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. 2026 Sep 23;68:974–987. doi: 10.1016/j.bioactmat.2026.09.017

Calcium dysregulation in synergy with sonodynamic therapy via medroxyprogesterone-targeted and ROS-responsive nanoparticles for endometrial cancer

Bing Xiao a,b,1, Jingyi Zhou a,b,1, Mao Tang c,d, Yanjiu Li c,d, Ting Huang a,b, Jinjian Liu e, Jianfeng Liu e, Lijun Yang e, Haihua Xiao c,d, Lingpu Zhang c,d,⁎, Jianliu Wang a,b,⁎⁎
PMCID: PMC13634293  PMID: 42830878

Abstract

Effective therapies that enable precise drug delivery and integrated monitoring are urgently needed for fertility-sparing management of endometrial cancer (EC). Although calcium channel blockers (CCBs) exhibit anti-proliferative potential in EC, their poor tumor selectivity and systemic toxicity limit clinical translation. To overcome these limitations, a nanoparticle-mediated sonodynamic therapy (SDT) strategy is employed to deliver CCBs while integrating real-time imaging. This study develops MPA-RSP/Ce6/MDP, medroxyprogesterone (MPA)-modified nanoparticles based on a reactive oxygen species (ROS)-sensitive polymer (RSP), co-incorporating the sonosensitizer chlorin e6 (Ce6) and CCB manidipine (MDP). Upon ultrasound activation, Ce6 generates cytotoxic ROS, which trigger degradation of the ROS-responsive shell and release of MDP. The combination of ROS generation and calcium influx inhibition synergistically induces severe endoplasmic reticulum stress, thereby triggering immunogenic cell death and subsequent activation of antitumor immunity. This SDT approach further synergizes with PD-1 checkpoint inhibition, significantly enhancing therapeutic outcomes in vivo. Additionally, single-photon emission computed tomography (SPECT) imaging with co-assembled MPA-RSP/99mTc-RSP confirms efficient tumor accumulation. In summary, this study introduces an ultrasound-responsive nanoparticle that integrates calcium-homeostasis disruption, sonodynamic therapy, and immune activation for non-surgical treatment of endometrial cancer. This strategy shifts from sustained hormonal exposure to spatiotemporally controlled physical intervention, thereby establishing a multimodal foundation for EC fertility-preservation treatment.

Keywords: Endometrial cancer, Ultrasound-responsive nanoparticle, Calcium dysregulation, SPECT imaging, Immunotherapy

Graphical abstract

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Highlights

  • •

    Medroxyprogesterone-targeted US-responsive nanoparticles (MPA-RSP/Ce6/MDP) codeliver manidipine/Ce6 for endometrial cancer.

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    Upon ultrasound activation, ROS and Ca2+ inhibition synergize to induce ER stress and immunogenic cell death.

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    ICD triggered by ultrasound with MPA-RSP/Ce6/MDP synergizes with PD-1 checkpoint blockade.

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    SPECT imaging with co-assembled MPA-RSP/99mTc-RSP confirms efficient tumor accumulation.

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    A novel multifunctional strategy for fertility-sparing endometrial cancer management is proposed.

1. Introduction

Endometrial cancer (EC) incidence is rising in young women with strong fertility intentions, underscoring the need for fertility-sparing management (FSM) that uses non-surgical, uterus-preserving treatments [1,2]. Current progestin-based therapy in FSM for EC achieves a response rate of approximately 70% but requires 9–12 months of treatment with assessments every 12 weeks. Moreover, due to high systemic exposure, oral progestin therapy causes significant systemic side effects including weight gain, hepatotoxicity, thromboembolic risk, and fluid retention, necessitating improved drug delivery or progestin sensitivity to reduce the required dose and adverse effects [3,4]. Besides, disease monitoring during FSM relies heavily on invasive hysteroscopy, with imaging playing only an adjunctive role. Given these limitations, alternative strategies that combine new therapeutic modalities with effective drug delivery and innovative non-invasive, real-time monitoring are urgently needed [4].

Previous studies have revealed that dysregulation of calcium homeostasis is a pivotal therapeutic target for endometrial cancer [5,6]. Elevated serum calcium correlates with poor prognosis in EC [7]. Calcium influx via the L-type channel Cav1.3 mediates estrogen-induced non-genomic effects, activating the ERK1/2/CREB pathway [8]. Calcium channel blockers (CCBs) inhibit cell proliferation and sensitize tumor cells to progestin therapy [9]. In patients, CCB use was associated with a potentially shorter time to complete remission. Furthermore, elevated serum free calcium levels are associated with advanced disease progression, and calcium channels regulate cell migration via the RhoA/ROCK1 pathway [10]. CCBs have also been identified as a potential therapeutic strategy in other tumor types, directly linking tumor progression to immune evasion. Calcium signaling orchestrates immunosuppression — elevated Ca2+ levels upregulate PD-L1 levels [11]. In contrast, CCBs have demonstrated the ability to enhance antitumor immunity and sensitize tumors to immune checkpoint blockade [12]. Based on the above mechanisms, targeting calcium homeostasis represents a dual-pronged strategy to sensitize EC to progestin therapy and enhance antitumor immunity. However, traditional CCBs face clinical limitations, including cardiovascular toxicity, poor tumor targeting, and reliance on suprapharmacological doses for pro-apoptotic effects [13,14].

