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• Accurate diagnosis and treatment of diseases is the ultimate goal of modern medical research and development. Accurate diagnosis can provide patients with the optimal treatment strategy in time, which depends on advanced imaging techniques and an in-depth understanding of the mechanism of disease development [1–4]. Precision therapy can effectively reduce the side effects of treatment and improve the patient's prognosis. Nowadays, the rapid development and complexity of cancer has become an urgent global problem. Compared to the traditional anticancer strategies, photothermal therapy (PTT), as one of the emerging therapeutic modality for precise cancer treatment, has attracted widespread interest due to its low invasiveness, superior spatiotemporal selectivity and high biosafety [5–9]. After light excitation, the hotosensitizer can transduce the absorbed energy into heat via nonradiative energy relaxation pathway, causing the hyperthermia of tumor tissue and effective elimination [10,11]. Moreover, the development of new fluorescent dyes and advanced fluorescence technology provides a new and effective strategy for accurate diagnosis of diseases. Compared with visible light and near-infrared Ⅰ (NIR-Ⅰ) fluorescence imaging (FLI), fluorescence imaging in the longer second near-infrared optical window (1000–1700 nm) has obvious advantages because of its noninvasiveness, deeper tissue penetration, less photo-scattering, and minimal unnecessary injury to healthy tissues [12–14]. NIR-Ⅱ fluorescent probes can not only be used in the diagnosis of early cancer, but also can visualize deep biological information in real-time with high sensitivity due to their high tissue penetration [15,16]. In addition, FLI-guided PTT can cooperate cancer diagnosis and treatment to further improve the treatment effect.

  Fluorophores with rigid oxanthracene structures have relatively long emission wavelength, good photostability, and high quantum yields, which is an excellent platform for the design of NIR-Ⅱ fluorescence probes. Therefore, they have been widely used in biomedical imaging and disease diagnosis [17–23]. Lei et al. reported a panel of fluorescent dyes (CX) characterized by two xanthene units linked by an odd number of methine units, which exhibited a wavelength tunability of cyanine dyes and chemo- and photostabllity in an aqueous environment [24]. Bian et al. reported five novel symmetric NIR-Ⅱ dyes (BHs), which are characterized by strategically conjugating dyad innovative xanthonium with sequentially extended polymethine bridges. The BHs exhibited moderate photothermal heating, and considerable fluorescence [25]. Inspired by the biomedical importance of oxanthracene structures as NIR-Ⅱ fluorophore, it was necessary for us to attempt alternative structural modification of oxanthracene structures to find favorable or different biological activities. As shown in Fig. 1, the asymmetric novel NIR-Ⅱ fluorophore Xan-OH based on xanthene structure was designed and synthesized in this work (Scheme S1 in Supporting information). Furthermore, upon conjugation with fetal bovine serum (FBS), the formation of Xan-OH/FBS complexes was induced, contributing to a significant enhancement in aqueous solubility, biocompatibility, and fluorescence emission. Under 808 nm laser irradiation, Xan-OH/FBS can perform real-time NIR-Ⅱ FLI of acute liver injury induced by carbon tetrachloride (CCl₄). Meanwhile, Xan-OH/FBS have good photothermal conversion efficiency, which realized NIR-Ⅱ FLI-guided PTT for xenograft tumor models.

  Figure 1

  

  Figure 1.  Schematic preparation of Xan-OH/FBS and illustration of the application of Xan-OH/FBS in NIR-Ⅱ FLI of acute liver injury and NIR-Ⅱ FLI-guided PTT.

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  As shown in Scheme S1, Xan-OH was synthesized by aldol condensation reaction of 6-(dimethylamino)−1,2,3,4-tetrahydroxanthium perchlorate and 6‑hydroxy-2,3-dihydro-1H-xanthene-4-carbaldehyde. Xan-OH was characterized by ¹H NMR, HR-MS, and X-ray (Figs. S1–S3 in Supporting information). Additionally, as shown in Fig. S3b, it is found that compound Xan-OH exhibits π-π stacking interactions with an intermolecular distance of 3.619 Å. This is beneficial for increasing the compound's Stokes shift, leading to fluorescence emission in the NIR-Ⅱ region [26,27]. Xan-OH/FBS complexes were obtained after combining Xan-OH with FBS (Fig. S4 in Supporting information). The size of Xan-OH/FBS was characterized to be 73 nm by dynamic light scattering (DLS) (Fig. 2a), and the zeta potential of Xan-OH/FBS was measured to be −12 mV (Fig. 2b). The zeta potential before and after encapsulation is basically consistent with FBS, suggesting that the structure of FBS has not been destroyed. Moreover, the particle size after encapsulation is more concentrated than that of FBS, indicating that the encapsulated particles are more uniform.

