Enzyme-enhanced albumin-stabilized nanopolymers for cascade catalysis-driven ferroptosis-immunotherapy

Abstract

Ferroptosis, an iron-dependent form of programmed cell death, holds significant promise for cancer therapy due to its unique redox-driven mechanism and immunogenic potential. However, its therapeutic efficacy is often compromised by inadequate catalytic kinetics and an unfavorable tumor microenvironment. Here, we develop a multifunctional nanocatalyst (BCuP@G) based on albumin-stabilized polypyrrole doped with multivalent Cu ions and conjugated with glucose oxidase (GOx) to enable enzyme-enhanced cascade catalysis for ferroptosis-immunotherapy. The embedded Cu⁺/Cu²⁺ redox centers catalyze glutathione depletion and ·OH generation, while GOx continuously supplies H₂O₂ through glucose oxidation. Upon NIR-II laser irradiation, the photothermal effect further accelerates both enzymatic and catalytic kinetics, triggering excessive redox imbalance, lipid peroxidation, and synergistic ferroptosis-apoptosis. Moreover, BCuP@G demonstrates effective tumor accumulation and potent antitumor efficacy, particularly in combination with anti-PD-1 therapy, which promotes tumor-associated macrophage repolarization and T cell infiltration. This study presents a robust cascade-catalytic nanoplatform that integrates redox modulation and immune activation for advanced ferroptosis-enhanced cancer immunotherapy.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12951-026-04781-9.

Introduction

Ferroptosis is a distinct form of programmed cell death characterized by iron-dependent accumulation of reactive oxygen species (ROS) and lipid peroxides (LPO), demonstrating significant promise for cancer therapy [1–3]. Initiated by intracellular redox imbalance, ferroptosis involves transition metal ions (e.g., Fe²⁺, Cu⁺, Mn²⁺) catalyzing a Fenton or Fenton-like reaction that converts hydrogen peroxide (H₂O₂) into toxic hydroxyl radicals (·OH), leading to lipid peroxidation of polyunsaturated fatty acids in membrane phospholipids, membrane rupture, and subsequent cell death [4–7]. Beyond its direct lethal effects, ferroptosis also exhibits immunogenic characteristics by releasing damage-associated molecular patterns and pro-inflammatory cytokines, which promote tumor-associated macrophages (TAMs) repolarization and T cell infiltration into the tumor microenvironment (TME) [8–10]. Notwithstanding these encouraging attributes, the efficacy of ferroptosis is impeded by two principal factors: (i) intracellular antioxidant defense systems, particularly the glutathione/glutathione peroxidase 4 (GSH/GPX4) axis, which counteracts free radicals and suppresses LPO accumulation [11–13], and (ii) suboptimal catalytic kinetics in the TME resulting from insufficient H₂O₂ concentrations, low temperatures, and mild acidity, which restrict Fenton-like reactions [14, 15].

To address these challenges, numerous ferroptosis inducers have been developed to boost catalytic activity and LPO accumulation [16–18]. In particular, multivalent copper (Cu⁺/Cu²⁺)-based catalysts have garnered significant interest for their capacity to catalyze ·OH generation in a pH-independent manner, while simultaneously depleting intracellular GSH via redox interactions, thereby intensifying redox imbalance and potentiating ferroptotic cell death [19–21]. In parallel, remodeling the TME is of paramount importance. Glucose oxidase (GOx), an oxidoreductase that catalyzes the oxidation of glucose into gluconic acid and H₂O₂, has become a valuable co-therapeutic enzyme [22–24]. This enzymatic process not only provides H₂O₂ as a substrate for ·OH generation but also decreases the local pH to enhance catalytic efficiency [25]. Moreover, moderate hyperthermia has been shown to expedite both enzymatic and Fenton-like reactions, potentially augmenting ferroptotic effectiveness [26, 27]. Despite the importance of optimizing cascade reaction kinetics and modulating TME, there exists a paucity of research on Cu-based cascade nanoreactors capable of attaining high catalytic activity, continuous H₂O₂ supply, low pH level, and heat generation, all of which are required for effective tumor ferroptosis therapy.

Albumin, a biocompatible, biodegradable, and non-immunogenic serum protein, has been extensively employed as a fundamental component in the design of nanomedicines for cancer therapy, as evidenced by clinically approved nanomedicines like Abraxane^® and Fyarro^® [28–30]. Leveraging its structural versatility and abundant functional groups, albumin readily forms nanocomplexes with drugs or polymers by non-covalent conjugation, including electrostatic and hydrophobic interactions as well as hydrogen bonding, without requiring harsh chemical synthesis [31, 32]. However, the inherently reversible nature of these physical interactions may jeopardize the stability and in vivo reproducibility of albumin-based nanocomplexes, representative of the rapid disintegration of Abraxane^® in circulation [33]. Recent research has demonstrated that integrating polymers into albumin-drug assemblies improves the stability and drug-loading capacity of the resulting nanocomplexes, providing a promising foundation for the advancement of next-generation nanotherapeutics with improved pharmacokinetics, controlled drug release, and enhanced therapeutic efficacy [34–36].

