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Published in final edited form as: ACS Appl Mater Interfaces. 2025 Jul 22;17(31):44178–44187. doi: 10.1021/acsami.5c08038

Tumor-Embedded Nerve Surgery Using Dual-Channel Intraoperative Imaging Probes

Eun Bi Jun 1,2,3,†, Kyungsu Kim 1,2,3,†, Jun Hee Lee 1,2,3, Seung Hun Park 4, Satoshi Kashiwagi 4, Kai Bao 4, Hyun Koo Kim 1,2,3,*, Hak Soo Choi 4,*
PMCID: PMC12952925  NIHMSID: NIHMS2147597  PMID: 40695740

Abstract

During cancer surgery, the identification of normal structures such as nerves is difficult owing to anatomical shifts around the tumor, which increases the risk of nerve injury. Accidental nerve damage can lead to severe complications, including chronic pain, numbness, motor dysfunction, aspiration and respiratory difficulties. Therefore, precise nerve identification and preservation are critical, even when nerves are located near the tumor. Current nerve-targeted fluorescent contrast agents designed for intravenous administration pose potential risks of systemic side effects. To address this, we developed nerve-specific near-infrared (NIR) fluorophores, including NTFP700, for local application at a wavelength of 700 nm and evaluated its ability to selectively image nerves. Additionally, we explored a dual-channel NIR imaging approach by combining NTFP700 with a tumor-targeted contrast agent (cRGD-ZW800-PEG, 800 nm) to enable clear differentiation of nerves and tumors. NTFP700 effectively highlighted nerve structures with strong fluorescence signals, and its application using a dyed-gauze method allowed for improved visualization, particularly in anatomically altered nerve structures. When combined with cRGD-ZW800-PEG, dual-channel NIR imaging successfully distinguished nerves from tumors with distinct fluorescent signals. These findings demonstrate the potential of NTFP700 as a nerve-targeting fluorophore for local application. The integration of NTFP700 with a tumor-targeting contrast agent, combined with the surgeon-friendly dyed-gauze method, provides a promising strategy for precise nerve and tumor differentiation, potentially reducing the risk of nerve-related complications in oncological procedures.

Keywords: nerve imaging, cancer targeting, fluorescence contrast agent, dual-channel imaging, dyed gauze method

Graphical Abstract

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INTRODUCTION

Iatrogenic nerve injury refers to unintended nerve damage resulting from medical or surgical interventions.1, 2 It can lead to severe complications, such as chronic pain, numbness, motor dysfunction, aspiration, and respiratory difficulties ultimately affecting post-surgical quality of life.3, 4 Nerve identification can be particularly challenging in cancer surgery owing to anatomical alterations caused by tumor growth, especially with the increasing adoption of minimally invasive techniques.5–7 For instance, recurrent laryngeal nerve injury occurs in up to 50% of esophagectomy and thyroidectomy cases, potentially leading to hoarseness, aspiration, and respiratory difficulties.8, 9 Similarly, cavernous nerve injury has been reported in up to 70% of prostatectomy cases, resulting in sexual dysfunction and urological disorders.10–12 Thus, precise intraoperative nerve identification and preservation are critical for improving patient outcomes.13

Several techniques, including electromyography, ultrasound, and optical coherence tomography, are currently used for intraoperative nerve identification.14, 15 However, these methods have limitations in terms of field of view, resolution, and real-time feedback, making them suboptimal for surgical guidance.5 Near-infrared (NIR) fluorescence imaging has emerged as a powerful tool for fluorescence-guided surgery (FGS), particularly in oncology.16–20 Nerve-specific fluorescent contrast agents allow real-time intraoperative visualization, reducing the risk of accidental nerve injury.7

Despite the increasing interest in nerve-targeting fluorescent contrast agents, most available agents are designed for intravenous administration.21–24 However, intravenous delivery poses risks, such as allergic reactions, nephrotoxicity, and pulmonary edema. In contrast, topical administration at the nerve site offers a faster, safer, and more targeted approach, minimizing systemic side effects, as demonstrated in previous studies using in situ spraying techniques for intraoperative fluorescence imaging.25, 26 Nonetheless, unintended diffusion beyond the nerve region may reduce visualization specificity and complicate surgical decision-making.27, 28 Furthermore, conventional topical application methods may be less effective in visualizing nerves located at steep angles or in anatomically distorted regions owing to tumor growth.

