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. 2026 Oct 30. Online ahead of print. doi: 10.1039/d6ra06275j

A GRPR-targeted near-infrared fluorescent probe for precise tumor imaging and intraoperative surgical navigation

Huihui Lu a,b, Caiting Deng b,c, Jiayao Zhao b, Jingjing Zhang b, Anping Cao b, Feifei An b,d,✉, Nandi Chen e,✉, Yichao Chai a,✉
PMCID: PMC13721913  PMID: 42965098

Abstract

Fluorescence-guided surgery requires tumor-imaging probes that provide reliable contrast while minimizing background interference. Herein, we developed a gastrin-releasing peptide receptor (GRPR)-targeted near-infrared (NIR) fluorescent probe, IR783-BBN, by covalently conjugating the bombesin peptide with the IR783 fluorophore. This molecular design was intended to combine the tumor-targeting potential of bombesin (BBN) with the NIR imaging capability of IR783. IR783-BBN was successfully synthesized and exhibited absorption and emission maxima at 801 and 820 nm, respectively. In vitro studies demonstrated that the probe cell viability remained above 82% under the tested conditions and was internalized by 4T1 breast cancer cells in a time- and concentration-dependent manner. In 4T1 tumor-bearing mice, IR783-BBN generated detectable tumor-associated fluorescence after systemic administration, with a tumor-to-muscle fluorescence ratio of 6.97 at 1 h post-injection (n = 3). Histological and fluorescence microscopic analyses of the excised tissues further supported the correspondence between fluorescence signals and tumor-containing specimens. Overall, these findings support the feasibility of IR783-BBN as a BBN-conjugated NIR fluorescent probe for tumor imaging and fluorescence-guided surgical navigation.

A GRPR-targeted NIR fluorescent probe, IR783-BBN, exhibits favorable optical properties and biocompatibility, enabling specific tumor imaging and fluorescence-guided intraoperative tumor resection in 4T1 breast tumor-bearing mice.graphic file with name d6ra06275j-ga.webp

1. Introduction

Precise tumor localization and complete tumor resection remain central to achieving favorable surgical outcomes. Intraoperative real-time near-infrared (NIR) fluorescence navigation has emerged as a valuable tool for image-guided surgery because of its high sensitivity, lack of ionizing radiation, real-time feedback, and relatively improved tissue penetration.1–3 This technique is particularly useful for tumors with indistinct margins and invasive growth patterns, such as breast cancer. However, currently available fluorescent probes still face several challenges, including insufficient tumor specificity, high background signals, limited imaging contrast, and difficulty in balancing favorable pharmacokinetics with biocompatibility.4–8 Therefore, the development of tumor-targeted NIR fluorescent probes with reliable imaging performance remains important for improving intraoperative tumor visualization.

Receptor-mediated targeting provides an effective strategy to enhance tumor-associated probe accumulation by exploiting the overexpression of specific receptors on tumor cells.9 Among these receptors, gastrin-releasing peptide receptor (GRPR) has attracted considerable attention because it is overexpressed in various solid tumors, including certain breast cancer subtypes. Bombesin (BBN) and BBN-like peptides can bind GRPR with high affinity and have therefore been widely investigated as targeting ligands for tumor imaging and drug delivery.10–13 In addition to the targeting ligand, the choice of fluorophore is also critical for achieving high-contrast tumor imaging. Small-molecule NIR fluorophores with suitable optical properties, modifiable structures, and favorable tumor-associated distribution characteristics can improve fluorescence imaging performance and tumor-to-background contrast.14–17 BBN-based probes targeting GRPR have been investigated using different imaging platforms, including fluorescent imaging approaches.18 Previous studies have established the feasibility of using BBN-derived ligands for visualization of GRPR-expressing tumors; however, probe performance is strongly influenced by the choice of fluorophore, conjugation strategy, and resulting pharmacokinetic properties.19 Therefore, the development of structurally simple BBN-conjugated NIR probes with suitable optical characteristics and applicability to fluorescence-guided surgery remains of interest. IR783 and its derivatives are heptamethine cyanine dyes with NIR fluorescence, functional modification sites, and moderate lipophilicity, making them useful scaffolds for constructing tumor-imaging probes.20–23 Moreover, rational molecular engineering of cyanine dyes has been reported to regulate their optical behavior, tumor microenvironment responsiveness, and in vivo imaging performance.24–27 Previous GRPR-targeted fluorescent probes have employed IRDye800CW-labeled RM26 and NIR fluorescent dye-labeled BBN antagonists for tumor imaging.28,29 These studies demonstrated the feasibility of BBN/GRPR-based fluorescence targeting, whereas differences in fluorophore structure, peptide conjugation, and imaging characteristics may substantially affect in vivo performance. In contrast, the present study employed IR783 as the NIR fluorophore and evaluates the resulting IR783-BBN conjugate from photophysical characterization and cellular uptake to in vivo imaging and fluorescence-guided sequential tumor resection.