In this study, we developed a novel albumin-stabilized polypyrrole (BCuP) through protein-assisted oxidative polymerization of pyrrole in the presence of CuCl₂ under mild conditions, allowing for simultaneous in situ encapsulation of bovine serum albumin (BSA) and effective doping of multivalent Cu ions (Scheme 1). Subsequently, GOx was covalently conjugated onto the BCuP through amide bonding, yielding a multifunctional nanocatalyst (BCuP@G) with enzyme-enhanced catalytic activity. Upon cellular internalization, the embedded Cu⁺/Cu²⁺ redox centers catalyzed GSH depletion and the conversion of H₂O₂ into ·OH, while GOx continuously oxidized glucose to replenish H₂O₂. This cascade exacerbated intracellular oxidative imbalances, leading to mitochondrial dysfunction, GPX4 downregulation, and LPO accumulation. Furthermore, moderate hyperthermia activated by polypyrrole conjugated structures under 1064 nm laser irradiation further accelerated both enzymatic and Fenton-like catalytic rates, ultimately triggering apoptosis and ferroptosis in tumor cells. Immunologically, BCuP@G synergized with anti-programmed cell death 1 (αPD-1) to stimulate immunogenic cell death, repolarized TAMs to the antitumoral M1 phenotype, and enhanced effector T cell infiltration, thereby potentiating systemic antitumor immunity. Altogether, this work underscores the potential of BCuP@G as a versatile therapeutic nanoplatform that integrates redox cascade catalysis and immune activation for synergistic ferroptosis-immunotherapy in cancer treatment.

Scheme 1.

Schematic illustration of the synthesis and therapeutic mechanism of BCuP@G. (A) Synthesis of BCuP@G via BSA-assisted polymerization followed by GOx conjugation. (B) Enzyme-enhanced catalytic activity of BCuP@G induced GSH depletion and ·OH generation, leading to tumor cell apoptosis and ferroptosis, thereby augmenting antitumor immunotherapy

Materials and methods

Synthesis of BCuP

50 mg of BSA was dissolved in 10 mL of deionized (Di) water under constant stirring at room temperature. After complete dissolution, 1 g of CuCl₂, pre-dissolved in 5 ml of Di water, was added and the solution was stirred for an additional 30 min. Subsequently, 50 µL of pyrrole monomer was introduced, and the reaction mixture was allowed to proceed for 6 h at room temperature. The resulting product was subjected to centrifugation at 6000 rpm for 5 min to remove large aggregates, followed by further purification via centrifugation at 20,000 rpm for 25 min. The final precipitate, designated as BCuP, was collected and stored at 4 °C for further use.

Synthesis of BCuP@G

To activate the carboxyl groups, 10 mg of BCuP was dissolved in Di water, followed by the addition of a mixture solution comprising N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC, 10 mg) and N-Hydroxysuccinimide (NHS, 6 mg). After stirring for 1 h at room temperature, different qualities of GOx (1, 2, or 5 mg) was added to the mixture and stirred overnight. The reaction product was collected by centrifugation and lyophilized to obtain GOx-loaded BSA-stabilized multivalence copper ion-doped polypyrrole (BCuP@G). The final product was stored at 4 °C for further use.

Photothermal performance of BCuP@G

BCuP@G aqueous solutions at various concentrations (0, 12.5, 25, 50, and 100 µg mL^− 1) were irradiated with 808 nm or 1064 nm lasers at different power densities (0.25, 0.5, 1.0, and 1.5 W cm^− 2) for 5 min. The temperature changes were recorded in real time using an infrared thermal imaging camera (Ti480U, Fluke, USA). To evaluate photothermal stability, a BCuP@G solution (50 µg mL^− 1) was subjected to five laser on/off cycles (808 nm or1064 nm, 1.0 W cm^− 2; on: 6 min, off: 9 min), and the corresponding heating-cooling curves were monitored.

Enzymatic activity of BCuP@G

BCuP@G (60 µg mL^− 1) was incubated in phosphate-buffered saline (PBS) with or without glucose (10 mM) at 25 °C or 45 °C under continuous stirring. At specified time intervals (0, 15, 30, 45, 60, 75, 90, 120, 150, and 180 min), aliquots were obtained and centrifuged, and the absorbance of the supernatant at 560 nm was measured using a commercial hydrogen peroxide detection kit to quantify H₂O₂ concentrations. The pH of each sample was concurrently measured with a calibrated pH meter (PHS-2 F, Leici, China).

GSH depletion in vitro

The depletion of GSH over time was evaluated using UV-vis spectroscopy. BCuP@G at varying concentrations (0, 6.25, 12.5, 25, and 50 µg mL^− 1) was incubated with 0.5 mM GSH at ambient temperature. At predetermined time points (0, 15, 30, 45, 60, 75, and 90 min), aliquots were withdrawn and reacted with 25 µL of 5 mg mL^− 1 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) for 5 min. The absorbance of the resulting mixture was measured using a UV-vis spectrophotometer to assess residual GSH levels.

