A pH-responsive layered double hydroxide nanoradiosensitizer for bone metastasis tumor

Abstract

Breast cancer bone metastasis causes severe complications, yet current radiotherapy has limitations in treating secondary metastases and protecting healthy tissues. This study introduces calcium gadolinium layered double hydroxide (CaGd-LDH), a novel nanoradiosensitizer combining gadolinium (Gd) and calcium (Ca²⁺) in a layered double hydroxide (LDH) structure. This design enables dual therapeutic functionalities to realize high Z element Gd-mediated radiosensitization and pH-responsive Ca²⁺ release to trigger pyroptosis-induced immunotherapy, addressing both local tumor control and metastatic spread in breast cancer bone lesions. In vitro, it induces reactive oxygen species production, DNA damage, and pyroptosis via caspase-1/GSDMD pathway activation, which can significantly suppresses breast cancer cell proliferation. In vivo, CaGd-LDH combined with X-ray irradiation inhibits tumor growth, reduces cell proliferation, activates pyroptosis, and suppresses lung metastasis in a breast cancer bone metastasis mouse model. This multifunctional CaGd-LDH integrates radiosensitization, pyroptosis induction, and good biocompatibility, offering a promising strategy to improve local therapy and inhibit distant metastasis in breast cancer bone metastasis.

Graphical abstract

Highlights

• •CaGd-LDH nanoplatform was firstly used in radiosensitzation.
• •It enables pH-responsive radiosensitization and pyroptosis.
• •Acidic tumor microenviroment triggers Ca²⁺ release, inducing calcium overload.
• •Calcium overload activates the caspase-1/GSDMD pathway, triggering pyroptosis.
• •It synergistically suppresses local and metastatic tumors.

1. Introduction

Breast cancer is the most prevalent form of malignancy among the global female population, with an estimated 2.3 million new cases reported worldwide in 2022 [1]. The skeleton is the most common site of distant metastasis in breast cancer, accounting for 65-75% of all metastatic cases [2]. Bone metastases often lead to skeletal-related events, such as spinal cord compression, hypercalcemia, and pathological fractures, significantly impairing patients’ quality of life [3]. Moreover, breast cancer bone metastases are characterized by high rates of recurrence and secondary metastasis, with tumor cells in approximately two-thirds of cases disseminating to other organs, ultimately contributing to patient mortality [4]. Current therapeutic strategies for breast cancer bone metastasis include surgery, radiotherapy, chemotherapy, and immunotherapy [5]. Among these, radiotherapy is frequently employed to alleviate pain, reduce fracture risk, and target solitary or localized bone metastases [6]. However, radiotherapy is limited in its scope, exhibits suboptimal efficacy against secondary metastases, and may cause collateral damage to surrounding healthy tissues [7]. Consequently, there is an urgent need to develop radiosensitization strategies that can simultaneously address local tumor control and metastatic spread in breast cancer bone lesions (see Scheme 1).

Scheme 1.

Schematic illustration of the therapeutic principle.

To enhance tumor radiosensitivity, numerous nanomaterials incorporating high atomic number (high-Z) elements (e.g., hafnium [8], tungsten [9], platinum [10], and gadolinium [11]) have been developed as radiosensitizers. These elements exhibit a strong absorption capacity for X-rays, and their accumulation in tumor tissues can significantly enhance localized radiation dose deposition, amplifying the cytotoxic effects on tumor cells while minimizing damage to adjacent healthy tissues [12]. For instance, AGuIX, a nanoparticle primarily composed of gadolinium (Gd) that has been granted Fast Track designation by the U.S. Food and Drug Administration for use as a next-generation radiosensitizer in the treatment of malignant gliomas [13]. With improved local therapeutic efficacy, the next challenge lies in improving prognosis and suppressing recurrence and metastasis. Activating the immune system provides a promising solution [14]. Among various immune activation mechanisms, pyroptosis has emerged as a novel effective strategy [15]. This new form of programmed cell death, facilitated by Gasdermin family proteins, is characterized by cell swelling, membrane rupture, and the release of pro-inflammatory intracellular contents, eliciting a robust immune response. In breast cancer, the expression of Gasdermin D (GSDMD) is particularly elevated compared to surrounding normal tissues [16]. Targeting GSDMD enables selective induction of pyroptosis in cancer cells while sparing normal. Metal ions overload-mediated pyroptosis, particularly calcium (Ca²⁺) overload, has emerged as an effective strategy to activate pyroptosis [17]. Studies have shown that high concentrations of Ca²⁺ activate the NLRP3/caspase-1/GSDMD pyroptosis pathway via reactive oxygen species (ROS) [18]. For instance, Li et al. developed CCAH (CUR@CaCO₃-PArg@HA), a nanomodulator that induces GSDMD-mediated pyroptosis via synergistic calcium regulation, offering a novel cancer therapy strategy [19]. Zhang et al. demonstrated that calcium overload can activate pyroptosis and exhibits promising therapeutic effects in the treatment of breast cancer [20]. Furthermore, Ca²⁺ overload exhibits a synergistic effect with radiotherapy. Mitochondrial Ca²⁺ overload disrupts the electron transport chain, driving ROS production and mitochondrial dysfunction in a feedback loop. In radiotherapy, X-ray irradiation generates ROS, which is synergistically amplified by Ca²⁺-induced mitochondrial ROS, overwhelming antioxidant defenses, enhancing DNA damage, and promoting cancer cell death. As Wang et al. designed a dual-channel calcium nanomodulator that induces Ca²⁺ overload, ultimately enhancing tumor radiosensitivity [21]. In the study by Qian et al., a synergistic enhancement of the efficacy of radiotherapy and immunotherapy was achieved by reprogramming the calcium dynamics within the tumor microenvironment [22]. This dual role of Ca²⁺ in both inducing pyroptosis and enhancing radiosensitization underscores its therapeutic potential in the context of breast cancer bone metastasis. Therefore, for the radiotherapy of breast cancer bone metastasis, combining high-Z elements with calcium ions to enhance local therapeutic efficacy, induce pyroptosis, and inhibit distal metastasis may serve as an effective strategy.

