Radiation-activated PD-L1 aptamer-functionalized nanoradiosensitizer to potentiate antitumor immunity in combined radioimmunotherapy and photothermal therapy

Abstract

Reactive oxygen species (ROS)-mediated immunogenic cell death (ICD) is crucial in radioimmunotherapy by boosting innate antitumor immunity. However, the hypoxic tumor microenvironment (TME) often impedes ROS production, limiting the efficacy of radiotherapy. To tackle this challenge, a combination therapy involving radiotherapy and immune checkpoint blockade (ICB) with anti-programmed death-ligand 1 (PD-L1) has been explored to enhance antitumor effects and reprogram the immunosuppressive TME. Here, we introduce a novel PD-L1 aptamer-functionalized nanoradiosensitizer designed to augment radiotherapy by increasing X-ray deposition specifically at the tumor site. This innovative X-ray-activated nanoradiosensitizer, comprising gold–MnO₂ nanoflowers, efficiently enhances ROS generation under single low-dose radiation and repolarizes M2-like macrophages, thereby boosting antitumor immunity. Additionally, the ICB inhibitor BMS-202 synergizes with the PD-L1 aptamer-assisted nanoradiosensitizer to block the PD-L1 receptor, promoting T cell activation. Furthermore, this nanoradiosensitizer exhibits exceptional photothermal conversion efficiency, amplifying the ICD effect. The PD-L1-targeted nanoradiosensitizer effectively inhibits primary tumor growth and eliminates distant tumors, underscoring the potential of this strategy in optimizing both radioimmunotherapy and photothermal therapy.

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1. Introduction

Over the past decades, radiotherapy has been indispensable in clinical cancer treatment due to its ability to induce reactive oxygen species (ROS) production, leading to DNA damage, tumor cell death, and release of antitumor-associated antigens.^1–6 However, the hypoxic conditions within the tumor microenvironment (TME) often restrict ROS generation, even at recommended radiation doses.^7–11 Furthermore, radiation-induced ROS typically trigger only a mild immune response, known as immunogenic cell death (ICD), which often falls short of therapeutic needs.^12–18 This suboptimal radiotherapy-mediated immunoactivation in solid tumors indicates that the immunosuppressive TME hampers the therapeutic effect of radiotherapy. Therefore, reprograming the immunosuppressive TME is a critical yet formidable challenge for effective tumor therapy.^19,20 To address this issue, various strategies have been developed to enhance radiotherapy outcomes, focusing on two key approaches: augmenting X-ray deposition within tumor sites and upregulating the ROS levels within the TME.^21–25

Local X-ray radiation can induce ICD and release damage-associated molecular patterns (DAMPs) such as calreticulin (CRT), high mobility group box 1 (HMGB1), and adenosine triphosphate (ATP).^26,27 Evidence suggests that ICD can boost the innate immune system, evoke immune memory effects, and induce an antitumor effect on distant tumors, known as the “abscopal effect”.^14,28,29 However, antitumor immunity within the TME is often hindered by immunosuppressive cell populations, notably tumor-associated myeloid cells (TAMCs) and regulatory T cells (Tregs). Effective ICD induction can promote M2 macrophage polarization, which is essential for reprogramming the immunosuppressive TME. Transforming a “cold” tumor, characterized by inadequate infiltrating effector T cells, into a “hot” tumor capable of releasing proinflammatory cytokines is a critical factor in achieving successful tumor immunotherapy.^19,30–32 Recently, combining radiotherapy and immunotherapy has emerged as a promising strategy to boost innate antitumor immunity. Immune checkpoint blockade (ICB), particularly targeting programmed death-ligand 1 (PD-L1), has exhibited remarkable progress in clinical trials.^33,34 The PD-L1 blockade can modulate the TME and enhance the activity of T cells, thereby synergizing with radiotherapy to inhibit tumor growth and metastasis.^{15,20,35–37} Further exploration of this combined approach holds great potential for optimizing tumor therapy.

