Construction Of Targeted Polymer Microbubble System For Gene Delivery And Its Correlation Property Research

ObjectiveTo prepare plasmid DNA-containing acoustic contrast agent encapsulating inert gases with high polymer material and targeting modify it.It aim to provide an therapeutic method by microbubbles(MB)with high stability for targeted gene therapy for breast cancer.It also provide a novel non-viral vector for gene transfection.Methods1.Immunofluorescence was used to identify HER-2 expression on the surface of the three breast cancer cell lines BT474,SK-BR-3 and MDA-MB-231.The recombinant plasmid pGPU6/GFP/Neo-survivin was obtained by alkaline lysis method,followed by analysis of its purification and concentration;Then X-tremeGENE HP DNA Transfection Reagent was used to in vitro transfect the three breast cancer cell lines,transfetion rate of them was compared as well.2.The DC-Chol/DNA complexes were prepared according to different +/-charge ratio,including 5:1,2:1,1:1,1:2 and 1:5.The binding ability of DC-Chol to plasmid DNA in the different charge ratio was analyzed by agarose gel eleetrophoresis.3.The polymer microbubbles encapsulating perfluoropentane were prepared by single emulsification method with mPEG-PLLA and DC-Chol as the shell material.DNA-PI was linked with MB via electrostatic interactions.Observation was made to analyse the form,density,particle size,distribution of MB and evaluate the ligation between MB and plasmid DNA.4.Film-forming material PLGA-PEG-COOH was prepared and then used for MB preparation as shell material together with mPEG-PLLA and DC-Chol.To be specific,perfluoropentane encapsulated polymer microbubbles were prepared firstly by single emulsification method,followed by ligation of PI labeled plasmid DNA to MB through electrostatic interactions.After carboxyl activation by carbodiimide method,the Herceptin was linked with MB.Then,the form,density,particle size and distribution of MB were observed by using optical microscope.Besides,the rate of MB connection with both plasmid and Herceptin,and the specific binding ability of Herceptin to HER-2 antigen was analyzed respectively.Results1.Immunofluorescence results showed that the BT474 cells can be combined With Bio-Herceptin and produce the strongest green fluorescence,followed by SK-BR-3 cells,but no fluorescence was found in MDA-MB-231 cells and controls.The recombinant plasmid can be used to transfection experiment based on its high-purity.The BT474 cells showed the highest transfection rate,followed by the MDA-MB-231 cells,then the SK-BR-3 cells after transfection by X-tremeGENE HP DNA Transfection Reagent.In addition,the expression of green fluorescence tend to increase over time.2.When the charge ratio was 2:1,the plasmid was totally loaded into DC-Chol.But when it was 1:1,the DNA ladder was shown.3.The polymer MB,on an average particle size of(3.2±1.2)?m and density of 4.23×108/ml,for carrying genes was well-dispersed.Fluorescence microscopic examination presented that red fluorescence was found on all the MB surface,indicating the MB carrying genes was well prepared.4.It's confirmed that PLGA-PEG-COOH was well prepared by NMR analysis.The targeted polymer MB carrying genes was well-dispersed,with an average particle size of(3.0±1.5)?m and the density of 8.8×107/ml.By coupling with antibody based on carbodiimide method,green fluorescence was observed on the MB surface with fluorescence microscope as compared to absent fluorescence in control groups,demonstrating that antibody was well linked with MB.Flow cytometry revealed that the MB with both green and red fluorescence accounted for 96.28%.It also showed that the targeted MB has a potential specific binding ability to HER-2(+)cells.Conclusion1.The BT474 cells,on which HER-2 antigen was highest expression and with the highest transfection efficiency,was an ideal subject for transfection research in vitro by ultrasound mediated the targeted polymer MB delivery system carrying genes.2.The genes carrying targeted polymer MB encapsulating perfluoropentane,with mPEG-PLLA,PLGA-PEG-COOH and DC-Chol as shell materials,was well prepared.The MB with high stability can be used to bind HER-2(+)cells specifically.It will provide a novel gene carrier for gene therapy in vitro for breast cancer.