Based on these considerations, we designed and synthesized a GRPR-targeted NIR fluorescent probe, IR783-BBN, by covalently conjugating the BBN peptide to the IR783 chromophore. In the molecular design, factors such as the conjugation site, linker structure, and hydrophilic/hydrophobic balance were considered to preserve the targeting function of BBN and the NIR optical properties of IR783.30–32 The resulting IR783-BBN probe was expected to combine BBN-mediated tumor targeting with IR783-based NIR fluorescence imaging. In this study, we systematically evaluated the synthesis and characterization, photophysical properties, cytotoxicity, cellular uptake, in vivo tumor imaging, biodistribution, and fluorescence-guided surgical performance of IR783-BBN in a 4T1 tumor-bearing mouse model. This work aimed to assess the feasibility of using a BBN-conjugated NIR fluorescent probe for tumor imaging and fluorescence-guided surgical navigation.

2. Material and methods

2.1. Materials

The DMEM, fetal bovine serum (FBS), and penicillin–streptomycin solution were purchased from Gibco. Cell culture dishes and other cell culture consumables were obtained from Sangon Biotech Co., Ltd (China). IR783 was purchased from Aladdin Biochemical Technology Co., Ltd (Shanghai, China). BBN-SH was purchased from Apeptide Co., Ltd (Shanghai, China).

2.2. Methods

2.2.1. Chemical synthesis of IR783-BBN

IR783-BBN was synthesized following the route illustrated in Fig. 1. Briefly, IR783 and BBN-SH were dissolved in anhydrous DMF, and triethylamine (TEA) was added as the base (Fig. S1). The mixture was stirred overnight at room temperature. Upon reaction completion, the crude mixture was redissolved in DMF and purified by preparative HPLC equipped with a C18 reversed-phase column (Luna 10 µM C18, 100 Å, LC column 250 × 21.2 mm). The mobile phase gradient was programmed as follows: water/acetonitrile (ACN), from 90/10 to 5/95 over 20 min at 12 mL min−1; isocratic elution at water/ACN = 5/95 for 10 min at 12 mL min−1; then ramped from 5/95 to 90/10 over 5 min at 12 mL min−1. Ultrapure water and HPLC-grade ACN served as mobile phases. Target fractions were pooled and lyophilized to afford powdered IR783-BBN. A total of 10 mg product was obtained, corresponding to an isolated yield of 72.3%. The conjugation was designed to proceed through nucleophilic substitution involving the thiol group of BBN-SH. The purified conjugate was characterized by analytical HPLC and HRMS. The observed molecular mass was consistent with the expected IR783-BBN conjugate; however, these analytical methods alone do not provide an unambiguous site-specific assignment of the conjugation linkage. Fluorescence emission spectra of IR783-BBN in DMSO and water were recorded on a Hitachi F-4700 fluorescence spectrophotometer (Hitachi, Tokyo, Japan).

Fig. 1. Preparation and characterization of IR783-BBN. (a) Schematic illustration of the synthetic route for IR783-BBN. (b) HPLC chromatogram of purified IR783-BBN, showing a major peak at 16.39 min. (c) LC-MS characterization of IR783-BBN. The observed molecular ion peak at m/z 1152.5370 was consistent with the expected product.

Fig. 1

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2.2.2. In vitro cytotoxicity determination by standard MTT assay

The cytotoxicity of IR783-BBN against 4T1 cells was determined via MTT assay. Briefly, 4T1 cells were seeded in 96-well plates at a density of 2 × 104 cells per well in 100 µL of complete culture medium and cultured for 24 h for cell adhesion. After medium removal, cells were incubated with IR783-BBN at gradient concentrations (0, 0.78, 1.55, 3.13, 6.25, 12.5, 25, and 50 µM) for another 24 h. Subsequently, 10 µL of MTT solution (5 mg mL−1) was added to each well, and the cells were incubated for 4 h in the dark. After the supernatant was discarded, 100 µL of DMSO was added to each well to fully dissolve the formazan crystals. The plates were gently shaken, and the absorbance at 490 nm was measured using a microplate reader (EPOCH 2, Agilent, USA). Cell viability was calculated based on the detected absorbance values. Each concentration was tested in four replicates (n = 4).

