General
Unless otherwise stated, all solvents and reagents were obtained from Sigma-Aldrich or Fisher Scientific and were used without further purification. Cyanine7.5 (Cy7.5) was purchased from Lumiprobe (Maryland, USA). Anti-NaV1.7 antibody [N68/6] was purchased from Abcam (ab85015). Water (>18.2 MΩ∙cm at 25°C) was obtained from an Alpha-Q Ultrapure water system (Millipore). Acetonitrile (AcN) was of high-performance liquid chromatography (HPLC) grade and was purchased from Fisher Scientific. Phosphate-buffered saline (PBS) without Ca2+ or Mg2+ was obtained from the Media Preparation Facility at Memorial Sloan Kettering Cancer Center (MSKCC) and used for all in vivo injections. Reverse-phase (RP) HPLC purifications were performed on a Shimadzu HPLC system equipped with a DGU-20A degasser, SPD-M20A UV detector, LC-20AB pump system, and a CBM-20A communication bus module using RP-HPLC columns (Atlantis T3 C18, 5 µm, 4.6 x 250 mm, P/N: 186003748). Epifluorescence imaging was performed on an IVIS Spectrum imaging system (PerkinElmer). Confocal microscopy images were captured using a Leica SP8 inverted-stand confocal microscope equipped a tunable white light laser that ranges from 470 to 670 nm. The microscope is also equipped with a 405 nm diode, argon laser (with 476 nm, 488 nm, 496 nm and 514 nm laser line) and a 725 nm laser for near infra-red NIR imaging coupled with avalanche photo diode detectors (APDs) which were used for detection of Hs1a-FL.
Synthesis of Hs1a
Recombinant Hs1a was produced via expression in the periplasm of E. coli using a protocol optimized for production of disulfide-rich peptides [37]. The recombinant peptide containing a non-native N-terminal glycine residue was purified by nickel affinity chromatography after liberation from the His6-MBP fusion tag via cleavage with tobacco etch virus protease. LC-ESI-MS (ES+), m/z calculated for [C164H251N49O47S6] 3850.74, [C164H251N49O47S6+3H]3+ 1284.58, found [M+3H]3+ 1285.00, [C164H251N49O47S6+4H]4+ 963.69, found [M+4H]4+ 964.20, [C164H251N49O47S6+5H]5+ 771.15, found [M+5H]5+ 772.60, [C164H251N49O47S6+6H]6+ 642.79, found 643.25.
Synthesis of Hs1a-FL
Hs1a was discovered in a high-throughput fluorescent-based assay to screen spider venoms against hNaV1.7, as previously described [38]. Recombinant Hs1a peptide (0.26 mM, 200 µg in 200 µL of AcN) and Na2CO3 (1M, 40 µL) were transferred into a 3 mL amber vial with a magnetic bar stirrer. Cy7.5-NHS (4 µL of a 24 mM solution) was dissolved in AcN and added dropwise to the reaction mixture. The final volume of the reaction mixture was 350 µL. The reaction mixture was stirred for at least 10 min before dilution with 100 µL of water. This reaction produced mono- and di- adducts of Cy7.5, which were purified and separated using RP-HPLC. Fractions containing the mono-adduct of Hs1a-FL were concentrated, then the solvent was removed in vacuo to afford a dark greenish powder (20 µg, 14% yield from Hs1a peptide). This purified compound was then formulated in 100% Ca2+/Mg2+-free PBS or 10% dimethyl sulfoxide (DMSO) and PBS. LC-ESI-MS (ES+), m/z calculated for [C209H298N51O48S6] 4482.12, [C209H298N51O48S6+3H]3+ 1495.04, found [M+3H]3+ 1495.45, [C209H298N51O48S6+4H]4+ 1121.53, found [M+4H]4+ 1121.75, [C209H298N51O48S6+5H]5+ 897.42, found [M+5H]5+ 897.75, [C209H298N51O48S6+6H]6+ 748.02, found [M+6H]6+ 748.25
Cell Lines
HEK293 cells stably expressing the human NaV channel b1 subunit (hNaVb1) in combination with the α subunit hNaV1.1, hNaV1.2, hNaV1.3, hNaV1.4, hNaV1.5, hNaV1.6 or hNaV1.7 (Scottish Biomedical, Glasgow, UK) were cultured in DMEM/F-12 media (1:1), supplemented with 10% fetal bovine serum, 400 mg/mL geneticin and 100 mM non-essential amino acids (all reagents from Invitrogen) at 37°C and in 5% CO2.
