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Previously, we developed a gene delivery system using Bubble liposomes (perfluoropropane gas-entrapping liposomes; BLs) and ultrasound (US) [1].The combination of BLs and US can deliver plasmid DNA into the cytoplasm via the formation of transient membrane pores by cavitation. To enhance the efficiency of gene delivery, it is important to induce the cavitation of BLs at the vicinity of cells. In brief, cell-binding BLs would be useful for gene delivery to induce the cavitation on the cell membrane. In this study, we prepared the BL conjugated cyclic RGD (cRGD) peptides which could bind to tumor neovessel [2], and we investigated gene transfection efficiency for Human Umbilical Vein Endothelial Cells (HUVECs) with cRGD conjugated BLs (cRGD-BLs) and US. Binding assay: Fluorescence labeled cRGD-BLs were incubated with HUVECs at 4 °C. After 1 hr, the cells were washed and the fluorescence intensity was measured with flowcytometer. Gene delivery: Luciferase coded plasmid DNA (pCMVLuc) and either cRGD-BLs or BLs (60 μg/mL) were added to HUVECs.Then, US (2 MHz, 2.5 W/cm, 10 sec.) was exposed. After that, HUVECs were washed with PBS and incubated for 24 hr. Finally, luciferase expression was measured. cRGD-BLs effectively bound to HUVECs compared to non-targeted BLs. Luciferase expression in the group of treatment with cRGDBLs and US was higher than that with BLs and US (Fig. 1). This result suggested that cRGD-BLs effectively delivered pCMVLuc into HUVECs. Although the mechanism of enhancement for gene delivery efficiency is not clear, it is thought that many transient pores as a route of gene delivery might be opened by the cavitation of cRGD-BLs after binding on cell membrane. We found out that cRGD-BLs could enhance the gene delivery efficiency with US. Therefore, the combination of celltargeting BLs and US would be an effective gene delivery system.

Development of cell-targeting bubble liposome for effective gene delivery with ultrasound