2.1 Characterization of TISN

TISN was synthesized according to Figure 1. Briefly, a heat shock protein inhibitor (Tanespimycin, Tane for short) and a photosensitizer (IR780) could self-assemble into uniform nanoparticles (Figure 2A). Since both Tanespimycin and IR780 have large conjugated electron clouds, they are expected to form nanoparticles through π–π stacking interaction, and the self-assembled nanoparticles were supposed to be obtained by adjusting the feed ratios of Tanespimycin and IR780.²⁶ To verify this hypothesis, their morphologies were observed by TEM. The particles with a 3:1 (Figure 2B) or 1:3 (Figure 2D) ratio displayed high morphological heterogeneity, while the 1:1 group (Figure 2C) exhibited a homogeneously dispersed spherical morphology, with a 67.30 nm hydrodynamic size detected and a 0.224 PDI value by dynamic light scatter (DLS), which was selected for further analysis (Figure 2G). To further explore this interesting phenomenon, we observed the detailed morphology of TISN by atomic force microscope (AFM) and found the uniform multilayer structures, maybe indicating a potential fabrication mechanism via π–π stacking²⁷ (Figure 2E). In Figure 2F, Tyndall's effect occurred in the TISN solution and further verified the nanostructure of TISN.

Moreover, we delved into the self-assembly mechanism of TISN, employing a diverse array of investigative techniques to elucidate the underlying processes. Figure 2H demonstrated the absorption spectra TISN, Tane, and IR780. TISN exhibited a wider peak due to the π–π stacking and hydrophobic interaction between the aromatic ring of Tanespimycin and IR780.²⁸ In Figure 2I, with the increase of sodium chloride ion strength in the solution, the UV–vis spectrum of TISN did not change significantly, indicating that the electrostatic interaction between molecules can be ignored. To delve deeper into the self-assembly mechanism, TISN was dispersed in solutions of 0.2% (w/v) sodium dodecyl sulfate (SDS; Figure 2J) and DMSO (Figure 2K) for subsequent UV–vis spectroscopy analysis. After dissolving in DMSO, the typical absorption peak of IR780 in TISN was restored due to the destruction of noncovalent interaction between molecules. Moreover, a significant change was observed in the TISN spectrum in 0.2% SDS, as a result, SDS was involved in the hydrophobic effect in TISN. The absorption peak of IR780 in TISN is restored after the addition of SDS, which also confirms the important role of noncovalent bond interaction in TISN. Moreover, we recorded the FTIR spectrum of IR780 (Figure S1), Tanespimycin (Figure S2), and TISN (Figure S3). TISN displayed a strong peak at 2923.57 cm⁻¹ characteristic of the intermolecular hydrogen bond. Additionally, a peak was observed at 1718.92 cm⁻¹ characteristic of the conjugated bond. Moreover, the characteristic peaks at 1487.68 and 1025.62 cm⁻¹ are indicative of C-C skeleton stretching vibration and C-O stretching vibration. These observations, coupled with the hydrophobic interactions and π–π stacking effects identified, suggest that hydrophobicity and π-π stacking are the primary driving forces behind the self-assembly of TISN.²⁸ Different concentrations of TISN were used to draw the standard curve according to the absorbance at 780 nm (Figure 2L). The fluorescence spectrum of TISN was recorded in the Supporting Information (Figure S4). Additionally, the diameters of TISN aggregates in PBS and serum were monitored daily using DLS, and it was observed that they exhibited stability over a period of 7 days (Figure 2M).

