A self-supplying H₂O₂ “nano-reaction engine” with photothermal boosting for cascade chemodynamic–immunotherapy

Abstract

Chemodynamic therapy (CDT) involves conversion of endogenous hydrogen peroxide (H₂O₂) into highly toxic hydroxyl radicals (•OH) for effective tumor ablation. However, the therapeutic efficacy of CDT is severely limited by the insufficient H₂O₂ levels and excessive glutathione (GSH) within the tumor microenvironment, which collectively establish a redox-protective state that resists oxidative stress. To overcome these intrinsic barriers, we developed a self-supplying H₂O₂ nanoplatform (CZPI) that enables synergistically enhanced tumor immunotherapy. The CZPI platform features a Cu-ZnO₂ core nanostructure as a “reaction engine”, and utilizes sequential surface modification by oxidative self-polymerization of polydopamine (PDA) and π–π stacking of indocyanine green (ICG) to introduce a high-efficiency photothermal “booster.” In the weakly acidic TME, ZnO₂ undergoes acid-triggered decomposition to release H₂O₂ and Cu²⁺ ions. The latter react with intracellular GSH to generate Cu ⁺ ions, which catalyze the Fenton-like decomposition of H₂O₂ to continuously produce •OH while regenerating Cu²⁺ for redox cycling. Furthermore, the synergistic photothermal effect of PDA and ICG increases local temperature in response to near-infrared (NIR) irradiation, thereby accelerating reaction kinetics and amplifying the cascade reaction. This photothermal-chemodynamic action of CZPI induces multiple forms of programmed cell death – including apoptosis, ferroptosis, and immunogenic cell death (ICD) – which subsequently activate systemic anti-tumor immune responses. Overall, this study presents a novel “reaction engine-booster” paradigm that integrates self-supplying H₂O₂ generation with photothermal acceleration, offering a powerful platform for multimodal synergistic cancer therapy and durable immune activation.

Graphical abstract

1. Introduction

Cancer remains a major threat to human health, and the current therapeutic strategies, including surgical resection, radiotherapy, chemotherapy and immunotherapy, present significant limitations despite having achieved remarkable clinical progress [[1], [2], [3], [4]]. Surgical treatment often fails to completely eliminate microscopic residual lesions, leading to tumor recurrence and metastasis [5]. Radiotherapy and chemotherapy, while capable of effectively eradicating cancer cells, inevitably damage healthy tissues and may induce drug resistance during prolonged treatment [6]. Immunotherapy has opened new avenues for cancer management; however, its efficacy largely depends on the patient's immune status and benefits only a subset of individuals [7,8]. Moreover, the intrinsic heterogeneity of tumors, the complexity of the tumor microenvironment (TME), and immune evasion mechanisms of tumor cells collectively undermine the effectiveness of conventional therapies, posing formidable challenges to achieving durable therapeutic outcomes [[9], [10], [11]].

Chemodynamic therapy (CDT) has attracted considerable attention in recent years as a non-invasive approach that relies on endogenous chemical reactions within tumor cells [12,13]. The core mechanism of CDT is based on the Fenton or Fenton-like reaction, which generates highly toxic hydroxyl radicals (•OH) in situ [14]. Specifically, CDT utilizes transition metal ions (e.g., Fe²⁺, Cu⁺, Mn²⁺) to catalyze the decomposition of endogenous hydrogen peroxide (H₂O₂) in the TME, thereby producing the highly reactive •OH species that trigger oxidative stress and subsequently induce multiple forms of programmed cell death, such as apoptosis, ferroptosis, and pyroptosis [[15], [16], [17]]. Furthermore, programmed cell death is often accompanied by the release of damage-associated molecular patterns (DAMPs), including ATP, high mobility group box 1 (HMGB1), and exposed calreticulin (CRT), which collectively promote immunogenic cell death (ICD) [[18], [19], [20]]. The antigens released from the dying tumor cells induce maturation of dendritic cells (DCs) and activate cytotoxic T lymphocyte (CTL) responses, thereby eliciting systemic anti-tumor immunity in addition to local tumor eradication [21,22]. This synergistic action of chemical cytotoxicity and immune activation endows CDT with direct tumoricidal efficacy, as well as potent immunotherapeutic potential.

However, despite these unique advantages, the efficacy of CDT remains constrained by several intrinsic factors [23]. First, the performance of CDT strongly depends on the concentration of endogenous H₂O₂ within the TME [24]. Although tumor cells exhibit elevated metabolic activity and oxidative stress, leading to relatively higher H₂O₂ levels compared to normal tissues, the actual concentration is still far below the threshold needed to efficiently trigger Fenton or Fenton-like reactions [25]. The insufficient supply of H₂O₂ limits continuous generation of •OH, thereby reducing the overall catalytic efficiency and anti-tumor potency of CDT. In addition, the high level of glutathione (GSH) in tumor cells also poses a major challenge. As a key endogenous antioxidant, GSH rapidly scavenges •OH and other reactive oxygen species (ROS) produced during CDT [26,27]. This intrinsic redox buffering system not only attenuates CDT-induced oxidative stress but also promotes tumor cell survival and resistance. Moreover, factors such as inefficient metal ion release, pH-dependent reaction activity, and inadequate tumor-targeting capability of delivery systems further compromise the therapeutic outcomes of CDT in vivo [[28], [29], [30]]. Consequently, it is challenging to achieve sustained and robust generation of free radicals, along with effective penetration into deep tumor tissues. Therefore, strategies aimed at enhancing H₂O₂ availability, depleting intracellular GSH, optimizing reaction efficiency, and improving tumor-targeted delivery are critical for improving CDT efficacy.

To overcome the limitations of CDT arising from insufficient H₂O₂ and excessive GSH in the TME, we constructed a self-supplying H₂O₂ nanoplatform with photothermal-enhanced catalytic activity – designated as Cu-ZnO₂@PDA/ICG (CZPI). The nanocomposite was fabricated by sequential oxidative self-polymerization of dopamine and π–π stacking of indocyanine green (ICG) on the Cu-ZnO₂ surface, endowing it with high catalytic activity and superior photothermal conversion efficiency. In essence, the Cu-ZnO₂ core structure serves as the “reaction engine,” while the polydopamine (PDA) shell and ICG function as photothermal boosters, enabling TME-specific and photothermally-enhanced catalysis. The Cu-ZnO₂ core undergoes acid-triggered decomposition in the weakly acidic TME, and gradually releases H₂O₂ and Cu²⁺ ions. The latter are subsequently reduced by intracellular GSH to Cu⁺, which catalyzes H₂O₂ decomposition via a Fenton-like reaction to continuously generate •OH, while maintaining a Cu²⁺/Cu⁺ redox cycle. This process ensures sustained ROS production and concurrent GSH depletion, thereby achieving a dual catalytic-oxidative effect within the tumor. Upon NIR irradiation, the synergistic photothermal effect of PDA and ICG markedly elevates local temperature, inducing direct thermal ablation and accelerating catalytic kinetics to promote rapid •OH generation. Consequently, the combined photothermal and chemodynamic action amplifies CDT and overcomes the resistance of tumor cells to oxidative stress, which in turn triggers multimodal programmed cell death, including apoptosis and ferroptosis. Moreover, the thermal ablation of tumors and programmed cell death processes induce ICD, which elicits a systemic anti-tumor immune response. Collectively, this study presents a novel multifunctional nanoplatform integrating a self-supplying H₂O₂ reaction engine with a photothermal reaction booster to achieve highly efficient CDT. By synergistically inducing programmed cell death and immune activation, the CZPI platform not only enables direct tumor eradication but also establishes durable immune memory to effectively suppress tumor growth and recurrence (Scheme 1).

