In vitrothrombolytic efficacy of echogenic liposomes loaded with tissue plasminogen activator and octafluoropropane gas

Abstract

Echogenic liposomes loaded with the thrombolytic recombinant tissue-type plasminogen activator (rt-PA) are under development for the treatment of ischemic stroke. These agents are designed to co-encapsulate cavitation nuclei to promote bubble activity in response to ultrasound exposure, and to enable localized delivery of thrombolytic. Stable cavitation improves the efficacy of the thrombolytic through enhanced fluid mixing. Echogenic liposomes that encapsulate air-filled microbubbles nucleate scant stable cavitation activity in response to 120 kHz intermittent ultrasound exposure, and have demonstrated thrombolytic efficacy equivalent to rt-PA alone. It was hypothesized that encapsulating octafluoropropane (OFP) gas within rt-PA-loaded liposomes instead of air will enhance ultrasound-mediated stable cavitation activity and increase thrombolytic efficacy compared to previous studies. The thrombolytic efficacy and cavitation activity nucleated from liposomes that encapsulate OFP microbubbles and rt-PA (OFP t-ELIP) was evaluated in vitro. Human whole blood clots were exposed to human fresh-frozen plasma alone, rt-PA (0, 0.32, 1.58, and 3.15 μg ml−1), or OFP t-ELIP at equivalent enzymatic activity, with and without exposure to intermittent ultrasound. Further, numerical simulations were performed to gain insight into the mechanisms of cavitation nucleation. Sustained ultraharmonic activity was nucleated from OFP t-ELIP when exposed to ultrasound. Furthermore, the thrombolytic efficacy was enhanced compared to rt-PA alone at concentrations of 1.58 μg ml−1 and 3.15 μg ml−1 (p< 0.05). These results indicate that OFP t-ELIP can nucleate sustained stable cavitation activity and enhance the efficacy of thrombolysis.

Tissue plasminogen activator-based nanothrombolysis for ischemic stroke

“…Results from these studies showed that: 1) tPA released from ELIP had similar enzymatic activity as free tPA 69 ; 2) encapsulating PFC gas other than air could enhance ultrasound-mediated stable cavitation activity and increase thrombolytic efficacy 73, 74 ; 3) thrombolytic activity of tPA loaded with ELIP was comparable to that of tPA alone, and addition of 120 kHz ultrasound significantly enhanced thrombolytic efficacy of both tPA and tPA-loaded ELIP 75 ; 4) entrapment of tPA into ELIP showed effective clot lysis and targeted ultrasound-facilitated drug release 75 ; 5) fibrin binding of tPA loaded with ELIP was twice that of free tPA 76 , and PPACK-inactivated tPA still maintained fibrin-binding activity 77 , which can be employed as a fibrin-targeting moiety for ELIP-based thrombolysis.…”

An in vitro proof-of-principle study of sonobactericide

“…In this study, a 120-kHz frequency was chosen considering that the majority of previous studies for treating bacterial biofilms and clots in the presence of rt-PA have been performed with low frequency US 10 – 14 , 21 – 24 , 64 . The intermittent exposure scheme has been reported previously for sonothrombolysis 29 , 65 . However, diagnostic cardiac imaging is typically performed using a 2-MHz centre frequency 66 .…”

“…Unlike other thrombolytics, rt-PA has a high affinity to fibrin and thus provides a more local activation of fibrin-bound plasminogen, therefore decreasing the risk of negative effects due to systemic plasminogen cleavage 61 , 62 . Localized delivery of rt-PA to IE biofilms using echogenic liposomes loaded with rt-PA in combination with an antibiotic could also be a promising strategy to reduce off-target effects 65 .…”

Nano-Medicine for Thrombosis: A Precise Diagnosis and Treatment Strategy

“…Meanwhile, the easy encapsulation of thrombolytic agents and surface targeting modification have made liposomes preferred candidates for thrombolysis [59][60][61][62]. In addition, the combined utilization of external irradiation could show effective release in thrombotic sites, as in the case of echogenic liposomes (ELIP) [63]. ELIP was prepared using a lipid monolayer shell, encapsulated with octofluoropropane and loaded with rt-PA to construct an acoustically activated drug-delivery system [64].…”