High-dose radiation therapy can adversely affect normal organs, whereas inadequate radiation fails to trigger an immune response. To minimize the side effects of high-dose radiation, radiosensitizers have been employed to enhance X-ray deposition. Radiosensitizers, typically small chemical drugs or nanoparticles, increase tumors' radiosensitivity to X-rays.³⁸ Notably, nanoradiosensitizers have significantly advanced in augmenting X-ray deposition within nanoparticles. High-Z metals, particularly gold, hafnium, and lanthanide elements, are extensively used in nanoradiosensitizer fabrication to improve the antitumor efficacy and stimulate the immune response.^39–43 Of special note, gold nanoparticles, as a radiosensitizer, engender broad opportunities for radiotherapy, which is ascribed to higher radiation deposition and good biocompatibility.^44,45 Moreover, gold nanoparticles serve as excellent photothermal agents in photothermal therapy (PTT), inducing the release of HMGB1, ATP, and heat shock proteins (HSPs) from dying tumor cells, thereby activating the immune system, promoting tumor antigen presentation, and enhancing the ICD effect. Hence, utilizing gold nanoparticles in combined PTT and radiotherapy holds great potential for synergistically enhancing the efficacy of ICD treatment.^46,47

In this study, a multifunctional X-ray-activated nanoradiosensitizer was designed to orchestrate radioimmunotherapy and PTT. The biomineralization-based method was employed to construct gold-coated MnO₂ nanoparticles (BAPBM) with enhanced X-ray absorption. The PD-L1 aptamer was utilized for specific targeting of cancer cells, while the ICB inhibitor BMS-202 was loaded onto MnO₂ NPs to block the PD-L1 receptor within cancer cells (Fig. 1). Previous studies have shown that oxygen produced by MnO₂ could relieve tumor hypoxia and increase ROS generation, which would enhance X-ray-induced cell apoptosis.⁴⁸ The PD-L1 aptamer-functionalized nanoradiosensitizer effectively inhibited tumor growth and metastasis even under single low-dose irradiation, promoting CD8⁺ T cell activation and reprogramming the immunosuppressive TME. Furthermore, the excellent photothermal conversion efficiency of BAPBM enhanced the ICD effect, triggering a strong abscopal effect. This study successfully developed an efficient nanoradiosensitizer, offering a promising strategy to combine radioimmunotherapy and PTT for the treatment of breast cancer.

Fig. 1 A schematic diagram of the preparation of BAPBM as a multifunctional nanoradiosensitiser to enhance radioimmunotherapy and photothermal therapy.

2. Materials and methods

2.1 Materials

AuNPs were purchased from XFNANO Company (Nanjing, China). 8-Arm-PEG-SG was purchased from Creative PEGWorks. Dopamine hydrochloride (DA.HCl) and KMnO₄ were obtained from Sigma-Aldrich. Antibodies (anti-CD11c-APC, anti-CD80-FITC, anti-CD86-PE, anti-CD45-percp, anti-CD3-PE/Cy7, anti-CD4-FITC, anti-CD8a-APC/Cy7, anti-CD69-APC, anti-CD62L-FITC, anti-CD44-PE, anti-CD-11b-FITC, anti-F4/80-PE and anti-CD206-APC) for flow cytometry analysis were purchased from Biolegend (California, USA). An anti-CRT recombinant monoclonal antibody and an anti-HMGB1 monoclonal antibody were purchased from HUABIO (Hangzhou, China). γ-H₂AX was obtained from CST. The PD-L1 aptamer (sequence: ACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGT-NH₂) was provided by Tsingke Biotech.

2.2 Preparation of BAPBM NPs

8-Arm-PEG-DA was prepared according to the following steps. Firstly, 400 mg of 8-arm-PEG-SG and 152 mg of DA.HCl were dissolved in 20 mL of anhydrous DMF under nitrogen protection. Subsequently, 80 µL of TEA was added to the above solution and stirred at room temperature for 24 h. The mixture was then purified by dialysis. To prepare Au-PEG NPs, 5 mg of 8-arm-PEG-DA was dissolved in 10 mL of AuNPs and stirred overnight at room temperature. Au-PEG NPs were obtained by centrifugation (12 000 rpm, 15 min) and washed with PBS twice. To prepare APBM NPs, 10 mL of Au-PEG NPs were ultrasonically dispersed in water. Then, 100 µL of KMnO₄ (10 mg mL⁻¹) was added to the above mixture and stirred for 2 h at room temperature. The mixture was obtained by centrifugation (12 000 rpm, 15 min) and dispersed in PBS (pH 7.4). The PD-L1 aptamer was added to the above mixture and incubated overnight. For the synthesis of BAPBM, 5 mg of BMS-202 was added to the above mixture and stirred for 12 h at room temperature.

2.3 Cellular uptake

4T1 cells were seeded into 24-well plates at a density of 4 × 10⁴ per well and cultured overnight. Then, the cells were incubated with BAPBM-Cy5 for 1, 2, and 4 h. Then, the 4T1 cells were washed with PBS and stained with DAPI and β-actin.