Scheme 1.

Illustration of the mechanism of self-supplying H₂O₂ “nano-reaction engine” with photothermal enhancement for cascade chemodynamic-immunotherapy.

2. Materials and methods

2.1. Chemicals and reagents

Copper chloride dihydrate (CuCl₂•2H₂O), zinc acetate (Zn (OAC)₂), H₂O₂ (30%), dopamine hydrochloride, polyvinylpyrrolidone (PVP, Mw = 10,000), ICG, potassium permanganate (KMnO4), GSH, 5,5′-dithiobis (2-nitrobenzoic- acid) (DTNB), 3,3′,5,5′-tetramethylbenzidine (TMB), Rhodamine B (RhB), Methylene blue (MB), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were purchased from Aladdin Bio-Chem Technology Co. Ltd (Shanghai, China). Cell Counting Kit- 8 assay kit, Annexin V-FITC/PI cell apoptosis assay kit, Lipid Peroxidation MDA Assay Kit, BCA protein assay kit, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1), C11 BODIPY 581/591 and Calcein-AM/PI cell viability/cytotoxicity assay kit were purchased from Beyotime Biotechnology (Shanghai, China). The 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Reduced GSH assay kit (Nanjing Jiancheng Bioengineering Institute) and antibodies for Western blotting were bought from Abmart Medical Technology (Shanghai) Co., Ltd. All chemicals and reagents were used as received according to the manufacturer's instructions.

2.2. Characterization instruments

The XRD patterns were obtained using a Rigaku D/Max 2550 X-ray diffractometer with Cu Kα (λ = 1.5418 Å) radiation (40 kV, 40 mA). The morphology of the NPs was examined with a JEOL JSM-6700 SEM instrument with a low accelerating voltage of 3 kV. TEM and TEM-mapping analysis were performed on an FEI Tecnai G2 S-Twin microscope, equipped with a field emission gun, operating at an accelerating voltage of 200 kV. The IR spectra of the samples were recorded in the range of 400-4000 cm⁻¹ with KBr pellets on the Bruker IFS 66v/S FT-IR spectrometer. Ultraviolet-visible (UV-vis) absorption spectra were recorded on a Shimadzu UV-2450 UV-vis spectrophotometer. EPR spectra were recorded on a Bruker A-300 EPR spectrometer. XPS was performed with an ESCALAB 250 spectrometer (Thermo Electron Corporation) using monochromatic Mg Kα as the X-ray source (1486.6 eV).

2.3. Synthesis of Cu-ZnO₂ (CZ)

ZnO₂ (Z) was first synthesized by dissolving 300 mg Zn (OAC)₂ and 300 mg PVP in 15 mL deionized water. Under vigorous stirring, 1.5 mL H₂O₂ was added. The mixture was stirred at room temperature for 24 h, and ZnO₂ was obtained. Finally, 15 mg ZnO₂ was dissolved in water, and 2.5 mg/mL aqueous CuCl₂ solution was added to produce Cu-doped ZnO₂ using the cation exchange method.

2.4. Synthesis of Cu- ZnO₂@PDA (CZP)

Cu-ZnO₂ (20 mg) was dispersed in a mixed solution of 15 mL water and 15 mL ethanol by ultrasonication, and 30 mg dopamine hydrochloride was added. After adding 200 μL Tris (pH = 8.5) to initiate polymerization, the mixture was stirred at room temperature for 8 h to obtain Cu-ZnO₂@PDA.

2.5. Synthesis of Cu-ZnO₂@PDA/ICG (CZPI)

To synthesize CZPI, 5 mg CZP was dispersed in 5 mL water via ultrasonication, and 1 mg ICG was added to the solution. The mixture was stirred at room temperature for 12 h, centrifuged, and washed with water. The supernatant was collected and freeze-dried to obtain CZPI. To determine the loading amount of ICG, the UV-visible absorbance of the supernatant was measured at 779 nm, and ICG concentration was calculated from its standard curve.

2.6. Detection of H₂O₂ production

Five milligrams of CZPI nanoparticles (NPs) were dispersed in 10 mL neutral (pH 7.4) and acidic (pH 6) PBS. 1 mL aliquots were aspirated at different time points and irradiated (1 W/cm² for 10 min). The solutions were centrifuged, and the supernatants were mixed with 0.1 mL (12.5 mg/mL) Ti (SO₄)₂. The UV-visible spectra of the different mixtures were analyzed.

For the colorimetric analysis, 100 μg/mL NPs was mixed with KMnO₄ (50 μg/mL) containing sulfuric acid (0.1 M), or deionized water as the control, and reacted for 30 min. Following laser irradiation (1 W/cm² for 10 min), the supernatants were collected by centrifugation, and the absorption peaks in the range of 400-700 nm were recorded by UV absorption spectroscopy.

2.7. Acid responsiveness of CZPI

CZPI (1 mg/mL) was dispersed in neutral (pH 7.4) and acidic (pH 6) PBS, stirred at room temperature for 24 h, and irradiated with 808 nm laser (1 W/cm² for 10 min). The morphology of the NPs was observed by TEM. To assess time-dependent dissolution of the NPs, 10 mg CZPI was dispersed in 10 mL PBS at different pH levels (6 and 7.4), and 1 mL aliquots were aspirated at different time points and irradiated (1 W/cm² for 10 min). The samples were centrifuged, and the content of Cu²⁺ and Zn²⁺ in the supernatants was measured by inductively coupled plasma optical emission spectrometry (ICP-OES).

2.8. Detection of •OH

The generation of •OH was detected by ESR spectroscopy using DMPO as the •OH trapping agent. The •OH signals in the ESR spectra were identified with the 1:2:2:1 signature. For colorimetric detection, 100 μL of the nanomaterials were dispersed in 1 mL PBS (pH = 6), and 100 μL H₂O₂ (10 mM) and 20 μL TMB (20 mM) were added. After 30 min of reaction, the absorbance of the mixed solution at 320-800 nm was recorded by UV-visible spectrometry. In addition, •OH generation was also measured by MB degradation. Briefly, different concentrations of CZPI were reacted with 8 μg/mL MB and 10 mM H₂O₂ in 1 mL PBS for 30 min. The absorbance at 662 nm was detected by UV-visible spectrometry. In all of the above assays, the NPs were irradiated with 808 nm laser at 1 W/cm² for 10 min.

2.9. GSH depletion assay

Different concentrations of CZPI were dispersed in 6 mL GSH solution (1 mM, pH = 6.) at 37 °C. Aliquots were taken at different time points (0, 1, 2, 3, 4, 6, 8 h) and centrifuged, and 0.5 mL of the supernatant was mixed with 50 μL DTNB (10 mM) in 2.5 mL PBS. The absorbance of the samples at 250-650 nm was recorded by UV absorption spectrometry.

