Synthesis and characterization of CICF
CpG oligonucleotide is a type of immune adjuvant that can activate Toll-like receptor 9 (TLR9) to trigger an immune response and enhance anti-tumor immune responses [32]. ICG is commonly used as a fluorescent dye in the medical field and can trigger its powerful photothermal and photodynamic effect upon irradiation with an NIR laser [33]. Previous studies reported that metal ions, DNA, and small molecule drugs can self-assemble into nanoparticles through coordination [28, 34]. In this study, with the aid of Cu2+ ions, CpG and ICG molecules were co-assembled into a uniform nanosphere, named Cu-ICG-CpG (CIC) (Fig. 1A). The element distribution of CIC was analyzed through the high-angle annular dark-field scanning transmission electron microscopy-energy-dispersive X-ray spectroscopy (HAADF-STEM-EDS) elemental mapping (Fig. 1F). P (derived from CpG), S (derived from ICG), and Cu elements were homogeneously distributed in the CIC nanoparticles, demonstrating the successful self-assembly of CpG, ICG, and Cu2+ ions.
A The efficient coordination-driven self-assembly of cargos during the synthesis process of CICF. TEM showed the morphology of CICF at B low magnification (scale bar: 200 nm) and C high magnification (scale bar: 100 nm). D EDS line scanning profiles of CICF. TEM element mapping images of C, N, O, P, S, and Cu in E CICF and F CIC. Scale bar: 200 nm. G DLS analysis of the hydrodynamic size of CIC and CICF. H Zeta potential of CIC and CICF. I UV–vis absorption spectra of FA, CIC, and CICF. Results are presented as means ± SD (n = 3)
As a vitamin, folic acid (FA) has become an important ligand in targeted cancer therapy because it can bind to a tumor-associated antigen called the folate receptor (FR). Here, FA was modified on the surface of CIC nanoparticles through coordination. After modification with FA, CICF binds specifically to folate receptors on the surface of tumor cells through a receptor-ligand interaction mechanism. This enables more effective recognition, binding, and entry into folate receptor-positive tumor cells, thereby enhancing targeting capability [35]. Representative transmission electron microscope (TEM) images of CICF (Fig. 1B, C). Monodisperse nanoparticles with a diameter of about 200 nm can be clearly observed. Scanning electron microscope (SEM) showed a similar morphology of CICF compared with CIC nanoparticles (Figure S1). Dynamic light scattering (DLS) results indicated the average diameter of CICF increased from 201.96 ± 1.95 nm to 216.10 ± 4.8 nm (Fig. 1G). The zeta potential of CICF decreased from −16 mV to −19 mV (Fig. 1H) due to the existence of carboxyl negative groups in FA. Furthermore, we observed characteristic peaks of FA in CICF (Fig. 1I), directly demonstrating the successful modification of FA. Therefore, there are certain differences in properties between CIC and CICF. The HAADF-STEM-EDS elemental mapping of CICF also illustrated a uniform distribution of P (derived from CpG), S (derived from ICG), and Cu elements (Fig. 1E). These homogeneously distributed elements within a single CICF nanoparticle are also confirmed by the EDS line scan profiles (Fig. 1D). Then the quantitative analysis of ICG molecules contained in the CICF nanoparticles was conducted. An absorbance-concentration plot was tested based on a series of ICG concentrations, resulting in a fitted straight line y = 0.04015x + 0.01111, R2 = 0.998 (Figure S2). According to the linear relationship of ICG, the loading content of ICG in CICF nanoparticles was 64%. Similarly, the loading content of CpG was calculated as 35% (Figure S3). The high loading efficiency of ICG and CpG in CICF was based on the efficient coordination-driven self-assembly of cargos during the synthesis process of CICF. In addition, to ascertain the acid-degradation capability of CICF, we conducted a comparative experiment utilizing solutions with pH values of 6.4 and 7.4 to monitor the release of ICG from CICF (Figure S6). Under acidic conditions (pH 6.4), there was a marked increase in the release of ICG. These findings demonstrate that CICF possesses robust acid-degradation properties, enabling the effective release of ICG in an acidic environment.
