A Multiple Enzyme‐Mimetic Platinum–Ruthenium Nanohybrid Prodrug Potentiates Chemo‐/Chemodynamic‐/Immuno‐Therapy of Hypoxic Tumors

ABSTRACT

Hypoxia in the tumor microenvironment (TME) is a hallmark of solid tumors and is tightly associated with the development of chemoresistance and immunosuppression, severely compromising the efficacy of mainstream clinical oncological treatments. Platinum‐based metallodrugs, especially oxaliplatin (Oxa), serve as first‐line chemotherapeutics in clinical practice. However, their clinical utility is greatly restricted by acquired drug resistance, insufficient tumor accumulation, and weak immunostimulatory capacity. Herein, we synthesize a platinum–ruthenium nanohybrid prodrug (denoted as PR) via self‐assembly, which integrates Oxa PR and ruthenium ions for synergistic chemo‐/chemodynamic‐/immunotherapy of hypoxic tumors. The PR nanohybrid possesses intrinsic multi‐enzyme activities (catalase, peroxidase, and glutathione peroxidase), enabling efficient oxygen generation, hydroxyl radical production, and glutathione depletion. These cascading events enhance chemosensitivity, trigger robust immunogenic cell death, and activate the cGAS‐STING signaling pathway. Furthermore, nanocatalytic modulation of the hypoxic TME alleviates hypoxia‐driven immunosuppression, downregulates PD‐L1 expression on cancer cells, and reinforces antitumor immune responses. In vitro and in vivo investigations demonstrate that PR nanohybrid exhibits superior anticancer efficacy over free Oxa and displays promising potential in combination with PD‐1 blockade therapy. These findings highlight the PR nanohybrid as a versatile TME‐modulating platform for hypoxic tumor treatment, offering a novel strategy to advance platinum‐based combination cancer therapy.

A nanohybrid prodrug (PR) with multi‐enzyme activities was synthesized to enhance chemo‐/chemodynamic‐/immuno‐therapy of hypoxic tumors. By generating oxygen and hydroxyl radicals while depleting glutathione, PR amplifies immunogenic cell death, activates the cGAS‐STING pathway, and modulates the immunosuppressive tumor microenvironment. This strategy achieves superior anticancer efficacy and synergizes with PD‐1 antibody therapy.

1. Introduction

Oxaliplatin (Oxa), a mainstream platinum chemotherapeutic drug for clinical use, suffers from prominent drawbacks including acquired drug resistance, poor tumor accumulation efficiency, and weak immunostimulatory capability [1, 2, 3, 4]. Elevated intracellular glutathione (GSH) further inactivates platinum drugs [5, 6, 7], while cancer stem cells (CSCs) also lead to tumor chemoresistance and postsurgical recurrence [8, 9, 10]. Moreover, the clinical performance of Oxa is further limited by inefficient tumor accumulation [11]. In contrast to conventional platinum(II) complexes, platinum(IV) prodrugs feature an octahedral coordination geometry with superior inertness and stability, which reduces undesired displacement reactions with biomolecules, minimizes off‐target toxicity, and prevents premature deactivation by proteins [12, 13, 14, 15]. Notably, platinum(IV) prodrugs can be selectively reduced to cytotoxic platinum(II) species in the reductive tumor microenvironment (TME). The presence of axial ligands also facilitates conjugation with diverse delivery systems, rendering platinum(IV) prodrugs a promising alternative to traditional platinum(II) drugs.

High intracellular glutathione (GSH) further inactivates platinum drugs, while cancer stem cells (CSCs) also lead to tumor recurrence. Additionally, the hypoxic TME upregulates HIF‐1α and PD‐L1, induces M2‐type macrophage polarization, and greatly hinders the outcome of immunotherapy. Platinum(IV) prodrugs with better stability can be selectively activated in tumor tissues, yet they still fail to reverse hypoxia and immune suppression.

Cancer immunotherapy has revolutionized clinical oncology by harnessing the host immune system to eradicate tumor cells, inhibit metastasis, and prevent recurrence [16, 17, 18]. Immune checkpoint blockade (ICB) targeting the PD‐1/PD‐L1 axis and chimeric antigen receptor (CAR)‐T cell therapy have achieved remarkable clinical success. However, their efficacy against solid tumors remains unsatisfactory [19, 20, 21]. This limitation is largely attributed to the immunosuppressive TME and the scarcity of tumor‐specific antigens [22, 23, 24]. Therefore, remodeling the immunosuppressive TME to boost antitumor immunity has become a central research focus. Among various strategies, immunogenic cell death (ICD) stands out as an effective approach to trigger antitumor immune responses by releasing tumor‐associated antigens (TAAs) and damage‐associated molecular patterns (DAMPs) [25, 26]. Although chemotherapeutics such as Oxa and doxorubicin can induce ICD [27, 28], their therapeutic outcomes are hampered by intrinsic immunosuppressive side effects.

Rapid proliferation of tumor cells causes excessive oxygen consumption, resulting in tumor hypoxia and aberrant vascularization [29, 30]. Hypoxia upregulates hypoxia‐inducible factor‐1α (HIF‐1α), a master transcription factor that mediates adaptive responses to low oxygen tension [31]. HIF‐1α drives both chemoresistance and immunosuppression, thereby attenuating the efficacy of immunotherapy [32, 33, 34, 35]. Hypoxia promotes the polarization of tumor‐associated macrophages (TAMs) from the pro‐inflammatory M1 phenotype to the immunosuppressive M2 phenotype. M1 macrophages exert antitumor effects by secreting pro‐inflammatory cytokines (e.g., tumor necrosis factor‐α, interleukin (IL)‐6), whereas M2 macrophages suppress immune activation via anti‐inflammatory factors (e.g., IL‐10). Recent studies also reveal that M2 macrophages contribute to tumor chemoresistance [36]. Furthermore, hypoxic TME upregulates PD‐L1 expression on tumor cells and PD‐1 on T cells, impairing cytotoxic T lymphocyte‐mediated tumor lysis and limiting ICB efficacy [37, 38]. Accordingly, strategies that normalize hypoxic TME hold great promise to overcome chemoresistance and enhance antitumor immunity, including oxygen delivery via perfluorocarbons and hydrogen peroxide (H₂O₂) decomposition using catalase (CAT) or CAT‐mimetic nanozymes [39, 40, 41, 42, 43].

Nanomaterial‐based drug delivery systems have emerged as powerful tools in biomedical research, enabling enhanced cellular internalization and preferential tumor accumulation of platinum drugs through passive (enhanced permeability and retention (EPR) effect) or active targeting [44, 45]. However, traditional nanocarriers suffer from drawbacks such as low drug loading, inherent toxicity, and uncontrolled drug release [46, 47]. Self‐assembly based on non‐covalent interactions (drug–drug or drug–metal ion) provides a feasible alternative for drug delivery [48, 49, 50]. Self‐assembled platinum(IV) prodrug (PR) nanosystems show great potential in improving cellular uptake and tumor accumulation. Among non‐platinum metallodrugs, ruthenium‐based complexes have attracted extensive attention owing to their favorable antitumor activity and unique catalytic properties [51, 52, 53]. Moreover, Ru‐based nanomaterials can function as nanozymes with diverse enzymatic activities, potentially enhancing cancer cell lethality through biocatalysis [54, 55, 56]. Despite these advances, the exploration of ruthenium‐coordinated self‐assembled nanodrugs remains largely underexplored.

In this work, we fabricate a multi‐enzyme‐mimetic nanohybrid PR via self‐assembly of platinum(IV) PR, ruthenium ions, polyvinylpyrrolidone (PVP), and Pluronic F127 (PF127) for hypoxic tumor therapy (Scheme 1). Upon GSH‐triggered reduction, PR degrades and releases active Oxa to induce DNA damage and cell death. The multivalent ruthenium ions endow PR with integrated CAT‐, peroxidase (POD)‐, and glutathione peroxidase (GPx)‐like activities, which catalyze H₂O₂ to generate hydroxyl radicals for chemodynamic therapy (CDT). PR reverses the hypoxic TME, depletes GSH, and eliminates CSCs, thereby overcoming Oxa resistance in colon cancer cells. Moreover, PR strongly amplifies Oxa‐induced ICD by promoting the release of TAAs and DAMPs. Mechanistically, PR remodels hypoxia‐induced immunosuppressive TME, downregulates PD‐L1 expression, and activates the cGAS‐STING pathway to enhance the infiltration of antitumor immune cells. RNA‐seq analysis systematically elucidates the antitumor mechanism of PR, involving TME improvement, chemoresistance reversal, and immune activation. Collectively, the multi‐enzyme‐mimetic platinum–ruthenium nanohybrid PR represents a promising multifunctional platform for enhanced hypoxic tumor combination therapy.