2.10. Photothermal performance

CZPI, free ICG (10 μg/mL), and water were irradiated with 808 nm laser (1 W/cm²) for 10 min, and the temperature change was recorded with a thermographic recorder every 30 s. In addition, CZPI solutions of different concentrations (20, 40, 60, 80, 100 μg/mL) were irradiated at 1 W/cm², and 100 μg/mL CZPI was irradiated with different laser power densities (0.5, 1, 1.5, 2 W/cm²), and the temperature changes in these samples were recorded. To evaluate the stability of the nanocomposite during the photothermal conversion process, 100 μg/mL CZPI was subjected to four cycles of irradiation, and the cooling curve of the first cycle was selected to calculate the photothermal conversion efficiency (η) using the following equation:

$$\eta =\frac{hs\left(T_{\mathit{max}}-T_{surr}\right)-Q_s}{I\left(1-{10}^{-A808}\right)}$$

In the equation, h was the heat transfer coefficient, s was the surface area of the sample container, Q_s was the heat absorbed by the solvent and the container, I was the laser power, and A808 was the absorbance of the sample at 808 nm; hs was calculated using the following equation:

$$hs=\frac{m_D{C}_D}{{\tau }_s}$$

Deionized water was used as the solvent; m_D and C_D were the mass and specific heat capacity of the solvent (4.2 J/g/K), and τ_s was the time constant of the sample; τ_s was calculated from the linear relationship between t and -lnθ during cooling.

$$t=-{\tau }_s\mathit{ln}\theta$$

$$\theta =\frac{T-T_{Surr}}{T_{\mathit{max}}-T_{Surr}}$$

$$Q_s=hs\left(T_{\left(H2O\right)\max }-T_{Surr}\right)$$

T, T_max, and T_Surr represented the real-time temperatures of the CZPI dispersion, the highest temperature of the CZPI dispersion, and the ambient temperature. Based on these formulae, η was calculated to be 38.39%.

2.11. Cell culture and cellular uptake of NPs

The murine melanoma B16F10 cell line was purchased from the Shanghai Institute of Cells, Chinese Academy of Sciences. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C under 5% CO₂. For the cellular uptake assay, B16 cells were seeded in confocal dishes. The culture medium was replaced with 1 mL fresh medium containing CZPI/RhB (50 μg/mL), and the cells were incubated for 0.5, 1, 2, 4 and 6 h. After washing thrice with PBS, the cells were fixed with 4% paraformaldehyde and stained with DAPI (2.5 μg/mL, 200 μL) for 15 min. The cells were observed with a confocal fluorescent microscope to track internalization of the NPs.

2.12. In vitro cytotoxicity assay

The B16 cells were seeded in 96-well plates and incubated for 24 h. After co-culturing with different concentrations of CZPI for 4 h, the cells were irradiated with 808 nm laser (1 W/cm², 5 min). The cells were cultured for 48 h, and the CCK-8 reagent was added to each well. The absorbance of the wells at 450 nm was measured using a microplate reader, and the viability rates were calculated.

2.13. Live-dead staining

The B16 cells were seeded in 48-well plates and incubated for 24 h. The culture medium was discarded and replaced with fresh medium containing the different nanomaterials. After 4 h of culture, the cells were irradiated with 808 nm laser (1 W/cm², 5 min) and cultured additional 24 h. Treated cells were washed thrice with PBS, and stained using the Calcein AM/PI Double Staining Kit at 37 °C for 15 min. The excess dye was washed off with PBS, and the cells were observed with an inverted fluorescence microscope. The live (green fluorescence) and dead (red fluorescence) cells were counted.

2.14. Apoptosis assay

The B16 cells were seeded in 6-well plates and incubated for 24 h. The culture medium was discarded, and fresh medium containing the different nanomaterials was added. After 4 h of culture, the cells were irradiated (1 W/cm², 5 min) and cultured additional 24 h. Treated cells were washed thrice with PBS. The cells were then stained with Annexin V-FITC/PI, and the apoptosis rates were analyzed by flow cytometry.

2.15. Detection of intracellular ROS

The B16 cells were seeded in 48-well plates and incubated for 24 h. The culture medium was replaced with fresh medium containing the different nanomaterials. After 4 h of culture, the cells were irradiated (1 W/cm², 5 min) and cultured additional 24 h. Treated cells were washed thrice with PBS, and then stained with DCFH-DA for 30 min. The excess dye was washed off with PBS, and the stained cells were observed under an inverted fluorescence microscope.

2.16. Measurement of mitochondrial membrane potential

The B16 cells were seeded in 6-well plates and incubated for 24 h. The medium was replaced with fresh medium containing the different nanomaterials, and the cells were cultured for 4 h. Following laser irradiation (1 W/cm², 5 min), the cells were cultured additional 24 h. Treated cells were washed thrice with PBS, and then stained with JC-1 dye solution for 30 min. The excess dye was washed off with PBS, and the cells were observed under an inverted fluorescence microscope.

2.17. Determination of GSH content

The B16 cells were seeded in 6-well plates and cultured for 24 h. Fresh medium containing different nanoparticles were added, and the cells were cultured for 4 h. Following laser irradiation (1 W/cm², 5 min), the cells were cultured additional 24 h. After washing thrice with PBS, the cells were lysed with 500 μL PBS (10X) for 20 min and then sonicated for 3 min. The lysates were centrifuged, and the GSH content in the supernatants was measured using a GSH + GSSG/GSH Assay Kit (Abcam, ab239709) according to the instructions. The assay is based on the specific reaction between the thiol (-SH) group of GSH and DTNB, generating the yellow-colored product TNB⁻, which exhibits a strong absorbance at 412 nm. The protein content of each sample was determined using the BCA protein assay kit. GSH depletion was calculated relative to the GSH content in the untreated cells.

2.18. Western blotting

The B16 cells were seeded in 6-well plates and incubated overnight, and then cultured with the different nanomaterials for 4 h. Following laser irradiation (1 W/cm², 5 min), the cells were cultured additional 24 h. The protein fraction was isolated from each sample, and quantified using the BCA protein assay kit. Equal amounts of proteins were separated using a 12% sodium dodecyl sulfate-polyacrylamide gel, and transferred to PVDF membranes. After blocking with skim milk for 2 h, the membranes were washed with TBST buffer 5 to 6 times, and then incubated overnight with the anti-GPX4 antibody. The membranes were then washed 5∼6 times and incubated with secondary antibodies at room temperature for 1 h. GPX4 protein expression was detected by chemiluminescence according to the ECL kit instructions.

2.19. Measurement of intracellular LPO levels

The B16 cells were seeded in 6-well plates and incubated for 24 h. The medium was replaced with fresh medium containing different nanomaterials. After 4 h of culture, the cells were irradiated (1 W/cm², 5 min) and cultured additional 24 h. Treated cells were washed thrice with PBS, and stained with C11 BODIPY 581/591 for 30 min. The excess dye was washed off with PBS, and the stained cells were observed under an inverted fluorescence microscope.

2.20. Determination of malondialdehyde (MDA) content

The B16 cells were seeded in 6-well plates and incubated for 24 h. The medium was replaced with fresh medium containing the different nanomaterials, and the cells were cultured for 4 h. Following laser irradiation (1 W/cm², 5 min), the cells were cultured additional 24 h. The treated cells were washed thrice with PBS, and lysed with 200 μL RIPA buffer (containing 1% phosphatase inhibitor) on ice for 20 min. The MDA content in the lysates was determined using a kit according to the instructions. The protein content of each sample was measured using the BCA protein assay kit.

