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• Cancer is one of the formidable and life-threatening diseases. According to the cancer statistics released by the International Agency for Research on Cancer, nearly 20 million new cancer cases and approximately 9.7 million cancer deaths occurred worldwide in 2022 [1]. Early diagnosis and precise treatment are efficient schedule for improvement the prognosis of cancer patients [2]. In conventional clinical application, hematoxylin and eosin (H&E) staining is an effective method for tumor diagnosis, but this method is invasive and cannot achieve real-time monitoring [3]. In addition, surgical resection is one of the most reliable methods to cure cancer patients if the cancerous tissues are radically removed [4]. However, during surgery, it is difficult to accurately distinguish the margins between cancerous and healthy tissues with the naked eye or tactile information, even for experienced surgeons. Overestimation of tumor margins can damage normal tissues that will lead to the loss of normal function, while underestimation can result in tumor residue that will cause local recurrence and poor prognosis [5,6]. For some tiny or residual tumor tissues which are difficult to remove by surgery, chemotherapy and radiotherapy are mainly used for treatment in clinical practice [7,8]. However, these methods show poor specificity and are harmful to healthy tissues that bring about undesired side effects to patients such as nausea, vomiting, alopecia, neuropathic pain while curing tumors [9]. Therefore, development of a powerful tool for diagnosis, assisting surgeons in accurately determining tumor boundaries in real time during surgical procedures, and enabling ablation the tiny or residual tumor tissues is conducive for radical treatment of cancer.

  Compared to the conventional clinical diagnostic and therapeutic techniques, fluorescence imaging (FL) provides great advantages in early detection and management of tumors due to its simple operation, lower cost-effectiveness, noninvasive real-time imaging [10-12]. As we all know, once a molecule is excited, part of the light energy will be converted into fluorescence by radiative transition, and the other part of the light energy will be converted into heat energy by non-radiative transition, which can be used for eradication tumor cells by generating in situ heat with high effectiveness [13,14]. Thus, by balancing the energy transfer between radiative and non-radiative transitions, a theranostic agent that integrates FL and photothermal therapy (PTT) can be constructed. This reagent can be used for clinical diagnosis, surgical navigation, and precision treatment of cancer, thereby arousing enormous attentions [15,16]. Recently, second near-infrared (NIR-Ⅱ) fluorescence agents present high spatial resolution, deep penetration depth, and minimal tissue autofluorescence which have aroused the attention of researchers [17-20]. At present, a variety of NIR-Ⅱ fluorescent theranostic agents, including metallic nanoparticles, quantum dots and rare earth nanomaterials and organic small molecules, have been extensively developed [21-25]. Among these, organic small-molecule NIR-Ⅱ fluorophores are regarded as ideal materials for clinical applications due to its easy metabolism, synthetic flexibility, and excellent processability [26-28]. However, the existing organic NIR-Ⅱ theranostic agents possessed always-on fluorescence behavior which lack enough specificity. These non-specific agents cannot effectively distinguish cancerous and noncancerous tissues, resulting in harm to normal tissues during treating tumors. Therefore, development of an activatable NIR-Ⅱ organic probe to improve the precise capability of cancer diagnosis and treatment is highly desirable. To the best of our knowledge, using the activatable NIR-Ⅱ organic theranostic agent for FL guided intraoperative resection and photothermal therapy of cancer has few reports.

  Herein, an activatable organic small molecule probe (NRS) was synthesized based on the incorporation of the rhodamine and xanthene derivatives for NIR-Ⅱ FL-guided diagnosis and treatment of tumor. In the presence of H₂S, the masking group 2,4-dinitrobenzenesulfonyl chloride can be removed to release the NIR-Ⅱ fluorescence signal. Under the guidance of NIR-Ⅱ FL modality of NRS, the cancerous tissue margin was successfully identified with high signal-to-background ratio. In addition, with the help of NIR-Ⅱ fluorescence surgery navigation, tumors tissues were precisely removed which avoided the severe damage to normal tissues. More importantly, NRS also exhibits good photothermal conversion efficiency. Upon 808 nm laser irradiation, the temperature of the tumor site can elevate to 52 ℃, which can induce tumor cells apoptosis. All these results indicate that NRS served as theranostic agent can be achieved to distinguish tumor from normal tissues, guide surgical resection, and perform PTT of cancer tumors under NIR-Ⅱ FL.

