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 1x108 MBs and by changing MB dose (1x107, 5x107, 1x108, or 5x108 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 1x108 MBs with no further significant increase with 5x108 MBs (P = 0.97). However, compared to neutral MBs, cationic MBs enhanced UMGD efficiency the most at low MB doses. Relative enhancement of UMGD efficiency using cationic over neutral MBs decreased from a factor of 27 for 1x107 MBs (P = 0.02) to a factor of 1.4 for 1x108 MBs (P < 0.01) and no significant difference for 5x108 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.

Development of therapeutic microbubbles for enhancing ultrasound-mediated gene delivery

“…Given that Definity MBs are already commonly used in clinical diagnostic US and in some therapeutic US research, we aim to improve gene delivery efficiency relative to this standard. Two custom-made MBs were designed to enhance their role as cavitation nuclei that facilitate sonoporation and DNA entry into cells: RN18 has a longer phospholipid acyl chain length of C18 than Definity (C16), which has shown superior stability due to increased shell cohesiveness [31], whereas RC5K has an added cationic surface charge that was hypothesized to further aid gene delivery by associating and protecting anionic genetic cargo by ways of electrostatic interaction [13, 34, 37, 39]. Although both types are similar to Definity in several basic features, including lipid composition, concentration, stability, and size, RC5K has a slightly lower concentration and stability, which is likely due to electrostatic lateral repulsion between cationic lipid head groups at submicrometer radii of curvature [46].…”

“…As the US intensity and MB dosage were increased, we found that transfection efficiency also increased significantly, consistent with results in the literature. [10–13, 15, 16, 47] However, higher intensities and MB dosages lead to more violent cavitations and energy release, which in turn may lead to wider perforations on the cell membranes. Presence of these holes along the membranes can then lead to increased DNA entry; but big holes (>5 μm) and too many perforations may lead to cell death[48].…”

“…Secondly, a cationic charge on the MB shell surface has several potential advantages. Recent studies have found that cationic MBs can electrostatically couple with anionic DNA, thus protecting it from premature degradation by nucleases while en route to the target location, as well as increasing the genetic payload in the vicinity of target cells, allowing amplified gene transfer once sonoporation is induced [13, 34, 37, 39]. The purpose of this study was to directly compare the effectiveness of the two customized MBs to that of Definity ® and further investigate parameters that can enhance the utility of neutral and cationic MBs in US-mediated gene delivery.…”

Monitoring/Imaging and Regenerative Agents for Enhancing Tissue Engineering Characterization and Therapies

“…4,79 Cationic particles carry higher payloads of nucleic acids and better prevent premature degradation compared to neutral particles. 136 In the case of drug delivery, loading is primarily restricted to the PFCp shell area since drugs have limited solubility in the hydrophobic and lipophobic PFC. 174 Therefore, drugs are loaded by conjugating them to the outer layer, loading them within the shell, or within an oil encapsulating layer.…”

Nucleic acid delivery with microbubbles and ultrasound

“…So, the plasmid or oligonucleotide should be placed on the microbubble surface, which is easily accomplished by electrostatic binding. Indeed, a direct comparison study has determined that microbubble-plasmid complexes provided significantly better transfection than co-administered plasmid and microbubbles incapable of complex formation [5, 28]. …”