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

Abstract

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-κB 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 μM) with Iowa Black FQ-ODN (0.1 μM) 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, 60 seconds.) 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 for gene therapy in atherosclerosis treatment.

Relationship between cavitation and loss of echogenicity from ultrasound contrast agents

“…ELIP are lipid bilayer vesicles, ranging from 40 nm to 6 μm in diameter (Kopechek et al 2011), each encapsulating an aqueous core and gas pocket (Huang 2008, Kopechek et al 2011). ELIP can be loaded with several cardiovascular therapeutic agents (Britton et al 2010, Buchanan et al 2010, Herbst et al 2010, Tiukinhoy-Laing et al 2007, Huang et al 2007, Huang et al 2009) and targeted to specific molecular receptors in diseased vasculature (Hamilton et al 2004, Laing et al 2011). In addition, ultrasound exposure has been shown to mediate delivery of therapeutic-loaded ELIP into vascular tissue (Herbst et al 2010, Hitchcock et al 2010).…”

Ultrasound-Mediated Drug Delivery for Cardiovascular Disease

“…Several vascular drug delivery vehicles have been proposed to encapsulate therapeutics, such as nanogels [102,103], micelles [104,105] and perfluorocarbon droplet emulsions [106][107][108]. Drugs and bioactive gases have also been encapsulated in nanometer and micron-sized echogenic liposomes [109][110][111][112][113][114]. To be an effective vehicle, the agent must maintain stability in vivo while protecting the drug against endogenous agents.…”

“…Kee et al [232] Liposome Papaverine 1.85 mg/ml 3 mg/ml [241] Buchanan et al [113] Liposome NF-κB 2 mM 10 mM [242] Rothdiener et al [234] Liposome siRNA 500 mmole/mole lipid 2.7 mM [243] Fabiilli et al [106] PFP droplet Chlorambucil 3.12 mg/ml 0.02 mg/ml [244] Fabiilli et al [107] PFP droplet Thrombin 3.1 IU/ml 1.3 IU/ml [245] Huang et al [ Wang et al [238] PFP droplet Doxorubicin 304 mg/ml 1.8 mg/ml [250] Jin et al [239] Nanogel Urokinase 500 mg/ml 4 mg/ml [251] Therapeutic concentrations calculated by assuming average human weight and blood volume.…”

Nucleic acid delivery with microbubbles and ultrasound

“…These particles have been tested for ultrasound-assisted plasmid delivery [62] and oligonucleotide delivery [63]. Particle size here is significantly larger than for eliposomes or bubble-liposomes.…”