Cancer-cell-mimicking MnO₂ nanoplatform delivers immunological adjuvants to enhance radio-immunotherapy

Abstract

The hypoxic and immunosuppressive microenvironment within tumors represents a fundamental mechanism contributing to radiotherapy (RT) resistance, which is closely linked to poor prognosis in cancer patients. To overcome these limitations, we constructed a tumor cell membrane-mimetic manganese-based nanoplatform for delivering immune adjuvants (MnO₂-R848@CM) to synergistically enhance the efficacy of clinical radiotherapy. In vitro experiments demonstrated that MnO₂-R848@CM is rapidly and specifically taken up by 4T1 cells. Upon X-ray irradiation, immunogenic cell death (ICD) and released Mn²⁺ activated the cGAS-STING pathway in dendritic cells (DCs), synergistically accelerating DCs maturation alongside R848 to trigger downstream immune response cascades. In breast cancer mouse models, MnO₂–R848@CM exhibited highly effective homotargeting properties, rapidly accumulating and persisting at tumor sites. It further established a long-lasting adaptive anti-tumor immune response via multiple pathways, effectively accelerating tumor ablation and significantly inhibiting tumor growth in tumor-bearing mice. These findings highlight the potential of the MnO₂–R848@CM nanoplatform in advancing synergistic cancer radioimmunotherapy, which effectively integrates the direct cytotoxic effects of radiotherapy with ongoing antitumor immune responses.

Graphical abstract

1. Introduction

Radiation therapy (RT) stands as a prevalent clinical modality for cancer treatment, applicable to nearly all solid tumors [1,2]. Beyond its direct cytotoxic effects on cancer cells, RT operates through two principal mechanisms. One is to promote reactive oxygen species (ROS) production via ionizing radiation, which directly damages cellular DNA and other organelles to kill cancer cells [3]. The other is to induce immunogenic cell death (ICD) and activate complement responses and adaptive immune responses [4,5]. Multiple immunotherapies have yielded encouraging outcomes in clinical cancer treatment [6]. For instance, cancer vaccines, characterized by their cost-effectiveness, high specificity, and minimal adverse effects, are under active clinical investigation [7]. Specifically, there is a growing interest in the clinical integration of RT with immunotherapy to target both localized and metastatic tumors [[8], [9], [10]]. Despite these advances, the tumor microenvironment (TME) presents significant challenges, with immunosuppressive cells and hypoxic conditions being major impediments [1,11]. Hence, there is an urgent need to devise effective strategies to overcome these barriers, thereby enhancing antitumor efficacy and improving the therapeutic outcomes of radioimmunotherapy.

Transition metal manganese (Mn), as an emerging radiosensitizer, demonstrates remarkable potential in bridging RT and immunotherapy [12]. Its core mechanism lies in activating the cGAS-STING innate immune pathway within tumor cells, thereby integrating the localized cytotoxic effects of radiotherapy with systemic antitumor immune responses [13,14]. Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) specifically recognizes damaged DNA and antigens in the cytoplasmic environment, promoting the synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). This activates the signaling adaptor protein (STING), initiating the production of type I interferons (IFNs) and pro-inflammatory cytokines to achieve tumor cell killing [15]. Emerging evidence indicates that Mn²⁺ can accelerate the overall catalytic activity of cGAS and enhance the binding affinity of cGAMP to STING [16]. Zhu et al. [17] reported that an endoplasmic reticulum-targeted Mn nanocomplex effectively activates STING, thereby achieving systemic tumor suppression. Furthermore, combining Mn²⁺ with STING agonists significantly enhances STING activation, thereby amplifying both local and systemic cancer eradication effects [13,18]. Nonetheless, free Mn²⁺ exhibits acute off-target toxicity during blood transport and rapid clearance, which restricts its broader clinical application.

Nanoformulations have emerged as a potent strategy to improve the local accumulation of anticancer drugs while minimizing off-target effects, primarily by leveraging the enhanced permeability and retention (EPR) effect associated with the aberrant vasculature of tumors [19]. This EPR effect is fundamentally influenced by several factors, including lymph angiogenesis, stromal response, intertumoral pressure, structural disorganization, irregular branching, irregular perfusion, uneven distribution density, and the intrinsic properties of the nanoparticles themselves [20,21]. To overcome these limitations, cell membrane-coated nanoparticles represent a novel drug delivery strategy offering prolonged circulation time, active targeting capabilities, and more efficient drug delivery [22]. Among various cell membranes employed in these systems, those derived from tumor cells offer unique advantages beyond mere homologous targeting [[23], [24], [25]]. For example, personalized vaccines encapsulated within tumor cell membranes can elicit specific immune responses tailored to individual tumors, effectively circumventing immune evasion mechanisms such as cytoplasmic protein interference and the downregulation of tumor antigen expression [26]. Moreover, tumor cell membranes endowed with specific ligands enhance the targeting of dendritic cells by vaccines, thereby intensifying the resultant immune responses [27,28].

