In vitro characterization of sonothrombolysis and echocontrast agents to treat ischemic stroke

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.

Echogenic exosomes as ultrasound contrast agents

“…24,53,58 Recently, Shekhar et al evaluated the nature of the echogenic behaviors of liposomes using differential interference contrast microcopy (DIC). 62 Their DIC images showed micrometer sized bubbles encapsulated inside liposome, but the typical size of the observable liposomes in the DIC images were far larger; the number weighted and volumeweighted diameters of these vesicles were 1.35 mm and 8.23 mm respectively. 62 The exosomes studied here, unlike previously studied echogenic liposomes and the polymersomes, are preformed vesicles, but they underwent the same freeze-drying procedure in presence of mannitol that was also part of the preparation protocol for echogenic liposomes and polymersomes.…”

“…62 Their DIC images showed micrometer sized bubbles encapsulated inside liposome, but the typical size of the observable liposomes in the DIC images were far larger; the number weighted and volumeweighted diameters of these vesicles were 1.35 mm and 8.23 mm respectively. 62 The exosomes studied here, unlike previously studied echogenic liposomes and the polymersomes, are preformed vesicles, but they underwent the same freeze-drying procedure in presence of mannitol that was also part of the preparation protocol for echogenic liposomes and polymersomes. 18,24,28,53,58,[63][64][65] However, unlike echogenic liposomes carrying encapsulated microbubble inside studied by Shekhar et al 62 the echogenic exosomes are much smaller in size-50-150 nm.…”

“…62 The exosomes studied here, unlike previously studied echogenic liposomes and the polymersomes, are preformed vesicles, but they underwent the same freeze-drying procedure in presence of mannitol that was also part of the preparation protocol for echogenic liposomes and polymersomes. 18,24,28,53,58,[63][64][65] However, unlike echogenic liposomes carrying encapsulated microbubble inside studied by Shekhar et al 62 the echogenic exosomes are much smaller in size-50-150 nm. The smaller nanometer size of the exosomes indicates similarity to nanocups and other such nanoparticles investigated by Kwan et al 66 which spontaneously grow surface trapped bubbles upon ultrasound excitation due to their geometry and surface properties.…”

Bactericidal Activity of Lipid-Shelled Nitric Oxide-Loaded Microbubbles

“…NO-OFP-MB, Nitric oxide and octafluoropropane microbubbles. Definity ® as previously reported (Shekhar et al, 2019a). A previous study employed the Griess assay in combination with serial dialysis for NO dose assessment within liposomes (Huang et al, 2009).…”

Advances in Sonothrombolysis Techniques Using Piezoelectric Transducers

“…Drug loaded microbubbles are of interest due to their potential to minimize non-site specific tPA or urokinase binding. Because the thrombolytic drug is primarily released when activated by ultrasound, and the ultrasound is only focused on the clot of interest, this may both minimize hemorrhage risks while also taking advantage of the synergistic improvements of thrombolysis outcomes with MBs and thrombolytic agents [87][88][89][90][91][92]. Both urokinase loaded echogenic liposomes and tPA loaded echogenic liposomes outperform using a thrombolytic agent alone or thrombolytic agent mediated sonothrombolysis.…”