2.21. In vivo fluorescence imaging

Female C57 mice (5∼6 weeks, 15∼20 g) were purchased from Charles River Company. All animal experiments were approved by the Experimental Animal Management and Ethics Committee of China Medical University.

For in vivo imaging, tumor-bearing mice were intravenously injected with CZPI (50 μg/mL, 100 μL), and fluorescence images were acquired at different time points post-injection using a small animal imaging instrument. The mice were euthanized 24 h later, and the tumor tissues and major organs, including heart, lungs, liver, spleen and kidneys, were collected for subsequent imaging analysis.

2.22. In vivo biocompatibility assessment

Healthy C57 mice (aged 4 - 6 weeks, n = 3) were injected with 100 μL of the CZPI solution (100 μg/mL) via the tail vein. Blood samples were collected 60 days later for routine hematology analysis. Subsequently, serum was isolated and the biomarkers of liver function, kidney function and myocardial injury, and the routine blood parameters were evaluated using specific ELISA kits (Leiden Company).

2.23. In vivo experiments with the B16 melanoma model

2.23.1. Evaluation of therapeutic efficacy

Female C57 mice were subcutaneously injected with B16 cells (5 × 10⁵) into their right thighs. Once the tumor volume reached ∼100 mm³, the mice were randomly divided into the PBS + L, CZ + L, ZPI + L, CZPI, and CZPI + L groups (n = 6 per group), and injected intravenously with the corresponding drugs (50 μg/mL, 100 μL). Depending on the treatment group, the tumors were irradiated with 808 nm laser (1W/cm², 10 min) 8 h after drug administration, and the temperature changes were recorded using an infrared camera. The entire treatment protocol was repeated every three days, and a total of four treatment cycles were carried out. Tumor volume and body weight of each mouse were recorded every two days during the treatment period. Tumor volume was calculated as (width)² × length/2. The mice were monitored for 60 days post-injection, and euthanized if the tumor volume exceeded 2000 mm³. Complete remission was defined as the tumor shrinking to less than 20 mm³, which is the minimum detectable size. The tumor tissues were harvested for downstream analyses. Note: All laser irradiations in both in vitro and in vivo experiments were maintained at approximately 50 °C.

2.23.2. Immune response analysis

The tumor tissues and lymph nodes harvested from the differentially treated mice were homogenized into single-cell suspensions. The lymph node cells were stained with anti-mouse CD86-PE, CD80-Percp/cy5.5, and CD11c-FITC for mature DCs, and with CD44-PE and CD62L-APC antibodies for effector memory T cells. The CTLs and helper T cells in the tumor and lymph nodes were co-stained with anti-mouse CD8-APC-Cy7, CD4-PE-Cy7, and CD3-FITC. The stained cells were analyzed by flow cytometry.

2.23.3. Immunohistochemistry, TUNEL and hematoxylin-eosin (H&E) staining

The tumors were fixed in 10% paraformaldehyde, embedded in paraffin, and cut into thin sections. H&E staining, immunostaining for Ki-67 and GPX4, and terminal deoxynucleotide transfer-mediated dUTP nickel-end labeling (TUNEL) were performed as per standard protocols. In addition, major organs (heart, liver, spleen, lungs, and kidneys) were stained with H&E, and observed for pathological changes.

2.23.4. Assessment of long-term immune memory

To assess immune memory, 5 × 10⁵ B16 cells were injected subcutaneously into the right flanks of female C57 mice. When the tumor volume reached ∼100 mm³, the mice were subjected to the different treatments with or without anti-PDL1 antibody (aPDL1). The tumors were irradiated with 808 nm laser (1 W/cm², 5 min) 8 h after injecting the different nanomaterials, and aPDL1 (10 mg/kg) was injected 12 h post-irradiation. The treatment was repeated every 3 days for a total of 4 cycles (1 cycle = 3 days). Two weeks later, 5 × 10⁵ B16 cells were injected subcutaneously into the left flank, and the tumor size was recorded every 5 days after rechallenge. The mice were monitored for 60 days to assess survival rates, and euthanized when the tumor volume exceeded 2000 mm³.

3. Results and discussion

Synthesis and characterization of CZPI. The procedure for synthesizing CZPI is illustrated in Fig. 1A. Briefly, zinc peroxide (ZnO₂) was first prepared using H₂O₂ and zinc acetate as precursors, and then doped with Cu (Cu-ZnO₂) through a cation-exchange process to endow the nanoplatform with CDT capability. Subsequently, dopamine hydrochloride was added to the Cu-ZnO₂ suspension, where it underwent oxidative self-polymerization under mildly alkaline conditions, allowing the catechol groups to firmly anchor a PDA shell onto the surface of the NPs. Finally, ICG was adsorbed onto the Cu-ZnO₂@PDA (CZP) NPs via π–π stacking interactions, yielding the final CZPI construct. As shown in the TEM images in Fig. 1B, the pristine ZnO₂ NPs were spherical with an average diameter of ∼40 nm, and had a rough surface texture. Furthermore, Cu incorporation did not alter the morphology of the ZnO₂ NPs (Fig. 1C). The spherical structure of CZP was retained after PDA coating, although the size of the NPs increased to ∼80 nm, corresponding to a uniform PDA layer of ∼20 nm thickness (Fig. 1D). On the other hand, ICG loading did not induce any observable morphological changes (Fig. 1E). Elemental mapping further confirmed the successful fabrication of the hierarchical nanostructure. As shown in Fig. 1F, Cu, Zn, and O were homogeneously distributed within the core, while C and N originating from PDA and S from ICG were predominantly localized on the outer shell.

Fig. 1.

Characterization of CZPI. (A) Schematic illustration of the synthesis route for CZPI NPs. (B–E) TEM images of Z, CZ, CZP, and CZPI. (F) Elemental mapping of CZPI. (G) XRD patterns of Z, CZ, CZP, and CZPI. (H) X-ray photoelectron spectrum of CZPI. (I, J) High-resolution XPS spectra of Cu 2p and Zn 2p. (K) FTIR spectra of Z, CZ, CZP, CZPI, and ICG. (L) Zeta potentials of Z, CZ, CZP, and CZPI. (M) UV-vis absorption spectra of Z, CZ, CZP, CZPI, and ICG.

X-ray diffraction (XRD) analysis showed that the characteristic diffraction peaks of ZnO₂ matched well with the standard reference pattern (JCPDS No. 13-0311) (Fig. 1G). Notably, the diffraction peak positions of CZ, CZP, and CZPI remained essentially unchanged compared to that of pristine ZnO₂, indicating that neither Cu incorporation, nor coating with PDA and ICG perturbed the intrinsic crystal structure of ZnO₂. X-ray photoelectron spectroscopy (XPS) was performed to further determine the oxidation states of Zn and Cu in CZPI (Fig. 1H). High-resolution Cu 2p spectra displayed two main peaks at 954.65 eV and 934.86 eV, accompanied by satellite peaks at 944.21 eV and 940.98 eV, thus confirming the presence of Cu²⁺ as the predominant oxidation state. Additional peaks at 952.21 eV and 932.15 eV, along with a satellite feature at 962.64 eV, revealed the coexistence of Cu⁺ (Fig. 1I) [31]. The partial reduction of Cu²⁺ to Cu⁺ is attributed to the reductive characteristics of catechol groups in PDA; catechols are readily oxidized to quinones under alkaline conditions, which release electrons that convert a portion of Cu²⁺ into Cu⁺ [32,33]. The Zn 2p spectra exhibited binding energies at 1045.37 eV (Zn 2p₁/₂) and 1022.29 eV (Zn 2p₃/₂), which was consistent with Zn²⁺ species (Fig. 1J) [34]. The amount of Zn and Cu in CZPI – as measured by ICP-OES – were 37.78% and 9.76% respectively.