SCHEME 1.

Schematic representation of (A) the synthesis process and (B) mechanism of PR nanohybrid. PR nanohybrid with CAT‐, POD‐, and GPx‐ like catalytic capability amplifies tumor efficacy and induces robust antitumor immune responses through synergy therapy as well as modulating hypoxia‐induced immunosuppression TME.

2. Results and Discussion

2.1. Preparation, Characterization, and Enzyme‐Mimetic Activity of PR Nanohybrid

The PR nanohybrid was synthesized via a facile self‐assembly process using Oxa PR, ruthenium ions, PVP, and PF127 [56]. Transmission electron microscopy (TEM) images show that PR presents an irregular granular morphology with diameters of 50–100 nm (Figure 1A), which favors passive tumor accumulation via the EPR effect. Energy‐dispersive X‐ray (EDX) spectroscopy and elemental mapping confirm the homogeneous distribution of C, N, O, Pt, and Ru within the nanoparticles (Figure 1A), verifying the successful incorporation of Oxa PR, ruthenium ions, PVP, and PF127. EDX line scans further validate the coexistence of C, N, Pt, and Ru (Figure S1), consistent with elemental mapping results. Inductively coupled plasma mass spectrometry (ICP‐MS) quantifies the mass fractions of Oxa PR and Ru in PR as 47.0% and 21.8%, respectively, indicating high drug‐loading efficiency. The hydrodynamic diameter of PR was measured to be 154 ± 28 nm with a polydispersity index (PDI) of 0.277 ± 0.06 (Figure S2). PR exhibits excellent stability in water and phosphate‐buffered saline (PBS) over 3 days (Figure 1B). The zeta potential of PR is ‐22.5 ± 0.4 mV, which is conducive to prolonged blood circulation [57]. X‐ray photoelectron spectroscopy (XPS) was employed to analyze the elemental composition and valence states (Figure S3). The Pt 4f peak at 78.5 eV is assigned to Pt⁴⁺ (Figure 1C), confirming the preservation of the platinum(IV) oxidation state in the nanohybrid. The Ru 3d spectrum reveals two pairs of characteristic peaks corresponding to Ru³⁺ (282.3 eV and 285.9 eV) and Ru⁴⁺ (281.5 eV and 284.5 eV), which underpin the multi‐enzyme catalytic activity of PR (Figure 1D) [58]. UV–vis absorption spectroscopy shows a characteristic absorption band of Oxa PR at 250–300 nm (Figure S4), further verifying successful self‐assembly.

FIGURE 1.

(A) TEM image and EDX element mapping of PR nanohybrids. Scale bars are 50 nm. (B) Change of hydrodynamic diameter of PR nanohybrids within 3 days. (C) Pt 4f and (D) Ru 3d XPS spectra of PR nanohybrids. (E) Dissolved oxygen concentrations of aqueous solution under indicated conditions. Unit: µg/mL. (F) Absorption spectra of TMB after incubation with different concentrations of PR nanohybrids containing H₂O₂. The release of Pt (G) and Ru (H) elements from PR in aqueous solution with (+)/without (−) GSH (10 mM). (I) GSH scavenging activity of Oxa prodrugs and PR nanohybrids with various concentrations. (J) Schematic representation of the catalytic oxygen and hydroxyl radical generation, GSH depletion mediated by PR nanohybrids.

The CAT‐like activity of PR was evaluated by monitoring oxygen generation from H₂O₂. PR induces a concentration‐dependent increase in dissolved oxygen, while negligible oxygen production is observed in the control group (Figure 1E). Visible bubble formation is only detected in the mixture of PR and H₂O₂ (Figure S5), confirming the CAT‐mimetic activity. Kinetic analysis yields a Michaelis–Menten constant (K _m) of 5.5×10⁻⁴s⁻¹ (Figure S6). The POD‐like activity was assessed using 3,3′,5,5′‐tetramethylbenzidine (TMB) as a chromogenic substrate. The absorbance intensity of oxidized TMB at 650 nm increases with PR concentration, demonstrating POD‐mimetic activity (Figures 1F and S7). The K _m values of PR for TMB and H₂O₂ are 0.2 mM and 8.0 mM (Figure S8A,B), respectively.

The response of PR to GSH was further investigated. The addition of GSH causes obvious morphological changes and size reduction of PR (Figures S9 and S10), indicating GSH‐triggered degradation. ICP‐MS reveals that GSH significantly accelerates Pt release from PR, with approximately 80% Pt released within 24 h (Figure 1G). Mass spectrometry confirms the release of active Oxa under reductive conditions (Figure S11). In contrast, Ru release is negligible (<5% within 12 h, Figure 1H), suggesting that high intracellular GSH levels can selectively activate Oxa release from PR. Both multivalent metal nanomaterials and platinum(IV) prodrugs are reported to deplete GSH. Spectroscopic analysis shows that PR exhibits stronger GSH‐scavenging capacity than free Oxa PR: 40 µM PR consumes 75.8% of GSH, while the same concentration of Oxa PR only depletes 35.0% of GSH (Figures 1I and S12), confirming the GPx‐like activity of PR. These results demonstrate that PR integrates biomimetic nanocatalysis, GSH depletion, activatable platinum(IV) PR, and biodegradability into a single nanoplatform for cancer therapy (Figure 1J).

2.2. In Vitro Cytotoxicity and Immune Activation

The In vitro antitumor efficacy of PR was quantitatively evaluated. After 48 h incubation, PR exhibits concentration‐dependent cytotoxicity against CT26 colon cancer cells, with superior potency over free Oxa. The half‐maximal inhibitory concentration (IC₅₀) values of PR and Oxa under normoxia are 6 and 13.4 µM, respectively (Figure 2A). Under hypoxic conditions, CT26 cells develop obvious resistance to Oxa, whereas PR maintains high cytotoxicity (IC₅₀: 11.5 µM for PR vs. 17.2 µM for Oxa). Live/dead staining shows a markedly higher proportion of dead cells in the PR group (Figure 2G). Flow cytometry reveals that PR induces a higher apoptosis rate (70.1%) than Oxa (43.5%) at 10 µM (Figure S13).

FIGURE 2.

(A) Cytotoxicity of Oxa and PR nanohybrid on CT26 cells with different concentrations as indicated under normoxia/hypoxia conditions. (B) The expression of HIF‐1α in cells when incubated with Oxa or PR nanohybrids at indicated concentrations under hypoxic conditions. (C) Intracellular GSH level in cells from various treatment groups as measured by the relevant assay kit. (D) Expression levels of cGAS, STING, and p‐STING with indicated treatments as measured by western blot assay. (E) Cytotoxicity of Oxa and PR nanohybrid on Oxa‐resistant CT26 cells with different concentrations as indicated under normoxia/hypoxia conditions. (F) Intracellular Pt content of normal CT26 cells and drug‐resistance CT26 cells was measured after incubation with PR nanohybrids and Oxa for 12 h. (G) Live/dead stained images of CT26 cells under indicated conditions. The scale bar is 50 µm. (H) Oxygen level in CT26 cells stained with Ru(dpp)₃Cl₂ after incubation with PR nanohybrids and Oxa under normoxia/hypoxia conditions. (I) ROS levels of various treatment groups stained with DCF probe. Representative immunofluorescence images of cells after treatment with PBS, Oxa, or PR nanohybrids stained with (J) γ‐H2AX, (K) CRT, (L) HMGB1, and (M) PD‐L1 antibodies, respectively. Scale bars are 20 µm in (H‐M). **indicates p < 0.01, *indicates p < 0.05, and ^nsindicates p > 0.05. Data are expressed as mean ± standard deviation.

