Abstract
The development of adjuvant techniques to improve thrombolytic efficacy is important for advancing ischemic stroke therapy. We characterized octafluoropropane and recombinant tissue plasminogen activator (rt-PA)-loaded echogenic liposomes (OFP t-ELIP) using differential interference and fluorescence microscopy, attenuation spectroscopy, and electrozone sensing. The loading of rt-PA in OFP t-ELIP was assessed using spectrophotometry. Further, it was tested whether the agent shields rt-PA against degradation by plasminogen activator inhibitor-1 (PAI-1). An in vitro system was used to assess whether ultrasound (US) combined with either Definity or OFP t-ELIP enhances rt-PA thrombolysis. Human whole blood clots were mounted in a flow system and visualized using an inverted microscope. The perfusate consisted of either (1) plasma alone, (2) rt-PA, (3) OFP t-ELIP, (4) rt-PA and US, (5) OFP t-ELIP and US, (6) Definity and US, or (7) rt-PA, Definity, and US (n = 16 clots per group). An intermittent US insonation scheme was employed (220 kHz frequency, and 0.44 MPa peak-to-peak pressures) for 30 min. Microscopic imaging revealed that OFP t-ELIP included a variety of structures such as liposomes (with and without gas) and lipid-shelled microbubbles. OFP t-ELIP preserved up to 76% of rt-PA activity in the presence of PAI-1, whereas only 24% activity was preserved for unencapsulated rt-PA. The use of US with rt-PA and Definity enhanced lytic efficacy (p < 0.05) relative to rt-PA alone. US combined with OFP t-ELIP enhanced lysis over OFP t-ELIP alone (p < 0.01). These results demonstrate that ultrasound combined with Definity or OFP t-ELIP can enhance the lytic activity relative to rt-PA or OFP t-ELIP alone, respectively.
Highlights
Stroke causes 6.5 million deaths worldwide every year[1] and remains the fifth leading cause of death in the United States[2]
We assessed the morphology of echocontrast agents using differential interference contrast (DIC) and fluorescence microscopy to ascertain the location of OFP and recombinant tissue plasminogen activator (rt-PA) within these agents
Spectrophotometric assessment revealed that undiluted OFP-tELIP contained 252 ± 9 μl of rt-PA per ml of solution in vial, which corresponds to a loading ratio of 84 ± 3%
Summary
Stroke causes 6.5 million deaths worldwide every year[1] and remains the fifth leading cause of death in the United States[2]. Subsequent studies showed that a subset of patients with large vessel occlusions could be treated with mechanical thrombectomy within 16–24 hours of symptom onset[17,18,19]. Our group has reported enhanced thrombolysis in the presence of 120-kHz US with echogenic liposomes[31] These agents contain rt-PA and octafluoropropane gas-loaded vesicles (OFP t-ELIP). Stable cavitation and acoustic radiation force were identified as the mechanisms for the lytic enhancement[22,32] These results were encouraging, large microbubbles (50 ± 19 μm diameter) were observed after 120 kHz exposures of Definity in vitro[32], which could pose an embolization risk[34]. We tested whether 220-kHz US in combination with cavitation nuclei – either OFP t-ELIP, or Definity enhances thrombolytic efficacy relative to treatment with rt-PA alone
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