Combining nanomedicine with sonodynamic therapy (SDT) offers a transformative approach to overcoming these barriers. By encapsulating hydrophobic therapeutic agents, reactive oxygen species (ROS)-sensitive polymer (RSP) nanoparticles significantly enhance drug solubility and stability. These engineered nanoparticles achieve preferential tumor accumulation via passive or active targeting strategies, thereby reducing both systemic and off-target toxicity and amplifying antitumor efficacy. SDT is an advanced physical modality that uses ultrasound (US) to activate sonosensitizers in deep-seated tumors with high spatial precision and tissue penetration [15]. When integrated with nanomedicine, US-generated ROS can trigger RSP degradation for controlled drug release and induce immunogenic cell death (ICD) through endoplasmic reticulum (ER) stress activation [[16], [17], [18]]. Thus, the integration of US-guided nanomedicine with CCBs offers a rational strategy for EC treatment that addresses both drug-delivery challenges and immune activation. Furthermore, as a versatile carrier, the polymeric nanoparticles could integrate multimodal imaging for real-time tumor monitoring, thereby optimizing therapeutic protocols and enhancing clinical translation.

To this end, medroxyprogesterone (MPA)-modified ROS-responsive nanoparticles (MPA-RSP/Ce6/MDP) were developed to integrate SDT, calcium dysregulation, and immune activation. MPA-mediated targeting enhances tumor accumulation, while US-triggered ROS-responsive shell degradation precisely releases sonosensitizer chlorin e6 (Ce6) and CCB manidipine (MDP), synergistically inducing ROS generation and calcium imbalance. Mechanistically, MDP-mediated calcium-channel blockade perturbs calcium homeostasis and alters the cellular proteostasis response to SDT-generated oxidative stress, thereby intensifying ER stress through p-eIF2α/LC3B/CHOP and p-IRE1α/p-NFκB pathways. Concurrently, ICD, manifested by surface calreticulin (CRT) exposure and high-mobility group box 1 (HMGB1) translocation, promotes dendritic cell (DC) maturation and cytotoxic T lymphocyte (CTL) infiltration, subsequently enhancing anti-PD-1 antibody efficacy. Additionally, SPECT imaging using co-assembled MPA-RSP/99mTc-RSP enables non-invasive visualization of the efficient tumor accumulation of MPA-RSP-based nanoparticles. The nanoparticle offers a promising multifunctional strategy for EC therapy, integrating targeted delivery, spatiotemporal control, and immune activation.

2. Results

2.1. Preparation and characterization of MPA-RSP/Ce6/MDP

Progesterone receptor (PR) expression is typically retained in well-differentiated EC, making it a therapeutic target for progestin therapy [19]. To confirm PR expression in our experimental models, we examined PR protein levels in the three endometrial cancer cell lines used in this study by Western blotting. All three cell lines (Ishikawa, Hec-1A, and LH01) were positive for PR expression, with A2780 ovarian cancer cells serving as a positive control (Fig. S1). Concurrently, sonosensitizers generate elevated levels of ROS under US stimulation, providing a mechanism for triggered drug release. To exploit these features, a ROS-responsive polymer was designed for targeted EC therapy (Scheme 1). The ROS-sensitive backbone, RSP-COOH, was synthesized through condensation polymerization of lysine diisocyanate and ROS-sensitive monomers (RSM), followed by terminal capping with OH-PEG5k-COOH [20]. RSP-COOH was end-functionalized with medroxyprogesterone through EDC/DMAP-mediated esterification to obtain MPA-RSP, which was characterized by 1H NMR and infrared (IR) spectroscopy (Fig. 1A and Figure S2-4). Quantitative 1H NMR analysis indicated an average MPA substitution of ∼1.4 groups per polymer chain (Fig. S3). This approach integrated progesterone receptor targeting with ROS-responsive drug release, aiming to enhance therapeutic efficacy in endometrial malignancies.

Scheme 1.

Scheme 1

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Design schematic of ROS-responsive nanoparticles for synergistic sonodynamic and calcium dysregulation therapy in EC. (A) The MPA-RSP polymer is designed for medroxyprogesterone-targeted tumor accumulation and ultrasound-responsive drug delivery by encapsulating sonosensitizer Ce6 and CCB manidipine. (B) US-triggered ROS generation cleaves the responsive shell, enabling synergistic SDT and calcium dysregulation, which amplifies ER stress, ultimately activating antitumor immunity and potentiating anti-PD-1 immunotherapy.

Fig. 1.

Fig. 1

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Characterization of MPA-RSP/Ce6 and MPA-RSP/Ce6/MDP. (A) Synthetic routes of RSP-COOH and MPA-RSP. (B) Design and controlled release mechanism of MPA-RSP/Ce6/MDP. (C, D) TEM images (scale bar: 200 nm) of MPA-RSP/Ce6/MDP (C) and MPA-RSP/Ce6 (D). (E, F) Hydrodynamic size distribution (E) and zeta potential (F) of MPA-RSP/Ce6 and MPA-RSP/Ce6/MDP. (G) DLS size monitoring over 7 days. (H) ESR spectroscopy of MPA-RSP/Ce6 and MPA-RSP/Ce6/MDP with TEMP under US, with water as control. (I, J) DLS-derived size distribution (I) and TEM image (scale bar: 250 nm, J) of MPA-RSP/Ce6/MDP following US. (K) Representative CLSM images of Ishikawa spheroids labeled with Calcein-AM (green) after 4 h incubation with MPA-RSP/Ce6 (Ce6: red; scale bar: 200 μm). (L, M) Cellular uptake kinetics of MPA-RSP/Ce6 analyzed by FCM (L) and quantitative comparison (M). (N) Comparative FCM quantification of cellular uptake efficiency for free Ce6, RSP/Ce6, and MPA-RSP/Ce6 after 4 h incubation. Data are presented as mean ± SD (n = 3); statistical analysis by one-way ANOVA with Bonferroni post-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

The amphiphilic MPA-RSP self-assembled into spherical nanoparticles for co-encapsulation of Ce6 and MDP, yielding MPA-RSP/Ce6/MDP (Scheme 1A, Fig. 1B). For comparative analysis, MPA-RSP/Ce6 (without MDP) and RSP/Ce6 (without MPA) were prepared under identical conditions. Transmission electron microscopy (TEM) verified that both MPA-RSP/Ce6/MDP (Fig. 1C) and MPA-RSP/Ce6 (Fig. 1D) possessed uniformly spherical morphologies. These observations were consistent with dynamic light scattering (DLS) results, indicating a narrow size distribution, with a mean diameter of 169 nm (polydispersity index, PDI = 0.07) for MPA-RSP/Ce6/MDP and 159 nm (PDI = 0.08) for MPA-RSP/Ce6 (Fig. 1E). Furthermore, the zeta potentials were measured at −20.0 ± 3.7 mV and −23.9 ± 0.7 mV, respectively (Fig. 1F), ensuring robust colloidal stability. Both MPA-RSP/Ce6/MDP and MPA-RSP/Ce6 demonstrated negligible size variation (<10%) over 7 days (Fig. 1G), indicating their suitability for systemic administration.