To this end, layered double hydroxides (LDHs), which consist of positively charged metal layers and interlayer anions, have garnered considerable attention in biomedicine due to their good biocompatibility and tunable physicochemical properties [23]. Initially applied in the early 21st century as the active component of the antacid and anti-pepsin drug Talcid, LDHs have since been extensively explored for applications in drug delivery, immunotherapy, and bone diseases treatment, among other applications [24]. The compositional versatility of LDHs allows the incorporation of different metal element, enabling the above dual functionalities: physical radiosensitization for radiotherapy and pyroptosis activation. Concomitantly, LDHs demonstrate sensitivity to acidic conditions, thereby enabling targeted delivery to the acidic tumor microenvironment. Specifically, this approach meets the dual objectives of treating breast cancer bone metastases while simultaneously inhibiting their further spread. Based on this rationale, we synthesized calcium gadolinium layered double hydroxide (CaGd-LDH) as a novel radiosensitizer, leveraging the properties of high-Z elements (Gd) and Ca overload-induced pyroptosis. The accumulation of Gd in tumor tissues enhances localized X-ray energy deposition, generating ROS upon irradiation, which significantly improves radiosensitivity. While Ca²⁺ release in the acidic tumor microenvironment induces calcium overload and pyroptosis. Specifically, the process activates caspase-1, leading to the cleavage of GSDMD and the initiation of pyroptosis, which plays a crucial role in improving the prognosis of bone metastasis and reducing secondary metastasis. In summary, we have for the first time developed this multifunctional CaGd-LDH as an efficient nanoradiosensitizer for both local tumor control and metastatic spread, which offers a promising strategy for enhancing cancer therapy.

2. Results and discussion

2.1. Preparation and characterization of CaGd-LDH

The distinctive ability of Gd³⁺ and Ca²⁺ to regulate cell death, coupled with their synergistic effect, provides ideas for the development of radiosensitizer and pyroptosis inducer. Based on this, we synthesized CaGd-LDH using a co-precipitation method (Fig. 1a) [25]. Scanning electron microscopy (SEM) revealed that CaGd-LDH exhibited the typical hexagonal morphology with an average diameter of ∼80 ± 10 nm (Fig. 1b). The elemental composition and elemental mapping analysis revealed the uniform distribution of Ca and Gd on the radiosensitizer (Fig. 1c). The X-ray diffraction (XRD) pattern of the CaGd-Cl-LDH exhibited a high degree of agreement with the pattern recorded in PDF 31-0245 in the database of the International Centre for Diffraction Data. This confirmed the successful synthesis of CaGd-LDH (Fig. 1d). X-ray photoelectron spectroscopy (XPS) also confirmed the presence of Ca and Gd within the material (Fig. 1e). The XPS spectrum of Ca 2p showed two major peaks with binding energies at ∼346.83 and ∼359.63 eV, corresponding to Ca 2p_3/2 and Ca 2p_1/2, respectively (Fig. 1f). The binding energy peaks at ∼141.70 and ∼148.10 eV in the Gd 4d spectrum were observed for Gd 4d_5/2 and Gd 4d_3/2, respectively (Fig. 1g). Furthermore, the results demonstrate that the material exhibits a zeta potential of approximately +20.2 mV, consistent with the characteristic positive charge of LDHs (Fig. 1h). The positively charged LDHs can interact with the negatively charged membranes of tumor cells, thereby enhancing their targeting ability toward tumor tissues. Moreover, dispersibility of CaGd-LDH in physiological solutions was also proved for subsequent biological application (Fig. S1, Supporting Information). The good dispersibility of CaGd-LDH in physiological solutions confirms its suitability for further biological applications. All these results indicated the successful formation of CaGd-LDH.

Fig. 1.

Preparation and characterization of CaGd-LDH. a) The synthesis of CaGd-LDH. b) SEM image of CaGd-LDH. c) Elemental mapping of CaGd-LDH. d) XRD patterns of CaAl-LDH and CaGdAl-LDH. e) Full XPS spectrum of CaGd-LDH. f) XPS spectra of the Ca 2p peaks. g) XPS spectra of the Gd 4d peaks. h) Hydrodynamic diameters and Zeta potential of CaGd-LDH. i) Accumulated release profiles of Ca²⁺ form CaGd-LDH at different pH values.