The successful fabrication of CZPI was further verified by Fourier-transform infrared (FTIR) spectroscopy (Fig. 1K). Compared to ZnO₂, both CZP and CZPI exhibited a distinct aromatic C=C stretching vibration band at 1600 cm⁻¹, which confirmed the presence of the PDA shell [35]. Characteristic absorption bands of ICG at 1500-1400 cm⁻¹ and 1100-900 cm⁻¹ – corresponding to aromatic C=C and alkenyl C=C stretching vibrations, respectively – in the CZPI spectrum indicated successful incorporation of the dye [36]. Zeta potential measurements revealed surface charge variations throughout the fabrication process. Z and CZ displayed zeta potentials of +22.2 mV and +11.7 mV, respectively; upon PDA coating, the surface charge of CZP shifted to −16.3 mV, and subsequent ICG loading resulted in a similar potential of −15.6 mV (Fig. 1L). The UV-vis absorption spectrum of free ICG exhibited a characteristic peak at 780 nm, whereas CZPI showed a red-shifted absorption band in the 800-900 nm region (Fig. 1M). This bathochromic shift is attributed to J-aggregation of ICG molecules arising from dimer or polymer formation [37], which further confirms the successful loading of ICG onto the nanoplatform. According to UV-vis quantification, the ICG loading content was 10% (Fig. S1). Collectively, these results demonstrated the successful synthesis of CZPI.

Photothermal-boosted H₂O₂ self-supply and GSH depletion amplified •OH production. The photothermal performance of CZPI was evaluated using an 808 nm laser within the NIR window, which is more suitable for clinical applications due to its superior tissue penetration. As shown in Fig. 2A, the temperature of CZPI suspension (100 μg/mL) rapidly increased from 21.8 °C to 58.4 °C within 10 min of irradiation (1 W cm⁻²). On the other hand, the temperature of free ICG reached only 45.2 °C under identical conditions, thus demonstrating the potent photothermal capability of CZPI. Furthermore, the heating rate of CZPI was significantly accelerated by increasing the concentration of the NPs (Fig. S2) and the laser power density (Fig. S3), indicating a clear concentration- and power-dependent photothermal conversion behavior. The photothermal stability of CZPI was assessed by subjecting the NPs to four cycles of laser on/off irradiation, which resulted in minimal signal attenuation (Fig. S4). The photothermal conversion efficiency (η) was quantified by monitoring the natural cooling curve following laser exposure and fitting the temperature decay profile (Fig. S5), which yielded an η value of 38.39%. We next evaluated the in vivo photothermal performance of CZPI in a B16 tumor-bearing mouse model through infrared thermal imaging. Following intravenous administration of CZPI, the tumor site was irradiated with 808 nm laser. The PBS-treated mice exhibited negligible temperature elevation, while the intra-tumor temperature in the ICG + L group rose to 44.4 °C after 10 min of irradiation. On the other hand, the combination of CZPI and laser irradiation achieved a significantly higher temperature of 53.1 °C under the same conditions, which surpasses the threshold required for effective tumor ablation.

Fig. 2.

Photothermally enhanced H₂O₂ self-supply and GSH depletion for cascade amplification of •OH generation. (A) Photothermal heating curves and infrared thermal images of PBS, ICG, and CZPI under 808 nm laser irradiation (1.0 W/cm², 10 min). (B) Infrared thermal images of B16 tumor-bearing mice subjected to different treatments under 808 nm laser irradiation (1.0 W/cm², 10 min), and (C) the corresponding temperature-time profiles. (D) Schematic illustration of the photothermal acceleration of catalytic reactions. (E, F) Release profiles of Zn²⁺ and Cu²⁺ from CZPI under various conditions. (G) H₂O₂ release from CZPI in response to different treatments, quantified using Ti(SO₄)₂ as the probe (1.0 W/cm², 10 min). (H) Absorption spectra of H₂O₂ production detected with KMnO₄ (100 μg/mL, 1.0 W/cm², 10 min). (I) Absorption spectra for GSH depletion measured using DTNB (pH = 6.0, 100 μg/mL, 1.0 W/cm², 10 min). (J) ESR spectra of different reaction systems using DMPO as the radical-trapping agent (pH = 6.0, 1 mM GSH). (K, L) Absorption spectra of •OH generation detected by TMB and MB under acidic conditions (pH = 6.0, 100 μg/mL, 1.0 W/cm², 10 min).

The mechanism underlying the photothermally-accelerated chemodynamic effects of CZPI is illustrated in Fig. 2D. Briefly, the continuous supply of H₂O₂ by CZPI generates Cu²⁺ in the acidic conditions of the TME. The abundant GSH reduces Cu²⁺ to Cu⁺, and the latter subsequently catalyzes a Fenton-like reaction with H₂O₂ to yield •OH, while regenerating Cu²⁺. This redox cycling continuously amplifies •OH production, and the photothermal effect induced by NIR irradiation further accelerates this catalytic loop (Fig. 2D). Accordingly, we investigated whether the near-infrared (NIR)–mediated photothermal effect could enhance the self-supplying H₂O₂ generation capacity of CZPI. Considering the mildly acidic nature of the tumor microenvironment, we first evaluated the acid-responsive degradation behavior of CZPI. As shown in Fig. S6–S7, CZPI retained its structural integrity after incubation in neutral PBS (pH 7.4) for 24 h, and its hydrodynamic size remained stable even after 7 days, indicating excellent stability under physiological conditions. In contrast, under acidic conditions (pH 6.0), although the polydopamine (PDA) shell remained largely intact, the internal core began to undergo gradual degradation. Upon 808 nm laser irradiation, this degradation was further accelerated, and some nanoparticles evolved into hollow structures. Moreover, time-dependent analysis under acidic conditions combined with NIR irradiation revealed progressive decomposition of CZPI; by 48 h, the outer PDA shell was almost completely degraded. To further assess the pH-dependent activity of CZPI, the amount of Zn²⁺ and Cu²⁺ released from the NPs under different pH conditions was measured by ICP-OES, and H₂O₂ production was quantified by titanium sulfate colorimetric assay (Fig. 2E–G, Fig. S8). CZPI rapidly released Zn²⁺ and Cu²⁺ under acidic conditions, and 808 nm laser irradiation significantly accelerated the dissociation of the ions, resulting in a pronounced increase in H₂O₂ generation. Conversely, the release of both metal ions and H₂O₂ was markedly slower in neutral PBS. The photothermally-enhanced H₂O₂ production was also measured by the KMnO₄ discoloration assay. KMnO₄ is reduced by H₂O₂ in acidic solutions, resulting in the loss of its characteristic purple-red color. As shown in Fig. 2H, the absorption peak of KMnO₄ decreased substantially in the presence of CZPI, and the reduction was even more pronounced under 808 nm irradiation, thus confirming enhanced H₂O₂ generation. Taken together, CZPI exhibits efficient photothermal conversion under 808 nm irradiation, which significantly enhances its H₂O₂ self-supply capability under acidic TME-mimicking conditions.