Combination strategies involving radiotherapy (RT) and immunotherapy have attracted considerable interest due to their clinical relevance and potential synergistic effects in tumor treatment [29,30]. In this study, we propose a simple synthesis of a manganese-based nanoplatform (MnO₂–R848@CM), designed for in situ targeted radiotherapy sensitization in conjunction with immunotherapy (Fig. 1). MnO₂ catalyzes the decomposition of tumor-derived H₂O₂ to produce oxygen, alleviating local tumor hypoxia and enhancing X-ray-induced cell death, as confirmed by prior studies. The subsequently released Mn²⁺, along with R848 (a Toll-like receptor agonist) and DAMPs (damage-associated molecular patterns), collectively enhances the activation of the cGAS-STING signaling pathway. This activation leads to the production of type I interferons (IFNs), accelerates dendritic cell (DC) maturation, and initiates adaptive immune responses following RT. In summary, this MnO₂-based nanoplatform induces potent antitumor immune responses by alleviating tumor hypoxic microenvironments, reversing immunosuppressive TME, promoting DC maturation, and enhancing immune cell (CD8⁺ T cell) infiltration. Consequently, this work provides a straightforward approach to prepare MnO₂-R848@CM nanoplatforms for enhancing radioimmunotherapy.

Fig. 1.

Schematic diagram of MnO₂–R848@CM nanoplatform-activated immunotherapy combined with radiotherapy for tumor treatment. (a) MnO₂ nanosheets and 4T1 cell membranes were co-extruded to fabricate MnO₂-R848@CM nanoplatform (Green dots represent R848; blue sheets represent MnO₂ nanosheets; purple represents 4T1 cell membrane). (b) This platform releases R848, ICD, and TAAS after radiotherapy, synergistically accelerating DC cell maturation. (c) Furthermore, the hydrolyzed nanoplatform effectively alleviates the tumor hypoxic microenvironment. By releasing Mn²⁺ that activate the cGAS/STING signaling pathway, it induces IFN release, directly killing tumor cells while synergistically promoting DCs maturation and activating long-lasting adaptive immunity.

2. Results and discussion

2.1. Construction and characterization of MnO₂-R848@CM nanoplatform

The synthesis of MnO₂-R848 followed previously reported procedures with some modifications [31]. Specifically, MnO₂ nanosheets were stirred with mPEG-NH₂ at 4 °C to allow preliminary modification of mPEG-NH₂ onto the MnO₂ nanosheet surface. Then, after centrifugation following blending with the hydrophobic small-molecule drug R848, the MnO₂-R848 composite was obtained. This process was accomplished through NH₂–mediated hydrophilic-hydrophobic interactions, Mn–N coordination, and π–π stacking interactions. With the increase in R848 content, the encapsulation efficiency (EE) of the prepared MnO₂-R848 complex gradually rose, but the drug loading (DL) reached its maximum of 35.70% at a feed ratio (MnO₂: R848) of 1:3 (Table S1). Then, high-purity cell membranes were extracted from 3t3 and 4t1 cells via repeated freeze-thaw cycles, and the tumor cell membranes were co-extruded with MnO₂-R848 complexes through a polycarbonate membrane filter to obtain tumor cell membrane-mimetic MnO₂-R848 nanoplatforms (MnO₂-R848@CM) (Fig. 2a). SDS-PAGE protein electrophoresis gel results displayed the absence of protein bands in pure MnO₂-R848, while 3T3 CM and 4T1 CM exhibited significantly distinct protein abundance patterns, indicating high heterogeneity between tumor cell membranes and normal cell membranes. The almost consistent band patterns between MnO₂-R848@CM and 4T1 CM indicate successful modification of 4T1 CM on the nanosheets' surface (Fig. S1).

Fig. 2.

Preparation and characterization of MnO₂-R848@CM. (a) Schematic illustration of the preparation of MnO₂-R848@CM. (b) The particle sizes and (c) zeta potentials of MnO₂, MnO₂-R848, and MnO₂-R848@CM. (d) Typical transmission electron microscope (TEM) and (e) Atomic force microscope (AFM) images of MnO₂, MnO₂-R848, and MnO₂-R848@CM and the line scan of thickness. Scale: 200 and 600 nm, respectively.