2.2.3. Cellular uptake characterization

The cellular uptake of IR783-BBN in 4T1 cells was evaluated by confocal microscopy and flow cytometry. For confocal microscopy, 4T1 cells were incubated with IR783-BBN (10 µM) for 2 h. Subsequently, the dishes were rinsed three times with PBS (pH = 7.4) and then stained with DAPI. The cells were imaged using a Leica STELLARIS 5 confocal microscope (Leica Microsystems, Wetzlar, Germany). DAPI fluorescence was excited with a 405 nm laser and collected at 425–500 nm, while IR783-BBN fluorescence was excited with a 638 nm laser and collected at 700–844 nm.

For flow cytometry analysis, 4T1 cells were incubated with IR783-BBN (10 µM) for different time periods (15 min, 0.5 h, 1 h, 2 h). After incubation, the cells were collected, washed three times with 1× PBS, and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Jersey City, NJ, USA). The fluorescence signal of IR783-BBN was detected using the APC-750 channel, with excitation at 638 nm and emission collected at 780/60 nm.

2.2.4. In vivo fluorescence imaging of IR783-BBN

All animal experiments have been approved by the Biomedical Ethics Committee of Health Science Center, Xi'an Jiaotong University (No. XJTUAE2024-1061). 4T1 tumor-bearing mice were established by subcutaneous injection of 4T1 cells (1 × 107) suspended in 100 µL PBS into the right dorsum of female BALB/c mice. Tumors were allowed to grow until reaching approximately 100–150 mm3 before in vivo imaging experiments. Subsequently, 4T1 tumor-bearing mice (n = 3) were intravenously injected with 150 µL IR783-BBN solution containing 5 nmol of probe. In vivo fluorescence imaging was performed at 1, 4, 8, 12, and 24 h post-injection using a real-time small-animal imaging system (VISQUE In vivo Optical Imager, Vieworks, Gyeonggi-do, Korea) with the Cy7 channel (Ex: 710 nm, Em: 810 nm). To evaluate the biodistribution of IR783-BBN, the mice were sacrificed at 24 h post-injection. The major organs, including the liver, intestine, stomach, lung, kidney, spleen, and heart, as well as tumor tissues were excised, rinsed with PBS and gently blotted dry for subsequent ex vivo fluorescence imaging. Regions of interest (ROIs) were manually drawn to encompass each tumour and adjacent peritumoral muscle. Fluorescence radiant efficiency within each ROI was quantified following background subtraction. The tumour-to-muscle (T/M) ratio was calculated by dividing the radiant efficiency of tumour tissue by that of peritumoral muscle.

2.2.5. Fluorescence guided intraoperative tumor resection mediated by IR783-BBN

A 4T1 tumor-bearing mouse was intravenously injected with 150 µL of IR783-BBN solution containing 5 nmol of probe. At 1 h post injection, the mice were anesthetized using isoflurane and subjected to imaging using the previously mentioned fluorescence system.

Fluorescence-guided tumor resection was then performed under NIR imaging guidance, with excitation at 780 nm and emission collected at 820 nm. All serially excised tissue specimens were collected for subsequent ex vivo fluorescence detection. After intraoperative operation, all harvested tissue slices were prepared for two parallel characterizations: hematoxylin and eosin (H&E) histological staining and tissue-section fluorescence microscopic imaging.