The intracellular catalytic activity of PR was verified using an oxygen‐sensitive probe tris(4,7‐diphenyl‐1,10‐phenanthroline)ruthenium(II) dichloride complex (Ru(dpp)₃Cl₂). PR treatment significantly reduces phosphorescence intensity, restoring cellular oxygen levels close to normoxic conditions (Figure 2H), confirming effective hypoxia relief by CAT‐like activity. Western blotting shows that PR downregulates HIF‐1α expression in a concentration‐dependent manner, while Oxa has no obvious effect (Figures 2B and S14). Besides, quantification of intracellular GSH levels through the corresponding kit and according to the reagent manufacturer specifications revealed that PR (20 µM) drastically depletes intracellular GSH from 48.8 µM to 20.6 µM (Figure 2C), whereas Oxa only causes a slight decrease (41.7 µM). Then, reactive oxygen species (ROS) levels were monitored using the fluorescence probe 2',7'‐dichlorodihydrofluorescein diacetate (DCFH‐DA). The result shows that PR significantly elevates intracellular ROS levels (Figure 2I). Similarly, the results of the flow cytometry assay revealed that PR led to an increase in the level of hydroxyl radicals within the cells and was more effective than Oxa (Figure S15), validating its POD‐like activity. Considering DNA as a primary target of Oxa, DNA damage was evaluated via immunofluorescence staining of γ‐H2AX. A discernible increase in red fluorescence signal was observed in both PR and Oxa groups compared to the PBS group (Figure 2J), signifying DNA damage, with the PR group exhibiting the most pronounced effect. Besides, wound healing and Trans‐well experiments were performed to evaluate whether PR allow suppress the metastasis of CT26 cells. PR obviously inhibited the migration of CT26 cells more effectively than Oxa at the same concentration, as shown in wound healing assay (Figure S16). Then, Trans‐well experiment further confirmed that PR exhibited better ability to restrict the migration of CT26 cells (Figure S17).

Oxaliplatin (Oxa) induced the translocation of calreticulin (CRT), an immunogenic biomarker, from the endoplasmic reticulum to the cell membrane surface, and PR nanohybrid potentiated this process (Figures 2K and S18A). Similarly, Oxa triggered the release of high‐mobility group box 1 (HMGB1), which was further amplified upon treatment with PR nanohybrid (Figures 2L and S18B). Intracellular ATP levels were also reduced following treatment with both PR nanohybrid and Oxa (Figure S19), decreasing from 4.4 ± 0.2 µM in the control group to 2.3 ± 0.3 µM and 2.8 ± 0.1 µM, respectively. These data collectively indicate that PR nanohybrid is capable of amplifying the ICD induced by Oxa. Extensive literature has documented the activation of the cGAS‐STING pathway following exposure to double‐stranded DNA, culminating in the release of interferon‐β (IFN‐β) and the initiation of antitumor immune responses. As presented in Figures 2D and S20, the expression of cGAS and p‐STING was increased 1.9‐fold and 3.9‐fold, respectively, under PR treatment (20 µM), compared to the 1.0‐fold and 1.8‐fold changes observed in Oxa‐treated cells. Subsequent enzyme‐linked immunosorbent assay (ELISA) analysis of culture medium supernatants showed that Oxa‐ and PR‐treated cells secreted approximately 2.2‐fold and 5.7‐fold increases in IFN‐β at a concentration of 20 µM (Figure S21), respectively, relative to PBS‐treated cells. Furthermore, the expression levels of PD‐L1, a key modulator of immunotherapy efficacy, were assessed. As illustrated in Figure 2M, PR nanohybrid treatment upregulated membrane‐localized PD‐L1 in CT26 cells, whereas minimal changes were observed following Oxa treatment. These cumulative results suggest that PR exhibits a superior therapeutic effect and induces a greater degree of ICD in CT26 cells compared to Oxa.

Oxaliplatin (Oxa)‐resistant cell lines were generated by continuous administration of Oxa to CT26 cells for 2 months. The therapeutic effect of PR and Oxa on Oxa‐resistant cell lines was further evaluated. Notably, at equivalent concentrations, PR exhibited higher treatment efficiency than Oxa. The IC₅₀ values of PR and Oxa against Oxa‐resistant CT26 cells were 14.6 µM and 27.2 µM under normoxia, and 23.6 µM and 36.8 µM under hypoxia, respectively (Figure 2E). Under a concentration of 50 µM, PR treatment resulted in the death of 86.4% of colon cancer cells, whereas only 57.3% of cells were killed following treatment with the same amount of Oxa. Notably, both CT26 and its drug‐resistant counterpart suggested a substantially higher degree of PR internalization compared to equivalent dosages of Oxa (Figure 2F). Specifically, following a 12‐h incubation period, quantification of Pt levels in PR‐treated CT26 cells yielded a concentration of 540.5 ng/10⁶ cells, whereas Oxa‐treated CT26 cells exhibited a concentration of only 31.3 ng/10⁶ cells. Similarly, in drug‐resistant cells, PR treatment resulted in Pt levels of 252.8 ng/10⁶ cells, while Oxa treatment resulted in a comparatively lower concentration of 20.6 ng/10⁶ cells. These data collectively demonstrate that PR possesses the capacity to reverse drug resistance in colon cancer.

2.3. Inhibition of CSCs

Cancer stem cells (CSCs) are critical mediators of tumor chemoresistance and recurrence. The effect of PR on CT26‐derived CSCs was investigated. Flow cytometric analysis (Figure 3A,B), utilizing the CSC biomarkers CD133, revealed that PR, at a concentration of 20 µM, significantly reduced the proportion of CSCs (8.5%) compared to both the PBS group (41.9%) and cells treated with Oxa at the same concentration (22.8%). Similarly, PR could down‐regulate the expression of CD44 and its effect was more significant than that of Oxa. Analysis of mammosphere cultures demonstrated that pre‐treatment with PR significantly inhibited mammosphere formation, both in terms of size and number, in contrast to PBS and Oxa treatment groups (Figure 3C,D). This observation was further supported by a cell viability test, which revealed that the survival rate of the mammospheres after PR treatment was significantly reduced (Figure 3E,F). Specifically, at a concentration of 40 µM, the survival rate of Oxa‐treated mammospheres was approximately 35%, while the survival rate of PR‐treated mammospheres was less than 10% (Figure 3G). These results indicate that PR effectively eliminates CSCs by reversing hypoxic TME, contributing to chemoresistance reversal.

FIGURE 3.

(A) Clustering diagram and (B) quantitative analysis of CD44⁺ cells in CSC mammospheres as measured by flow cytometry. (C) Clustering diagram and (D) quantitative analysis of CD133⁺ cells in CSC mammospheres as measured by flow cytometry. (E) Representative images and (F) diameter of CSC mammospheres at day 1 and 7 with indicated treatments. Scale bar: 100 µm. (G) Quantitative analysis in CSC mammospheres after incubation with PBS, Oxa, or PR.

2.4. Modulation of Immune Cells by PR Nanohybrid

Prodrug (PR)‐induced ICD might facilitate the tumor antigen presentation of mature dendritic cells (mDCs), thereby activating antitumor immunity. In vitro experiments using bone marrow‐derived dendritic cells (BMDCs) treated with colon cancer cells demonstrated a dose‐dependent increase in the proportion of mDCs (CD11c⁺/CD86⁺) upon exposure to both PR and Oxa, with PR exhibiting a more pronounced effect. As shown in Figure 4A,E, at a concentration of 20 µM, PR resulted in a significantly higher proportion of mDCs (66.8%) compared to Oxa (56.1%). Subsequently, co‐culture assays involving activated T lymphocytes and colon cancer cells revealed that pretreatment with the PR nanohybrid led to substantial tumor cell killing, attributable to both nanocatalysis‐mediated TME modulation and tumor‐associated antigen release. Quantification of cell death confirmed that the PR group (98%) exhibited a significantly higher percentage of colon cancer cell death compared to the Oxa (82%) group at 20 µM (Figure 4B,F).