The initial MnO₂ nanosheets exhibited a typical 2D plate-like morphology with an average lateral dimension of approximately 84 nm and a thickness of about 1.5 nm. After loading R848 and membrane modification, these dimensions increased to 107 nm and 170 nm, respectively, while the thickness increased to 6 nm and 15 nm (Fig. 2b–e, and S2). Notably, the cell membrane modification not only enabled MnO₂–R848@CM to exhibit excellent dispersion properties, larger dimensions, and a potential folded/curled structure (Fig. 2d), but also conferred higher electronegativity (Fig. 2c). This increased electronegativity is generally beneficial for nanodrug hematogenous transport, as evidenced by the more stable size of MnO₂–R848@CM in simulated body fluids, consistently remaining below 200 nm (Fig. S3). Furthermore, the cell membrane coating provides additional protective effects, significantly enhancing the feasibility of MnO₂–R848@CM as a nanomedicine delivery system for effective in situ tumor targeting within the hemotransport. Given the weakly acidic tumor microenvironment, the pH-responsive release behavior of R848 from MnO₂-R848@CM was further evaluated. As mentioned earlier, the tumor microenvironment exhibits unique features compared with normal tissues, such as a weakly acidic environment. Consequently, in vitro drug release studies were carried out at 37 °C under different pH conditions. As shown in Fig. S4, the release of R848 from MnO₂-R848@CM was largely controlled at pH 7.4. In this case, the cumulative release was only 19.12 ± 0.69% within 12 h, indicating a slow and sustained release profile. This could be attributed to the stable coordination between Mn and the amino groups of mPEG-NH₂, as well as the π–π stacking and Mn–N interactions between R848 and the MnO₂ nanosheets. Interestingly, the release of R848 from MnO₂-R848@CM was apparently accelerated at pH 5.0 compared with that at pH 7.4, probably as a result of the dissolution of MnO₂ in acidic conditions and the weakening of the interactions between R848 and the nanosheets. Specifically, approximately 45% of R848 was released within the first 0.5 h at pH 5.0, and the cumulative release reached about 77% at 3 h, approaching a plateau thereafter. In contrast, less than 20% of R848 was released at pH 7.4 over 12 h. Therefore, when the drug-loaded MnO₂ nanosheets accumulate at tumor tissues, the weakly acidic environment of tumors may substantially promote the breakup of the nanosheets and rapid release of the loaded R848. This acid-triggered drug release profile indicates that MnO₂-R848@CM can act as a smart drug delivery system for tumor-specific therapy.

2.2. MnO₂–R848@CM exhibits specific targeting towards breast cancer

Cell membrane biomimetic modification endows nanomedicines with highly efficient internalization capabilities, thereby enhancing their therapeutic efficacy. We encapsulated coumarin 6 (C6) within MnO₂–R848@CM nanoparticles to investigate its uptake behavior by 4T1 cells. The fluorescence intensity within 4T1 cells was detected at different time points using inverted fluorescence microscopy and flow cytometry. Results demonstrated a rapid increase in green fluorescence intensity within the first hour, confirming the rapid internalization of MnO₂–C6@CM by cancer cells (Fig. 3a). Over the subsequent 7 h, fluorescence intensity continued to rise, reaching a plateau at approximately the sixth hour (Fig. 3b–e). To further validate the specific targeting of this internalization phenomenon, the fluorescence intensity of C6-labeled nanoparticles was compared between 3T3 cells (normal) and 4T1 cells. Results demonstrated significantly enhanced fluorescence intensity in 4T1 cells after 6 h of co-incubation, with flow cytometry confirming consistent findings (Fig. 3c and d). These results indicate that the cancer cell-mimetic coating system substantially enhances the internalization and homologous targeted delivery efficacy of the nanoplatform.

Fig. 3.

In vitro and in vivo verification of MnO₂-R848@CM for specific tumor accumulation. (a) Representative captured images, (b) corresponding flow cytometry analysis, and (e) quantitative analysis of 4T1 cells incubated with MnO₂-C6@CM at different times, MnO₂-C6@CM and nuclei were labeled with C6 and Hoechst33342, respectively. (c) Flow cytometry quantitative analysis and (d) representative fluorescence images of the cell membrane-targeting ability of C6-labeled MnO₂-C6@CM in 3T3 and 4T1 cells. (f) Real-time in vivo imaging of mice bearing subcutaneous 4T1 tumors at 1, 6, and 12 h after intravenous injection of free DiR and MnO₂-R848@CM. (g) Ex vivo DiR signals of the harvested major organs (heart, liver, spleen, lung, and kidney) and tumor tissues at 1, 6, and 12 h after administration. (h) Mean fluorescence intensity (MFI) of DiR signals in harvested tumor tissues at 1, 6, and 12 h post-injection. (i) The analysis of mean fluorescence intensity in tumor sites at various time points post-injection, ****p < 0.0001.