Upon US stimulation, the generated ROS cleaves the responsive shell, leading to drug release and direct cytotoxic effects (Fig. 1B). Singlet oxygen (1O2) production by MPA-RSP/Ce6/MDP and MPA-RSP/Ce6 under US was assessed using electron spin resonance (ESR) spectroscopy with 2,2,6,6-tetramethylpiperidine (TEMP) as a trapping agent, which exhibited the characteristic 1:1:1 triplet signal (Fig. 1H) [21]. In parallel, gel permeation chromatography (GPC) confirmed ROS-sensitive backbone cleavage of the polymer after H2O2 treatment. While the untreated MPA-RSP polymer showed a dominant elution peak at approximately 22.5 min, a later-eluting peak emerged at around 32.5 min following H2O2 exposure, indicating the generation of degradation fragments (Fig. S6). After US stimulation, DLS revealed a shift toward a broader, multimodal size distribution with increased polydispersity, consistent with nanoparticle disassembly (Fig. 1I). Furthermore, TEM images directly visualized the dissociation of nanoparticles into fragmented aggregates (Fig. 1J). In parallel, the release kinetics of manidipine from MPA-RSP/Ce6/MDP were evaluated under different conditions. Approximately 17.2% of MDP was spontaneously released from MPA-RSP/Ce6/MDP in PBS over 24 h. In the ROS group, the cumulative release increased to 26.6%. In contrast, US promoted a robust release, with the cumulative percentage reaching ∼68.6%. These results confirm the ROS-responsive release behavior of MPA-RSP/Ce6/MDP, demonstrating that US enables spatiotemporally controlled drug delivery (Fig. S7). Together, these results validate the controlled, ROS-mediated release mechanism of the nanoparticles.

The MPA moiety significantly enhanced tumor-specific uptake. Confocal laser scanning microscope (CLSM) imaging revealed uniform distribution of MPA-RSP/Ce6 nanoparticles within Ishikawa spheroids (Fig. 1K, Fig. S8). Flow cytometry (FCM)-based quantification demonstrated that intracellular fluorescence intensity reached its maximum at 4 h after incubation (mean fluorescence intensity, MFI = 17.7 ± 0.06 × 105, Fig. 1L, M, Fig. S9). MPA-RSP/Ce6 exhibited 4.8-fold higher uptake than free Ce6 (p < 0.0001) and 2.7-fold higher uptake than non-targeted RSP/Ce6 (p < 0.0001, Fig. 1N, Figs. S9 and 10). A similar enhancement in cellular uptake was also observed in Hec-1A cells (Figs. S11 and S12). To further verify that the enhanced uptake was specifically mediated by MPA-recognizing interactions rather than nonspecific nanoparticle surface effects, we performed an MPA competition assay. Pre-treatment with free MPA inhibited MPA-RSP/Ce6 uptake in a concentration-dependent manner (Fig. S13), confirming the specificity of MPA-mediated targeting. Taken together, MPA-driven cellular uptake and US-triggered nanoparticle disassembly laid the foundation for spatiotemporally controlled combination therapy.

2.2. In vitro sonodynamic performance and calcium influx inhibition of MPA-RSP/Ce6/MDP

The in vitro sonodynamic performance of MPA-RSP/Ce6 and MPA-RSP/Ce6/MDP was evaluated. ROS generation, assessed by CLSM (Fig. 2A and B) and FCM (Fig. 2C), demonstrated that MPA-RSP/Ce6/MDP+US induced the pronounced cytoplasmic ROS elevation (MFI = 11.5 ± 0.5 × 105), comparable to MPA-RSP/Ce6+US (MFI = 14.7 ± 0.9 × 105) and significantly surpassing all controls (Fig. 2D). This US-triggered cellular ROS ensured spatially controlled drug release.

Fig. 2.

Fig. 2

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ROS generation and calcium dysregulation induced by MPA-RSP/Ce6/MDP+US synergistically enhanced in vitro anticancer efficacy. (A) Schematic diagram depicting the detection of intracellular mechanisms for US-activated MPA-RSP/Ce6 and MPA-RSP/Ce6/MDP. (B) CLSM images representing intracellular ROS generation (red fluorescence) of Ishikawa cells under the indicated conditions. Blue: DAPI (nuclei); Red: Actin (cytoskeleton); scale bar: 20 μm. (C) Intracellular ROS measured by FCM in Ishikawa cells exposed to different treatments. (D) Quantification of ROS levels from (C). (E) Apoptosis detected by Annexin V-FITC labeling through FCM in Ishikawa cells subjected to the same treatment groups as in (C, D). (F) Quantification of apoptosis from (E). (G) Fluo-4 AM-loaded cells were imaged by CLSM every 15 s under the indicated conditions, and representative images captured at key time points (0, 1, 6, 12, and 15 min) are shown. Scale bar: 100 μm. (H) Calcium influx traces quantified from the time-lapse CLSM recordings in (G). Images were acquired every 15 s. The fluorescence intensity of each region of interest (ROI) within individual cells was normalized as (Ft − F0)/F0, where F0 is the mean baseline fluorescence intensity of each ROI at 0 min, and Ft represents the mean intensity of the same ROI at each subsequent time point. Data are presented as mean ± SD (n = 10 ROIs per group, each corresponding to an individual cell). Extracellular calcium solution was administered at 1 min. (I) Cell viability of Ishikawa cells under different treatments (n = 4). Data: mean ± SD (n = 3); statistical analysis by one-way ANOVA with Bonferroni post-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. US parameters: 3.0 MHz, 1.5 W/cm2, 50% duty cycle, 3 min.

