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• Phototherapy, such as photothermal therapy (PTT) and photodynamic therapy (PDT), is an emerging therapy with advantages of minimal invasiveness, negligible systemic toxicity, and high selectivity [1–5]. Moreover, the produced heat in PTT and reactive oxygen species (ROS) in PDT lead to in situ tumor antigens exposure, causing immunogenic cell death (ICD) which further activates the systemic immunity to inhibit the tumor growth and recurrence [6,7]. However, the therapeutic efficacy is limited due to the weak tissue penetration ability of the light [8–11]. The combination of phototherapy and other light-independent treatment methods is very beneficial for the treatment of deep tumors [12,13].

  Chemodynamic therapy (CDT) is a light-independent cancer treatment which utilizes the cytotoxic hydroxyl radical (^•OH) produced by the Fenton-like reaction to kill tumor cells [14–16]. The ^•OH enhances oxidative stress of the cells, thereby activating immune response [17]. Cascading CDT and phototherapy is usually achieved with a metal-containing phototherapy platform [18,19]. For example, Cai et al. prepared nanocomplexes of dual photosensitizers (merocyanine 540 and chlorin-e6)-loaded upconversion nanoparticles (NPs) and ferric-tannic acid for synergetic PDT and CDT [20]. Jiang et al. synthesized Mn²⁺- and oligodeoxynucleotides-modified phosphorus nanosheets for combining PDT, PTT, and CDT. The enhanced ICD by PTT and CDT was able to increase T lymphocyte infiltration and induce strong immune response [21]. While the limited H₂O₂ within tumor limits the advantages of CDT.

  Fortunately, glucose oxidase (GOx) induced starvation therapy can validly convert endogenous glucose to gluconic acid and H₂O₂, which exacerbates the oxidative stress and acidic tumor microenvironment (TME) [22–25]. Thus, starvation therapy cascaded CDT is widely used in combining with other therapeutic modalities. For example, Lin et al. prepared copper nano-enzymes (named as Cu-MCGH) by using dendritic mesoporous copper carbon nanospheres as a carrier, loaded with GOx and then modified with hyaluronic acid. The copper nano-enzymes possessed peroxidase and glutathione oxidase mimetic activities to enhance the production of ^•OH. Under 1064 nm laser irradiation, both ^•OH production and GOx-induced blockage of energy supply reduced the expression of heat shock proteins, which enhanced PTT (η = 34.9%), and the synergistic effect of multiple therapies resulted in a tumor inhibition rate of up to 93.4% [26]. Zhang et al. prepared a novel iron-based POF material named IPM, which has a porous structure that facilitates drug loading; the chemiluminescent material-luminol and GOx were loaded into IPM, and then assisted by N-methylethylenediamine (T^ER) and hyaluronic acid (HA), IPM@Luminol@GOx/HA-T^ER (PLGH-T^ER) was obtained. PLGH-T^ER exhibited endoplasmic reticulum (ER)-targeted accumulation after being internalised by the cells. GOx converted glucose overexpressed in the tumor to H₂O₂, cutting off the nutrient supply of starvation therapy and increasing the concentration of H₂O₂, and the reaction of luminol with H₂O₂ triggered chemically-excited PDT, which facilitated the production of singlet oxygen (¹O₂) [27]. However, the enhanced acidity of the TME due to gluconic acid produced by the reaction of GOx with glucose has not been effectively exploited.

  Considering the advantages of the combination therapy mentioned above, herein, we designed and prepared MICG NPs by assembling GOx and indocyanine green (ICG) with MnCO₃ NPs for starvation therapy cascaded CDT, enhanced PDT and immune activation. As shown in Scheme 1, the GOx consumes intratumoral glucose resulting in starvation therapy, and simultaneously increases H₂O₂ concentration and exacerbates the acidic TME. The decreased pH in tumor is beneficial for degrading MnCO₃ NPs to release Mn²⁺ which can catalyze H₂O₂ to generate ^•OH for CDT. Under laser irradiation, ICG generates cytotoxic ¹O₂ and heat to kill cancer cells through PDT and PTT mechanism, respectively. Additionally, the ^•OH and ¹O₂ would accelerate the oxidative stress to strengthen ICD and induce the maturity of dendritic cells (DCs), and in turn activated the systemic immunity. This work provides a new therapeutic platform for combining therapy of tumor.

