Optimized cationic lipid microbubbles for ultrasound-triggered gene delivery: formulation, characterization, and in vitro evaluation

The clinical efficacy of non-viral gene therapy is often limited by the low loading capacity of delivery vectors. To address this, we engineered an ultrasound-responsive cationic lipid microbubbles (CLMBs) platform from clinically accepted biocompatible components to maximize nucleic acid encapsulation. The system, featuring a C3F8 gas core, was optimized via thin-film hydration-mechanical agitation, with an optimized DSPC:DSPE-PEG2000:DPPC:Dc-Chol mass ratio of 3:1:3:6 proving optimal for adsorbing the plasmid pEGFP-N1. Physicochemical characterization confirmed nanobubbles with a zeta potential of (44.5 ± 1.8) mV and a mean diameter of (459.6 ± 27.1) nm, achieving a maximum plasmid-loading capacity of 31.30 µg per 106 CLMBs, corresponding to a milligram-scale payload of 1.13 mg of plasmid DNA per mL of CLMB suspension. Stability assessments demonstrated no plasmid leakage over nine days across various temperatures (4 °C–37 °C). Notably, while structurally stable at pH 7.0–7.4, the CLMBs showed partial plasmid release at pH 5.4, indicating acid-sensitive release behavior under an acidic endolysosomal-relevant condition. In vitro assays in HeLa cells confirmed excellent biosafety and a significantly superior transfection efficiency under ultrasound irradiation (1 MHz, 1.8 W/cm2, 20% duty cycle). This work advances high-payload gene delivery by providing a robust technical foundation for next-generation non-viral platforms. By combining intelligent stimulus-triggered release with exceptional stability and loading efficiency, this strategy enables precise, ultrasound-guided therapeutic interventions.