FIGURE 4.

(A) Flow cytometric and (E) quantitative analysis of the population of mDCs (CD11c⁺, CD86⁺). (B) CT26 cells were pretreated with PBS, Oxa, or PR before cultured with T cells. Scale bar is 200 µm. (F) Quantitative analysis of the impact of PR and Oxa on the T cell killing of CT26 cells. (C) Flow analysis and (G) histogram of the proportion of M1‐type macrophages (CD86⁺, F4/80⁺) after treatment with PR or Oxa at different concentrations. (D) Flow analysis and (H) histogram of the proportion of M2‐type macrophages (CD206⁺, F4/80⁺) after treatment with PR or Oxa at different concentrations. The secretion of (I) IL‐6 and (J) IL‐10 from macrophages. ***indicates p < 0.001, and **indicates p < 0.01. Data are expressed as mean ± standard deviation.

TAMs also play a crucial role in tumor immunotherapy. Immunosuppressed M2‐type TAMs can reduce the efficiency of immunotherapy, while immune activation of M1‐type TAMs is able to promote immunotherapy. Thus, the conversion of macrophages from the immunosuppressive M2 type to the immunostimulatory M1 type can convert the “cold” tumor to a “hot” tumor. Subsequently, the M1‐type macrophages with biomarker CD86 and M2‐type macrophages with biomarker CD206 were constructed via incubating RAW264.7 cells with lipopolysaccharide (LPS) and IL‐4, respectively. The assay results indicated that PR nanohybrid effectively polarized macrophages from the immunosuppressive M2‐type (CD206⁺) to the immunostimulatory M1‐type (CD86⁺). Flow cytometry analysis revealed a concentration‐dependent increase in CD86‐positive macrophages and a decrease in CD206‐positive macrophages upon exposure to both PR and Oxa (Figure 4C,G), with PR demonstrating superior efficacy. Specifically, PR nanohybrid increased the percentage of CD86‐positive macrophages from 43.9% (PBS) to 79.8%, while Oxa increased it to 71.2%. Conversely, PR reduced CD206‐positive macrophages from 51.4% (PBS) to 28.0%, while Oxa reduced them to 40.4% (Figure 4D,H). Furthermore, PR treatment led to a decrease in the secretion of the anti‐inflammatory cytokine IL‐10 and an increase in the secretion of the pro‐inflammatory cytokine IL‐6, suggesting that PR facilitates the conversion of macrophages from an M2 to an M1 phenotype In vitro (Figure 4I,J). Taken together, the PR nanohybrid combined chemotherapy, CDT, and nanocatalytic therapy effectively overcomes Oxa resistance, induces enhanced ICD, and reverses hypoxia‐induced immunosuppressive TME to boost antitumor immune responses.

2.5. Transcriptomic Analysis of Antitumor Mechanism

RNA‐seq was performed to elucidate the antitumor mechanism of PR. Volcano plots show dramatic transcriptomic differences between PR and Oxa groups: PR upregulates 1931 genes and downregulates 1655 genes compared to Oxa, and upregulates 381 genes and downregulates 265 genes compared to PBS (Figure 5A,B). Heatmap visualization also illustrated differential gene expression patterns across the various treatments (Figure 5C). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis reveals that differentially expressed genes are mainly enriched in cell cycle regulation, DNA replication, p53 signaling pathway, cancer pathways, and platinum drug resistance (Figure 5D). Gene Set Enrichment Analysis (GSEA) confirms that PR significantly upregulates genes related to cell death (p53, apoptosis), hypoxia response, DNA repair, and immune activation (IFN‐β, antigen processing and presentation), as shown in Figure 5E–J. These results validate that PR modulates genes involved in hypoxia‐induced chemoresistance and immunosuppression.

FIGURE 5.

(A, B) Volcano plot of differentially expressed genes after treatment with PBS, Oxa, or PR nanohybrids (20 µM). Green plots represent the downregulated genes, and red plots represent the upregulated genes, and black plots represent invariant genes. (C) Heat map of the gene variation among PBS, Oxa, and PR nanohybrid groups. (D) KEGG enrichment analysis on dramatically varied genome products with various treatments. (E–J) GSEA reveals positive and negative enrichment of PR‐altered genes in indicated cellular processes.

2.6. In Vivo Antitumor Efficacy

Inspired by the excellent antitumor activity of PR and its effective immune‐boosting capacity In vitro, the therapeutic effect of PR In vivo was evaluated. CT26 tumor‐bearing mice were injected venously with PBS, Oxa, or PR and performed according to the flow chart of Figure 6A. As presented in Figures 6B‐D and S22, Oxa treatment had only a slight inhibitory effect, while PR treatment had a significant antitumor effect. Oxa and PR treatment reduced tumor volume in the PBS group from 1409 mm³ to 1093 mm³ and 378 mm³, while reducing tumor mass in the PBS group from 1.16 g to 0.9 g and 0.5 g. Then, the tumor sections were obtained for staining analysis, including hematoxylin and eosin (H&E), Terminal deoxynucleotidyl transferase dUTP nick end labeling‌ (TUNEL), HIF‐1α, and dihydroethidium (DHE) staining. Histological analysis of H&E staining (Figure 6E), reveals amplified tumor cell damage, characterized by nuclear shrinkage and cytoplasmic lysis, in the PR‐treated group, contrasted with only scattered destruction in the Oxa group. TUNEL staining indicates a marked increase in apoptotic cells following PR treatment, whereas a slightly increased number of apoptotic cells following Oxa treatment (Figure 6F). Further mechanistic investigation demonstrates that PR treatment effectively alleviates hypoxic TME, as shown by reduced expression of HIF‐1α (Figure 6G) and improved oxygen saturation levels measured via photoacoustic imaging (Figure 6I,J). The oxygen saturation increased to a maximum of 76% at 12 h post‐injection compared to 34% before injection, while the oxygen saturation of the tumor tissue changed little after Oxa injection. This effect is attributed to the catalytic therapeutic properties of PR, which also lead to an increased burst of ROS as analyzed by DHE staining (Figure S23), further contributing to cell damage and enhanced treatment efficiency. ICP‐MS result confirms higher Pt accumulation and retention within the tumor tissue in the PR‐treated group compared to the Oxa group, correlating with the superior therapeutic outcome. At 24 h post‐injection of drugs, the Pt content in Oxa and PR groups was 449 ng/g and 1146 ng/g tissues (0.76 and 1.93%ID/g), respectively (Figure 6K). Besides, flow cytometric analysis was conducted on tumor‐isolated cells, utilizing CD44, CD24, and CD133 antibodies to characterize the population of CSCs within CT26 tumor tissues and to assess the impact of the PR and Oxa on this subpopulation. The observation demonstrates a notable reduction in the proportion of CSCs following PR treatment (Figure 6H). Specifically, the percentage of CSCs decreased from 9.3% and 7.4% in the PBS‐ and Oxa‐treated groups, respectively, to 5.6% in the PR‐treated group (Figure 6L), suggesting a potential therapeutic effect of PR on CSC populations within the TME. Importantly, the In vivo toxicity of PR was also assessed. Hemolysis tests revealed that PR caused a hemolysis rate lower than 3% across all concentrations ranging from 0 to 2.8 mg/mL (Figure S24), which possesses good blood compatibility. Data indicates that PR treatment does not induce significant changes in body weight (Figure S25), nor does it cause apparent histological damage or inflammation in major organs, as assessed by H&E staining (Figure S26). Furthermore, liver and kidney function indicators remain largely undisturbed post‐treatment (Figure S27), supporting the conclusion that PR exhibits good biosafety. Importantly, by collecting and testing the Ru elements in urine and feces, it could be observed that Ru element is mainly excreted through feces and its content also increases over time. For mice that were injected for 48 h, the Ru content in their feces and urine reached 64.1% and 5.7% of the injection dose respectively (Figure S28), indicating that PR are mainly excreted from the body through the liver. All those data suggest that PR represents a promising therapeutic agent for tumor treatment, demonstrating superior efficacy compared to Oxa in inhibiting local tumor growth while exhibiting a favorable safety profile in the murine model.

FIGURE 6.