In comparison to normal tissues, the tumor microenvironment displays distinctive acidic characteristics, a pathological feature driven by metabolic reprogramming, inadequate vascular perfusion, and the accumulation of acidic metabolites such as lactate. Consequently, the utilisation of the acidic nature of tumors to precisely and controllably release drug has been the subject of significant research interest [26]. Research has indicated that breast cancer bone metastases exhibit pronounced acidic properties [27]. LDHs, being essentially a type of hydroxide, are sensitive to acidic environments. In the presence of an acidic environment, LDHs undergo a gradual degradation process that is characterized by hydrolysis. Based on this, we performed inductively coupled plasma optical emission spectrometry (ICP-OES) to analyze the time-dependent release profile of Ca²⁺ and Gd³⁺ under different pH conditions. As shown in Fig. 1i and S2, the release of Ca²⁺ and Gd³⁺ ions increases while pH level decreases. The results revealed that CaGd-LDH released a significantly higher amount of Ca ions in acidic environments. This pH-responsive ion release behavior not only highlights the acid-triggered degradation of CaGd-LDH but also further confirms the successful synthesis of the acid-responsive CaGd-LDH material. These findings underscore the potential of CaGd-LDH as a pH-responsive nanomaterial for targeted applications in acidic microenvironments, such as bone metastasis tumor.

2.2. In vitro therapeutic efficacy of CaGd-LDH

In order to evaluate the biocompatibility of CaGd-LDH, toxicity assays were conducted on healthy human umbilical vein endothrlial cells (HUVEC) and mouse mammary epithelial cells (HC11). These assays demonstrated negligible cytotoxicity even at concentrations of up to 800 μg/ml. Conversely, CaGd-LDH exhibited a substantial concentration-dependent inhibitory effect on the proliferation of 4T1 cells (Fig. 2a). Furthermore, we then studied its radiosensitizing ability using live/dead staining assay (Fig. 2b and c). Treatment of cells with 100 μg/mL CaGd-LDH in conjunction with X-rays (4 Gy) resulted in a marked red fluorescence (dead cells) and a significant decrease in green fluorescence (live cells). These results support the hypothesis that CaGd-LDH is an effective radiosensitizer of breast tumor cells. Next, the radiosensitizing potential of CaGd-LDH on breast tumor cells was then evaluated using clonogenic assays, where CaGd-LDH significantly suppressed 4T1 cell proliferation post-irradiation (Fig. 2d and g). To quantitatively assess the radiosensitization efficacy of CaGd-LDH, we calculated the sensitizer enhancement radio (SER) by comparing the survival curves of 4T1 cells irradiated with various doses of X-ray (0, 2, 4 and 6 Gy), which was determined to be 1.593 (Fig. S3, Supporting Information). The results demonstrated that CaGd-LDH not only could realize tumor-responsive killing effect but also enhanced the radiosensitivity of tumor cells. To further elucidate the underlying mechanisms of CaGd-LDH-induced cell death, we conducted a reactive oxygen species (ROS) fluorescence assay (Fig. 2e and h). The results demonstrated that the CaGd-LDH plus X-ray group generated significantly higher levels of ROS compared to the other groups. This enhanced ROS production under X-ray irradiation indicated that CaGd-LDH effectively amplifies oxidative stress within cancer cells, thereby enhancing the therapeutic efficacy of radiotherapy. In light of the observed ROS production capacity of CaGd-LDH under X-ray irradiation, a subsequent evaluation of the DNA double-strand (DSBs) breaks of CaGd-LDH in vitro was undertaken. It is widely accepted that DSBs are the most severe form of damage caused by radiotherapy [28]. The level of DSBs was assessed by means of an analysis of the phosphorylated histone H2AX (γ-H2AX) in the nucleus after irradiation (Fig. 2f and i). The results demonstrated that the group treated with CaGd-LDH plus X-ray exhibited a greater number of severe DSBs in comparison to the CaGd-LDH or X-ray-alone group. These findings provide substantial support for the potential of CaGd-LDH as a promising radiosensitizer in cancer treatment.

Fig. 2.

In vitro radiosenstization outcomes with CaGd-LDH. a) Cytotoxicity of CaGd-LDH for HUVEC cells, HC11 cells and 4T1 cells. b) Live/dead staining analysis and c) images for 4T1 cells treated with different treatments. d) Colony formation images and g) analysis for 4T1 cells treated with different treatments. e) Intracellular ROS fluorescence images and h) intensity analysis for 4T1 cells treated with different treatments. f) Fluorescence images of DSBs detected by γ-H2AX foci. i) Counts of γ-H2AX foci per cell in f. All experiments are biologically independent. All data are expressed as mean ± SD. n = 5 per group in (a). n = 3 per group in (b, g, h, i). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