  Scheme 1

  

  Scheme 1.  Schematic diagram of the therapeutic mechanism of MICG NPs.

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  The MnCO₃ NPs were prepared following previous report [28]. The scanning electron microscopic (SEM) images shown in Fig. 1a revealed that the prepared MnCO₃ NPs have the uniform cubic morphology, with a size of about 100 nm. Interestingly, this morphology is stable in neutral solution (pH 7). However, MnCO₃ NPs could be decomposed in acid solution (pH 5), and completely broken down after 6 h, suggesting MnCO₃ NPs can serve as the TME responsive drug carrier. The X-ray photoelectron spectroscopic (XPS) spectrum shown in Fig. S1 (Supporting information) revealed that Mn mainly existed as Mn²⁺, enabling them the potential application in CDT. Next, the zeta potentials of GOx, ICG, and MnCO₃ NPs were tested separately (Fig. S2 in Supporting information), the potential of GOx, ICG, and MnCO₃ NPs is 20, −4, and −58 mV. Therefore, GOx and ICG can be co-loaded on the surface of MnCO₃ NPs through electrostatic interaction and prepared as MICG NPs. As verified by Fig. S2, the successful drug loading is proved by the zeta potential of MICG NPs increased from −58 mV (MnCO₃ NPs) to −29 mV.

  Figure 1

  

  Figure 1.  (a) Time-dependent SEM images of MnCO₃ NPs in pH 7 and pH 5 buffer solutions. (b) Absorbance spectra of ICG, MnCO₃, and MICG NPs aqueous solutions. (c) Fluorescence spectrum of MICG NPs aqueous solution. (d) The temperature increasing curve of H₂O, GOx, MnCO₃ NPs, ICG, and MICG NPs aqueous solutions under laser irradiation. (e) Linearity of absorbance at 400 nm of MICG NPs, ICG and H₂O in ABDA-Na₂ aqueous solution with irradiation time. (f) Time-dependent changes of pH in groups. (g) H₂O₂ concentration-dependent linear plot of fluorescence ratios of MICG NPs, MnCO₃ and H₂O in the presence of TA (60 µmol/L).

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  Moreover, the overlap in the ultraviolet-visible absorption spectra of the ICG, MnCO₃ NPs, and MICG NPs shown in Fig. 1b indicating the successful loading of all the components. Based on the photochemical properties of ICG, MICG NPs retain the near-infrared fluorescence, photothermal conversion and photodynamic properties of ICG. As shown in Fig. 1c, MICG NPs have strong fluorescence emission at 810 nm. Fig. 1d demonstrated the temperature rise curves of MICG NPs and ICG with time, under 808 nm laser irradiation, the temperatures of MICG NPs and ICG both rapidly increased to about 45 ℃ at 240 s (Fig. 1d). The temperature of MICG NPs was slightly faster than that of ICG, which may be caused by the aggregation of ICG. ICG have the ability to produce ¹O₂ effectively under 808 nm laser, and similarly, the photodynamic effect of ICG was also retained in MICG NPs, a significant decrease in the absorption peak of the ¹O₂ trapper–sodium 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA-Na₂) with irradiation time was observed for MICG NPs (Fig. 1e and Fig. S3 in Supporting information), indicating that MICG NPs have the capability to produce ¹O₂ under 808 nm laser. Compared with H₂O and ICG, MICG NPs have a slightly stronger ¹O₂ production ability. Although, ICG has poor photostability, the photostability of MICG improved after ICG loading on MnCO₃, as shown in Fig. S4 (Supporting information), which is favorable for the next in vivo applications.