Apoptosis analysis (Fig. 2E and F) revealed that US-activated MPA-RSP/Ce6/MDP induced the highest apoptosis rate (60.2% ± 1.1%), significantly exceeding MPA-RSP/Ce6+US (47.6% ± 0.9%) and MDP monotherapy (25.3% ± 0.7%) (p < 0.001). Similar trends were observed in Hec-1A cells (Fig. S14). By blocking L-type voltage-dependent calcium channels, MDP disturbs intracellular calcium homeostasis [22]. Calcium influx assays (Figs. S15 and S16) confirmed effective calcium channel blockade by MPA-RSP/Ce6/MDP+US, comparable to MDP alone (Fig. 2G and H).

Cell viability assays (Fig. 2I) revealed distinct cytotoxic profiles across treatment groups. MDP monotherapy exhibited moderate anticancer activity (∼65% viability at 20 μM), consistent with its calcium channel-blocking function. In contrast, MPA-RSP/Ce6/MDP+US exhibited potent concentration-dependent cytotoxicity, reducing cell viability to below 50% at ≥10 μM. Crucially, negligible cytotoxicity was observed without US activation (>75% viability at 40 μM). Consistent results were obtained in Hec-1A cells (Fig. S17). Collectively, these findings demonstrated the spatiotemporally controlled therapeutic effects of MPA-RSP/Ce6/MDP with US-triggered ROS generation and synergistic calcium modulation, amplifying tumor-specific cytotoxicity.

2.3. MPA-RSP/Ce6+US amplified ER stress by disrupting calcium homeostasis

To elucidate the synergistic mechanisms between calcium dyshomeostasis and ROS, transcriptomic profiling was performed across four experimental groups: PBS (control), MDP, MPA-RSP/Ce6+US, and MPA-RSP/Ce6/MDP+US. Differential expression gene (DEGs) analysis identified treatment-specific gene signatures (Fig. S18A–C), with partial overlap between the combination therapy and other groups (Fig. S18D). Pathway enrichment analysis of DEGs using the KEGG database revealed three major categories (Fig. 3A and Fig. S18E and F). Protein metabolism and degradation pathways were significantly enriched, with ER protein processing and proteasome ranking among the top pathways. Correspondingly, cell death regulation pathways, including apoptosis, ferroptosis, p53 signaling, and cell cycle, were also enriched, suggesting coordinated tumor suppression through programmed cell death mechanisms. Meanwhile, metabolic and signaling pathways, such as mTOR, FoxO, and AMPK pathways, were enriched as well, underscoring ROS-driven oxidative stress in metabolic reprogramming.

Fig. 3.

Fig. 3

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Transcriptomic analysis of proteostasis-related responses in Ishikawa cells treated with MPA-RSP/Ce6/MDP+US. Ishikawa cells were incubated with MPA-RSP/Ce6/MDP for 6 h, then exposed to US (3.0 MHz, 1.5 W/cm2, 3 min) and further incubated for 12 h before RNA extraction. (A) KEGG pathway enrichment analysis of DEGs in the MPA-RSP/Ce6/MDP+US group compared with the PBS control. The Protein processing in endoplasmic reticulum and Proteasome pathways are highlighted in blue and bold font to indicate the proteostasis-related pathways further examined in subsequent analyses. (B, C) GSEA enrichment plots for the “Protein processing in endoplasmic reticulum” (B) and “Proteasome” (C) pathways. (D, E) Protein–protein interaction (PPI) networks constructed from DEGs enriched in the “Protein processing in endoplasmic reticulum” (D) and “Proteasome” (E) pathways. Node color represents log2(fold change), and node size represents –log10(q-value).

Gene Set Enrichment Analysis (GSEA) was further performed to investigate the synergistic effects on ER stress induction. The proteasome pathway was significantly downregulated in the MDP group (p < 0.001; Normalized Enrichment Score [NES] = −2.1; Fig. S18G and K; Fig. S19A), indicating suppression of a proteasome-associated transcriptional program. In the MPA-RSP/Ce6+US group, the ER protein processing pathway was significantly upregulated (NES = 1.85, p = 7.4 × 10−6, Figs. S18H, L, Figure S19B). Strikingly, the combination therapy group (MPA-RSP/Ce6/MDP+US) exhibited an ER stress “overload” phenotype. The ER protein processing pathway was significantly inhibited (NES = −1.59, p = 0.0007, Figure S18I, Fig. 3C and D), while the proteasome pathway showed a positive enrichment trend (NES = 1.46, p < 0.05, Fig. 3B and E). This positive transcriptional shift may represent a compensatory response to the increased proteotoxic burden. The marked negative enrichment of the proteasome gene set in the MDP group indicates that calcium-channel blockade is associated with suppression of a proteasome-related transcriptional program. SDT-generated ROS increase oxidative and proteotoxic stress. Under the additional proteotoxic burden, the positive proteasome enrichment observed in the combination group may represent a stress-driven compensatory transcriptional response aimed at restoring proteostasis [23,24]. Thus, these treatment-dependent transcriptional patterns suggest that calcium dysregulation alters the proteostasis response to SDT, which may contribute to intensified ER stress.

2.4. ER stress and resultant ICD induced by MPA-RSP/Ce6+US boosted tumor immunogenicity

To validate the transcriptomic findings at the protein level, we examined ER stress markers in Ishikawa cells across treatment groups. Western blot results demonstrated that the MPA-RSP/Ce6/MDP+US combination exhibited intensified ER stress responses, with markedly strong p-eIF2α/CHOP/p-IRE1α/p-NFκB band intensities (Fig. 4A). Additionally, the combined treatment resulted in increased p-PERK and BiP expression together with the highest ATF4 level (Fig. S20). Collectively, these results demonstrate a pronounced and sustained ER stress response following MPA-RSP/Ce6/MDP+US treatment.