2.3. Cell pyroptosis induced by CaGd-LDH

In the material characterization section, the ICP-OES experiments have already confirmed the release of Ca²⁺ ions from CaGd-LDH under acidic conditions, providing a potential mechanism for calcium overload (Fig. 1i). This pH-responsive ion release behavior lays the foundation for further exploring the role of CaGd-LDH inducing calcium overload. Furthermore, the intracellular Ca ions concentration was assessed using Ca²⁺ ions probe Fluo-4 Acetoxymethyl Ester (Fluo-4-AM) (Fig. 3a and b). The green fluorescence substantially increased in the cells treated with CaGd-LDH plus X-ray, indicating calcium overload in these treatment groups. Mitochondrial calcium overload disrupts electron transport chain function, leading to ROS production and further mitochondrial dysfunction in a positive feedback loop [29]. Excessive Ca²⁺ uptake also induces opening of the mitochondrial permeability transition pore (mPTP), resulting in loss of mitochondrial membrane potential (ΔΨm), uncoupling of oxidative phosphorylation, and ultimately cell death [30]. To assess this, we evaluated ΔΨm in 4T1 cells using the JC-1 assay. As shown in Fig. 3c and d, the X-ray, CaGd-LDH, and CaGd-LDH plus X-ray groups all exhibited varying degrees of increased green fluorescence and decreased red fluorescence compared to the Control group, indicating decreased ΔΨm. Notably, the CaGd-LDH plus X-ray combination group displayed the most significant changes, characterized by the strongest green fluorescence and the weakest red fluorescence, suggesting the most severe mitochondrial damage in this group. Collectively, these results demonstrate that the combination of CaGd-LDH and X-ray synergistically induces calcium overload, thereby exacerbating mitochondrial dysfunction and providing the upstream signaling basis for subsequent ROS generation and pyroptosis activation.

Fig. 3.

CaGd-LDH-induced cell pyroptosis. a) Intracellular Fluo-4 AM fluorescence images and b) intensity analysis for 4T1 cells treated with different treatments. c) Intracellular JC-1fluorescence intensity images and d) analysis for 4T1 cells treated with different treatments. e) Biological transmission electron microscopy images. f) Western blot of pyroptosis-associated proteins in each condition. g) lactate dehydrogenase (LDH) release levels of 4T1 cells following different treatments. h) Intracellular HMGB1 fluorescence images for 4T1 cells treated with different treatments. i) ATP levels in the cell supernatant. j) Intracellular CRT fluorescence images for 4T1 cells treated with different treatments. All experiments are biologically independent. All data are expressed as mean ± SD. n = 3 per group in (b, d, g, i). ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

During radiotherapy, X-ray generates ROS both directly via water radiolysis and indirectly through mitochondrial damage [31], while Ca²⁺-induced mitochondrial ROS amplifies oxidative stress, overwhelms antioxidant defenses, and enhances DNA damage. Reduced ΔΨm further promotes mPTP opening, releasing mitochondrial DNA and other damage-associated molecular patterns (DAMPs) that activate inflammatory pathways [32]. As demonstrated by the experimental results described above, the CaGd-LDH combined with radiotherapy group exhibited higher ROS production, more severe DNA damage, and greater 4T1 cell death. Therefore, CaGd-LDH-induced calcium overload synergizes with radiotherapy. Notably, calcium overload is well documented to impair mitochondrial integrity and initiated NLRP3 inflammasome [33], which in turn activates caspase-1, cleaves GSDMD, and ultimately triggers pyroptosis [34]. Collectively, these findings provide direct evidence that the increased intracellular calcium leads to mitochondrial damage, thereby supporting the conclusion that CaGd-LDH induces tumor cell pyroptosis via the mitochondrial calcium overload-mitochondrial dysfunction-pyroptosis pathway.