Based on the encouraging results from in vitro experiments, we evaluated the tumor-targeting efficiency of MnO₂-R848@CM in vivo. Efficient in situ accumulation at tumor sites is a critical prerequisite for reducing off-target effects of nanoplatforms and enhancing precision therapy efficacy. The hemodynamic safety of nanoplatforms has been validated through in vitro blood compatibility testing, confirming that the nanosystem does not induce hemolysis within the concentration gradient range (Fig. S5). The biodistribution of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR)-labeled MnO₂-R848@CM was tracked in 4T1 tumor-bearing mice via fluorescence imaging. The mice were intravenously injected with free DiR or DiR-labeled MnO₂-R848@CM, and fluorescence imaging was performed at various time points post-injection. As shown in Fig. 3f and g, the fluorescence intensity of MnO₂-R848@CM at the tumor site was significantly higher than the free DiR group. Notably, the fluorescence signal of MnO₂-R848@CM gradually accumulated within the tumor, peaking at 12 h post-injection (Fig. 3i). At the 12-h time point, the fluorescence intensity of MnO₂-R848@CM was 73.27-fold higher than the free DiR group, indicating its superior systemic distribution capability and tumor targeting ability (Fig. 3h). To further confirm that the observed tumor targeting is specifically mediated by homologous 4T1 membrane coating rather than a general membrane effect, an additional biodistribution study including free DiR, 3T3 membrane-coated MnO₂-R848, and 4T1 membrane-coated MnO₂-R848@CM was performed (Fig. S6). As shown in Fig. S6, free DiR showed strong signals in the liver and spleen at 1 h and in the liver, spleen, and lung at 12 h. The 3T3 membrane-coated group showed strong signals in the lung and kidney at 1 h and in the liver, spleen, and lung at 12 h, with no obvious tumor enrichment. In contrast, the 4T1 membrane-coated group showed strong signals in the liver, spleen, lung, and tumor at 1 h and in the liver, spleen, and tumor at 12 h 3T3 cells are normal mouse embryonic fibroblasts and share the same species background as 4T1 cells but lack tumor-specific membrane adhesion molecules; therefore, 3T3 cell membranes were used as an irrelevant membrane control. The lack of tumor enrichment in the 3T3 membrane-coated group, together with the reduced off-target uptake in non-tumor tissues such as the lung, confirms that the enhanced tumor accumulation and retention are specifically mediated by the 4T1 cancer cell membrane coating rather than by a general membrane effect.

2.3. In vitro synergistic antitumor effect of MnO₂–R848@CM and radiotherapy

To systematically evaluate the biocompatibility of the nanoplatform, we treated 3T3 cells with different concentrations of various materials. Results showed that the survival rate of 3T3 cells in all treatment groups exceeded 80% at a MnO₂ concentration of 2.5 μg/mL. Furthermore, the CM-modified group exhibited higher cell viability. Specifically, at a MnO₂ concentration of 5 μg/mL, the survival rate in the control group decreased to 74.58%, while the treatment group exhibited a higher survival rate of 83.52%. This indicates that cell membrane encapsulation enhances the stability of nanosheets, reduces their tendency for massive aggregation, and thereby mitigates their cytotoxic effects on 3T3 cells (Fig. S7). Similar experiments conducted on 4T1 cells revealed that at a MnO₂ concentration of 5 μg/mL, relative cell viability dropped below 70%. This is attributed to the inherent instability of tumor cells, which renders them more susceptible to initiating apoptosis upon exogenous stimulation (Fig. 4a).

Fig. 4.

