pH-responsive CaCO3 nanoplatform amplifies SDT via calcium overload-ROS loop for deep tumor therapy

TL;DR

A pH-sensitive nanoparticle boosts sonodynamic therapy for deep tumors by triggering a calcium and ROS feedback loop, improving treatment effectiveness.

Contribution

A novel pH-responsive nanoplatform that synergizes calcium overload with sonodynamic therapy via a self-amplifying ROS loop.

Findings

The nanoplatform achieved 90.9% tumor inhibition and 80% 60-day survival rate in vivo.

Mitochondrial calcium overload caused a 71% collapse in membrane potential and a 1.6-fold increase in ROS.

The therapy enhanced immunogenic cell death markers like CRT exposure and HMGB1 release.

Abstract

Sonodynamic therapy (SDT) for deep-seated tumors is limited by tumor microenvironment (TME) barriers. We developed a hyaluronic acid (HA)-modified mesoporous calcium carbonate nanoplatform (HA/CaCO3@Ce6) to synergistically enhance calcium overload and SDT. The CD44-targeted nanoplatform demonstrated pH-responsive degradation in acidic TME, resulting in the release of Ca2+ and chlorin e6 (Ce6). The released Ca2+ induced mitochondrial calcium overload, causing 71% collapse in membrane potential and 1.6-fold increase in reactive oxygen species (ROS) generation, establishing a “Ca2+-ROS positive feedback loop.” This synergy triggered robust immunogenic cell death (ICD), enhancing CRT exposure by 94.2%, HMGB1 release by 46.2%, and ATP decrease by 74.5%. In vivo, it achieved 90.9% tumor inhibition and 80% 60-day survival rate, alleviated tumor hypoxia, and inhibited tumor proliferation and angiogenesis. This “ion-interference SDT” paradigm overcomes the limitations of SDT through self-amplified ROS cycle and provides an effective strategy for treating deep-seated solid tumor with significant clinical translation potential.

•Calcium overload synergizes with SDT for deep tumors•pH-responsive nanoplatform targets tumors and degrades in acidic microenvironment•Mitochondrial Ca2+ overload triggers a self-amplifying ROS loop, enhancing SDT efficacy•Nanotherapy induces mitochondrial dysfunction and immunogenic cell death

Calcium overload synergizes with SDT for deep tumors

pH-responsive nanoplatform targets tumors and degrades in acidic microenvironment

Mitochondrial Ca2+ overload triggers a self-amplifying ROS loop, enhancing SDT efficacy

Nanotherapy induces mitochondrial dysfunction and immunogenic cell death

Nanoparticles; Drug delivery system; Applied science

Figures

Characterization and property evaluation of HA/CaCO_3_@Ce6(A)TEM images of CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6. Scale bars, 50 nm.(B) Hydrodynamic diameters of CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6 measured by DLS method. Data are expressed as the mean ± SD (*n* = 3).(C) Zeta potentials of CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6 measured by DLS method. Data are expressed as the mean ± SD (*n* = 3).(D) UV-vis spectra of free Ce6, CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6.(E) Fluorescence spectra of CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6.(F) N_2_ adsorption/desorption isotherm of CaCO_

Evaluating Ca^2+^ overload and immunogenic cell death in tumor cells induced by HA/CaCO_3_@Ce6 + US(A) Alizarin red S staining showed the localize Ca^2+^ deposits forming a purplish-red complex, respectively. Scale bars, 100 μm.(B) Bio-TEM images of Hepa1-6 cells treated with HA/CaCO_3_@Ce6 + US and PBS control. Scale bars, 2 μm and 500 nm.(C) CLSM images of Hepa1-6 cells stained with CRT antibody after different treatments. Scale bars, 100 μm.(D) Quantitative immunofluorescence intensity of Hepa1-6 cells stained with CRT antibody after different treatments. Data are expressed as the mean ± SD

Blood and histological biological safety of HA/CaCO_3_@Ce6(A) Microscopic images of H&E-stained tissue slices of different organs harvested from mice after caudal intravenous injections of different nanoparticles. Scale bars, 100 μm.(B) The liver function (ALT, AST, TBIL, ALB, and ALP) and renal function (BUN, CREA, and UA) indices of mice in each group after caudal intravenous injections of different nanoparticles. Data are expressed as the mean ± SD (*n* = 3). G1–G5 represent PBS, Ce6, CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6, respectively. The consistent Ce6 equivalent dose of 3.5 mg/kg was

Theoretical investigation of ·O_2_^−^ production(A) Electrostatic potential map of the Ce6 molecule.(B) PDOS of the Ce6 molecule.(C) TD-DFT plot of the Ce6 molecule.(D) LUMO distribution of the Ce6 molecule.(E) HOMO distribution of the Ce6 molecule.(F) HOMO/LUMO distributions of Ce6@O_2_ molecules.(G) HOMO/LUMO distributions of Ce6@·O_2_^−^ molecules.PDOS, projected density of states; TD-DFT, time-dependent density functional theory; HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital.

