Doctoral Dissertations

Date of Award

12-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Major Professor

Michael D. Best

Committee Members

John E. Bartmess, Brian Long, Todd B. Reynolds

Abstract

Lipids control a variety of complex biological processes. Bulk lipids such as phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) represent the major components of cellular membranes. In addition, unilamellar vesicles composed of lipids (liposomes) are valuable for delivery applications since they can encapsulate and transport drugs and other agents. In order to maximize delivery efficiency and target specific membranes, the ability to trigger and control vesicle-vesicle fusion is desirable. Such approaches generally seek to mimic the membrane fusion machinery present in nature while imparting specificity in the membranes that undergo fusion. The goal of this work is selective drug delivery to diseased cells. We have explored the copper-free click reaction as a bioorthogonal means to drive fusion between membranes containing cyclooctyne-tagged and azido-tagged lipids.

We synthesized three novel lipids containing either the cyclooctyne or azide functional group at the headgroup. In chapter one, we describe the synthesis of Oxy-dibenzocyclooctyne (ODIBO) lipids 1 and 15 and azido-lipid 18, which contain reactive partners for copper-free click chemistry. In these compounds, the phosphate headgroup typically seen in phospholipids is substituted for a triazole ring. In chapter two, we describe the analysis of these compounds for membrane derivatization and fusion. We first set out to confirm the successful derivatization of liposomes containing ODIBO lipids 1 and 15 using a Förster resonance energy transfer (FRET) assay. Next, we investigated membrane fusion by mixing complementary reactive liposomes including ODIBO 1 and azido-lipids, which was again studied through FRET. We studied the effects of liposome composition on fusion, including the PC / PE ratio and the structures of the cyclooctyne-lipids (1- 4) and azido-lipids (5, 36). Through these studies, we identified that ODIBO-lipid 1 and azido-lipid 5 yielded the greatest amount of fusion when incorporated into opposing liposomes containing a 45% / 45% PC/PE ratio. We also attempted to facilitate fusion by the addition of oppositely charged lipids and cholesterol into liposomes, although we were unsuccessful in seeing anything meaningful. This provides, to our knowledge, the first example of exploiting copper-free click chemistry to drive membrane fusion.

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