Ultrasound-mediated gene delivery with cationic versus neutral microbubbles: Effect of DNA and microbubble dose on In Vivo transfection efficiency

Abstract

Objective: To assess the effect of varying microbubble (MB) and DNA doses on the overall and comparative efficiencies of ultrasound (US)-mediated gene delivery (UMGD) to murine hindlimb skeletal muscle using cationic versus neutral MBs. Materials and Methods: Cationic and control neutral MBs were characterized for size, charge, plasmid DNA binding, and ability to protect DNA against endonuclease degradation. UMGD of a codon optimized firefly luciferase (Fluc) reporter plasmid to endothelial cells (1 MHz, 1 W/cm2, 20% duty cycle, 1 min) was performed in cell culture using cationic, neutral, or no MBs. In vivo UMGD to mouse hindlimb muscle was performed by insonation (1 MHz, 2 W/cm2, 50% duty cycle, 5 min) after intravenous administration of Fluc combined with cationic, neutral, or no MBs. Gene delivery efficiency was assessed by serial in vivo bioluminescence imaging. Efficiency of in vivo UMGD with cationic versus neutral MBs was systematically evaluated by varying plasmid DNA dose (10, 17.5, 25, 37.5, and 50 μg) while maintaining a constant MB dose of 1×108 MBs and by changing MB dose (1×107, 5×107, 1×108, or 5×108 MBs) while keeping a constant DNA dose of 50 μg. Results: Cationic and size-matched control neutral MBs differed significantly in zeta potential with cationic MBs being able to bind plasmid DNA (binding capacity of 0.03 pg/MB) and partially protect DNA from nuclease degradation while neutral MBs could not. Cationic MBs enhanced UMGD compared to neutral MBs as well as no MB and no US controls both in cell culture (P < 0.001) and in vivo (P < 0.05). Regardless of MB type, in vivo UMGD efficiency increased dose-dependently with DNA dose and showed overall maximum transfection with 50 μg DNA. However, there was an inverse correlation (ρ = -0.90; P = 0.02) between DNA dose and the degree of enhanced UMGD efficiency observed with using cationic MBs instead of neutral MBs. The delivery efficiency advantage associated with cationic MBs was most prominent at the lowest investigated DNA dose (7.5-fold increase with cationic versus neutral MBs at a DNA dose of 10 μg; P = 0.02) compared to only a 1.4-fold increase at a DNA dose of 50 μg (P < 0.01). With increasing MB dose, overall in vivo UMGD efficiency increased dose-dependently with a maximum reached at a dose of 1×108 MBs with no further significant increase with 5×108 MBs (P = 0.97). However, compared to neutral MBs, cationic MBs enhanced UMGD efficiency the most at low MB doses. Relative enhancement of UMGD effi-ciency using cationic over neutral MBs decreased from a factor of 27 for 1×107 MBs (P = 0.02) to a factor of 1.4 for 1×108 MBs (P < 0.01) and no significant difference for 5×108 MBs. Conclusions: Cationic MBs enhance UMGD to mouse skeletal muscle relative to neutral MBs but this is dependent on MB and DNA dose. The enhancement effect of cationic MBs on UMGD efficiency is more evident when lower doses of MBs or DNA are used, whereas the advantage of cationic MBs over neutral MBs is substantially reduced in the presence of excess MBs or DNA. © Ivyspring International Publisher.