  Figure 2

  

  Figure 2.  (a) Zeta potential of FBS and Xan-OH/FBS. (b) Hydrodynamic diameters of FBS and Xan-OH/FBS. (c) Absorption and fluorescence spectra of Xan-OH and Xan-OH/FBS in CH₃CN/PBS (v/v = 4/6, pH 7.4) (10 µmol/L, Ex = 808 nm). (d) Normalized fluorescence intensity of Xan-OH/FBS and ICG upon being exposed to continual irradiation by 808 nm laser (0.5 W/cm²) for a total period of 60 min.

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  As shown in Fig. 2c, Xan-OH and Xan-OH/FBS showed strong absorbance in the NIR region (600–900 nm). The emission wavelength of Xan-OH and Xan-OH/FBS was extended to 1100 nm and centered at 902 and 897 nm, respectively. Although Xan-OH exhibits excellent near-infrared fluorescence properties in organic solvents, its solubility decreases in aqueous solutions, which limit its biological application. In contrast, Xan-OH/FBS increases water solubility after binding with FBS, has good absorption at 808 nm, and is well matched with 808 nm laser, giving Xan-OH/FBS excellent NIR-Ⅱ FLI capability. Interestingly, the fluorescence intensity of Xan-OH/FBS is superior to that of Xan-OH. It may due to the aggregation and restrict of Xan-OH after binding with FBS. It encourage us to explore the optical response of Xan-OH/FBS to viscosity. As shown in Fig. S5a (Supporting information), the emission intensity of Xan-OH/FBS increased significantly at 900 nm in H₂O-glycerol mixture solution, with a good linear dependence between log I₉₀₀ and logη (viscosity), evidenced by the correlation coefficient of 0.92787 (Fig. S5b in Supporting information). This suggests that Xan-OH/FBS can be used as a sensitive viscosity probe in biomedical imaging.

  Photostability is an important index of photosensitizer. The photostability of Xan-OH/FBS was investigated upon being exposed to continual irradiation by 808 nm laser (0.5 W/cm²) for a total time period of 60 min. As shown in Fig. 2d, Xan-OH/FBS shows only a slight loss of fluorescent signals even after 60 min of continuous laser irradiation, while ICG is photobleached rapidly, suggesting the good photostability of Xan-OH/FBS and portending its potential visualized and therapeutic application in NIR-Ⅱ window.

  For further biomedical applications, we quantitatively tested the cytotoxicity of Xan-OH and Xan-OH/FBS on human cancer of the HeLa cells by cell counting kit-8 (CCK 8) method. When HeLa cells were treated with different concentrations of Xan-OH and Xan-OH/FBS (0–50 µmol/L), the survival rate of the cells treated with Xan-OH/FBS was higher than that of Xan-OH (Fig. S6 in Supporting information). The results showed that Xan-OH/FBS had low cytotoxicity and good biocompatibility and was more suitable for biological research.

  Spatial resolution is an important evaluation metric for fluorescence imaging, which depends on the penetration depth of the excitation and emission of the fluorophore. As shown in Fig. 3a, the fluorescence of Xan-OH/FBS was still detectable through chicken tissue with a thickness of 6 mm, demonstrating its deep NIR-Ⅱ light penetration ability and providing a high imaging depth for use in fluorescence imaging.