3. Results and discussion

3.1. Design, synthesis and characterization of IR783-BBN

BBN is a tumor-targeting peptide that can bind to gastrin-releasing peptide receptor (GRPR) with high affinity.33,34 In this study, IR783-BBN was designed by covalently conjugating a BBN peptide to the chlorinated IR783 indocyanine dye, in which IR783 served as the NIR fluorescent chromophore and BBN functioned as the GRPR-targeting ligand (Fig. 1a). The conjugation was achieved through a nucleophilic substitution reaction. The purified product was first characterized by high-performance liquid chromatography (HPLC). Similar to previously reported NIR dye-BBN conjugates, the successful formation of IR783-BBN was confirmed by HPLC and HRMS, where the observed molecular ion peak matched the theoretical mass of the target conjugate.34 As shown in Fig. 1b, IR783-BBN exhibited a major sharp peak with a retention time of 16.39 min, indicating that the product had been effectively purified. LC-MS analysis further confirmed the successful formation of IR783-BBN. A dominant molecular ion peak was observed at m/z 1152.5370, which was consistent with the expected molecular structure of the designed conjugate (Fig. 1c). Together, these results demonstrated the successful synthesis and purification of IR783-BBN, providing a reliable chemical basis for the subsequent cellular uptake experiments and in vivo fluorescence imaging studies.

3.2. Photophysical properties and in vitro cytotoxicity of IR783-BBN

The photophysical properties of IR783-BBN were evaluated by UV-vis absorption and fluorescence spectroscopy. As shown in Fig. 2a, IR783-BBN exhibited a maximum absorption peak at 801 nm and a fluorescence emission maximum at 820 nm in DMSO. The absorbance-based standard curve of IR783-BBN was further generated (Fig. S2). Both the absorption and emission wavelengths were located within the NIR region of 700–900 nm, which is advantageous for fluorescence imaging because this optical window can reduce tissue autofluorescence and improve imaging contrast. The fluorescence quantum yield of IR783-BBN in DMSO was calculated to be 0.314, higher than that of ICG (Φ = 0.13) (Fig. S3).35 The photostability of IR783-BBN was assessed under continuous light irradiation, with commercial ICG used as a parallel control (Fig. S4) Fluorescence attenuation over time was fitted with a single-exponential decay function. The photobleaching rate constant of IR783-BBN was 0.08947 min−1, lower than that of ICG (0.1571 min−1) (Fig. S5). This slower decay indicates that IR783-BBN exhibits better photostability, which is beneficial for long-term NIR fluorescence imaging.

Fig. 2. Optical properties and in vitro cytotoxicity of IR783-BBN. (a) Normalized absorption and fluorescence emission spectra of IR783-BBN in DMSO. (b) Relative viability of 4T1 cells after incubation with serially diluted IR783-BBN at different concentrations for 24 h. Data are presented as mean ± SD, n = 4.

Fig. 2

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The in vitro cytotoxicity of IR783-BBN was further assessed in 4T1 breast cancer cells using a standard MTT assay. After incubation with IR783-BBN at concentrations ranging from 0 to 50 µM for 24 h, the relative cell viability remained above 82% in all treatment groups (Fig. 2b). Although a slight concentration-dependent decrease in cell viability was observed as the probe concentration increased, no obvious cytotoxicity was detected even at the highest tested concentration of 50 µM. These results indicate that IR783-BBN has acceptable in vitro biocompatibility under the tested conditions, supporting its subsequent use in cellular uptake assays and in vivo NIR fluorescence imaging.

3.3. Cellular uptake characterization of IR783-BBN in 4T1 breast cancer cells

Cellular uptake assays were carried out in 4T1 murine breast cancer cells. 4T1 murine breast cancer cells have previously been reported to express GRPR, which can bind bombesin and bombesin-derived peptides.36 Fluorescence microscopy was used to assess the cellular uptake of IR783-BBN. As shown in Fig. 3, cell nuclei were counterstained with DAPI and displayed blue fluorescence, whereas the intracellular IR783-BBN signal was colored red. After incubation with IR783-BBN, red fluorescence was mainly observed in the cytoplasmic region of 4T1 cells, indicating that the probe could be internalized by the cells.

Fig. 3. In vitro cellular uptake of IR783-BBN in 4T1 breast cancer cells. (a) Concentration-dependent cellular uptake. Cells were incubated with serial concentrations of IR783-BBN (0.5, 2.5, 5, 10 µM) for a fixed incubation time of 2 h. (b) Time-dependent cellular uptake. Cells were incubated with IR783-BBN at a fixed concentration of 10 µM for 15 min, 0.5 h, 1 h and 2 h, respectively. Cell nuclei were stained with DAPI and are shown in blue, while IR783-BBN fluorescence is pseudo-colored red. Merged images show the overlay of DAPI and IR783-BBN fluorescence signals. Scale bar: 100 µm.