To address these challenges, we developed NTFP700 (Nerve Targeting Fluorescence Probe-700nm), a novel nerve-targeted fluorescent contrast agent optimized for intraoperative nerve visualization with minimal systemic toxicity. NTFP700 operates at 700 nm within the NIR spectrum (650–900 nm), enabling clear nerve identification during FGS. To improve staining efficiency, particularly in complex anatomical regions, we introduced a dyed-gauze method, which enhances visualization using readily available clinical materials. Additionally, we developed cRGD-ZW800-PEG, a tumor-targeting NIR fluorophore operating at 800 nm, allowing precise tumor delineation during surgery.29

We integrated NTFP700 and cRGD-ZW800-PEG to establish a dual-channel FGS, enabling simultaneous visualization of nerves and tumors in distinct colors. This approach enhances surgical precision by reducing the risk of nerve injury while ensuring complete tumor resection. This study aimed to assess the feasibility and effectiveness of this novel FGS in preclinical models.

RESULTS

Synthesis and Characterization of NIR Fluorophores

The NIR fluorophores utilized in this study, including NTFP700, Oxazine 1, Oxazine 4, and cRGD-ZW800-PEG, were successfully synthesized and characterized (Figure 1A). High-purity levels of all compounds were confirmed using Waters LC-MS, ensuring their suitability for physicochemical and optical analyses. To ensure chemical integrity and reproducibility, LC-MS and HPLC analyses were performed for all fluorophores. Each compound exhibited a sharp, single peak at the expected retention time in HPLC chromatograms, and purity was confirmed to be ≥90% (Figure S1). Corresponding mass spectra matched the expected molecular ions or adducts, validating their identity and consistency with supplier specifications.

Figure 1. Chemical structure and in vivo imaging of nerve-targeting fluorescent contrast agents.

Figure 1.

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(A) Chemical structure and molecular weight (MW) of NTFP700, Oxazine4 (Ox4), Oxazine1 (Ox1) and Indocyanine Green (ICG). (B) In vivo fluorescence imaging of the mouse sciatic nerve was performed using topical administration and intravenous injection of NTFP700, Ox4, Ox1, and ICG. NTFP700 and Ox4 were imaged in the 700 nm NIR channel, Ox1 was imaged in the 600 nm NIR channel, and ICG was imaged in the 800 nm NIR channel, respectively. White arrowheads indicate nerve bundles. Scale bars = 2mm. Images are representative of n = 3 independent experiments. All NIR fluorescence images have identical exposure times (200 ms) and normalizations.

In silico calculations of physicochemical properties, performed using MarvinSketch and InstantJChem, indicated that NTFP700 (MW: 383.55), Oxazine 1 (MW: 395.84), and Oxazine 4 (MW: 423.89) are similar in size and possess a positive charge. Notably, the logD values at pH 7.4 for NTFP700 and Oxazine 4 were 3.56 and 3.38, respectively, whereas Oxazine 1 exhibited a logD of 0.33 (Figure S2A). The blood-brain barrier scores, calculated based on five physicochemical descriptors, were 4.76 for NTFP700, 4.69 for Oxazine 4, and 5.20 for Oxazine 1, suggesting potential differences in their nerve-targeting behaviors. Absorbance and fluorescence emission spectra further confirmed their suitability for dual-channel intraoperative imaging (Figure S2B, C).

In vivo Screening of Nerve-Specific Fluorophores

The nerve specificity of NTFP700 was assessed by comparing its fluorescence performance with Oxazine 4, Oxazine 1, and Indocyanine green (ICG) following both topical administration to the sciatic nerve and intravenous injection. Upon topical application, NTFP700 distinctly highlighted the sciatic nerve (SN) with minimal background fluorescence. This superior nerve contrast can be attributed to its high lipophilicity (logD = 3.56) and positive charge, which enable strong hydrophobic and electrostatic interactions with the negatively charged, lipid-rich myelin sheath. These interactions enable selective accumulation at nerve surfaces, particularly under topical application, leading to high signal-to-background contrast.

