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• Breast cancer (BC) is a malignant tumor that poses a significant threat to women's health and lives, ranking as the most common malignancy among females [1,2]. In fact, BC has surpassed lung cancer as the most prevalent cancer among women [3]. Despite advancements in treatment, the recurrence and metastasis of tumors remain the primary factors contributing to lower survival rates [4,5]. Currently, there is no effective postoperative treatment plan for malignant tumor metastasis and recurrence [6]. Thus, preventing or impeding tumor recurrence and metastasis presents a critical challenge for enhancing clinical effectiveness and patient survival rates.

  To date, in addition to standard surgery and chemotherapy, photothermal therapy (PTT) has garnered substantial attention due to its noninvasive, highly targeted, and convenient properties. PTT is a therapeutic approach that entails the introduction of photothermal agents with a high conversion efficiency for light-to-heat energy into the human body, achieved through near-infrared (NIR) light irradiation [7]. Graphene oxide (GO), a two-dimensional nanomaterial with an atomic-thin layered structure, exhibits unique physicochemical properties in electrical, optical, and thermal domains, rendering it a vital component of functional materials [8,9]. Thanks to its remarkably high specific surface area and potent NIR light absorption capacity, GO serves as an effective photothermal agent, efficiently converting light energy into heat energy and yielding a substantial photothermal effect on tumor cells. The precision, controllability, high efficiency, and minimal side effects in normal tissue make PTT a formidable asset in tumor therapy.

  Nevertheless, the heterogeneity and diversity of tumors significantly impede the effectiveness of PTT [10,11]. Particularly, limitations imposed by the size of the photothermal excitation light source and tissue penetration depth invariably leave a portion of tumor tissue after PTT, contributing to recurrence and metastasis [12-14]. Therefore, this study aims to combat residual tumor recurrence and metastasis stemming from singular therapy and enhance the prognostic survival rate of malignant tumor patients through a chemodynamic therapy (CDT)-enhanced PTT system. Leveraging the exceptional catalytic potential of nanomaterials, CDT prompts hydrogen peroxide (H₂O₂) in the tumor microenvironment to generate hydroxyl free radicals (^•OH) via a Fenton-like reaction, thereby inducing tumor cell apoptosis. This approach is expected to further eliminate remaining tumor tissue post-PTT treatment, thereby suppressing tumor metastasis and recurrence.

  Common metal-based nanomaterials like iron oxide [15-18], molybdenum sulfide [19,20], and copper sulfide [21-25] exhibit favorable catalytic capabilities for PTT and/or CDT. Nevertheless, their limited biochemical stability and potential metal toxicity hinder their broader application in clinical practice. In contrast, non-metallic carbon-based catalysts, typified by GO, not only possess excellent photothermal properties but also harbor CDT therapeutic potential. Although GO's catalytic activity may not match that of conventional metal catalysts, it can be enhanced by doping the graphene's surface or edges with various elements [26,27]. The introduction of heteroatoms (such as nitrogen and sulfur) to the carbon-carbon double bond edge in the graphene lattice disrupts its original symmetry, alters electron spin density, and changes charge distribution, creating active sites that effectively enhance the catalytic activity of graphene derivatives [28-30]. Thus, a high-performance CDT-enhanced PTT system can be established based on N-containing graphene derivatives, offering a novel solution for the effective treatment of malignant tumors and an improvement in long-term patient survival rates.

  Considering the challenges associated with the persistent difficulty in completely eradicating malignant tumors and their tendency to relapse and metastasis, we have devised a metal-free nitrogen containing polymer-modified GO enzyme system. In this endeavor, GO functions as a nano template, while nitrogen-rich polyethyleneimine (PEI) serves as a highly branched and monodisperse synthetic polymer. Its precise molecular structure and abundant surface amino groups make it a valuable nitrogen source and catalyst carrier. To enhance its biocompatibility and prolong its circulation time in the bloodstream, poly(ethylene glycol) (PEG) was conjugated to PEI via a coupling reaction with 1-(3-dimethylaminopropyl)−3-ethyl-carbodiimide hydrochloride (EDC).