To determine whether CZPI could enhance GSH depletion and amplify the •OH-generation cascade upon photothermal activation, we quantified GSH consumption by CZPI using the DTNB probe. DTNB reacts with the thiol group of GSH to form the yellow product 5-thio-2-nitrobenzoic acid (TNB), which exhibits a characteristic absorption peak at 412 nm. Briefly, CZPI was added to GSH solutions, and the change in absorbance at 412 nm was measured at pre-determined time points. As shown in Fig. S9, CZPI accelerated GSH consumption in a concentration-dependent manner. Moreover, when different concentrations of CZPI were added to GSH solutions, the absorbance at 412 nm gradually decreased over time (Fig. S10), confirming continuous GSH depletion. These results indicate that CZPI-driven GSH consumption is both time- and concentration-dependent. Furthermore, photothermal activation of CZPI significantly enhanced GSH depletion (Fig. 2I). DMPO was used as a trapping agent for ESR analysis to detect •OH generation (Fig. 2J). Under acidic conditions, co-incubation of CZPI with H₂O₂ produced weak •OH signals, which became stronger in the presence of GSH and reached their maximum intensity under NIR irradiation. This indicated that NIR irradiation activates CZPI to amplify GSH depletion-dependent •OH production. Enhanced •OH generation was further verified by TMB oxidation (Fig. 2K, Fig. S11) and MB degradation assays (Fig. 2L, Fig. S12).

Cellular targeting of CZPI. Efficient intracellular uptake is a prerequisite for the anti-cancer effects of nanomaterials. CZPI was labeled with the red fluorescent dye rhodamine B and incubated with B16 murine melanoma cells to track cellular internalization; a marked increase in intracellular red fluorescence intensity was observed with prolonged incubation (Fig. 3A), indicating a time-dependent uptake process that peaked at 4 h. Furthermore, Bio-TEM imaging confirmed the presence of CZPI within tumor cells after 4 h of incubation (Fig. 3B). The cytotoxicity of CZPI against tumor cells was next evaluated by the CCK-8 assay. While CZPI alone exhibited only weak cytotoxicity toward tumor cells, NIR irradiation significantly decreased the viability of the CZPI-treated cells in a concentration-dependent manner (Fig. 3C). To clarify the contribution of each component, CZ and ZPI were included as controls. As shown in Fig. 3D, CZ reduced the viability of tumor cells by only 28%, and 808 nm irradiation did not improve its cytotoxic activity. On the other hand, ZPI achieved 19% growth inhibition, which increased to 34% under NIR irradiation, reflecting its intrinsic photothermal effect. Notably, CZPI induced up to 68% inhibition, and the tumor cells were nearly completely eradicated upon NIR irradiation (Fig. 3D), indicating that the cytotoxic effect of CZPI is dependent on NIR-induced photothermal activation (Fig. 3E). To further validate the photothermally mediated killing of tumor cells, B16 cells subjected to different treatments were stained using Calcein-AM and PI. As shown in Fig. 3F, only minimal cell death was observed in the CZ + L and ZPI + L groups, whereas CZPI + L treatment resulted in widespread cell death. Taken together, CZPI exhibits potent tumoricidal activity upon NIR irradiation.

Fig. 3.

Photothermally-induced targeted ablation of tumor cells by CZPI. (A) CLSM images of B16 cells incubated with RhB-labeled CZPI (50 μg/mL) for the indicated durations (scale bar: 20 μm). (B) Bio-TEM images showing intracellular internalization of CZPI after 4 h of incubation. (C) Viability rates of B16 cells treated with CZPI with or without 808 nm laser irradiation (1 W/cm², 5 min). (D) Viability rates of B16 cells exposed to various nanoformulations under the same irradiation conditions. (E) Infrared thermal images of B16 cells subjected to different treatments following NIR exposure. (F) Fluorescence images of B16 cells stained with calcein-AM (green) and PI (red) after 24 h of treatment (scale bar: 50 μm). Statistical analysis was performed using one-way ANOVA with Tukey's post hoc test (∗P < 0.05, ∗∗P < 0.01).

CZPI induces multiple forms of programmed tumor cell death under NIR activation. The mechanisms underlying the NIR-mediated cytotoxic effects of CZPI were also investigated. Excessive •OH generation disrupts the mitochondrial membrane by causing membrane potential loss, and the subsequent release of cytochrome c into the cytoplasm activates the downstream caspase cascades and triggers apoptosis [38]. During ferroptosis induction, •OH is predominantly produced through Fenton-type reactions of H₂O₂ with metal ions such as Fe²⁺, Mn²⁺, or Cu²⁺, which promotes peroxidation of polyunsaturated fatty acids in cellular membranes [39,40]. This leads to lipid peroxide accumulation and membrane destabilization, which culminates in iron-dependent and lipid peroxidation-driven cell death. The ferroptotic cascade is further amplified by GSH depletion and inactivation of GPX4. Based on these mechanistic insights, we hypothesized that CZPI can induce both apoptosis and ferroptosis in tumor cells upon NIR activation. As expected, the CZPI + L group exhibited a markedly high apoptosis rate of 52.7% (Fig. 4A). We performed western blot analysis to evaluate the expression of cleaved caspase-3 in B16 cells treated with different materials. The results showed that cleaved caspase-3 expression was highest in the CZPI + NIR group, indicating a significant activation of apoptosis (Fig. S13). Intracellular ROS production was quantified using the DCFH-DA probe, which is oxidized in the presence of ROS into the green-fluorescent product DCF. As shown in Fig. 4B, both CZPI and CZPI + L induced strong green fluorescence relative to the control cells, and the fluorescence signals were more intense in the CZPI + L group compared to that in the CZPI group. These results indicate that CZPI elevates intracellular ROS levels, and NIR-induced photothermal activation further amplifies this effect. The JC-1 probe was used to measure changes in mitochondrial membrane potential (ΔΨm). CZPI + L treatment led to a marked reduction in the red-fluorescent JC-1 aggregates and a concomitant increase in the green-fluorescent monomers (Fig. 4C), indicating severe mitochondrial damage due to loss of ΔΨm. Furthermore, CZPI significantly reduced intracellular GSH levels, and GSH depletion was accelerated under NIR activation (Fig. 4D). GPX4 utilizes GSH as a substrate to catalyze lipid repair; thus, GSH depletion suppresses GPX4 activity and prevents the conversion of lipid peroxides into non-toxic lipid alcohols, eventually triggering ferroptosis [41]. Consistent with this mechanism, CZPI treatment markedly downregulated GPX4 expression, and a greater reduction was observed in the CZPI + L group (Fig. 4E and F). We further assessed lipid peroxidation using a lipid ROS-sensitive fluorescent probe; cells treated with CZPI + L exhibited a significantly enhanced green fluorescence signal (Fig. 4G), indicating elevated lipid peroxidation. MDA, a degradation product of lipid peroxides, is a hallmark indicator of ferroptosis. As shown in Fig. 4H, CZPI significantly increased MDA levels NIR irradiation, which confirmed robust intracellular lipid peroxide accumulation.

Fig. 4.

