Ultrasound Combined With Cationic Microbubble Loading Gene Enhanced Gene Transfection In Vitro

Objective To prepare a new type of self-made cationic lipidultrasound microbubble. To assess its physical and chemical properties, highgene loading ability and its enhancement effects on contrast imaging in vivo.Methods Cationic lipid ultrasound microbubble was prepared byfilmy dispersion and mechanical oscillation with DC-cholesterol, andmodified by PEG. Its appearance, distribution, zeta potential, stability weredetected; and its mean diameter, concentration and zeta potential wereobserved during two weeks at different points(6h,12h,24h,2d,7d,14d);resistance ability of DNAseI,toxicity,gene loading ability and enhancementeffects on contrast imaging of microbubble were tested. All the character-istics were compared with those of the common microbubbles.Results The mean diameter of cationic lipid microbubble varied from 1201.4±0.47to1405.3±0.25nm, and concentration varied from（4.13±0.13）×108/ml to（3.85±0.05）×108/ml during two weeks; the meanzeta potential was (29.02±1.30) mV. The maximum ratio and amount ofloading gene of microbubble was39.7%and4g/108microbubble (MB)respectively. Contrast imaging and agarose gel electrophoresis showed thatthe loaded gene cationic microbubble could significantly enhance echointensity of rabbit liver and resist DNAseI degradation.Conclusions The self-made cationic microbubble as an idealultrasound contrast agent could improve gene loading ability and protectDNA from nuclease. It may hopefully improve the transfection efficiency totargeted cells and become a promising material for gene vector. Objective To optimize the parameters of ultrasound mediatedmicrobubble destruction (UMMD) for gene transfection.To observe theefficiceny of UMMD enhanced gene transfer with self-made cationicmicrobubble, and compare with common lipid microbubble and liposome2000, to analysis the cell survival rate after treatment.Methods Find the best combination of ultrasound intensity and microbubble concentration according to cell survival rate. Microbubble,liposome and DNA with HUVEC were divided into six groups:①plasmidonly;②plasmid and ultrasound;③plasmid and cationic microbubble;④plasmid,common microbubble and ultrasound;⑤plasmid and liposome;⑥plasmid, cationic microbubble and ultrasound. Cell viability andtransfection efficiency of different concentration microbubbles wereassessed together in Group⑥; transfection efficiceny of HEK293cells in④⑤⑥groups were compared with HUVEC. Expression of the reportgene EGFP was observed on fluorescent microscopy and quantified byFACS analysis. The cell viability was measured using MTT assayResults The best combination of Ultrasound intensity andmicrobubble concentration was0.5W/cm2,3108/ml. UMMD enhancedthe gene transfer HUVEC, which was significantly different fromnoncationic microbubble, and significantlly different from liposome2000inHEK293cell.Decrease in viability of HUVEC was observed with increasingMB concentration of group⑥.For achieving effecttive transfection,microbubble concentration between (2.5～5.0)×108/ml was better.Conclusion Cationic microbubble enhance the efficiency of HUVECgene transfer, which is much higher than common microbubble. By findingbest parameters and seeking high transfection, more cells death should beavoided.