  Moreover, the MICG NPs exhibit similar pH-dependent enzyme activity with GOx. As shown in Fig. 1f, the pH changes little after the interaction of GOx and glucose under neutral conditions, while the pH of the mixture of GOx and glucose decreases faster in acidic solution, which indicates that the presence of MnCO₃ and ICG has little effect on the enzymatic activity of GOx, and also proves that GOx and MICG NPs produce a large amount of gluconic acid when interacting with glucose under acidic conditions. Moreover, the produced gluconic acid is beneficial for decomposing MnCO₃ NPs to Mn²⁺, which could further catalyze H₂O₂ and generate ^•OH. Next, the ability of the chemodynamics of MnCO₃ and MICG NPs to generate ^•OH were verified by simulating TME at the solution level. As shown in Fig. 1g and Fig. S5 (Supporting information), the fluorescence intensity of the ^•OH trapper-terephthalic acid (TA) increased gradually with the addition of H₂O₂ when MnCO₃ and MICG NPs were present in an acidic solution (pH 5), whereas there was no change in the fluorescence peaks of only TA in an acidic solution. The results showed that MICG NPs were able to be decomposed into Mn²⁺ and catalyze the generation of ^•OH from H₂O₂ under acidic conditions.

  The excellent photochemical properties and GOx enzyme activity of MICG NPs suggesting they could be used for multimode therapy of tumor. The ROS generation capability of MICG NPs was confirmed in cells firstly. As shown in Fig. 2a and Fig. S6 (Supporting information), the obvious green fluorescence of O22 in the cells treated with MICG NPs + laser (L) indicated the effective cellular ¹O₂ production, while the phosphate buffered solution (PBS) and MICG NPs groups showed no fluorescence. The O27 (^•OH probe) staining is depicted in Fig. 2b. The images showed clear green fluorescence, which is the indication of the effective ^•OH production in cells by Mn²⁺-catalyzed Fenton-like reaction. Next, the intracellular treatment was verified using calcein-AM/propidium iodide (AM/PI), as shown in Fig. 2c, where the starvation effect or chemodynamic efficacy resulting from the treatment of GOx, MnCO₃ NPs, and MICG NPs led to a certain degree of cell death, whereas the tumor cells were completely dead after the treatment of MICG NPs + L, which confirmed the efficacy of combined multimodal treatment.

  Figure 2

  

  Figure 2.  Images of (a) O22 staining for cellular ¹O₂ production, and (b) O27 staining for cellular ^•OH production, (c) AM/PI staining for live and dead cells, and (d) JC-1 staining for mitochondrial dysfunction detection. Scare bar: 100 µm. Concentration-dependent viability of cells treated with (e) GOx, MnCO₃ NPs, MICG NPs, and (f) ICG, ICG +L, MICG NPs + L. Data are presented as mean ± standard deviation (SD) (n = 6). (g) CRT and (h) HMGB1 exposure shown by green fluorescence. Scare bar: 50 µm.

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  It has been reported that Mn²⁺ has the potential to cause the dysfunction of mitochondria [29,30]. As verified by the JC-1 staining shown in Fig. 2d, the cells exhibited green fluorescence in GOx and MnCO₃ NPs groups. The JC-1 probe also emitted green fluorescence in 4T1 cells after GOx incubation, which was attributed to the fact that the starvation treatment cut off the energy supply to the cells, leading to a certain degree of mitochondrial dysfunction, which may be related to the effect of the excess H₂O₂ and gluconic acid produced in cells as well. While the green fluorescence was further enhanced in the cells treated with MICG NPs or MICG NPs + L, indicating the mitochondrial dysfunction as a result of Mn²⁺, GOx and ROS. As shown in Fig. 2e, the viability of cells incubated with GOx, MnCO₃ NPs, and MICG NPs decreased in a concentration-dependent manner, which is an indication of the effective starvation therapy and CDT. ICG showed negligible cytotoxicity, and after 808 nm laser irradiation, cell viability was further decreased (Fig. 2f) as a result of phototherapy, whereas the cell viability of MICG NPs + L treatment was significantly lower than that of ICG + L, which again demonstrated the advantage of starvation therapy/CDT/PDT/PTT combination therapy for MICG NPs + L.

  By assessing changes in intracellular fluorescein isothiocyanate (FITC)-labelled high mobility group protein B1 (HMGB1) and calreticulin (CRT) expression, the ability of different treatment groups to induce ICD was investigated. During ICD, HMGB1 migrates outward from the nucleus and excludes the extracellular area, and the expression of HMGB1 in the nucleus is reduced; whereas CRT is expressed in the endoplasmic reticulum under normal conditions, and under immune stress, CRT translocates and exposes itself to the surface of the cell membrane, releasing the "eat-me" signals to the immune cells, which activate the immune response. As shown in Fig. 2g, the green fluorescence was observed in the cells treated by MICG NPs and laser irradiation, suggesting the expression of CRT was increased. At the same time, the fluorescence intensity of HMGB1 in the nucleus was significantly reduced after treatments (Fig. 2h). All the above observations are the evidence of ICD at the cell level.

