The Fundamental Study Of Self-made Nano-liposomal Bubbles As An Ultrasound Contrast Agent And A Transgene Media

Part1Ultrasound-mediated Microbubble DestructionAccompanied with Cationic Liposome Enhanced GeneTransfection in VitroObjective To investigate whether ultrasound-mediated microbubble destruction(UMD) could enhance cationic liposome (CL) induced plasmid DNA delivery or not,and optimize the transfection conditions.Methods Multiple parameters were explored to obtain the optimal transgeneefficiency by means of with or without serum in culture medium, various CL ornano-liposomal bubble(NB) concentrations, different time points of ultrasonicirradiation. The transfection efficiency was assessed by fluorescence microscopy andflow cytometer, and cell viability was evaluated by Cell Counting Kit-8(CCK-8)assay. Results The serum could protect the cells but show little impact on transfectionefficiency induced by CL. CL and plasmid DNA at a weight ratio of4:1exhibitedhigh transfection efficiency of (17.71±0.79)%and high cell viability of (91.28±0.76)%. CL combining with ultrasonic irradiation at the time point of1hour couldincrease the transfection efficiency to (24.85±0.78)%(P<0.01). Higher transfectionrate (32.47±4.01)%was obtained by adding NB at the concentration of10%(P<0.05).Conclusion UMD accompanied with CL could enhance gene delivery effectively,which would provide a new method for gene therapy. Part2Technological Optimization of the Nano-liposomalBubbles and Assessment of Their Ultrasound Contrast Abilityand Transgene EffectObjective To compare the imaging and gene transfer effects of self-madenano-liposomal bubbles(NB) with commercial ultrasound contrast agent, SonoVue.Methods PEG-liposomes were placed in vials supercharged with perfluoropropanegas, then sonicated in two different methods to prepare NB. One was sonicated in abath sonicator(Ⅰ), and the other was sonicated with a probe sonicator(Ⅱ). Threenormal rabbits and three rabbits implanted VX2tumors in the livers were included in this study. After the contrast agents (SonoVue, NBⅠor NBⅡ) injected, the imagesand videos were acquired with the contrast mode ultrasonography, then recorded andanalyzed. Moreover, the effects of the different contrast agents onultrasound-mediated gene transfer in HepG2cells were compared. The transfectionefficiency was assessed by fluorescence microscopy and flow cytometer, and cellviability was evaluated by Cell Counting Kit-8(CCK-8) assay.Results After the contrast agents injected, the echo enhancement durations of NBⅠand NBⅡ in the rabbit liver parenchyma were longer than SonoVue(P<0.01), andthe peak intensity of NBⅡ was higher than Sonovue or NBⅠ(P<0.05). Theperipheral regions of VX2tumor was ring-shaped high enhancement in arterial phase,and no enhancement in the internal of VX2tumor in parenchymal phase while thenormal liver parenchyma was high enhancement. At the concentration of10%, NBⅡcombined with ultrasound exposure induced gene transfection rate at (12.54±2.13)%,which was higher than the other groups(P<0.05).Conclusion The NBⅡ shows advantage comparing with NBⅠand SonoVue, nomatter in ultrasound contrast imaging or transgene. Therefore, the self-made NBⅡcould be used as a potential agent for ultrasound molecular imaging and targeted geneor drug delivery in the future.