·OH generation in vitro

The catalytic activity of BCuP@G for ·OH generation was assessed by incubating different formulations with glucose (10 mM) and H₂O₂ (1 mM), followed by methylene blue (MB) degradation analysis. For the laser-treated groups, the mixtures were exposed to 1064 nm laser irradiation (1.0 W cm^− 2) for 5 min at 0, 1, and 2 h post-incubation. The degradation of MB was quantified by measuring absorbance changes using a UV-vis spectrophotometer. Additionally, ·OH production was further verified via electron spin resonance (ESR) spectroscopy (A300, Bruker, USA) using 5,5′-dimethyl-1-pyrroline N-oxide as a spin-trapping agent.

Cell viability assays

The cytotoxicity of BCuP@G was evaluated using the CCK-8 assay. 4T1 or MCF-7 cells were seeded into 96-well plates at a density of 5 × 10³ cells per well and incubated overnight at 37 °C in a humidified incubator with 5% CO₂. The culture medium was then replaced with fresh medium containing varying concentrations of BCuP (0, 6.25, 12.5, 25, 50, 100, 200, and 400 µg mL^–1) or BCuP@G (0, 30, 40, 50, 60, 70, and 80 µg mL^–1). After 24 h of incubation, cell viability was determined using the CCK-8 assay according to the manufacturer’s instructions.

To assess laser-enhanced cytotoxicity, 4T1 or MCF-7 cells were first incubated with free medium, BCuP, or BCuP@G for 4 h, followed by 1064 nm laser irradiation (1.0 W cm^–2) for 5 min. Cells were then cultured for an additional 24 h. Afterward, the cells were washed with PBS, and cell viability was measured using the CCK-8 assay. For apoptosis analysis, 4T1 cells were subjected to identical conditions, with or without co-incubation with the ferroptosis inhibitor Liproxstatin-1 (Lip-1, 50 nmol/L), and then stained with Annexin V-FITC and propidium iodide (PI) following the manufacturer’s protocol. The stained cells were washed with PBS and immediately analyzed using flow cytometry (Celesta, BD Biosciences, USA).

Intracellular GSH, ROS, LPO, Fe²⁺, and MDA level assessment

4T1 cells (1.5 × 10⁵ cells per dish/well) were seeded in confocal dishes or 6-well plates and incubated overnight. Subsequently, fresh DMEM containing BCuP or BCuP@G was added to the cells and co-incubated for 12 h. Cells in laser-treated groups were irradiated with a 1064 nm laser (1.0 W cm^–2) for 5 min. For intracellular GSH detection, cells were washed three times with PBS and incubated with ThiolTrackerTM Violet (10 µM) for 30 min. For intracellular ROS detection, the old medium was replaced with fresh medium containing DCFH-DA (10 µM), and the cells were incubated for 30 min. For intracellular LPO detection, cells were incubated with BODIPY581/591-C11 (5 µM) for 30 min. For intracellular Fe²⁺ detection, cells were incubated with FerroOrange (1 µM) for 30 min. Finally, cells were monitored by confocal laser scanning microscopy (CLSM, LSM710, Carl ZEISS, Germany) and flow cytometry. In addition, intracellular malondialdehyde (MDA) levels were quantified following the same treatments using a commercial lipid peroxidation MDA assay kit, according to the manufacturer’s protocol.

Apoptosis and ferroptosis pathway-related indicators

Mitochondrial membrane potential was determined by JC-1 mitochondrial membrane potential assay kit. 4T1 cells (1.5 × 10⁵ cells per dish) were seeded in confocal dishes and incubated overnight. Subsequently, fresh DMEM containing BCuP or BCuP@G was added to the cells and co-incubated for 4 h. For laser-treated groups, cells were irradiated with a 1064 nm laser (1.0 W cm^–2) for 5 min, followed by incubation for an additional 12 h. After treatment, all cells were stained with JC-1 dye for 30 min and imaged using CLSM. To evaluate protein expression of GPX4, cytochrome c (Cyt-c), and cleaved caspase-3 (Cle-Casp3), treated cells were lysed with radioimmunoprecipitation assay lysis buffer to extract intracellular proteins. Next, protein samples were subjected to SDS-PAGE and transferred to PVDF membranes, followed by incubation with anti-GPX4, anti-Cyt-c, or anti-Cle-Casp3 antibodies at 4 °C. Protein bands were visualized using an enhanced chemiluminescence detection kit.

Animal model

Female Balb/c mice (4–6 weeks old) were obtained from Hunan SJA Laboratory Animal Co., Ltd. All animal experiments were approved by the Experimental Ethics Committee of Jiujiang University (JJU20250115). To establish the orthotopic breast cancer model, 100 µL of 4T1 cell suspension (1 × 10⁷ cells mL^–1) was injected into the mammary fat pad beneath the fourth mammary gland on the left side of each mouse.

In vivo fluorescence imaging and biological distribution study

4T1 tumor-bearing mice were intravenously injected with either free Cy5 or Cy5-labeled BCuP@G (^Cy5BCuP@G) at an equivalent Cy5 dose of 1.5 mg kg^–1. At predetermined time points (3, 6, 9, 12, and 24 h) post-injection, the mice were imaged using an In-Vivo Imaging System (IVIS Spectrum, PerkinElmer, USA) to monitor biodistribution. At the end of the imaging period, all mice were euthanized, and tumors along with major organs (heart, liver, spleen, lung, and kidney) were harvested for ex vivo fluorescence imaging.