  Figure 3

  

  Figure 3.  (a) In vitro penetration depth of Xan-OH/FBS using chicken tissue as tissue phantoms imitator. (b) Fluorescence imaging pseudocolor images and (c) fluorescence intensity of liver at 30 min after intravenous injection of Xan-OH/FBS (200 µL, 200 µmol/L) with different treatment methods (n = 3). Control group: no treatment; 50 µL CCl₄, once group: inject 50 µL of CCl₄ once; 50 µL CCl₄, twice group: injection of 50 µL CCl₄ twice; NAC group: intraperitoneal injection of NAC (300 mg/kg), followed by intraperitoneal injection of 50 µL of CCl₄ once after 2 h. (d) Fluorescence intensity of liver at 30 min after intravenous injection of Xan-OH/FBS (200 µL, 200 µmol/L) with different treatment methods. Data are presented as mean ± standard deviation (SD) (n = 3). (e) Major organs (heart, liver, spleen, lung, and kidney) of mice at 1 h after intravenous injection of Xan-OH/FBS were stained with hematoxylin and eosin (scale bar: 200 µm).

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  To assess the biodistribution of Xan-OH/FBS, a phosphate buffer saline (PBS, pH 7.4) solution of Xan-OH/FBS (120 µL, 200 µmol/L) was injected into the normal nude mice through a tail vein. As shown in Figs. S7a and b (Supporting information), the Xan-OH/FBS in nude mice showed a clear and bright NIR-Ⅱ fluorescence signal, and the fluorescence intensity in the liver reached a maximum for 20 min after intravenous injection and then dropped gradually. With the extension of time, the nude mice at 2 h after Xan-OH/FBS injection displayed significant NIR-Ⅱ signals in digestive system. Subsequently, we dissected nude mice at 20 min after injection to obtain optical pictures and NIR-Ⅱ fluorescence images of isolated organs (Fig. S7b). The fluorescence imaging of the isolated organs displayed that the fluorescence intensity was high in the liver and spleen, while no obvious fluorescence signals were observed in the heart, lungs, and kidneys. The above result indicated that Xan-OH/FBS were mostly concentrated in the liver and spleen (Fig. S7b). The fluorescence was disappeared after 48 h, indicating that the drug has been fully excreted from the body (Figs. S7a and c in Supporting information). All experimental procedures were conducted in accordance with institutional guidelines for the care and use of laboratory animals, and protocols were approved by the Institutional Animal Care and Use Committee in Southwest Medical University.

  Liver injury often leads to changes in the cellular microenvironment, particularly abnormal alterations in viscosity. Therefore, real-time monitoring of viscosity changes is of great significance for the early diagnosis and treatment of liver injury. Guided by the above results, we hypothesized Xan-OH/FBS could be used for NIR-Ⅱ diagnostic fluorescence imaging of drug induced acute liver injury. The intracellular fluorescence response of Xan-OH/FBS was first measured in two different groups of hepatocytes (control group and lipopolysaccharide (LPS) group), where LPS induced acute damage to BRL cells (rat liver cells) resulting in enhanced cell viscosity. As shown in Fig. S8 (Supporting information), compared with the control, the fluorescence intensity of the LPS group was increased. The results showed that Xan-OH/FBS could be used to monitor the change of cellular viscosity by NIL-Ⅱ fluorescence manner.

