Intellectual Merits: Electrotransfection, also known as electroporation or electric field-mediated DNA delivery, is a technology that has been widely used for introducing specific genes into cells. Its applications cover both basic research and clinical treatment and prevention of diseases. However, the technology has two obstacles that are currently limiting its further development. One is the lack of design principles for guiding development of optimal electrotransfection protocols; and the other is the poor understanding of mechanisms of plasmid DNA (pDNA) transport into cells during electrotransfection. As a result, most investigators in previous studies have to rely on the trial and error approach to protocol development, which requires measurement of electrotransfection efficiency (eTE) under different experimental conditions, in terms of electric pulse sequences and electrotransfection buffers, to identify an optimal set of conditions for each type of cells. This process is inefficient and can be expensive. Furthermore, some recent studies suggest that eTE is only partially dependent on electric pulse parameters. Other parameters, such as properties of cell membrane and pathways for intracellular transport, may be more crucial for determination of eTE. To this end, the goal of the proposed study is to investigate novel mechanisms of pDNA transport during electrotransfection. The central hypothesis is that adsorptive endocytosis is a key mechanism of electrotransfection for cellular uptake and intracellular transport of pDNA. To test the hypothesis, different types of mammalian cells will be treated with molecular inhibitors or enhancers, either prior to or post their exposure to pulsed electric fields. The treatments will alter electric field-induced binding between pDNA and cell membrane, or change activities in endocytic pathways. After each treatment, kinetics of cellular uptake and pDNA transport will be measured with confocal and electron microscopy techniques, and the kinetic data will be analyzed quantitatively using various mathematical models to determine mechanisms of cellular uptake and intracellular transport of pDNA.
Broader Impact: Improvement in electrotransfection technology can broadly impact basic biomedical research since the technology has been widely used for gene transfer into target cells. It may also impact clinical applications of gene therapy and DNA vaccination, which will lead to improvement in healthcare in both developed and developing countries because electrotransfection systems can be constructed inexpensively. The proposed study will show that the efficiency of gene/vaccine delivery can be improved through optimization of DNA attachment to cell membrane and enhancement of DNA transport via endocytic pathways. The research findings are crucial for development of novel design principles and strategies for improving the electrotransfection technology in future studies. In terms of education, results from the research project will be used to improve an existing biomedical engineering (BME) course on drug and gene delivery, currently taught by the PI at Duke University, and to develop a new elective course on intracellular transport of molecules and nanoparticles. In addition, the project will be used to train individual students to do research. Graduate students, including at least one minority student, will use the project as the basis of their thesis/dissertation. Undergraduate students will be recruited to the project through the independent studies and the Pratt Research Fellow program at Duke University. Furthermore, the PI will mentor underrepresented students in an outreach program. These students will be paired with graduate students to do summer research related to the proposed study.