In vivo antitumor therapy and immune response

When the tumor volumes of 4T1 tumor-bearing mice reached approximately 80 mm³, the mice were randomly divided into five groups (n = 5): (G1) Control, (G2) BCuP, (G3) BCuP@G, (G4) BCuP@G + L, and (G5) BCuP@G + L + αPD-1. Intravenous injections were administered on day 0 at doses of 17.48 mg kg^–1 for BCuP and 20 mg kg^–1 for BCuP@G. For immune checkpoint blockade therapy, αPD-1 (100 µg) was administered intraperitoneally on days 1, 4, and 7. For groups requiring laser, a 1064 nm laser (1.0 W cm^–2) was exposed to tumor tissues for 5 min at 12 h post-injection. Body weights and tumor volumes were recorded every two days over a 16-day period. Tumor volumes were calculated using the formula: Tumor volume = 1/2 × (tumor width)² × (tumor length). On day 16, tumors were excised for histological and immunofluorescence analysis, including hematoxylin and eosin (H&E), Ki67, DCFH-DA, and GPX4 staining. Major organs (heart, liver, spleen, lung, and kidney) were extracted for H&E staining to evaluate systemic toxicity. Meanwhile, blood samples were collected for routine hematology and serum biochemical analysis.

Following euthanasia, 4T1 tumors were excised, minced, and homogenized in PBS to obtain single-cell suspensions via filtration. For macrophage phenotype analysis, cell suspensions were stained with PE anti-CD11b and FITC anti-CD86 (M1 marker), or PE anti-CD11b and FITC anti-CD206 (M2 marker) antibodies. T cell activation was assessed by staining with APC anti-CD3, PE anti-CD8a, BV421 anti-GmzB, Percp-Cy5.5 anti-IFN-γ, and FITC anti-CD4 antibodies. Regulatory T (Treg) cells were identified using APC anti-CD3, FITC anti-CD4, and PE anti-Foxp3 antibodies. Myeloid-derived suppressor cells (MDSCs) were stained with PE anti-Ly6C and APC anti-Ly6G. All samples were analyzed using flow cytometry. In addition, serum sample were collected, and the levels of cytokines including IL-6, IL-12, and IFN-γ were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer’s instructions.

Statistical analysis

All data were presented as mean ± standard deviation (SD). For statistical comparisons, one-way analysis of variance (ANOVA) followed by Tukey’s Honestly Significant Difference (HSD) post hoc test was applied. Statistical significance was defined as follows: ***p < 0.001, **p < 0.01, *p < 0.05.

Results and discussion

Preparation and characterization of BCuP@G

BSA-stabilized polypyrrole (BCuP) nanoparticles were synthesized using a moderate protein-assisted oxidative polymerization strategy, with BSA functioning as a stabilizer and CuCl₂ serving as both the oxidant and copper donor. Transmission electron microscopy (TEM) images revealed that the resulting BCuP had a nearly spherical morphology with an average diameter exceeding 25 nm (Fig. 1A). High-resolution TEM and element mapping images further confirmed the homogeneous distribution of Cu, C, N, and O elements throughout the nanostructure (Fig. 1B). Structurally, the successful doping of Cu ions was verified by X-ray photoelectron spectroscopy (XPS), which displayed distinct Cu 2p peaks for both Cu⁺ and Cu²⁺ oxidation states (Fig. 1D and Fig. S1). Specifically, peaks at 932.3 and 934.5 eV were attributed to Cu⁺ 2p_3/2 and Cu²⁺ 2p_3/2, while peaks at 952.4 and 954.3 eV corresponded to Cu⁺ 2p_1/2 and Cu²⁺ 2p_1/2, respectively, indicating a Cu⁺/Cu²⁺ ratio of approximately 2:5. The existence of multivalent Cu species was critical for enabling redox cycling and Fenton-like catalytic activity. To confirm protein incorporation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was then conducted. The electrophoretic profile of BCuP showed a distinct band pattern similar to that of native BSA, suggesting the effective integration of BSA into the BCuP framework (Fig. 1C). These findings together indicated the successful construction of BCuP with defined morphology, stable protein incorporation, and Cu⁺/Cu²⁺ redox centers, suitable for subsequent catalytic and biological applications.

Fig. 1.

Preparation and characterization of BCuP@G. (A) TEM image of BCuP, scale bar: 100 nm. (B) High-resolution TEM and element mapping images of BCuP, scale bar: 200 nm. (C) SDS-PAGE analysis of BSA and BCuP. (D) High-resolution XPS spectra of Cu 2p in BCuP. (E) FTIR spectra of BCuP and BCuP@G. (F) TGA curves of BCuP and BCuP@G with different GOx feed ratios