Given the presence of calcium overload, we further investigated whether it could activate pyroptosis. GSDMD-mediated pyroptosis usually goes through activation by external stimuli, inflammasomes assemble, caspase-1 activation, and finally GSDMD cleavage. The N-terminal domain of GSDMD (N-GSDMD) forms pores in the cell membrane, disrupting its integrity and releasing inflammatory mediators that promote adaptive immune responses. At the cellular level, we observed a “blowout” phenomenon (Fig. S4, Supporting Information), which hinted at the occurrence of pyroptosis. To confirm this, we employed bio-transmission electron microscopy (Bio-TEM) (Fig. 3e) and found that the CaGd-LDH Plus X-ray group exhibited characteristic features of pyroptosis, including cell membrane rupture, leakage of cellular contents, and blurred organelles. Subsequently, Western blot analysis revealed the high expression of cleaved-caspase-1 and N-GSDMD in the CaGd-LDH plus X-ray group, further supporting pyroptosis activation mechanism (Fig. 3f). Damage to cell membranes results in the release of cytoplasmic enzymes into the surrounding medium [35]. An analysis was conducted of the levels of lactate dehydrogenase (LDH) in the cellular supernatant after a series of treatments. The results demonstrated that the level of LDH in the supernatant of 4T1 cells treated with CaGd-LDH plus X-ray was considerably elevated in comparison to the other groups (Fig. 3g). Moreover, an examination was conducted into the proportion of Annexin V/propidium iodide (PI) double-positive cells by means of flow cytometry. The results indicated that the proportion of double-positive cells increased significantly in 4T1 cells treated with CaGd-LDH plus X-ray (Fig. S5, Supporting Information). In normal cells, neither Annexin V nor PI can penetrate the cell membrane, leaving cells unstained. However, during pyroptosis, altered membrane permeability allows PI to enter the cell and bind to DNA, while Annexin V bind to phosphatidylserine on the inner leaflet of the membrane. Consequently, pyroptotic cells can transition rapidly from being unstained to double-positive for PI and Annexin V. Thus, the flow cytometry results suggest that the cell membrane permeability underwent rapid changes under this condition [36]. These results demonstrated that CaGd-LDH plus X-ray could successfully induce pyroptosis in 4T1 cells. To evaluate the immunogenic cell death (ICD) effect induced by pyroptosis, we examined key ICD markers: adenosine triphosphate (ATP), high mobility group box 1 (HMGB1) and calreticulin (CRT) [37]. During pyroptosis, ATP is released into the extracellular environment and acts as a damage-associated molecular pattern, promoting inflammatory responses. Therefore, we measured intracellular ATP levels under different treatment conditions (Fig. 3i). The results showed that the CaGd-LDH plus X-ray group exhibited the lowest levels of intracellular ATP, confirming substantial ATP release from cells. HMGB1 is an endonuclear protein that has been shown to bind to pattern recognition receptors located on the surface of myeloid cells. It has been shown to serve as an adjuvant to activate anti-tumor immunity [38]. The release of HMGB1 from CaGd-LDH plus X-ray treated cancer cells was pronounced (Fig. 3h). During the process of ICD, ecto-CRT functions as a “eat me” signal, thereby stimulating antigen-presenting cells to phagocytose the tumor. This, in turn, results in increased tumor antigen presentation and tumor-specific cytotoxic T lymphocyte responses [39]. CaGd-LDH plus X-ray successfully promoted the translocation of CRT to the cancer cell membrane (Fig. 3j). These findings indicate that the CaGd-LDH plus X-Ray is effective in triggering immunogenic pyroptosis.

2.4. In vivo therapeutic efficacy of CaGd-LDH

The acidic tumor microenvironment is one of the most prominent pathophysiological hallmarks of malignant tumors. It refers to a persistent state in which the pH within solid tumor tissues-particularly in the extracellular space-is lower than that of normal tissues. Typically, the extracellular pH of normal tissues is maintained at approximately 7.4, whereas in many solid tumors, it can drop to between 6.5 and 7.0, with even lower values possible in hypoxic core regions. Based on the characteristically acidic microenvironment of breast cancer bone metastases, we designed and constructed a layered double hydroxide (LDH)-based CaGd-LDH composite nanomaterial. This material leverages the acidic microenvironment to achieve specific targeting of tumor tissue, promoting the efficient accumulation of the high Z element Gd within the tumor. This process significantly enhances the energy deposition rate of radiation in the tumor region during radiotherapy, thereby improving therapeutic efficacy. Furthermore, under acidic conditions, CaGd-LDH enables the controlled release of Ca²⁺ ions, inducing intracellular calcium overload in tumor cells, which in turn activates the pyroptosis pathway and triggers inflammatory cell death. Previous studies have confirmed that the Ca²⁺ release rate from CaGd-LDH under acidic conditions is significantly higher than under neutral conditions, and in vitro experiments have preliminarily validated its antitumor efficacy.

Concomitantly, inspired by the positive in vitro therapeutic effect, we further conducted in vivo experiments to systematically evaluate the therapeutic effect of CaGd-LDH on breast cancer bone metastases. A mouse model of bone metastasis of breast cancer was established, and treatment was initiated when the tumor size reached approximately ∼70 mm³. The mice were randomly split into four groups: (1) Control, (2) CaGd-LDH, (3) X-ray, (4) CaGd-LDH + X-ray. Control groups was substituted by phosphate buffer solution instead of CaGd-LDH (Fig. 4a). Given that Gd-based radiosensitization strongly depends on tumor accumulation, we assessed the intratumoral retention of CaGd-LDH using micro-computed tomography (micro-CT) imaging (Fig. S6, Supporting Information). Following intratumoral injection, CaGd-LDH exhibited sustained Gd signal within the tumor and negligible accumulation in off-target organs, confirming efficient tumor-specific localization. Thus, CaGd-LDH achieves efficient tumor-specific retention, making it a promising and safe radiosensitizing agent. Tumor size and mouse weight were measured every 3 days after treatment. At experimental dose, both CaGd-LDH and X-ray showed limited impacts on inhibiting tumor growth. In contrast, CaGd-LDH plus X-ray had the most significant tumor suppressive effect, achieving a tumor inhibition of 54% on day 27 (Fig. 4b). Throughout the treatment, the mouse maintained a stable body weight, confirming the biocompatibility of CaGd-LDH (Fig. 4c). Additionally, the tumor inhibitory effects were further assessed at the histological level. Compared to other groups, Hematoxylin & Eosin (H&E)-stained tumor tissues treated with CaGd-LDH plus X-ray exhibited minimal hematoxylin staining (Fig. 4d). In contrast, tumors in the control group display intense hematoxylin staining, indicative of a high proliferation rate of cancer cells. Ki67 fluorescence staining results, which is a tumor proliferation maker, revealed a significant reduction in the proliferation of tumor cells in tissues treated with CaGd-LDH plus X-ray, as evidenced by a weaker fluorescence signal when compared to the other groups (Fig. 4e and f). Bone destruction has been identified as a hallmark feature of breast cancer metastases to the bone. Accordingly, we evaluated tibial bone quality using micro-CT imaging (Fig. S7, Supporting Information). As shown in Fig. S7, the control group exhibited severe osteolytic lesions, whereas the CaGd-LDH plus X-ray group exhibited substantially milder bone damage. These findings confirmed the prominent efficacy of CaGd-LDH plus X-Ray in killing breast cancer bone metastatic cells.

