We are proposing to develop new methods for optimal delivery of cytotoxic drugs and other macromolecules to specific tumor cells in vitro, by the use of antibody-targeted liposomes. We have planned a three-pronged approach which involves both the development of new liposome systems and their application to specifically-selected tumor cells. 1. pH-sensitive, fusogenic liposomes: We are planning to optimize the behaviour of pH-sensitive liposomes for obtaining maximal cytoplasmic release following endocytosis. Specifically, we will accomplish this goal by: (a) formulation of appropriate fatty-acid/phosphatidylethanolamine (FA/PE) liposomes that fuse without leakage by avoiding the Hexagonal phase transition of PE; (b) synthesis of new pH-sensitive lipids of appropriate molecular architecture. The extent of cytoplasmic release will be followed by fluorescent probes, inhibition of protein synthesis by toxin A fragments and infectivity of viral DNA. 2. New Assays for endocytosis and lysosomal processing: Liposomes will be used as carriers of fluorescent or fluorogenic molecules capable of responding either to low pH or to the presence of specific hydrolytic enzymes. To achieve this we will use the following types of probes: (a) fluorescent probes that show an easily measurable pH-induced spectral shift; (b) fluorogenic probes that show an easily measurable pH-induced spectral shift; (b) fluorogenic probes attached to liposome-encapsulated ligands via a pH-sensitive covalent bond; (c) fluorogenic probes attached by a bond cleavable by specific enzymes, by elastase, or other proteases. Judicious use of such assays will give valuable quantitative information on the kinetics of endocytosis andphage-lysosome fusion applicable both to drug delivery via liposomes but also to studies on the fate of pathogenic viruses and bacteria. 3. Targeting to specific tumor cells: We have selected as model systems human (ovarian carcinoma) and murine (liver metastatic) tumor cell lines for specific reasons relating to intended future therapeutic applications in animals and ultimately in humans. We plan to maximize the cytotoxicity of drug-loaded liposomes by using targeted liposomes that have the necessary properties for efficient cytoplasmic delivery of the encapsulated drug.
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