Polycation-based gene carriers, though promising as safer alternatives to viral gene carriers, have been limited in large part due to their poor biological activities. Future design and development of better polycation gene carriers will be greatly facilitated by an improved understanding of the relationship between the polycation chemistry and performance mechanisms. Using combined state-of-the-art polymer synthesis and biological imaging techniques, the proposed research aims to address the gap that exists in our understanding of how the chemical and molecular properties of polycation carriers influence their biological activities and delivery performances in overcoming the two important cellular-level transport barriers, i.e., endosomal escape and nuclear import. Two associated specific aims will be addressed. First, the PI will investigate how the polycation molecular characteristics (in particular, the pKa of the amine group in the polycation, and the spacing between the amine groups along the polycation chain) impact the pathways and kinetics of the early intracellular trafficking processes (endocytosis and endosome escape); for this purpose, polycations with systematically varying chemical/molecular structures will be designed and synthesized, and the endosomal trafficking processes of the DNA complexes based on these polycations will be examined by confocal microscopy. Second, the PI will study the interrelationship between the exact subcellular location (cytosol or nucleus) of partial/complete DNA dissociation from the polycation complex and the nuclear import/transcription of the delivered DNA; for this study, a chemically cross-linked polycation carrier that is degradable upon exposure to mild UV irradiation will be developed and utilized to precisely control the timing and location of the DNA release.
The proposed research will use a unique combination of polymer science and cell biology methodologies, this research will provide useful information toward understanding (i) the exact chemical and molecular factors that promote the timely escape of polycation/DNA complexes from endosomes and (ii) the exact mechanisms by which certain polycations (such as polyethylenimine) so effectively enhance the nuclear localization and release/transcription of the delivered DNA.
A precise molecular-level understanding of these polycation chemistry vs. performance relationships will provide a fundamental basis for developing new materials and strategies for vastly improved efficiencies of non-viral gene delivery systems, and will therefore help further vitalize the gene therapy field toward realizing the full potential of the technology in both conventional and emerging areas of its applications. The proposed research will provide integrated training for graduate and undergraduate students in a multidisciplinary, collaborative and intellectually stimulating environment to learn skills necessary for the future generation of chemical and biological engineering researchers. Aspects of the proposed research will be used to enhance curricula in the area of nanomedicine.
