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Targeted liposomes

University of British Columbia

This thesis presents an optimized and general procedure for coupling proteins to liposomes and investigates certain aspects of the interaction of liposomes with components of the circulation. The object of these studies was to develop straightforward methods for the preparation of well characterized protein-liposome conjugates which exhibit extended circulation half-lives in the blood. These favorable properties should potentiate the use of protein coupled vesicles in in vivo applications such as targeting or diagnostic protocols. A general approach for the preparation of protein-liposome conjugates was developed which employs the high affinity binding of streptavidin for biotinated proteins. Streptavidin was initially attached in a non-covalent manner (via biotin phosphatidylethanolamine) or covalently (via maleimidophenyl-butyryl phosphatidyl-ethanolamine, MPB-PE or pyridyldithio-propionyl phosphatidylethanolamine, PDP-PE) to pre-formed liposomes containing the various lipid derivatives. It was shown that the procedure based on the maleimide derivative MPB-PE, was the most efficient coupling method. Standard procedures for the preparation of MPB-PE however, were found to result in an impure product. Recently a new method for the synthesis of a pure SMPB derivative of phosphatidylethanolamine was developed (Lewis Choi, unpublished). Efficient coupling of proteins to liposomes containing low amounts of pure MPB-DPPE was achieved. Subsequently it was shown that gentle incubation with biotinated proteins results in the rapid and efficient generation of protein coupled vesicles. These retain their ability to interact with defined target celte. Aggregation of liposomes during the coupling reaction is a common consequence of the efficient coupling of protein to liposomes. A method was therefore developed for the preparation of small homogeneously sized protein-liposome conjugates by an extrusion process which does not denature the attached protein. These extruded samples exhibited extended blood circulation times and were stable for significant periods in vivo. The second part of this thesis investigated the in vitro interaction of liposomes of various lipid compositions with platelets. It was demonstrated that large liposomes (> 200 nm in diameter) containing negatively charged lipids (such as EPG) or thiol reactive lipid derivatives (such as MPB-PE) can induce aggregation of platelets in vitro. This interaction was mediated by complement. It is suggested that the formation of platelet-liposome microaggregates in vivo on intravenous administration of negatively charged liposomes, resulted in the transient thrombocytopenia observed in rats. This adhesion event may have also contributed to the rapid removal of aggregated protein-liposome conjugates (containing MPB-PE) from the circulation.

Text

2010-10-14

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http://hdl.handle.net/2429/29180

Biochemistry and Molecular Biology

University of British Columbia

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