Synthesis, characterization, and evaluation of photo-active amphiphiles for gene delivery applications
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Abstract
Gene therapy has the potential to alter the landscape of medical therapeutic techniques by offering a means of introducing or knocking out genes to treat a number of diseases. Both viral and nonviral vectors are currently being utilized in gene therapy clinical trials. To overcome the obstacles in the cellular uptake and transfection pathways which impede nonviral gene delivery, novel lipids, polymers, and dendrimers are being engineered.
Cationic lipid vectors have been widely characterized as gene delivery tools as they electrostatically interact with the anionic nucleic acid backbone to form a supramolecular structure (lipoplex). This complex allows the nucleic acid to be protected from enzymatic degradation during transport and interacts with the cell membrane to facilitate internalization by endocytosis. A limitation of current systems is a lack of mechanism for release of the nucleic acid, which is an integral step toward transcription and translation. The use of a charge-reversal or charge-switching amphiphile has been previously described by which the amphiphile initially has a net positive charge and is rendered negatively charged upon enzymatic removal of a terminal ester group. In order to further improve the transfection efficacy of cationic lipids and to impart an externally controlled release mechanism, we have developed a library of novel photo-active chargereversal lipids which can be triggered by ultraviolet (UV) light.
In this work, we describe the synthesis and characterization of photo-active lipids for binding and releasing deoxyribonucleic acid (DNA) and evaluate the cellular uptake kinetics and transfection efficiency in vitro. The binding, release, and cellular uptake behaviors of lipoplexes were found to be dependent on lipid composition and resulting supramolecular structures. The transfection efficiency of the photo-active lipoplexes was further affected by variables associated with cellular incubation and UV exposure. Continued development of controlled release gene delivery vectors, including photoactive lipids, will enhance the understanding and utility of gene therapy by providing spatiotemporal control of the process.
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Thesis (Ph.D.)--Boston University
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