Electrophysiology
Whole-cell patch-clamp experiments were performed at room temperature using a QPatch 16x automated electrophysiology platform (Sophion Bioscience, Denmark) using 16-channel planar patch-chip plates (QPlates) with a patch-hole diameter of 1 μm and resistance of 2 MΩ. Whole-cell currents were filtered at 5 kHz (8-pole Bessel) and digitized at 25 kHz. A P4 online leak-subtraction protocol was used with non-leak-subtracted currents acquired in parallel. The extracellular solution was 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 4 mM KCl, 145 mM NaCl at pH 7.4 and the intracellular solution was 140 mM CsF, 1 mM/5 mM EGTA/CsOH, 10 mM HEPES, 10 mM NaCl at pH 7.3. Hs1a-FL were dissolved in extracellular solution with 0.1% bovine serum albumin (BSA). Concentration-response data were obtained using five concentrations of peptide (2 nM to 10 μM). HEK293-hNaV cells were clamped at a holding potential of –60 mV for NaV1.1, –65 mV for NaV1.2, –60 mV for NaV1.3, –75 mV for NaV1.4, –105 mV for NaV1.5, –60 mV for NaV1.6 and –75 mV for NaV1.7. For each concentration, 10 μL of peptide was added for 6 s before applying the following voltage protocol: –80 mV for 10 ms, –120 mV for 200 ms, 0 mV for 20 ms, then return to –80 mV potential. This was repeated once every 60 s during liquid applications. Cells were otherwise held at the holding potential when the above voltage protocol was not executed. Upon establishment of the whole cell recording configuration, a total of five applications of the extracellular solution (1x control buffer, 3x test compound/control, 1 μM tetrodotoxin (TTX; positive control)), all containing 0.1% BSA (except for the TTX solution) were made on each cell. The voltage protocol was executed 10 times after each application. Currents were sampled at 25 kHz and filtered at 5 kHz with an 8-pole Bessel filter. The series resistance compensation level was set at 80%. All experiments were performed at room temperature (~22 °C). IC50 values were determined from non-linear regression of concentration-response data using GraphPad Prism.
Animal Studies
Female athymic nude mice (4–8 weeks old, athymic-Nude (outbred) (Stock#:088; Envigo, USA) were allowed to acclimatize at the MSKCC vivarium for 1 week with ad libitum food and water prior to the experimental procedure. For imaging experiments, animals were sacrificed 30 min post-tail vein injection of Hs1a-FL, Hs1a/Hs1a-FL or PBS. All animal experiments were performed in accordance with institutional guidelines and approved by the MSKCC Institutional Animal Care and Use Committee, following NIH guidelines for animal welfare.
Mouse Cryosectioning and Image-based Reconstruction
Post-euthanasia, a representative mouse was fast frozen in hexanes with dry ice. Coronal cryosectioning and white-light imaging were performed by EMIT using a Xerra imager; following each sequential removal of 50 μm thick slices, the tissue-embedded block was imaged at 30 μm in-plane resolution. A 3D image volume of the mouse was generated through multiplanar reformation using 3D Slicer software for anatomic visualization (Fig. 1a).
Immunohistochemistry
Immunohistochemical (IHC) staining experiments were used to detect the expression and abundance of sodium channel NaV1.7 in mouse sciatic nerve tissue. Anti-NaV1.7 antibody [N68/6] (Abcam ab85015) was found to specifically bind to mouse NaV1.7 (0.5 µg/mL). Paraffin-embedded formalin-fixed 5 µm sections were deparaffinized with EZPrep buffer. For IHC detection, a 3,3'-diaminobenzidine (DAB) detection kit (Ventana Medical Systems, Tucson, AZ) was used according to the manufacturer’s instructions. These experiments were performed at the MSKCC Molecular Cytology Core Facility using the Discovery XT processor (Ventana Medical System, Tucson, AZ). Adjacent sections were stained against IgG, to control for non-specific binding to NaV1.7. Sections were counterstained with hematoxylin and eosin (H&E) and coverslipped with Permount (Fisher Scientific, Pittsburgh, PA) for morphological evaluation of tissue characteristics.
Confocal Microscopy
For the confocal microscopy experiments, 5 µm cryosections of sciatic nerve tissues embedded in optimal cutting temperature compound (OCT) were used to determine the distribution and localization of Hs1a-FL from mice previously injected with the fluorescent agent Hs1a-FL (4 nmol, 45 µM of Hs1a-FL in 100 µL of PBS), the blocking solution Hs1a/Hs1a-FL (Hs1a-FL, 45 µM, 4 nmol and Hs1a 120 µM, 12 nmol in 100 µL PBS), or 100 µL of PBS. These resected nerves were incubated with Hoechst 33342 (20 µM, 1 nmol in 50 µL of PBS) to counterstain nuclei, which were subsequently embedded in Mowiol mounting medium. Fresh tissues were counterstained with Hoechst 33342 (20 µM, 1 nmol in 50 µL of PBS) and samples placed directly on a microscope slide for detection of the fluorescence signal of the fluorescent peptide.
Epifluorescence Imaging
One group of animals was intravenously injected with Hs1a-FL (4 nmol, 45 µM of Hs1a-FL in 100 µL of PBS, n = 3). A second group of animals was injected with Hs1a and Hs1a-FL (Hs1a-FL, 45 µM, 4 nmol and Hs1a, 120 µM, 12 nmol in 100 µL PBS, n = 3) or PBS (n = 3). Animals were sacrificed 30 min post-injection and epifluorescence images obtained. Epifluorescence images of the right sciatic nerve (RSN) and left sciatic nerve (LSN) were obtained in situ from all the mice in the study. Epifluorescence images of the biodistribution included RSN, LSN, muscle, heart, kidney, liver and brain, and were acquired with an IVIS Spectrum imaging system (PerkinElmer) using a predetermined filter set (excitation = 710/45 nm, emission = 800-820 nm). Autofluorescence was removed through spectral unmixing. Semiquantitative analysis of the Hs1a-FL signal was conducted by measuring the average radiant efficiency (in units of [p/s/cm2/sr]/[μW/cm2]) in regions of interest (ROIs) that were placed on all resected nerves and as well in all organs from the biodistribution under white light guidance.
Statistical Analyses
Statistical analyses were performed using GraphPad Prism 8. Unless otherwise stated, data points represent mean values, and error bars represent standard deviations of biological replicates. All p-values were calculated using an unpaired t-test. Statistical significance was considered for p-values < 0.05 and as follows: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.