Fig. 4.

Fig. 4

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ER stress and resultant ICD triggered by MPA-RSP/Ce6/MDP+US. (A) Western blot analysis of ER stress markers (eIF2α, p-eIF2α, LC3B, CHOP, p-IRE1α, p-NFκB) in Ishikawa cells after different treatments. β-Tubulin served as a loading control. (B) Schematic Illustration of the in vitro assays for detecting ICD markers and BMDC maturation. (C) Membrane translocation of CRT (green) in Ishikawa cells following the indicated treatments was visualized by CLSM. Scale bar: 20 μm. (D, E) FCM quantification of surface-exposed CRT. (F) CLSM images of HMGB1 release (green) in Ishikawa cells under different treatments. Scale bar: 20 μm. (G, H) BMDC maturation (CD11c+CD80+CD86+) was assessed by FCM, showing representative plots (G) and quantitative analysis (H). (I) Schematic illustration of the mechanism by which synergistic sonodynamic and calcium dysregulation therapy enhances ER stress-mediated ICD activation. Data: mean ± SD (n = 3). One-way ANOVA with Bonferroni post-test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. US parameters: 3.0 MHz, 1.5 W/cm2, 50% duty cycle, 3 min.

ER stress and ROS production could trigger ICD, which features damage-associated molecular patterns (DAMPs) emission from cells undergoing apoptosis. Surface-exposed CRT acts as an “eat-me” signal to facilitate DC maturation. The release of HMGB1 activates immune receptors, thereby enhancing antigen presentation. Adenosine triphosphate (ATP), another key DAMP, acts as a “find-me” signal to recruit immune cells and promote inflammatory responses [25,26]. Therefore, CRT exposure and HMGB1 release were assessed in Ishikawa cells treated respectively with PBS, MDP, MPA-RSP/Ce6+US, and MPA-RSP/Ce6/MDP+US (Fig. 4B). CLSM images revealed gradually increased CRT membrane exposure in the MDP, MPA-RSP/Ce6+US, and MPA-RSP/Ce6/MDP+US groups compared with the PBS control (Fig. 4C), and FCM further confirmed that MPA-RSP/Ce6/MDP+US induced the most pronounced CRT membrane exposure, significantly exceeding PBS controls, MDP monotherapy, and MPA-RSP/Ce6+US (Fig. 4D and E). In parallel, MPA-RSP/Ce6/MDP+US induced robust HMGB1 release as observed by CLSM (Fig. 4F). In addition, ATP levels increased progressively from PBS controls to MDP alone, MPA-RSP/Ce6+US, and MPA-RSP/Ce6/MDP+US groups, with the combination treatment showing the highest ATP release (Fig. S21). Collectively, these results demonstrated that US combined with calcium channel blockade induced profound ICD.

To functionally validate ICD induction, murine EC cells subjected to the same treatments as in the ICD experiment were co-cultured with bone marrow-derived dendritic cells (BMDC, Fig. 4B). Consistently, MPA-RSP/Ce6/MDP+US induced the highest DC maturation levels, achieving 1.1-fold and 1.7-fold increases over MPA-RSP/Ce6+US and MDP groups, respectively (Figure S22, Fig. 4G and H).

Collectively, these results demonstrated synergistic amplification of ER stress by MPA-RSP/Ce6/MDP+US, leading to robust ICD and enhanced tumor immunogenicity (Fig. 4I).

2.5. Dual-modal imaging with MPA-RSP/Ce6 and MPA-RSP/99mTc-RSP

Targeting efficiency was evaluated in an Ishikawa EC xenograft mouse model. After intravenous administration of MPA-RSP/Ce6, real-time in vivo imaging (Fig. 5A) showed a time-dependent rise in fluorescent signal within the tumor region. At 24 h, MPA-RSP/Ce6 achieved a tumor fluorescence signal 3.9-fold that of RSP/Ce6, verifying the active targeting capability mediated by medroxyprogesterone modification (Fig. 5B and C). Ex vivo organ imaging at 48 h further confirmed that the fluorescence intensity in tumor tissue was exceeded only by that of the liver and was markedly higher than in other major organs (Fig. 5D and E), highlighting the exceptional tumor-specific accumulation potential of MPA-RSP/Ce6.

Fig. 5.

Fig. 5

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In vivo dual-modal imaging of the biodistribution of MPA-RSP/Ce6 and MPA-RSP/99mTc-RSP. (A) Schematic illustrating the in vivo fluorescence imaging experiment. (B) In vivo fluorescence imaging of Ishikawa tumor-bearing mice at multiple time points after intravenous delivery of MPA-RSP/Ce6 (Ex/Em 640 nm/700 nm). (C) Semiquantitative fluorescence intensity at tumor sites over time (MPA-RSP/Ce6 vs. RSP/Ce6 control). (D) Ex vivo fluorescence imaging of harvested tissues (heart [H], lung [Lu], kidney [K], liver [Li], intestine [I], spleen [S], tumor [T]) at 48 h post-injection. (E) Semiquantitative fluorescence intensity of organs and tumors from (D). (F) Synthesis route of 99mTc-RSP and schematic illustrating the preparation of MPA-RSP/99mTc-RSP for SPECT Imaging, with free Na99mTcO4 as a control. (G) SPECT images after intravenous injection of free Na99mTcO4 or MPA-RSP/99mTc-RSP. (H) Semiquantitative analysis (n = 1) of SPECT signals in tumor sites at indicated time points from (G). Data: mean ± SD (n = 3); statistical analysis by one-way ANOVA with Bonferroni post-test in E and two-way ANOVA with Tukey multiple comparison test in C. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

For more precise imaging, SPECT technology was introduced. Although 99mTc is a commonly used radionuclide in nuclear medicine, its application is often limited by the poor targeting and in vivo instability of traditional carriers [15]. To address this, we designed a structurally stable radiolabeled polymer, 99mTc-RSP (Fig. S23), and co-assembled it with MPA-RSP into nanoparticles (MPA-RSP/99mTc-RSP, Fig. 5F). Successful co-assembly was confirmed by DLS and thin-layer chromatography (TLC) (Fig. S24). SPECT imaging revealed minimal uptake of free Na99mTcO4 in tumors, whereas MPA-RSP/99mTc-RSP achieved significant tumor enrichment and retention as early as 4 h post-injection, substantially improving tumor targeting efficiency (Fig. 5G and H).

