626. Ultrasound-Triggered/Enhanced Delivery of Oligonucleotides into Aorta

Abstract

Synthesized oligonucleotides (ODNs) are used in anti-sense and anti-gene technology to control gene expression. As tissues and cells do not easily take up ODNs, the development of inexpensive and effective approaches to transiently decrease gene expression in vivo would be useful clinically. These data demonstrate that ultrasound can release ODNs from acoustically-active cationic liposomes and increase the delivery of ODNs into cultured rat aorta tissue. Cationic liposomes were made using the component lipids 1, 2-Myristoyl-sn-glycero-3-ethylphosphocholine(EDMPC), phosphotidylcholine and cholesterol (CH). Liposomes were made acoustically-active by sonication in water, addition of mannitol, freezing, lyophilization, and rehydration. Acoustically-active lipoplexes were made by mixing ODNs with acoustically-active liposomes in a ratio of 1:2. Ultrasound triggered-ODN release was measured as follows: acoustically-active liposomes/FITC-ODNs complex were incorporated with cultured cells, ultrasound (1 MHz, 2W/cm2,10% duty cycle for 10 seconds) was applied after washing out all free ODNs and unattached lipoplexes. The release of ODNs into solution was measured before and after the ultrasound application. ODN release was calculated by subtracting the fluorescence of the background media from the cellular fluorescence. One application of ultrasound caused a 45% release of attached FITC-ODNs from cationic liposomes. Nuclear localization of FITC-ODNs can be seen immediately after ultrasound application in about 3% of the cells. This delivery method was also tested on arterial tissue using freshly-removed rat aorta. Acoustically-active lipoplexes were injected into the endothelium of the aorta in a perfusion system with 20 cm H2O pressure. Ultrasound (1 MHz, 2W/cm2,100% duty cycle) was applied to the aorta for 60 seconds. Aortas were then washed and frozen-sectioned. The distribution of FITC-ODNs was assessed by fluorescent microscopy. Ultrasound enhanced the transport of ODNs through endothelium and the FITC-ODNs was seen extending into the arterial media. These results suggest that ultrasound can both increase the release of nucleic acid from acoustically-active liposome complexes as well as increase the penetration of oligonucleotides into a complex tissue such as arterial muscle. These data indicate that therapeutic transfer using these methods is possible. (See Figure)