Preparation And Characterization Of Targeted Microbubbles Carrying Eukaryotic Expression Vector Of Survivin SiRNA

ObjectiveDerivatives of polyethylene imine cholesterol connected eukaryoticexpression vector with siRNA about silencing human survivin gene to thecarrier lipid microbubbles.Targeted microbubbles modification withherceptin specifically binded to the surface of breast cancer cell throughHER-2, which would be a novel gene transfection tools for RNA interferencetherapy for breast cancer.Methods1. Immunofluorescence to detect HER-2on the surface of breast cancer cell;the lipid microbubbles were prepared by the method of mechanicaloscillation, and Herceptin was linked to the lipid microbubbles withBiotin-Avidin system. The form, particle size and distribution wereobserved by light microscope, and then specifically binded to SK-BR-3cell.2. Built recombinant plasmid pGPU6/GFP/Neo-survivin with hairpin siRNAtemplate targeting human survivin gene which was cloned into theeukaryotic expression vector pGPU6/GFP/Neo.Transformed therecombinant plasmid into competent cells, amplified and plasmids wereextracted.The recombinant plasmid was restriction enzyme digestionand sequencing.3. PEI-Chol liPoPolymer was synthesized by link in crolesterylchloroformate to the amino groups of branehed poly(ethylenimine)(PEI).The binding ability of eondense DNA and against RNaseI degrade withPEI-Chol were investigated by agarose gel eleetrophoresis.4. Microbubbles prepared by optimizing conditions were linked withmicrobubbles to be lipid bubble/PEI-Chol/DNA complexes. The form, particle size and distribution were observed by light microscope, andthen specifically binded to SK-BR-3cells.Results1. Immunofluorescence results show that the SK-BR-3cells fluoresce green,while the MDA-MB-231and no fluorescence can be combined withHerceptin.The prepared Trastuzumab loaded lipid microbubbles weresmooth, and the mean particle size was detected as (3.1±0.7) um, atthe same time specifically combined to SK-BR-3cells byHER2-Herceptin.2. The recombinant plasmid pGPU6/GFP/Neo-survivin were successfullyconstructed, which was proved by restriction enzyme and sequencingofnucleic acid.3. PEI graft cholesterol improved the ability of compression DNA andprovided with the ability to anti-enzyme.4. The lipid microbubbles contained siRNA eukaryotic expression vectorwere made successfully, which were smooth, and the mean particle sizewas detected as (3.8±0.9) um, and specifically combined to SK-BR-3cells.ConclusionPEI-Chol can be combined and protected of plasmid DNA, whichconstructed gene delivery system with neutral lipid microbubblessuccessfully.The lipid microbubbles contained siRNA eukaryoticexpression vector specific binding to SK-BR-3cells were preparedsuccessfully,which will may be a novel gene transfection tools for RNAinterference therapy for breast cancer.