2.6. In vivo antitumor efficacy with US stimulation of MPA-RSP/Ce6/MDP

To evaluate therapeutic efficacy, Ishikawa tumor-bearing mice were randomized to receive various treatments: (1) saline (i.v.), (2) MDP (i.p.), (3) MPA-RSP/Ce6+US (i.v.), and (4) MPA-RSP/Ce6/MDP+US (i.v.). Treatments were performed every other day, and tumor growth was followed over 15 days (Fig. 6A). Over the 15-day treatment period, saline-treated mice exhibited aggressive tumor progression (1278.5 ± 105.7 mm3), whereas MDP monotherapy showed moderate growth suppression (1002.5 ± 106.8 mm3; p < 0.0001 vs control), validating calcium modulation as a viable therapeutic axis. The MPA-RSP/Ce6+US group showed significantly enhanced efficacy (807.8 ± 63.5 mm3, p < 0.0001). Strikingly, the MPA-RSP/Ce6/MDP+US combination achieved superior tumor control (438.8 ± 53.7 mm3, 65.7% reduction vs control, p < 0.0001), outperforming all monotherapies (p < 0.0001) (Fig. 6B–D). All treatments were well tolerated with no significant weight loss (Fig. 6C). Furthermore, routine serum indices were all within the normal reference intervals, and H&E-stained sections from main organs exhibited no visible pathological changes, collectively confirming the good biocompatibility of the regimens (Fig. S25).

Fig. 6.

Fig. 6

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In vivo antitumor efficacy of MPA-RSP/Ce6/MDP under US stimulation. (A) Experimental scheme of the various treatments for Ishikawa tumor-bearing mice. (B, C) Growth profiles of tumors (B) and body weight monitoring (C) of mice (n = 5 in each group). (D) Representative photograph of excised tumors from mice treated with saline (control), MDP, MPA-RSP/Ce6+US, and MPA-RSP/Ce6/MDP+US. Scale bar: 1 cm. (E) H&E staining of tumor sections for morphological evaluation. Scale bar: 100 μm. (F, G) IHC staining of e-IF2α (F) and CHOP (G). Scale bar: 100 μm. Data: mean ± SD (n = 5); statistical analysis by two-way ANOVA with Tukey multiple comparison test in B and C. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. US parameters: 3.0 MHz, 1.5 W/cm2, 50% duty cycle, 5 min.

To validate the tumor-suppressive effects mediated by synergistic ER stress, hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis were performed on tumor tissues. H&E staining revealed nuclear fragmentation and cellular swelling in US-treated tumors, with the MPA-RSP/Ce6/MDP+US group exhibiting more severe morphological alterations than the MPA-RSP/Ce6+US group. Minimal changes were observed in saline- and MDP-treated groups (Fig. 6E). IHC analysis of ER stress markers eIF2α (Fig. 6F, Fig. S26A) and CHOP (Fig. 6G, Fig. S26B) revealed the strongest ER stress activation in the MPA-RSP/Ce6/MDP+US group. These results collectively indicated that the synergistic effects of calcium homeostasis disruption and US-induced ROS generation enhanced ER stress-mediated antitumor mechanisms, effectively suppressing tumor proliferation in vivo.

2.7. MPA-RSP/Ce6/MDP+US enhanced antitumor immune responses and synergized with immune checkpoint blockade

To systematically validate the US-triggered tumor immune activation of MPA-RSP/Ce6/MDP, murine endometrial carcinoma (LH01) tumor-bearing models were established. Given that ICD-driven antitumor immunity is often limited by PD-1-mediated immune evasion, we assessed whether combining this therapy with α-PD1 blockade enhances efficacy. Mice were randomized into four groups: control, α-PD1 alone, MPA-RSP/Ce6/MDP+US, and MPA-RSP/Ce6/MDP+US+α-PD1. Tumor growth and body weight were monitored. At the endpoint, tumors and spleens were collected for FCM analysis (Fig. 7A). Tumor growth monitoring revealed that MPA-RSP/Ce6/MDP+US with α-PD1 therapy achieved the most potent tumor suppression (Fig. 7B). US-activated MPA-RSP/Ce6/MDP therapy alone slowed tumor progression by 61.2% (day 15 vol: 188.5 ± 75.4 mm3 vs saline 485.9 ± 345.5 mm3, p < 0.001), while combined α-PD1 amplified this effect to 75.0% suppression (121.7 ± 55.8 mm3), suggesting synergistic immune reactivation (Fig. 7B). Importantly, none of the treatments induced notable fluctuations in body weight, indicating a favorable safety profile (Fig. 7C). Quantification of tumor weight confirmed that this dual therapy reduced tumor mass by 81.3% (p < 0.001), significantly exceeding the tumor inhibition achieved by individual monotherapies (MPA-RSP/Ce6/MDP+US: 69.1%; α-PD1: 52.3%) (Fig. 7D and E).

Fig. 7.