CZPI induces multiple forms of programmed cell death under photothermal activation. (A) Flow cytometry plots of Annexin V-FITC/PI dual-staining showing percentage of apoptotic B16 cells in the indicated groups. (B) Fluorescence images of B16 cells stained with DCFH-DA after the indicated treatments (scale bar: 50 μm). (C) Fluorescence images of JC-1 staining showing changes in the mitochondrial membrane potential of B16 cells following different treatments (scale bar: 25 μm). (D) Intracellular GSH levels in B16 cells after the specified treatments. (E) Immunoblot showing GPX4 expression in B16 cells subjected to the indicated treatments. (F) Quantitative analysis of GPX4 expression in B16 cells following treatment with different samples, as determined by Western blot. (G) Fluorescence images of B16 cells stained with C11-BODIPY 581/591 after the indicated treatments (scale bar: 25 μm). (I) Confocal images showing CRT exposure and HMGB1 release in B16 cells under the indicated conditions (scale bar:10 μm). (H) The relative content of MDA was measured in B16 cells with indicated treatment. ∗P < 0.05, ∗∗P < 0.01, one-way ANOVA with Tukey's post hoc test. (J) Schematic illustration of the multiple programmed cell death pathways triggered by CZPI upon photothermal activation.

Previous studies have shown that programmed cell death can initiate immunogenic death of tumor cells – a process characterized by the surface translocation of CRT and the release of HMGB1 – which can trigger anti-tumor immune responses. Confocal laser scanning microscopy showed weak CRT fluorescence in the CZ + L and ZPI + L groups, whereas cells treated with CZPI + L exhibited robust CRT signals on the plasma membrane (Fig. 4I). Furthermore, HMGB1 release into the cytoplasm was evaluated by immunofluorescence staining; as shown in Fig. 4I, HMGB1 fluorescence largely overlapped with DAPI staining in cells treated with PBS + L, indicating nuclear retention. While both CZ + L and ZPI + L induced HMGB1 translocation from the nucleus, HMGB1 was almost completely redistributed to the cytoplasm in the CZPI + L group, thus confirming ICD. Taken together, CZPI induces apoptosis and ferroptosis upon NIR activation, which subsequently trigger robust ICD (Fig. 4J).

NIR-driven photothermal therapeutic effects of CZPI. The NIR-mediated photothermal therapeutic efficacy of CZPI was validated in a B16 tumor-bearing mouse model. To assess biodistribution of CZPI, the mice were injected intravenously with the formulation, and the fluorescence signal of ICG was monitored using an in vivo imaging system. As shown in Fig. 5A and Fig. S14, the fluorescence intensity within the tumor region progressively increased over time and peaked at 8 h post-injection, indicating effective tumor-targeted accumulation of CZPI. These results also suggested that the optimal time point for implementing photothermal therapy was approximately 8 h after administration of CZPI. Ex vivo fluorescence imaging at 24 further confirmed the preferential accumulation of CZPI in the tumor tissues (Fig. 5B). As shown in Fig. S15, the fluorescence signals in the heart, spleen, lungs, and kidneys were significantly weaker compared to that in the tumors. However, the liver exhibited comparatively higher fluorescence intensity, suggesting that CZPI is primarily metabolized via hepatic pathways.

Fig. 5.

In vivo therapeutic efficacy of photothermally activated CZPI in tumor-bearing mice. (A) Representative ex vivo fluorescence images showing the biodistribution of CZPI in B16 tumor-bearing mice. (B) Fluorescence images of major organs and tumor tissues were collected 24 h after intravenous administration of CZPI. (C) Schematic illustration of the B16 tumor model establishment and CZPI-mediated photothermal therapy. (D) Tumor growth curves of mice in the indicated treatment groups. (E) Survival curves of mice from the indicated groups. (F) Tumor growth rates in each treatment group. (G) Representative images showing H&E staining and Ki67 immunostaining of tumor tissues from the indicated groups (scale bar: 50 μm). (H) Percentage of Ki67-positive regions in the tumor sections from indicated groups. (I) Representative TUNEL staining images of tumor tissues (scale bar: 50 μm) and (J) quantification of TUNEL-positive regions in the indicated groups. (K) Immunohistochemical staining of GPX4 in tumor tissues (scale bar: 50 μm) and (L) quantification of GPX4 expression in the indicated groups. (M) Intratumoral GSH levels in each treatment group. (N) Serum HMGB1 levels and (O) serum CRT levels in tumor-bearing mice from the indicated groups. Data are presented as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, one-way ANOVA followed by Tukey's post hoc test (n = 6).

We further evaluated the in vivo photothermal therapeutic efficacy of CZPI in the B16 tumor-bearing mouse model. The experimental workflow is illustrated in Fig. 5C. Tumor-bearing mice were monitored every two days for body weight, tumor volume, and survival to assess treatment outcomes. As shown in Fig. 5D–F, tumors in the PBS + L group grew rapidly, reaching nearly 1500 mm³ within 15 days, which indicated that NIR irradiation alone had no therapeutic effect. Not surprisingly, all mice in this group died within 17 days. The tumor inhibition rate in the CZ + L and ZPI + L groups were only 25.7% and 24.2%, respectively; however, mice in both groups died within 25 days. Notably, CZPI exhibited a markedly enhanced tumor-suppression effect of up to 49.2% (P < 0.01), while the combination of CZPI and NIR irradiation led to near complete tumor ablation. In addition, all mice in the CZPI + L group survived for at least 45 days. To further verify the therapeutic efficacy of CZPI + L, we analyzed the histopathological changes in the tumor tissues from different groups. H&E staining revealed extensive nuclear fragmentation and pronounced karyolysis in tumor sections from the CZPI + L group, indicating severe tissue damage consistent with the tumor-ablation (Fig. 5G). Furthermore, a significant reduction in the proportion of Ki67+ proliferating cells in the CZPI + L-treated tumors (Fig. 5G and H) confirmed the effective eradication of tumor cells upon NIR activation. To elucidate the mechanisms underlying cell death, we next analyzed the markers of apoptosis, ferroptosis, and ICD in tumor tissues. Consistent with the in vitro findings, TUNEL staining (Fig. 5I and J) demonstrated a marked increase in apoptotic cells in the CZPI + L group compared to that in the other treatment groups. In addition, the reduced expression of GPX4 (Fig. 5K and L) and decreased GSH levels (Fig. 5M) were indicative of ferroptosis, and the significant elevation of CRT and HMGB1 in the serum confirmed that CZPI + L effectively triggers ICD (Fig. 5N and O).

Notably, no significant body-weight loss was observed in any treatment group throughout the study (Fig. S16), and H&E staining of major organs revealed no evident pathological abnormalities (Fig. S17). To further assess the in vivo biosafety of CZPI, healthy mice were intravenously injected with a high dose of CZPI, and hematological parameters and serum biochemical indicators were evaluated after 60 days. Liver function and kidney function indices were normal in the control (PBS-treated) and CZPI-treated mice, and no significant differences were observed between the two groups (Fig. S18). In addition, no discernible hemolysis was observed after incubating red blood cells with different concentrations of CZPI at 37 °C for 24 h, which further verify the biocompatibility of CZPI (Fig. S19).