In contrast, although Oxazine 4 shares a similar lipophilicity (logD = 3.38), its polar and partially ionized structure at physiological pH facilitates nonspecific uptake in adipose and other off-target tissues, leading to diffuse background fluorescence.5 While Oxazine 4 did succeed in visualizing nerves after intravenous administration, consistent with previous reports, its specificity was inferior to NTFP700 under topical conditions.24 Oxazine 1, with the lowest logD value of 0.33, is relatively hydrophilic and distributes broadly in vascular and aqueous compartments. As a result, it displays high background fluorescence and poor nerve contrast, failing to provide effective nerve-targeted imaging.

ICG, despite its high lipophilicity, contains negatively charged sulfonate groups that hinder electrostatic interactions with the negatively charged myelin sheath due to charge repulsion. In addition, rapid binding to plasma proteins contributes to nonspecific distribution and poor nerve retention, ultimately limiting its utility for nerve-specific imaging. Following intravenous injection, NTFP700 did not generate sufficient contrast for nerve delineation, further supporting the importance of topical administration to fully leverage its physicochemical advantages (Figure 1B).

Optimization of NTFP700 Topical Administration Protocol

Dose optimization for topical administration in mice demonstrated that higher doses (50–100 μM) enhanced nerve fluorescence but also increased background staining in surrounding muscle and fat, whereas lower doses (1–10 μM) produced weak nerve signals (Figure 2A). A concentration of 25 μM yielded the highest SBR in both the sciatic nerve (SN: 3.93 ± 0.55) and brachial plexus (BP: 4.18 ± 0.29) compared to other doses (****P < 0.0001) (Figure 2B).

Figure 2. Optimization of NTFP700 uptake for topical administration in mice.

Figure 2.

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(A) Representative sciatic nerve (SN) and brachial plexus (BP) images following staining with various NTFP700 concentrations (1–100 μM). White arrowheads indicate nerve bundles. (B) Quantified fluorescence intensity and SBR (mean ± SD) for SN and BP. (C) Representative SN and BP images after staining with different incubation times (1, 3, and 5 min). (D) Quantified fluorescence intensity and SBR (mean ± SD) for different incubation times. (E) Representative SN and BP images after varying washing times (0–10 washes). (F) Quantified fluorescence intensity and SBR (mean ± SD) after different washing conditions. All images represent data from n = 4 nerve sites. ****P < 0.0001. Scale bars = 2 mm. All NIR fluorescence images were acquired with identical exposure times (200 ms) and normalization settings.

Optimization of the incubation time (1, 3, and 5 min) indicated that prolonged exposure led to nonspecific staining, thereby reducing nerve contrast (Figure 2C). An incubation period of 1 min provided the highest SBR (SN: 3.81 ± 0.45, BP: 3.92 ± 0.19, ****P < 0.0001) (Figure 2D). Further refinement of the washing protocol involved incubating 25 μM NTFP700 for 1 min, followed by 1–10 washes with PBS (Figure 2E). A single wash effectively reduced background fluorescence while maintaining nerve signal integrity (SBR: SN: 4.46 ± 0.22, BP: 3.67 ± 0.45) (Figure 2F). As additional washes did not significantly improve the SBR, a single wash was deemed the optimal protocol, providing a practical and efficient method for intraoperative use.

Retention Time of NTFP700 in Nerve Tissues

Following topical administration, NIR fluorescence imaging was performed at multiple time points (1–600 min) (Figure 3A). The highest SBR (4.25 ± 0.19) was observed at 1 min, ensuring optimal nerve visualization. The contrast remained sufficient at 60 min (3.38 ± 0.02) and 120 min (2.97 ± 0.15) but gradually declined beyond 240 min (Figure 3B). Exponential decay curve fitting estimated a fluorescence signal half-life of 181.4 min (Figure 3C). Additionally, no toxicity was observed in blood samples or major organs 2 weeks post-administration (Figures S3, S4A–B).

Figure 3. Time-dependent fluorescence imaging of NTFP700.

Figure 3.

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(A) Representative color and NIR fluorescence images captured at various time points (1, 15, 30, 60, 120, 240, 360, 480, and 600 min) following topical administration of NTFP700 to the sciatic nerve. (B) Quantification of fluorescence intensity and SBR (mean ± SD, n = 3). **P < 0.01. (C) Analysis of fluorescence signal decay, showing the calculated half-life of NTFP700 in the mouse sciatic nerve (n = 3). Scale bars = 2 mm. All NIR fluorescence images have identical exposure times (200 ms) and normalizations.