The antitumor activity of MnO₂-R848@CM and RT in vitro. MTT assay for cell viability of 4T1 cells after 24 h co-culture with MnO₂, MnO₂-R848, and MnO₂-R848@CM, (a) without and (b) with X-ray. (c) Immunofluorescence staining of γ-H2AX (blue: nucleus; green: γ-H2AX) with (+) and without (−) X-ray of different groups and (d) corresponding flow cytometry analysis. (e) Quantitative analysis of apoptosis in groups treated without or with X-ray and (f) representative flow cytometry images. (g) DCFH-DA staining images of in vitro generation of ROS without or with X-ray of different groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Following previous literature reports [1], a 6 Gy irradiation dose was used to simulate radiotherapy on 4T1 cells. Results indicated that at a MnO₂ concentration of 5 μg/mL, the viability of cells treated with radiotherapy decreased by approximately 20%, suggesting that the MnO₂–R848@CM nanosystem can enhance the efficacy of radiotherapy (Fig. 4b). The apparent difference in cytotoxicity between MnO₂ and MnO₂-R848@CM without and with X-ray irradiation can be explained by their distinct colloidal and biological behaviors. Without irradiation, bare MnO₂ nanosheets tend to aggregate and interact non-specifically with cell membranes, causing non-specific oxidative damage and thus lower cell viability. In contrast, the cell membrane coating improves colloidal stability and reduces non-specific interactions, resulting in higher cell viability in the MnO₂-R848@CM group. Upon X-ray irradiation, however, MnO₂-R848@CM benefits from homologous targeting and enhanced cellular uptake, and the membrane coating protects MnO₂ from premature degradation before internalization, allowing more MnO₂ to enter 4T1 cells. Irradiation generates ROS and H₂O₂, while MnO₂ consumes GSH and releases Mn²⁺, collectively amplifying DNA damage and ROS-mediated cell death. Consequently, MnO₂-R848@CM exhibits stronger radiosensitization and higher cytotoxicity than bare MnO₂ under X-ray irradiation. It is known that high-energy ionizing radiation damage and excessive ROS production are primary mechanisms by which RT kills tumor cells [32]. RT-generated ionizing radiation directly attacks DNA, inducing double-strand breaks and producing phosphorylated γ-H2AX. γ-H2AX immunofluorescence staining revealed significant fluorescence within cells following X-ray treatment compared to the control group, indicating RT-induced DNA damage (Fig. 4c and d). The MnO₂–R848@CM and RT co-treatment group exhibited the highest fluorescence intensity, suggesting that the presence of Mn²⁺ exacerbated DNA damage, which was attributed to the ROS-generating amplification effect of Mn²⁺ (Fig. 4g). The damaged DNA further triggered cell cycle arrest and apoptosis in tumor cells, accelerating tumor ablation (Fig. 4e and f). These results demonstrate that the MnO₂–R848@CM nanoplatform can provide broader synergistic benefits to RT.

2.4. In vitro synergistic antitumor effect of MnO₂–R848@CM and RT

Previous studies indicate that RT enhances tumor immune responses primarily through the induction of immunogenic cell death (ICD) [33]. This process leads to the emergence of Damage-Associated Molecular Patterns (DAMPs), primarily manifested by the translocation of calreticulin (CRT) and heat shock proteins (HSPs) towards the cell surface, along with the extracellular release of adenosine triphosphate (ATP), high-mobility group box 1 (HMGB1), and others. These DAMPs serve as danger signals recognized by pattern recognition receptors (PRRs) in the innate immune system, thereby activating anti-tumor immune responses and transforming “cold” to “warm” tumors [34]. To investigate the synergistic role of MnO₂-R848@CM in this critical event, we separately assessed CRT membrane translocation, HMGB1 and ATP release under different treatment conditions. Results showed minimal red fluorescence on untreated 4T1 cell surfaces, indicating baseline CRT expression. Significant increases in red fluorescence were observed in groups treated with MnO₂, MnO₂-R848, MnO₂-R848@CM, and radiation alone. Notably, the group which received combined RT and nanomedication treatment exhibited the highest levels of translocated CRT on the cell surface, confirming that the synergistic application of Mn-containing nanoparticles significantly enhanced the induction of ICD (Fig. 5a and b). We subsequently examined two major DAMP signaling molecules, HMGB1 and ATP. Normally, HMGB1 exists as a non-histone chromatin protein within the nucleus, acting as a DNA chaperone and interacting with chromatin. During ICD, HMGB1 is released from the nucleus and plays a crucial role in promoting dendritic cells (DCs) maturation and then activates cytotoxic T lymphocytes (CTLs) to target and kill cancer cells. HMGB1 immunofluorescence staining revealed the strongest nuclear expression in untreated 4T1 cells. Following radiation or nanomedicine treatment, HMGB1 signal intensity significantly decreased, with the MnO₂–R848@CM and RT combination group displayed the lowest nuclear HMGB1 expression (Fig. 5c and d). To further investigate the translocation of HMGB1 from the nucleus after ICD induction, the cytoplasmic fluorescence intensity of PE-labeled anti-HMGB1 antibody was quantified via post-treatment flow cytometry. Compared to the untreated group, significant shifts in fluorescence intensity peaks were observed following both single and combined treatments, confirming that the combined MnO₂–R848@CM and RT treatment not only induces ICD but also promotes HMGB1 release from the nucleus (Fig. 5e). Additionally, the released ATP content similarly increased significantly (Fig. 5f). In summary, MnO₂–R848@CM effectively internalizes into cancer cells and enhances radiosensitivity under X-ray irradiation, thereby inducing ICD. This process lays the basis for promoting DC maturation and activating immune responses.