  To verify the in vivo real-time fluorescence monitoring capabilities of Xan-OH/FBS, different doses of CCl₄ was selected to establish acute liver injury mouse model with varying severities and N-acetylcysteine (NAC) was used to mitigated the liver injury [28]. Subsequently, liver tissues from each group were stained with hematoxylin-eosin staining (H&E), confirming the establishment of liver injury models with varying degrees induced by CCl₄ (Fig. S9 in Supporting information). In the 50 µL CCl₄ group, nuclear loss and incomplete hepatocyte morphology were observed, confirming the occurrence of inflammatory responses and pathological damage. With increased dosing frequency or concentration, the severity of liver injury was progressively worsened, with more pronounced inflammatory responses and pathological changes. The successful establishment of the model was further validated by immunohistochemical analysis using Bax protein (Fig. S10 in Supporting information). Additionally, in Fig. 3e, significant inflammatory changes in hepatocytes were also shown for the 50 µL CCl₄ group, whereas cells in the 50 µL CCl₄ + NAC group displayed intact morphology, with no notable inflammatory response or pathological damage, suggesting that liver injury was alleviated by NAC. In the control group, strong nuclear staining and intact cell morphology indicated robust hepatocyte growth. Then, Xan-OH/FBS (200 µL, 200 µmol/L) was administered via tail vein injection to the mice model. As shown in Fig. 3b, NIR-Ⅱ fluorescent signals at hepatic region become clearly visible at 1 min after intravenous injection and lasted for 30 min. The result showed that Xan-OH/FBS display a strong NIR-Ⅱ window real-time fluorescence imaging capability for acute liver injury. Furthermore, Xan-OH/FBS was used to explore the different degrees of acute liver injury model established by CCl₄. As shown in Figs. 3c and d, NIR-Ⅱ fluorescent signals were observed in the liver after Xan-OH/FBS's intravenous, and the fluorescence signal enhanced with the increasing CCl₄ dose. In addition, an decreased fluorescence signal was observed with further treatment by NAC, which is related to the amelioration of liver injury by NAC. Furthermore, the mice were sacrificed after in vivo imaging and the major organs were harvested. The ex vivo fluorescence imaging results confirmed that the fluorescence intensity of CCl₄-induced liver injury groups was higher than those of control and NAC group (Figs. S11a and b in Supporting information). Finally, we investigated the biosafety of Xan-OH/FBS. According to the H&E staining experiment (Fig. 3e), the major organs of the mouse (heart, spleen, lungs, kidneys) did not show significant lesions, indicating that Xan-OH/FBS has good biocompatibility. Simultaneously, we performed in vivo fluorescence imaging on both normal and acute liver injury mice using Xan-OH, and compared the results with those obtained from Xan-OH/FBS (Fig. S11c in Supporting information). These results further prove that Xan-OH/FBS is suitable for the exploration of acute liver injury with NIR-Ⅱ fluorescence.

  In light of excellent photophysical properties, the photothermal performance of Xan-OH/FBS under 808 nm laser was further determined. The temperature is positively associated with the concentration of Xan-OH/FBS and the power density of laser (Figs. 4a and b). After 10 min irradiation, the temperature maxima of Xan-OH/FBS reached 61.5 ℃, which was basically consistent with that of Xan-OH (Fig. 4c). According to the heating-cooling curves, Xan-OH/FBS exhibit a commendable photothermal conversion efficiency (η) of 26.77% (Fig. 4d), comparable to that of Xan-OH, which was 27.53% (Fig. S12a in Supporting information). Moreover, both Xan-OH and Xan-OH/FBS presented better photostability than commercial photosensitizer indocyanine green (ICG). In the process of five heating-cooling cycles, the peak temperature of ICG showed a significant decrease after just two cycles, while both Xan-OH and Xan-OH/FBS were observed to decline moderately (Figs. S12b–d in Supporting information).

  Figure 4

  

  Figure 4.  (a) Concentration-dependent temperature increases of Xan-OH/FBS under 808 nm (1.5 W/cm²). (b) Laser power-dependent temperature increases of Xan-OH/FBS (50 µmol/L) under 808 nm laser. (c) Photothermal heating images of Xan-OH (50 µmol/L, PBS) and Xan-OH/FBS (50 µmol/L, PBS) under 808 nm laser (1.5 W/cm²). (d) The heating-cooling curve and time constant of Xan-OH/FBS (τs).