Comparison of Topical Administration and Dyed Gauze Methods

The efficacy of the topical application and dyed gauze methods for nerve staining was compared (Figure 4A). NTFP700 exhibited differences in staining efficiency, particularly in regions with steep anatomical angles. In the topical administration method, weaker and uneven staining was observed at steep angles due to the downward flow of NTFP700 (Figure 4B, red arrowhead). Conversely, the dyed gauze method provided uniform staining across the nerve, even in regions with steep angles (Figure 4C, yellow arrowhead).

Figure 4. Comparison of topical administration and dyed gauze methods for nerve staining.

Figure 4.

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(A) Schematic representation of the topical administration and dyed gauze methods for nerve staining. (B, C) Representative sciatic nerve (SN) fluorescence images obtained using NTFP700 with each method. The red arrowhead highlights a nerve imaged at a steep anatomical angle using the topical administration method, whereas the yellow arrowhead indicates the corresponding nerve visualized using the dyed gauze method. Yellow lines denote the nerve angles. (D) Quantification of fluorescence intensity, expressed as tumor-to-background and nerve-to-background signal ratios (mean ± SD). Data are representative of N = 3 independent experiments. (E, F) Simultaneous intraoperative dual-channel fluorescence imaging of tumor (cRGD-ZW800-PEG, 800 nm NIR) and nerve (NTFP700, 700 nm NIR) in a mouse tumor model. The white dotted line outlines the tumor. White arrowheads indicate nerves imaged at flat anatomical angles, while yellow arrowheads highlight nerves imaged at steep angles due to the presence of the tumor.

Quantitative analysis confirmed that both methods produced sufficient SBR (> 3.0) at flat angles. However, at steep angles, the dyed gauze method significantly improved nerve contrast (SBR: 3.91 ± 0.25) compared to topical administration (Figure 4D). These findings suggest that the dyed gauze method is superior for nerve visualization, particularly in challenging anatomical regions (Figure 4E, F).

Simultaneous Visualization of Nerve and Tumor Using Dual-Channel Fluorescence Imaging in a Rabbit Cancer Model

The clinical feasibility of using NTFP700 for nerve recognition and cRGD-ZW800-PEG for tumor detection was evaluated in a rabbit subcutaneous cancer model. To enable tumor visualization, cRGD-ZW800-PEG was intravenously injected into the VX2 rabbit model 4 h prior to surgery. Dual-channel fluorescence imaging using NTFP700-dyed gauze facilitated the simultaneous visualization of nerves (700 nm) and tumors (800 nm) during surgery (Figure 5A). The merged imaging channel distinctly differentiated nerves (blue) from tumors (green), allowing for precise nerve preservation and tumor resection (Figure 5B, Movie S1).

Figure 5. Precision tumor surgery guided by dual-channel fluorescence imaging in a rabbit tumor model.

Figure 5.

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(A) Schematic illustration of dual-channel fluorescence-guided surgery using cRGD-ZW800-PEG for tumor visualization and NTFP700 for nerve identification. cRGD-ZW800-PEG was administered intravenously 4 h before surgery, while NTFP700 was applied intraoperatively using the dyed gauze method. (B) Representative intraoperative dual-channel fluorescence imaging demonstrating simultaneous tumor (green) and nerve (blue) visualization. The white dotted line outlines the tumor, while the yellow arrowhead indicates the nerve. (C) Histological analysis of the rabbit sciatic nerve using H&E, NeuroTrace, FluoroMyelin, and NTFP700 staining. Shown are representative H&E and fluorescence microscopy images of the sciatic nerve (top), with squares highlighting regions enlarged at 20× magnification (bottom). Scale bars: 200 μm (top) and 50 μm (bottom). All fluorescence images were acquired under identical exposure times and normalization settings for consistency.

Histological analysis of sciatic nerve sections stained in vivo using the dyed gauze method confirmed nerve-specific fluorescence. H&E, NeuroTrace, and FluoroMyelin staining verified nerve morphology and co-localization of NTFP700 with myelin-rich areas. NeuroTrace and FluoroMyelin, which specifically stain Nissl substance and myelin, respectively, demonstrated strong correlation with NTFP700 fluorescence signals (Figure 5C).