Fig. 5.

In Vitro immunotherapy of MnO₂-R848@CM. (a) Immunofluorescence staining of CRT (blue: DAPI; red: CRT) with (+) and without (−) X-ray and (b) ImageJ relative quantitative fluorescence intensity analysis of CRT. (c) Immunofluorescence staining of HMGB1 (blue: DAPI; red: HMGB1) with (+) and without (−) X-ray and (d) ImageJ relative quantitative fluorescence intensity analysis of HMGB1 and (e) flow cytometry of extra-nuclear HMGB1 expression. (f) Quantitative calculation of ATP levels in 4T1 cells with and without X-ray. (g) Schematic illustration of the synergistic effects of MnO₂-R848@CM and RT on BMDC maturation in transwell co-incubation experiment. (h) Relative quantitative histogram of MnO₂-R848@CM on the maturation of BMDCs by flow cytometry. (i) The TNF-α secretion levels in supernatants of various treatments from BMDCs after incubation with 4T1 tumor cells. (j) Relative quantitative histogram of MnO₂-R848@CM and RT on the maturation of BMDCs by flow cytometry. (k) Elisa measurement of TNF-α levels in supernatants of various treatments. *p < 0.05, **p < 0.01, ****p < 0.0001.

DCs are classic antigen-presenting cells (APCs) that play crucial roles in activating innate and adaptive immunity, but only mature DCs can promote antigen presentation and activate T cells. MnO₂ nanoparticles have been reported to accelerate DC maturation through the cGAS-STING signaling pathway, thereby enhancing subsequent immune responses [1]. R848 is an immunological adjuvant with a well-defined mechanism of action, primarily accelerating DC maturation by activating TLR7 and TLR8 receptors [35]. Consequently, MnO₂-R848@CM exhibits potential synergistic effects in activating immune responses. Immature CD11c⁺ dendritic cells (iBMDCs) were isolated from mouse bone marrow and induced to differentiate, exhibiting a semi-adherent and clustered phenotype suitable for subsequent experiments (Fig. S8). Compared to the control group, iBMDCs treated with MnO₂ or R848 alone exhibited significantly increased maturation rates (CD80⁺/CD86⁺), rising from 59.2% to 76.2% and 78.0%, respectively (Fig. 5h). MnO₂–R848 exhibited enhanced effects compared to either reagent alone, suggesting a potential synergistic interaction between MnO₂ and R848 (Fig. S9). Notably, the difference in promoting BMDC maturation between MnO₂-R848 and MnO₂-R848@CM was not statistically significant, showing only a slightly elevated trend. This was attributed to the potent activation effect of MnO₂-R848 itself overshadowing the effect of CM, yet both were superior to the positive control group (LPS-induced). Next, we assessed functional activity by detecting the release of the tumor necrosis factor-α (TNF-α) following BMDC maturation. Results showed significantly increased TNF-α secretion after treatment with different nanoparticles, indicating that MnO₂–R848@CM effectively stimulates DC maturation and induces robust immune responses (Fig. 5i).

To demonstrate the superiority of combined therapy, we established an in vitro Transwell model to simulate the interaction between 4T1 cells and BMDCs exposed to radiation and various nanomedicines. Specifically, 4T1 cells were treated and placed in the upper chamber, while iBMDCs were incubated in the lower chamber. After 24 h, analysis was performed via flow cytometry. Results showed that X-ray treatment increased BMDC maturation from 36.3% to 43%, and MnO₂ treatment further elevated maturation to 77.5%. This effect significantly surpassed the individual treatments, with the initial reduction in maturation attributed to batch variations and indirect contact (Fig. 5j and S10). Consistent with these findings, BMDCs treated with combined RT and MnO₂-R848@CM secreted the highest levels of TNF-α, further supporting the potential of combined therapy to enhance antitumor immunity (Fig. 5k). This powerful effect was attributed to Mn²⁺ binding to the cGAS enzyme, altering its conformation and enhancing its sensitivity to DNA, thereby activating the STING signaling pathway and initiating potent downstream events. Western blot results showed that MnO₂ nanosheets could activate the cGAS-STING signaling pathway in DCs, and the addition of R848 did not result in an additional increase in p-STING protein, suggesting the existence of an alternative activation mechanism. MnO₂-R848@CM exhibited the most effective activation, indicating that the membrane modification contributes to DC maturation and is more effective in activating DC maturation following RT (Fig. S11).