Evaluation of therapeutic effect on tumors in CT26‐bearing BALB/c mice. (A) Schematic illustration of the in vivo treatment experiment timeline. (B) Tumor volume changes of mice (n = 5) in different treatment groups within 14 days. (C) Photographs and (D) weights of the tumor tissues obtained from different groups after 14 days of treatment. Representative images of the tumor region from the indicated groups stained (E) H&E, (F) TUNEL, and (G) HIF‐1α. Scale bars are 100 µm. (H) Clustering diagram and (L) quantitative analysis of CD44⁺CD133⁺ cells in tumor tissues as measured by flow cytometry. (I) Photoacoustic images and (J) quantitative results of oxygen saturation in the tumor region after injection with PR or Oxa. (K) Accumulation of Pt element in tumor area after injection with PR. ***indicates p < 0.001, **indicates p < 0.01, and *indicates p < 0.05. Data are expressed as mean ± standard deviation.

2.7. In Vivo Antitumor Immune Responses

Following treatment, an analysis of immune responses was conducted. Immunofluorescence staining was employed to evaluate CRT exposure and HMGB1 release from cancer cells. The PR treatment group exhibited the most substantial surface CRT exposure and nuclear HMGB1 leakage (Figure 7A,B), indicating effective activation of ICD In vivo. Consistent with these findings, cGAS activation was significantly elevated in the PR group compared to the Oxa and PBS groups (Figure 7C). Due to the observed reversal of the hypoxic TME, PD‐L1 expression in tumor cells within the PR group was significantly lower than that in the PBS group, whereas Oxa treatment demonstrated minimal impact (Figure 7D). Flow cytometric analysis revealed that PR treatment markedly promoted the maturation of DCs in the spleen. The proportion of mDCs in the spleen increased from 8.2% in the PBS group to 9.0% and 14.4% in the Oxa and PR groups, respectively (Figure 7E,I). It was shown that PR treatment, in contrast to Oxa, significantly increased the frequency of killer CD8+ T lymphocytes within the total T lymphocyte population (19.8% for PBS, 18.7% for Oxa, and 32% for PR, Figure 7F,J). Furthermore, PR (47.3%) significantly elevated the content of helper CD4+ T lymphocytes compared to the PBS (25.2%) and Oxa (27.9%) groups (Figure 7G,K).

FIGURE 7.

Assessment of systemic immune responses on tumors in CT26‐bearing BALB/c mice. Representative images of the tumor region from the indicated groups stained with (A) CRT, (B) HMGB1, (C) cGAS, and (D) PD‐L1 antibodies. Scale bars are 50 µm. (E) Contour chart and (I) quantitative result of DC maturation status (CD80/CD86/CD11c) in lymph gland as analyzed via the flow cytometry. (F‐G) Contour chart and (J–K) quantitative result of CD3⁺/CD8⁺ T cells and CD3⁺/CD4⁺ T cells from the indicated groups as analyzed via the flow cytometry. (H) Contour chart and (L) proportion of M1 macrophages (CD86⁺/CD11b⁺/F4/80⁺) from the indicated groups as analyzed via the flow cytometry. The serum levels of (M) IL‐6 and (N) IL‐10 from indicated treatment groups. ***indicates p < 0.001, **indicates p < 0.01, *indicates p < 0.05, and ^nsindicates p > 0.05. Data are expressed as mean ± standard deviation.

In addition, macrophage polarization, shifting from the immunosuppressive M2‐type to the immunostimulatory M1‐type, enhanced the efficacy of tumor immunotherapy. Following PR treatment, the proportion of M1‐type macrophages increased from 1.3% in the control group and 2.4% in the Oxa group to 7.8% (Figure 7H,L). Concurrently, the proportion of M2‐type macrophages decreased to 1.2% in the PR group, approximately 3‐fold lower than the PBS group. In contrast, the proportion of M2‐type macrophages in the Oxa treatment group only decreased to 2.7% (Figures S29 and S30). Serum cytokine levels were also measured. The serum level of the immunostimulatory cytokine IL‐6 increased from 31.1 pg/mL in the PBS group to 55.7 and 126.5 pg/mL in the Oxa‐ and PR‐treated mice, respectively (Figure 7M). Conversely, the serum level of the immunosuppressive cytokine IL‐10 decreased from 52.3 pg/mL in the PBS group to 37.1 and 17.8 pg/mL in the Oxa‐ and PR‐treated mice (Figure 7N). Thus, the combination of PR with chemotherapy, CDT, and nanocatalysis‐mediated modulation of the TME effectively activated robust In vivo antitumor immune responses.

2.8. In Vivo Antimetastasis Effect

In order to assess the impact on immunological memory and therapeutic effectiveness, a CT26 lung metastasis mouse model was employed to evaluate the combined effect of PR and PD‐1 antibody (anti‐PD‐1), as shown in Figure 8A. The murine subjects were stratified into five distinct cohorts: control (PBS) group, Oxa treatment group, PR treatment group, Oxa+anti‐PD‐1 combination group, and PR+anti‐PD‐1 combination group. PBS, Oxa, and PR were administered intravenously at two‐day intervals for a total of three administrations. Anti‐PD‐1 was administered intravenously every three days for a total of two administrations. Consistent with In vivo therapeutic observations, subcutaneous tumor suppression following drug treatments exhibited similar trends (Figure 8B). Oxa treatment resulted in only marginal inhibition of subcutaneous tumor growth. While PR and Oxa+anti‐PD‐1 treatments exhibited partial inhibitory effects, the combined administration of PR+anti‐PD‐1 significantly suppressed subcutaneous tumor growth. Flow cytometric analysis of splenocytes revealed that the combination of PR+anti‐PD‐1 elicited a substantial increase in the proportion of CD8‐positive T cells, exhibiting a 4.9‐fold increase compared to the PBS group and surpassing the effect observed in the PR group (3.0‐fold), thereby promoting enhanced antitumor immune responses (Figure 8C,E). Following the final anti‐PD‐1 injection, CT26 cells were intravenously introduced into each treatment group. At the end of the treatment regimen, lung tissue samples were harvested from each treatment group to quantify metastatic lesions (Figure 8D,F). It was indicated a high prevalence of metastatic lesions in the PBS and Oxa groups. Treatment with PR and Oxa+anti‐PD‐1 resulted in a reduction in metastatic lesions. Notably, the combined PR+anti‐PD‐1 treatment exhibited a near‐complete absence of discernible metastatic lesions in lung tissues. Histological analysis of lung sections on day 16 stained with H&E further substantiated these findings (Figure 8G). These results demonstrate that PR, in conjunction with anti‐PD‐1, effectively stimulates long‐term immunological memory, leading to potent suppression of CT26 tumor growth and metastasis, as well as recurrence.

FIGURE 8.

Evaluation of the anti‐metastasis effect on lung metastasis of CT26‐bearing BALB/c mice. (A) Schematic representation of suppressing lung metastasis by antitumor immune responses induced by various treatments. (B) Tumor volume changes of mice (n = 5) in different treatment groups within 16 days. (C) Quantitative result and (E) contour chart of CD3⁺/CD8⁺ T cells from the indicated groups as analyzed via the flow cytometry. (D) The number of metastases in each group. (F) Photograph and (G) H&E staining of lung tissues in each group. The bottom image provides a detailed view of the area outlined by the red box in the top image, with metastatic tumors indicated by red arrows. Scale bar is 500 µm. ***indicates p < 0.001, *indicates p < 0.05, and ^nsindicates p > 0.05. Data are expressed as mean ± standard deviation.

3. Conclusion

In summary, we have developed a GSH‐responsive, biodegradable multi‐enzyme‐mimetic platinum–ruthenium nanohybrid PR for synergistic chemo‐/chemodynamic‐/immunotherapy of hypoxic tumors. PR is uniformly nanostructured with excellent stability and tumor‐targeting accumulation via the EPR effect. It exhibits intrinsic CAT‐, POD‐, and GPx‐like activities, enabling hypoxia relief, ROS generation, GSH depletion, and DNA damage. These cascading effects activate the cGAS‐STING pathway, trigger robust ICD, reverse Oxa resistance, and eliminate CSCs. PR remodels the hypoxic immunosuppressive TME, downregulates PD‐L1, promotes DC maturation, enhances T cell infiltration, and induces M1‐type macrophage polarization. When combined with anti‐PD‐1 immunotherapy, PR elicits strong immunological response and superior anti‐metastasis efficacy. Meanwhile, PR shows favorable biosafety and biodegradability In vivo.