  Subsequently, we enriched GO with nitrogen-rich PEI-PEG using the marginal ball milling method, imparting exceptional catalytic activity to GO. Recognizing that intracellular hydrogen peroxide levels may not suffice as a catalytic substrate to generate a substantial quantity of hydroxyl radicals, we introduced glucose oxidase (GOD) into the nanosystem through electrostatic interactions. GOD catalyzes the production of ample hydrogen peroxide from the abundant glucose in tumor cells. In the acidic microenvironment, GO-PEI-PEG (GPP) catalyzes hydrogen peroxide to generate a significant volume of hydroxyl radicals with anti-tumor properties. This approach, to some extent, mitigates the cytotoxicity resulting from normal cell phagocytosis and enhances the specificity of the nanomaterials.

  The developed GO-PEI-PEG/GOD (GPP/G) non-metallic nanobionic enzyme exhibited remarkable lethality against tumors under NIR irradiation. Remaining tumors beyond the laser's reach and deep within the tumor were eradicated by the Fenton-like reaction of GPP, catalyzed by the tumor's slightly acidic environment. In this study, we realized a groundbreaking anti-tumor strategy by creating a non-metallic nanobionic enzyme based on CDT sensitized PTT. This approach holds promising implications for the treatment of BC and the prevention of its recurrence and metastasis.

  The preparation of GPP/G is detailed in Fig. 1a. Initially, nitrogen-containing graphene was obtained through a physical ball-milling process involving PEI-PEG and GO. Covalent interaction between GO and PEI-PEG was achieved via a solid-state mechanochemical reaction as reported elsewhere [31]. PEI-PEG was expected to functionalize the GO nanosheets via the active nitrogen sites (from PEI) during the ball-milling process. Subsequently, the GPP/G system was crafted via the electrostatic interaction between the positively charged GPP and the negatively charged GOD. To examine the morphology of the prepared GPP, we conducted atomic force microscopy (AFM) observations, as depicted in Fig. 1b. GPP exhibits a distinctly uniform lamellar structure with bright spots interspersed throughout the graphene sheet. The thickness of GO was measured at 1.28 nm, while that of PEI-PEG was approximately 6.28 nm. This data indicates that the ball-milled GO is in a monolithic state, with successful doping of PEI-PEG onto the GO sheet. The results of Raman spectroscopy for both GO and GPP reveal three evident absorption signals, corresponding to the absorption peak (D peak at 1350 cm⁻¹), the vibration peak (G peak at 1595 cm⁻¹), and the second-order Raman peak (2D peak at 2680 cm⁻¹) of the graphite structure on GO (Fig. S1 in Supporting information). This indicates that the ball milling of PEI-PEG and GO does not significantly alter the GO's structure.

  Figure 1

  

  Figure 1.  (a) Schematic illustration of the preparation of GPP/G. (b) AFM micrograph of GPP/G. (c) UV–vis spectra representing the absorption of MB in the presence of H₂O₂, with variations upon the addition of GO and GPP. (d) The surface zeta potential of GPP and GPP/G. Data are presented as mean ± standard deviation (SD) (n = 3).

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  Fig. S2 (Supporting information) presents the X-ray diffraction (XRD) patterns of the resulting GPP, where the intense diffraction peaks at 10.5° and 21.5° represent GO and PEI, respectively, validating the successful formation of GPP. The PEI-PEG embedded in GO was further characterized via the Fourier transform infrared (FTIR) spectrum (Fig. S3 in Supporting information). Key bands include a strong peak at 1655 cm⁻¹ associated with N–H bending vibration, the absorption band at 2915 cm⁻¹ due to the presence of -CH₂ groups, and the peak at 1650 cm⁻¹ indicating the amide bond in the PEI. The peak at 1105 cm⁻¹ is linked to the stretching vibration of the C–N bond, while the 1630 cm⁻¹ peak is likely associated with the bending vibration of C–OH in the GO. The FTIR spectroscopy confirms the successful doping of PEI-PEG nanoparticles on GO. To assess the NIR absorption properties of GPP, ultraviolet-visible (UV–vis) spectra were performed (Fig. S4 in Supporting information), revealing distinct NIR absorption characteristics. Consequently, GPP exhibits significant potential for PTT.