2.4 Intracellular ROS generation

4T1 cells were seeded into 24-well palates at a density of 6 × 10⁴ per well and cultured overnight. After this, PBS, free BMS-202, APBM, and BAPBM (with 4 Gy RT and PTT) were added and stained with DCFH-DA (10 µM) in the dark for 30 min. The green fluorescence images were obtained by confocal microscopy.

2.5 Cell viability, apoptosis, and colony formation

For the CCK-8 assay, 4T1 cells were seeded into 96-well plates at a density of 3 × 10³ per well and cultured overnight. Then, the cells were incubated with free BMS-202 and BAPBM (with 4 Gy RT and 808 nm laser) and cultured for 24 h. After this, the cell viability was performed using the CCK-8 assay. For cell apoptosis, the 4T1 cells were pretreated with PBS, free BMS, APBM, and BAPBM (with RT and PTT) and cultured for 24 h. Finally, the 4T1 cells were stained with Annexin-V FITC and 7AAD and detected using a flow cytometer. 4T1 cells were seeded into 24-well plates at a density of 2 × 10⁴ per well and cultured overnight. The cells were incubated with PBS, free BMS-202, APBM, and BAPBM (with 4 Gy RT and PTT). After 6 h co-culturation, 4T1 cells were stained with Calcein-AM and propidium iodide (PI) for 30 min at room temperature. The cells were observed using a fluorescence microscope.

4T1 cells were seeded into 6-well plates at a density of 600 per well and cultured for 24 h. After this, the groups were incubated with PBS, free BMS, APBM, and BAPBM (with 4 Gy RT and PTT) and cultured for 7 days. The cell clones were fixed with 4% paraformaldehyde and stained with crystal violet.

2.6 DNA damage detection

The levels of DNA double-strand breaks were detected by γ-H₂AX immunofluorescence staining. 4T1 cells were added into a confocal dish at a density of 2 × 10⁴ per well and cultured overnight. The cells were incubated with PBS, free BMS-202, APBM, and BAPBM (with 4 Gy RT and PTT). After this, 4T1 cells were stained by a γ-H₂AX mouse monoclonal antibody (CST, USA) and a secondary antibody conjugated with Alexa Fluor 594 (HUABIO, China) for 1 h and 12 h, respectively. DNA damage images were collected by laser scanning confocal microscopy.

2.7 Biodistribution of BAPBM NPs

4T1 tumor-bearing mice were treated with free Cy5, BAPBM(n)-Cy5 (without the PD-L1 aptamer) and BAPBM-Cy5 (with the PD-L1 aptamer) via intravenous injection. The biodistribution images were collected using an IVI Spectrum system (Caliper, Hopkinton, MA, USA) at different time points.

2.8 In vivo antitumor effects and immune responses

4T1-bearing tumor models were established by injecting 1 × 10⁶ cells into the fat pad of the breast. To evaluate the synergistic antitumor effect of RT and PTT, mice were divided into eight groups (n = 6): G1: PBS, G2: PBS + RT, G3: BMS-202, G3: BMS-202 + RT, G5: APBM + RT, G6: APBM + RT + PTT, G7: BAPBM + RT, and G8: BAPBM + RT + PTT. The doses of BMS-202 and APBM were 10 mg kg⁻¹ and 5 mg kg⁻¹, respectively. The mice were only treated once, and tumor volume and weight were recorded every two days. The tumor tissues were collected for H&E, Ki-67, γ-H₂AX, and TUNEL. All animal experiments have been approved by the Sichuan University Animal Care and Use Committee and conformed to the Principles of Laboratory Animal Care formulation by The National Society for Medical Research.

To investigate the DC maturation induced by BAPBM (with 4 Gy X-ray RT and PTT), tumor-draining lymph nodes (TDLNs) were collected and blocked with a goat serum for 30 min. Single-cell suspensions were stained with anti-CD11b-APC, anti-CD80-FITC, and anti-CD86-PE (Biolegend, USA). To detect the CD8⁺ T cell infiltration, tumor tissues of the treated mice were harvested with collagenase (1 mg mL⁻¹) containing DNase I (2 U mL⁻¹) to prepare single-cell suspensions. The single cell suspensions were treated with red blood cell lysis buffer for 15 min. Then, tumor cells were stained with the Zombie Red Fixable Viability Kit, anti-CD45-Percp, anti-CD3-PE-Cy7, anti-CD4-FITC, anti-CD8a-APC/Cy7, and anti-CD69-APC (Biolegend, USA) for flow cytometry analysis. Besides, tumor tissues were collected for immunofluorescence to further evaluate tumor-infiltrating T lymphocytes.