2.2 GSH-stimulated released and tumor penetration of TISN

Due to the competition of intracellular reducing agent GSH, the π–π stacking may be destroyed and thus lead to loading drug release in tumor cytoplasm.^{29, 30} Once TISN penetrated tumor cells, part of the TISN consumed the intracellular GSH and thus increased the ROS generation via mitochondria-targeted PDT³¹ (Figure 3A). To verify this hypothesis, we performed a variety of in vitro and in vivo experiments. In Figure 3B, IR780 fluorescence was partly quenched after self-assembly into TISN in the absence of GSH. After GSH was added, the fluorescence was recovered. It is well established that tumor cells exhibit a higher concentration of GSH compared with their normal counterparts. After incubation with AGS cells, the IR780 fluorescence appeared indicating the stimulated released behavior of TISN. Furthermore, TISN was observed to depolymerize upon the addition of GSH, which is associated with alterations in its size (Figure 3C) and zeta potential (Figure 3D), highlighting the role of GSH in the self-assembly process of TISN. Moreover, the GSH consumption also indicates the disassembly of TISN and the release of Tanespimycin. We also observed the recovery of IR780 fluorescence to demonstrate the release of Tanespimycin after cellular uptake of TISN (Figures S5 and S6). After coincubation with IR780 and TISN for 0.5 h, the tumor cells were washed three times and observed in 0.5, 1, 2, and 4 h. As shown in the IR780 group, the fluorescence was stable from 0.5 to 4 h. In the TISN group, as IR780 and Tanespimycin were released, the fluorescence of IR780 gradually enhanced. To further quantify the consumption of GSH, we evaluated the GSH concentration at different time points after TISN was added (Figure 3E). Using the GSH/GSSG quantification kit, we found that GSH concentration was significantly consumed once TISN was incubated for 4 h. Though GSH concentration gradually decreased within 24 h, the residual concentration (66.62%) showed no significant difference compared with that at 4 h (51.92%). The consumption curve of GSH by Tanespimycin, IR780, and TISN (Figure S7) and the GSH concentration in tumor cells after different treatments (Figure S8) also proved the GSH depletion by TISN. To detect the Intracellular GSH depletion by TISN, we used FreSHtracer to evaluate the change in GSH concentration in tumor cells.³² As depicted in Figure S9, there were few red fluorescence signals (F580) before 0 h. After coincubation with TISN for 4 h, the fluorescence signals of F580 increased, indicating the intracellular depletion of GSH by TISN.

Next, we assessed the tumor penetration ability of TISN in 3D AGS tumor cell spheroids.³³ As described in Figure 3F, after 3 and 6 h of incubation, IR780 was mainly located in the margin areas of the tumor spheroids and indicated a limited ability to penetrate deeper into the tumor mass. However, upon incubation for 3 and 6 h, the green fluorescence of TISN was gradually distributed throughout whole spheroids. Besides, the surface plots also displayed a stronger signal of TISN in AGS tumor spheroids than IR780 (Figure 3H). Moreover, we detected the tumor penetration of IR780 (Figure 3I) and TISN (Figure 3J) in vivo. 24 h after tail injection of IR780 and TISN (200 µL, 100 µg/mL IR780, tail vein injection), we discovered an enhanced fluorescence signal from TISN concentrated at the tumor's core, underscoring the superior penetrating capability of this self-assembled system. This deeper penetration is a significant advantage, as it suggests that TISN can effectively reach the inner regions of tumors. In conclusion, these studies confirm the proficient tumor-infiltrating ability of TISN. Such characteristics are instrumental in enhancing drug delivery efficiency, which is paramount for advancing cancer treatment strategies.

2.3 PDT and PTT effect of TISN

To verify the PTT effect of TISN, different concentrations of TISN solutions were prepared with sterile PBS, including 0.125, 0.25, 0.5, 1, 2, and 4 µg/mL, respectively. They were irradiated by 808 nm laser and image acquisition was carried out every 1 min with a thermal image. As shown in Figure 4A, which refers to its capacity to convert light into heat, has been observed to escalate with an increase in concentration. At the concentration of 0.125 µg/mL, laser irradiation for 6 min can only reach about 40°C. If the concentration reaches 1 µg/mL above, the temperature can reach higher than 50°C after 6 min’ irradiation. It is worth noting that in a concentration of 4 µg/mL, 3 min’ irradiation reached a high temperature.

To further investigate whether TISN exhibited a better photothermal effect than IR780, a thermocouple thermometer was utilized to measure different samples (PBS, 4 µg/mL Tane, 4 µg/mL IR780, 4 µg/mL TISN, the concentration of TISN was calculated according to IR780). In Figure 4B, the temperature curve of TISN and IR780 increases rapidly with the duration of laser irradiation. The temperature increased by more than 25°C in 3 min and increased by about 35°C in 6 min. The temperature changes in PBS and Tanespimycin groups were much smaller. The cyclic heat generation effect of TISN was also detected (Figure 4C), in which TISN recovered to room temperature after irradiation for 3 min, and continued another irradiation for 3 min. After several irradiation-cooling loops, TISN can still maintain the effect of photothermal for 3 min to raise the temperature to about 35°C. Moreover, the photothermal conversion efficiency (PCE) of TISN was shown. We drew the temperature curve of TISN (Figure 4D) and IR780 (Figure 4E) in water with an 808 nm laser for 210 s followed by natural cooling. According to our previous study,³⁴ we calculated the PCE of TISN, IR780, and TISN+GSH (Figure 4F). Due to unfavorable dispersal in water, the PCE of IR780 was 12.47%, while the PCE of TISN significantly increased and reached 36.16%. Once GSH was introduced, the nanostructure of TISN was destroyed and the PCE decreased to 20.14%. These data indicated that TISN could serve as a favorable PTT agent.