Next, we evaluated the photothermal ability of CICF under NIR laser irradiation. Under the irradiation of the NIR laser, the temperature of the CICF solution (50 μg/mL) rises rapidly, reaching the highest temperature of 61 °C at about 5 min light irradiation (1.0 W/cm2) (Fig. 2A). An increase in the concentration of CICF will result in the generation of more heat when exposed to near-infrared laser irradiation. Notably, the aqueous solutions containing free ICG, CIC, and CICF presented similar temperature profiles, indicating that the self-assembly of nanoparticles and the FA modification cannot impact the photothermal effect of ICG molecules (Fig. 2B). The temperature increase curves of different concentrations of CICF showed a concentration-dependent photothermal behavior (Fig. 2A). To determine the photothermal conversion efficiency of CICF, the heating and cooling curve of the aqueous solution of CICF was tested (Fig. 2D). As presented in Fig. 2D, E, the photothermal conversion efficiency was calculated to be 33.35%. In addition, CICF still showed a high photothermal conversion effect after 5 ON/OFF irradiation cycles (1 W/cm2), which proved that CICF had excellent photothermal stability (Fig. 2C). The photothermal effect of CICF is similar to previously reported results of materials constructed with ICG [36, 37]. Both the high photothermal conversion efficiency and the good photothermal stability suggest that CICF holds great promise for tumor photothermal therapy.
A Temperature curves with CICF at different concentrations under NIR laser irradiation (1.0 W/cm2). B Temperature curves of different solutions with the same amount of ICG upon the NIR laser irradiation (1.0 W/cm2). C Temperature curves of ICG and CICF (100 µg/mL) over five laser irradiation ON/OFF cycles. D Photothermal effect CICF with NIR laser irradiation (1.0 W/cm2). E Linear time data versus − ln(θ) obtained from the cooling period of (Fig. 2D). F DPBF was incubated with different groups and the UV–vis absorption spectra after NIR laser irradiation were compared to the generation of 1O2. G UV–vis absorption spectra of DPBF after incubation with CICF at different times following NIR laser irradiation for comparison of 1O2 generation. H UV–vis absorption spectra of MB aqueous solution under different treatment conditions. I UV–vis spectra of MB aqueous solution treated with CICF and GSH at different concentrations for 30 min. Results are presented as means ± SD (n = 3)
Methylene blue (MB) can be degraded by·OH and is considered as an indicator for Fenton reaction [11]. Herein, we chose MB as an indicator to evaluate the ability of CICF to produce·OH via a Fenten-like reaction. After incubation of CICF with H2O2 and GSH (CICF + H2O2 + GSH) for 30 min in MB solution, the absorbance of MB significantly decreased (Figs. 2H and S4). By contrast, the absorption intensity of MB remained unchanged after CICF, laser, or CICF + H2O2 treatments. These results indicate that GSH reduces Cu2+ to Cu+. Subsequently, the reduced Cu + reacts with H2O2, catalyzing the decomposition of H2O2 and generating hydroxyl radicals (·OH) with strong oxidizing properties. Moreover, the decrease in absorbance of MB in the CICF solution exhibited a concentration-dependent catalytic behavior (Fig. 2I). To verify that CICF could effectively convert GSH to GSSG, the GSH indicator of 5,5′-dithiobenzene 2-(nitrobenzoic acid) (DTNB) was used to test the GSH consumption during the ·OH generation. The colorless DTNB can be reacted with GSH to generate a yellow compound of TNB, therefore providing a means for assessment of GSH consumption [38]. GSH levels decreased with increasing concentrations of CICF, indicating the effective reduction of GSH induced by CICF (Figure S5).