2.9 Statistical analysis

A two-tailed Student's t-test was used to analyze the differences between the two groups. Data were shown as means ± SD and analyzed by GraphPad Prism. A p-value of < 0.05 represents statistically significant.

3. Results and discussion

3.1 Preparation and characterization of BAPBM NPs

In this study, we investigated the formation of BAPBM NPs using a simple biomineralization method. Initially, 8-arm-PEG-DA was covalent to gold nanoparticles (AuNPs) to form Au-PEG-DA, which was subsequently employed in the fabrication of BAPBM NPs. Transmission electron microscopy (TEM) analysis demonstrated that BAPBM NPs had an average size of approximately 72.5 nm (Fig. 2A and Fig. S1, ESI†). The gold nanoparticles were uniformly distributed on the surface of MnO₂, resulting in a distinctive nanoflower-like morphology (Fig. 2A and B). High-resolution TEM (HRTEM) was employed to observe the lattice parameters of the AuNPs, which corresponded to the (111) plane (Fig. 2B and Fig. S2, ESI†). Dynamic light scattering (DLS) analysis revealed that the average hydrodynamic diameters of AuNPs, Au-PEG, and BAPBM were approximately 19.8 nm, 50.4 nm, and 86 nm, respectively (Fig. 2C). EDS elemental mapping images showed the uniform distribution of Au, Mn, O, and P, confirming the successful synthesis of BAPBM NPs (Fig. 2D).

Fig. 2 Synthesis and characterization of the BAPBM nanoradiosensitizer. (A) and (B) High-resolution transmission electron microscopy (HRTEM) images of BAPBM and AuNPs. (C) Hydrodynamic diameters of AuNPs, Au-PEG, and BAPBM NPs. (D) High angle annular dark field-scanning TEM (HAADF-STEM) image and EDS mapping images of BAPBM NPs, including Au, Mn, O, and P. The scale bar is 20 nm. (E) H-NMR spectra of 8-arm-PEG-DA. (F) Agarose gel electrophoresis images of AuNPs, Au-PEG, and BAPBM NPs. (G) In vitro and (H) in vivo photothermal images of PBS, APBM, and BAPBM NPs.

Moreover, X-ray photoelectron spectroscopy (XPS) analysis of BAPBM revealed the typical peaks ascribed to Au 4f, Mn 2p, C 1s, N 1s, and O 1s, further confirming the presence of Mn(IV) dominated materials and gold nanoparticles within the sample (Fig. S3, ESI†). As shown in Fig. S4 (ESI†), the zeta potential values of AuNPs, Au-PEG, and BAPBM were observed to be −36.8 mV, −12.6 mV, and −3.8 mV, respectively. The increase in the zeta potential value of BAPBM indicated the successful biomineralization process of Au-PEG. Besides, BAPBM NPs maintained long-term stability under physiological conditions (Fig. S5, ESI†). The structure of 8-arm-PEG-DA was confirmed by H-NMR spectroscopy, and the modification efficacy was assessed by comparing the integral value of methylene to the aromatic protons of catechol (Fig. 2E). These findings provided robust evidence for the successful synthesis and characterization of the BAPBM NPs.

To further verify the successful fabrication of BAPBM NPs, agarose gel electrophoresis was employed. As shown in Fig. 2F, bare AuNPs appeared as a black color and aggregated in the origin point. In contrast, the red color exhibited by Au-PEG NPs indicated the successful modification of 8-arm-PEG-DA onto AuNPs, demonstrating excellent dispersibility. Interestingly, the negatively charged Au-PEG migrated from the positive electrode to the negative electrode, which could be attributed to the synergistic influence of coupling forces and hydrodynamic drag. The photothermal efficiency of the nanoradiosensitizer was investigated by recording thermal images to monitor the temperature changes of APBM and BAPBM NPs under 808 nm laser irradiation (Fig. 2G, H, and Fig. S6, ESI†). Compared to the PBS control group, both APBM and BAPBM NPs exhibited rapid temperature increases, reaching 43.8 °C and 44.2 °C, respectively. BAPBM NPs showed excellent photothermal stability, as evidenced by the consistent temperature increase (Fig. 2G). Furthermore, in vivo thermal images of APBM and BAPBM NPs revealed a significant temperature increase at the tumor region, reaching 44.3 °C and 44.7 °C, respectively, within 5 minutes (Fig. 2H). These results confirm the excellent photothermal stability and conversion efficiency of the BAPBM nanoradiosensitizer.