  Extending the conjugated structures can significantly enlarge the absorption and emission wavelengths of fluorophores which is one of widely employed strategy for designing long-wavelength fluorescent sensors [29-31]. Meanwhile, expanding the wavelength can narrow the energy gap, thereby endowing photothermal therapy capability [15]. In addition, it is reported that overexpression of the H₂S-producing enzymes cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST) in some cancers, including melanoma, prostate cancer and colon cancer, could produce increased amounts of H₂S [32]. Thus, H₂S can be recognized as a biomarker for distinguishing these tumors from normal tissues. Undoubtedly, H₂S-activated NIR-Ⅱ FL integrating photothermal therapy could accurately diagnose and ablate these tumors. Based on these principles, electron-rich xanthene derivatives was introduced into the rhodamine skeleton to construct a NIR-Ⅱ fluorophore. Meanwhile, a 2,4-dinitrobenzenesulfonyl chloride group (Fig. 1A), acting as the H₂S specific identification moiety as well as the fluorescence quencher, was integrated into the fluorophore to establish a probe NRS. We conjectured that the probe can generate the NIR-Ⅱ fluorescence in the presence of H₂S and can be used for fluorescence-image-guided surgical excision and photothermal therapy (Scheme 1). The synthesis and characterization of NRS were detailed in Supporting information.

  Figure 1

  

  Figure 1.  (A) The cleavage mechanism of 2,4-dinitrobenzenesulfonate in the presence of HS⁻. (B) The normalized absorption spectra of NRS (10 µmol/L) with or without 6 equiv. of HS⁻ in aqueous solution (pH 7.4, PBS buffer solution, 20% DMSO). (C) Fluorescence titration experiment of NRS (10 µmol/L) in the absence or presence of various concentrations of HS⁻ (0–60 µmol/L) in PBS buffer (pH 7.4, 20% DMSO). λ_ex = 808 nm. (D) The linear fitting curve of NRS against the concentration of HS⁻. (E) Reaction time profile of probe NRS (10 µmol/L) for HS⁻ (6 equiv.). (F) The fluorescence intensity of NRS in the presence of H₂S and other analytes. (G) The normalized fluorescence intensity scale diagram of the probe in the presence of various molecules. λ_ex = 808 nm.

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  Scheme 1

  

  Scheme 1.  The design and application of NRS.

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  The absorption spectra were firstly performed. As illustrated in Fig. 1B, the absorption band of NRS was centered at 650–900 nm. With the addition of HS⁻, the absorption band increased obviously. Under excitation at 808 nm, the free probe exhibited a weak fluorescence, while with the increasing of HS⁻, the NIR-Ⅱ fluorescence emission was activated and enhanced gradually at 900–1000 nm (Fig. 1C). These changes were attributed to the transformation of NRS into NR, which confirmed by high resolution mass spectrum (HRMS) (Fig. S1 in Supporting information). Moreover, the fluorescence intensity of I₉₂₅ afforded a good linear relationship with the concentration of HS⁻ range from 4 µmol/L to 10 µmol/L (Fig. 1D). Accordingly, the detection limit was calculated to be 1.19 µmol/L, demonstrating the probe has a high sensitivity for detection of HS⁻. Moreover, the fluorescence intensity of the probe in the presence of 6 equiv. of HS⁻ reached a plateau within about 10 min with about 13-fold enhancement, suggesting that the probe can quickly detect HS⁻ with high sensitivity (Fig. 1E). These favorable results show the probe possesses superior detection performance for HS⁻ with NIR-Ⅱ fluorescence signal.

  In order to validate the specificity of the probe for recognizing HS⁻, the fluorescence changes of NRS in the presence of several representative biological analytes were evaluated. As depicted in Fig. S2 (Supporting information), the fluorescence intensity of NRS barely changed in the presence of these interfering agents. Inversely, a remarkable emission enhancement of NRS can be observed in the presence of HS⁻, suggestting that the probe NRS has superior selectivity for HS⁻ over other biological analytes (Figs. 1F and G). In ddition, the effect of pH for the probe in the absence or presence of HS⁻ was also measured. As shown in Fig. S3 (Supporting information), the free probe NRS displayed weak fluorescence at various pH values. However, the obvious enhancement of fluorescence signals can be obtained after the probe NRS interacted with HS⁻ at physiological pH. In addition, the probe NRS possesses a good photo-stability (Fig. S4 in Supporting information). These data indicate that probe has a high selectively for HS⁻ and is suitable for visualization HS⁻ in physiological pH.

