Self-made Cationic Nanobubbles To Enhance Gene Delivery By Ultrasound In Vitro

Part Ⅰ Experimental studies on preparing of cationic nanobubble and evaluating its propertiesObjective A nanoscale cationic lipid ultrasound contrast agent was prepared to evaluate its physical and chemical properties and stability,evaluate its maging effects both,in vitro and in vivo,to verify its passive targeting effect on tumor and evaluate its toxicity both in vivo and in vitro.Methods Film hydration method was used,by mixing stearic acid modified polyethylenimine600 with the lipidic materials to prepare PEI600 cationic nanobubbles,microscope was used to observe its morphology and calcμlate the concentrations,surface potential,particle size was measure by Zetasizer Nano particle and potential instrument,to investigate the variation of concentrations and particle size with time,verifing the surface positive charge of the of the nanobubble by cell adhesion experiment,evaluate its maging effects both in vitro and in vivo,Immunofluorescence staining was taken to observe the passive targeting of tumor,CCK8 assay was used to evaluate its toxicity in vitro,After injection of cationic nanobubbles through the tail vein,the toxicity of SD rats in vivo was observed by tissue section.Resμlts The cationic nanobubble has a good stability,the average particle size of the nanobubble is 484.8 ± 8.5 nm,and the average surface potential is 24.78 ± 3.52 m V.The average particle diameter and concentration was not significantly changed in one week,the cationic nanobubble demonstrate great contrast effect in vitro,constrast enhancement in vivo show that cationic nanobubble can significantly enhance the heart and subcutaneous transplantation tumor,imaging in heart can last for more than 20 min.Conclusion The zeta potential of self-made nano bubble is positive and has good particle size and concentration stability;(2)The self-made cationic nano bubbles have good acoustic character;(3)The self-made cationic nanobubbles have good passive targeting ability of tumor.;(4)The self-made cationic nanobubble has low toxicity and is a safe ultrasound contrast agent.Part Ⅱ Enhanced gene transfection to MCF-7 cells mediated by Stearic-PEI600 cationic nanobubblesObjective The in vitro gene loading capacity and gene carrying efficiency of cationic nanbubbles were measured,and the ability of anti DNA enzyme digestion after cationic nanobubble DNA binding was evaluated.The best acoustic condition of μltrasound combined with cationic nanobubble explosion for MCF-7 cells transfection was explored.Methods Microplate reader was used to measure the gene loading capacity of nanobubbles in vitro,Flow cytometric wasused to measure gene efficiency of nanobubbles in vitro,gel electrophoresis experiment was taken to elevate the ability of cationic nanobubble protecting the DNA from digesting by DNAseⅠ,the method of transfection experiment is as follows: It is divided into 6 groups:(1)Simple plasmid group;(2)plasmid + ultrasonic irradiation group;(3)plasmid + nanobubble group;(4)plasmid +cationic nanobubble group;(5)plasmid + ultrasonic irradiation + nanobubble group;(6)plasmid + ultrasonic irradiation + cationic nanobubble group;the transfection efficiency of each group was compared.The expression of green fluorescent protein(EGFP)was observed by fluorescence microscope,and the gene transfection rate was measured by flow cytometry.Resμlts Cationic nanbubbles can effectively combine DNA with electrostatic interaction,while reducing DNA enzyme digestion.Cationic nanbubbles can enhance gene transfection efficiency of MCF-7 cells,which is significantly higher than that of normal nanobubbleConclusion The self-made cationic nanofole is a good carrier for carrying genes,which can protect the DNA and can significantly improve the transfection efficiency.