Nevertheless, this work still has several limitations for clinical translation. We have not systematically explored the long‐term biosafety, metabolic distribution and excretion profiles of PR. In addition, the laboratory‐scale synthesis and single administration route used in this study need further optimization for large‐scale production and clinical application. Future work will focus on addressing these issues, verifying the therapeutic effect of PR on various solid tumors, and exploring its combination with other clinical therapies. Overall, this multi‐enzyme‐mimetic nanoplatform presents a promising candidate to optimize platinum‐based chemotherapy and treat hypoxic solid tumors.

4. Experimental Section/Methods

4.1. Materials

Ruthenium trichloride hydrate (RuCl₃·xH₂O, 99.9%), Oxa (98%), succinic anhydride (99.5%), triethylamine (99.5%), GSH (98%), TMB (98%), 5,5’‐dithio bis(2‐nitrobenzoic acid) (DTNB, 98%), thiazolyl blue (MTT, 99.9%), and Ru(dpp)₃]Cl₂ (95%) were obtained from Macklin (China). IL‐4 and H₂O₂ (30 wt%, analytical reagent) were purchased from Novoprotein Technology Co., LTD (China) and Sinopharm Group Chemical Reagent Co., Ltd. (China), respectively. PBS was obtained from Labgic Technology Co., Ltd. (China). PVP and PF127 were purchased from Sigma‐Aldrich (USA), respectively. LPS was obtained from Yuanye Biotechnology Co., Ltd. (China). DCFH‐DA was purchased from Solarbio Technology Co., Ltd. (China). Live/death staining detection kit was obtained from Beyotime Biotechnology Co., Ltd. (China). Antibodies against HIF‐1α, HMGB1, CRT, γ‐H2AX, PD‐L1, and PD‐1 were boughted from Cell Signaling Technology (USA) and Signalway Antibody (USA).

4.2. Preparation and Characterization of PR Nanohybrids

Oxa PR was synthesized by the previous method [59]. 16 mg of Pt(IV) PR and 6 µL of triethylamine were added in 6 mL of ultrapure water within a round‐bottom flask. The resulting solution was then stirred continuously for a duration of 24 h. Next, 6.4 mg of RuCl₃·xH₂O, 7.5 mg of PF127, and 12 mg of PVP in 3.7 mL of ultrapure water were slowly added dropwise to the above solutions, and after which the mixture was continuously stirred for an additional 24 h. The supernatant was removed via centrifugation at 12000 rpm for 10 min. The resulting precipitate was then washed three times with ultrapure water to remove residual reactants, thereby yielding the self‐assembled nanohybrids.

TEM (JEOL, JED‐2300T, Japan) with an energy spectrometer (JEOL, JEM‐F200, Japan) was implemented for the characterization of particle size, morphology and elemental composition. Furthermore, XPS (Thermo scientific k‐alpha, USA) was utilized to ascertain the elemental composition and valence state of PR nanohybrids. Pt and Ru contents were detected by ICP‐MS (Agilent 7700, USA). The hydrodynamic diameter and zeta potential of PR solution were measured by using a particle size analyzer (Malvern, Nano‐ZS ZEN 3600, England). The absorption spectra were acquired by using a ultraviolet and visible spectrophotometer (UV2600, shimadzu, Japan).

4.3. CAT‐Like Activity of PR

Dissolved oxygen concentrations in H₂O₂ (0.3 mM) solutions containing different concentrations of PR (0, 1.25, 2.5, and 5 µg/mL) were quantitatively assessed using a dissolved oxygen meter (Rex Electric Chemical, JPBJ‐608, China). Furthermore, the visual appearance of four solution groups (control, H₂O₂, PR, and a combined PR+H₂O₂ solution) were documented through photographic records acquired within centrifuge tubes.

The kinetic assays of PR were conducted in a PBS (pH 7.4) at 25°C with PR (5 µg/mL) in the presence of H₂O₂ (5, 7.5, 10, 12.5, and 15 mM). The concentration of H₂O₂ was monitored in real‐time by measuring absorbance at a wavelength of 240 nm using a spectrophotometer. The K _m was obtained using the Lineweaver‐Burk plot, 1/V = 1/V _max + K _m(V _max C), V: initial velocity, V _max: maximum rate of conversion, C: substrate concentration.

4.4. POD‐Like Activity of PR

The POD‐mimicking activity of the PR nanohybrids was assessed by TMB chromogenic assay. Different concentrations of PR nanohybrids (0‐8 µg/mL) were added to the reaction system containing TMB (0.4 mM) and H₂O₂ (1 mM), mixed thoroughly and incubated for 5 min at room temperature. Subsequently, the absorption spectra of the solutions were recorded using a UV‐vis spectrophotometer.

The kinetic analysis of PR were executed in a PBS (pH 7.4) at 25°C with PR (2 µg/mL) in the presence of H₂O₂ and TMB. To determine the enzyme kinetics, two sets of experiments were conducted. In the first set, the concentration of H₂O₂ (0.75, 1, 1.5, 2, 3, and 4 mM) was varied while maintaining a constant TMB concentration (2 mM). Conversely, in the second set, the concentration of TMB (0.25, 0.5, 0.75, 1, 1.5, and 2 mM) was varied while maintaining a constant H₂O₂ concentration (1 mM). The oxidation of TMB was continuously monitored via spectrophotometry by measuring the absorbance at a wavelength of 652 nm. The K _m was determined by the Michaelis‐Menten equation: V = V _max C/(K _m+C), where V: initial velocity, V _max: maximum rate of conversion, C: substrate concentration.

4.5. GPx‐Like Activity of PR

The GSH‐depleting capacity of PR and Oxa PR was quantified using the Ellman method, a colorimetric assay predicated on the reaction of DTNB with free thiol groups. Solutions of the PR and the Oxa PR, ranging in concentration from 0 to 40 µM, were incubated with GSH in PBS. Following this incubation, DTNB was introduced to initiate the chromogenic reaction. Subsequently, the resultant absorbance values at 412 nm, acquired via UV‐vis spectrophotometry, were utilized to determine the residual GSH concentration. The GSH‐depleting capacity of PR and an equal concentration of Oxa PR was calculated.

4.6. GSH‐Responsive Release

To investigate the GSH‐responsive behavior of the PR nanohybrids, a PR solution was incubated for 12 h at room temperature in both a 10 mM GSH solution and ultrapure water as a control. Following incubation, TEM images were obtained to analyze the morphology and size of the resultant structures. The centrifuged supernatant was analyzed to determine whether the Oxa PR was reduced to Oxa. Meanwhile, the centrifuged supernatant was also tested for the levels of Ru and Pt released.

4.7. Cell Culture Conditions and Construction of Oxa‐resistant CT26 cells

The murine colon cancer (CT26) and RAW264.7 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with high glucose, 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin, and cultured in a humidified incubator at 37°C. DCs were cultured in RPMI‐1640 high‐glucose medium containing 10% FBS and 1% penicillin/streptomycin under identical incubation conditions. Under normoxic conditions, the oxygen concentration was 21%, while under hypoxic conditions the oxygen concentration was only 1%.

The murine colon cancer (CT26) cells were seeded in cell culture bottles and cultured in a incubator. Then, Oxa (4 µM) was added to the culture medium for 24 h. Then, the cells were replaced with fresh medium. When the cells recovered to 90% confluence, they were digested with trypsin and sub‐cultured. After the cells grew stably in the presence of Oxa, the drug concentration was gradually increased to 10 µM and the same treatment method was used for two months to obtain Oxa‐resistant CT26 cell line.

