Development and Validation of Nerve-Targeted Bacteriochlorin Sensors

Abstract

We report an innovative approach to producing bacteriochlorins (bacs) via formal cycloaddition by subjecting a porphyrin to a trimolecular reaction. Bacs are near-infrared probes with the intrinsic ability to serve in multimodal imaging. However, despite their ability to fluoresce and chelate metal ions, existing bacs have thus offered limited ability to label biomolecules for target specificity or have lacked chemical purity, limiting their use in bio-imaging. In this work, bacs allowed a precise and controlled appending of clickable linkers, lending the porphyrinoids substantially more chemical stability, clickability, and solubility, rendering them more suitable for preclinical investigation. Our bac probes enable the targeted use of biomolecules in fluorescence imaging and Cerenkov luminescence for guided intraoperative imaging. Bacs' capacity for chelation provides opportunities for use in non-invasive positron emission tomography/computed tomography. Herein, we report the labeling of bacs with Hs1a, a (NaV1.7)-sodium-channel-binding peptide derived from the Chinese tarantula Cyriopagopus schmidti to yield Bac-Hs1a and radiolabeled Hs1a, which shuttles our bac sensor(s) to mouse nerves. In vivo, the bac sensor allowed us to observe high signal-to-background ratios in the nerves of animals injected with fluorescent Bac-Hs1a and radiolabeled Hs1a in all imaging modes. This study demonstrates that Bac-Hs1a and [⁶⁴Cu]Cu-Bac-Hs1a accumulate in peripheral nerves, providing contrast and utility in the preclinical space. For the chemistry and bio-imaging fields, this study represents an exciting starting point for the modular manipulation of bacs, their development and use as probes for diagnosis, and their deployment as formidable multiplex nerve-imaging agents for use in routine imaging experiments.

Figure 1.

Photochemical features, affinity, and validation of bacteriochlorin-based tracers. (A) Absorbance spectra of 0.1 μM compound 6 (dashed blue) and Bac-Hs1a (solid blue), observed from 400–800 nm, and where the classical 730 nm bacteriochlorin peak was detected. (B) Fluorescence spectra of 0.1 μM Hs1a (black), 0.1 μM Bac-FL (dashed blue), and 0.1 μM Bac-Hs1a (solid blue), observed at a range of 700–800 nm, and the classical bacteriochlorin emission wavelength at 730 nm, where the excitation was 680 nm. (C) RP-HPLC chromatograms of the Hs1a peptide (black) and the fluorescent Bac-Hs1a tracer (blue), with retention times of 20 and 22 min, respectively, observed at 280 nm absorption. (D) LC–MS spectra of Bac-Hs1a and Hs1a peptides, showing major ion species that correspond to the calculated mass of the synthetic Hs1a and the fluorescent Bac-Hs1a. (E) Effect of Hs1a and Bac-Hs1a peptides on human NaV1.7. Representative currents of human NaV1.7 (left) in the presence of 500 nM Hs1a (top) or 500 nM Bac-Hs1a (bottom). In addition, a fit of the Hill equation to concentration–response curves (right) showing inhibition by Hs1a (IC₅₀ = 34.9 ± 7.0 nM; n = 7) and Bac-Hs1a (IC₅₀ = 45.2 ± 14.0 nM; n = 6). The peak current amplitude recorded after peptide treatment was normalized to the maximum peak current from the same cell in the absence of the peptide. (F) Exclusive features of compound 5, Bac-FL (6), and Bac-Hs1a comprising absorbance and emission maxima, quantum yields, individual yields, intensity of Q bands, and obtained yields.

Figure 2.

Radiolabeling, characterization, and stability of bacteriochlorin-based tracers. (A) Radiochemical synthesis via the chelation of the bacteriochlorin with ⁶⁴Cu to afford the [⁶⁴Cu]Cu-Bac-Hs1a radiotracer, a radiolabeling that takes place in slightly acidic pH in around 30 min. (B) Radio-HPLC chromatogram of a labeled [⁶⁴Cu]Cu-Bac-Hs1a at 37 °C for 30 min with a corresponding HPLC chromatogram of the cold Bac-Hs1a standard at 280 nm absorption. (C) Radio-HPLC experiments for stability of [⁶⁴Cu]Cu-Bac-Hs1a in saline and serum solution(s), (RCP = the radiochemical purity of [⁶⁴Cu]Cu-Bac-Hs1a, n = 3). (D) Cerenkov luminescence of the formulated [⁶⁴Cu]Cu-Bac-Hs1a and block formulation after radiochemical synthesis before injection.

Figure 3.

