Construction Of ICAM-1Targeted Microbubble With Angiopoietin-1and Transient Transfection Of Angiopoietin-1into Vascular Endothelial Cells By Ultrasound Combined With Microbubble

Part1Construction of ICAM-1targeted microbubble with angiopoietin-1geneObjective To construct a ICAM-1targeted microbubble containing angiopoietin-1(Ang-1) gene and to develop a novel approach of transfecting therapeutic gene effectively in293cells mediated by ultrasound-mediated microbubble destruction.Methods Subclone a recombinant plasmid containing angiopoietin-1(Ang-1) gene from a adeno-associated virus containing Ang-1gene.Plasmids encoding green fluorescent protein and Ang-1were mixed with SonoVue microbubbles.293T cells suspension of six orifice plates were divided into4groups by different ways:group A (the contrast group)was given lOug Ang-1plasmid only, group B(the plasmid-ultrasound group)was given lOug Ang-1plasmid, group C(the plasmid-microbubbles-ultrasound group) given mixture including10ug Ang-1plasmid and200ug SonoVue microbubbles, group D(the plasmid—liposome group)was was given mixture including4ug liposome and lOug Ang-1plasmid. Ultrasound exposure was administrated in group B and group C (1.5W/cm2) for30s.Forty-eight hours later, the transient expression rate was observed by fluorescence microscopy and flow cytometry. The ICAM-1targeted microbubbles were prepared by conjugating the SonoVue microbubbles and anti-ICAM-1antibody labeled by EGFP in pH value4.0-5.0or8.0-9.0by ratio2:1.The combination was proved by fluorescence microscope, and the combination effect was surveyed by flow cytometry quantitively.Results Fluorescence microscopy observed green fluorescence in group A, B, C and D. The transfection efficiency was(0.26±0.16)%in the group A,(1.38±0.22)%in the group B,(15.17±2.32)%in the group C and(7.17±2.64)%in the group D, respectively. Fluorescence microscope observed the green Fluorescene in the shell of microbubbles, indicating the combination of anti-ICAM-1antibody labeled by EGFP and microbubbles. Flow cytometry results were (54.97±9.51)%in pH value4.0-5.0and (3.48±1.53)%in pH value8.0-9.0.Conclusion Ultrasound can encourage gene transfection in cells, whereas microbubbles combined with ultrasound exposure can improve thetransfection efficiency obviously. ICAM-1-targeted microbubbles can be prepared successfully with high combinant efficiency. Part2Transient transfection of angiopoietin-1into vascular endothelial cells by ultrasound combined with microbubblesObjective To investigate the effects of transient transfection of angiopoient-1(Ang-1) into vascular endothelial cells CRL1730,and the effects for cell migration.Methods cells were divided into four groups:control group(neither Ang-1nor microbubbles), Ang-1group(added with Ang-1only), microbubble group(added with microbubble only), ultrasound group(both Ang-1and microbubble, with ultrasound irradiation), all cultivated for24hours.The Ang-1mRNA and protein levels were measured by RT-PCR and western-blot methods, and cell migraton ability were detected by transwell method.Results There were no difference between the control group, Ang-1group, microbubble group in Ang-1mRNA and protein levels.but the target gene expression was increased by exposed to1MHz ultrasound for30S in ultrasound group after24hours (p<0.05).Moreover cell migration ability is also enhanced in ultrasound group.Conclusion Combination of ultrasound and microbubbles might successfully transient transfect Ang-1gene into vascular endothelial cells, the transfected cells migration ability was enhanced also.