ICG can be used not only as a photothermal agent but also as a photosensitizer for photodynamic therapy (PDT). To study the ability of NIR to produce singlet oxygen (1O2), the production efficiency of 1O2 was evaluated by using 1, 3-diphenylisobenzofuran (DPBF). DPBF generally has a characteristic absorption peak near 420 nm, and its intensity decays irreversibly in the presence of 1O2 [9]. CICF exhibits a similar reduction in absorbance values at NIR laser compared to free ICG, indicating effective 1O2 production (Fig. 2F). In addition, after NIR laser irradiation of CICF, the absorbance value of DPBF significantly decreased with the increase of time (Fig. 2G).
Cellular internalization and 1O2 generation of CICF
Encouraged by the effectiveness of PTT, PDT, and CDT, we further validated the effectiveness of CICF in vitro. Efficient internalization of materials by tumor cells is a prerequisite for achieving therapeutic effects. CICF specifically binds to folate receptors on the surface of tumor cells, thereby more effectively recognizing, binding to, and entering folate receptor-positive 4 T1 cells (Figs S7–S10). So, we evaluated the intracellular uptake efficiency of CIC and CICF by 4 T1 cells (expressed FA receptor) using confocal laser scanning microscopy (CLSM) and flow cytometry. The cells treated with CICF (CpG was labeled with Cy5) showed more red fluorescence signals than those incubated with CIC (Fig. 3A), validating that FA ligands can effectively drive the accumulation of CICF into tumor cells. In addition, flow cytometry analysis (Fig. 3B, C) showed that the fluorescence intensity of 4 T1 cells incubated with CICF was 2.4-fold higher than that of cells treated with CIC. We further investigated the subcellular localization of CpG internalization. Lysosomes were labeled with LysoTracker Green. As shown in Figure S11, the CpG in CICF was delivered to the lysosomes of the cells, and a portion of CICF escaped from the lysosomes, which may contribute to the functional efficacy of CICF.
A CLSM images of 4 T1 cells incubated with CIC-Cy5 or CICF-Cy5 for 4 h. Scale bar: 50 μm. B Flow cytometric analysis of the cellular uptake of 4 T1 cells treated with CIC-Cy5 or CICF-Cy5 for 4 h and C the corresponding normalized mean fluorescence intensity (MFI) of (B). D CLSM images of ROS in 4 T1 cells treated with or without NIR by different components of drugs. Scale bar: 50 μm. E The corresponding quantifications of ROS (D). F Flow cytometric analysis of ROS accumulation after treatment with CIC or CICF and G the corresponding normalized mean fluorescence intensity (MFI). H Intracellular relative GSH levels after CICF incubation. Results are presented as means ± SD (n = 3). The difference was statistically significant: **p < 0.01, ***p < 0.001, ****p < 0.0001
Subsequently, we identified the mechanism of killing tumor cells (Fig. 3I). However, the therapeutic efficiency of CDT often suffers from high concentrations of glutathione (GSH). Cu2+ can be reduced to Cu+ by depleting overexpressed GSH, thereby enhancing CDT [39]. To verify that CICF can effectively reduce the concentration of GSH, a GSH assay kit was utilized to test the intracellular GSH consumption. The intracellular GSH content was reduced to ~ 80% after 12 h treatment with CICF, indicating effective intracellular GSH consumption (Fig. 3H). Then the intracellular ·OH generation by CICF was investigated by using dichlorofluorescein diacetate (DCFH-DA) as ·OH indicator. CIC and CICF produce only weak green fluorescence signals under non-laser conditions. The 4 T1 cells incubated with ICG, CIC, and CICF following laser irradiation all showed significant green fluorescence compared to without irradiation. CICF-treated cells showed the strongest green fluorescence, indicating that targeted CICF produced more reactive oxygen species (ROS) (Fig. 3D, E). The production of ROS was further verified by flow cytometry. The production of ROS induced by CICF + NIR was significantly higher than that of other groups, and the flow cytometry results were consistent with the CLSM experimental results (Fig. 3F, G). Therefore, CICF could produce more ROS under NIR laser irradiation.