To endow BCuP with enzyme-enhanced catalytic activity, GOx was next covalently conjugated to the BCuP surface through a classical EDC/NHS-mediated amidation reaction, resulting in the final construct, BCuP@G. Fig. S2 showed that GOx modification had little effect on the morphology or TEM size of nanoparticles. However, dynamic light scattering (DLS) analysis indicated an increase in hydrodynamic diameter from ~ 43 nm for BCuP to ~ 79 nm for BCuP@G, along with a decrease in zeta potential from 2.81 ± 0.37 mV to − 7.22 ± 3.57 mV, corroborating successful surface modification with GOx (Fig. S3). Moreover, BCuP@G exhibited similar particle size, polydispersity index, and zeta potential during 7 days of co-incubation with serum-containing medium, confirming its favorable colloidal stability (Fig. S4). Fourier transform infrared (FTIR) spectra was further employed to examine the structural composition. Both BCuP and BCuP@G displayed characteristic peaks at 1186 and 1457 cm^− 1, corresponding to the stretching vibrations of the pyrrole ring and C-N bonds in the traditional polypyrrole framework, which confirmed the successful oxidative polymerization of pyrrole under CuCl₂ initiation (Fig. 1E). Furthermore, amide I (1660 cm^− 1) and amide II (1539 cm^− 1) bands characteristic of protein secondary structures were observed in both samples, verifying the structural integrity of BSA post-polymerization and successful GOx conjugation. To quantify the GOx loading capacity, BCuP was incubated with varying mass ratios of GOx (BCuP: GOx = 10:1, 5:1, and 2:1), while maintaining all other parameters constant. Thermogravimetric analysis (TGA) indicated that the corresponding GOx loading capacities were 12.6%, 16.2%, and 18.3% (Fig. 1F).

Enzyme-enhanced catalytic activity and underlying mechanism

Considering that polypyrrole is a commonly used photothermal agent, the optical and photothermal properties of BCuP@G were first evaluated [37]. As shown in the UV-vis-NIR absorption spectra, BCuP@G aqueous solution exhibited broad and strong optical absorbance from 300 to 1100 nm, with absorbance intensity increasing proportionally with concentration (Fig. 2A and Fig. S5). Afterward, the photothermal conversion and catalytic performance of BCuP@G were investigated. As shown in Fig. 2B and Fig. S6,S7, a rapid and significant temperature elevation was achieved by regulating the concentration of BCuP@G or the power density of the laser irradiation (either 808 nm or1064 nm). Specifically, the temperature change (ΔT) of BCuP@G solution at 100 µg mL^{− 1} rapidly rose to 58.5 °C after 5 min of 1064 nm laser (1 W cm^{− 2}) irradiation. The photothermal effect remained highly stable over five consecutive on/off irradiation cycles, demonstrating excellent photothermal stability (Fig. 2C). Furthermore, based on the thermal decay profile and heat-transfer time constant, the photothermal-conversion efficiency of BCuP@G at 1064 nm was calculated to be 47.68%, highlighting its efficient light-to-heat transformation (Fig. 2D).

Fig. 2.

The catalytic performance of BCuP@G. (A) UV-vis absorption spectra of BCuP@G at various concentrations. (B) The photothermal profiles of BCuP@G at various concentrations upon 1064 nm laser (1 W cm^− 2) irradiation. (C) Photothermal stability of BCuP@G under five successive laser on/off cycles (1064 nm, 1 W cm^− 2). (D) Linear fitting of −lnθ versus time during the cooling period. (E) pH variation and (F) H₂O₂ generation after different treatments. (G) GSH depletion caused by BCuP@G at various concentrations. (H) Time-dependent GSH depletion by BCuP@G at 50 µg mL^–1. (I) MB degradation by ·OH generation catalyzed by different treatments. (J) Time-dependent MB degradation by BCuP@G + L. (K) ESR spectra of the ·OH generation after various treatments. (L) Diagram illustrating the cascade catalytic mechanism of BCuP@G upon 1064 nm laser irradiation

Encouraged by the favorable photothermal conversion performance of BCuP@G, we investigated its enzymatic activity at varying temperatures, both in the presence and absence of glucose. As shown in Fig. 2E, F, co-incubation of BCuP@G with glucose led to a time-dependent decrease in pH and a concurrent increase in H₂O₂ production, which was further amplified at elevated temperatures, suggesting that GOx-mediated enzymatic reactions could effectively remodel TME. Benefitting from the embedded Cu⁺/Cu²⁺ redox centers in BCuP@G mentioned above, we subsequently evaluated its enzyme-enhanced catalytic activity using DTNB and MB as probes for GSH depletion and ·OH generation, respectively. A rapid and concentration-dependent depletion of GSH was observed as a result of the redox reaction between GSH and Cu²⁺ redox centers within the BCuP@G nanostructure (Fig. 2G, H and Fig. S8). In parallel, in the presence of glucose (10 mM) and H₂O₂ (1 mM), Cu⁺ redox centers within the BCuP nanostructure catalyzed the Fenton-like decomposition of H₂O₂ into ·OH and Cu²⁺ (Fig. 2I and Fig. S9). Comparatively, increased ·OH generation was found in the BCuP@G group, attributed to continuous replenishment of H₂O₂ through GOx-mediated enzymatic reactions. Treatment with BCuP@G coupled with 1064 nm laser irradiation resulted in a peak in ·OH generation, demonstrating the benefit of mild hyperthermia in accelerating both enzymatic and Fenton-like catalytic kinetics (Fig. 2J). Meanwhile, ESR spectroscopy further validated ·OH production, displaying consistent results with MB degradation assays (Fig. 2K). Based on those findings, an underlying cascade catalytic mechanism for BCuP@G upon 1064 nm laser irradiation was proposed and illustrated in Fig. 2L. The embedded Cu⁺/Cu²⁺ redox centers within the BCuP@G nanostructure facilitated cyclic ·OH generation and GSH depletion, while GOx continuously catalyzed H₂O₂ synthesis and acidified the local microenvironment. Simultaneously, laser-activated hyperthermia accelerated both enzymatic and Fenton-like kinetics, resulting in persistent redox imbalance, LPO accumulation, and ultimately, apoptosis and ferroptosis.