Fig. 3

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The concentration-dependent uptake of IR783-BBN was first investigated after a fixed incubation period of 2 h. Weak intracellular fluorescence was observed at relatively low probe concentrations, such as 0.5 and 1 µM. As the probe concentration increased, the intracellular fluorescence signal gradually became stronger and reached the highest level at 10 µM (Fig. 3a). These results suggest that IR783-BBN uptake by 4T1 cells increased with probe concentration under the tested conditions. The time-dependent uptake of IR783-BBN was further examined at a fixed probe concentration. A weak red fluorescence signal was detected after 15 min of incubation, whereas the intracellular fluorescence gradually increased with prolonged incubation time and became most pronounced at 2 h (Fig. 3b). This time-dependent increase indicates progressive cellular accumulation of IR783-BBN in 4T1 cells.

Together, these fluorescence microscopy results demonstrate that IR783-BBN can be efficiently internalized by 4T1 breast cancer cells in both concentration- and time-dependent manners. This uptake behavior is consistent with the expected contribution of the BBN targeting moiety to cellular internalization. However, further competitive inhibition assays using excess free BBN or comparison with GRPR-low/negative cells would be required to directly confirm GRPR-specific uptake.

Additional control experiments were performed using free IR783 and free BBN under the same incubation conditions (Fig. S6). Compared with free IR783, IR783-BBN exhibited a stronger intracellular fluorescence signal under the tested conditions. Because free BBN does not contain the IR783 fluorophore, no corresponding Cy7 fluorescence was expected from the peptide-only control. These observations indicate that conjugation with BBN alters the cellular uptake behavior of IR783; however, they do not by themselves establish a GRPR-specific or receptor-mediated uptake mechanism.

3.4. Quantitative analysis of IR783-BBN uptake by cells by flow cytometry

The cellular uptake of IR783-BBN in 4T1 cells was further quantified by flow cytometry. As shown in Fig. 4a, untreated 4T1 cells exhibited only weak background fluorescence, whereas IR783-BBN-treated cells showed a pronounced rightward shift in the fluorescence intensity distribution. Consistently, the mean fluorescence intensity (MFI) of the IR783-BBN-treated group was significantly higher than that of the untreated control group (P < 0.0001), confirming efficient cellular uptake of the probe (Fig. 4b). Time-dependent uptake was then evaluated at a fixed probe concentration. The intracellular fluorescence signal increased progressively with prolonged incubation time from 0 to 2 h, indicating gradual accumulation of IR783-BBN in 4T1 cells over time (Fig. 4c). In addition, concentration-dependent uptake was assessed after incubation with different concentrations of IR783-BBN for 2 h. The mean fluorescence intensity increased steadily as the probe concentration increased from 0 to 20 µM, demonstrating concentration-dependent cellular uptake under the tested conditions (Fig. 4d).

Fig. 4. Flow cytometric quantification of IR783-BBN cellular uptake in 4T1 cells. (a) Representative flow cytometry histograms, fluorescence intensity in untreated 4T1 cells and 4T1 cells incubated with 10 µM IR783-BBN for 2 h. (b) Quantitative analysis of mean Data are presented as mean ± SD (n = 3). Statistical comparisons were performed via one-way ANOVA. (c) Time-dependent cellular uptake of IR783-BBN in 4T1 cells. (d) Concentration-dependent cellular uptake of IR783-BBN in 4T1 cells.

Fig. 4

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Together, these quantitative flow cytometry results are consistent with the fluorescence microscopy observations shown in Fig. 3 and further demonstrate that IR783-BBN can be internalized by 4T1 cells in both time- and concentration-dependent manners. These findings support the potential role of the BBN moiety in promoting cellular uptake of the probe. However, additional competitive inhibition experiments using excess free BBN or comparison with GRPR-low/negative cells would be required to directly verify GRPR-mediated specificity.

3.5. In vivo fluorescence imaging of IR783-BBN

The in vivo tumor-imaging performance of IR783-BBN was evaluated in 4T1 tumor-bearing mice. After tail-vein injection of 150 µL of IR783-BBN solution containing 5 nmol of probe, whole-body fluorescence imaging was performed at 1, 4, 8, 12, and 24 h post-injection. As shown in Fig. 5a, fluorescence signals could be detected at the tumor region after systemic administration of IR783-BBN. Quantitative analysis of the tumor region showed that the fluorescence intensity reached its maximum at 1 h post-injection and then gradually decreased over time (Fig. 5d), with a tumor to muscle (T/M) ratio of 6.97 (n = 3) (Fig. S7). Although the signal declined after the initial peak, tumor-associated fluorescence remained detectable up to 24 h post-injection, indicating that IR783-BBN could provide sustained tumor contrast within the observation period.