Full text

Introduction

The abnormal characteristics of the tumor microenvironment (TME) provide significant possibilities for the development of targeted tumor therapy strategies. In recent years, therapeutic strategies based on disrupting the oxidative stress homeostasis of tumor cells have become a focus of research in the field of tumor therapy.1^,^2^,^3^,^4 Sonodynamic therapy (SDT), as an emerging non-invasive treatment modality, generates a large amount of reactive oxygen species (ROS) including superoxide anion (・O_2_^−^), hydrogen peroxide (H_2_O_2_), singlet oxygen (^1^O_2_), and hydroxyl radical (・OH) through ultrasound-activated sonosensitizers, which are cytotoxic substances that induce programmed death of tumor cells.5^,^6^,^7 Compared with photodynamic therapy (PDT), SDT has significant advantages such as greater tissue penetration depth and wider application range, showing good therapeutic potential for most deep-seated solid tumors.8^,^9^,^10 However, the common characteristics in the TME such as hypoxia, acidification, and interstitial hypertension severely limit the clinical efficacy of SDT.11^,^12^,^13 Therefore, designing nanoplatforms that can respond to the abnormal characteristics of the TME and effectively overcome these limiting factors is of great significance for improving the precise killing efficiency of SDT.

Mitochondria, as the “powerhouse” of cells, not only provide a continuous source of energy for cells but also participate in regulating various signaling pathways, including apoptosis, calcium ion (Ca^2+^) homeostasis, and redox balance.14^,^15^,^16 Studies have shown that mitochondrial Ca^2+^ overload can disrupt the oxidative stress homeostasis of tumor cells, producing effective “destructive factors” that induce tumor cell death.17^,^18^,^19^,^20^,^21 Currently, various antitumor strategies based on Ca^2+^ overload mechanism have been developed, such as calcium peroxide (CaO_2_) nanoparticles,20^,^22 calcium-iron synergistic delivery systems,18 and calcium carbonate-based PDT platforms.23 However, these studies have mainly focused on chemotherapy, photodynamic therapy, or immunotherapy, and only a few reports have focused on combining the Ca^2+^ overload mechanism with SDT. In addition, existing calcium-based nanomaterials mostly use CaO_2_ as a calcium source, which can provide both Ca^2+^ and H_2_O_2_, but its rapid decomposition and potential risk of excessive oxidative stress limit its clinical application.24^,^25^,^26^,^27

Notably, mitochondrial Ca^2+^ overload can not only directly induce tumor cell death but also enhance the production of ROS by disrupting mitochondrial function.28^,^29^,^30^,^31 Recent studies have revealed that there is a complex interaction between Ca^2+^ overload and ROS: on one hand, ROS can cause abnormal function of Ca^2+^ channels, leading to uncontrolled accumulation of Ca^2+^, and on the other hand, Ca^2+^ overload can further promote ROS outburst, forming a vicious cycle that ultimately results in cell death.25^,^30 However, the application of the synergistic effect in SDT has not been fully explored. In particular, the key scientific question of whether the small amount of ROS produced by Ca^2+^ overload can exert a synergistic amplification effect with the ROS produced by SDT, thus significantly improving the therapeutic effect, remains to be addressed.