In vitro therapeutic efficacy and underlying antitumor molecular mechanism

To assess cellular internalization, 4T1 cells were incubated with Cy5-labeled BCuP@G and imaged via CLSM at various time points. As shown in Fig. S10, intracellular fluorescence intensity progressively increased with prolonged incubation, confirming time-dependent cellular uptake of BCuP@G. The cytotoxicity of BCuP and BCuP@G was next evaluated in 4T1 cells using the cell counting kit-8 assay. BCuP treatment led to a concentration-dependent reduction in cell viability, decreasing to 78.34% and 65.30% at 200 and 400 µg mL^− 1, respectively, which was attributed to the oxidative stress induced by the embedded Cu⁺/Cu²⁺ active centers (Fig. 3A). Notably, BCuP@G exhibited significantly enhanced cytotoxicity, owing to GOx-mediated enzymatic activity that elevated intracellular H₂O₂ levels and exacerbated redox imbalance (Fig. 3B). This effect was further amplified under 1064 nm laser irradiation. As illustrated in Fig. 3C, laser-actived hyperthermia markedly boosted the therapeutic efficacy of BCuP@G, implying that the synergistic strategy of oxidative stress and hyperthermia was more effective than any single therapy. These cytotoxicity results were also validated in MCF-7 human breast cancer cells (Fig. S11). Subsequently, the live/dead staining assay was performed to visually validate these findings. The images in Fig. S12 demonstrated that BCuP@G + L induced the most extensive damage and mortality in 4T1 cells.

Fig. 3.

In vitro therapeutic efficacy of laser-enhanced BCuP@G against breast cancer. Cytotoxicity of (A) BCuP and (B) BCuP@G at various concentrations toward 4T1 cells without laser irradiation. (C) Relative viability of 4T1 cells after various treatments. (D) Representative CLSM images and (E) flow cytometry analysis of GSH and ·OH levels in 4T1 cells after different treatments. (F) Flow cytometry analysis of LPO levels in 4T1 cells after different treatments. (G) Quantification of intracellular MDA levels in 4T1 cells after different treatments. (H) Western blot analysis of GPX4, Cyt-C, and Cle-Casp3 expression in 4T1 cells after different treatments. (I) Flow cytometry of cell apoptosis after different treatments in the presence or absence of ferroptosis inhibitor Lip-1. Data are expressed as means ± SD (n = 4). Statistical significances were calculated via one-way ANOVA (with Tukey’s HSD post hoc test). *p < 0.05, **p < 0.01, and ***p < 0.001

To investigate the enzyme-enhanced catalytic activity of BCuP@G at the cellular level, 2’,7’-dichlorofluorescein diacetate (DCFH-DA) and ThiolTrackerTM Violet probes were employed to detect ROS and GSH changes in 4T1 cells. Compared to BCuP, BCuP@G treatment resulted in a markedly elevated ROS level and reduced GSH content within 4T1 cells, as visualized by CLSM and quantified by flow cytometry (Fig. 3D, E). These redox alterations were further amplified upon 1064 nm laser irradiation, confirming the synergistic enhancement of catalytic activity by hyperthermia stimulation. More importantly, this redox imbalance simultaneously caused Fe²⁺ increase, LPO accumulation, and MDA production, indicative of ferroptotic cell death (Fig. 3F, G and Fig. S13). Given the known interplay between oxidative stress and mitochondrial dysfunction, we further evaluated mitochondrial membrane potential using JC-1 staining. As shown in Fig. S14, the green/red fluorescence ratio increased significantly following BCuP@G treatment, with a further rise observed upon laser irradiation, indicating substantial mitochondrial membrane depolarization. Western blot analysis demonstrated diminished GPX4 expression, elevated Cyt-c release, and increased Cle-Casp3 expression in the BCuP@G + L group, signifying the simultaneous activation of the mitochondrial apoptosis pathway and ferroptosis (Fig. 3H). Annexin V-FITC/PI staining further supported these findings, showing 12.0% ferroptosis, 66.84% apoptosis, and 0.57% necrosis in the BCuP@G + L group (Fig. 3I).

To further elucidate the underlying antitumor molecular mechanism of BCuP@G, transcriptome sequencing was conducted on 4T1 cells following BCuP@G + L treatment. A total of 311 differentially expressed genes (DEGs) were identified in comparison to the control group, comprising 223 upregulated and 88 downregulated genes (Fig. 4A). Gene ontology (GO) enrichment analysis revealed that these DEGs were significantly associated with pathways including response to copper ion, cellular response to heat, MAPK cascade, and apoptotic process, confirming the cellular oxidative imbalance induced by the Cu⁺/Cu²⁺ redox centers of BCuP@G under hyperthermic conditions (Fig. S15). Additionally, kyoto encyclopedia of genes and genomes (KEGG) analysis indicated prominent activation of ferroptosis, apoptosis, and necroptosis signaling pathways in the BCuP@G + L group, with notable alterations in ferroptosis-related genes such as Slc7a11 and Hmox1 (Fig. 4B, C). Gene set enrichment analysis (GSEA) further corroborated the enrichment of these death-related signaling pathways (Fig. 4D).