The excellent PTT effect of TISN inspired the exploration of its potential in PDT. As suggested in Figure 4G, the singlet oxygen sensor green (SOSG) signal remained almost unchanged in the absence of photosensitizer of laser irradiation, indicating a negligible generation of singlet oxygen (¹O₂), like the blank control. Once the laser irradiation was introduced, TISN illustrated a similar fluorescence increase to IR780, displaying a dependence of ¹O₂ production on constant illumination. Moreover, the fluorescence signals increased with the concentration of TISN (Figure 4H) or irradiation time (Figure 4I), indicating that TISN generated ¹O₂ under NIR laser irradiation in a dose-dependent manner.

2.4 Synergistic mitochondria-targeted phototherapeutic efficiency of TISN

Tanespimycin inhibited Hsp90 and was applied to destroy the intracellular resistance against PTT and improve curative effect.^{35, 36} Moreover, HIF1α is a client protein of Hsp90 that could be downregulated by Tane.^{25, 37} Thus, the combination of Tanespimycin and mitochondria-targeted photosensitizer IR780 was supposed to achieve strong photocytotoxicity against tumor cells. As described in Figure 5A, we detected the Hsp90 expression level in tumor cells under PTT by immunofluorescence. As a molecular chaperone, Hsp90 is present in various tumor cells for fast proliferation and hypermetabolism under normal conditions. Moderate green fluorescence was witnessed in the control group. Comparatively, in the presence of Tane, the weak green fluorescence of the Tanespimycin group indicated that the expression of Hsp90 was inhibited. However, a strong fluorescence signal was discovered in the IR780+L group, and the Hsp90 expression level was upregulated under stress conditions. As expected, the TISN+L group exhibited a low intensity of green fluorescence of Hsp90, suggesting the destruction of the intracellular defense against PTT.

We previously reported that IR780 could specifically combine with mitochondria.^{14, 31} We evaluated the subcellular localization and TISN. As illustrated in Figure 5B, different subcellular localization between the green fluorescence of lysosome and the red fluorescence of TISN. On the contrary, the red fluorescence exhibited almost the same subcellular localization as the green fluorescence of mitochondria. According to the colocation scatterplots, the Pearson Correction (PC) coefficient of TISN and lysosome was 0.23, but the PC coefficient of TISN and mitochondria was 0.76. Additionally, the colocalization analysis of lysosome tracker and TISN suggested a different trend (Figure 5C), but mitochondria tracker and TISN indicated a similar trend (Figure 5D). Furthermore, biological transmission electron microscopy was applied to analyze the subcellular localization of TISN in the revised manuscript (Figure S10). Treatment of tumor cells with TISN+L leads to conspicuous damage to mitochondrial structures. Moreover, we employed the JC-1 assay kit to demonstrate the impact of various treatments on mitochondrial function within tumor cells, as depicted in Figure S11. Typically, under normal physiological conditions, mitochondria display red fluorescence, signifying a high mitochondrial membrane potential. In the control groups, which included treatments with PBS, IR780, Tanespimycin, and TISN, the majority of mitochondria preserved a healthy membrane potential, showing the persistent red signal. However, in the IR780 group, we discovered a significant decrease in red signals. Conversely, the TISN+L group displayed green fluorescence, a hallmark of a disrupted mitochondrial membrane potential. Based on the above results, we could conclude that the TISN showed specifically mitochondria-targeted intracellular localization.

