Encapsulation of NF-kappaB decoy oligonucleotides within echogenic liposomes and ultrasound-triggered release

Echogenic liposomes (ELIP) have additional promise, beyond diagnostic agents, as vehicles for delivering oligonucleotides (ODN), especially if the release of the agent can be triggered and its uptake can be enhanced by ultrasound application at a specific site. The purpose of this study was to co-encapsulate air and NF-kappaB decoy ODN within ELIP allowing ultrasound to release encapsulated ODN from ELIP, and to accurately quantify release of encapsulated ODN from ELIP upon ultrasound application. FITC-labeled sense ODN (2 mM) was incorporated within ELIP using freeze/thaw method. Encapsulation efficiency of FITC-ODN was spectrofluorometrically analyzed by quenching fluorescence of unencapsulated FITC-ODN using a complementary strand tagged with Iowa Black FQ-ODN. Quenching of FITC-ODN (0.05 microM) with Iowa Black FQ-ODN (0.1 microM) was found to be efficient (92.4+/-0.2%), allowing accurate determination of encapsulated ODN. Encapsulation efficiency of ODN was 14.2+/-2.5% in DPPC/DOPC/DPPG/CH liposomes and 29.6+/-1.5% in DPPC/DOPE/DPPG/CH liposomes. Application of ultrasound (1 MHz continuous wave, 0.26 MPa peak-to-peak pressure amplitude, 60s.) to the latter formulation triggered 41.6+/-4.3% release of ODN from ODN-containing ELIP. We have thus demonstrated that ODN can be encapsulated into ELIP and released efficiently upon ultrasound application. These findings suggest potential applications to gene therapy for atherosclerosis as well as a variety of other diseases.