3.2 In vitro therapeutic efficacy

To investigate the temporal cellular uptake of BAPBM NPs in 4T1 cells, Cy5-labeled BAPBM was employed. Confocal laser scanning microscopy (CLSM) images revealed the distribution of Cy5-BAPBM NPs in the cytoplasm, with the red fluorescence gradually increasing over time. This finding indicated that BAPBM NPs were effectively absorbed by 4T1 cells (Fig. 3A and Fig. S7, ESI†). The radiosensitization efficacy of BAPBM NPs was evaluated by measuring intracellular ROS generation in 4T1 cells using the DCFH-DA fluorescence probe. As depicted in Fig. S8 (ESI†), the fluorescence intensity of ROS in the BAPBM group (with X-ray and PTT) was significantly higher than that in the other groups, indicating that the combination of low-dose X-ray and PTT augmented the production of toxic ROS. Moreover, DNA damage, a primary characteristic induced by X-ray, was measured using the γ-H₂AX immunofluorescence assay. Red fluorescence was observed in the cell nucleus of the group treated with X-ray alone, whereas BAPBM NPs (with X-ray and PTT) exhibited strong γ-H₂AX fluorescence, suggesting that the nanoradiosensitizer significantly increased DNA damage (Fig. 3D). These results indicated that BAPBM NPs effectively enhanced the radiosensitization of cancer cells and increased the production of toxic ROS, underscoring their potential utility in cancer therapy.

Fig. 3 In vitro antitumor effects of BAPBM NPs. (A) Cellular uptake of Cy5-labeled BAPBM NPs. The scale bar is 50 µm. (B) Cell apoptosis analysis in 4T1 cells after treatment with radiotherapy and PTT. (C) Live/dead double staining of 4T1 cells; the scale bar is 100 µm. (D) Representative immunofluorescence images of γ-H₂AX in 4T1 cells treated with radiotherapy and PTT; the scale bar is 25 µm. (E) Representative colony formation images of 4T1 cells under different treatments. (F) Cellular ATP levels and (G) cell viability of 4T1 cells under different treatments. G1: PBS, G2: PBS + X-ray (4 Gy), G3: BMS, G4: BMS + X-ray, G5: APBM + X-ray, G6: APBM + X-ray + NIR (0.5 W cm⁻², 5 min), G7: BAPBM + X-ray, and G8: BAPBM + X-ray + NIR. Data were given as mean ± S.D. Two-tailed Student's t-test, *P < 0.05. **P < 0.01. ***P < 0.001.

The release of DAMPs, including ATP, is a key marker of ICD and can activate an immune response. The secretion of ATP was significantly increased in the BAPBM group (with X-ray and PTT), suggesting its potential contribution to triggering ICD (Fig. 3F). Moreover, oxygen can amplify the interaction between DNA molecules and X-ray radiation, thereby impeding DNA repair and improving the therapeutic efficacy of radiotherapy in cancer cells. The oxygen-generating potential of the nanoradiosensitizer was evaluated using the [Ru(dpp)₃]Cl₂ (RDPP) probe, which undergoes fluorescence quenching in the presence of O₂. As displayed in Fig. S9 (ESI†), the fluorescence intensity of RDPP in the BAPBM and APBM NPs significantly decreased, indicating the generation of O₂ facilitated by the reaction between MnO₂ and endogenous H₂O₂.

To assess the in vitro anticancer effects of BAPBM NPs, cytotoxicity was detected using the CCK-8 assay. The BAPBM group (with X-ray and PTT) effectively reduced the viability of 4T1 cells, and the addition of a single low-dose X-ray enhanced the cytotoxic effects of the nanosystem (Fig. 3G and Fig. S10, ESI†). An apoptosis assay measured by flow cytometry demonstrated that BAPBM NPs (with X-ray and PTT) exhibited an apoptosis rate of 95%, higher than that observed in the other groups (Fig. 3B). Similar results were observed in the live/dead double-staining assay (Fig. 3C). The combination of radioimmunotherapy and PTT effectively promoted the anticancer effect. The therapeutic efficacy of the nanoradiosensitizer was further assessed through a colony formation assay, which showed that only a few colonies were present in the BAPBM group (with X-ray and PTT), suggesting its excellent ability to inhibit proliferation (Fig. 3E). These findings demonstrate the strong and effective anticancer effects of BAPBM NPs, especially when combined with X-ray radiation and PTT, highlighting their potential as a promising therapeutic strategy for cancer treatment.

