Catching bubbles: targeting ultrasound microbubbles using bioorthogonal inverse-electron-demand Diels-Alder reactions.

Abstract

Ultrasound imaging remains one of the most extensively used medical imaging methods because of its high spatial and temporal sensitivity, low cost, and portability and accessibility of equipment. Contrast-enhanced ultrasound using gas-filled microbubbles (MBs) has further enhanced the utility of ultrasound and created the opportunity to employ biomolecule-targeted derivatives for molecular imaging applications. We describe here a new approach to ultrasound molecular imaging that employs the covalent and highly selective capture of functionalized MBs in vitro and in vivo through bioorthogonal inverse-electron-demand Diels–Alder reactions. While pretargeting methods for nanometer-sized materials, such as nanoparticles and liposomes, have been published recently, the work reported herein is, to our knowledge, the first example of the bioorthogonal capture of micron-sized materials and the employment of pretargeting strategies for ultrasound molecular imaging. Ultrasound contrast agents are generally comprised of an inert gas, such as a perfluorocarbon, surrounded by a lipid, synthetic polymer, or protein shell. The traditional approach to targeting MBs, which are typically 1–8 mm in diameter and therefore restricted to intravascular targets, has been to link biomolecules with a high affinity for a specific protein to the outer shell through covalent bonds (e.g., amide bonds) or strong noncovalent interactions such as biotin–streptavidin binding. These approaches, which have largely exploited antibody and peptide vectors, have demonstrated the ability to selectively localize MBs to sites of angiogenesis, inflammation, and intravascular thrombus formation. Rather than using targeting vectors to localize conjugated prosthetic groups, new strategies for creating molecular imaging probes are being exploited that employ pretargeting and bioorthogonal coupling chemistry. Here, a targeting vector is administered first, allowing time for localization and clearance from nontarget organs, followed by a fluorescent or radiolabeled coupling partner that provides a readout for the molecular signal. The inverse-electron-demandDiels–Alder reaction between tetrazines and trans-cyclooctene (TCO) is an example of a highly selective and rapid bioorthogonal coupling reaction that has been used successfully to prepare targeted nuclear and optical molecular imaging probes. A comparable strategy for localizing MBs has not been reported. Such a method could offer a way to overcome obstacles to targeting ultrasound contrast agents whose large size and ability to bind only intravascular targets where blood flow rates and shear stress are high, make it particularly challenging to achieve and maintain good contrast in a timeframe that aligns with the limited in vivo stability of MBs. To test the feasibility of capturing micron-sized bubbles, a novel tetrazine-tagged MB (MBTz) was developed, and its reactivity towards cells treated with a TCO-conjugated antivascular endothelial growth factor receptor 2 (VEGFR2) antibody evaluated (Figure 1). VEGFR2 is overexpressed on tumor cells and upon activation triggers multiple signaling pathways that contribute to angiogenesis. The choice of target also allows the use of anti-VEGFR2-tagged MBs (MBV), which were developed by Rychak, Foster, and coworkers for evaluation in preclinical models, to validate the tetrazine–TCO capture methodology. Tetrazine-functionalized bubbles were prepared using commercially available streptavidin-coated MBs (micromarker target-ready contrast agents, VisualSonics) and a biotinylated tetrazine. The biotin–tetrazine derivative 5 was synthesized from biotin in four high-yielding steps (Scheme 1). The desired product was ultimately obtained by coupling commercially available 4-(1,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride with 6-biotinamidohexanoic tetrafluorophenyl (TFP) ester (4) at room temperature. After semipreparative HPLC, compound 5 was isolated in 75% yield and the product was stable in the freezer for more than six months. The TCO-conjugated antibody (TCO–antiVEGFR2) was prepared by combining an excess (20 equiv) of commercially available (E)-cyclooct-4-enyl-2,5-dioxopyrrolidin-1-yl carbonate (TCO–NHS) with antiVEGFR2 (eBioscience) at 4 8C overnight at pH 9.0–9.5. After purification using a 30 kDa centrifugal filter (Amicon Ultra-0.5) MALDITOF MS showed an average of 2.8 TCO groups per antibody in the product. The derivatized bubbles MBTz and MBV were prepared by adding 5 or biotinylated antiVEGFR2, respectively, to freshly reconstituted streptavidin-coated MBs. Isolation of the bubbles from the biotin-containing reagents was accomplished by treating the solution with streptavidin-coated magnetic beads (New England Biolabs), which bound residual tetrazine and [*] A. Zlitni, N. Janzen, Dr. J. F. Valliant Department of Chemistry and Chemical Biology, McMaster University 1280 Main St W., Hamilton, Ont., L8S 4M1 (Canada) E-mail: valliant@mcmaster.ca