Objective： To investigate the feasibility of ultrasound （US） mediated enhanced green fluorescent protein （EGFP） gene delivery in subcutaneous transplanted tumors of human cervical carcinoma （He/a） and the contribution of lipid shell microbubble （LSMB） on gene transfection. Methods： LSMB and plasmid were injected into nude mice by tail vein followed local US irradiation （P ＋ LSMB ＋ US group）. US exposure parameter was set at 2.0 W/cm2, 2 rain, duty cycle 20%. EGFP expression was evaluated by imaging for 7 days. Nude mice undergoing plasmid injection alone （P group）, plasmid injection and US exposure （P ＋ US group）, plasmid and LSMB injection （P ＋ LSMB group） were used as controls. Frozen section and histological examinations were conducted. Expression of EGFP was scored. Kinetics of protein expression post transfection and localization in vivo were evaluated. Results： Plasmid injection with LSMB plus US exposure strongly increased gene transfer efficiency. Strong EGFP expression was mainly seen in LSMB ＋ P ＋ US group. It was significantly higher than any of the following groups, P group, US ＋ P group, or LSMB ＋ P group （P 〈 0.01）./n vivo expression level of post-US 3 days was significantly higher than any other time points （P 〈 0.01）. There was not significant expression level of EGFP in other organs or tissues regardless of US exposure. No tissue damage was seen histologically. Conclusion： The combination of LSMB and US exposure could effectively transfer plasmid DNA to transplanted tumors without causing any apparently adverse effect. LSMB could be effective as a non-viral vector system in in vivo gene delivery. It would be a safe gene delivery method and provide an alternative to current clinical gene therapy.
Zhiyi Chen, Mingxing Xie, Xinfang Wang, Qing Lv, Shangwei Ding Department of Ultrasonography, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology
Objective： To investigate the effect of different therapeutic ultrasound （TUS） parameters and culture conditions on the cell viability and transfection efficiency of human cervical cancer cells （HeLa）. Methods： HeLa cells were cultured using two different protocols （in suspension or in monolayer）. Subsequently, cells were exposed to different TUS intensity （0.4 W/cm^2, 1.0 W/cm^2, 1.6 W/cm^2, 2.2 W/cm^2）, duty cycle （DC）（10%, 20%, 50%）, exposure time （1 min or 3 min）. Cell viability was analyzed by flow cytometry. Gene transfection of red fluorescent protein （DsRED） was detected. Results： TUS intensity and duty cycle had a great impact on the overall results （P〈0.01）. Cell injury were found to increase progressively with intensity （1.6 W/cm^2, 2.2 W/cm^2） and duty cycle （50%） and cell detachment was accompanied by ultrasound exposure in adherent cells Results of factorial design showed that the fashion of cell culture and the TUS parameters had interaction （P〈0.01）. The ideal conditions that cell viability above 80% producing maximum efficiency were noted to be at 1.0 W/cm^2 irradiated 3 min with a duty cycle of 20% in cell suspension. Conclusion： TUS parameters and transfection conditions have a great impact on the gene transfection and cell viability. Optimal parameters could enhance cell inembrane permeability, which facilitate to delivering the macromolecules into cells.
