Ultrasound-Triggered Release of Recombinant Tissue-Type Plasminogen Activator from Echogenic Liposomes

Abstract

Echogenic liposomes (ELIP) were developed as ultrasound-triggered targeted drug or gene delivery vehicles (Lanza et al., 1997;Huang et al., 2001). Recombinant tissue-type Plasminogen Activator (rt-PA), a thrombolytic, has been loaded into ELIP (Tiukinhoy-Laing et al., 2007). These 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. A clinical diagnostic ultrasound scanner (Philips HDI 5000) equipped with a linear array transducer (L12-5) was employed for in vitro studies using rt-PA-loaded ELIP (T-ELIP). The goal of this study was to quantify ultrasound-triggered drug release from rt-PA-loaded echogenic liposomes. T-ELIP samples were exposed to 6.9-MHz B-mode pulses at a low pressure amplitude (600 kPa) to track the echogenicity over time under four experimental conditions: 1) flow alone to monitor gas diffusion from the T-ELIP, 2) pulsed 6.0-MHz color Doppler exposure above the acoustically driven threshold (0.8 MPa) to force gas out of the liposome gently, 3) pulsed 6.0-MHz color Doppler above the rapid fragmentation threshold (2.6 MPa), or 4) Triton X-100 to rupture the T-ELIP chemically as a positive control. Release of rt-PA for each ultrasound exposure protocol was assayed spectrophotometrically. T-ELIP were echogenic in the flow model (5 ml/min) for thirty minutes. The thrombolytic drug remained associated with the liposome when exposed to lowamplitude B-mode pulses over 60 min and was released when exposed to color Doppler pulses or Triton X-100. The rt-PA released from the liposomes had similar enzymatic activity as the free drug. These T-ELIP are robust and echogenic during continuous fundamental 6.9-MHz B-mode imaging at a low exposure output level (600 kPa). Furthermore, a therapeutic concentration of rt-PA can be released by fragmenting the T-ELIP with pulsed 6.0-MHz color Doppler ultrasound above the rapid fragmentation threshold (1.59 MPa).

Passive imaging with pulsed ultrasound insonations

“…[30][31][32] Echogenic liposomes were used solely as cavitation nuclei in this work. However, others have demonstrated that ELIP can be used for localized ultrasound-mediated drug release 43 and delivery. 51 Passive cavitation imaging could be used in these contexts to obtain spatially resolved feedback during a treatment.…”

“…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.…”

Construction of thrombus-targeted microbubbles carrying tissue plasminogen activator and their in vitro thrombolysis efficacy: a primary research

“…Taking lipid as the shell, microbubbles has several qualities such as good deformability, stability, biological compatibility, safety, no immunogenicity and no danger of infection, which endow it with the wide prospect of clinical application. Compared with the other ways of preparation of thrombolytics-loaded microbubbles [14,18,19], the lyopyilization we employed to encapsulate tPA into the lipid shell is operated under the condition of low temperature, which is suited for the temperature sensitive material such as tPA, and can embed the drugs in the shell, which can effectively protect them from the outside disturbance. The results showed that the envelope rate of tPA is satisfactory, and the agarose fibrin plate process proved that the encapsulated tPA kept good activity under the condition of both self-lysis and ultrasound exposure.…”

In Vivo Gene Transfer in the Female Bovine: Potential Applications for Biomedical Research in Reproductive Sciences

“…This could be easily circumvented with the bovine model as intraovarian injections could be carried out at least twice per week (Velazquez et al, 2009a). Advances in ultrasound-triggered targeted gene delivery vehicles such as echogenic liposomes (Smith et al, 2010) could provide an efficient OTU in vivo vector delivery system in cattle. Following gene delivery, integration of the transgene could be analyzed in primordial and preantral follicles (Aerts et al, 2005), oocytes from antral follicles (Zaraza et al, 2010) and granulosa cells (Wells et al, 1999) with minimal discomfort for the carrier animal.…”