4.8. In Vitro Cytotoxicity

MTT Assay

The cytotoxicity of Oxa and PR nanohybrids against CT26 cells or Oxa‐resistant CT26 cells was quantitatively assessed via MTT assay. CT26 cells were seeded at a density of 3×10³ cells per well in 96‐well plates. Following a 48 h incubation period wherein CT26 cells were exposed to varying concentrations of Oxa or PR nanohybrids (ranging from 0 to 50 µM) under both normoxic and hypoxic conditions, MTT was introduced into the culture medium and incubated for an additional 4 h. Subsequently, the supernatant was removed, dimethyl sulfoxide was added, and the absorbance of the resulting assay solution was measured at a wavelength of 490 nm to determine the toxicity of the drug to the cells.

Live and Dead Cell Staining Assay

In the experimental procedure, CT26 cells were cultured in 6‐well plates at a density of 2×10⁴ cells/well and subsequently exposed to either Oxa or a PR nanohybrids at equivalent Pt concentrations (10 µM) for 24 h. Following incubation, cells were stained with PI and Calcein‐AM, and fluorescence imaging was performed to assess and quantify the proportion of live and dead cells under the different treatment conditions.

Apoptosis Detection Assay

The murine colon cancer (CT26) cells were seeded in 6‐well plates at a density of 2×10⁴ cells/well and treated with either Oxa or a PR nanohybrids (10 µM Pt) for 24 h. Following incubation, cells were harvested, stained with Annexin V‐FITC and PI, and analyzed by flow cytometry (Luminex, amnis ImageStreamX Mark II, USA), adhering strictly to the manufacturer's protocol for the apoptosis detection kit (Beyotime, China).

4.9. Measurement of Oxygen Level

To detect the intracellular oxygen level, the oxygen‐sensitive phosphorescence probe Ru(dpp)₃Cl₂ was used for cell staining. CT26 cells were seeded in confocal dishes at a density of 5×10⁵ cells/well and then placed in a hypoxic incubator. Next, 20 µM of Oxa or PR nanohybrids were added to the cell culture medium and incubated for 6 h under normoxic and hypoxic conditions. Subsequently, cells were stained with Ru(dpp)₃Cl₂ probe and imaged by confocal laser scanning microscopy (CLSM, Zeiss LSM900, Germany) to analyze the intracellular oxygen levels.

4.10. GSH Level Evaluation

To assess the impact of Oxa and PR nanohybrids on intracellular GSH levels, CT26 cells were cultured in 6‐well plates at a density of 2×10⁵ cells/well. Subsequently, cells were treated with either Oxa or PR nanohybrids, maintaining equivalent Pt concentrations ranging from 0 to 20 µM, within the cell medium. Following a 24‐h incubation period, a commercially available GSH detection kit (Beyotime, China) was employed to quantify the intracellular GSH concentration.

4.11. Measurement of ROS Level

The murine colon cancer (CT26) cells were seeded into confocal dishes at a density of 1×10⁵ cells/well. Cells were then treated with either Oxa or the PR nanohybrids (20 µM) for 24 h. Subsequently, DCFH‐DA probe was added to the cell culture medium. Following a 20‐min incubation period in the dark, the cells were washed with serum‐free medium for 3 times. Finally, cells were stained with Hoechst 33342 (Beyotime, China) to visualize nuclei and imaged using CLSM.

4.12. DNA Damage Evaluation

The murine colon cancer (CT26) cells were treated with Oxa or PR nanohybrids (20 µM) for 24 h. Then, the cells were stained with γ‐H2AX and a secondary antibody conjugated to Alexa Fluor 488, and the cell nuclei was stained with Hoechst 33342. Subsequently, the cells were imaged by CLSM.

4.13. Evaluation of ICD

Immunofluorescence staining was employed to assess the impact of Oxa and PR nanohybrids on the expression of CRT and HMGB1 within CT26 cells. Following a 24 h incubation period with 20 µM concentrations of Oxa or PR nanohybrids, cells were subjected to a standardized protocol involving fixation, permeabilization, and blocking. Primary and secondary antibody labeling, in conjunction with Hoechst 33342 nuclear staining, facilitated visualization via CLSM. Quantitative image analysis, performed using ImageJ software (version 1.8.0, USA), provided a means to determine the relative expression levels of CRT and HMGB1 under each experimental condition.

The influence of Oxa and PR nanohybrids on intracellular ATP levels in CT26 cells was quantitatively assessed using an ATP detection kit. Cells were cultured and subsequently exposed to varying concentrations (0, 10, and 20 µM) of Oxa or PR nanohybrids for a duration of 24 h. Following incubation, ATP levels were determined according to the manufacturer's instructions for the ATP detection kit (Beyotime, China). In addition, the levels of IFN‐β in cell supernatant were measured by the IFN‐β ELISA kit.

4.14. Assessment of PD‐L1 Expression

The murine colon cancer (CT26) cells were cultured in confocal dishes at a density of 1×10⁵ cells/well, and the cells were treated with Oxa or PR nanohybrids (20 µM). After treatment for 24 h, cells were fixed, stained with a PD‐L1 antibody and Hoechst 33342, and subsequently imaged via CLSM.

4.15. Cellular Uptake of Oxa and PR Nanohybrids

The murine colon cancer (CT26) and Oxa‐resistant CT26 cells were seeded in 6‐cm dishes at a density of 5×10⁵ cells/well and incubated overnight. The cell culture medium was then supplemented with either Oxa or PR nanohybrids (20 µM). Following 12 h incubation under hypoxic conditions, cells were collected and counted, and subjected to aqua regia digestion. The content of Pt was analyzed by ICP‐MS.

4.16. Western Blotting

The murine colon cancer (CT26) cells or Oxa‐resistant CT26 cells were seeded in 6‐cm dishes and cultured overnight. Next, cells were treated with either Oxa or PR nanohybrids (0‐20 µM) for 24 h. Following incubation under hypoxic conditions, cells were washed thrice with PBS, and lysed using RIPA lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors for 15 min. The lysate was then centrifuged at 12000 rpm for 10 min at 4°C. The supernatant, containing the extracted proteins, was collected and mixed with SDS buffer, followed by denaturation through boiling for 10 min. Protein quantification was performed using a bicinchoninic acid protein assay kit. For electrophoresis, protein samples were initially concentrated at 80 kV for 30 min, followed by separation at 100 kV. Resolved proteins were then transferred to a 0.45 µm polyvinylidene difluoride (PVDF) membrane using a semi‐dry transfer apparatus at 100 kV for 60 min, the PVDF membranes blocked with TBS‐T containing 5% bovine serum albumin (BSA) for 1.5 h. Following blocking, membranes were incubated overnight at 4°C with the appropriate primary antibody solution. The next day, membranes were incubated with a goat anti‐rabbit secondary antibody solution for 1.5 h at room temperature. After secondary antibody incubation, membranes were developed using an enhanced chemiluminescence kit to visualize target protein bands. Protein band intensities were quantified through grayscale analysis using ImageJ software. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) or β‐tubulin was used as an internal loading control to normalize protein expression levels.

4.17. Establishment of CSC Mammospheres

The murine colon cancer (CT26) cells were seeded in low‐attachment 6‐ or 96‐well plates (Corning, USA) to promote the formation of mammospheres. The culture medium consisted of serum‐free DMEM/F12 (Invitrogen, USA) supplemented with specific growth factors and nutrients, including 4 mg/mL insulin (Sigma‐Aldrich, USA), 0.4% low‐endotoxin bovine serum albumin (Sangon Biotech, China), 20 ng/mL human epidermal growth factor (hEGF, BD Biosciences, USA), and B27 supplement (Invitrogen, USA).

4.18. Therapeutic Evaluation on CSC Mammospheres

To assess the therapeutic efficacy of Oxa and PR on CSCs, a series of in vitro experiments were conducted under hypoxic conditions. Specifically, mammospheres derived from CSCs were subjected to treatments of PBS (control), Oxa, or PR (20 or 40 µM) for 48 h. Flow cytometry analysis (Beckman CytoFLEX S.4, USA) was then performed using antibodies CD44, and CD133 (Biolegend, USA) to quantify changes in CSC marker expression. Furthermore, mammosphere size was monitored and quantified on days 1, 3, 5, and 7 post‐treatment. For the survival assay, a Cell Counting‐Lite3D Luminescent Kit (Vazyme, China) was employed to evaluate cell survival following 48 h of exposure to the aforementioned treatments in a 96‐well plate format.