DISCUSSION

Minimally invasive resection aims to enhance the quality of life for cancer patient’s post-surgery;30, 31 however, nerve injuries remain an unavoidable complication.8, 32 The intricate anatomical structures surrounding nerves pose substantial challenges for their identification during oncologic surgery.33 Accurate intraoperative nerve visualization is crucial for minimizing iatrogenic nerve damage and preserving function.34 The development of effective NIR fluorophores for nerve-specific imaging represents a significant advancement in intraoperative visualization, providing surgeons with a powerful tool to enhance surgical precision.

In this study, we developed NTFP700, a 700-nm-emitting NIR fluorophore specifically designed for fluorescence image-guided surgery, and evaluated its clinical feasibility. Additionally, we introduced a dyed gauze method to improve the practical application of NTFP700 in surgical settings, enabling more effective and consistent nerve staining.

Previously reported topical administration methodologies optimized fluorophores, such as Oxazine 4, Oxazine 1, and ICG, for nerve visualization.24, 35 While Oxazine 4 demonstrated nerve visualization via intravenous injection, topical administration of NTFP700 provided significantly higher contrast, leading to clearer and more precise nerve identification. Dose optimization studies revealed that topical administration of 25 μM NTFP700, followed by 1-min incubation and a single wash, achieved the highest SBR while minimizing nonspecific staining. Furthermore, retention analysis demonstrated that NTFP700 maintained effective contrast for nerve visualization for up to 240 min, with a fluorescence half-life exceeding 180 min. These findings indicate that NTFP700 is well-suited for intraoperative use and has strong potential for clinical translation.36–39

To further evaluate its feasibility in cancer surgery, we compared the dyed gauze method with direct topical administration. The dyed gauze method provided more uniform staining across the entire nerve, particularly in anatomically complex regions where topical application resulted in uneven staining owing to steep angles. These findings highlight the superiority of the dyed gauze approach for achieving consistent and reliable nerve visualization, especially in challenging surgical scenarios.

Moreover, dual-channel fluorescence imaging using NTFP700 for nerve visualization and cRGD-ZW800-PEG for tumor identification enabled simultaneous differentiation of nerves and tumors. This approach facilitated precise nerve preservation while guiding tumor resection, demonstrating its efficacy in both mouse and rabbit tumor models. These results underscore the potential of dual-channel fluorescence imaging for improving surgical precision in oncologic procedures. By integrating this imaging strategy, surgeons can simultaneously visualize nerves and tumors, minimizing the risk of nerve injury and optimizing surgical outcomes.

Despite these promising findings, certain limitations of this study warrant further investigation. The lipophilic nature of NTFP700 resulted in some non-specific staining in the adipose tissue, which should be addressed in future fluorophore modifications. Additionally, as this study was conducted exclusively in mouse and rabbit models, further preclinical and clinical evaluations in human nerves are essential to validate the specificity, efficacy, and safety of NTFP700 before its widespread clinical adoption.

CONCLUSIONS

This study establishes NTFP700 as a promising nerve-specific NIR fluorophore, optimized for intraoperative nerve visualization via a topical administration protocol. The dyed gauze method enhances nerve staining in complex anatomical regions, while dual-channel imaging with cRGD-ZW800-PEG enables simultaneous nerve and tumor differentiation. By integrating optimized fluorophore design, administration protocols, and imaging techniques, we have developed a powerful tool for improving surgical precision and patient outcomes. These findings lay the groundwork for the clinical translation of FGS techniques, ultimately advancing surgical precision and postoperative quality of life for patients with cancer.

METHODS

Preparation of NIR Fluorophores

NTFP700 (1,1’,3,3,3’,3’-hexamethylindodicarbocyanine iodide), Oxazine1 (3,7-bis(diethylamino)phenoxazin-5-ium perchlorate), and Oxazine4 (3,7-bis(ethylamino)-2,8-dimethyl-phenoxazin-5-iuperchlorate) were synthesized following previously reported protocols with minor modifications,24, 40 while ICG (sodium; 4-[(2Z)-2-[(2E,4E,6E)-7-[1,1-dimethyl-3-(4-sulfonatobutyl)benzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonate) was purchased from TCI America (Portland, OR). cRGD-ZW800-PEG was synthesized as described in a prior study.29 All chemicals were obtained from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich (St. Louis, MO). Solvents used in this study were of reagent grade or higher and used without further purification. The purity of the compounds was assessed using Waters liquid chromatography-mass spectrometry (LC-MS), incorporating an Alliance e2695 separation module, a 2998 PDA detector (212–800 nm), and an Acquity QDA detector.