3.3 In vivo fluorescence targeting of tumors

Accurately distinguishing tumors from their boundaries is crucial for maximizing cancer cell killing and minimizing side effects. To determine tumor-targeting specificity in vivo, 4T1 tumor-bearing mice were intravenously injected with Cy5-labeled BAPBM NPs. As shown in Fig. S11 (ESI†), BAPBM NPs displayed efficient accumulation at tumor sites, with a gradual increase in fluorescence intensity over time. Ex vivo images of major organs (heart, liver, spleen, lungs, kidneys, and tumor) revealed the highest fluorescence intensity in the tumor, indicating effective targeting by the PD-L1 aptamer-modified nanoradiosensitizer. In contrast, mice treated with free Cy5 did not show noticeable accumulation in the tumor (Fig. S12, ESI†). Subsequently, the tumors collected at 24 h were stained with DAPI to assess the distribution of the nanoradiosensitizer within the tumor tissue (Fig. S13, ESI†). The results above confirmed that the PD-L1 aptamer-modified nanoradiosensitizer possesses effective tumor-targeting ability.

3.4 Antitumor effects of BAPBM NPs

To evaluate the in vivo antitumor effects of BAPBM NPs and assess the therapeutic efficacy of combined radioimmunotherapy and PTT, a 4T1-bearing tumor model was established. Mice were randomly divided into eight groups and intravenously injected with PBS, PBS + RT, BMS-202, BMS-202 + RT, APBM + X-ray, APBM + X-ray + PTT, BAPBM + X-ray, and BAPBM + X-ray + PTT. As shown in Fig. 4A, a single low-dose 4 Gy X-ray radiation was administered on day 1 to boost the innate immune response. The results revealed that BAPBM NPs with radiotherapy and PTT effectively suppressed the growth of 4T1-bearing tumors. Conversely, the low-dose X-ray radiation treatment in the PBS group did not effectively retard tumor growth due to the inadequate activation of the immune system (Fig. 4B).

Fig. 4 In vivo antitumor effects of BAPBM NPs. (A) A schematic diagram of combined radioimmunotherapy and PTT. (B) Ex vivo images of tumors after different treatments. (C) Tumor growth curves and (D) the weight of mice after different treatments. (E) Immunohistochemistry (HE, Ki-67, and γ-H₂AX) and immunofluorescence (TUNEL) images of tumor sections; the scale bar is 100 µm. G1: PBS, G2: PBS + X-ray, G3: BMS, G4: BMS + X-ray, G5: APBM + X-ray, G6: APBM + X-ray (4 Gy) + NIR (0.5 W cm⁻², 5 min), G7: BAPBM + X-ray, and G8: BAPBM + X-ray + NIR. Data were given as mean ± S.D. Two-tailed Student's t-test, *P < 0.05. **P < 0.01. ***P < 0.001.

Notably, BAPBM NPs combined with radiotherapy and PTT exhibited a remarkable reduction in tumor growth (Fig. 4C). No significant weight loss was observed following the administration of the various treatments (Fig. 4D). To further evaluate the in vivo antitumor effects, tumor tissues were collected and stained with H&E, Ki-67, TUNEL, and γ-H₂AX, respectively (Fig. 4E). These results highlighted that the BAPBM group (with X-ray and PTT) effectively suppressed cancer cell proliferation and induced substantial cell apoptosis in the tumors. In the BAPBM group (with X-ray and PTT), a high percentage of γ-H₂AX positive cells was observed in tumor slices, indicating significant DNA damage through radioimmunotherapy. Additionally, no noticeable side effects were observed in the major organs, highlighting the excellent biosafety of the treatments (Fig. S14, ESI†). These findings demonstrate the strong synergistic effects of BAPBM NPs with radioimmunotherapy and PTT in suppressing tumor growth, highlighting their potential as a promising strategy for cancer treatment with excellent safety profiles.

3.5 Antitumor immunity of BAPBM NPs

To investigate the underlying mechanism of antitumor immunity evoked by radioimmunotherapy and PTT, dendritic cells (DCs) were identified as key players in initiating and maintaining antitumor T cell immunity.^49,50 Upon maturation, DCs migrate to the lymph nodes and present cancer antigens to CD8⁺ T cells. The maturation of DCs in the lymph node was measured by flow cytometry. These results showed that mice treated with BAPBM (with X-ray and PTT) promoted the highest levels of DC maturation in the lymph nodes, thereby amplifying the antigen presentation capacity of DCs, as demonstrated in Fig. 5A and E. The immunosuppression of the TME is closely related to immune evasion and tumoral progression, ultimately restricting the therapeutic effectiveness of antitumor immunity. Converting a “cold tumor” into a “hot tumor” optimizes the immune response, leading to strong cytotoxic effects on the tumor, favorable prognosis, and improved response to immunotherapy.^51,52 Following treatment with BAPBM NPs (with X-ray and PTT), a significant reduction in the intratumoral populations of M2 phenotype macrophages was observed, reversing the immunosuppressive TME within the tumor (Fig. 5B and E).

