno-MSN particle. First, drug release from the Fe@MSN particle was triggered using an external radiofrequency (RF) field to enhance the distribution of DOX and/or BTIC inhibitor across the partially intact BBB and into the tumor interstitium. The effective delivery of drugs facilitated by Fe@MSN particles translated into suppressed GBM growth, depleted stem-like cell phenotypes in hypoxic regions, and prolonged or cancer-free survival. Second, towards further improving GBM treatment strategies, the immuno-MSN particle delivered immune-stimulating agonists to dysfunctional immune cells in GBM to reverse the effects of immunosuppression. Immuno-MSN particles facilitated the recruitment of key immune cells to the tumor microenvironment (TME) while sparing healthy brain tissue and peripheral organs, resulting in elevated circulating CD8+ T cells. The findings from this dissertation provide key insights into overcoming physical and cellular barriers to drug delivery in GBM using systemically administered nanoparticles and have potential to improve patient responses to systemic treatments that are otherwise ineffective. Future work includes refining the mesoporous silica nanoparticles and experimenting with combination therapy.

Although the average cancer mortality rate has steadily declined over the past 25 years, the majority of advanced disease remains resistant to current treatment methods and yields dismal prognoses.1 Novel immunotherapies such as CAR T-cell transfer therapy and ICB provide promising avenues to overcome tumor-mediated immune evasion and initiate an adaptive immune response towards cancer cells. The strongly immunosuppressive microenvironment in advanced tumors, however, can limit the downstream potential of these therapies, and to date these therapies have provided mixed clinical results.2,3 In an attempt to reverse the immunosuppressive tumor microenvironment, we have designed a MSN that can traffic a STING agonist—cdGMP—into innate immune cells and produce a nearly 9-fold increase in pro-inflammatory IFN-훽 secretion compared to the free agonist. In a metastatic 4T1 model, the surface density of PEG molecules dictates what innate immune cells have access to systemically injected MSNs. Following treatment with cdGMP-loaded MSNs, this differential distribution and uptake of particles leads to differences in innate immune cell recruitment at metastatic sites and in the blood. It is displayed that an immunostimulatory nanoparticle can be engineered to enhance innate immune cell activation in advanced, metastatic disease, providing a tool that may be able to work in combination with other current immunotherapies to yield more robust and consistent therapeutic responses.

Mesoporous silica nanoparticle (MSNP) supported-lipid bilayers, termed ‘protocells,’ represent a potentially transformative class of therapeutic and theranostic delivery vehicles. The field of targeted drug delivery poses considerable challenges that cannot be addressed with a single ‘magic bullet’. Consequently, the protocell has been designed as a modular platform composed of interchangeable biocompatible components. The mesoporous silica core can have variable size and shape to direct biodistribution and controlled pore size and surface chemistry to accommodate diverse cargos. The encapsulating supported lipid bilayer can be modified with targeting and trafficking ligands as well as polyethylene glycol (PEG) to effect selective binding, endosomal escape of cargo, drug efflux prevention, and potent therapeutic delivery, while maintaining in vivo colloidal stability. Many nanocarrier cancer therapeutics currently under development, as well as those used in the clinical setting, rely upon the enhanced permeability and retention (EPR) effect to passively accumulate in the tumor microenvironment and kill cancer cells. In leukemia, where leukemogenic stem cells and their progeny circulate within the peripheral blood or bone marrow, the EPR effect may not be operative. Thus, for leukemia therapeutics, it is essential to target and bind individual circulating cells. Here, we investigate protocells, an emerging class of nanocarriers, and establish the synthesis conditions and lipid bilayer composition needed to achieve highly monodisperse protocells that remain stable in complex media as assessed in vitro by dynamic light scattering and cryo-electron microscopy and ex ovo by direct imaging within a chick chorioallantoic membrane (CAM) model. We show that for vesicle fusion conditions where the lipid surface area exceeds the external surface area of the MSNP and the ionic strength exceeds 20 mM, we form monosized protocells (polydispersity index < 0.1) on MSNP cores with varying size, shape, and pore size, whose conformal zwitterionic supported lipid bilayer confers excellent stability as judged by circulation in the CAM and minimal opsonization in vivo in a mouse model. Having established protocell formulations that are stable colloids, we further modified them with anti-EGFR antibodies as targeting agents and re-verified their monodispersity and stability. Then using intravital imaging in the CAM we directly observed in real time the progression of selective targeting of individual leukemia cells (using the established REH leukemia cell line transduced with EGFR) and delivery of a model cargo. Overall we have established the effectiveness of the protocell platform for individual cell targeting and delivery needed for leukemia and other disseminated disease.

The work covered in this thesis focuses on the development of mesoporous silica nanoparticle-based platforms for the controlled delivery of therapeutic agents. The first part of the thesis discusses the physical chemical properties of mesoporous silica nanoparticles, including the studies on their uptake and release capacities and the physical states of the encapsulated cargo molecules using spectroscopic methods. The second part of the thesis describes the organic functionalizations of the mesoporous silica nanoparticles with novel cyclodextrin-based nanogate systems, achieving pH and redox responsive controlled release mechanisms, as well as cargo size selectivity for the release from a dual-loaded system. In the third part of this thesis, several examples of the biomedical applications of the mesoporous silica nanoparticle based drug delivery systems are shown. This includes the in vitro delivery of small interference RNA to shutdown endogenous and exogenous gene expressions in cells using the polyethyleneimine coated nanoparticles, the in vivo delivery of anti-cancer therapeutic to achieve tumor suppression effects on mice models, and the in vivo delivery of anti-microbial therapeutic to achieve animal protection from anthrax lethal toxins. A combination of all these areas of research demonstrate the advancement of the mesoporous silica nanoparticle based drug delivery system towards utilization within living organisms, and realization of medicine on the nanoscale.