Antitumor performance of CICF in vitro
The in vitro cytotoxicity of 4 T1 cells irradiated at a power of 2 W/cm2 for 5 min was evaluated using CCK-8 assay (Figure S12). At this power, not only did each treatment group almost completely kill all tumor cells, but the control group also showed significant cell damage. Therefore, while maintaining the drug concentration in each treatment group, we adjusted the irradiation power to 1 W/cm2, with the irradiation time remaining at 5 min, to re-evaluate the anti-tumor efficacy of each group. As shown in Fig. 4A, control or free ICG treatments did not show an obvious cell-killing effect, demonstrating the negligible toxicity of the NIR light irradiation or free ICG to 4 T1 cells. The cells treated with CIC or CICF without NIR induced a low cytotoxicity because of the generation of ·OH. In contrast, the cells treated with CIC + NIR or CICF + NIR showed significant cytotoxicity compared with other groups, demonstrating that combined therapy could effectively improve the therapeutic effect. Notably, NIR-irradiated CICF showed higher cytotoxicity compared to that of the cells treated with CIC + NIR, indicating an enhanced antitumor efficiency of CICF through the FA targeting effect. A dose-dependent cytotoxicity of CICF upon NIR laser irradiation was also tested (Fig. 4B), verifying the excellent cell-killing effect of CICF as an anti-tumor agent. As shown in Figure S13, the survival rate of normal 16HBE cells was significantly higher than that of breast cancer 4 T1 cells at the same concentration of CICF, especially at 10 and 20 μg/mL concentrations. This finding suggests that CICF has good anti-tumor selectivity, which may be due to the fact that the modified FA on the surface of CICF can specifically bind to the overexpressed FA receptor on the surface of 4 T1 cells to promote the cellular uptake of CICF (Figs S9 and S10). The cell-killing efficiency of free ICG, CIC, and CICF against 4 T1 cells was further confirmed using live/dead cell staining of calcein acetoxymethyl ester (Calcein-AM) and propyl iodide (PI). The killing effect of CICF + NIR on 4 T1 cells is more significant than that of other treatments (Fig. 4C, E).
A Cell viability of 4 T1 cells treated with ICG, CIC, and CICF under laser or no laser conditions. B Cell viability of 4 T1 cells treated with different concentrations of CICF (from 5 to 20 μg/mL) under laser or no laser conditions. C Quantification of PI +/Annexin V-FITC + 4 T1 cells after the indicated treatment. D CLSM images of 4 T1 cells under different treatments were stained with Calcein-AM (green, live cells) and PI (red, dead cells). Scale bar: 50 μm. E Apoptosis of 4 T1 cells stained with Annexin V-FITC/PI was analyzed by flow cytometry. Results are presented as means ± SD (n = 3)
CICF-induced immunogenic death
PTT, PDT, and CTD were able to trigger host immunity, which provided promising potential in combination with immunotherapy [40]. Considering the functions of CICF contain PTT/PDT/CDT, we verified whether CICF could induce immunogenic cell death (ICD). The occurrence of ICD is accompanied by the release of a series of ICD-related molecules, including high mobility group box 1 (HMGB1) and calreticulin (CRT) [41]. CRT translocates from the endoplasmic reticulum to the cell membrane surface, a process that facilitates antigen presentation. Simultaneously, HMGB1 is released from the nucleus to the extracellular space, acting as a damage-associated molecular pattern (DAMP), which activates immune cells (Fig. 5I). The extracellular release of HMGB1 was first detected by CLSM. During ICD, tumor cells would translocate HMGB1 from the nucleus to the cytoplasm, followed by its secretion into the extracellular milieu. As shown in Fig. 5A, the levels of HMGB1 were marginally reduced in cells treated with CIC or CICF compared to the control group. The cells treated with CIC + NIR showed a weak fluorescence intensity, indicating a more efficient secretion of HMGB1 through combination therapy. It’s worth noting that the NIR-irradiated CICF exhibited the lowest levels of green fluorescence of HMGB1, indicating an enhanced secretion of HMGB1 from CICF due to the targeting effect of FA (Fig. 5A and C). CRT represented another significant feature of ICD. The cells treated with CICF in combination with NIR exhibited a pronounced red fluorescence of CRT, surpassing that observed in cells treated with other groups. This suggests that CICF-mediated PTT and CDT can effectively induce an immunogenic response (Fig. 5B and D).
