Date of Award

Spring 1-1-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Christopher N. Bowman

Second Advisor

Andrew P. Goodwin

Third Advisor

Daniel K. Schwartz

Fourth Advisor

Veronica Vaida

Fifth Advisor

Michael R. Shirts

Abstract

This thesis investigated the coupling of lysolipids and functionalized tails for in situ formation of synthetic liposomes designed to enable specific characteristics or behaviors in applications ranging from drug delivery to the advancement of artificial cell development. Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) mediated lipid coupling was improved via incorporation of a photoinitiation system. Here, photo-CuAAC enabled spatiotemporal control over liposome assembly, an over 400-fold increase in formation density, and control over the maximal cross-sectional area of the liposomes formed.

Thiol-Michael mediated lipid coupling was enabled using thiol-functionalized lysolipids and acrylate tails where phospholipids were produced over 48 hours with approximate 90% conversion. Coupling was achieved using the thiol-Michael addition reaction for designer lipid synthesis by forming lipids bearing terminal alkyne functionalities in the presence of a visible light-sensitive photoinitiator over 48 hours to reach approximately 90% conversion followed by irradiation to homopolymerize the lipid tails.

Dynamic lipid bilayers were formed using thiol-thioester exchange for in situ liposome formation between thiol-functionalized lysolipids and phenyl thioester-functionalized aliphatic tails. Two tails, a C7 phenyl thioester and a C11 phenyl thioester, reacted with thiol lysolipid to greater than 90% conversion over 48 hours and 12 hours, respectively. These phospholipid products produced liposomes with differences in self-assembly behavior and enhanced permeability was found in the C11 thioester-containing phospholipid system. Following phospholipid formation with the C11 tail, addition of C7 phenyl thioester enabled exchange to convert between 30 and 40% of C11 thioester-containing phospholipids into C7 thioester-containing phospholipids over 96 hours.

Finally, photo-cleavable lipids synthesized using CuAAC-mediated coupling were mixed with natural lipids to enable photo-induced pinocytosis behavior in liposomes formed either via lipid film hydration or the pull-down technique. The lipid film hydration method liposomes displayed consistent pinocytosis in liposomes with pearled structures or aspect ratios greater than 2, indicating that they possessed a critical volume-to-surface area ratio. Morphological transitions and 31x greater liposome formation using an asymmetric formation technique with photocleavable lipid in the outer leaflet indicate a dependence upon asymmetric lipid distribution, decreasing the outer leaflet to inner leaflet ratio during irradiation, to cause engulfment. Pinocytosis in spherical, unilamellar systems using osmotic pressure followed by irradiation lead to an average of 44% of the imaged population undergoing pinocytosis, making this approach potentially applicable to protocell and artificial cell systems.

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