Fig. 4.

In vivo antitumor therapy. a) Schematic illustration of therapeutic design. b) Tumor volume growth curves of mice. c) Body weight curves of mice. d) Images of H&E staining of tumor slices. e) Ki-67 fluorescence analysis and f) images of tumor slices. g) N-GSDMD fluorescence images and h) analysis of tumor slices. i) Cleaved-Caspase-1 fluorescence images and j) analysis of tumor slices. All experiments are biologically independent. All data are expressed as mean ± SD. n = 6 per group in (b, c, e, h, j). ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

To confirm the mechanism underlying its tumor-inhibitory effect at in vitro level, immunostaining was employed. The CaGd-LDH plus X-Ray treatment group exhibited intense fluorescence signals for N-GSDMD (Fig. 4g and h) and cleaved-caspase-1 (Fig. 4i and j), confirming the activation of the pyroptosis pathway. Immunofluorescence staining revealed that the combined CaGd-LDH and X-Ray treatment group exhibited markedly increased IL-1β expression in tumor tissue compared to the monotherapy or control groups (Fig. S8, Supporting Information), consistent with the induction of pyroptosis-associated inflammatory response. Moreover, fluorescence staining of HMGB1 (Fig. 5a and c) CRT (Fig. 5b and d) highlighted the occurrence of immunogenic cell death. To evaluate immune cell infiltration and activation, triple immunofluorescence staining for CD 8 (cytotoxic T cells), CD 11c (dendritic cells), and CD86 (activation marker) was performed on tumor tissues. As shown in Fig. S9, the combined treatment group exhibited not only increased infiltration of CD8⁺ T cells but also significantly enhanced activation of CD11c⁺ dendritic cells, as evidenced by elevated CD86 expression. These findings suggest that pyroptosis-associated immunogenic cell death promotes antitumor immune responses. Studies have shown that approximately two-thirds of patients with bone metastases will eventually develop distant metastases [40]. Among these, the lungs are one of the most common sites for distant metastasis. Once pulmonary metastasis occurs, it disrupts the normal function of the lungs, ultimately leading to respiratory failure and death [41]. Consequently, we observed the occurrence of tumor metastasis in the lung of mice after 27 days of treatment. As demonstrated in Fig. 5e and f, prominent metastatic nodules were observed in the lung tissues of mice from the Control group. The CaGd-LDH group and the X-Ray group also exhibited varying degrees of pulmonary metastatic lesions. Conversely, a limited number of metastatic foci were identified in the lung tissues of mice treated with CaGd-LDH combined with X-Ray irradiation. The findings were consistent with the results of the H&E staining of mouse lung tissues (Fig. 5g), in which the combination therapy group displayed the mildest pulmonary metastasis among the treatment groups. These results suggest that pyroptosis induction may counteract metastatic progression. These findings indicated that radiation-induced pyroptosis based on CaGd-LDH can effectively sensitize local radiotherapy of breast cancer bone metastasis, as well as significantly reduce the risk of tumor metastasis by inducing immunogenic cell death-mediated immune effect.

Fig. 5.

In vivo antitumor therapy. a) HMGB1 fluorescence images and c) analysis of tumor slices. b) CRT fluorescence images and d) analysis of tumor slices. e) Statistics and (f) images of the lungs at the end of treatment. g) Images of H&E staining of lung slices. All experiments are biologically independent. All data are expressed as mean ± SD. n = 6 per group in (c, d, e) ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

In addition to its therapeutic and antimetastatic effects, CaGd-LDH also enables imaging-guided therapy owing to its Gd³⁺-based MRI contrast property. To investigate this potential, in vivo Magnetic Resonance Imaging (MRI) was performed after intratumoral injection of CaGd-LDH. As shown in Fig. S10, a sustained and localized T1-weighted hyperintense signal was observed in the tumor region, indicating prolonged Gd³⁺ retention. This real-time visualization capability supports the feasibility of using CaGd-LDH for MRI-guided radiotherapy.

2.5. The biosafety of CaGd-LDH

It is imperative to ensure the biosafety of nanomaterials is for their utilisation in biomedical applications. In order to this end, a comprehensive evaluation of the biosafety of CaGd-LDH was conducted in vitro and in vivo. Initially, a vitro safety evaluation of CaGd-LDH was conducted using a hemolysis assay (Fig. 6a), which confirmed that CaGd-LDH did not cause hemolysis. Subsequently, the in vivo safety of CaGd-LDH was ecaluated by monitoring and analyzing blood and major organs from mice. As demonstrated in Fig. 6b and c, there was no significant difference in blood routine and routine biochemical between the groups that received CaGd-LDH and the control group. In addition, we conducted an evaluation of the safety of the major organs using H&E staining. The findings demonstrated taht there was no statistically significant difference in the staining outcomes of the heart, liver, spleen, lung, and kidney between the groups. This observation auggests that CaGd-LDH exhibits good biological safety in vivo (Fig. 6 d).

