| dc.description.abstract | Gene therapy holds great promise to treat a variety of human diseases, including diabetes. The development of a new gene delivery system called Ultrasound Targeted Microbubble Destruction (UTMD) has been developed to enhance in vivo gene delivery using non-viral vectors. A new family of cationic lipids called gemini surfactants have been synthesized for their use as gene carriers due to their small particle size, increased surface charge, superior surface binding capabilities, reduced toxicity, and economic advantages, resulting in their increased safety for in vivo application. Gemini surfactants have not been evaluated with UTMD. The purpose of this study was to assess their transfection capabilities as microbubble gene carriers.
First, gemini surfactants 16-3-16 and 16-7NH-16 were assessed and compared to three commercially used monovalent cationic lipids: 1) Lipofectamine 2000, 2) Fugene, and 3) DOTAP microbubbles. Colloidal stability was assessed using dynamic light scattering to measure size and electrophoresis for charge. Gemini surfactants 16-3-16 and 16-7NH-16 displayed a small particle size of 2.33 µm (16-3-16), 0.74 µm (16-7NH-16) and increased surface charge of +34.8 mV (16-3-16), +37.74 mV (16-7NH-16) when compared to commercially used monovalent cationic lipid Lipofectamine 2000 (size 3.9 µm and charge -6.2 mV). Gemini surfactant 16-7NH-16 was chosen for further investigation due to its reduced particle size and increased surface charge and was investigated for its DNA binding and release capabilities using UTMD though gel electrophoresis. Gel electrophoresis analysis determined gemini surfactant 16-7NH-16 was capable of fully binding 25 µg of pAMAXA plasmid GFP DNA, but not able to release DNA when exposed to ultrasound. Transfection efficiency of gemini surfactant 16-7NH-16 microbubbles with 25 µg of pAMAXA plasmid GFP DNA was additionally investigated in HEK 293 (human embryonic kidney cells) and INS-1 832/13 cells (rat insulinoma cells) using UTMD, and determined with florescent microscopy. Gemini surfactant 16-7NH-16 microbubbles resulted in a 5 % transfection rate and a 90 % death rate in vitro when exposed to UTMD. Commercially used monovalent cationic lipid microbubbles exhibited higher transfection rates and lower death rates overall in vitro when compared to gemini surfactant 16-7NH-16 microbubbles and exposed to UTMD. (Lipofectamine 2000 70 % transfection rate, 5 % death rate, Fugene 70 % transfection rate, 5 % death rate, DOTAP 30 % transfection rate, 90 % death rate). Cationic lipid microbubbles most influenced in vitro using the UTMD technique for transfection was DOTAP in combination with neutral lipids (DOTAP + neutral lipids). Transfection rates of DOTAP + neutral lipids increased as ultrasound intensities increased (10 % transfection at intensity 1.5 W/cm2 and 30 % transfection at intensity 2.0 W/cm2), demonstrating a direct correlation. In vitro gene delivery demonstrated gemini surfactant 16-7NH-16 microbubbles did not significantly enhance transfection capabilities when incorporated with the UTMD technique when compared to commercially used monovalent cationic lipids. Cationic lipid microbubble most influenced by ultrasound was DOTAP + neural lipid microbubbles when compared to all experimental groups tested with the UTMD technique.
These results indicate that gemini surfactants, a new family of cationic lipids could not significantly enhance cellular transfection rates in vitro with the incorporation of the UTMD technique; despite their small particle size, increased surface charge, superior surface binding capabilities and economic advantage for in vivo application when compared to commercially used cationic monovalent lipids.
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