A Images of CLSM released by HMGB1 in 4 T1 cells after different treatments. Scale bar: 50 μm. B Images of CLSM expressed by CRT in 4 T1 cells after different treatments. Scale bar: 50 μm. The corresponding quantifications of CLSM images fluorescence intensity of HMGB1 (C) and CRT (D). The cytokine concentrations of E TNF-α and F IL-6 in the supernatant of RAW264.7 cells cultured under different treatment conditions were detected by ELISA. G Flow cytometry assay of mature DCs (CD11c + CD80 + CD86 +) in the different groups. H Corresponding quantitative percentages of mature DCs in different groups. I immunogenic cell death activates DC cell maturation. Results are presented as means ± SD (n = 3). The difference was statistically significant: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
DCs are indispensable protagonists in anti-tumor immune response [42]. During ICD, signaling molecules such as CRT and HMGB1 are exposed on the surface of tumor cells, where they can be recognized by specific receptors on DCs, thereby facilitating the maturation of DCs [43]. To investigate the immunostimulatory effect of CICF on DCs maturation, we co-cultured mouse bone marrow-derived dendritic cells (BMDCs) with pretreated 4 T1 cells. The typical markers CD80 and CD86 were detected by flow cytometry (Fig. 5G, H). The CICF + NIR group elicited the most pronounced maturation of DCs, achieving a level 3.51 times greater than that observed in the control group. It is noteworthy that the DCs maturity in the CICF + NIR group is 1.52 times greater than that observed in the CICF group. This finding suggests that NIR can more effectively facilitate the release of tumor antigens in conjunction with CpG, thereby enhancing the efficacy of immunotherapy. Furthermore, the combination of CICF and NIR demonstrated a higher efficacy compared to CIC and NIR, providing additional evidence that CICF exhibits enhanced immunostimulatory activity following FA targeting. Mature DCs are capable of secreting immune-related cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). We subsequently assessed the secretion levels of TNF-α and IL-6 using enzyme-linked immunosorbent assays (ELISA) (Fig. 5E, F). The results indicated that the secretion levels of TNF-α and IL-6 induced by CICF were significantly elevated compared to those in cells treated with free CpG or CIC. This finding suggests that CIC and CICF effectively facilitate the intracellular delivery of CpG, thereby promoting the maturation of DCs and playing a crucial role in anti-tumor immunity.
Targeting ability of CICF in vivo and photothermal imaging
The favorable therapeutic outcomes in vitro motivated us to investigate the tumor accumulation capacity and photothermal properties of CICF in vivo. CICF (CpG was labeled with Cy5) was intravenously injected into the 4 T1 tumor-bearing mice with CIC as the control. The fluorescence intensity at the tumor site peaked at 1 h and then started to decline at 3 h (Figs. 6A and S14). Additionally, the fluorescence intensity of CICF was higher than that of CIC, demonstrating that the FA modification conferred better targeting ability. By euthanizing the mice, the major organs and tumor tissues were obtained for ex vivo imaging (Fig. 6B, C). The semi-quantitative findings from ex vivo imaging, as illustrated in Fig. 6E, F, were consistent with the whole-body fluorescence imaging shown in Fig. 6A. These results demonstrated that CICF achieved approximately a 2.2-fold enhancement in tumoral fluorescence intensity relative to CIC at 1 h post-injection, thereby indicating the effective tumor-targeted delivery capability of CICF. Similar results were reported for in vivo bioimaging evaluation of nanoparticles [44].