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  As depicted in Fig. S13 (Supporting information), we quantitatively assessed the photothermal cytotoxicity of Xan-OH/FBS on HeLa cells using CCK-8 assay. Compared to HeLa cells treated with Xan-OH/FBS (0–50 µmol/L) in the dark, cells treated with Xan-OH/FBS under 808 nm laser irradiation for 5 min showed significant lower survival rate. Meanwhile, the live/dead cell assay results showed that only the Xan-OH/FBS + laser group realized significant PTT-induced cell death. These findings suggested that Xan-OH/FBS exhibits excellent PTT efficacy (Fig. S14 in Supporting information). In order to further investigate the photothermal effect of Xan-OH/FBS, we inoculated HeLa cells into the right hind limb of BALB/c mice to establish a xenograft tumor mouse model. After intratumoral injection of Xan-OH/FBS, the tumor site was irradiated by 808 nm laser (1 W/cm²) and the temperature change was monitored (Figs. 5a and b). It can be clearly seen that the temperature of the tumor regions in Xan-OH/FBS-treated mice rapidly increased from 31.4 ℃ to 48.7 ℃ within 2 min and stabilized at approximately 54 ℃ after 10 min of continuous laser irradiation. Excitingly, the increased temperature was sufficient to eliminate the tumor. In contrast, tumors in PBS-treated mice showed no temperature change after laser exposure. Subsequently, we evaluated the FLI capabilities of Xan-OH/FBS. After intratumoral injection of Xan-OH/FBS (200 µL, 200 µmol/L), NIR-Ⅱ FLI was performed in vivo and monitored for 120 min (Fig. S15 in Supporting information). The imaging results indicated that Xan-OH/FBS was uniformly distributed within the tumor, exhibiting strong NIR-Ⅱ fluorescence that clearly delineated the tumor's contours. This suggested that Xan-OH/FBS has great potential to precision tumor imaging and further FLI-guide PTT.

  Figure 5

  

  Figure 5.  (a) Photothermal image of tumor region on HeLa-tumor-bearing mice and (b) corresponding heating curves under 808 nm laser irradiation (1 W/cm²) for 10 min after 30 min of the injection of PBS or Xan-OH/FBS (200 µmol/L). (c) Tumor volume and (d) body weight of mice during PTT after different treatments. Data are presented as mean ± SD (n = 3). (e) Typical photographs of ex vivo tumor after 16 days of treatment. (f) Major organs (heart, liver, spleen, lung, and kidney) from the tumor-bearing mice in different treatment groups were stained with hematoxylin and eosin (scale bar: 200 µm).

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  Encouraged by the above results, we further investigate the PTT effect of Xan-OH/FBS against tumors in vivo. The HeLa tumor-bearing mice were classified into four groups randomly (PBS, PBS + laser, Xan-OH/FBS, Xan-OH/FBS + laser). After 30 min of intratumor injection of PBS or Xan-OH/FBS (200 µL, 200 µmol/L), the tumors of the laser groups were irradiated under 808 nm laser (1 W/cm²) for 10 min. The tumor volume and body weight of these four groups of mice were then monitored for 16 days (Figs. 5c and d). During the treatment, the tumor volumes displayed rapid increase rates in the comparative groups, while the tumor volumes of the PTT group (Xan-OH/FBS + laser) showed distinct growth inhibition. In the meantime, the region of the tumors in Xan-OH/FBS + laser group showed severe scalding after being irradiated with laser, indicating that the tumor cells were killed rapidly by hyperthermia generated by Xan-OH/FBS through the photothermal conversion (Fig. S16 in Supporting information). After 16 days of treatment, all mice were euthanized and the major organs including tumors were obtained. The results showed that Xan-OH/FBS displayed the outstanding tumor suppression behavior with laser irradiation, and the tumors were completely eradicated (Fig. 5e). In addition, the weights of the four groups of mice did not change noticeably during treatment. Furthermore, no obvious tissue lesions such as injury or inflammation were found in the major organs (heart, liver, spleen, lung, and kidney) of the four groups through the H&E staining assay (Fig. 5f), revealing the excellent biologically safe of Xan-OH/FBS and laser therapy.

  In this study, we successfully constructed the asymmetric novel NIR-Ⅱ fluorophores Xan-OH and Xan-OH/FBS based on xanthene core. Under 808 nm laser irradiation, Xan-OH/FBS displayed deep NIR-Ⅱ light penetration ability and performed real-time NIR-Ⅱ FLI of acute liver injury induced by CCl₄. Moreover, Xan-OH/FBS exhibited excellent performances on NIR-Ⅱ FLI-guided photothermal ablation of tumor in HeLa tumor-bearing mice models. This study provides a novel NIR-Ⅱ FLI-guided theranostic agent for both inflammatory and cancer diseases.

  In this paper, the fluorophores Xan-OH and Xan-OH/FBS, based on xanthene structure, possess an effective near-infrared absorption, NIR-Ⅱ fluorescent imaging ability, and excellent photothermal property. It is indicated that Xan-OH/FBS is an efficient NIR-Ⅱ fluorescent imaging agent for acute liver injury and a potential photothermal therapeutic agent.