Physicochemical and Optical Property Measurements

In silico physicochemical properties, including molecular weight (MW), distribution coefficient (LogD at pH 7.4), surface molecular charge, hydrophobicity, hydrogen bond acceptors/donors, acid dissociation constant (pKa), and topological polar surface area, were calculated using the MarvinSketch (ver. 23.11) and InstantJChem (ver. 23.17) calculator plug-ins (ChemAxon, Budapest, Hungary). The absorbance and fluorescence emission spectra of each fluorophore were measured using an HR2000 absorbance spectrometer (200–1,100 nm) and a USB2000FL fluorescence spectrometer (350–1,000 nm) from Ocean Optics (Dunedin, FL). Fluorescence emission spectra were recorded under NIR excitation using 660 nm and 760 nm laser pointers (Opcom Inc., Xiamen, China). The quantum yield (QY) of the fluorophores was determined using ICG in DMSO (QY = 13%) as the reference standard.

Tumor-Bearing Xenograft Mouse Model

All animal experiments, including care and handling, were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Korea University (Approval No. KOREA-2023–0189). Five-week-old C57BL/6 mice (20–25 g) were obtained from Orient Biotech (Seoul, South Korea) and housed individually with ad libitum access to food and water. Mice were acclimated for 1–2 weeks under humane animal care protocols.

For the xenograft model, luciferase-transfected Lewis lung carcinoma (LL/2-Luc2) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Waltham, MA) supplemented with 10% fetal bovine serum and 1% antimycotic (Thermo Fisher Scientific, Waltham, MA) under standard conditions (37 °C, 5% CO2, humidified atmosphere). A suspension of 2 × 105 cells in 100 μL was injected into the sciatic nerve region of 6-week-old C57BL/6 mice. Tumor growth was monitored 2–3 weeks post-inoculation via intraperitoneal injection of luciferin (15 mg/mL), followed by luminescence imaging using the In Vivo Imaging System (Davinch-K, South Korea).

VX2 Tumor-Bearing Rabbit Model

For the VX2 tumor model, fresh VX2 carcinoma tissue was harvested using a surgical blade and rinsed with phosphate-buffered saline (PBS) to remove necrotic material. The tissue was minced, passed through a 100-μm cell strainer to create a suspension, centrifuged at 1,200 rpm for 3 min, and re-suspended in PBS at 1 × 107 cells/mL. The VX2 cell suspension was mixed with 100 μL of Matrigel and loaded into a 1-mL syringe with a 23-gauge needle.

Female New Zealand white rabbits (2.0–2.5 kg) were obtained from Koatech (Seoul, South Korea). Anesthesia was induced via intramuscular administration of xylazine (5 mg/kg; Rompun, Bayer, Seoul, South Korea) and tiletamine-zolazepam (10 mg/kg; Zoletil 50, Virbac Korea Inc., Seoul, South Korea). The thigh region was shaved and disinfected with povidone-iodine. A mixture of VX2 cells and Matrigel was injected into the muscle adjacent to the sciatic nerve, and the VX2 thigh tumor model was established within 2–3 weeks post-injection.

NIR Fluorescence Imaging System

For dual-channel NIR fluorescence imaging, 630 nm excitation light (700 nm NIR) at 1.0 mW/cm2 and 760 nm excitation light (800 nm NIR) at 1.0 mW/cm2 were used, alongside white light illumination (400–650 nm) at 5,500 lux. The dual-channel NIR FLARE imaging system was previously described in detail.29 Custom software controlled the imaging system at up to 15 Hz, with the field of view manually adjustable using a macro zoom lens (0–10×; Navitar Zoom 7000 with SWIR coating).