4.19. Maturation of DCs

The BMDCs were obtained by reported methods [37], based on IL‐4 (20 ng/mL) and dranulocyte‐macrophage colony‐stimulating factor (GM‐CSF, 20 ng/mL) induction. Next, CT26 cells were pre‐treated with varying concentrations of PR nanohybrids or Oxa (0‐20 µM) for 24 h. Then, the cell supernatant was transferred to cultures of DC for 24 h. Subsequently, DCs were harvested, stained with CD11c (PE‐Cy7, BioLegend)/CD86 (APC, BioLegend) antibodies, and analyzed via flow cytometry.

4.20. The cytotoxic Efficacy of the PR Nanohybrids and Oxa Mediated by T‐cell Immunity

CD8⁺ T‐cells were obtained by activation as described in the literature [37]. CT26 cells were seeded in 24‐well plates and incubated overnight. Then, cells were treated with varying concentrations (0‐20 µM) of PR nanohybrids or Oxa for 24 h. Subsequently, pre‐activated T‐cells were then introduced at a 5:1 ratio and co‐incubated for an additional 24 h. Following co‐incubation, the remaining cells were fixed with 4% paraformaldehyde and stained with crystal violet for visualization. Quantitative analysis of the resulting images was conducted utilizing ImageJ software to assess the extent of cell death.

4.21. Macrophage Polarization

The RAW264.7 cells were cultured in 12‐well plates at a seeding density of 3×10⁵ cells/well. To induce M1 and M2 macrophage polarization, cells were pre‐treated with LPS (200 ng/mL) or IL‐4 (100 ng/mL), for 24 h, respectively. Following polarization, cells were incubated with Oxa or PR nanohybrids (0‐20 µM) for 24 h. Post‐incubation, cells were labeled with fluorochrome‐conjugated antibodies against F4/80 (BV421, BioLegend), CD86 (APC, BioLegend), and CD206 (FITC, BioLegend). Flow cytometry was then employed to assess the resulting phenotypic modulation of the macrophages in response to the drugs. In addition, the levels of IL‐10 and IL‐6 in cell supernatant were measured by the corresponding ELISA kits.

4.22. RNA Sequencing

CT26 cells were seeded in 10‐cm dishes and allowed to adhere for 5 h prior to being transferred to a hypoxic incubator for 24 h. Once cells reached 80% confluency, they were exposed to either Oxa or PR nanohybrids (20 µM). Following a 24‐h incubation, the cells were lysed and extracted mRNA by Trizol for transcriptome sequencing analysis. Transcriptome data were analyzed using the OMICSHARE platform (https://www.omicshare.com/tools/) to identify differentially expressed genes, which were subsequently utilized to construct heatmaps for inter‐group correlation analysis. Additionally, KEGG pathway analysis and GSEA were performed to elucidate biological pathways significantly enriched among genes displaying the highest variability. Genes with a p value less than 0.01 and an absolute fold change of ≥ 1.5 were considered to have significantly different expression.

4.23. Animals

Animal studies were performed in accordance with protocols approved by the Animal Ethics and Use Committee of Guangzhou Medical University (license number: SCXK 2022‐0063), ensuring adherence to ethical guidelines for animal research. Six‐week‐old female Balb/c mice, obtained from Guangdong Vitong Lihua Laboratory Animal Technology Co., LTD.

4.24. In Vivo Biodistribution

To establish the tumor‐bearing mouse model, 6–8 week‐old female Balb/c mice were subcutaneously injected with CT26 cells (2×10⁶). The mice were intravenously administrated with Oxa (6 mg/kg) or an equal amount of Pt in PR nanohybrids. The tumor tissues were collected at 12‐ and 24 h post‐injection. Then, ICP‐MS was employed to determine Pt levels after tissue digestion with aqua regia.

4.25. In Vivo Antitumor Effect and Biosafety Analysis

After the tumor volume was about 80 mm³ (designated as day 0), tumor‐bearing mice were randomized into 3 groups (n = 6): control (PBS) group, Oxa treatment group, and PR nanohybrid treatment group. Interventions were administered intravenously on days 0, 3, 6, and 9. Specifically, the Oxa treatment group received Oxa at a dosage of 8 mg/kg, and the PR nanohybrid treatment group was given PR containing 8 mg/kg Oxa. The PBS group received an equivalent volume of PBS (200 µL). Tumor size and mouse weight were monitored every two days over a 14‐day period. On day 14, major organs were harvested and subjected to histological analysis via H&E, TUNEL, HIF‐1α antibody, and DHE staining. Blood samples were collected for biochemical analyses. Furthermore, single‐cell suspensions were prepared from spleen tissues, stained with CD4 (FITC, BioLegend), CD8 (PE‐Cy7, BioLegend), CD3 (PE, BioLegend) antibodies to test T cells, stained with F4/80 (BV421, BioLegend), CD11b (PE‐Cy7, BioLegend), CD206 (FITC, BioLegend), CD86 (APC, BioLegend) antibodies to test macrophages, and stained with CD80 (APC, BioLegend) and CD11c (PE‐Cy7, BioLegend) antibodies to test DCs via flow cytometry. Moreover, the levels of IL‐10 and IL‐6 in serum were measured by the corresponding ELISA kits.

4.26. Measurement of Oxygen Saturation

The tumor‐bearing mice were intravenously injected with Oxa (6 mg/kg) or an equal amount of Pt in PR nanohybrids. The oxygen saturation of tumor tissues at different time were measured by photoacoustic imager (Vevo, LAZR‐X, USA).

4.27. In Vivo CSCs Assay

The tumors were harvested after treatment to evaluate the CSCs. Then, the cut tumors were digested by collagenase IV (Biosharp, USA) to obtain the single cell. The harvested cells were stained with antibodies, including CD24, CD44, and CD133 via flow cytometry analysis.

4.28. Establishment of CT26 Metastatic Cancer Model and Efficacy Study

Following subcutaneous tumor implantation, mice were stratified into five cohorts (n = 5): (i) control (PBS) group; (ii) Oxa treatment group; (iii) PR nanohybrid treatment group; (iv) Oxa and PD‐1 antibody (Oxa+anti‐PD‐1) treatment group; and (v) PR nanohybrid and PD‐1 antibody (Oxa+anti‐PD‐1) treatment group. Oxa and PR nanohybrid (8 mg/kg) were intravenously administered on days 0 and 3 to the indicated treatment groups, while the control group received an equivalent volume of PBS (200 µL). Subsequent to these administrations, the anti‐PD‐1 (1 mg/kg) was intravenously injected into groups (iv) and (v) on days 1 and 4. Tumor volume was monitored every two days over a 16‐day observation period. Upon study termination on day 16, lung tissues were harvested, fixed in 4% paraformaldehyde, and processed for H&E staining. Concurrently, splenocytes were isolated to generate single‐cell suspensions, which were then subjected to flow cytometric analysis to assess alterations in CD8⁺ T cell populations.

4.29. Statistical Analysis

Data in this study are presented as means ± standard deviations from at least three independent replicates. The significant differences were obtained by the Origin95 software, ***p < 0.001, **p < 0.01, *p < 0.05, and ^nsindicates p > 0.05. The quantitative results were analyzed via ImageJ software analysis.

Author Contributions

Yue Zheng: Writing – original draft, methodology, project administration. Huixi Yi: Data curation, investigation, visualization. Jingyao Li: Project administration. Zhixiong Zhan: Data curation. Jianliang Shen: Conceptualization, project administration, writing – original draft. Liyou Guo: investigation. Shanshan Xue: funding acquisition. Dong‐Yang Zhang: Conceptualization, writing – original draft, writing – review and editing, project administration, funding acquisition. Xiyong Yu: Conceptualization, project administration, writing – review and editing. Nannan Fu: Data curation, visualization. Qiuhua Li: Investigation, visualization.

Funding

This work is financially supported by National Natural Science Foundation of China (22207019) and Natural Science Foundation of Shandong Province (2023HWYQ‐073).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: adhm71484‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.