A The fluorescence distribution of 4 T1 tumor-bearing mice was observed after intravenous injection of CICF-Cy5 and CIC-Cy5 at 1, 3, 6, and 12 h. B Ex vivo fluorescence images of resected tumors and major organs at 1, 3, 6, and 12 h after CICF-Cy5 injection. C Ex vivo fluorescence images of tumors and major organs harvested 1 h after injection of CICF-Cy5 or CIC-Cy5. D Near-infrared thermal imaging of 4 T1 tumor-bearing mice at different time points after different treatments. E Semi-quantitative analysis of fluorescence intensity of Figure (C). F Semi-quantitative analysis of fluorescence intensity of Figure (B). Results are expressed as mean ± SD (n = 3). The difference was statistically significant: ****p < 0.0001
Given the satisfactory tumor-targeting capacity of CICF in vivo, we subsequently evaluated the photothermal effect of CICF on 4 T1 tumor-bearing mice. When the tumor volume reached 150–300 mm3, PBS, ICG, CIC, and CICF were intravenously injected into the mice. One hour after intravenous injection, the tumors were irradiated with an NIR laser, and the surface temperature of the tumors was monitored by infrared thermal imaging at 0, 1, 2, 3, 4, 5, and 10 to assess the PTT effect of each group (Figs. 6D and S15). Compared with the Control group, the tumor temperatures in the ICG group, CIC group, and CICF group significantly increased over time. Furthermore, the temperature observed in the CICF group exceeded that of the other groups, suggesting an enhanced potential for PTT in tumor eradication.
In vivo therapeutic effects of CICF
Encouraged by the efficient tumor accumulation capacity and excellent photothermal properties of CICF, we further evaluated the anti-tumor efficacy and in vivo safety of CICF in 4 T1 tumor-bearing mice (Fig. 7A). Mice were randomly divided into eight groups and treated with Control, NIR, ICG, ICG + NIR, CIC, CIC + NIR, CICF, and CICF + NIR, respectively. NIR laser irradiation was applied to mice at 1 h after tail vein injection. The changes in tumor volumes and body weights of the mice were monitored every 2 days for 15 consecutive days. The tumors in the Control, NIR, and ICG groups exhibited rapid growth, whereas tumor proliferation in the CIC and CICF groups was moderately suppressed. (Fig. 7B–D). This indicated that the laser irradiation and free ICG had no effect on tumor growth, and the CIC or CICF treatments had a poor therapeutic effect on tumors through CDT. The inhibitory effect in the CIC + NIR group was moderate, suggesting that the combined therapy of CDT and PTT had a better anti-tumor effect. The treatment regimen combining CICF with NIR demonstrated a significantly greater inhibition of tumor growth, suggesting that the introduction of tumor-targeting capabilities substantially enhanced antitumor efficacy. On the sixteenth day of the study, the mice were euthanized, and histopathological analyses were performed on their major organs, including tumor, heart, liver, spleen, lung, and kidney. The therapeutic efficacy of CICF was further assessed through hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (Fig. 7F), which revealed significant nuclear necrosis or dissociation of tumor cells in the CICF + NIR group. Therefore, both H&E staining and TUNEL assay demonstrate that the CICF + NIR group exhibits significant anti-tumor efficacy. Additionally, to further assess the potential inhibition of lung metastasis, the lungs of 4 T1 tumor-bearing mice were harvested after 30 days of various treatments to evaluate treatment efficacy (Fig. 7G). No significant lung metastases were observed in the CICF + NIR-treated mice compared to other groups. Therefore, under near-infrared laser irradiation, CICF not only exerts chemodynamic therapy (CDT) effects but also induces photodynamic therapy (PDT) and photothermal therapy (PTT), thereby achieving a synergistic effect of CDT and PDT/PTT. This significantly enhances the efficiency of tumor growth inhibition and more effectively prevents lung metastasis. CICF system, combining CDT/PDT/PTT under NIR, aligns with multimodal nanotherapeutics (e.g., CuS nanoflakes, catalytic scaffolds) that disrupt tumor immunosuppression via synergistic ROS/hyperthermia-driven antigen release and immune activation. This nanoengineered strategy enhances tumor suppression and metastasis prevention, exemplifying the power of material-driven synergy in advancing precision cancer immunotherapy [29,30,31, 45].
