Liposomal modular complexes for simultaneous targeted delivery of bioactive gases and therapeutics

Abstract

Intrinsically echogenic liposomes (ELIP) can be adapted to encapsulate nitric oxide to facilitate ultrasound-enhanced delivery of therapeutic agents to atherosclerotic plaques. However, the NO loading of targeted ELIP caused a 93% decrease of antibody (Ab) immunoreactivity. The following hypothesis was tested: biotin/avidin-mediated coupling of NO-ELIP and Ab-conjugated ELIP will enable co-delivery of bioactive gases and ELIP that can encapsulate other agents without loss of targeting efficiency. Complex formation was initiated by addition of excess streptavidin to equal proportions of biotinylated Ab-ELIP and NO-ELIP. Fluorescence deconvolution microscopy, Coulter Multisizer 3 analysis and flow cytometry demonstrated that the ELIP coupling procedure formed mixed aggregates of ≥10 liposomes within 1 minute. Intravascular ultrasound imaging and ELISA showed that echogenicity and targeting efficiency were completely and 69-99% retained, respectively. When complexed to NO-ELIP, ELIP bifunctionally targeted to both CD34 and ICAM-1 (BF-ELIP) increased human mononuclear cell migration through human coronary artery endothelial cell monolayers in transwell plates 4-fold relative to a nonspecific IgG-ELIP control and 2-fold relative to BF-ELIP alone. It was concluded that this novel multi-functional conjugation methodology provides a platform technology for site-specific co-delivery of bioactive gases and other agents.

Passive imaging with pulsed ultrasound insonations

“…Signals in the frequency domain, S n ðxÞ, were obtained by insonifying an experimental ultrasound contrast agent, echogenic liposomes (ELIP), 37,38 in a flow phantom with spectral Doppler pulses (center frequency of 6 MHz) from a CL15-7 transducer driven by an HDI-5000 clinical scanner (Philips Medical Systems, Bothell, WA). The acoustic properties and morphology of ELIP have been recently studied by Kopechek et al 39 and Paul et al 40 ELIP are of interest for both their diagnostic 37,38,41,42 and therapeutic [43][44][45][46][47] capabilities. The flow phantom consisted of a reservoir connected to a peristaltic pump (Rabbit, Rainin, Oakland, CA), which pumped a solution of ELIP (0.1 mg/ml lipid concentration) in 0.5% (wt./vol) bovine serum albumin (Sigma-Aldrich Co., St. Louis, MO) in phosphate buffered saline (Sigma-Aldrich Co.), through a low-density polyethylene tube (2.7 mm inner diameter, 4.0 mm outer diameter, McMaster-Carr, Aurora, OH) at a 2.0 mL/min flow rate.…”

Acoustic characterization of echogenic liposomes: Frequency-dependent attenuation and backscatter

“…Both techniques have been used previously to size liposomes and ultrasound contrast agents. 19,[29][30][31][32][33][34][35][36] Transmission electron micrograph (TEM) images of ELIPs were acquired for comparison. ELIPs were reconstituted in water at a concentration of 1 mg/ml and negatively stained using 1% uranyl acetate on 300 mesh formvar carbon grids (EMS, Hatfield, PA).…”

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.…”

“…Acoustic cavitation is hypothesized to expel the contents of the vesicle within diseased portions of the vasculature, enhancing the therapeutic index and mitigating non-specific cytotoxicity. Gasencapsulating liposomes are particularly suited for US-triggered release because the cavitation nucleation site is built into the vesicle [114]. Gas-free liposomes have also been shown to release their contents during acoustic cavitation [120].…”