  To investigate the Fenton-like catalytic performance of GPP, we employed methylene blue (MB) as a color reagent to monitor the generation of ^•OH. In this process, GPP functions as a Fenton-like catalyst, catalyzing H₂O₂ to produce copious ^•OH, which then interacts with MB, quenching its characteristic absorption peak at 660 nm. Fig. 1c illustrates a significant reduction in the absorption value of MB with the addition of GO. However, the introduction of GPP results in the complete disappearance of the absorption value of MB, indicating superior Fenton-like catalytic performance, establishing it as an effective Fenton catalyst.

  Furthermore, the positively charged GPP can sequester the negatively charged GOD through electrostatic interaction. We evaluated the hydrodynamic diameter and zeta potential of both GPP and the GPP/G complex. As depicted in Fig. S5 (Supporting information), GPP exhibited a hydrodynamic diameter of 208.4 nm, attributed to the gentle curling of graphene oxide sheets in water. In contrast, the GPP/G complex exhibited a slightly larger size of 224.2 nm. This suggests that the entrapment of GOD does not significantly alter the volume of GPP, and variations in pH do not substantially affect the size of the complex. The zeta potential also demonstrates a noticeable contrast between GPP (~30 mV) and the GPP/G complex (~25 mV) (Fig. 1d). Evidently, the loading of GOD partially neutralizes the positive charge of GPP, rendering it less biologically toxic during circulation.

  We evaluated the photothermal property of GPP/G at different concentrations. By monitoring the temperature change in a GPP/G solution under NIR laser irradiation (1.2 W/cm², 5 min), we observed that higher concentrations of GPP/G exhibited a more pronounced temperature increase. The temperature increased from 26.9 ℃ to 71.5 ℃ within 5 min of laser irradiation for GPP/G with a higher concentration (Fig. 2a). In contrast, the temperature of the control group remained stable under the same conditions. Furthermore, the temperature changes (ΔT) in 5 min were recorded as 17.2, 29.3 and 44.4 ℃, respectively, with increasing concentrations of GPP/G (Fig. 2b). These data highlight GPP/G's rapid conversion of NIR light into heat, showcasing its remarkable photothermal conversion performance. The photothermal conversion efficiency (η) of GPP/G was obtained from Fig. S6 (Supporting information). It was calculated to be 58.3%.

  Figure 2

  

  Figure 2.  (a) Temperature variation curves for GPP/G at different concentrations under 808-nm laser irradiation (1.2 W/cm²) for 5 min. (b) Temperature changes (ΔT) for GPP/G under laser irradiation for 5 min. (c) Michaelis–Menten kinetic curve indicating the catalytic activity of GPP/G in TMB upon the addition of glucose. (d) Lineweaver–Burk plotting curve illustrating the catalytic activity of GPP/G in TMB after the addition of glucose.

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  To assess the Fenton-like catalytic performance of GO and GPP, we conducted a 3,3′,5,5′-tetramethylbenzidine (TMB) chromogenic reaction, utilizing H₂O₂ as the catalytic substrate. In this process, TMB can be oxidized by highly reactive ^•OH, resulting in a color change from colorless to blue. The maximum blue absorbance was detected by scanning at 652 nm using an ultraviolet spectrophotometer. The absorbance indicates the ability of the nanocatalyst (e.g., GO or GPP) to produce hydroxyl radicals. Fig. S7 (Supporting information) illustrates that the characteristic peak becomes increasingly prominent with higher concentrations and extended reaction times. Notably, GPP exhibits a more distinct and rapid color change compared to GO, underscoring its excellent Fenton-like catalytic performance. This could be attributed to the physical ball grinding and free radical collision processes, which replace weakly oxygenated groups on GO with the active amino groups present on the surface of the nitrogenous cationic polymer. The strong electron-absorbing capacity of nitrogen leads to electron cloud migration towards it, causing an electron deficiency on graphene and a corresponding need for electron absorption, thus endowing GPP with superior Fenton-like catalytic performance.

