Influence of DNA-Microbubble Coupling on Contrast Ultrasound–Mediated Gene Transfection in Muscle and Liver

Background Contrast ultrasound–mediated gene delivery (CUMGD) is a promising approach for enhancing gene therapy that relies on microbubble (MB) cavitation to augment complementary deoxyribonucleic acid (cDNA) transfection. The aims of this study were to determine optimal conditions for charge-coupling cDNA to MBs and to evaluate the advantages of surface loading for gene transfection in muscle and liver. Methods Charge coupling of fluorescently labeled cDNA to either neutral MBs (MB_N) or cationic MBs (MB₊) in low- to high-ionic conditions (0.3%-1.8% NaCl) was assessed by flow cytometry. MB aggregation from cDNA coupling was determined by electrozone sensing. Tissue transfection of luciferase in murine hindlimb skeletal muscle and liver was made by CUMGD with MB_N or MB₊ combined with subsaturated, saturated, or supersaturated cDNA concentrations (2.5, 50, and 200 μg/10⁸ MBs). Results Charge-coupling of cDNA was detected for MB₊ but not MB_N. Coupling occurred over almost the entire range of ionic conditions, with a peak at 1.2% NaCl, although electrostatic interference occurred at >1.5% NaCl. DNA-mediated aggregation of MB₊ was observed at ≤0.6% NaCl but did not reduce the ability to produce inertial cavitation. Transfection with CUMGD in muscle and liver was low for both MBs at subsaturation concentrations. In muscle, higher cDNA concentrations produced a 10-fold higher degree of transfection with MB₊, which was approximately fivefold higher (P < .05) than that for MB_N. There was no effect of DNA supersaturation. The same pattern was seen for liver except that supersaturation further increased transfection with MB_N equal to that of MB₊. Conclusions Efficient charge-coupling of cDNA to MB₊ but not MB_N occurs over a relatively wide range of ionic conditions without aggregation. Transfection with CUMGD is much more efficient with charge-coupling of cDNA to MBs and is not affected by supersaturation except in the liver, which is specialized for macromolecular and cDNA uptake.