  To evaluate the photothermal generation ability of the probe after identifying HS⁻, various concentrations of NR were irradiated at an 808 nm laser with different power densities, and the changes of temperature were then recorded. As expected, under irradiation of an 808 nm laser, the temperature of NR was closely connected with the laser power density and the concentration, indicating the manageable photothermal behavior (Fig. 2A). Specifically, the temperature of 100 µmol/L NR rapidly increased from 25 ℃ to 63 ℃ (∆T = 38 ℃) under the 808 nm laser irradiation for 8 min with a power density of 1.2 W/cm² (Fig. S5 in Supporting information). Moreover, to assess the photothermal stability, NRS was irradiated in the presence of HS⁻ under 808 nm laser (1.2 W/cm²) and the temperature was recorded during the heating and cooling cycles. As presented in Figs. 2B and C, the degradation of heating behavior was negligible after four cycles heating/cooling processes under laser irradiation, guaranteeing constant thermal outputs in the implementation of PTT. Based on the curve relationship between solution cooling time and temperature driving force (−lnθ) (Fig. 2D), the photothermal conversion efficiency of NRS in the presence of HS⁻ was calculated as 43%. All of these results indicate that the probe after identifying HS⁻ displays excellent photothermal stability and high photothermal conversion efficiency.

  Figure 2

  

  Figure 2.  (A) The time-dependent photothermal images of NR with different concentrations under 808 nm laser irradiation with various powers. (B) Photothermal stability of NRS in the presence of HS⁻ during four consecutive laser irradiation (808 nm, 1.2 W/cm²) and cooling cycles. (C) Temperature fluctuations curves of NRS in the presence of HS⁻ or only PBS during laser irradiation and cooling. (D) Linear correlation of the cooling time versus negative logarithm of temperature driving force (−lnθ).

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  Melanoma is the most malignant type of skin tumor, which is prone to distant metastasis. Once the metastatic process has begun, the tumor becomes difficult to cure and has a high mortality rate. Primary melanoma usually displays dark, but can also present amelanotic, making it difficult to be distinguished with the naked eye [33]. It often occured on the skin, but can be also found in other areas such as oral, eyes, gastrointestinal tract and ears [34]. Therefore, it is important for early detection and accurate treatment of melanoma. Since overexpressed H₂S-producing enzymes produce high levels of H₂S in melanoma, the increased H₂S production can be considered as a marker for distinguishing melanoma from normal tissues [35]. Encouraged by the excellent fluorescence capabilities of NRS for detection of H₂S in vitro, the feasibility of NRS to identify melanoma was investigated by tracking the increased H₂S production in NIR-Ⅱ imaging modality. All animal experiments were conducted in accordance with the Animal Management Rules of the Ministry of Health in People's Republic of China (document No. 55, 2001) and approved by the Medical Ethic Committee of Shandong Provincial ENT Hospital (protocol number: 2023-039-01). B16 tumor-bearing mice were administered NRS by intratumoral injection and then imaged at various times by NIR-Ⅱ FL system (Fig. 3A). As presented in Fig. 3B, an obvious fluorescence signal was specifically observed in the tumor region with high signal-to-background ratio (10–49.7) after the administration of NRS within 3 min, and the fluorescence signal was gradually enhanced with time increasing (Figs. 3C and D). While barely detectable fluorescence was obtained in normal mice injected with the NRS or phosphate buffered saline (PBS). In addition, the biological toxicity of NRS in living mice was evaluated by H&E staining analysis. As shown in Fig. S6 (Supporting information), there was no obvious lesions in the heart, liver, spleen, lung and kidney, indicating that the probe has a good biocompatibility. These results indicated that NRS can be served as a reliable tool for recognition melanoma by imaging H₂S.

  Figure 3

  

  Figure 3.  (A) Schematic diagram of NIR-Ⅱ FL in tumor bearing mice. (B) NIR-Ⅱ fluorescence images of normal and B16 tumor-bearing mice at pointed times after injected with NRS. (C) The mean fluorescence intensity (FI) of living mice at various times after treated with NRS and PBS. (D) The mean fluorescence intensity of B16 tumor-bearing mice treated with NRS at different times. Data are presented as mean ± standard deviation (SD) (n = 3).

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  Motivated by these promising results, the surgical excision of melanoma was performed under the guidance of NIR-Ⅱ FL of NRS (Fig. 4A). As demonstrated in Fig. 4B, an obvious fluorescnece signal could be observed in B16 bearing mice which is the location of the tumor tissue. Then the tumor was resected using surgical instruments under the guidance of NIR-Ⅱ FL (Figs. 4C and D). After the operation, there was no fluorescence signal in living mice could be observed, suggesting the tumor tissue was completely removed (Fig. 4E). The excised tumor tissue was then imaged and a bright fluorescent signal could be obtained (Figs. 4F and G), indicating the tumor tissue was successfully resected under NIR-Ⅱ FL-guided of NRS. The surgical procedure was provided in Video S1 (Supporting information). All results illustrate that the probe can be used as a surgical navigation reagent to accurately remove tumor under NIR-Ⅱ FL.