  Moreover, Michaelis-Menten steady-state kinetics calculations provide more intuitive confirmation of these results, translating absorbance values into initial velocities (V₀) in accordance with the Beer-Lambert law and plotting them against the nanocatalyst concentration. As depicted in Fig. S8 (Supporting information), the Michaelis-Menten constant (K_m) and the maximum reaction velocities (V_m) for GPP are 39.26 mmol/L and 6.68 × 10^–7 mol L⁻¹ s⁻¹, respectively, demonstrating their superior performance compared to GO (K_m = 60.52 mmol/L, V_m = 3.77 × 10^–7 mol L⁻¹ s⁻¹).

  The catalytic activity of glucose as a substrate at various concentration gradients was studied under 100 µg/mL GPP/G catalysis. This catalysis primarily involves the following process: GOD within the GPP/G complex catalyzes the production of H₂O₂ from glucose in the tumor micro acidic environment, and GPP then converts H₂O₂ to ^•OH through Fenton-like catalysis. As shown in Figs. 2c and d, GPP/G demonstrates an impressive catalytic effect, with a K_m value of 15.85 mmol/L and a V_m value of 2.70 × 10^–7 mol L⁻¹ s⁻¹. These results suggest that GPP/G can produce a sufficient amount of ^•OH through exogenous catalytic supplementation to overcome the issue of low endogenous H₂O₂ concentrations in cells, effectively enhancing CDT.

  The cytotoxicity of the as-prepared GPP was assessed using a cell counting kit-8 (CCK-8) assay with 4T1 cells. It is evident that GPP does not induce any significant toxic effects on cells within the concentration range of 0–100 µg/mL under both culture conditions (pH 7.4/6.0) (Fig. S9 in Supporting information). Only when the concentration reaches 150 µg/mL does GPP begin to exhibit a slight degree of cytotoxicity. This demonstrates that within a given concentration range, GPP exhibits good biological safety. Additionally, the viability of GOD in 4T1 cells was evaluated to determine the appropriate concentration for preparing the GPP/G complex. The results in Fig. S10 (Supporting information) indicate that GOD remains safe for cellular use within the given concentration range (0–100 ng/mL). Hence, the concentrations of 100 µg/mL GPP and 100 ng/mL GOD were selected for further use to minimize the individual materials' cytotoxicity to cells.

  Fig. 3a illustrates the cytocompatibility of 4T1 cells cultured with GPP/G under specific pH conditions. Clearly, cell viability exhibits a concentration-dependent and pH-sensitive response. Under acidic conditions, GPP/G displays cell viabilities of 100.3%, 94.9%, 78.8%, 64.5%, 43.3%, 31.1%, and 27.9% at descending concentrations, while under neutral conditions, significantly higher cell viabilities of 98.6%, 97.1%, 93.4%, 77.3%, 70.1%, 67.6%, and 52.2% are observed at corresponding concentrations. This indicates that GPP/G exhibits higher tumor-suppressing capacity in an acidic pH environment compared to a neutral one. For better understanding the cytotoxicity of GPP/G, normal L929 mouse fibroblast cells were also used for the cytotoxicity test. As shown in Fig. S11 (Supporting information), no visible declined cell viability of L929 cells in the neutral culture media (pH 7.4) was observed. The cell viability remains higher than 90% even 200 µg/mL GPP/G was applied. This supports that the GPP/G nanocomposite's good pH sensitivity is beneficial to reduce non-specific damage on normal cells/tissues during the antitumor treatment.

