Study On The Preparation Of Liposomal Nanobubbles And Its Ultrasound Contrast Enhancement And Induced Gene Transfection

Objective In this experiment,we prepared perfluoropropane-filled nanobubbles to investigated its physical characteristics,transfection efficiency and the ability of contrast-enhanced ultrasound imaging.We prepared liquid-gas phase transition nanoemulsions to explore the conditions of phase transition,so as to provide reference for the subsequent preparation of targeted liquid-gas phase transition nano-emulsions.Methods In vitro,the perfluoropropane-filled nanobubbles were prepared by the method of thin-film hydration.The morphology,distribution,average diameter and surface potential were detected by the Dynamic light scattering(DLS).Next,we delivered the EGFP plasmid DNA into HepG2 human liver cancer cells with ultrasound irradiation and nanobubbles.The expression of green fluorescent protein in HepG2 cells was observed by inverted fluorescence microscope,and the transfection efficiency was determined by flow cytometry.The cells activity and apoptosis were tested by Cell Counting Kit-8 and Annexin V-FITC Apoptosis Detection Kit respectively.Finally,we diluted the nanobubbles and commercial contrast agent(SonoVue)in equal proportion and observed the capability of contrast-enhanced ultrasound(CEUS)in vitro and in vivo and analyzed the effect of contrast enhancement by time-intensity curve(TIC).Perfluoropentane-filled nanoemulsions were prepared by ultrasonic emulsification.The morphology,distribution,average diameter,and surface potential of the nanoemulsions were measured.The stability and liquid-gas phase transition conditions of the nanoemulsions were observed and evaluated by heating.Results(1)The perfluoropropane-filled nanobubbles we prepared were uniformly spherical particles without obvious aggregation under the light microscope and transmission electron microscope.(2)The concentration of perfluoropropane-filled nanobubbles was about 1.5-3.2×108/ml,the average diameter was about(291.3±5.3)nm and the surface potential was(-1.42-3.05 mv).(3)Compared with SonoVue,there was no significant difference in the beginning of enhanced time and enhanced peak time（P>0.05),but the enhanced peak intensity was significantly lower than SonoVue and the fading time was earlier than SonoVue through TIC analysis(P<0.05).(4)The transfection efficiency was significantly improved in nanobubbles and ultrasound irradiation group compared with pure ultrasound irradiation group(P<0.05).The cell apoptosis rate increased after the treatment of ultrasound(P<0.05).(5)The liquid perfluoropentane-filled nanoemulsion we developed were uniformly spherical particles.(6)The average diameter of nanoemulsion was about(168.0±20.0 nm),the surface potential was about(-4.82-4.19 mv)before heating.However,the average diameter and the surface potential were risen to(303.1±10.3 nm)and(-5.9-4.98 mv)respectively in 50℃ water bath,besides the average diameter and the surface potential were risen to(592.6±31.2 nm)and(3.73-5.20 mv)respectively in 80℃ water bath.The concentration and side scattering intensities were 2.1×1011/ml,1.7×1011/ml,1.5×1011/ml and 1614.13,3235.94,6456.54 respectively by the high sensitivity flow cytometry.(7)The intensity of contrast-enhanced imaging was significantly increased at 80℃ water bath compared with 50℃ water bath in vitro.Conclusion Perfluoropropane-filled nanobubbles had the ability of contrastenhanced imaging and could promote the effect of genes delivery with ultrasound irradiation.The stability of nanoemulsions was better and intensity of contrast-enhanced imaging increased apparently after 80℃ water bath.