  Figure 4

  

  Figure 4.  NIR-Ⅱ FL-guided tumor resection on living mice. (A) The schematic diagram of surgical resection under NIR-Ⅱ FL guidance. (B) The precisely located tumor labeled with NRS under 808 nm laser excitation (yellow circle: the position of tumor). (C, D) The processes of tumor resection. (E) NIR-Ⅱ FL image of living mice after tumor removal. (F) The bright field of removed tumor tissue. (G) The NIR-Ⅱ FL image of removed tumor tissue.

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  Given the desirable photothermal capacity and deep tissue penetration of NRS, the photothermal therapeutic ability of NRS against B16 cells was evaluated. As depicted in Fig. 5A, the cell viability exhibited negligible decreased at a concentration of 40 µmol/L of NRS, indicating that NRS possesses a good biocompatibility without laser irradiation. By contrast, the cell viability significantly decreased as the concentration of NRS increased under 808 nm laser irradiation (1.2 W/cm²). These results display that NRS can ablate the tumor cells under the exposure of an 808 nm laser (1.2 W/cm², 5 min).

  Figure 5

  

  Figure 5.  (A) The cell viability of B16 cells incubated with various concentrations of NRS with or without 808 nm laser radiation. (B) Photothermal therapy of NRS for tumor mice. (a) The schematic diagram of photothermal treatment for tumor mice. (b) Photothermal imaging of B16 tumor-bearing mice under 808 nm laser irradiation with a power density of 1.2 W/cm² for 8 min after the post-injection of the probe NRS and PBS, respectively. (c) The temperature change curves of melanoma tumor at various 808 nm laser irradiation times after injection of NRS or PBS. (d) The tumor volume changes of mice following different therapeutic interventions at diverse times. (e) Body weights curves of mice during different treatments within 10 days. (f) H&E staining analysis of the tumor sections dissected from B16 tumor bearing mice that injected with NRS (upper row) and PBS (bottom row) with laser irradiation. Data are presented as mean ± SD (n = 3).

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  According to the above-mentioned PTT effect of NRS on cancer cells, we proceeded to assess the photothermal ablation capability of NRS in an orthotopic B16 tumor-bearing mice (Fig. 5B). After intratumoural injection of NRS or PBS, the photothermal imaging was performed to study the changes of temperature in tumor region at different time points under 808 nm laser irradiation (1.2 W/cm²) (Fig. 5B-a). As demonstrated in Figs. 5B-b and c, the temperature of NRS-labeled tumor mice increased significantly as laser irradiation time increasing, and reached about 52 ℃ after irradiation for 8 min. By sharp contrast, the temperature in PBS injected tumor mice exhibitd marginal changes under 808 nm irradiation, indicating that the 808 nm irradiation alone did not cause the evident overheating. Subsequently, the tumor volume and weight of tumor mice were recorded for 10 consecutive days. As depicted in Figs. 5B-d and Fig. S7 (Supporting information), the tumor volumes increased constantly in NRS-labeled tumor mice without irradiation and PBS-treated tumor mice with 808 nm laser irradiation. However, the growth of tumors in NRS-injected tumor mice with 808 nm laser irradiation was conspicuously suppressed and presented no clue of recurrence. In addition, the mice weight in all groups revealed subtle changes after 10 days (Fig. 5B-e). To further verify the therapeutic effect of NRS against tumor, histological examination by H&E staining was performed on tumors excised from laser-irradiated mice. As shown in Fig. 5B-f, the tumor slices of PBS-injected mice demonstrated a normal nuclear morphology and without notable abnormalities. On the contrary, apparent tumor region destruction and substantial apoptosis of tumor cells could be obversed in NRS therapy group. These results demonstrate that NRS holds an outstanding PTT performance to suppress tumor growth and ablate tumor.

  In summary, an activatable NIR-Ⅱ theranostic agent for visualizing melanoma with overexpression of H₂S and efficient photothermal therapy was developed. The free probe NRS exhibits weak fluorescence, while in the presecnce of H₂S, a largely enhanced fluorescence emission can be generated in NIR-Ⅱ region. With the desirable attributes of deep-tissue penetration and high selectivity for detection H₂S, the theranostic agent NRS can accurately delineate melanoma margins with overexpression of H₂S in vivo and surgically resect it under the guidance of NIR-Ⅱ FL. More importantly, with the good photothermal conversion efficiency, NRS possesses high tumor suppression capacity, and successfully achieved tumor ablation under 808 nm laser irradiation without apparent biological toxicity. Taken together, the diagnostic reagent NRS can be used to distinguish tumor from normal tissues, guide intraoperative precise resection and implement kill tumors effectively under NIR-Ⅱ FL, which may have translational potential in biological and clinical systems.