  Figure 3

  

  Figure 3.  (a) Cytotoxicity of 4T1 cells exposed to varying concentrations of GPP/G under pH 7.4/6.0 conditions for 24 h. (b) The quantitative analysis of ROS fluorescence in 4T1 cells after co-incubation with nanomaterials. (c) The results of L-ascorbic acid-assisted cell rescue in 4T1 cells induced by GPP/G. (d) The viability of 4T1 cells under the different treatments (mean ± SD, n = 3). **P < 0.01, ***P < 0.001. ns, not significant.

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  The therapeutic effect of CDT on cancer cells was further assessed through a reactive oxygen species (ROS) assay. As shown in Fig. 3b and Fig. S12 (Supporting information), the green fluorescence is barely observable in the GPP group under different pH conditions, indicating insignificant ROS generation by GPP alone. The Fenton-like reaction is initiated by the interaction between glucose and GOD, producing ample H₂O₂. In the absence of H₂O₂, GPP cannot exert its catalytic ability. Conversely, strong green fluorescence is evident in the fluorescence image of GPP/G-treated cancer cells under pH 6.0, suggesting significant intracellular ROS production. In comparison, GPP/G under pH 7.4 displays much weaker fluorescence intensity than that under pH 6.0, indicating less efficient ROS production in a neutral condition compared to an acidic one. The ROS results align with previous toxicity findings.

  To further elucidate the role of the catalytic moiety, cell viability was investigated for cells co-cultured with the GPP/G complex and l-ascorbic acid. The results indicate that the cytotoxicity of the GPP/G complex towards cancer cells results from oxidative damage caused by active radicals (Fig. 3c). Furthermore, the cytotoxicity of the GPP/G complex can be induced by the sequential catalytic effects of GOD and GPP.

  The viability of 4T1 cells treated with GPP, with or without laser irradiation (NIR− and NIR+), under standard culture conditions was examined to assess the independent photothermal effect on cancer cells. Clearly, without the presence of GOD, the cytotoxicity of GPP under different pH values exhibits no significant differences (Fig. S13 in Supporting information). Cell survival rates are higher for cells treated without NIR laser irradiation than for those treated with laser irradiation. Under NIR laser irradiation, cell viability decreases notably with increasing GPP concentration, especially at higher concentrations. Nearly 75.0% of cells are killed with GPP treatment (NIR+) at a concentration of 100 µg/mL, demonstrating a good tumor-destruction effect. This suggests that the prepared GPP can inhibit tumor growth through PTT but the survival tumor cells are still visible.

  To evaluate the therapeutic impact of CDT and PTT, the viability of 4T1 cells treated with the GPP/G complex was assessed, with phosphate buffered saline (PBS) and GPP serving as controls (Fig. 3d). In comparison to the PBS (NIR− and NIR+) groups under pH 7.4/6.0 and the GPP (NIR−) group under pH 7.4, the cells exhibited good viability (>90%). However, cells cultured with GPP/G (NIR−) under pH 6.0 displayed a significantly reduced survival rate (27.7%), albeit higher than that of the GPP/G (NIR−) group under pH 7.4 (65.8%). It is evident that CDT exhibits a pronounced pH-dependent effect. Furthermore, cells treated with GPP (NIR+) under pH 7.4/6.0 displayed high cell mortality (32.9% and 28.8% survival rates, respectively). This indicates that photothermal treatment is not pH-dependent. Remarkably, the GPP/G (NIR+) group under pH 6.0 exhibited the lowest survival rate (13.8%), higher than that of the GPP/G (NIR+) group under pH 7.4 (29.8%). Clearly, CDT significantly enhances the photothermal ablation effect under acidic conditions (pH 6.0), demonstrating an excellent outcome in tumor treatment.