In merged color-NIR images, the 700-nm fluorescence signal was pseudo-colored blue, while the 800-nm fluorescence signal was pseudo-colored green. The imaging head was positioned 9 inches from the surgical field, and all NIR fluorescence images were captured under identical exposure times and normalized for consistency.

Comparison of NIR Nerve Fluorophores

The neural specificity of NTFP700 was evaluated against Oxazine4, Oxazine1, and ICG using both topical and intravenous administration methods. For topical administration, the sciatic nerve of anesthetized mice was surgically exposed, and 100 μL of each fluorophore (62.5 μM in 1X PBS) was applied directly to the nerve. After a 1-min incubation, excess fluorophore was removed using gauze. Fluorescence images were acquired using the In Vivo Imaging System (Davinchi-K) under each fluorophore’s optimal excitation and emission conditions. For IV administration, 100 μL of each fluorophore (2 mM in 1X PBS) was injected via the tail vein. After a 4-h circulation period, the sciatic nerve was surgically exposed, and fluorescence images were captured using the same imaging system.

Optimization of Administration Protocol

To optimize NTFP700 administration, experiments were conducted to determine the ideal dose, incubation time, and washing protocol using C57BL/6 mice (n = 4). To optimize the dose, NTFP700 was applied directly to the exposed nerve at concentrations ranging from 1–100 μM. After a 1-min incubation, the fluorophore was removed using clean gauze, followed by five PBS washes (200 μL per wash). To optimize the incubation time, 25 μM NTFP700 was applied and incubated for 1, 3, or 5 min, followed by five PBS washes. The washing protocol was refined by varying the number of PBS washes from 1–10 to reduce non-specific staining in surrounding tissues. Fluorescence images were captured at each step, and the signal-to-background ratio (SBR) was calculated to assess nerve-specific contrast. The optimal dose, incubation time, and washing protocol were determined based on the highest SBR.

Evaluation of NTFP700 Retention Time

To assess the retention time of NTFP700 in the sciatic nerve, the optimized topical administration protocol was applied. Fluorescence images were captured at predefined intervals (1, 60, 120, 240, 360, 480, and 600 min post-application) using both color and NIR imaging. Fluorescence intensity and SBR were quantified at each time point to evaluate the temporal stability of NTFP700 over a 10-h period.

Comparison of Topical Administration and Dyed Gauze Methods

The optimized protocol for NTFP700 was used to compare the topical administration method with the dyed gauze method, focusing on nerves located at different angles. For topical administration, NTFP700 was applied directly to the exposed sciatic nerve as described previously. In the dyed gauze method, a 25 μM NTFP700 solution (25 mL) was prepared, and a sterile gauze (1.5 × 1.5 cm) was immersed in the solution until it was completely saturated. After thorough soaking, the dyed gauze was carefully removed and placed on the target nerve for visualization. NIR fluorescence images were acquired for nerves positioned at both flat and steep angles. The fluorescence intensity and SBR of nerve tissues relative to the surrounding muscle were measured to evaluate the efficacy of each method.

Simultaneous Dual-Channel Imaging of Nerves and Tumors

Dual-channel fluorescence imaging was performed in tumor-bearing mouse and VX2 rabbit models to simultaneously visualize nerves and tumors. Animals were anesthetized with xylazine and tiletamine-zolazepam, and the tumor-targeting contrast agent cRGD-ZW800-PEG was administered intravenously (50 nmol for mice, 0.1 mg/kg for rabbits). The sciatic nerve region was surgically exposed 4 h post-injection. Tumors near the nerve were identified using a color and fluorescence-merged NIR imaging system (800 nm). Subsequently, NTFP700 was applied to the exposed sciatic nerve using both the optimized topical administration and dyed gauze methods. NIR imaging at 700 nm enabled clear differentiation of nerve and tumor signals. SBR values for both fluorophores were analyzed to evaluate imaging performance, facilitating real-time tumor resection guided by simultaneous visualization of nerves and tumors.

Histological Confirmation of NTFP700

To confirm nerve-specific staining, sciatic nerves from rabbit models were harvested after in vivo application of NTFP700 using the dyed gauze method. The tissues were flash-frozen in optimal cutting temperature (OCT) compound and sectioned at 10-μm thickness. Slides were washed in PBS to remove residual OCT, fixed with 2% paraformaldehyde for 15 min, and washed three times in PBS. One section was stained with hematoxylin and eosin (H&E), while serial sections underwent fluorescence microscopy after staining with NeuroTrace, FluoroMyelin, and NTFP700. Fluorescence images were acquired using an LSM 900 confocal microscope (Zeiss, Oberkochen, Germany) at 2.5× and 10× magnifications.

