Click‐Chemistry‐Mediated Rapid Microbubble Capture for Acute Thrombus Ultrasound Molecular Imaging

Abstract

Bioorthogonal coupling chemistry has been studied as a potentially advantageous approach for molecular imaging because it offers rapid, efficient, and strong binding, which might also benefit stability, production, and chemical conjugation. The inverse-electron-demand Diels-Alder reaction between a 1,2,4,5-tetrazine and trans-cyclooctene (TCO) is an example of a highly selective and rapid bioorthogonal coupling reaction that has been used successfully to prepare targeted molecular imaging probes. Here we report a fast, reliable, and highly sensitive approach, based on a two-step pretargeting bioorthogonal approach, to achieving activated-platelet-specific CD62p-targeted thrombus ultrasound molecular imaging. Tetrazine-modified microbubbles (tetra-MBs) could be uniquely and rapidly captured by subsequent click chemistry of thrombus tagged with a trans-cyclooctene-pretreated CD62p antibody. Moreover, such tetra-MBs showed great long-term stability under physiological conditions, thus offering the ability to monitor thrombus changes in real time. We demonstrated for the first time that a bioorthogonal targeting molecular ultrasound imaging strategy based on tetra-MBs could be a simple but powerful tool for rapid diagnosis of acute thrombosis.

Biomedical applications of copper-free click chemistry: in vitro, in vivo, and ex vivo

“…Using a similar strategy, Wang et al performed successful ultrasound imaging of acute thrombus in rats. 55 The results of the studies discussed are promising and demonstrate that click chemistry has great potential in biomedical imaging in vivo. However, strategies and the click molecules used need to be selected carefully because required doses of probes and reaction times will vary according to the types of imaging modalities and disease models.…”

“…Tumor VEGF (vascular endothelial growth factor receptor 2) imaging by ultrasound 54 Antivascular endothelial growth factor receptor 2 antibody (TCO-antiVEGFR2)/Tz-modied microbubbles iEDDA Acute thrombus imaging by ultrasound 55 AntiCD62p-TCO/Tz-modied microbubbles iEDDA Tumor-targeted liposomes for drug delivery 25 Ac 4 ManNAz/DBCO-modied liposome SPACC Tumor-targeted photosensitizer delivery 27 Ac 4 ManNAz-loading chitosan nanoparticles/DBCO-modied chitosan nanoparticles SPACC Tumor-targeted doxorubicin delivery 56 Peptide-modied Ac 4 ManNAz/DBCO-modied doxorubicin SPACC Upconversion nanoparticle-based photodynamic therapy 57 Upconversion NP modied with Tz groups/norborane (NB)-conjugated rose bengal (RB) iEDDA Endothelial progenitor cell therapy for myocardiac infarction 58 Azide-modied CD34 antibody/DBCO-modied CD41 antibody SPACC Ex vivo Glycoproteomic analysis of intact and hypertrophy hearts 59 Ac 4 ManNAz/DBCO-Fluor 488 SPACC…”

Current Strategies for Microbubble-Based Thrombus Targeting: Activation-Specific Epitopes and Small Molecular Ligands

“…Other moieties, such as small peptides (Modery et al, 2011) and constructs based on bio-orthogonal coupling chemistry (Wang et al, 2017), have been developed while each of these methods has its own limitations compared to polysaccharides. Mechanisms of the latter approach are based on the rapid and selective Diels-Alder reaction between trans-cyclooctene (TCO)-tagged P-selectin-specific antibodies and tetrazine-tagged microbubbles, which empowered the ultrasound imaging with a specific, rapid, and sensitive contrast agent (Wang et al, 2017). However, additional studies including pharmacokinetics and pharmacodynamics of the targeted moieties are required.…”

“…For example, fibrin as an important epitope of thrombus would decrease, while collagen deposition would increase in the aging thrombus. Most of the studies available examined the targeting efficacy of ligands with thrombi of the acute phase (Wang et al, 2012(Wang et al, , 2017Lu et al, 2019), while the efficacy in subacute or chronic thrombi remained unknown (Wu et al, 2013). Therefore, the influence of the age of thrombus on the targeting capability of ligands has to be systemically evaluated further.…”

Antibody Pretargeting Based on Bioorthogonal Click Chemistry for Cancer Imaging and Targeted Radionuclide Therapy

“…The theranostic ability of IEDDA pretargeting makes it a promising strategy, not only for human cancer management but also for many fields of application. Indeed, IEDDA (and also some other bioorthogonal click reactions) was otherwise successfully investigated for the modifications of nanoparticles, 77 the biosynthesis of antifungals, 78 labeling the HIV-1 envelope glycoproteins, 79 to microtubules, 80 the preparation of targeted thrombus ultrasound molecular probes, 81 or even the activation of antiinflammatory drugs. 82 However, despite the encouraging results observed in the past few years, the IEDDA approach has surprisingly not reached clinical trials yet.…”