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Echogenic immunoliposomes (ELIP) are under development to enable ultrasound-controlled drug delivery. Mechanistic studies in vitro have revealed that stable cavitation is correlated with enhanced recombinant tissue Plasminogen Activator (rt-PA) thrombolysis, yet strategies to optimize the occurrence of such bubble activity and avoid potential harmful bioeffects have yet to be identified. Stable cavitation is characterized by bubbles pulsating gently in response to the time-varying acoustic pressure in an ultrasound field. A review of in vitro sonothrombolysis studies utilizing commercial US contrast agent or echogenic liposomes loaded with rt-PA to nucleate stable cavitation will be presented. Strategies for the development of ultrasound-enhanced thrombolysis and drug delivery will be discussed.
Targeted echogenic immunoliposomes (ELIP) are under development to highlight thrombus or atheroma components. Liposomes, or phospholipid bilayer vesicles enclosing gas and fluid, are novel agents that may permit evaluation of vasoactive and pathologic endothelium. Since first examined by Bangham et al. , liposomes have been developed for a wide range of technical and medical applications. Echogenic liposomal dispersions can be prepared by dispersing a lipid in water, adding mannitol, and lyophilizing [2-4]. The echogenicity of these preparations is due to the presence of gas, which is entrapped and stabilized by the lipid during the rehydration process following lyophilization .
ELIP can be targeted to certain tissues by attaching specific ligands and antibodies to the surface of liposomes [6, 7]. For example, ELIP coupled to anti-VCAM-1 antibodies could be used to identify pathologic endothelium at early stages of atherosclerosis development. Similarly, the linkage of a liposome with antifibrin or D-Dimer antibody may identify and highlight thrombus or plaque rupture . Lanza et al.  have also demonstrated that a multi-step acoustic biotinylated, lipid-coated, perfluorocarbon nanoemulsion could be successfully targeted to thrombi in vitro while maintaining ultrasound contrast. They have additionally demonstrated that this ultrasound agent can infiltrate arterial walls and localize tissue factor expression . Unger et al. [11, 12] created targeted microbubbles containing perfluorobutane (aerosomes) that were approximately 2 microns in diameter and were able to bind to thrombus in vitro. Echogenic liposomes, additionally, have the potential to encapsulate a drug for targeted therapeutic delivery.
Providing efficient and safe methods for the delivery of drugs to target cells is the principal goal of a clinically useful pharmacotherapeutic strategy. When a pharmaceutic is administered systemically, only a small fraction of the drug may reach the target tissue, necessitating large systemic doses to achieve effective local concentrations. Hence, systemic toxicity is usually the dose-limiting factor . Localized pharmacotherapeutic delivery has distinct advantages, which include increased concentrations at the specific tissue site and decreased toxicities . Direct delivery of a drug to the target tissue results in a high ratio of local to systemic bioavailability, with the result of a decreased required total dose. As such, specific delivery may result in increased tolerable concentrations of the drug. In addition, the use of ultrasound to fragment all of the liposomes simultaneously near the target, rather than relying on a more gradual passive therapeutic delivery, has the potential to produce a large temporal peak in drug or therapeutic effect. This is particularly important at the endothelium where the constant flow of blood may carry away the released drug rapidly, making it unavailable for ultrasound-induced uptake across the endothelium.
Additional benefits of liposomes are that lipids are small molecular structures and the lipid complexes can be made smaller by filtering or sonication techniques. Proteins and delivery agents made of proteins tend to break if they are manipulated to make them smaller. The difference lies in the rigidity of the protein, which has covalent bonds, versus the lipid, which is composed of small molecules held together by hydrophobic interactions. An example would be the rigidity of mayonnaise (a lipid formulation) vs. a hard-boiled egg (a protein formulation). Liposomes are ideal targeted delivery systems as they are nontoxic and can carry either hydrophilic or hydrophobic compounds, either in the aqueous compartment or within the phospholipid bilayers, respectively, and can be targeted to specific tissues. Hence, the development of a liposome-drug delivery mechanism that can be manipulated for site-specific tissue targeting may provide improved specificity of drug delivery and more efficient and enduring effects at the target site.
Several studies have demonstrated that low intensity (< 2 W/cm2) ultrasound used as an adjuvant to recombinant tissue Plasminogen Activator (rt-PA), a thrombolytic, can increase thrombus dissolution in an in vitro model [16, 17]. Significant enhancement of thrombolysis correlates with the presence of stable cavitation  and this type of gentle bubble activity, can be sustained using an intermittent infusion of a commercial contrast agent, Definity® . In addition, inertial cavitation, which elicits broadband acoustic emissions, has been shown to be counter-productive for enhanced thrombolysis. Rather, the most effective form of bubble activity is stable cavitation, which elicits ultrasonic subharmonic generation. Increased penetration of rt-PA and plasminogen has been observed in clots exposed to stable cavitation nucleated by Definity®.
The thrombolytic, rt-PA, has been successfully loaded into ELIP and Smith et al.  have quantified ultrasound-triggered release of rt-PA in an in vitro flow model. These rt-PA-loaded ELIP are robust and echogenic during continuous fundamental 6.9-MHz B-mode imaging using a clinical diagnostic scanner at a low exposure output level (MI = 0.04). Furthermore, a therapeutic concentration of rt-PA can be released from the ELIP with pulsed 6.0-MHz color Doppler ultrasound at an MI above 0.43 . Tiukinhoy et al.  and Klegerman et al.  have demonstrated specific fibrin binding of rt-PA-loaded ELIP using an in vitro porcine clot model. In addition, Shaw et al. have shown that the lytic efficacy of rt-PA-loaded echogenic liposomes is comparable to that of tPA alone . Exposure of these ELIP to pulsed 120 kHz ultrasound (0.35 MPa peak to peak pressure amplitude, 50% duty cycle, 1667 Hz pulse repetition frequency) significantly enhanced lytic treatment efficacy for both rt-PA and rt-PA-loaded ELIP. These drug-loaded vesicles have the potential to be used for ultrasound-enhanced thrombolysis in the treatment of acute ischemic stroke, myocardial infarction, deep vein thrombosis, or pulmonary embolus.
This work was supported by NIH 1RO1 NS047603.