  The therapeutic effect of CDT/PTT was visually examined through fluorescence microscopic observation of the cytoskeleton and cell nuclei of 4T1 cells (Fig. 4a). As cell functions are reflected by the cytoskeleton and cell nuclei, PTT/CDT in cancer cells may lead to cytoskeleton disruption and oxidative DNA damage due to intracellular hyperthermia and the inhibition of hydroxyl radicals. It is evident that cells treated with PBS, GPP (NIR−), and GPP/G (NIR−) exhibit regular cell morphology. In contrast, cells in the GPP/G (NIR−) group under neutral conditions exhibit a noticeable disruption of actin stress fibers. The results suggest that the produced hydroxyl radicals damage cell morphology to a certain extent. Moreover, the GPP/G (NIR+) group under pH 7.4/6.0 shows an almost completely destroyed cytoskeleton structure. The data indicate that PTT eliminates cancer cells by disrupting the cytoskeleton, while CDT damages cancer cells by generating hydroxyl radicals. The visual damage aligns with the results of previous cell tests.

  Figure 4

  

  Figure 4.  (a) Skeleton fluorescence staining of 4T1 cells treated with GPP/G. Green represents Alexa Fluor 488-Phalloidin stained skeletons, while blue represents 4′,6-diamidino-2-phenylindole (DAPI) stained nuclei. (b) Fluorescence microscopic images of live/dead stained 4T1 cells treated with GPP/G. Green indicates live cells, and red indicates dead cells.

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  A live/dead staining method was employed to visually observe the distribution of viable and dead cells. 4T1 cells were stained with calcein-AM (green) and propidium iodide (PI) (red) solution after co-incubation with GPP/G under neutral or acidic conditions for 24 h. The fluorescence images reveal that no portion of 4T1 cells is damaged when treated with PBS (Fig. 4b). However, when incubated with GPP/G (NIR−) under acidic conditions, some dead cells are observed, likely due to the production of ROS. A large number of dead cells are observed when incubated with GPP/G (NIR+) under neutral conditions, a result of the photothermal effect. Notably, cells treated with GPP/G (NIR+) under acidic conditions are almost entirely killed by the highly toxic hydroxyl radicals generated in situ and the effective photothermal properties. It is evident that the prepared GPP/G serves as an outstanding agent for tumor treatment.

  To assess the in vivo application potential of the GPP/G complex, its hemolytic performance was tested when co-cultured with red blood cells. All experimental procedures involving animals were approved by the Wenzhou Medical University Animal Care and Use Committee (No. wydw2022–0119). Our animal experiments strictly adhered to the principles outlined in the Declaration of Helsinki. As shown in Fig. 5a, the GPP/G complex exhibits a low hemolysis rate (<3%) within the identified concentration range (<1000 µg/mL), establishing a basis for in vivo applications.

  Figure 5

  

  Figure 5.  (a) Hemolysis percentages of red blood cells (RBC) treated with various amounts of GPP/G (mean ± SD, n = 3). (b) NIR thermal images of 4T1 tumor-bearing BALB/c nude mice injected with different nanomaterials after 4 h. and irradiated by an 808-nm laser (1.2 W/cm²) for 5 min. (c) The relative tumor volumes of the tumor-bearing mice after different treatments (mean ± SD, n = 4, ***P < 0.001). (d) Digital photos of the harvested tumors after different treatments. (e) Digital photos of the tumor site changes after different treatments.

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  The photothermal properties of the GPP/G complex in tumor-bearing mice were assessed through photothermal imaging of the tumor site. It is evident that the temperature at the tumor site significantly increases after laser irradiation compared to the control group, reaching 47.5 ℃ in the GPP group and 45.5 ℃ in the GPP/P group (Fig. 5b). Under such elevated temperatures, most tumor cells undergo apoptosis due to heat-induced damage.

