Date of Award

Summer 2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering

First Advisor

Shengnian Wang

Abstract

Nonviral gene delivery methods have been explored as the replacement of viral systems for their low toxicity and immunogenicity. However, they have yet to reach levels competitive to their viral counterparts. Electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection efficiency and cell viability, no restrictions of probe or cell type, and operation simplicity. The commercial electroporation systems have been widely adopted in the past two decades but still carry drawbacks associated with the high applied electric voltage, unsatisfied delivery efficiency, and/or low cell viability. What we did was to improve electroporation performance by application of gold nanoparticles (AuNPs). By adding highly conductive AuNPs in an electroporation buffer solution, we demonstrated an enhanced electroporation performance (i.e., better DNA delivery efficiency and higher cell viability) on mammalian cells from two different aspects: the free, naked AuNPs reduced the resistance of the electroporation solution so that the local pulse strength on cells was enhanced; targeting AuNPs (e.g., Tf-AuNPs) were brought to the cell membrane to work as virtual microelectrodes to porate the cells with a limited area from many different sites.

The enhancement was confirmed with leukemia cells in both a commercial batch electroporation system and a home-made flow-through system using gWizGFP plasmid DNA probes. Such enhancement depends on the size, concentration, and the mixing ratio of free AuNPs/Tf-AuNPs. An equivalent mixture of free AuNPs and Tf-AuNPs exhibited the best enhancement with the transfection efficiency increasing 2-3 folds with minimum sacrifice of cell viability. This new delivery concept — the combination of nanoparticles and electroporation technologies — could be widely applied in various in vitro and in vivo delivery routes of nucleic acids, anticancer drugs, or other therapeutic materials. In the second part of this dissertation, we further demonstrated its success in the enhancement of polyplex delivery of DNA. Specifically, AuNPs were used to carry polyplex (a chemical approach) while electroporation (a physical approach) was applied for fast and direct cytosolic delivery. AuNPs of various sizes were first coated with polyethylenimine, which were further conjugated with DNA plasmids to form AuNPs-polyplex. The hybrid nanoparticles were then mixed with cells and introduced into cell cytosol by electroporation.

In this hybrid approach, cationic polymer molecules condense and/or protect genetic probes, while AuNPs help fix polycations to reduce their cytotoxicity and promote the transfection efficiency of electroporation. The delivery efficiency was evaluated with model adherent cells (i.e., NIH 3T3) and suspended cells (i.e., K562) together with their impact on cell viability. We found that AuNP-polyplex showed 1.5-2 folds improvement on the transfection efficiency with no significant increase of toxicity when compared to free plasmid delivery by electroporation alone. Such a combination of physical and chemical delivery concepts may be further developed for the delivery of various therapeutic materials for both in vitro and in vivo applications. Thirdly, we tried nanoparticle enhanced delivery of small nucleotide including siRNA and miRNA as further proof of our concept. AuNPs are used to enhance the strength of the local electric field and conjugated with the polyplex to reduce the cytotoxicity. The RNA release, expression, and their effect in regulating the target genes were justified.

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