In Vivo Toxicity Tests

To evaluate the toxicity of NTFP700, C57BL/6 mice (5 weeks old) were randomly divided into four groups and injected with NTFP700 at concentrations of 25, 100, and 250 μM into the muscles near the nerves. Blood samples were collected 2 weeks post-injection for serum biochemical analysis. Major organs (liver, spleen, kidney, heart, and lung), muscles, and sciatic nerves were harvested, fixed in 10% neutral-buffered formalin, and processed into paraffin sections (8 μm). Histological examination was conducted using H&E staining and digital microscopy.

Statistical Analysis

Results are presented as means ± standard deviations (SDs) for all analyses. Statistical analyses were conducted using GraphPad Prism 9 (GraphPad, San Diego, CA) through one-way or two-way analysis of variance (ANOVA), followed by Sidak’s multiple comparisons test. Statistical significance was denoted according to GraphPad conventions: not significant (ns), P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; and ****P ≤ 0.0001. Fluorescence intensity (%) and colocalizing pixels (%) were quantified using the ImageJ software (US NIH, Bethesda, MD) based on the measured values.

Supplementary Material

Supplementary Information
Movie S1

Movie S1. Real-time dual-channel intraoperative fluorescence imaging of nerve and tumor

Download video file (1.9MB, mp4)
Figure S4

Figure S4. In vivo toxicity assessment of NTFP700

Figure S2

Figure S2. Physicochemical and optical properties of nerve-targeted NIR fluorophores

FIgure S3

Figure S3. Biodistribution and clearance of nerve-targeted NIR fluorophores

Figure S1

Figure S1. HPLC-MS characterization of fluorophores

Acknowledgements:

Figures 4 and 5 were created using BioRender.com under an academic license. All other images, including those in the TOC graphic and Supporting Information, were created or captured directly by the authors. The authors thank Editage (www.editage.com) for English language editing support.

Funding:

This study was supported by KHIDI #RS-2024-00436472 and KUCRF #RS-2024-00466887, funded by the Ministry of Health and Welfare and the Ministry of Science and ICT, Republic of Korea. This work was also supported by Korea University Guro Hospital and a grant funded by Korea University Medicine (#K2407281 and #K2507521) and NCI #R01CA280968, funded by U.S. National Institute of Health (NIH). The content expressed is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Abbreviations

FGS

fluorescence-guided surgery

H&E

hematoxylin and eosin

ICG

indocyanine green

LC-MS

liquid chromatography-mass spectrometry

MW

molecular weight

NIR

near-infrared

OCT

Optimal Cutting Temperature

PBS

phosphate-buffered saline

QY

quantum yield

SBR

signal-to-background ratio

SD

standard deviation

SN

sciatic nerve

Footnotes

Competing interests: The authors declare no conflict of interest. HSC reports a relationship with Nawoo Vision and Ferrex Therapeutics: Consulting, stock, and royalty.

Data availability:

All data included in this study are available from the corresponding authors on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information
NIHMS2147597-supplement-Supplementary_Information.docx (7.7MB, docx)
Movie S1

Movie S1. Real-time dual-channel intraoperative fluorescence imaging of nerve and tumor

Download video file (1.9MB, mp4)
Figure S4

Figure S4. In vivo toxicity assessment of NTFP700

NIHMS2147597-supplement-Figure_S4.tif (38.8MB, tif)
Figure S2

Figure S2. Physicochemical and optical properties of nerve-targeted NIR fluorophores

NIHMS2147597-supplement-Figure_S2.tif (2.4MB, tif)
FIgure S3

Figure S3. Biodistribution and clearance of nerve-targeted NIR fluorophores

NIHMS2147597-supplement-FIgure_S3.tif (9.7MB, tif)
Figure S1

Figure S1. HPLC-MS characterization of fluorophores

NIHMS2147597-supplement-Figure_S1.tif (2.7MB, tif)

Data Availability Statement

All data included in this study are available from the corresponding authors on reasonable request.

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