Fig. 6.

Biosafety assessment of CaGd-LDH. a) Hematolysis rate of CaGd-LDH at different concentrations. b) Heatmap of hematological parameters of mice. c) Blood biochemical parameters of mice injected with or without CaGd-LDH. N = 3. d) Images of H&E staining of heart, liver, spleen, lung and kidney slices. All experiments are biologically independent. All data are expressed as mean ± SD. n = 3 per group in (a-c).

3. Conclusion

The data presented in this study demonstrates the successful synthesis of CaGd-LDH as a novel radiosensitizer, marking the first application of LDHs in radiotherapy. This design combines the unique properties of high-Z elements and pH-responsive ion release to achieve dual therapeutic effect. On one hand, the accumulation of Gd in tumor tissues enhances localized X-ray energy deposition, generating ROS to improve radiosensitivity. On the other hand, the release of Ca²⁺ in the acidic tumor microenvironment induces calcium overload, activating pyroptosis through the caspase-1/GSDMD pathway. This pyroptotic cell death plays a crucial role in improving the prognosis of bone metastasis and reducing secondary metastasis, offering a promising strategy to address the challenges of tumor recurrence dissemination. Furthermore, micro-CT evaluation confirms that CaGd-LDH plus X-ray alleviates osteolytic bone destruction compared to the control group. Meanwhile, the intrinsic Gd³⁺-based MRI contrast capability enables real-time visualization of tumor retention, supporting the feasibility of MRI-guided radiotherapy. In conclusion, CaGd-LDH integrates radiosensitization, pyroptosis induction, MRI-guided imaging, and bone protection within a single platform, paves the way for a promising strategy to improve the treatment of breast cancer bone metastasis. Notably, this work represents the first utilisation of LDHs as radiosensitizers in radiotherapy. Given the rich composition of LDH materials, this study is expected to provide new ideas for the application of such materials in the field of radiosensitization.

4. Experimental section

4.1. Preparation of CaGd-LDH

CaGd-LDH was synthesized using the co-precipitation method c), a). Specifically, a salt solution was prepared by dissolving 3 mM CaCl₂, 0.5 mM GdCl₂, and 0.5 mM AlCl₃ in 20 mL of deionized water. Meanwhile 10 mL of 1 mM NaOH solution was used as the alkaline source. Both solutions were simultaneously added dropwise into a flask containing 35 mL of deionized water under constant stirring at 400 rpm in a 60 °C water bath. The pH of the reaction mixture was maintained between 11 and 12 by controlling the dripping rate of the alkaline solution. After the addition, the reaction was continued for 6 h until precipitate formation was observed. The suspension was then aged at 50 °C for 12 h. The resulting product was washed with deionized water, dried at 80 °C for 12 h, and finally ground into a fine powder for further use.

4.2. DNA double-strand breaks detection

The DNA double-strand breaks were visualized via immunofluorescence analysis. Cells were inoculated on a cell slide (2 × 10⁴ per well) and then treated with various treatments (PBS, CaGd-LDH, X-ray, CaGd-LDH plus X-ray). The cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in phosphate buffered saline (PBS) buffer, and blocked with 5% fetal bovine serum (FBS). Subsequently, anti-γ-H2AX and Cy3-labeled anti-rabbit IgG antibodies were incubated, and the cell nuclei were stained with Hoechst 33342. Fluorescence images were captured using a confocal laser sacnning microscope (CLSM). In all subsequent experiments, unless otherwise specified, the irradiation parameters were set to 160 kV, 15 mA and 4 Gy, and the concentration of CaGd-LDH was 100 μg/mL.

4.3. Intracellular Ca²⁺ detection

The intracellular Ca ions concentration was assessed using Ca²⁺ ions probe Fluo-4 AM. 4T1 cells were seeded in confocal dishes and cultured for 24 h. Subsequently, cells were treated with CaGd-LDH (100 μg/mL) or left untreated for 8 h, irradiated with X-ray (160 kV, 15 mA, 0 or 4 Gy), washed with PBS, and incubated with Fluo-4-AM for 30 min. Fluorescence images were captured using CLSM.

4.4. Bio-TEM assay

After culturing the cells under normal conditions, they were exposed to X-rays (160 kV, 15 mA, 0 or 4 Gy) following incubation with PBS or CaGd-LDH for 8 h 24 h post-irradiation, the cells were detached using trypsin, collected through centrifugation, and subsequently fixed in a 2.5% solution of glutaraldehyde. Following fixation, the cellular samples were embedded and sectioned for analysis. Transmission electron microscopy (TEM) was then conducted to visualize the cells.