  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

  Mingrui Zhang: Methodology, Investigation, Formal analysis. Lingyu Jin: Visualization, Validation, Software. Yuda Zhu: Visualization, Validation, Software. Junfeng Kou: Validation, Investigation, Data curation. Bo Liu: Validation, Investigation. Jing Chen: Visualization, Validation, Software. Xiaolin Zhong: Visualization, Validation, Software. Xianghua Wu: Writing – original draft, Validation, Investigation. Junfeng Zhang: Writing – review & editing, Supervision, Project administration, Funding acquisition. Wenxiu Ren: Writing – review & editing, Supervision, Project administration, Funding acquisition.

  Acknowledgments

  This work was supported by the National Natural Science Foundation of China (Nos. 22067021, 22367024, and 22267023), Innovation Team of Yunnan Education Department, the "Youth Talent of Wan Ren Project", the Sichuan Science and Technology Program (No. 2022YFS0633). This work also was technically supported by the Public Platform of Advanced Detecting Instruments, Public Center of Experimental Technology, Southwest Medical University.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110772.

  
  
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  Figure 1  Schematic preparation of Xan-OH/FBS and illustration of the application of Xan-OH/FBS in NIR-Ⅱ FLI of acute liver injury and NIR-Ⅱ FLI-guided PTT.

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  Figure 2  (a) Zeta potential of FBS and Xan-OH/FBS. (b) Hydrodynamic diameters of FBS and Xan-OH/FBS. (c) Absorption and fluorescence spectra of Xan-OH and Xan-OH/FBS in CH₃CN/PBS (v/v = 4/6, pH 7.4) (10 µmol/L, Ex = 808 nm). (d) Normalized fluorescence intensity of Xan-OH/FBS and ICG upon being exposed to continual irradiation by 808 nm laser (0.5 W/cm²) for a total period of 60 min.

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  Figure 3  (a) In vitro penetration depth of Xan-OH/FBS using chicken tissue as tissue phantoms imitator. (b) Fluorescence imaging pseudocolor images and (c) fluorescence intensity of liver at 30 min after intravenous injection of Xan-OH/FBS (200 µL, 200 µmol/L) with different treatment methods (n = 3). Control group: no treatment; 50 µL CCl₄, once group: inject 50 µL of CCl₄ once; 50 µL CCl₄, twice group: injection of 50 µL CCl₄ twice; NAC group: intraperitoneal injection of NAC (300 mg/kg), followed by intraperitoneal injection of 50 µL of CCl₄ once after 2 h. (d) Fluorescence intensity of liver at 30 min after intravenous injection of Xan-OH/FBS (200 µL, 200 µmol/L) with different treatment methods. Data are presented as mean ± standard deviation (SD) (n = 3). (e) Major organs (heart, liver, spleen, lung, and kidney) of mice at 1 h after intravenous injection of Xan-OH/FBS were stained with hematoxylin and eosin (scale bar: 200 µm).

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  Figure 4  (a) Concentration-dependent temperature increases of Xan-OH/FBS under 808 nm (1.5 W/cm²). (b) Laser power-dependent temperature increases of Xan-OH/FBS (50 µmol/L) under 808 nm laser. (c) Photothermal heating images of Xan-OH (50 µmol/L, PBS) and Xan-OH/FBS (50 µmol/L, PBS) under 808 nm laser (1.5 W/cm²). (d) The heating-cooling curve and time constant of Xan-OH/FBS (τs).

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  Figure 5  (a) Photothermal image of tumor region on HeLa-tumor-bearing mice and (b) corresponding heating curves under 808 nm laser irradiation (1 W/cm²) for 10 min after 30 min of the injection of PBS or Xan-OH/FBS (200 µmol/L). (c) Tumor volume and (d) body weight of mice during PTT after different treatments. Data are presented as mean ± SD (n = 3). (e) Typical photographs of ex vivo tumor after 16 days of treatment. (f) Major organs (heart, liver, spleen, lung, and kidney) from the tumor-bearing mice in different treatment groups were stained with hematoxylin and eosin (scale bar: 200 µm).

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