Purpose: Previously, we developed lipid bubbles (LBs) which were prepared from liposomes, as an ultrasound (US) imaging agent. We found out that cyclic RGD peptides (cRGD) conjugated LBs (cRGD-LBs) accumulated at the site of blood clots and the tumor vessels. Therefore, we expected that these LBs could be useful as active-targeting US contrast agents for diagnostics of blood clot and tumor. In addition, we have studed gene delivery using the combination of LBs and US. Utilizing this delivery system, plasmid DNA was delivered into the cytoplasm of cells via the formation of transient pores in the membrane due to bubble cavitation. To enhance the efficiency of gene delivery, it is important to induce the cavitation of LBs in the vicinity of the target cells. In brief, cell-targeting LBs could be useful for gene delivery by inducing bubble cavitation at the cell membrane. In this study, we prepared cRGD-LBs which could bind to tumor endothelial cells. We investigated the gene transfection efficiency for Human Umbilical Vein Endothelial Cells (HUVECs) as a neovessel model in vitro using cRGD-LBs and US.
Methods: Binding assay: Fluorescence labeled cRGD-LBs were incubated with HUVECs at 4 °C. After 1 hr, the cells were washed and the fluorescence intensity of the cells was measured with flowcytometer. Gene delivery: Luciferase coded plasmid DNA (pCMVLuc) and either cRGD-LBs or LBs (60 μg/mL) were added to HUVECs. Then, US (2 MHz, 2.5 W/cm2, 10 sec.) was insonated on the cells. After that, HUVECs were washed with PBS and incubated for 24 hr. Finally, luciferase expression was measured.
Results: cRGD-LBs effectively bound to HUVECs compared with non-targeted LBs. Luciferase expression in the group treated with pCMVLuc, cRGD-LBs and US was higher than that with pCMVLuc, LBs and US. This result suggests that cRGD-LBs effectively delivered pCMVLuc into HUVECs. Although the mechanism of enhancement for gene delivery efficiency is not clear, it is thought that many transient pores opened in the cell membranes by the cavitation of cRGD-LBs after binding to the cell membranes. We found out that cRGD-LBs could enhance the gene delivery efficiency with US.
Conclusion: cRGD-LBs could bind to target cells and result in delivering plasmid DNA into cells by using US. Therefore, the combination of cell-targeting LBs and US could be an effective gene delivery system.
Daiki Omata– Faculty of Pharma-Science, Teikyo University
Johan Unga– Faculty of Pharma-Science, Teikyo University
Lisa Munakata– B.A., Teikyo University, Tokyo
Tadamitsu Shima– Faculty of Pharma-Science, Teikyo University, Tokyo
Saori Kageyama– Faculty of Pharma-Science, Teikyo University, Tokyo
Mostafa Soleyman– Faculty of Pharma-Science, Teikyo University, School of Pharmacy, Utrecht University, Tokyo
Kazuo Maruyama– Faculty of Pharma-Science, Teikyo University, Tokyo