Introduction-Thrombolytics such as recombinant tissue plasminogen activator (rt-PA) have advanced the treatment of ischemic stroke, myocardial infarction, deep vein thrombosis and pulmonary embolism.
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).
Stroke is the third leading cause of death and the leading cause of disability in the United States. For patients with ischemic stroke, the thrombolytic drug tissue plasminogen activator (tPA) is the only FDA-approved treatment. To aid in the development of a stroke therapy, the synergistic thrombolytic effect of tissue plasminogen activator (t-PA) and ultrasound was assessed in vitro in a porcine clot model. Whole blood clots were prepared from fresh porcine blood by aliquoting 1.5 ml into 8-mm-i.d. glass tubes, immersing the tubes in a 37<th>°C water bath for 3 h and storing the clots at 5<th>°C for at least 3 days prior to use in comparative ultrasound and t-PA studies, which ensured complete clot retraction. The 120-kHz or 1-MHz ultrasound peak-to-peak pressure amplitude used for exposures was 0.35, 0.70, or 1.00 MPa. The range of duty cycles varied from 10% to 100% (continuous wave) and the pulse repetition frequency was 1.7 kHz. Clot mass loss was measured as a function of t-PA concentration (without ultrasound) from 0.25 to 8.5 times the human clinical dose. The mass loss increased monotonically as a function of [t-PA] and saturated at 4 times the clinical dose. The degree of the ultrasound enhancement of t-PA was also explored at 120 kHz and 1 MHz. With ultrasound exposure, clot mass loss increased by as much as 250% over sham (t-PA alone). A weak dependence of clot mass loss on duty cycle was noted. We conclude that 120-kHz and 1-MHz ultrasound enhances thrombolysis. [Work supported by Senmed Medical Ventures.]
A recently developed ultrasound contrast agent, rt-PA-loaded echogenic liposomes (TELIP), was assessed in vitro using a clinical diagnostic ultrasound scanner (Philips HDI 5000) equipped with a linear array (L12-5). The stability and echogenicity of static TELIP suspensions were determined using 4.5-MHz harmonic B-mode pulses (Pr=120 kPa; MI=0.04) in an anechoic chamber. An in vitro flow phantom with a flow rate of 5 ml/min at 37<th>°C was also used to assess TELIP for ultrasonically-triggered drug release. TELIP samples were exposed to: (1) Fundamental 6.9-MHz B-mode pulses (Pr=600 kPa; MI=0.04) where diffusion of gas out of the liposomes occurs over 60 min, or (2) 6.0-MHz color Doppler pulses (PD=3.33<th>μs, PRF=1 kHz) at two exposure levels, 0.8 MPa (MI=0.22) for which acoustically driven diffusion was evident or 2.6 MPa (MI=0.7), for which rapid fragmentation was confirmed. Exposure of TELIP to Triton-X, a nonionic detergent, served as a positive control for drug release. Release of rt-PA for each ultrasound exposure protocol was assayed spectrophotometrically (Shimadzu UV-1700). The thrombolytic drug remained associated with the lipid bilayer when exposed to B-mode pulses over time and was released when exposed to color Doppler pulses. [This work was supported by NIH 1RO1 NS047603 and NIH 1R01 HL074002.]
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