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To achieve successful gene therapy, safe and efficient gene delivery vectors are needed. As an alternative to viral vectors, non-viral vectors, incorporating compounds such as cationic polymers and lipids have been widely studied. Much effort has been made to enhance transgene delivery efficiency, such as development of more effective cationic lipids or polymers, optimization of transfection formulations, and investigation on structural-activity of delivery vectors. Gemini surfactant, consisting of two surfactant monomers linked by a spacer group, is a thrust research area for gene therapy as non-viral vectors due to their high stability, longer storage on shelves, easiness to produce. A series of phytanyl substituted asymmetric gemini surfactants, phy-3-m (m = 12, 16, and 18) and phy-7NH-m (m = 12, 16, and 18), were rationally designed and synthesized. Due to the bulky nature and increased hydrophobicity of phytanyl branch, phy-3-m surfactants showed much lower values of critical micelle concentration (CMC) compared to their corresponding symmetric m-3-m. Particle size and transmission electron microscopy (TEM) imaging indicate that this type of gemini surfactants tends to form stacked bilayers rather than spherical or rod-like micelles which are typically observed in gemini surfactants with shorter spacers. Phy-3-m surfactants have higher degree of micelle ionization, indicating that the counter ions of the gemini surfactants can be easily replaced by other anionic ions, such as DNA, which is an advantage of phy-3-m used as transgene vectors. To evaluate transfection ability, transfection assays were carried out in OVCAR-3 cells. Transfection complexes formed by a plasmid pVGtelRL, coding enhanced green fluorescence protein (EGFP) gene, phy-3-m, and a neutral lipid, 1,2-Dioleyl-sn-glycerophosphatidylethanolamine (DOPE), at the charge ratios (+/-) of 2:1, 5:1, 10:1, and 20:1, were incubated with OVCAR-3 cells. Treated cells at all charge ratios except 20:1 showed EGFP signals under fluorescence microscopy. Meanwhile, EGFP expression and cell toxicity was quantified using fluorescence-activated cell sorting (FACS). For each gemini surfactant complex, the transfection efficiency and cytotoxicity go through a maximum, occurring at different values of the charge ratio. Considering both transfection efficiency and cytotoxicity, the optimal charge ratio to formulate the complexes containing phy-3-m was found to be 5:1 for in vitro transfection. Compared to a positive control, 16-3-16, phy-3-m showed higher transfection ability and lower cytotoxicity to OVCAR-3 cells. Initial characterization of transfection complexes was investigated by measuring particle size and zeta potential. At all charge ratios, transfection complexes were positively charged, and greater than +30 mV at 5:1 and 10:1, indicating that the complexes would be stable in solution at the ratio above 2:1. Transfection complexes were larger at lower charge ratio, but particle size dropped with increasing charge ratio (+/-). Comparing particle size and zeta potential with transfection efficiency, no correlation between size/zeta potential and transfection ability was observed. The larger particles may enter cells through caveolin-mediated pathway or phagocytosis, and smaller ones through a clathrin-mediated endocytosis. In addition, phase structures of the complexes were investigated using small angle X-ray scattering (SAXS). The complexes containing phy-3-m gemini surfactants were found to be able to adopt multiple phase structures, such as L, HII, and other highly ordered unidentified phase structures. By contrast, L structure was dominant in the transfection complexes formed by 16-3-16. The ability of phy-3-m system to adopt multiple phases appears correlated with their higher transfection efficiency in OVCAR-3 cells.

Including inherited genetic diseases, like lipoprotein lipase deficiency, and acquired diseases, such as cancer and HIV, gene therapy has the potential to treat or cure afflicted people by driving an affected cell to produce a therapeutic protein. Using primarily viral vectors, gene therapies are involved in a number of ongoing clinical trials and have already been approved by multiple international regulatory drug administrations for several diseases. However, viral vectors suffer from serious disadvantages including poor transduction of many cell types, immunogenicity, direct tissue toxicity and lack of targetability. Non-viral polymeric gene delivery vectors (polyplexes) provide an alternative solution but are limited by poor transfection efficiency and cytotoxicity. Microfluidic (MF) nano-precipitation is an emerging field in which researchers seek to tune the physicochemical properties of nanoparticles by controlling the flow regime during synthesis. Using this approach, several groups have demonstrated the successful production of enhanced polymeric gene delivery vectors. It has been shown that polyplexes created in the diffusive flow environment have a higher transfection efficiency and lower cytotoxicity. Other groups have demonstrated that charge-stabilizing polyplexes by sequentially adding polymers of alternating charges improves transfection efficiency and serum stability, also addressing major challenges to the clinical implementation of non-viral gene delivery vectors. To advance non-viral gene delivery towards clinical relevance, we have developed a microfluidic platform (MS) that produces conventional polyplexes with increased transfection efficiency and decreased toxicity and then extended this platform for the production of ternary polyplexes. This work involves first designing microfluidic devices using computational fluid dynamics (CFD), fabricating the devices, and validating the devices using fluorescence flow characterization and absorbance measurements of the resulting products. With an integrated separation mechanism, excess polyethylenimine (PEI) is removed from the outer regions of the stream leaving purified polyplexes that can go on to be used directly in transfections or be charge stabilized by addition of polyanions such as polyglutamic acid (PGA) for the creation of ternary polyplexes. Following the design portion of the research, the device was used to produce binary particle characterization was carried out and particle sizes, polydispersity and zeta potential of both conventional and MS polyplexes was compared. MS-produced polyplexes exhibited up to a 75% reduction in particle size compared to BM-produced polyplexes, while exhibiting little difference in zeta potential and polydispersity. A variety of standard biological assays were carried out to test the effects of the vectors on a variety of cell lines – and in this case the MS polyplexes proved to be both less toxic and have higher transfection efficiency in most cell lines. HeLa cells demonstrated the highest increase in transgene expression with a 150-fold increase when comparing to conventional bulk mixed polyplexes at the optimum formulation. A similar set of experiments were carried out with ternary polyplexes produced by the separation device. In this case it was shown that there were statistically significant increases in transfection efficiency for the MS-produced ternary polyplexes compared to BM-produced poyplexes, with a 23-fold increase in transfection activity at the optimum PEI/DNA ratio in MDAMB-231 cells. These MS-produced ternary polyplexes exhibited higher cell viability in many instances, a result that may be explained but the reduction in both free polymer and ghost particles.