  Next, we evaluated the imaging and therapeutic efficacy of MICG NPs by intratumorally injecting them into in 4T1 tumor-bearing mice. All animal experiments conducted in this study have been approved by the Ethics Committee of Hunan Normal University (No. D2022024). As shown in Fig. 3a, strong NIR fluorescence signal was observed at the tumor site. The infrared thermal images were recorded during 808 nm laser irradiation. The images illustrated that the temperature of MICG NPs + L increased to 70.5 ℃ after laser irradiation for 10 min, while that of PBS + L increased to only 39.3 ℃ (Fig. 3b). This result illustrates the good photothermal ability of MING NPs in vivo. Then, the tumor volume (Fig. 3c) and mice weight (Fig. 3d) were recorded every 2 days for 2 weeks. The results showed that the tumor volume of MICG NPs-treat mice was smaller than that of GOx- and MnCO₃ NPs-treated mice. After laser irradiation, the tumors of mice in the MICG NPs + L group were largely eliminated. This shows that MICG NPs can effectively inhibit the tumor growth, which is very more than groups of GOx and MnCO₃ NPs. Moreover, these treatments did not result in any obvious weight changes (Fig. 3d).

  Figure 3

  

  Figure 3.  (a) NIR fluorescent image of MICG NPs in mouse. (b) Time-dependent infrared thermal images of mouse intratumorally injected with MICG NPs or PBS under 808 nm laser irradiation for 10 min. Time-dependent (c) tumor volume and (d) mouse weight variation after different treatments. Data are presented as mean ± SD (n = 5). (e) Images of H&E-, TUNEL- and Ki67-stained tumors with different treatment. (f) Images of H&E staining of major organs from different treated mice. Scare bar: 200 µm.

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  Besides, hematoxylin and eosin (H&E) staining (Fig. 3e) images of tumors illustrated that MICG NPs and MICG NPs + L could effectively destroy tumor tissues. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining (Fig. 3e) showed obvious green fluorescence, which is indicative of cell apoptosis promoted by MICG NPs + L. Moreover, the Ki67 staining images illustrated that the cell proliferation of MICG NPs- and MICG NPs + L-treated tumors was obviously inhibited (Fig. 3e). All the above are evidence contributing to the multi-mode therapy of MICG NPs. The mouse weight also was not obviously changed, an indication of the good biosafety of the therapy. This is in line with the results from H&E staining (Fig. 3f) of major organs from different treated mice.

  The immune response caused by different treatment groups was further studied using 4T1 tumor-bearing mice. The results of flow cytometry showed that the percentage of T cells in the spleen increased significantly from 19.4% to 49% (Fig. 4a and Fig. S7a in Supporting information) and the percentage of DCs increased from 45.7% to 63.2% (Fig. 4b and Fig. S7b in Supporting information) after MICG NPs + L treatment compared with that of the PBS group, which implies that MICG NPs + L treatment led to an increase in the percentage of immune cells in the spleen and an effective activation of the immune system. DCs is a class of immune cells in macrophages associated with antigen presentation and delivery. Next, the immune response in the tumor was investigated by flow cytometry. Unsurprisingly, as shown in Figs. 4c, d and Figs. S7c, d (Supporting information), a significant elevation in the percentage of T cells and DCs was also observed in MICG NPs + L-treated tumors compared to the PBS group (T cells, 24.4% vs. 47.4%; DCs, 48.8% vs. 73.6%). A significant immune response occurred in the tumors after MICG NPs + L treatment, and these immune cells were generated by the activated immune cells migrated from the spleen. This means that the systemic immunity of the mice was enhanced by the drug treatment, and the immune environment of the tumors was also strengthened.

  Figure 4

  

  Figure 4.  Quantitative analysis of cells densities of CD3⁺ T cells and CD80⁺ CD86⁺ DCs in (a, b) spleen and (c, d) tumor (n ≥ 3). Data are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA test with the Tukey's test. *P < 0.05, **P < 0.01, ****P < 0.0001. ns, no significance.