Fig. 7

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Antitumor immune responses induced by MPA-RSP/Ce6/MDP+US and combination with anti-PD1 therapy. (A) Schematic overview of the treatment protocols applied to LH01 tumor-bearing mice. (B, C) Tumor growth kinetics (B) and body weight monitoring (C) across various treatment groups (n = 5). (D, E) Weight (D) and representative photograph (E) of resected tumors (Scale bar: 1 cm). (F, H, J) FCM analysis of tumor-infiltrating mature DC (CD11c+CD80+CD86+, F), tumor-infiltrating cytotoxic T lymphocytes (CD3+CD8+CD4−, H), and spleen-derived central memory T cells (TCM) (CD62L+CD44+, J). (G, I, K) Percentage of corresponding cells from (F, H, J). (L-O) H&E staining (L) and IF staining for TUNEL (M), CRT (N), and CD8 (O) of tumors extracted from the various groups. Scale bar: 100 μm. Data: mean ± SD (n = 5); statistical analysis by one-way ANOVA with Bonferroni post-test in d, g, i and k, two-way ANOVA with Tukey multiple comparison test in B and C. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. US parameters: 3.0 MHz, 1.5 W/cm2, 50% duty cycle, 5 min.

Given the robust tumor suppression observed, the immune mechanism was further analyzed. FCM analysis revealed that MPA-RSP/Ce6/MDP+US significantly increased tumor-infiltrating mature DCs to 67.6% ± 3.4% (p < 0.001 vs saline controls [39.5% ± 6.1%]), with further enhancement upon α-PD1 combination (70.3% ± 2.4%, Figure S27, Fig. 7F and G). Augmented DC maturation reflected improved antigen presentation. CD8+ T cell infiltration surged to 39.4% ± 3.4% in the MPA-RSP/Ce6/MDP+US+α-PD1 group (p < 0.0001 vs saline group [22.6% ± 4.6%]), demonstrating US-driven ICD as a potent activator of cytotoxic lymphocytes. Notably, monotherapy with either MPA-RSP/Ce6/MDP+US (28.5% ± 3.5%) or α-PD1 alone (28.8% ± 2.4%) induced only moderate CD8+ T cell infiltration, highlighting the necessity of dual therapy for maximal immune activation (Figure S28, Fig. 7H and I). Splenic central memory T cells (TCM) rose from 7.2% ± 1.6% (saline) to 14.5% ± 2.2% in the combination group (p < 0.001; Figure S29, Fig. 7J and K). This 2.0-fold increase in TCM cells highlights the regimen's potential to establish long-term immune surveillance. Notably, while MPA-RSP/Ce6/MDP+US alone elevated TCM to 12.6% ± 2.6%, α-PD1 alone resulted in only 8.7% ± 2.7%, indicating the augmentation of memory cells by US.

To validate the tumor-suppressive effects and microenvironment remodeling, histological and immunofluorescence (IF) analyses were performed. Although H&E staining revealed no overt morphological alterations (Fig. 7L), TUNEL assay demonstrated pronounced DNA fragmentation in both the MPA-RSP/Ce6/MDP+US monotherapy and combination (MPA-RSP/Ce6/MDP+US+α-PD1) groups, confirming US-induced apoptosis (Fig. 7M, Fig. S30A). Supporting in vitro findings that ROS and calcium dyshomeostasis in MPA-RSP/Ce6/MDP+US synergistically amplify ER stress to trigger ICD (Fig. 4C–F), IF staining revealed significantly enhanced CRT exposure, in tumors treated with either MPA-RSP/Ce6/MDP+US monotherapy or its combination with α-PD1 (Fig. 7N, Fig. S30B), correlating with increased mature DC infiltration observed by flow cytometry (Fig. 7F and G). CD8 IF staining further validated that combination therapy achieved the highest cytotoxic T cell infiltration (Fig. 7O, Fig. S30C).

Collectively, these findings demonstrated that MPA-RSP/Ce6/MDP+US elicited the tumor immune microenvironment by promoting ICD-driven DC maturation and CD8+ T cell recruitment, thereby increasing tumor responsiveness to checkpoint inhibition and supporting further exploration of this combinatorial strategy for EC immunotherapy.

3. Discussion

Recent studies have demonstrated rapid advances in multifunctional sonodynamic therapy (SDT) nanoplatforms that integrate multiple therapeutic modalities to enhance antitumor efficacy. Semiconductor-based nanomaterials, metal–organic frameworks, and organic sonosensitizers have been developed to improve US-triggered ROS generation [27,28], while the incorporation of immunomodulation [29,30], ferroptosis induction [31], and multimodal imaging [28] has further expanded SDT-based therapeutic potential. However, many existing multifunctional SDT systems primarily focus on increasing ROS production or combining additional cytotoxic mechanisms, whereas disease-specific targeting, programmable control of synergistic therapeutic components, and real-time monitoring remain relatively underexplored. In this study, the MPA-RSP/Ce6/MDP nanoplatform provides a multifunctional SDT strategy by integrating MPA-mediated tumor targeting, ROS-triggered CCB release, calcium homeostasis disruption, and SPECT-guided imaging, thereby providing a precise and controllable therapeutic strategy with potential relevance to fertility-sparing treatment of endometrial cancer.

Although inhibiting calcium influx is a potential strategy for endometrial cancer, the clinical use of CCBs is limited by the need for systemic administration at high doses and by potential cardiovascular toxicity [5,9,13,14]. In this study, we encapsulated manidipine into a ROS-responsive nanoparticle to achieve controlled release with SDT. Our findings suggest that calcium dysregulation and SDT-induced ROS synergistically amplify ER stress. Transcriptomic analysis of Ishikawa cells under different treatment conditions revealed negative enrichment of the proteasome pathway in the MDP group, suggesting that inhibition of calcium signaling may lead to transcriptional downregulation of proteasome-related genes [32]. Meanwhile, SDT-generated ROS increased oxidative and proteotoxic stress. In the MPA-RSP/Ce6/MDP+US combination group, the positive enrichment of the proteasome pathway may represent a stress-driven compensatory transcriptional response to the oxidative burden [23,24]. Consistently, WB analysis demonstrated enhanced p-eIF2α, CHOP, and p-IRE1α signaling in the combination group, revealing amplified ER stress at the protein level. In addition, transcriptional alterations in apoptosis, ferroptosis, p53, mTOR, FoxO, and AMPK pathways reflect broader cellular responses. These findings provide additional transcriptomic context for the coordinated effects of calcium dysregulation and SDT-induced oxidative stress.

