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Melanoma is the most deadly form of skin cancers, with an incidence increasing more rapidly than any other malignant cancer in the past 40 years. Metastatic melanoma is resistant to conventional treatments, such as chemotherapy and radiation therapy. Our lab has previously demonstrated that Mcl-1 is a key contributor in protecting melanoma from therapy-induced cell death. RNAi therapeutics was employed as a novel way to silence the anti-apoptotic protein by using Mcl-1 mRNA sequence-specific siRNAs in vitro. In our hands, passive non-targeted delivery of RNAi therapy into melanoma tumours has been shown to be neither effective, nor selective in vitro and in vivo. Consequently, in this study, siRNA was linked to a delivery system which expressed a ligand specifically targeting melanoma cells. Previously shown, melanoma overexpresses the cell surface ganglioside GD3, thus it is my belief that the anti-GD3 R24 monoclonal antibody can function as a targeting molecule. The antibody was linked to coated cationic liposomes (CCLs) carrying siRNA molecules. Our first step was to confirm R24 ligation to CCLs. Untargeted CCLs showed insignificant values of antibody, whereas antibody-conjugated CCLs presented approximately 30 antibodies per liposome. I also confirmed that siRNA was internalized within CCLs using spectrometry, with an encapsulation efficiency of approximately 80%. Since liposomes need to be small to be effective in vitro and in vivo, CCLs were confirmed to be less than 100nm in diameter. In vitro studies using fluorescent microscopy demonstrated greater binding to melanoma cells with antibody-conjugated CCLs as compared to untargeted CCLs. In vivo experiments showed specific localization of targeted CCLs to induced subcutaneous mouse xenograft tumours. Western blotting demonstrated greater Mcl-1 knockdown using GD3-targeted CCLs. Taken together, these results suggest that anti-GD3 antibodies can serve as targeting molecules to deliver siRNA to melanoma cells and furthermore, GD3-targeted CCLs can promote siRNA-mediated gene silencing. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2010-09-02 10:29:37.944

Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.

Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.