Scott A. Hilderbrand

Near-infrared fluorescence: application to in vivo molecular imaging

Molecular imaging often relies on the use of targeted and activatable reporters to quantitate and visualize targets, biological processes, and cells in vivo. The use of optical probes with near-infrared fluorescence allows for improved photon penetration through tissue and minimizes the effects of tissue autofluorescence. There are several parameters that define the effectiveness of imaging agents in vivo. These factors include probe targeting, activation, pharmacokinetics, biocompatibility, and photophysics. Recent advances in our understanding of these variables as they pertain to the application of optical reporters for in vivo imaging are discussed in this review.

Tetrazine-Based Cycloadditions: Application to Pretargeted Live Cell Imaging

Bioorthogonal tetrazine cycloadditions have been applied to live cell labeling. Tetrazines react irreversibly with the strained dienophile norbornene forming dihydropyrazine products and dinitrogen. The reaction is high yielding, selective, and fast in aqueous media. Her2/neu receptors on live human breast cancer cells were targeted with a monoclonal antibody modified with a norbornene. Tetrazines conjugated to a near-infrared fluorochrome selectively and rapidly label the pretargeted antibody in the presence of serum. These findings indicate that this chemistry is suitable for in vitro labeling experiments, and suggests that it may prove a useful strategy for in vivo pretargeted imaging under numerous modalities.

Fast and Sensitive Pretargeted Labeling of Cancer Cells through a Tetrazine/trans-Cyclooctene Cycloaddition†

There is considerable interest in the use of bioorthogonal covalent chemistry such as “click” chemistries to label small molecules located on live or fixed cells.[1] Such labeling has been used for the visualization of glycans, activity based protein profiling, site-specific tagging of proteins, detection of DNA and RNA synthesis, revealing the fate of small molecules in plants, and detection of post-translational modification in proteins.[2-4] Most reported applications rely on either the copper catalyzed azide-alkyne cycloaddition, which is limited to in vitro application due to the cytotoxicity of copper, or the elegant strain-promoted azide-alkyne cycloaddition, which permits live cell and in vivo application use but is hindered by relatively slow kinetics and often difficult synthesis of cyclooctyne derivatives.[4-5] New bioorthogonal reactions that do not require catalyst and show rapid kinetics are therefore of interest for different molecular imaging applications at the cellular level. In this report we demonstrate the use of inverse electron demand Diels-Alder cycloaddition between a serum stable 1,2,4,5 tetrazine and a highly strained trans-cyclooctene to covalently label live cells. This chemistry has been applied to the pretargeted labeling of Cetuximab (Erbitux) tagged epidermal growth factor receptor (EGFR) on A549 cancer cells. We find that the tetrazine cycloaddition to trans-cyclooctene labeled cells is fast and can be amplified by increasing the loading of dienophile on the antibody. This results in a highly sensitive targeting strategy that can be used to label proteins using nanomolar concentrations of a secondary agent for short durations of time.

Upconverting Luminescent Nanomaterials: Application to In Vivo Bioimaging

In this report, the development of multi-channel anti-Stokes luminescent Y2O3 nanoparticles for application to in vivo upconversion imaging is detailed.

Bioorthogonal chemistry amplifies nanoparticle binding and enhances the sensitivity of cell detection

Nanoparticles have emerged as key materials for biomedical applications because of their unique and tunable physical properties, multivalent targeting capability, and high cargo capacity. Motivated by these properties and by current clinical needs, numerous diagnostic and therapeutic nanomaterials have recently emerged. Here we describe a novel nanoparticle targeting platform that uses a rapid, catalyst-free cycloaddition as the coupling mechanism. Antibodies against biomarkers of interest were modified with trans-cyclooctene and used as scaffolds to couple tetrazine-modified nanoparticles onto live cells. We show that the technique is fast, chemoselective, adaptable to metal nanomaterials, and scalable for biomedical use. This method also supports amplification of biomarker signals, making it superior to alternative targeting techniques including avidin/biotin.