  Declaration of competing 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.

  CRediT authorship contribution statement

  Yanyan Ma: Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Lizhen Xu: Validation, Software, Methodology, Investigation, Formal analysis, Data curation. Muxin Xu: Validation, Investigation, Formal analysis. Jie Niu: Writing – review & editing, Validation, Software, Resources, Investigation, Funding acquisition. Wei Xu: Writing – review & editing, Resources, Funding acquisition. Weiying Lin: Writing – review & editing, Resources, Funding acquisition.

  Acknowledgments

  This work was financially supported by National Natural Science Foundation of China (Nos. 22406099, 82172961, 22077048 and 22277014), the Special Fund of Taishan Scholars Project of Shandong Province (No. tsqnz20231253), Guangxi Natural Science Foundation (Nos. 2021GXNSFDA075003, AD21220061), the startup fund of Guangxi University (No. A3040051003) and the Major Science and Technology Plan Project of Hainan Province (No. ZDKJ202005).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.110850.

  
  
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• 

  Figure 1  (A) The cleavage mechanism of 2,4-dinitrobenzenesulfonate in the presence of HS⁻. (B) The normalized absorption spectra of NRS (10 µmol/L) with or without 6 equiv. of HS⁻ in aqueous solution (pH 7.4, PBS buffer solution, 20% DMSO). (C) Fluorescence titration experiment of NRS (10 µmol/L) in the absence or presence of various concentrations of HS⁻ (0–60 µmol/L) in PBS buffer (pH 7.4, 20% DMSO). λ_ex = 808 nm. (D) The linear fitting curve of NRS against the concentration of HS⁻. (E) Reaction time profile of probe NRS (10 µmol/L) for HS⁻ (6 equiv.). (F) The fluorescence intensity of NRS in the presence of H₂S and other analytes. (G) The normalized fluorescence intensity scale diagram of the probe in the presence of various molecules. λ_ex = 808 nm.

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  Scheme 1  The design and application of NRS.

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  Figure 2  (A) The time-dependent photothermal images of NR with different concentrations under 808 nm laser irradiation with various powers. (B) Photothermal stability of NRS in the presence of HS⁻ during four consecutive laser irradiation (808 nm, 1.2 W/cm²) and cooling cycles. (C) Temperature fluctuations curves of NRS in the presence of HS⁻ or only PBS during laser irradiation and cooling. (D) Linear correlation of the cooling time versus negative logarithm of temperature driving force (−lnθ).

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  Figure 3  (A) Schematic diagram of NIR-Ⅱ FL in tumor bearing mice. (B) NIR-Ⅱ fluorescence images of normal and B16 tumor-bearing mice at pointed times after injected with NRS. (C) The mean fluorescence intensity (FI) of living mice at various times after treated with NRS and PBS. (D) The mean fluorescence intensity of B16 tumor-bearing mice treated with NRS at different times. Data are presented as mean ± standard deviation (SD) (n = 3).

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  Figure 4  NIR-Ⅱ FL-guided tumor resection on living mice. (A) The schematic diagram of surgical resection under NIR-Ⅱ FL guidance. (B) The precisely located tumor labeled with NRS under 808 nm laser excitation (yellow circle: the position of tumor). (C, D) The processes of tumor resection. (E) NIR-Ⅱ FL image of living mice after tumor removal. (F) The bright field of removed tumor tissue. (G) The NIR-Ⅱ FL image of removed tumor tissue.

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  Figure 5  (A) The cell viability of B16 cells incubated with various concentrations of NRS with or without 808 nm laser radiation. (B) Photothermal therapy of NRS for tumor mice. (a) The schematic diagram of photothermal treatment for tumor mice. (b) Photothermal imaging of B16 tumor-bearing mice under 808 nm laser irradiation with a power density of 1.2 W/cm² for 8 min after the post-injection of the probe NRS and PBS, respectively. (c) The temperature change curves of melanoma tumor at various 808 nm laser irradiation times after injection of NRS or PBS. (d) The tumor volume changes of mice following different therapeutic interventions at diverse times. (e) Body weights curves of mice during different treatments within 10 days. (f) H&E staining analysis of the tumor sections dissected from B16 tumor bearing mice that injected with NRS (upper row) and PBS (bottom row) with laser irradiation. Data are presented as mean ± SD (n = 3).

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