After proving the Hsp90 inhibition and mitochondria-targeted features of TISN, the Mt-PIT effect was further examined. After treatment with PBS, IR780, Tanespimycin, and TISN with or without 808 nm laser irradiation, H₂DCFDA was utilized to detect ROS production (Figure 5E). In the PBS, IR780, and Tanespimycin without 808 nm laser irradiation, there was nearly no green fluorescence. In PBS+L, Tanespimycin+L, and IR780+L groups, in which ROS generated for GSH consumption or laser irradiation, there was a moderate level of ROS production. Compared with the IR780+L group, the TISN+L group showed the strongest green signal, indicating superior ROS production by enhanced PDT. Next, the synergistic mitochondria-targeted phototherapeutic efficacy was measured by Calcein-AM/PI double staining (Figure 5F). In the 3D spheroid tumor cells after different treatments, most of the living cells (marked as green) were found in the PBS, IR780, Tanespimycin, and TISN group, suggesting little cytotoxicity. Compared with the IR780+L group, a higher proportion of dying cells (red) in the TISN+L group were observed, suggesting a better curative effect. CCK-8 kit was also applied for TISN treatment examination in different concentrations. In the absence of 808 nm laser irradiation, the relative cell viability showed no significant difference, suggesting a favorable biocompatible out-of-laser-directing tumor area (Figure 5G). In Figure 5H, the relative cell viability of TISN with an 808 nm laser was significantly lower than the other two groups. The IC50 of Tanespimycin is 2.1331 nM while the IC50 of TISN is 0.7361 nM. We could conclude that TISN demonstrated both superior biosafety and potent phototherapeutic effect in the synergistic way of Hsp90 inhibition and GSH consumption.

2.5 Biodistribution and enhanced photothermal efficiency of TISN in vivo

TISN was able to be tracked by real-time NIR equipment for biodistribution evaluation due to the outstanding NIR imaging property of IR780 in vivo.³⁸ The mice injected with IR780 via tail intravenous were set as the control. The fluorescence of TISN was first detected at the tumor site by 6 h after tail intravenous injection of TISN, and the peak value was observed at 24 h after injection (Figure S12A). The fluorescence aggregated continuously on the tumor site, suggesting a considerable accumulation of TISN for the enhanced permeating and retention (EPR) effect. The fluorescence signals of TISN were stronger than IR780 due to deeper tumor penetration (Figure S12B). Ex vivo fluorescence results were reflected in Figure S13. A preferential accumulation of TISN was observed in tumor tissues rather than other organs. These different biodistribution profiles indicated that TISN exhibited an optimized tumor accumulation and prolonged circulation time, which may account for the improved tumor penetration after self-assembly.

We used an infrared thermal imager to analyze the photothermal effects of TISN in vivo (Figure S12C). Tumor-bearing mice in IR780+L and TISN+L groups were irradiated by 808 nm laser 24 h after intravenous injection. Different groups (Figure S12D) showed that the temperature of tumors in the IR780+L and TISN+L groups exhibited a mild temperature PTT effect. The temperature of tumors treated with other groups nearly did not change.

2.6 In vivo Hsp90 inhibition and antitumor efficacy

Next, the dual-mode phototherapeutic effect of TISN was examined. Tumor-bearing mice in different groups (PBS, IR780, Tanespimycin, TISN, IR780+L, and TISN+L) were randomly assigned and subsequently evaluated the efficacy of TISN (Figure 6A). No significant differences in body weight were observed among the groups (Figure 6B), suggesting no significant acute toxicity. Tumor volume curves were demonstrated in Figure 6C, upon irradiation, TISN-mediated mitochondria-targeted PTT/PDT dual mode phototherapy exhibited extraordinary treatment effect. Tumors in the TISN+L group gradually subsided and one of them was completely ablated without recurrence in the examination period. Ultimately, the mice were euthanized on day 13, and the tumors were subsequently excised, weighted (Figure 6D), and photographed (Figure 6E). Compared with the PBS group, TISN-mediated PIT displayed a 95% inhibition rate toward primary tumors. Furthermore, histological assessments, including hematoxylin and eosin (H&E), Hsp90, Ki67, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, were presented in Figure 6F. In the TISN+L group, the majority of cancer cells exhibited severe cellular damage, characterized by karyorrhexis, karyopyknosis, and karyolysis. The Hsp90 inhibition ability of TISN was also detected. Strong red fluorescence signals of Hsp90 were found in the IR780+L group, indicating a strong resistance in tumor cells against heat. However, the Hsp90 expression level in the TISN+L group was significantly inhibited, which led to a potent anticancer activity by way of Hsp90-inhibited PTT. The long-range antitumor effect was further verified in another survival analysis (Figure 6G). These above results demonstrated that the TISN exhibited a strong curative effect via Hsp90-inhibited and mitochondria-targeted synergistic phototherapy.

