483. Acoustically Active Liposomes for Oligonucleotide/Drug Encapsulation; Delivery Agents with Capacity for Ultrasound-Triggered Release

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Liposomes can carry many different kinds of therapeutic agents. Acoustically active liposomes (AAL), which contain small amounts of air within their bilayers, were previously developed by our group. These AAL also have the potential to carry oligonucleotides and their acoustic activity could enable them to respond to local ultrasound stimulation by releasing their contents at a disease site. Liposomes were composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and cholesterol at a molecular ratio of 69:8:8:15 and made acoustically active with a procedure involving hydrating the lipid film, sonication, freezing in the presence of mannitol, lyophilization, and rehydration. A FITC-labeled oligonuleotide (ODN, 12 bp) was added at the hydration step. Encapsulation efficiency was measured after Sepharose 4B chromotography. For other encapsuation as well as for release measurements, calcein was used as a test molecule because it is a convenient fluorescent probe for uptake and leakage determinations. The procedure of freeze-drying in the presence of mannitol has been found to be a superior method for oligonucleotide encapsulation. Approximally 25% encapsulation was obtained with one freeze-thaw cycle (Figure 1), which is similar to the encapsulation of calcein (20%). One MHz ultrasound at 2 W/cm2, 100% duty cycle, was applied to loaded, acoustically active liposomes for 10s intervals and caused 32% release of calcein. Six such applications released 62% of original contents. Release of contents was highly correlated with the loss of air (Figure 2) induced either by ultrasound or rapid pressure reduction, indicating that pressure-induced stress on liposome-associated air pockets is responsible for ultrasound-triggered drug release. This encapsulation and release technique should also be applicable to siRNA's, since recent research has found that delivery methods developed for ODN are also efficient for siRNA. The ultrasound-sensitive liposomes provides the distinct advantage of allowing control and localization of the release of nucleotide-based and other hydrophilic therapeutic agents. Furthermore, the destruction of microbubbles may facilitate nucleotide transport across biological membranes. Ultrasound image enhancement by these liposomes may also facilitate identification of regions of disease, allowing further improvements in site-specific delivery.