CREATING SYNTHETIC MEMBRANES USING TA PROTEIN CHIMERAS

Abstract

Liposomes can be used as drug delivery vehicles and like other nanoparticles, it offers a beneficial distribution mechanism for in vivo therapeutics. Although therapeutic liposomes reach tissues through entirely passive means it is possible to increase liposome uptake by a specific target cell type. Cell specificity in liposomal drug delivery can be achieved by displaying receptor specific targeting peptides on the liposome surface. These ligands evoke endocytic uptake of the liposome upon binding their cognate cell surface receptor and the liposome cargo is subsequently delivered to an intracellular compartment. The methodology used to attach such targeting peptides to liposomes is currently limited by the chemistry of conjugation reactions with functional groups that are attached to the lipid constituents. This project set out to explore an alternative method of ligand attachment using novel chimeric tail-anchored (TA) proteins. Several studies suggest that the transmembrane domain of the TA protein cytochrome b5 is capable of spontaneous insertion into synthetic membranes. Hence, artificial protein chimeras containing the transmembrane domain from cytochrome b5 were created and I showed that these polypeptides provided a robust membrane tether for the incorporation of three different targeting moieties into preformed liposomes. These TA protein chimeras were used to investigate their effect on liposome uptake using two cell culture models. When liposomes loaded with a model fluorescent cargo were modified with the chimeric polypeptides, one of the targeting ligands chosen for study significantly enhanced the association of the liposomes with both cell types. Furthermore, in the case of HeLa M cells I was also able to show that the liposome cargo was successfully internalised. However, when the liposomes were loaded with the cytotoxic drug doxorubicin, the effects of the ligands, as judged on the basis of cell viability and caspase activation, were comparatively modest. I speculate about the likely basis for these findings and discuss whether the attachment of targeting ligands to liposomes through tailanchoring could play a role in the design of future drug delivery systems.

Date of Award	31 Dec 2017
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Simon Webb (Supervisor), Stephen High (Supervisor) & Lynda Harris (Supervisor)