Fig. 5. In vivo fluorescence imaging and biodistribution of the IR783-BBN probe in 4T1 tumor-bearing mice. (a) Representative whole-body fluorescence images of 4T1 tumor-bearing mice at 1, 4, 8, 12, and 24 h after tail-vein injection of IR783-BBN (5 nmol in 150 µL). (b) Ex vivo fluorescence images of major organs and tumor tissues harvested at 24 h post-injection. (c) Quantitative analysis of the mean fluorescence radiant efficiency of isolated organs and tumor tissues. (d) Time-dependent fluorescence intensity profile of the tumor region within 24 h after IR783-BBN administration. Data are presented as mean ± SD, n = 3.

Fig. 5

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At 24 h post-injection, the mice were sacrificed, and the major organs and tumor tissues were collected for ex vivo fluorescence imaging. As shown in Fig. 5b and c, the tumor tissue exhibited evident fluorescence signal compared with several non-target organs, such as the heart, spleen, and lung. Relatively strong fluorescence signals were also observed in the liver and kidneys, suggesting that hepatic and renal pathways may be involved in the in vivo distribution and clearance of IR783-BBN. These pharmacokinetic characteristics further confirm that BBN driven GRPR specific recognition reduces off target probe deposition in healthy tissues and facilitates preferential tumor enrichment. From a translational perspective, the NIR fluorescence characteristics of IR783-BBN and its ability to generate tumor-to-surrounding-tissue contrast are relevant to fluorescence-guided surgery, where real-time visualization of tumor-associated tissue may assist intraoperative localization and resection. The use of BBN as a targeting ligand may provide a molecular strategy for imaging GRPR-expressing tumors. Nevertheless, the present study represents a preliminary proof-of-concept evaluation in a 4T1 mouse model. Further studies involving larger animal cohorts, additional GRPR-positive and GRPR-low/negative tumor models, pharmacokinetic characterization, dose optimization, and comprehensive safety evaluation are required to establish its translational potential.

Overall, the in vivo and ex vivo imaging results demonstrate that IR783-BBN can accumulate at the tumor site and generate detectable NIR fluorescence contrast in 4T1 tumor-bearing mice. The rapid tumor fluorescence peak at 1 h post-injection also suggests that this time point may be suitable for subsequent fluorescence-guided surgical imaging.

3.6. IR783-BBN-mediated fluorescence-guided intraoperative tumor resection

Based on the in vivo fluorescence imaging results, the tumor fluorescence signal reached its maximum at 1 h after IR783-BBN administration, Therefore, fluorescence-guided surgery was performed at this time point to evaluate the ability of IR783-BBN to assist intraoperative tumor localization and margin assessment (Fig. 6).

Fig. 6. IR783-BBN-mediated fluorescence-guided tumor resection and histopathological validation in vivo. (a) Representative intraoperative NIR fluorescence images showing sequential tumor resection guided by IR783-BBN fluorescence signals from the 1st to the 6th resection. (b) Ex vivo fluorescence imaging of the corresponding serially excised tissues. (c) H&E staining of sequentially resected specimens. The first five specimens showed tumor tissues, whereas the sixth specimen showed normal tissue, indicating a negative surgical margin. Scale bar: 200 µm. (d) Fluorescence microscopy images of the corresponding tissue sections. Cell nuclei were stained with DAPI and are shown in blue, while IR783-BBN fluorescence is shown in red. Merged images show the overlay of DAPI and IR783-BBN fluorescence signals. Scale bar: 200 µm.

Fig. 6

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As shown in Fig. 6a, the tumor region exhibited an obvious NIR fluorescence signal during surgery, allowing visual discrimination between fluorescent tumor-associated tissue and adjacent non-fluorescent tissue. Under real-time fluorescence guidance, the fluorescent lesions were sequentially resected from the 1st to the 6th specimen. Ex vivo fluorescence imaging of the excised tissues further supported the intraoperative observations (Fig. 6b). Strong fluorescence was observed in the initially resected specimens, whereas the fluorescence signal gradually decreased in subsequently excised tissues. In the sixth specimen, only minimal or no detectable fluorescence was observed, suggesting that the fluorescence-positive tissue had been largely removed.