A Diagram of treatment in vivo in mice. B Representative photographs of removed tumors obtained on day 15 of mice in different treatment groups. C Average tumor weight after 15 days of treatment with different groups. D Tumor growth curves of 4 T1 tumor-bearing mice within 15 days after treatment with different groups. E Body weight changes of the mice during therapy. F H&E and TUNEL staining images of tumor sections of 4 T1 tumor-bearing mice. Scale bar: 200 μm. G Photographic and H&E staining images of the isolated lungs. Scale bar: 100 μm. Results are expressed as mean ± SD (n = 3). The difference was statistically significant: **p < 0.01, ****p < 0.0001
The antitumor immune response of CICF in vivo
Subsequently, we evaluated the antitumor immune response elicited by CICF through immunofluorescence staining of tumor sections. The tumor tissue treated with CICF + NIR showed a strong green fluorescence of CRT and low red fluorescence of HMGB1, which is consistent with the results at the cellular level (Fig. 8A). These results suggested that CICF possessed the capability to not only eradicate tumors but also effectively induce ICD in tumors. Subsequently, we analyzed the maturation of DCs in tumor tissues by flow cytometry (Fig. 8D, E). After NIR laser irradiation, the CICF + NIR group induced the highest level of DCs maturation thanks to the synergistic effect of CpG and ICG. The percentage of mature DCs (CD80 + CD86 + in CD11c +) increased from 37.2% to 76.8%. DCs maturation can further stimulate cytotoxic T lymphocytes (CTLs) and improve anti-tumor immune response. Next, we tried to determine the activation status of T cells in mice (Fig. 8C). The CICF + NIR group significantly increased the number of tumor-infiltrating CD4 + and CD8 + T cells compared to the Control group. In addition, the secretion of cytokines, such as IL-6 and TNF-α, is a key indicator of anti-tumor immune response. The levels of IL-6 and TNF-α in the serum of mice treated with CICF + NIR were significantly upregulated (Fig. 8F, G). The findings suggested that the NIR-irradiated CICF treatment was capable of eliciting a robust immunogenic response in vivo.
A HMGB1 and B CRT immunofluorescence staining images of tumor sections of 4 T1 tumor-bearing mice. Scale bar: 100 μm. C Immunofluorescence images of CD4 +, CD8 +, CD80 +, and CD86 + in tumor tissues. Scale bar: 100 μm. D The maturation of DCs in tumor tissues was detected by flow cytometry. E Quantification of the proportion of mature DCs within tumor tissue. Serum levels of F IL-6 and G TNF-α were detected after 15 days of treatment with different groups. Results are expressed as mean ± SD (n = 3). The difference was statistically significant: **p < 0.01, ***p < 0.001, ****p < 0.0001
Biosafety of CICF in vivo
The safety of CICF was further evaluated. All groups showed negligible changes in mouse weight for 15 days after treatments (Fig. 7E). Subsequently, to further assess the potential toxicity of CICF, we conducted histological examinations and hematological analyses. The results of H&E staining (Figure S16) revealed that there were no apparent pathological lesions in the vital organs of the treated mice compared to the control group, indicating that CICF treatment caused no significant damage to the organ structure of the mice. Furthermore, the serum biochemical indices (Figure S17) and complete blood cell counts (Figure S18) showed no significant differences between the treatment groups and the control group, further demonstrating that CICF not only possesses excellent anti-tumor efficacy but also exhibits good biocompatibility.