This aligns with reports that calcium chelation with ethylene glycol tetraacetic acid (EGTA) reduces intracellular Ca2+ to enhance cancer cell death [33], and that loss of calcium homeostasis synergistically exacerbates proteasome dysfunction and ROS-mediated cell death [34]. However, the relative contribution of calcium dysregulation versus ROS-mediated damage requires further clarification. Future studies incorporating calcium-rescue experiments, genetic modulation of calcium channels, proteasome activity assays, or ubiquitinated protein accumulation analysis will be important to establish the causal role of calcium dysregulation in this therapeutic strategy. Notably, calcium inhibition has also been shown to sensitize photothermal therapy and thermo-immunotherapy by disrupting calcium-dependent stress responses [33,35]. Since calcium acts as a master second messenger in cellular homeostasis, CCBs may represent a potential sensitizer for energy-based interventions [36].

Recent studies in nanomedicine have adopted “in situ vaccination” strategies to induce durable systemic antitumor immunity [37,38]. In addition to initial cytotoxicity, persistent antitumor immune activity plays an important role in tumor control [39,40]. SDT generates ROS to trigger ICD, promoting DC maturation and T cell infiltration [41,42]. Reprogramming tumor-associated macrophages (TAMs) can suppress distant tumors [43]. At the molecular level, several mechanisms have been implicated in SDT, including the cGAS–STING axis, ROS-mediated pyroptosis [44], cuproptosis [45], metabolic reprogramming [46], and immune checkpoint regulation. In this study, calcium inhibition and ROS generation amplified ER stress, which triggered CRT exposure and the release of ATP/HMGB1, thereby enhancing DC maturation and CD8+ T cell infiltration, and synergizing with PD-1 blockade. The increased proportion of TCM cells in the MPA-RSP/Ce6/MDP+US+α-PD1 group provides suggestive evidence for a potential memory-associated immune response, which requires further validation through tumor-rechallenge experiments and longitudinal functional immune profiling.

For fertility-sparing management of endometrial cancer, progesterone-based therapy still faces problems, including drug insensitivity and the lack of non-invasive monitoring options [19,47]. Our SDT-CCB nanoplatform was designed to enable ultrasound-responsive drug release and real-time SPECT imaging. However, challenges in endometrial cancer nanomedicine remain unresolved and were not addressed in this study [[48], [49], [50]]. The preclinical status of endometrial cancer nanomedicine and SDT can be attributed to several unresolved issues, including manufacturing reproducibility of polymers and nanoparticles, long-term biosafety of repeated sonication on reproductive tissues, consistent pharmacokinetic profiles, regulatory approval pathways, and compatibility with existing progestin-based regimens. Moreover, more detailed mechanistic exploration will be needed to guide patient selection based on PR expression and tumor differentiation status [19,51,52]. Direct reproductive-safety evaluation and fertility-outcome studies will be necessary to further determine the clinical potential of this strategy for fertility-sparing treatment of endometrial cancer.

This study has several limitations. Subcutaneous models do not fully recapitulate the native tumor microenvironment. The treatment period was relatively short, and long-term safety and fertility outcomes remain unassessed. In addition, immune profiling should extend beyond DCs and T cells to include other relevant immune populations.

In summary, this study represents an ultrasound-responsive nanoparticle that integrates calcium inhibition, SDT, and immune activation. It offers a therapeutic strategy that shifts the focus from sustained hormonal exposure to spatiotemporally controlled physical intervention, thereby providing a potential multimodal strategy for fertility-preserving management of endometrial cancer.

CRediT authorship contribution statement

Bing Xiao: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Jingyi Zhou: Validation, Investigation, Funding acquisition, Conceptualization. Mao Tang: Writing – review & editing, Validation, Investigation, Formal analysis. Yanjiu Li: Writing – review & editing, Validation, Investigation, Formal analysis. Ting Huang: Funding acquisition, Conceptualization. Jinjian Liu: Validation, Resources, Investigation, Funding acquisition. Jianfeng Liu: Supervision, Resources, Funding acquisition. Lijun Yang: Validation, Resources, Methodology, Investigation, Funding acquisition. Haihua Xiao: Writing – review & editing, Supervision, Resources, Project administration, Conceptualization. Lingpu Zhang: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Conceptualization. Jianliu Wang: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Data availability statement

The RNA sequencing data of Ishikawa cells treated with control or various treatment conditions have been deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession number GSE325161.

Ethics approval and consent to participate

All animal experiments described in this work were conducted under guidelines evaluated and approved by the Peking University Institutional Animal Care and Use Committee (2022PHE099).

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.

Acknowledgements

This work was supported by National Natural Science Foundation of China (82230050, 82372621, 82403899, 32571626 and 52503213), the Special Scientific Research Project for Health Development in the Capital under Grant number 2022-1-4081, Tianjin Municipal Science and Technology Commission Grant (24ZXKJSY00050-7), and the Natural Science Foundation of Tianjin (24JCZDJC00150, 24JCZDJC00170).

Footnotes

Peer review under the responsibility of editorial board of Bioactive Materials.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioactmat.2026.09.017.

Contributor Information

Lingpu Zhang, Email: lpzhang@suda.edu.cn.

Jianliu Wang, Email: wangjianliu@pkuph.edu.cn.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (9.9MB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
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Data Availability Statement

The RNA sequencing data of Ishikawa cells treated with control or various treatment conditions have been deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession number GSE325161.

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