Fig. 4.

Transcriptome sequencing analysis of 4T1 cells after various treatments. (A) Volcanic plot revealing DEGs between the BCuP@G + L and control groups. (B) KEGG pathway enrichment analysis in the BCuP@G + L group versus control group. (C) Enriched chord diagram of the KEGG pathways. (D) GSEA for the altered gene sets in the BCuP@G + L group versus control group

In vivo biological biodistribution

To evaluate the in vivo biodistribution and tumor-targeting capability of BCuP@G, 4T1 tumor-bearing mice were intravenously (i.v.) injected with either free Cy5 or Cy5-labeled BCuP@G (^Cy5BCuP@G). As shown in Fig. 5A, B, ^Cy5BCuP@G exhibited progressively increased fluorescence intensity at the tumor site, peaking at 6 h and remaining substantial intensity up to 24 h post-injection, indicating efficient tumor accumulation and retention. Conversely, free Cy5 displayed weak and rapidly diminishing tumor accumulation. Ex vivo imaging of major organs and tumors at 24 h further confirmed the predominant accumulation of ^Cy5BCuP@G within tumor tissues, highlighting its favorable tumor-targeting and retention properties (Fig. 5C, D). Next, the photothermal conversion capacity of BCuP@G in vivo was assessed using thermal imaging. Upon 1064 nm laser irradiation (1.0 W cm^− 2, 5 min), the temperature at the tumor site in BCuP@G-treated mice increased rapidly and reached 53.3 °C (Fig. 5E, F). In contrast, mice injected with PBS showed negligible temperature elevation under identical conditions.

Fig. 5.

In vivo biodistribution of BCuP@G. (A) Representative fluorescence images and (B) corresponding fluorescence intensity of tumors in mice at various time points following i.v. injection of free Cy5 or ^Cy5BCuP@G. (C) Representative fluorescence images and (D) corresponding fluorescence intensity of dissected organs and tumors collected 24 h post-injection. (E) Tumor temperature changes and (F) corresponding thermal images in mice following i.v. injection of PBS or BCuP@G with 1064 nm laser irradiation (1 W cm^− 2, 5 min). Data are expressed as means ± SD (n = 3). Statistical significances were calculated via one-way ANOVA (with Tukey’s HSD post hoc test). *p < 0.05, **p < 0.01, and ***p < 0.001

In vivo antitumor therapy and immune response

To further investigate therapeutic efficacy of BCuP@G in vivo, 4T1 tumor-bearing mice were randomly assigned to five groups (n = 5): G1: Control, G2: BCuP, G3: BCuP@G, G4: BCuP@G + L, and G5: BCuP@G + L + αPD-1 (Fig. 6A). BCuP or BCuP@G were administered i.v. on day 0, followed by 1064 nm laser irradiation 12 h post-injection. For immunotherapy, αPD-1 was administered intraperitoneally on days 1, 4, and 7. Over a 16-day observation period, both BCuP and BCuP@G groups exhibited different degrees of tumor inhibition due to their differential catalytic characteristics (Fig. 6B, C). Comparatively, the BCuP@G + L and BCuP@G + L + αPD-1 groups achieved almost complete tumor eradication, highlighting the advantages of hyperthermia-accelerated and enzyme-enhanced catalytic therapy. Notably, no discernible differences in body weight were observed among those groups, suggesting minimal systemic toxicity (Fig. 6D). Post-treatment, the analysis of tumor weight further validated the superior antitumor efficacy of BCuP@G + L (Fig. 6E). Moreover, hematoxylin and eosin (H&E) staining showed that BCuP@G induced slight tumor cell death and apoptosis, whereas BCuP@G + L and BCuP@G + L + αPD-1 caused enhanced apoptosis and necrosis as well as inhibited proliferation of 4T1 tumor cells (Fig. 6F and Fig. S16). The ROS generation and GPX4 downregulation were also identified in the Cu-containing groups embedded with Cu⁺/Cu²⁺ redox centers, and further amplified by GOx encapsulation and laser irradiation. Importantly, histological examination of major organs confirmed the absence of pathological abnormalities, supporting the favorable biocompatibility of BCuP@G (Fig. S17).

Fig. 6.

