Graduation Year

2010

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Chemical and Biomedical Engineering

Major Professor

Mark Jaroszeski, Ph.D.

Committee Member

Richard Gilbert, Ph.D.

Committee Member

Richard Heller, Ph.D.

Committee Member

Andrew Hoff, Ph.D.

Committee Member

Anthony Llewellyn, Ph.D.

Committee Member

Karl Muffly, Ph.D.

Keywords

Nonequilibrium Plasma, Ion Delivery, Electroporation, DNA Delivery, GeneDelivery, Gene Therapy, DNA Vaccines

Abstract

Non-viral delivery of plasmid DNA has traditionally relied upon physical forces applied directly to target tissues. These physical methods typically involve contact between an applicator and the target tissue and often cause transient patient discomfort. To overcome the contact-dependent limitations of such delivery methodologies, an atmospheric direct current plasma source was developed to deposit ionized gas molecules onto localized treatment sites. The deposition of charged species onto a treatment site can lead to the establishment of an electric field with strengths similar to those used for traditional electroporation. In vitro experiments proved that this technology could transiently permeabilize cell membranes and that membrane restabilization followed first order kinetics. Optimum delivery of tracer molecules to cell suspensions occurred after 10 minutes of plasma exposure and was attained without adversely effecting cell viability.

In vivo testing of the plasma discharge demonstrated the capability of this system to deliver plasmid DNA to murine skin. Initial experiments involved the injection of plasmid DNA encoding luciferase into the dermis of C57BL/6J mice and then exposing the tissue to plasma discharge for 10 mintues. Delivery by this method resulted in increased luminescence that was as much as 19-fold greater than DNA injection alone. Follow-up optimization experiments demonstrated it was possible to obtain luminescence results that were similar in magnitude to those obtained using electroporation, which under optimum conditions resulted in about a 40-fold increase in peak luminescence. Finally, optimum conditions were used to deliver a plasmid DNA encoding for the 120 kilodalton glycoprotein present on the surface of a macrophage tropic HIV. Results from this vaccination experiment indicated this method was capable of producing antigen specific humoral immune responses at similar levels as when electroporation was utilized as the delivery method.

Download

Share

COinS