The latter, typically 100-200 nm PEG-coated liposome preparations [23] would circulate for many hours, and eventually accumulate in the liver. resulted in the release of ~30% of entrapped calcein, as estimated by the fluorescence quenching assay. Thrombin release from liposomes complexed with Adriamycin microbubbles (11% of entrapped material) due to ultrasound treatment was estimated by a chromogenic substrate study. Prior to insonation, substrate hydrolysis was at background level. SCDO3 Ultrasound-triggered release of thrombin from the pendant complexes caused acceleration of blood clotting. Keywords:Microbubbles, liposomes, ultrasound, triggered release, thrombin, clotting, calcein == INTRODUCTION == Ultrasound-assisted drug delivery has been investigated as a targeting mechanism that would allow enhancement of drug action in the desired regions. The strategy is to focus ultrasound field and insonate the drug-carrying bubbles that are in the target region, destroy them and release the drug. Until recently, the drug was either incorporated in the microbubble shell (preferred for hydrophobic drugs, such as paclitaxel [1], [2]) or attached to the microbubble shell directly (e.g., plasmid DNA that is bound to microbubble surface by electrostatic interaction [3],[4],[5]). The drug loading capacity for the drug-on-shell approach is limited, Adriamycin especially if a lipid monolayer is used as a microbubble shell, therefore it is desirable to increase the drug payload carried by each bubble. Earlier, we have prepared conjugates of liposomes and gas-filled microbubbles, as pendant-like structures, where liposomes decorated the surface of each microbubble[6],[7] to be applied as drug delivery vehicles. Ultrasound-triggered release of liposome-entrapped dye during insonation of microbubble located near the giant liposome has been reported [8]. Therefore, we can expect that liposomes that are directly attached to microbubble surface may also be ruptured when ultrasound treatment is applied. Recently, such microbubble-liposome complexes loaded with an anticancer antibiotic doxorubicin, [9] were prepared; successfulin vitrodelivery of doxorubicin in a cell culture model in response to ultrasound treatment was reported. The use of liposome-microbubble complexes for drug delivery, in comparison with plain microbubble preparations, widens the range of substances that could be incorporated, including proteins (e.g., enzymes or antibodies) or other hydrophilic drugs that could not otherwise be associated with the microbubble shell in a stable fashion. In this study we describe preparation of the microbubble-liposome complexes that entrap a model hydrophilic dye, calcein, and an enzyme thrombin. Ultrasound-mediated contents release is also investigated. == MATERIALS AND METHODS == == Microbubble preparation and characterization == Microbubbles were prepared from decafluorobutane gas and stabilized with a lipid monolayer that contained biotin binding groups for liposome attachment, in accordance with published procedures [10]. Briefly, 2 mg/ml phosphatidylcholine (Avanti Lipids, Alabaster, AL), 2 mg/ml PEG 150 stearate (Stepan Kessco, Northfield IL) and 0.1 mg/ml biotin-PEG3400-phosphatidylethanolamine (Avanti) were dispersed in 20 ml normal saline with a probe-type sonicator (XL2020, Misonix, Farmingdale NY, with a probe, at 40% power setting) to prepare aqueous micellar dispersion. For microbubble preparation, decafluorobutane gas (F2 Chemicals, Lancashire UK) was sparged through the aqueous medium, and sonication performed for 30 sec with the same XL2020 instrument operated at maximum power. Resulting aqueous dispersion of microbubbles was then aliquotted in small vials, sealed under decafluorobutane atmosphere and stored refrigerated until use. Microbubble size distribution was evaluated with Coulter counter equipped with a 50 um Adriamycin sensor orifice (Multisizer 3, Beckman Coulter, Miami FL): 10-20 ul of microbubble dispersion was added to 200 ml normal saline and particle size measured immediately for a 1-30 um range. == Liposome preparation and calcein entrapment == Small liposomes (~0.1 um) were prepared by the proliposome technique [11]: 10 ul of ethanol solution containing 4 mg phosphatidylcholine (Avanti), 0.7 mg cholesterol (Sigma) and 5 ug biotinamidocaproyl-phosphatidylethanolamine (Avanti) was mixed with 10 ul 0.2 M calcein aqueous solution to produce proliposome gel. The gel was rapidly mixed with 2 ml saline, resulting liposomes immediately vortexed and subjected to repeated centrifugation using either an Eppendorf microfuge (14,000g) or a Sorvall RC5C centrifuge with an angular rotor (40,000g). Liposome size was evaluated with a Nicomp 370 dynamic laser light scattering system (Particle Sizing Systems, Santa Barbara, CA). Large liposomes were prepared by reverse-phase evaporation technique [12]. Briefly, lipid solution of phosphatidylcholine, cholesterol and biotin-amidocaproyl-phosphatidylethanolamine in chloroform and ethyl ether was mixed with the aqueous medium containing 0.2 M calcein dye Adriamycin (Sigma, St. Louis, MO) at the 3:1 volume ratio between organic and aqueous phase. Water-in-oil emulsion was formed by brief (30 sec).
