Fibrin-targeted phase shift microbubbles for the treatment of microvascular obstruction

Abstract

Rationale: Microvascular obstruction (MVO) following percutaneous coronary intervention (PCI) is a common problem associated with adverse clinical outcomes. We are developing a novel treatment, termed sonoreperfusion (SRP), to restore microvascular patency. This entails using ultrasound-targeted microbubble cavitation (UTMC) of intravenously administered gas-filled lipid microbubbles (MBs) to dissolve obstructive microthrombi in the microvasculature. In our prior work, we used standard-sized lipid MBs. In the present study, to improve upon the efficiency and efficacy of SRP, we sought to determine the therapeutic efficacy of fibrin-targeted phase shift microbubbles (FTPSMBs) in achieving successful reperfusion of MVO. We hypothesized that owing to their much smaller size and affinity for thrombus, FTPSMBs would provide more effective dissolution of microthrombi when compared to that of the corresponding standard-sized lipid MBs. Methods: MVO in the rat hindlimb was created by direct injection of microthrombi into the left femoral artery. Definity MBs (Lantheus Medical Imaging) were infused through the jugular vein for contrast-enhanced ultrasound imaging (CEUS). A transducer was positioned vertically above the hindlimb for therapeutic US delivery during the concomitant administration of various therapeutic formulations, including (1) un-targeted MBs; (2) un-targeted phase shift microbubbles (PSMBs); (3) fibrin-targeted MB (FTMBs); and (4) fibrin-targeted PSMBs (FTPSMBs). CEUS cine loops with burst replenishment were obtained at baseline (BL), 10 min post-MVO, and after each of two successive 10-minute SRP treatment sessions (TX1, TX2) and analyzed (MATLAB). Results: In-vitro binding affinity assay showed increased fibrin binding peptide (FBP) affinity for the fibrin clots compared with the untargeted peptide (DK12). Similarly, in our in-vitro model of MVO, we observed a higher binding affinity of fluorescently labeled FTPSMBs with the porcine microthrombi compared to FTMBs, PSMBs, and MBs. Finally, in our hindlimb model, we found that UTMC with FTPSMBs yielded the greatest recovery of blood volume (dB) and flow rate (dB/sec) following MVO, compared to all other treatment groups. Conclusions: SRP with FTPSMBs achieves more rapid and complete reperfusion of MVO compared to FTMBs, PSMBs, and MBs. Studies to explore the underlying physical and molecular mechanisms are underway.

Keywords: cavitation; fibrin-targeted microbubbles; microvascular obstruction; phase-shift microbubbles; sonoreperfusion.

Figure 1

Experimental setup. (A) The rat hind limb microvascular obstruction (MVO) protocol includes an ultrasound (US) therapy and perfusion imaging time points. (B) A therapeutic probe was positioned perpendicular to the imaging plane of a clinical probe operated in contrast mode, which helps to guide and monitor US therapy. (C) Contrast imaging microbubbles (Definity^Ⓡ) were infused via jugular access and were utilized for burst replenishment imaging to record perfusion. (D) A contralateral femoral arterial line was placed for microthrombi injection to create MVO and to deliver the therapeutic contrast agents for direct first-pass arterial delivery. (E) Therapeutic US. (F) Imaging US. (Figure created by BioRender).

Figure 2

Fibrin Binding Peptide with an amine functional group (A) conjugated to DSPE-PEG5000-NHS Ester to make a product with an amide linker (B).

Figure 3

Illustration of phase shift in the presence of ultrasound: (A) Ingredients of fibrin-targeted microbubbles (FTMBs) in solution inactive state. (B) Upon vial mix for 45 sec results in FTMBs having a gaseous core shell of octafluoropropane. (C) Chilled pressure condensation of FTMBs results in the production of FTPSMBs. (D) Under the influence of ultrasound FTPSMBs phase shift to FTMBs. (Figure created by BioRender)

Figure 4

In-vitro fibrin binding affinity assay for fibrin thrombi with fluorescently labeled control peptide (DK12, black bars) and fluorescently labeled fibrin-binding peptide (grey bars).

Figure 5

In-vitro flow loop fibrin binding assay: (A) Fluorescent images of the in-vitro flow loop mesh following 10 min of perfusion with saline (control), DiO tagged microbubbles (MBs), DiO tagged phase shift microbubbles (PSMBs), DiO tagged fibrin-targeted microbubbles (FTMBs), and DiO tagged fibrin-targeted phase shift microbubbles (FTPSMBs). (B) Bar graph representing the fluorescence intensity of three independent studies. Data expressed as mean ± standard deviation (n=3). *p<0.05, **p<0.001.

Figure 6

Comparison between microbubbles (MBs) Vs. phase shift microbubbles (PSMBs). (A) Contrast-enhanced ultrasound images of rat hindlimb. (B) Peak plateau video intensity which reflects the vascular cross-sectional area and is directly proportional to blood volume (dB). (C) Flow rate (dB/sec). Data expressed as mean ± standard deviation (n=5 for MBs and n=6 for PSMBs). *p < 0.05, **p<0.001, ***p<0.0001.

Figure 7

Comparison between phase shift microbubbles (PSMBs) Vs. fibrin-targeted phase shift microbubbles (FTPSMBs). (A) Contrast-enhanced ultrasound images of rat hindlimb. (B) Peak plateau video intensity which reflects the vascular cross-sectional area and is directly proportional to blood volume (dB). (C) Flow rate (dB/sec). Data expressed as mean ± standard deviation (n=6). Data expressed as mean ± standard deviation (n=6). *p < 0.05, **p<0.001.

Figure 8

Comparison between fibrin-targeted microbubbles (FTMB) Vs. fibrin-targeted phase shift microbubbles (FTPSMBs). (A) Contrast-enhanced ultrasound images of rat hindlimb. (B) Peak plateau video intensity which reflects the vascular cross-sectional area and is directly proportional to blood volume (dB). (C) Flow rate (dB/sec). Data expressed as mean ± standard deviation (n=6). Data expressed as mean ± standard deviation (n=6). *p<0.05, **p<0.001 and ****p<0.0001.

Figure 9

Histology. Hematoxylin and eosin staining of the rat hind limb muscle following ultrasound-targeted microbubble cavitation (UTMC) treatment with microbubbles (MBs), fibrin-targeted microbubbles (FTMBs), phase shift microbubbles (PSMBs), and fibrin-targeted phase shift microbubbles (FTPSMBs). Black arrows indicate microvascular patency compared to matching control; red arrows represent occluded microvessels. Scale bar = 200 μm.