Synthesis and Evaluation of a Series of 1,2,4,5-Tetrazines for Bioorthogonal Conjugation

1,2,4,5-Tetrazines have been established as effective dienes for inverse electron demand [4 + 2] Diels-Alder cycloaddition reactions with strained alkenes for over 50 years. Recently, this reaction pair combination has been applied to bioorthogonal labeling and cell detection applications; however, to date, there has been no detailed examination and optimization of tetrazines for use in biological experiments. Here, we report the synthesis and characterization of 12 conjugatable tetrazines. The tetrazines were all synthesized in a similar fashion and were screened in parallel to identify candidates most ideally suited for biological studies. In depth follow-up studies revealed compounds with varying degrees of stability and reactivity that could each be useful in different bioorthogonal applications. One promising, highly stable, and water-soluble derivative was used in pretargeted cancer cell labeling studies, confirming its utility as a bioorthogonal moiety.

Development of a Bioorthogonal and Highly Efficient Conjugation Method for Quantum Dots using Tetrazine-Norbornene Cycloaddition

We present a bioorthogonal and modular conjugation method for efficient coupling of organic dyes and biomolecules to quantum dots (QDs) using a norbornene-tetrazine cycloaddition. The use of noncoordinating functional groups combined with the rapid rate of the cycloaddition leads to highly efficient conjugation. We have applied this method to the in situ targeting of norbornene-coated QDs to live cancer cells labeled with tetrazine-modified proteins.

Bioorthogonal reaction pairs enable simultaneous, selective, multi-target imaging.

Mutually orthogonal tetrazine–transcyclooctene and azide–cyclooctyne cycloaddition reactions were used simultaneously for the bioorthogonal labeling of two different live cell populations in the same culture. These small-molecule probes show good chemical reactivity and can be readily incorporated into biological systems.

BODIPY–Tetrazine Derivatives as Superbright Bioorthogonal Turn-on Probes†

The fastest and the brightest: A new design that intimately connects tetrazine to a BODIPY fluorophore enables exceptionally efficient energy transfer and quenching. Upon reaction of the tetrazine, the brightness of the fluorophore increases more than a thousand-fold, a fluorogenic activation up to two orders of magnitude greater than previously described.

Modular Strategy for the Construction of Radiometalated Antibodies for Positron Emission Tomography Based on Inverse Electron Demand Diels–Alder Click Chemistry

A modular system for the construction of radiometalated antibodies was developed based on the bioorthogonal cycloaddition reaction between 3-(4-benzylamino)-1,2,4,5-tetrazine and the strained dienophile norbornene. The well-characterized, HER2-specific antibody trastuzumab and the positron emitting radioisotopes 64Cu and 89Zr were employed as a model system. The antibody was first covalently coupled to norbornene, and this stock of norbornene-modified antibody was then reacted with tetrazines bearing the chelators 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) or desferrioxamine (DFO) and subsequently radiometalated with 64Cu and 89Zr, respectively. The modification strategy is simple and robust, and the resultant radiometalated constructs were obtained in high specific activity (2.7–5.3 mCi/mg). For a given initial stoichiometric ratio of norbornene to antibody, the 64Cu-DOTA- and 89Zr-DFO-based probes were shown to be nearly identical in terms of stability, the number of chelates per antibody, and immunoreactivity (>93% in all cases). In vivo PET imaging and acute biodistribution experiments revealed significant, specific uptake of the 64Cu- and 89Zr-trastuzumab bioconjugates in HER2-positive BT-474 xenografts, with little background uptake in HER2-negative MDA-MB-468 xenografts or other tissues. This modular system—one in which the divergent point is a single covalently modified antibody stock that can be reacted selectively with various chelators—will allow for both greater versatility and more facile cross-comparisons in the development of antibody-based radiopharmaceuticals.