2.5. In vivo synergistic antitumor effect of MnO₂–R848@CM and RT

The in vivo synergistic radioimmunotherapy efficacy of the MnO₂–R848@CM nanoplatform was evaluated using a 4T1 tumor-bearing Balb/c mouse model. Preliminary in vivo biosafety was assessed by tail vein injection of saline, MnO₂, R848, and MnO₂–R848@CM into healthy mice followed by 7 days. All groups’ mice exhibited good physiological status. No pathological changes or inflammatory infiltration were observed in major internal organs (heart, lungs, liver, spleen, and kidneys). Key blood biochemical parameters (ALT, AST, ALP, CREA, and BUN) showed no significant differences compared to normal mice, indicating the excellent biosafety of the MnO₂–R848@CM nanoplatform (Figs. S12 and S13). Next, mice were randomly divided into seven groups: G1, Saline; G2, R848; G3, X-ray; G4, MnO₂–R848; G5, MnO₂–R848 with X-ray; G6, MnO₂–R848@CM; and G7, MnO₂–R848@CM with X-ray. As previously reported, mice underwent a single drug injection and three radiation treatments throughout the therapeutic cycle [1]. The initial treatment demonstrated the most pronounced combined therapeutic effect, while subsequent treatments highlighted the convenience and persistence of the MnO₂–R848@CM nanoplatform (Fig. 6a). No significant changes in body weight were observed throughout the therapy cycle, indicating excellent tolerability of this treatment approach, which is considered beneficial for improving patient compliance (Fig. 6b). Treatment results showed that mice injected with saline exhibited rapid tumor growth, indicating no discernible tumor-suppressive effect. R848 group demonstrated a trend toward tumor reduction, attributed to its immune-activating properties, with enhanced efficacy when combined with RT. The MnO₂-R848 group did not demonstrate remarkable efficacy when not combined with RT, potentially because MnO₂ enhanced the anti-apoptotic effects in 4T1 cells. However, following RT administration, an extreme oxidative stress microenvironment emerged. Mn²⁺ induced complex metabolic reprogramming, rendering 4T1 cells more sensitive to ROS-induced death, thereby causing potent cytotoxic effects. Furthermore, the cell membrane-coated MnO₂–R848@CM enhanced targeted accumulation and retention at tumor sites, significantly boosting antitumor efficacy. It markedly suppressed tumor growth, achieving a tumor inhibition rate of 92.30% (Fig. 6c–e). H&E staining revealed irregular cellular arrangement and necrosis within treated tumors. The MnO₂-R848@CM-treated group exhibited the maximum number of apoptotic cells and the strongest suppression of cell proliferation within tumors (Fig. 6h–m). Previous in vitro cell experiments had indicated that this nanoplatform significantly promotes BMDC maturation and antigen presentation, thereby inducing antitumor immune responses. In downstream activities, CD8⁺ T cells serve as primary effectors of immune resistance, possessing the ability to directly recognize tumor-associated antigens and lysed altered cells. The frequency of CD8⁺ T cell infiltration and functional status within the tumor microenvironment correlate closely with overall tumor treatment efficacy. Immunofluorescence staining of CD8⁺ T cells in tumor tissues from different treatment groups revealed increased parenchymal CD8⁺ T cell infiltration across all groups compared to the saline control, with the most pronounced increase observed in the MnO₂–R848@CM plus RT group (Fig. 6f and g). The increased CD8⁺ T cells further accelerated tumor ablation directly by secreting factors such as interferon-γ (IFN-γ). To further investigate whether MnO₂-R848@CM alleviates tumor hypoxia in vivo, HIF-1α immunohistochemical staining was performed on tumor tissues from different treatment groups. As shown in Fig. S14, HIF-1α expression was markedly reduced in the MnO₂-R848@CM plus RT group compared with the saline and RT-alone groups, indicating effective relief of tumor hypoxia. This hypoxia alleviation may contribute to the enhanced radiosensitivity and improved antitumor immune response observed in the combination group.

