ABSTRACT

0933153
Kumta


Gene delivery using non-viral (plasmid) techniques are very desirable due to their economic, convenience, ease of manufacturing, cost-effectiveness, and safe characteristics. The techniques are currently limited by low transfection efficiencies due to lack of a suitable carrier. A number of cationic synthetic polymers have been studied as non-viral gene delivery agents. Calcium phosphate (CaP) commercial kits have also been known as standard non viral gene delivery vectors although there have been limited studies reported to ascertain their efficacy as non viral gene delivery agents. Nano-sized CaPs called "NanoCaPs" developed by the PI are novel delivery agents for plasmid DNA (pDNA) transfection. However, successful transfection is dependent on maintaining the complex size of 30-50 nm. It is hence critical to identify methods to stabilize the NanoCaPs. Novel CaP aquagels have been developed by the PI that display polymeric hydrogel characteristics. These CaP aquagels can be used directly as gene delivery agents or can serve as a matrix for embedding the synthesized p-DNA-NanoCaPs complex. The composite CaP aquagels can also provide controlled release of pDNA. These aquagels have the potential to provide nucleic-acid based therapeutics that closely resembles traditional pharmaceuticals and gene delivery for tissue engineering.

The overall objective is to engineer a safe and versatile plasmid gene delivery system for tissue engineering. The goal is to demonstrate that CaP aquagels will enhance the in vitro transfection efficiency of pDNA by increasing the uptake and expression of marker genes (Luciferase and/or GFP). This will be achieved using quantitative digital imaging methods. The synthesized CaP aquagels are biocompatible and the in vitro data is in excellent agreement with the objective to design and develop an efficient plasmid gene therapy for gene replacement therapy and tissue engineering. The present study will provide the foundation for conducting further research related to plasmid gene therapy. The specific objectives of the research have been formulated to provide solutions to fundamental questions related to in vitro pDNA transfection efficiency and the application of the aquagels in bone tissue engineering thus providing key information currently not available. The proposed research will enable the generation of novel CaP based aquagel carriers for plasmid gene delivery, the benefits of which will be seen in tissue engineering applications. The CaP aquagels comprising various Ca/P ratios and CaP phases provide the unique ability to not only serve as biocompatible scaffolds but also matrices for binding plasmid DNA. In addition, these novel gels can be synthesized to contain nano-structured carriers of pDNA thus serving the multiple roles of biocompatible, bioresorbable, safe scaffolds and non-viral gene delivery systems.

The intellectual merits of the proposed study are the following. A new class of biocompatible CaP based composite aquagels will be synthesized exhibiting efficient non-viral gene transfection. The studies will transform the current status of CaP based non-viral gene delivery by obtaining a good understanding of the underlying molecular processes involved in synthesizing the composite gels. The proposed studies will also provide fundamental insight into the influence of nanoscale interaction of the binding, condensation and release of pDNA. The proposed studies will help tailor safe and effective non viral gene delivery agents matching efficiencies of polymeric counterparts. The broader impacts of the proposed activity are the following. The proposed research will advance the science and technology of CaP aquagel systems for non viral gene delivery. The studies will also pave the way for the identification and fabrication of new biocompatible CaP based aquagels that will exhibit characteristics similar to organic hydrogels. The existing North Carolina Agriculture and Technical University (NCA&T) collaboration through the newly funded Engineering Research Center (ERC) will offer an excellent opportunity for minority women and individuals from underrepresented groups to participate in the research activity. Moreover, local high school students will engage in hands on laboratory courses and a summer symposia. The PI has continuously integrated women and individuals of underrepresented minority groups into past NSF research programs and will continue to do so. Minority students from the Society for Women Engineers (SWE) will also be activity recruited to participate in the research program. Students will participate in summer symposia and the best paper presenters will be given the opportunity to participate and attend national society meetings. Results of the proposed research study will be published in leading peer reviewed scientific journals and will also be periodically presented at national and international conferences. Thus significant benefits to society can be envisaged since the proposed research could offer a feasible solution to the development of efficient non-viral gene delivery agents.

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