2.7 TISN mediated ICD and systemic antitumor immunity

Since TISN-based phototherapy exhibited the potential to prompt the activation of immune responses, we attempted to investigate ICD in TISN treatment³⁹ (Figure 7A). With the introduction of TISN, the HIF-1α level was significantly downregulated in TISN and TISN+L groups, as a result of Hsp90 inhibition (Figure 7B). CT26-bearing mice treated with PBS, IR780, Tanespimycin, TISN, and IR780+L showed less exposure to calreticulin (CRT), but tumor cells treated with TISN in combination with laser irradiation exhibited significantly enhanced CRT expression on the cell surface, a key indicator of ICD. Quantitative analysis further substantiated the superiority of TISN over other treatments in inducing ICD, as evidenced by the pronounced cellular responses observed in the TISN group (Figure 7B,G). Compared with the groups treated with PBS, IR780, Tanespimycin, TISN, and IR780+L group, TISN+L group demonstrated a significantly enhanced release of high mobility group protein B1 (HMGB1) and ATP from CT26 tumor cells. This suggests that the TISN+L treatment notably potentiated the emission of key mediators associated with ICD (Figure 7C,E). Moreover, WB analysis of HMGB1 reflected the amplified HMGB1 release in CT26-bearing mice of TISN+L treatment (Figure 7D). To elucidate the impact of DAMP release on dendritic cell (DC) maturation, we collected tumor-draining lymph nodes from CT26 tumor-bearing mice and performed flow cytometry analysis. As displayed in Figure 7F, treatment with IR780+L in colorectal tumor-bearing mice elevated the proportion of mature DCs, characterized by CD80⁺ and CD86⁺ expression in CD11c⁺ DCs. Moreover, the TISN+L treatment markedly increased the ratio of mature DCs (CD80⁺ CD86⁺ CD11⁺) surpassing the effects observed in the IR780+L group. We previously found that highly immunogenic photosensitizers induced systemic antitumor immunity via CD8⁺T cell activation,^40-42 thus we profiled the infiltrating CD8⁺T cells (Figure 7G). In the tumor microenvironment, the infiltration of CD8+ T cells was minimal in groups treated with PBS, IR780, Tanespimycin, TISN, and IR780+L, indicating that conventional phototherapy may be inadequate to modulate the immunological microenvironment effectively.^{43, 44} However, phototherapy sensitized with TISN+L notably intensified the infiltration of CD8+ T cells within the tumors (Figure 7H). Given that cytotoxic T lymphocytes can emit interferon-γ (IFN-γ), which is instrumental in modulating the tumor microenvironment, we proceeded to examine IFN-γ in CT26 tumors in vivo (Figure 7I). The release of IFN-γ in the TISN+L group was significantly more than in other groups, demonstrating its potential for optimal tumor immunotherapy.

2.8 Local and abscopal effects of TISN-mediated synergistic PIT in vivo

We sought to examine whether this synergist PIT strategy could inhibit tumor metastasis (Figure 8A). The body weight of tumor-bearing mice indicated there was no acute biotoxicity in this TISN-mediated Hsp90 inhibition and mitochondria-targeted synergist PIT strategy (Figure 8B). Remarkably, TISN+L treatment significantly inhibited primary tumor growth (Figure 8C,D,H). The tumors were collected on day 17 and weighed (Figure 8E). Furthermore, TISN-mediated PIT also significantly decreased the growth of untreated abscopal tumors, and tumors in two mice were eradicated (inhibition rate: 97.4%; Figure 8F,I). The tumor weights further confirmed the superior anticancer effect of this synergistic PIT strategy (Figure 8G). Additionally, the immunofluorescence images of tumors demonstrated increased tumor destruction and CD8⁺ T cells infiltrating into both local and abscopal tumors after TISN+L intervention (Figure 8J). These results displayed the TISN-mediated synergist PIT strategy could initiate a system immune response and generate an abscopal effect.

2.9 Biosafety evaluation

H&E staining was conducted on key organs, encompassing the heart, liver, spleen, lung, and kidney, to assess the safety (Figure S14A). In comparison with the control group, no significant signs of inflammatory lesions, hydropic degeneration, or histopathological necrosis were observed post-treatment, underscoring the excellent systemic biocompatibility of TISN. Furthermore, hematological and serological assessments were undertaken to scrutinize the potential long-term biosafety of TISN (Figure S14B–L). These findings underscore the high therapeutic biosafety of TISN and suggest its suitability for future clinical applications.