Surgical exposure and Bac-Hs1a accumulation in mouse peripheral nerves and corresponding microscopy. (A) Workflow for the fluorescence in vivo experiments of the engineered contrast agent based on a bacteriochlorin molecule. Bac-Hs1a targets sodium channel NaV1.7 highly expressed on peripheral nerves. (B) Epifluorescence images (pre- and post-nerve-exposure) of animals injected with fluorescent agent Bac-Hs1a (1 nmol, 11 μM of Bac-Hs1a in 100 μL PBS) show highlighted nerve tissue on the upper and lower body. Images were taken after 30 min of tail vein injection. (C) Fluorescence intensity semi-quantification of Figure 3B. Statistics were calculated with parametric Student’s t test. **P < 0.01; ****P < 0.0001; and NS, not significant. (D) Biodistribution quantification of mouse peripheral nerves and corresponding organs that were injected with PBS and Bac-Hs1a. High fluorescence intensities, due to bacteriochlorin-Hs1a conjugate accumulation, were only observed in peripheral nerves injected with Bac-Hs1a. No fluorescence was observed after 30 min in mice injected with the vehicle, except for Bac-Hs1a injected spleen, kidney, and liver. (E) Bac-Hs1a uptake was validated in mouse sciatic nerve (first row) fresh tissue from mice injected with Bac-Hs1a (1 nmol, 11 μM of Bac-Hs1a in 100 μL of PBS), where the control was animals injected with 100 μL of PBS. Hoechst 33342 (blue, 20 μM, 1 nmol in 50 μL of PBS) was used to light up Schwann cell nuclei. Adjacent slides were stained with H&E (second row) and anti-NaV1.7 (third row) to validate the topology of the mouse peripheral sciatic nerve. Fluorescence elongated neurons lighted up (fourth row).

Figure 4.

Cerenkov luminescence of [⁶⁴Cu]Cu-Bac-Hs1a, quantification, and biodistribution. (A) Cerenkov luminescence images of mice injected with a block formulation (Hs1a and [⁶⁴Cu]Cu-Bac-Hs1a (left) or [⁶⁴Cu]Cu-Bac-Hs1a alone (right)). High radiance was observed in peripheral nerves (PN) after 30 min in mice injected with [⁶⁴Cu]Cu-Bac-Hs1a alone. (B) Cerenkov luminescence quantification of Figure 4A. (C) Cerenkov luminescence radiance quantification of peripheral nerves, muscle, spleen, heart, kidney, liver, and brain of mice injected with a block formulation and [⁶⁴Cu]Cu-Bac-Hs1a. Statistics were calculated with parametric Student’s t test. ****P < 0.0001.

Figure 5.

PET imaging of [⁶⁴Cu]Cu-Bac-Hs1a in mice, PET signal of resected nerves, and biodistribution quantification. (A) Representative coronal (top) and single transversal (bottom, fused) PET/CT images of mice injected with [⁶⁴Cu]Cu-Bac-Hs1a (3.2–4.2 MBq in 150 μL of PBS) or a block formulation (Hs1a and [⁶⁴Cu]Cu-Bac-Hs1a in 150 μL of PBS). A high %ID/g is observed after 60 min in mice injected with a radiotracer alone. In addition, for the block formulation, some %ID/g is observed in nerves, spleen, and liver after 60 min as well. (B) PET images of resected nerves from animals injected with radiotracer [⁶⁴Cu]Cu-Bac-Hs1a. (C) PET imaging quantification (%ID/g) of sciatic nerves, muscle, spleen, heart, kidney, liver, and brain of mice injected with the block formulation and [⁶⁴Cu]Cu-Bac-Hs1a. Statistics were calculated with parametric Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Scheme 1.. Mechanism To Form Conjugable Bacteriochlorin Sensors via Formal Cycloaddition^a

^aThis synthetic scheme mechanism for bacteriochlorin formation includes a transformation that generates solvent effects and resonance for inducement of formal cycloaddition. This trimolecular reaction begins with the Y1 attack and resonance on the porphyrin core to generate an aromatic substitution attack to a second Y1, which forms and initiates the first pyrrolidine and resonance, finally closing the second pyrrolidine ring and generating the bacteriochlorin. The compound formed is 2, which loses an aldehyde and water. The final step is a disproportionation reaction that affords compound 4. In this reaction, (i) mixed-grinded ylide comprised of glycine/paraformaldehyde (1:2), with aliquots added constantly over at least 10 h in DMF, 150–170 °C, 24–36 h, 20% yield.

Scheme 2.. Synthesis and Characterization of Bac-FL, Bac-Hs1a, and Amino Acid Sequence^a

^a(A) Synthetic scheme of a bacteriochlorin sensor and the activation with an NHS-ester moiety that provides target specificity when conjugated to a targeting molecule. To compound 4, (ii) 6 equiv succinic anhydride, DCM, NEt₃, room temperature, 45 min, 90% yield. (iii) 1.2 equiv N-hydroxysuccinimide, THF, 1.1 equiv DCC, r.t., 30% yield. (B) Bacteriochlorin-based molecules under white light (left) and under excitation at 680 nm (right), formulated in PBS for intravenous injection. (C) Reaction scheme for the covalent conjugation of the bacteriochlorin moiety to the Hs1a peptide under basic conditions to afford Bac-Hs1a. The ribbon model of Bac-Hs1a shows disulfide bridges (in orange), the amino acid sequence (purple), and the bacteriochlorin (black), a conjugation that takes place in at least 10 min. (D) Amino acid sequences of Hs1a and Bac-Hs1a peptides containing 35 amino acids; Bac-Hs1a contains a bacteriochlorin molecule at K₁₄. The connectivity (in orange) for the cystines in Hs1a is C₄–C₁₉, C₁₁–C₂₄, and C₁₈–C₃₁.