Echogenic liposomes loaded with recombinant tissue-type plasminogen activator (rt-PA) for image-guided, ultrasound-triggered drug release

Abstract

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.]

Ultrasound-Mediated Release of Hydrophilic and Lipophilic Agents From Echogenic Liposomes

“…The results of our study suggest that water-soluble compounds might be efficiently released from ELIPs by ultrasound. Indeed, rt-PA release from ELIPs by ultrasound has been shown 12,13. Hydrophilic compounds such as calcein are likely to be trapped within the aqueous core of liposomes.…”

“…Continuous wave ultrasound (2–7 W/cm 2 ) has been investigated as a means of triggering calcein or recombinant tissue plasminogen activator (rt-PA) release from ELIP in nonflowing solutions 4,11,12. Recently, pulsed color Doppler ultrasound was used to trigger release of rt-PA from rt-PA-loaded ELIPs in flowing solutions 13. The use of color Doppler ultrasound (a scanned mode) enables a larger number of ELIPs to be exposed per unit of time than spectral Doppler ultrasound (an unscanned mode).…”

Ultrasound-Assisted Thrombolysis for Stroke Therapy

“…Studies in vivo have demonstrated successful targeting of the ELIP to a thrombus surface using inactivated t-PA 44,46. An in vitro study demonstrated effective use of selectively tuned clinical diagnostic ultrasound to release drug from rt-PA-loaded ELIP 47,48. The rt-PA-loaded ELIP will be tested in the ex vivo artery system to determine optimal ultrasound parameters for delivery of the lytic drug and for promotion of stable cavitation to enhance efficacy.…”

Echogenic lipsomes for targeted drug delivery

“…The thrombolytic, rt-PA, has been successfully loaded into ELIP and Smith et al [20] 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).…”

“…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 [20]. Tiukinhoy et al [21] and Klegerman et al [22] have demonstrated specific fibrin binding of rt-PA-loaded ELIP using an in vitro porcine clot model.…”