4.5. Western blot analysis

Changes in Cleaved-Caspase-1 and N-GSDMD expression were assessed through Western blot analysis. After sequential treatments, 4T1 cells were harvested and cleaned with PBS and then lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors to prevent protein degradation and dephosphorylation. After centrifugation, the protein concentration was determined using the Bradford assay kit. SDS polyacrylamide gel electrophoresis was conducted with 30 μg of denatured protein per lane, and the protein was transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were then subjected to an overnight incubated at 4 °C, during which they were sealed with 5% skim mike powder and incubated with primary antibodies directed against the following proteins: β-actin, N-GSDMD, and cleaved-caspase-1. Finally, the membranes were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) overnight. Detection was performed using the ECL-plus system.

4.6. Immunogenic cell death

The phenomenon of ICD was evaluated by the exposure of CRT and the release of HMGB1. Cells were inoculated on a cell slide (2 × 10⁴ per well) and incubated with PBS or CaGd-LDH (100 μg/mL) for 24 h, followed by X-Ray irradiation (160 kV, 15 mA, 0 or 4 Gy). Following fixation, standard cell immunofluorescence staining techniques were employed to detect CRT or HMGB1. Fluorescence images were captured using CLSM.

4.7. Animal fedding

The experimental animals used were female BALB/c mice (16-18 g, 6 weeks old), sourced from Beijing SipeiFu Biotechnology Co., Ltd. The mice were housed under controlled conditions: 50 ± 5% relative humidity, 25 ± 2 °C ambient temperature, a 12-h light/dark cycle, with free access to a standard pellet diet and purified water. The “Guide for the Care and Use of Laboratory Animals” was approved by the Institute of High Energy Physics, Chinese Academy of Sciences (approval number: IHEPLLSC-064). It is asserted that all animal experiments were carried out in strict accrodance with this guideline.

4.8. In vivo tumor radiotherapy

Female BALB/c mice (16–18 g, 6 weeks old) were chosen for the establishment of the breast cancer bone metastasis model. The lateral and medial malleoli and the lower half of the tibia were gently grasped with the forefinger and thumb. The leg was then bent to combine flexion and lateral rotation, exposing the knee. The leg was then firmly held while a needle, without the cell suspension, was inserted under the patella. It passed through the middle of the patellar ligament and into the anterior intercondylar area at the top of the tibia. A blank syringe was used first to prevent blockage of the needle by bone tissue. Gentle lateral movements were then used to confirm the needle's position within the tibia and through the growth plate. Limited movement indicated correct placement. The blank syringe was then replaced with one containing 4T1 cells (5 × 10⁵ cells) suspension, following the same pathway. The needle was moved gently again to ensure proper placement, and then the cell solution was slowly injected to ensure that no resistance was felt [42]. After the mouse model of bone metastasis of breast cancer was established, treatment was initiated when the tumor volume reached approximately 70 mm³. The mice were randomly split into four groups: (1) Control, (2) CaGd-LDH, (3) X-ray(6Gy), (4) CaGd-LDH + X-ray. The mice received intratumoral injections of PBS or CaGd-LDH (750 μg), followed by X-Ray (160 kV, 15 mA, 6 Gy) irradiation at the right limb. The body weight and tumor volume were monitored at 2-day intervals. On day 27, the mice were euthanized and the tumors were surgically removed. Tumor tissues were fixed in 4% paraformaldehyde solution for subsequent paraffin embedding, sectioning and immunofluorescence staining of Ki67, N-GSDMD, cleaved-caspase-1, CRT, HMGB1, Il-1β, CD8⁺, CD11c⁺ and CD86. Upon the demise of the mice or their euthanized, the lungs were dissected for the purpose of pathological analysis. The intratumoral retention of CaGd-LDH was assessed using micro-CT imaging (65 kV, 200 μA). Separately, tibial bone quality was analyzed by same micro-CT system in conjunction with 3D reconstruction.

4.9. Data analysis

Statistical analysis was conducted using GraphPad Prism. The data presentation was as mean ± SD. Differences in data were evaluated using ordinary one-way analysis of variance (ANOVA) with Tukey's multiple comparisons post-test. Statistical significance was defined as a P value less than 0.05.

CRediT authorship contribution statement

Chen Yang: Writing – original draft, Visualization, Investigation, Formal analysis. Xue Wang: Writing – review & editing, Investigation, Formal analysis. Miao Zhu: Writing – review & editing, Visualization, Data curation. Shuo Shi: Writing – review & editing, Visualization. Shuang Zhu: Writing – review & editing, Supervision, Funding acquisition. Zhanjun Gu: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. Jiangfeng Du: Writing – review & editing, Supervision, Project administration, Funding acquisition.

Declaration of artificial intelligence tools

During the preparation of this work, the authors utilized DeepSeek for language refinement. The utilisation of these tools by the authors is accompanied by a comprehensive review, editing process, and absolute acceptance of responsibility for the published content.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Zhanjun Gu reports financial support was provided by Ministry of Science and Technology of the People's Republic of China. Zhanjun Gu reports was provided by National Natural Science Foundation of China. Shuang Zhu reports was provided by Beijing Natural Science Foundation. Zhanjun Gu reports was provided by Chinese Academy of Sciences. Jiangfeng Du reports was provided by Shanxi Applied Basic Research Project. If there are other authors, they 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.