NIR-mediated immunotherapeutic mechanism of CZPI. To elucidate the NIR-mediated immunotherapeutic mechanism of CZPI, we analyzed the immune landscape of the tumor and lymph nodes, which are the principal sites for antigen processing and lymphocyte activation (Fig. S20). As shown in Fig. 6A and B, the proportion of mature DCs (CD80⁺CD86⁺) in the CZPI + L group increased by 4.1-fold (to ∼21.0%) compared to that in the control group, indicating that the combination treatment can efficiently promote DC maturation. Given that mature DCs initiate adaptive immune responses through antigen presentation, we further evaluated the frequencies of CD3⁺CD8⁺ T cells and CD3⁺CD4⁺ T cells in the tumor and lymph nodes. The proportion of intra-tumor CD8⁺ T cells (4.17%) and CD4⁺ T cells (7.47%) respectively increased to 13.4% and 15.5% following CZPI + L treatment, confirming robust activation of both CD4⁺ and CD8⁺ T cell subsets (Fig. 6C and E). A similar increase in CD4⁺ and CD8⁺ T cell populations was observed in the lymph nodes as well (Fig. 6G and I).

Fig. 6.

In vivo immunotherapeutic effects of photothermally activated CZPI. (A, B) Proportion of mature DCs (CD80⁺CD86⁺) in the lymph nodes of mice from the indicated groups. (C-F) Proportion of tumor-infiltrating CD8⁺ T cells and CD4⁺ T cells in the mice from the indicated groups. (G-L) Proportion of CD8⁺ T cells, CD4⁺ T cells, and effector memory T cells in the lymph nodes of mice from the indicated groups. Data are presented as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, one-way ANOVA with Tukey's post hoc test (n = 6).

Memory T cells, which provide durable antigen-specific immune surveillance and protection against tumor recurrence, are considered a hallmark of successful anti-tumor immunity. Notably, CZPI + L treatment promoted the differentiation of CD8⁺ T cells in the lymph nodes into effector memory T cells (CD44⁺CD62L⁻). As shown in Fig. 6K, the proportion of memory T cells in the CZPI + L group was 4.2-fold higher than that in the control group. Collectively, these results demonstrate that the DAMPs released from the dying tumor cells following PTT are internalized by antigen-presenting cells, which subsequently migrate to the lymph nodes to drive DC maturation, T-cell priming, and effector T-cell expansion. This cascade initiates a systemic anti-tumor immune response, encompassing both innate immune activation, and the amplification and memory formation of effector T cells.

NIR-mediated suppression of tumor recurrence by CZPI. Long-term immune memory is essential for sustained tumor suppression. Central memory T cells exhibit prolonged persistence in vivo, and can rapidly proliferate and differentiate into effector memory T cells upon re-encounter with tumor antigens, thereby playing a pivotal role in preventing tumor recurrence. In light of the immunological effects and memory induction elicited by NIR-activated CZPI, we established a B16 melanoma rechallenge model to evaluate the therapeutic efficacy of CZPI against recurrent tumors (Fig. 7A). The PD-1/PD-L1 immune checkpoint frequently contributes to T-cell exhaustion within the TME, subsequently weakening anti-tumor immunity and hindering the establishment of durable immune memory. Studies show that PD-1/PD-L1 blockade can restore T-cell effector function, enhance memory responses, and improve control over tumor relapse [42]. To explore the translational potential of combining CZPI-based therapy with immune checkpoint blockade (ICB), the tumor-bearing mice were treated with aPD-L1 along with the PTT regimen.

Fig. 7.

Long-term immune memory induced by photothermally activated CZPI. (A) Schematic illustration of the establishment of the B16 tumor rechallenge model and the treatment regimen. (B) Tumor growth curves, (C) tumor growth ratios, and (D) Kaplan-Meier survival curves of mice in the indicated treatment groups (n = 6; ∗P < 0.05, ∗∗P < 0.01, one-way ANOVA followed by Tukey's post hoc test). (E) Schematic representation of the in vivo immunotherapeutic mechanism of CZPI under photothermal activation.

As shown in Fig. 7B and C, there was no significant inhibition of tumor growth in the PBS + L, CZ + L, ZPI + L, or aPD-L1+L groups following rechallenge. In sharp contrast, CZPI + NIR moderately delayed the progression of re-challenged tumors with an inhibition rate of 57.5%, whereas the combination of CZPI, NIR irradiation and aPD-L1 completely prevented tumor recurrence and achieved the most pronounced survival benefit (Fig. 7D). Collectively, these results demonstrate that CZPI-mediated photothermal/chemodynamic/immunological cascade can effectively stimulate robust adaptive immune responses against both primary and metastatic tumors when integrated with ICB. Furthermore, this combined strategy can not only enhance anti-tumor efficacy but also establish durable immune memory capable of preventing tumor recurrence (Fig. 7E).

4. Conclusion

We successfully developed a multifunctional nanoplatform that integrates self-supplied H₂O₂ generation with photothermal enhancement to overcome key limitations of CDT in tumor treatment. CZPI employs a Cu-ZnO₂ core as a catalytic reaction engine, and PDA and ICG as efficient photothermal boosters. CZPI is selectively degraded in the mildly acidic TME, enabling continuous release of H₂O₂ and Cu²⁺. Through the GSH-triggered Cu²⁺/Cu⁺ redox cycling, CZPI efficiently catalyzes Fenton-like reactions to generate highly cytotoxic •OH, while concurrently depleting intracellular GSH and thereby reversing tumor resistance to oxidative stress. Upon NIR irradiation, the photothermal effect of CZPI not only induces direct tumor cell ablation but also acts as a potent “reaction accelerator,” which enhances Cu-ZnO₂ acidolysis and the kinetics of Fenton-like catalysis, ultimately amplifying CDT efficacy. This synergistic photothermal-chemodynamic mechanism triggers apoptosis and ferroptosis, and robustly induces ICD, which is accompanied by the release of DAMPs such as CRT and HMGB1. The tumor-associated antigens released from the dying tumor cells effectively activate innate anti-tumor immunity, promote DC maturation, and subsequently stimulate the proliferation and infiltration of CTLs, thus establishing a strong adaptive immune response in vivo. Furthermore, the synergistic therapy facilitates the generation of effector memory T cells, providing a basis for long-term immune surveillance. In a tumor rechallenge model, CZPI combined with NIR irradiation markedly delayed tumor regrowth, while PD-L1 blockade almost completely prevented recurrence, thereby underscoring the potential of this synergistic strategy for establishing durable immune memory and preventing tumor relapse.

Overall, this study presents a new strategy that synergistically enhances CDT through a “self-supplied H₂O₂ engine” and a “photothermal booster,” and further pioneers a multimodal therapeutic paradigm integrating local physical ablation, intracellular catalytic therapy, and systemic immune activation. This platform offers a highly promising solution for effective tumor eradication and recurrence prevention with strong translational potential.

CRediT authorship contribution statement

Beibei Sun: Investigation, Methodology, Validation, Writing – original draft. Tingsong Zhang: Investigation. Ruoxi Wan: Methodology. Leijie Tong: Methodology. Tao Li: Methodology. Jinyan Wang: Validation. Runwei Wang: Conceptualization, Funding acquisition, Supervision. Fei Yan: Conceptualization, Supervision, Validation, Writing – original draft, Writing – review & editing. Shilun Qiu: Conceptualization, Supervision.

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.

Appendix A. Supplementary data

The following is/are the supplementary data to this article:

Data availability

Data will be made available on request.