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  In summary, we constructed multi-functional nano-platform MICG NPs for TME-responsive starvation cascaded CDT that increases ROS production with phototherapy for antitumor immune response. These NPs could reserve their acid-responsive enzyme activity, which in turn allowed GOx to produce H₂O₂ and gluconic acid in the tumor. The produced acid could then help to decrease MnCO₃ NPs to release Mn²⁺ for CDT. The ICG endowed NPs with phototherapy and NIR fluorescent imaging ability. Besides, the prepared NPs were found to possibly activate the immune response. All these properties of MICG NPs contribute to their multi-mode therapy capability, and the assessments in cells and mice illustrated the anti-tumor capability of MICG NPs, demonstrating that they are promising candidates for NIR imaging-guided synergistic antitumor therapy.

  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

  Qiuxia Tan: Writing – original draft, Formal analysis, Data curation, Conceptualization. E Pang: Writing – review & editing. Qin Wang: Data curation. Yuanyu Tang: Data curation. Pan Zhu: Data curation. Shaojing Zhao: Writing – review & editing, Resources, Funding acquisition, Data curation. Jianing Yi: Data curation. Shiguang Jin: Writing – review & editing, Resources, Funding acquisition, Data curation. Minhuan Lan: Writing – review & editing, Supervision, Resources, Funding acquisition, Data curation.

  Acknowledgments

  This work was supported by the National Key Research and Development Program of China (No. 2022YFA1207600), the National Natural Science Foundation of China (Nos. 62375289, 62175262), the Science and Technology Innovation Program of Hunan Province (No. 2022RC1201), the Scientific Research Fund of Hunan Provincial Education Department (No. 22B0081), Postdoctoral Funding Project of Jiangsu Province (No. 2019Z156).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110770.

  
  
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  Scheme 1  Schematic diagram of the therapeutic mechanism of MICG NPs.

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  Figure 1  (a) Time-dependent SEM images of MnCO₃ NPs in pH 7 and pH 5 buffer solutions. (b) Absorbance spectra of ICG, MnCO₃, and MICG NPs aqueous solutions. (c) Fluorescence spectrum of MICG NPs aqueous solution. (d) The temperature increasing curve of H₂O, GOx, MnCO₃ NPs, ICG, and MICG NPs aqueous solutions under laser irradiation. (e) Linearity of absorbance at 400 nm of MICG NPs, ICG and H₂O in ABDA-Na₂ aqueous solution with irradiation time. (f) Time-dependent changes of pH in groups. (g) H₂O₂ concentration-dependent linear plot of fluorescence ratios of MICG NPs, MnCO₃ and H₂O in the presence of TA (60 µmol/L).

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  Figure 2  Images of (a) O22 staining for cellular ¹O₂ production, and (b) O27 staining for cellular ^•OH production, (c) AM/PI staining for live and dead cells, and (d) JC-1 staining for mitochondrial dysfunction detection. Scare bar: 100 µm. Concentration-dependent viability of cells treated with (e) GOx, MnCO₃ NPs, MICG NPs, and (f) ICG, ICG +L, MICG NPs + L. Data are presented as mean ± standard deviation (SD) (n = 6). (g) CRT and (h) HMGB1 exposure shown by green fluorescence. Scare bar: 50 µm.

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  Figure 3  (a) NIR fluorescent image of MICG NPs in mouse. (b) Time-dependent infrared thermal images of mouse intratumorally injected with MICG NPs or PBS under 808 nm laser irradiation for 10 min. Time-dependent (c) tumor volume and (d) mouse weight variation after different treatments. Data are presented as mean ± SD (n = 5). (e) Images of H&E-, TUNEL- and Ki67-stained tumors with different treatment. (f) Images of H&E staining of major organs from different treated mice. Scare bar: 200 µm.

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  Figure 4  Quantitative analysis of cells densities of CD3⁺ T cells and CD80⁺ CD86⁺ DCs in (a, b) spleen and (c, d) tumor (n ≥ 3). Data are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA test with the Tukey's test. *P < 0.05, **P < 0.01, ****P < 0.0001. ns, no significance.

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