To further verify the pathological nature of the sequentially resected tissues, H&E staining and fluorescence microscopy were performed on the corresponding tissue sections. H&E staining showed that the first five specimens contained malignant tumor tissue, whereas the sixth specimen mainly consisted of normally organized striated muscle and connective tissue without obvious tumor cell infiltration (Fig. 6c). These histopathological results indicated that tumor cells were absent in the assayed final tissue section. Consistently, fluorescence microscopy revealed strong IR783-BBN fluorescence signals in the earlier tumor-containing sections, while the signal gradually decreased in later specimens and became negligible in the final normal tissue section (Fig. 6d). The agreement between intraoperative fluorescence imaging, ex vivo fluorescence imaging, H&E staining, and tissue fluorescence microscopy supports the feasibility of IR783-BBN for fluorescence-guided tumor localization and margin evaluation. Overall, these results demonstrate that IR783-BBN can provide useful NIR fluorescence contrast for guiding tumor resection in the 4T1 tumor-bearing mouse model, suggesting its potential application in fluorescence-guided surgical navigation. Nevertheless, the in vivo experiments were performed using a limited number of animals (n = 3), and the present findings should therefore be considered preliminary. Further studies with larger animal cohorts are required to establish the reproducibility of the imaging performance and to more rigorously evaluate the in vivo targeting characteristics of IR783-BBN.

4. Conclusions

In summary, we successfully synthesized a GRPR-targeted NIR fluorescent probe IR783-BBN via covalent linkage between IR783 fluorophore and bombesin targeting peptide. This conjugate inherits the superior NIR optical performance of IR783 and the specific tumor recognition capacity of BBN peptide, which relies on specific BBN-GRPR binding and subsequent receptor-mediated cellular internalization to realize targeted tumor fluorescence imaging. In vitro studies showed that IR783-BBN exhibited low cytotoxicity toward 4T1 cells under the tested conditions and could be efficiently internalized by these cells in both time- and concentration-dependent manners. In vivo imaging showed detectable tumor-associated fluorescence after systemic administration of IR783-BBN, while ex vivo biodistribution also revealed fluorescence in clearance-associated organs, particularly the liver and kidneys. The observed tumor-to-surrounding-tissue contrast supported fluorescence-guided visualization in the 4T1 model, although the present data do not establish exclusive tumor accumulation or GRPR-specific targeting. Overall, these findings demonstrate the feasibility of using a BBN-conjugated NIR fluorescent probe for tumor imaging and fluorescence-guided surgical navigation. Although further studies are required to establish receptor-specific targeting, pharmacokinetic behavior, safety, and performance in larger animal cohorts, the present results provide a basis for further development of IR783-BBN as an intraoperative tumor-imaging probe.

Author contributions

Huihui Lu: investigation, methodology, conceptualization, writing – original draft, visualization, writing – review & editing. Caiting Deng: methodology, investigation, conceptualization. Jiayao Zhao: methodology, investigation, writing – original draft. Jingjing Zhang: methodology, conceptualization. Anping Cao: methodology, investigation. Feifei An: conceptualization, supervision, project administration, funding acquisition, writing – review & editing. Nandi Chen: supervision, funding acquisition, writing – review & editing, project administration. Yichao Chai: conceptualization, supervision, project administration, writing – review & editing.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supplementary Material

RA-OLF-D6RA06275J-s001

Acknowledgments

This work was supported by the Natural Science Foundation of Shaanxi Province (2023-YBSF-270) and Natural Science Foundation of Top Talent of SZTU (grant no. GDRC202433).

Data availability

The data supporting the findings of this study are available within the article and its supplementary information (SI). Additional raw data, including fluorescence imaging data, flow cytometry data, and histological images, are available from the corresponding author upon reasonable request. Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra06275j.

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Associated Data

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

Supplementary Materials

RA-OLF-D6RA06275J-s001
RA-OLF-D6RA06275J-s001.pdf (1.1MB, pdf)

Data Availability Statement

The data supporting the findings of this study are available within the article and its supplementary information (SI). Additional raw data, including fluorescence imaging data, flow cytometry data, and histological images, are available from the corresponding author upon reasonable request. Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra06275j.

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