In vivo antitumor therapy. (A) Schematic illustration of the timeline for BCuP@G-based antitumor therapy. (B) Individual tumor growth curves in each treatment group. (C) Tumor volume and (D) body weight during the treatment period. (E) Tumor weight of mice after the indicated treatments. (F) H&E, Ki67, DCFH-DA, and GPX4 staining images of tumor sections after various treatments, scale bars: 200, 100, 50, and 50 μm, respectively. Data are expressed as means ± SD (n = 5). Statistical significances were calculated via one-way ANOVA (with Tukey’s HSD post hoc test). *p < 0.05, **p < 0.01, and ***p < 0.001

Given the notable antitumor effects, we next explored the underlying antitumor immunity activation triggered by BCuP@G in combination with αPD-1. Compared with the control group, tumor tissues from the BCuP@G + L and BCuP@G + L + αPD-1 groups exhibited a significantly elevated ratio of M1-like TAMs, suggesting hyperthermia-enhanced immune modulation (Fig. 7A, B,E). This immune remodeling was accompanied by alterations in tumor-infiltrating T cell populations. As shown in Fig. 7C, D and Fig. S18, BCuP@G + L + αPD-1 exhibited higher proportion of intratumoral CD4⁺ and CD8⁺ T cells than the BCuP@G + L group, along with reduced proportions of Treg and granulocytic myeloid-derived suppressor cells (G-MDSCs), confirming potent antitumor immune response mediated by αPD-1. Moreover, BCuP@G + L + αPD-1 facilitated the secretion of cytokines IFN-γ and GzmB by CD8⁺ T cells within tumors, with levels higher than those in the other treatment groups. Subsequently, serum levels of pro-inflammatory cytokines IL-6, IL-12, and IFN-γ were elevated in the combination group (Fig. 7F), indicating a robust systemic immune activation. BCuP@G also exhibited excellent hemocompatibility, as evidenced by minimal hemolysis and normal complete blood counts (Fig. S19,S20). Meanwhile, biochemical analysis revealed no significant differences in liver or kidney function indicators among the treatment groups (Fig. 7G). Collectively, these results demonstrated that BCuP@G, when combined with αPD-1 and laser irradiation, effectively induced ferroptosis and immunogenic cell death, reprogramed the immunosuppressive TME, and potentiated antitumor immunity.

Fig. 7.

In vivo antitumor immunity. Flow cytometric analysis of tumor-infiltrating (A) M2-like TAMs, (B) M1-like TAMs, (C) CD8⁺ T cells, and (D) Treg cells after different treatments. (E) The corresponding quantification of tumor-infiltrating TAMs, CD8⁺ T cells, IFN-γ⁺CD8⁺ T cells, GzmB⁺CD8⁺ T cells, and Treg cells after different treatments. (F) ELISA analysis of IL-6, IL-12, and IFN-γ after different treatments. (G) Blood biochemical analysis of the mice after different treatments. Data are expressed as means ± SD (n = 3). Statistical significances were calculated via one-way ANOVA (with Tukey’s HSD post hoc test). *p < 0.05, **p < 0.01, and ***p < 0.001

Conclusion

In summary, we have developed a multifunctional nanocatalyst (BCuP@G) by integrating BSA-stabilized polypyrrole with multivalent Cu ions and covalently conjugated GOx via a mild, protein-assisted polymerization strategy. BCuP@G harnessed the redox activity of embedded Cu⁺/Cu²⁺ centers for GSH depletion and ·OH generation, while GOx continuously oxidized glucose to supply H₂O₂ and acidify TME, sustaining and accelerating cascade catalytic reactions. Photothermal activation under 1064 nm laser irradiation further amplified both enzymatic and cascade catalytic kinetics, leading to extensive oxidative damage, mitochondrial dysfunction, and concurrent induction of ferroptosis and apoptosis. Beyond direct tumor cytotoxicity, BCuP@G remodeled the immunosuppressive microenvironment by reprogramming TAMs toward the M1 phenotype, suppressing Treg and G-MDSCs, and promoting cytotoxic T cell infiltration. The combination of BCuP@G with anti-PD-1 therapy elicited a strong systemic immune response and achieved near-complete tumor eradication with minimal systemic toxicity. Altogether, this work not only introduces a rationally designed enzyme-enhanced nanocatalyst but also provides valuable insights into ferroptosis-driven immune activation, offering a promising strategy for next-generation cancer nanomedicine.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

P.Y. and S.C.Y. contributed equally to this work. P.Y. conducted the investigation and revised the manuscript. S.C.Y. prepared materials, conducted experiments, curated data, performed formal analysis, and wrote the original draft. X.P.Y. and Z.H.Z. made adjustments to the figures and participated in discussions. Y.X.C., P.P.X., Y.H.D., and X.H.Z. assisted with in vivo experiments and checked the formal analysis. J.T.L. checked the formal analysis. P.Y., W.W.Z, and J.L. supervised the research and provided funding. All authors reviewed the manuscript.

Funding

This research was supported by the Natural Science Foundation of China (82360162, 82160371), Young Elite Scientists Sponsorship Program by JXAST (2023QT05), Natural Science Foundation in Jiangxi Province grant (20224ACB216009), the Jiangxi Province Thousands of Plans (jxsq2023201105), the Hengrui Diabetes Metabolism Research Fund (Z-2017-26-2202-4) and the China Postdoctoral Science Foundation (2024M750950, GZC20230844). The scheme was created with http://biorender.com.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The animal experiments were performed in accordance with the China Animal Protection Law. All animal treatments or procedures were approved by the Experimental Ethics Committee of Jiujiang University.

Consent for publication

The authors approve to publish on Journal of Nanobiotechnology if the manuscript can become acceptable.

Competing interests

The authors declare no competing interests.