1024. Encapsulation of the NF-kB Decoy Oligonucleotide in Echogenic Liposomes and Its Ultrasound-Triggered Release

BACKGROUND: Echogenic immunoliposomes (ELIP) may be useful tools for early detection of atherosclerosis. Encapsulation of drugs and genes in ELIP can improve their therapeutic efficacy, especially if their release can be triggered at a specific site. NF-kB protein is an important molecule in the regulation of the expression of genes involved in inflammation and vascular smooth muscle cell proliferation. Efficient delivery of various NF-kB inhibitors to proliferating cells should therefore contribute to early treatment of atheroma. The aim of this study was three-fold: 1) To develop a method to accurately measure oligonucleotide (ODN) encapsulation within conventional liposomes; 2) To demonstrate encapsulation of NF-kB decoy ODN within ELIP; and 3) To detect release of encapsulated ODN from ELIP upon application of ultrasound.METHODS: FITC-labeled ODN (2 μM) was incorporated within DPPC:DOPC:DPPG:CH liposomes (36:36:8:20, mol ratio) during hydration. The samples were made echogenic using a previously published freeze-dry method. Encapsulation of FITC-ODN was analyzed spectrofluorometrically. The initial fluorescence of the sample was measured (F b ). The fluorescence of unencapsulated FITC-ODN was quenched by a complementary strand tagged with Iowa BlackTM FQ (IB-ODN), added at a molar ratio of 2:1 (IB- ODN:FITC-ODN). The sample was reanalyzed (Fa ). Finally, fluorescence was measured after adding TritonX-100 to release encapsulated FITC-ODN (Ftotq ). Encapsulation of FITC-ODN was calculated using the following equation: [(Fa -Ftotq )/(Fb -Ftotq )] × 100%. To initiate release, 1 MHz ultrasound was applied at 2.0 W/cm2 for 60 seconds.RESULTS: FITC-ODN encapsulation was effected and quantified. Quenching of FITC-ODN (0.05 μM) by IB-ODN (0.1 μM) was efficient (95 ± 1.5%), allowing accurate determination of encapsulated FITC-ODN. Based on this method, the encapsulation of FITC-ODN was 18 ± 3% in DPPC:DOPC:DPPG:CH liposomes and 29 ± 7% in DPPC/DOPE/DPPG/CH liposomes. In addition, application of ultrasound to the former formulation triggered 50 ± 4% release of the encapsulated FITC-ODN from echogenic liposomes but, only 1 ± 2% from nonechogenic vesicles (n=3, p < 0.05).CONCLUSIONS: According to the procedure described, ODNs can be readily encapsulated in echogenic liposomes and released efficiently by ultrasound treatment. These NF-kB ODN- encapsulated vesicles show potential as tools for early detection and treatment of atherosclerosis. .