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UBC Theses and Dissertations

Characterization of liposomal drug delivery systems utilizing cell culture methods Shew, Clifford R.


This thesis deals with the characterization of liposomal drug delivery systems utilizing cell culture methods. Drug bioavailability from liposomes is believed to be an important factor in determining the therapeutic activity of a drug. Therefore, the development of liposomes that ensure full drug bioavailability or deliver their contents to the cell cytoplasm are under investigation. To develop liposomes capable of cytoplasmic delivery, it is necessary to have an assay procedure capable of distinguishing between cell surface binding and intracellular delivery. The research presented in this thesis addresses this central problem by developing assays that characterize liposomes specifically designed for cytoplasmic delivery. One liposomal formulation designed for improved cytoplasmic delivery, termed programmable fusogenic vesicles (PFVs), has shown promising preliminary results in vivo. Initial studies examined the bioavailability of mitoxantrone, an anticancer agent, encapsulated in PFVs and conventional liposomes to assess drug-induced toxicity against a human cell line (HEK 293) and a murine cell line (L1210) in vitro. These studies demonstrated greater mitoxantrone cytotoxicity when the drug was encapsulated in PFVs as compared to conventional liposomes. Subsequent studies utilized a lipid-mixing assay employing the lipid probe Rhodamine-PE (Rh-PE) to characterize fusion of PFVs with cellular membranes. These studies demonstrated that PFVs were capable of fusion with HEK 293 cells. An assay employing the nucleic acid dye, YOYO-1 iodide, was developed to quantitate cytoplasmic delivery to cultured cells. This novel assay was then used to compare the ability of PFVs to achieve cytoplasmic delivery. The results also demonstrated that cytoplasmic delivery of YOYO was greater when encapsulated within PFVs compared to other liposomal formulations. Moreover, the importance of individual lipid components in the PFV formulation to the cytoplasmic delivery of YOYO was established. To assess the therapeutic potential of these PFV formulations, studies with antisense oligonucleotides (ASO) were completed. Intracellular delivery of fluorescentlabeled ASO encapsulated in PFVs was quantitated by flow cytometry and confirmed using fluorescence microscopy. Furthermore, an antisense oligonucleotide to the bcl-2 gene encapsulated in PFVs demonstrated down-regulation of mRNA levels by 20% compared to empty PFVs and free antisense in a human melanoma cell line (518A2). The development of PFVs for use in the cytoplasmic delivery of toxic drugs or biologically active nucleotide sequences is discussed.

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