  To comprehensively assess the antitumor efficacy of the GPP/G complex in 4T1 tumor-bearing mice, it is essential to explore the effects of CDT/PTT. Notably, the relative tumor growth volume curves within 21 days post-injection reveal that the NS control group and GPP (NIR−) group had significantly larger tumors (Fig. 5c). In contrast, the tumor volumes in the comprehensive treatment group (GPP/G, NIR+) gradually diminish over time, demonstrating the most effective anti-tumor outcome. While PTT alone and CDT alone can temporarily delay tumor growth, they are unable to completely eradicate the tumor. During the treatment period, there was no significant weight loss observed in any of the mice, indicating that the material group did not cause substantial toxicity (Fig. S14 in Supporting information). Photographs of the tumors (Fig. 5d) after 3 days of therapy illustrate a similar therapeutic effect to that shown in Fig. 5c, with the following order: GPP/G (NIR+) group > GPP (NIR+) group > GPP/G (NIR−) group > other groups. Images of the mice taken every seven days revealed significant differences in tumor size with different treatment strategies. As illustrated in Fig. 5e, the size of the tumor increased sharply for the PBS and GPP (NIR−) group. In the GPP/G (NIR‒) group, the treated tumors grew slowly, though with some inhibition. What is interesting is the GPP (NIR+) group. On 7 and 14 days after treatment, the tumor seemed to disappear completely, as it did in the GPP/G (NIR+) group. However, over time, the tumors recurred in the single PTT group (as indicated in Fig. 5e), while this phenomenon was not observed in the GPP/G (NIR+) group. These macroscopic results indicate that PTT can indeed cause a significant number of tumor cell deaths in the early stage. However, some residual tumor cells continue to reappear at a later stage, which can only delay tumor progression to a certain extent. Notably, the additional CDT compensates for the deficiency of PTT and continues to kill the remaining tumor cells, thus enhancing the efficacy of PTT. The apoptosis of cancer cells was further tested using hematoxylin & eosin (HE) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining (Figs. S15–S17 in Supporting information). These results are consistent with the other results obtained from Fig. 5. This supports that the CDT after PTT can efficiently promote apoptosis of tumor cells.

  To investigate the effects of different treatments on mice in the later stage, blood routine and HE staining of major body organs were evaluated. The blood routine results demonstrate that the white blood cell (WBC) levels in the NS (NIR−), NS (NIR+), and GPP (NIR−) groups substantially increase in the late stage of tumor development (Fig. S18 in Supporting information). This suggests that as the tumor grows, the mice's immune system adapts in response to combat the disease. Tumors in the other treatment groups do not provoke significant increases in WBC count due to their varying degrees of treatment. The least WBC level was obtained in the GPP/G (NIR+) group, confirming the best antitumor effect of the GPP/G system. In addition, other blood routine data are within the normal range, suggesting that the treatments had little impact on the mice's blood status.

  The HE staining images of major organs (Fig. S19 in Supporting information) reveal no obvious damage to any organs in the GPP/G (NIR+) group. In contrast, three groups of mice (NS and GPP, NIR−) developed significant lung metastases, while the other two groups (GPP, NIR+ and GPP/G, NIR−) also exhibited mild metastases. This indicates that PTT is a useful but inefficient method for treating tumors, and CDT alone cannot completely eliminate tumors but can significantly enhance the therapeutic effect of PTT, effectively preventing tumor metastasis. Additionally, no significant tissue damage was observed in other major organs. This preliminary in vivo safety test also suggests that the prepared GPP/G complex possesses good biocompatibility, ensuring its potential clinical application.

