Fluorescence labeling of a NaV1.7-targeted peptide for near-infrared nerve visualization

Abstract

Background: Accidental peripheral nerve injury during surgical intervention results in a broad spectrum of potentially debilitating side effects. Tissue distortion and poor visibility can significantly increase the risk of nerve injury with long-lasting consequences for the patient. We developed and characterized Hs1a-FL, a fluorescent near-infrared molecule for nerve visualization in the operating theater with the aim of helping physicians to visualize nerves during surgery. Hs1a was derived from the venom of the Chinese bird spider, Haplopelma schmidti, and conjugated to Cy7.5 dye. Hs1a-FL was injected intravenously in mice, and harvested nerves were imaged microscopically and with epifluorescence.

Results: Hs1a-FL showed specific and stable binding to the sodium channel Na_V1.7, present on the surface of human and mouse nerves. Hs1a-FL allowed epifluorescence visualization of sciatic mouse nerves with favorable nerve-to-muscle contrast.

Conclusions: Fluorescent Na_V1.7-targeted tracers have the potential to be adopted clinically for the intraoperative visualization of peripheral nerves during surgery, providing guidance for the surgeon and potentially improving the standard of care.

Keywords: Hs1a-FL; Intraoperative; Near-infrared imaging; Nerve imaging.

Fig. 1

Ion channel selectivity and chemical synthesis of Hs1a-FL. a Representative view of the experimental settings. A 3D rendering of a frozen and sliced mouse. White arrows show the left sciatic nerve. Right sciatic nerve magnification shows the fluorescent Hs1a-FL agent bound to the nerve surface. b Selectivity of Hs1a towards human Na_V channels stably expressed in HEK293 cells. Calculated IC₅₀ values were hNa_V1.1; 19.4 nM, hNa_V1.2; 81.2 nM, hNa_V1.3; 106.8 nM, hNa_V1.4; > 3000 nM, hNa_V1.5; > 3000 nM, hNa_V1.6; 19.2 nM, hNa_V1.7; 26.9 nM. Each point on the curve is an average of 3–11 cells. c Reaction scheme for conjugation of Hs1a peptide with Cyanine7.5-NHS ester dye. The ribbon model of Hs1a-FL shows disulfide bridges (in yellow) and shows the attachment of one dye to the peptide (orange/blue)

Fig. 2

Chemical characterization of Hs1a-FL. a RP-HPLC chromatograms of Hs1a (black) and Hs1a-FL (pink) with absorbances observed at 280 nm. b LC-MS spectrum of Hs1a and c of Hs1a-FL. The mass spectra show four major ion species that correspond to the calculated mass of Hs1a peptide and four major ion species that confirm the calculated mass of Hs1a-FL after dye conjugation. d Fluorescence spectra (Ex/Em 720/835 nm) of 0.1 μM Hs1a peptide (black) and 0.1 μM Hs1a-FL (pink)

Fig. 3

Ex vivo microscopy imaging of Hs1a-FL in mouse sciatic nerve. a Fluorescence of Hs1a-FL-stained mouse sciatic nerves compared to mice injected with vehicle (PBS) or co-injected with Hs1a (Hs1a-FL, 45 μM, 4 nmol and Hs1a 120 μM, 12 nmol in 100 μL PBS). H&E staining of adjacent nerve tissue and IHC staining, confirming the expression of Na_V1.7. b Quantification of total detected fluorescence. Unpaired t test. *p value < 0.05

Fig. 4

Epifluorescence imaging of fresh, unprocessed mouse sciatic nerves with Hs1a-FL. a Epifluorescence images of resected sciatic nerves from animals injected with PBS, Hs1a-FL (4 nmol, 45 μM of Hs1a-FL in 100 μL of PBS), and a Hs1a/Hs1a-FL mixture (Hs1a-FL, 45 μM, 4 nmol and Hs1a 120 μM, 12 nmol in 100 μL PBS). Images were taken 30 min after tail vein injection. b Fluorescence intensity quantification. Unpaired t test. *p value < 0.05; **p value < 0.01