Fig. 5 In vivo antitumor immunity. (A) Quantitative analysis of matured DCs (CD86⁺CD80⁺ in CD11c⁺ cells) in tumor-draining lymph nodes. (B) Quantitative analysis of the M2 phenotype macrophage (CD11b⁺F4/80⁺CD206⁺) in tumors after different treatments. (C) Quantitative analysis of CD8⁺ T cells and CD4⁺ T cells in tumors after different treatments. (D) The expression levels of CD69 on CD8⁺ T cells. (E) Percentages of matured DCs (CD86⁺CD80⁺CD11c⁺), the M2 phenotype macrophage (CD11b⁺F4/80⁺CD206⁺), CD8⁺ T cells, and CD4⁺ T cells. (F) Immunofluorescence images of CRT and HMGB1; the scale bar is 100 µm. G1: PBS, G2: PBS + X-ray, G3: BMS, G4: BMS + X-ray, G5: APBM + X-ray, G6: APBM + X-ray (4 Gy) + NIR (0.5 W cm⁻², 5 min), G7: BAPBM + X-ray, and G8: BAPBM + X-ray + NIR. Data were given as mean ± S.D. Two-tailed Student's t-test, *P < 0.05. **P < 0.01. ***P < 0.001.

The activation of CD8⁺ T cells plays a vital role in antitumor immunity. Tumor-infiltrating CD8⁺ T cells were measured in each group by flow cytometry assays. Remarkably, the BAPBM group (with X-ray and PTT) showed the highest proportion of CD8⁺ T cells, with over 50%, while the PBS group exhibited less than 9.8% (Fig. 5C and E). Besides, the combination of radioimmunotherapy and PTT increased the infiltration of CD8⁺ T cells within the tumor (Fig. S15, ESI†). Furthermore, the activation marker, CD69, which plays an important role in stimulating CD8⁺ T cells and promoting antitumor immune responses, is considered an important target for immunotherapy.⁵¹ The population of CD69⁺ CD8⁺ T cells was significantly enhanced in the BAPBM group (with X-ray and PTT), indicating a stronger activation of the immune response. These results confirmed that the combination of radioimmunotherapy with PTT effectively triggered robust antitumor immunity (Fig. 5D).

The strategy of radiotherapy-induced ICD holds considerable promise for enhancing immunotherapy efficacy, improving tumor responsiveness to treatment, and reducing the risk of tumor recurrence and metastasis.^53,54 Moreover, it represents a compelling approach for eliciting an innate systemic immune response by enhancing radiotherapy-induced ICD. Notably, CRT and HMGB1 have been identified as important biomarkers for inducing ICD. Immunofluorescence images demonstrated that radioimmunotherapy significantly induced surface expression of CRT on tumor cells, while HMGB1 was detected in the extracellular space after being released from the nucleus (Fig. 5F). These results above suggest that BAPBM NPs (with X-ray and PTT) have strong potential to induce antitumor immunity by enhancing DC maturation, reversing the immunosuppressive TME, promoting CD8⁺ T cell activation, and inducing ICD.

3.6 Abscopal effect

Tumor metastasis is a common and life-threatening complication of cancer that can be effectively mitigated by the abscopal effect induced by radiotherapy. This phenomenon activates the immune system to target tumor cells beyond the treatment site.⁵⁰ To evaluate the impact of the abscopal effect on distant tumors, we established the 4T1 bilateral tumor model, as shown in Fig. 6A. Consistent with the observations from the unilateral tumor model, mice treated with BAPBM (X-ray and PTT) displayed suppression of primary tumor growth and effectively inhibited the growth of untreated distant tumors. In contrast, the PBS + RT, BMS, and BMS + RT groups showed no inhibitory effect on both primary and distant tumors (Fig. 6B–D). To investigate the mechanism underlying the abscopal effect, the immune responses elicited by different treatments were studied. Lymph nodes near the primary tumor were collected and analyzed by flow cytometry. Notably, the BAPBM group (with X-ray and PTT) exhibited a markedly higher capacity for stimulating DC maturation compared to the other groups (Fig. 6E and H).