  This study introduces CDT as a complementary approach to PTT, effectively coordinating the two treatments. The multifunctional GPP/G bionic nanozyme has been developed for pH-directed, tumor-specific therapy. The nitrogen doping enhances the chemodynamic catalytic properties of GO, and the GPP vector effectively protects GOD, allowing it to maintain its bioactivity and catalyze the production of hydrogen peroxide in the acidic tumor microenvironment. This is followed by a Fenton-like reaction that generates a large quantity of hydroxyl radicals. This treatment approach significantly addresses the limitations of PTT, eliminating residual tumor cells and effectively preventing tumor recurrence. The systematic in vitro and in vivo collaborative therapeutic results demonstrate that the combination of CDT and PTT is a mutually beneficial solution. This combined approach reduces the risk of BC recurrence and metastasis. By leveraging the physicochemical properties of nanomaterials in conjunction with the unique characteristics of the tumor microenvironment, this study provides an efficient alternative strategy for cancer treatment.

  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

  Lingdan Kong: Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Pingping Huang: Methodology, Investigation, Formal analysis, Data curation. Feng Yuan: Methodology, Data curation. Yue Zhang: Methodology. Xiaoqian Shi: Methodology. Kang Han: Methodology. Keke Liu: Investigation. Qing Xu: Investigation. Wenjing Zhang: Funding acquisition, Formal analysis. Tom Lawson: Writing – review & editing. Xiaoru Xia: Writing – review & editing, Supervision. Yong Liu: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. Yuepeng Jin: Writing – review & editing, Supervision, Conceptualization.

  Acknowledgments

  This research received financial support from the National Natural Science Foundation of China (Nos. 82202354, U20A20338, 82201247), The Summit Advancement Disciplines of Zhejiang Province (Wenzhou Medical University-Pharmaceutics), and the Key R&D Program of Zhejiang Province (No. 2021C04019). The valuable consultation and instrument support from Scientific Research Centre of Wenzhou Medical University are acknowledged.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.111030.

  
  
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  Figure 1  (a) Schematic illustration of the preparation of GPP/G. (b) AFM micrograph of GPP/G. (c) UV–vis spectra representing the absorption of MB in the presence of H₂O₂, with variations upon the addition of GO and GPP. (d) The surface zeta potential of GPP and GPP/G. Data are presented as mean ± standard deviation (SD) (n = 3).

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  Figure 2  (a) Temperature variation curves for GPP/G at different concentrations under 808-nm laser irradiation (1.2 W/cm²) for 5 min. (b) Temperature changes (ΔT) for GPP/G under laser irradiation for 5 min. (c) Michaelis–Menten kinetic curve indicating the catalytic activity of GPP/G in TMB upon the addition of glucose. (d) Lineweaver–Burk plotting curve illustrating the catalytic activity of GPP/G in TMB after the addition of glucose.

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  Figure 3  (a) Cytotoxicity of 4T1 cells exposed to varying concentrations of GPP/G under pH 7.4/6.0 conditions for 24 h. (b) The quantitative analysis of ROS fluorescence in 4T1 cells after co-incubation with nanomaterials. (c) The results of L-ascorbic acid-assisted cell rescue in 4T1 cells induced by GPP/G. (d) The viability of 4T1 cells under the different treatments (mean ± SD, n = 3). **P < 0.01, ***P < 0.001. ns, not significant.

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  Figure 4  (a) Skeleton fluorescence staining of 4T1 cells treated with GPP/G. Green represents Alexa Fluor 488-Phalloidin stained skeletons, while blue represents 4′,6-diamidino-2-phenylindole (DAPI) stained nuclei. (b) Fluorescence microscopic images of live/dead stained 4T1 cells treated with GPP/G. Green indicates live cells, and red indicates dead cells.

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  Figure 5  (a) Hemolysis percentages of red blood cells (RBC) treated with various amounts of GPP/G (mean ± SD, n = 3). (b) NIR thermal images of 4T1 tumor-bearing BALB/c nude mice injected with different nanomaterials after 4 h. and irradiated by an 808-nm laser (1.2 W/cm²) for 5 min. (c) The relative tumor volumes of the tumor-bearing mice after different treatments (mean ± SD, n = 4, ***P < 0.001). (d) Digital photos of the harvested tumors after different treatments. (e) Digital photos of the tumor site changes after different treatments.

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