5.1 Materials

Tanespimycin and H₂DCFDA were purchased from MedChemExpress (Shanghai, China). IR780, Calcein AM, and PI were purchased from Sigma–Aldrich (Missouri, USA). DMSO was purchased from Sinopharm Chemical Reagent Co. DAPI and SOSG were purchased from Keygen Biotech. Anti-CD11c, anti-CD80, anti-CD86, and anti-CD8 antibodies were purchased from Proteintech.

5.2 Synthesis of TISN

TISN NPs were synthesized as previously reported. Briefly, Tanespimycin and IR780 were dissolved in 100 µL DMSO, respectively. Next, the two solutions at different volume ratios were mixed. Then pure water was added gradually under high-speed dispersion to form TISN NPs. The resulting solution was put in a dialysis bag (1000 kDa) for 2 h, to remove free Tanespimycin and IR780.

5.3 Characterization

The morphology and size of TISN with different ratios of Tanespimycin and IR780 were observed by TEM (JEOL, Japan). DLS (90Plus; Brookhaven Instrum. Corp) was used to detect the particle diagram and zeta potential. The absorption spectrum was evaluated by UV-2450 (Shimadzu, Japan).

5.4 Cells culture and animal model

AGS and CT26 cells were obtained from Pricella Biotechnology Co., Ltd at 37°C in 5% CO₂. Cell construction and experiment were conducted once the cells reached 80% confluence. AGS cells were suspended in PBS (1 × 10⁷ cells in 100 µL each mouse) and then subcutaneously injected into the right upper extremity area of nude mice. Tumor volume was evaluated by (length × width × width × 0.5). CT26 colorectal bilateral cancer model was established by a similar method.

5.5 Measurement of penetration of TISN NPs

5 × 10³ per well of tumor cells seeded into ultralow attachment round-bottom 96-well plates to achieve 3D cell spheroids. While the spheroids formed, IR780 and TISN NPs were added and incubated for 0, 3, and 6 h, respectively. The penetration depth of IR780 and TISN NPs was detected by a confocal microscope.

5.6 Detection of the photothermal effect of TISN

To ascertain the photothermal capabilities of PBS, IR780, Tanespimycin, and TISN, a range of concentrations were utilized to evaluate the PTT effect across different groups. The in vitro PTT effect was quantified using a thermocouple thermometer (TAIS 600), with concurrent documentation of thermal images via a Visual IR Thermometer (FLUKE VT02). For in vivo assessments, a similar methodology was employed, utilizing the same Visual IR Thermometer. AGS tumor-bearing mice were injected with 200 µL of the respective solutions at a concentration of 4 µg/mL IR780, with or without exposure to an 808 nm laser at an intensity of 1 W/cm². Thermal images and temperature recordings were captured accordingly. The PCE of TISN was determined using the formula as described by Wang et al. Following a 24-h incubation period with the aforementioned solutions, the expression of Hsp90 in AGS cells was analyzed through western blotting.

5.7 Subcellular localization of TISN and in vitro antitumor efficacy

Subcellular localization was conducted in the previous method. For the assessment of cell death using the Calcein-AM/propidium iodide double staining method, cells were coincubated with 100 µL of the staining solution in confocal wells (20 mm glass-bottom dishes; NEST Biotechnology Co. Ltd.) for 30 min. After this incubation, the cells were washed three times to remove excess stains and then examined using a confocal microscope. Live cells were stained with Calcein-AM, while dead cells were identified by propidium iodide fluorescence. The quantitative analysis of stained cells was performed using ImageJ software.

5.8 In vivo biodistribution

After being injected with TISN (200 µL, 100 µg/mL IR780), NIR fluorescence images were acquired by the CRI maestro system (λ_ex/λ_em = 740/810 nm) at different time points (3, 6, 12, 24, and 36 h). Another group of mice was euthanized for fluorescence imaging of tumors and organs.

5.9 In vivo phototherapy and systemic antitumor immune effect

Mice were divided into PBS, IR780, Tane, TISN, IR780+L, and TISNL groups (n = 5). After treatment for 12 days, the mice were sacrificed and tumor tissue was collected for further histopathology analysis. In vitro and in vivo experiments were conducted as previous publication.⁵⁹ Once the immune cells in the spleen were obtained, then resuspended into the 24-well plate with 24 h pre-seeded CT26 cells with 2 × 10⁵ cells per well. After a 72-h incubation, the concentrations of TNF-α and IFN-γ in the supernatant were measured.

5.10 Statistical analysis

Statistical analyses were done via GraphPad Prism software with a significance level for ns p > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001. Data were shown in the mean ± standard deviation (SD) method.