Fig. 6 The abscopal effect of the bilateral tumor models after different treatments. (A) A schematic diagram of the establishment of 4T1 bilateral tumor models and therapeutic outcomes. Tumor growth curves of (B) primary tumors and (C) distant tumors after different treatments. (D) Tumor weights of excised primary tumors after different treatments. (E) Quantitative analysis of matured DCs (CD86⁺CD80⁺ in CD11c+ cells) in tumor-draining lymph nodes. (F) Representative flow cytometry plots of T_EM (CD44⁺CD62L⁻ in CD8⁺ T cells). (G) Representative flow cytometry plots of CD8⁺ T cells and CD4⁺ T cells in primary and distant tumors. (H)–(J) Percentages of matured DCs (CD86⁺CD80⁺CD11c⁺), T_Em (CD44⁺CD62L⁻ in CD8⁺ T cells), and CD8⁺ T cells. G1: PBS, G2: PBS + X-ray, G3: BMS, G4: BMS + X-ray, G5: APBM + X-ray, G6: APBM + X-ray + NIR, G7: BAPBM + X-ray, and G8: BAPBM + X-ray + NIR. Data were given as mean ± S.D. Two-tailed Student's t-test, *P < 0.05. **P < 0.01. ***P < 0.001.

Next, the proportion of effector memory T cells (T_Em, CD3⁺CD8⁺CD44⁺CD62L⁻) in splenocytes was analyzed across different experimental groups. The synergistic combination of radioimmunotherapy and PTT elicited a robust and sustained immune response against tumor recurrence, highlighting the potential of long-term immune surveillance (Fig. 6F and I). The infiltration of cytotoxic T cells (CD8⁺ T) plays an indispensable role in regulating the TME and converting a “cold” tumor to a “hot” tumor. As shown in Fig. 6G and J, the combination of radioimmunotherapy and PTT significantly increased the proportion of CD8⁺ T cells in both primary and distant tumors. Collectively, the combined therapy of radioimmunotherapy and PTT effectively triggered an abscopal effect to suppress tumor metastasis. These findings hold significant potential for developing effective cancer treatments with improved outcomes.

4. Conclusions

In conclusion, we have developed a novel nanoplatform, termed BAPBM, for the specifically targeted delivery of the nanoradiosensitizer and ICB inhibitors BMS-202. This nanoplatform has shown remarkable efficacy as a nanoradiosensitizer, enhancing X-ray deposition within the cancer cells and increasing ROS production, thus exhibiting tremendous potential in ICD-induced radioimmunotherapy. Activated by a single low-dose X-ray, the BAPBM nanoradiosensitizer effectively triggered an antitumor immune response and alleviated the immunosuppressive characteristics of the TME. This multifunctional nanoradiosensitizer not only activates cytotoxic immune cells but also repolarizes M2-like macrophages to an M1-like phenotype, fostering an environment conducive to antitumor activity. Importantly, this novel nanoradiosensitizer efficiently eliminated primary tumors and prevented tumor metastasis by stimulating the immune system. By reprogramming the immunosuppressive TME, it facilitated the infiltration of cytotoxic CD8⁺ T cells, resulting in a substantial suppression of both primary and distant tumor growth. This approach induces a robust antitumor immune response, significantly enhancing therapeutic outcomes and achieving long-term tumor control. In summary, the innovative integration of radioimmunotherapy with PTT using the BAPBM nanoradiosensitizer offers promising prospects for breast cancer treatment.

Author contributions

Bo Chen, Bingwen Zou and Gang Guo: conceptualization, methodology and writing – original draft. Yinbo He and Long Bai: performed the radiotherapy experiments. Bo Chen, Shulin Pan and Yinggang Wang: investigation, methodology, and data curation. Rangrang Fan and Min Mu: performed the animal experiments. Bo Han and Gang Guo: supervision and funding acquisition. Peter Huber, Bingwen Zou and Gang Guo: supervision, project administration and writing – review and editing.

Data availability

All relevant data are within the manuscript and its ESI.† And detailed data are available from the corresponding author upon reasonable request.

Conflicts of interest

The authors declare no competing interests.

Acknowledgements

This work was financially supported by National Natural Sciences Foundation of China (31971308 & 82102767), National S&T Major Project (2019ZX09301-147) and Sichuan Science and Technology Program (2022YFS0007 & 22GJHZ0015), Natural Science Foundation of Sichuan Province (2023NSFSC1866).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb01831a

‡ These authors contributed equally to this work.

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