Fig. 6.

The in vivo antitumor efficacy of MnO₂-R848@CM and RT. (a) Schematic schedule of the 4T1 tumor-bearing mouse model. (b) Body weight of 4T1 hormonal mice treated with groups. (c) In vivo tumor images and (d) tumor weight statistics of 4T1 hormonal mice treated with various groups. (e) Average tumor growth curves after intravenous injection of different formulations. (f) Immunofluorescence staining of CD8 in isolated tumors and (g) ImageJ relative quantitative fluorescence intensity analysis. (h) Hematoxylin-eosin (H&E) staining images of tumor tissues from 4T1 tumor-bearing mouse models after different treatments and (i) ImageJ relative quantitative fluorescence intensity analysis. Representative images of tumor sections stained with (j) TUNEL and (l) Ki67 from 4T1-bearing mice in different groups and corresponding quantitative analysis: (k) TUNEL⁺ cells (%) and (m) Ki67⁺ cells (%). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Overall, the combination of MnO₂-R848@CM and RT achieved excellent tumor suppression in both in vitro and in vivo 4T1 models, with complete tumor ablation observed in some mice. Nevertheless, a degree of “synergistic overflow” was observed in the final combination, meaning that the combined effect did not provide significant additional therapeutic benefit beyond the individual components while increasing the complexity of preparation. Therefore, future studies will optimize the system design to better support further preclinical antitumor evaluation. In addition, previous reports suggest that tumor cell membranes themselves may act as adjuvants to initiate downstream immune responses, because their unique surface membrane proteins can be exposed after freeze-thaw or ultrasonic treatment. However, this aspect was not fully explored in the present study. Future work will investigate the additional benefits of the cell membrane coating and the downstream immune events initiated by the MnO₂-R848@CM nanoplatform. Finally, primary resistance to immune checkpoint inhibitors (ICIs) remains a major clinical challenge. The MnO₂-R848@CM platform may sensitize tumors to ICIs by relieving hypoxia, inducing ICD, activating the cGAS-STING pathway, and delivering R848, thereby promoting DC maturation and CD8⁺ T-cell infiltration and converting immunologically “cold” tumors into “hot” tumors [34]. Although this platform was not directly combined with anti-PD-1/PD-L1 therapy in the present study, future studies will evaluate its potential to overcome ICI resistance in appropriate tumor models.

3. Conclusions

In summary, this study introduces a biomimetic MnO₂ nanosheet platform, coated with cancer cell membranes and loaded with the immunological adjuvant R848 (MnO₂–R848@CM), designed for the radioimmunotherapy of in situ breast cancer. This nanoplatform exhibits high homogenous targeting toward breast cancer cells both in vivo and in vitro. Evidence supports that MnO₂–R848@CM synergistically amplifies ROS production following RT, causing more severe DNA damage to tumor cells and promoting tumor antigen presentation by DCs through increased ICD levels. Notably, R848 and Mn²⁺ released from MnO₂–R848@CM in the tumor acidic environment further enhance adaptive immune responses through cGAS-STING signaling pathways, mediating tumor ablation in tumor-bearing mice. The MnO₂–R848@CM designed in this study offers a novel strategy to circumvent hypoxic tumor microenvironments and overcome immune tolerance at tumor sites. Collectively, these findings demonstrate that the MnO₂–R848@CM nanoplatform can significantly enhance the initial efficacy of clinical RT while eliciting sustained immunological effects, thereby improving overall antitumor outcomes and providing a promising approach for combined RT and immunotherapy in breast cancer.

CRediT authorship contribution statement

Zhen Zhang: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. Ping Liu: Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis. Weike Zeng: Writing – review & editing, Validation, Software, Methodology, Investigation, Formal analysis. Chunlin Huang: Writing – review & editing, Validation, Software, Methodology, Formal analysis. Siqing Wang: Visualization, Validation, Methodology, Investigation. Shiyao Guan: Validation, Methodology, Investigation. Yixi Duan: Validation, Methodology, Investigation. Dalin Wu: Writing – review & editing, Supervision, Project administration, Funding acquisition. Yi Zhao: Writing – review & editing, Supervision, Project administration, Funding acquisition. Jun Wu: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Experimental section

Full materials and experimental details can be found in the Supporting Information. All animal experiments procedures were performed according to the protocols approved by the Laboratory Animal Facility at the Hong Kong University of Science and Technology and the Laboratory Animal Guidelines of Welfare and Ethics (HKUST(GZ)-IACUC-2024-A0062).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.