In response to the above challenges, this study designed a hyaluronic acid (HA)-modified mesoporous nano-calcium carbonate platform (HA/CaCO_3_@Ce6) to enhance the efficacy of SDT. As shown in Scheme 1, compared with existing studies, this work has the following significant advantages: First, the combination of Ca^2+^ overload mechanism and SDT not only broadens the research direction of Ca^2+^ overload in the field of SDT but also provides more insights for the treatment of deep tumors. Second, it discovers and confirms the significant synergistic effect between Ca^2+^ overload and the ROS produced by SDT. Third, as the core carrier, mesoporous nano-calcium carbonate offers performance superior to that of traditional solid nano-calcium carbonate or CaO_2_: (1) The mesoporous structure can directly serve as a loading platform for the sonosensitizer chlorin e6 (Ce6), eliminating the need for additional porous carriers such as metal-organic framework (MOFs) or polymers, thereby significantly reducing preparation costs and complexity; (2) The mesoporous structure has a larger specific surface area and pore volume, which improve the loading efficiency of Ce6; (3) The mesoporous structure enables faster acid-responsive degradation, overcoming the limitation of traditional nanoparticles that require layer-by-layer exfoliation, allowing for faster release of Ca^2+^ and enhancing the Ca^2+^ overload effect. Finally, the nanoplatform achieves CD44 receptor targeting through HA modification, specifically degrading in the TME, releasing Ca^2+^ and Ce6, effectively regulating the acidic microenvironment of the tumor, alleviating hypoxia, and simultaneously inducing mitochondrial Ca^2+^ overload, which significantly enhance the ROS production capacity of SDT.Scheme 1. Synthesis process and mechanism diagram of HA/CaCO_3_@Ce6(A) Schematic of the functional pattern of HA/CaCO_3_@Ce6.(B) Tumor microenvironment-responsive calcium-based nanoplatform enables CD44-targeted modulation, improves acidic tumor niche, and amplifies mitochondrial Ca^2+^ overload-mediated ROS production to trigger immunogenic cell death, thereby potentiating SDT efficacy.

This study not only expands the application scope of the “ion-interference therapy” strategy32 but also successfully introduces this strategy into the field of SDT, providing new insights to address the limitations of SDT in the TME. In vitro and in vivo experiments confirmed that the HA/CaCO_3_@Ce6 nanoplatform, according to the synergistic action of Ca^2+^ overload and SDT, improves the TME and ROS production, effectively inducing immunogenic cell death (ICD) in tumor cells, manifested by the exposure of calreticulin (CRT), the release of high-mobility group box 1 (HMGB1), and the decrease in the intracellular adenosine triphosphate (ATP) level, thereby significantly enhancing antitumor efficacy. This research provides a safe and effective solution for non-invasive treatment of deep-seated solid tumors, holding great theoretical importance and clinical translational value, and is expected to provide a new strategic approach for future tumor treatment.

Results

Synthesis of HA/CaCO3@Ce6 nanoparticles

In this study, we developed a HA-functionalized mesoporous calcium carbonate nanoplatform loaded with Ce6 for SDT. Mesoporous CaCO_3_ nanoparticles with uniform spherical morphology (approximately 185 nm) were synthesized via a controlled co-precipitation method. Transmission electron microscopy (TEM) and elemental mapping (Figures 1A and S1) confirmed the homogeneous distribution of Ca, C, and O elements within the nanospheres, indicating a composite structure comprising an inorganic CaCO_3_ phase and an organic carbon-rich skeleton. X-ray photoelectron spectroscopy (XPS) survey and high-resolution spectra (Figures S2 and S3) identified characteristic peaks for Ca 2p, C 1s, O 1s, and N 1s, verifying the coexistence of HA, CaCO_3_, and Ce6 in the final HA/CaCO_3_@Ce6 nanospheres. Brunauer–Emmett–Teller (BET) analysis revealed a mesoporous structure with a specific surface area of 10.856 m^2^/g, pore volume of 0.03 cm^3^/g, and average pore diameter of 11.05 nm (Figures 1F and 1G), providing substantial capacity for drug loading. Ce6 was efficiently loaded into the mesopores via electrostatic adsorption, achieving a loading efficiency of 73.20% and a loading capacity of 12.3% (Figure 1H). This integrated mesoporous design eliminates the need for additional carriers and enables rapid degradation in acidic environments.Figure 1. Characterization and property evaluation of HA/CaCO_3_@Ce6(A)TEM images of CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6. Scale bars, 50 nm.(B) Hydrodynamic diameters of CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6 measured by DLS method. Data are expressed as the mean ± SD (n = 3).(C) Zeta potentials of CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6 measured by DLS method. Data are expressed as the mean ± SD (n = 3).(D) UV-vis spectra of free Ce6, CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6.(E) Fluorescence spectra of CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6.(F) N_2_ adsorption/desorption isotherm of CaCO_3.(G) Pore diameter distribution of CaCO_3.(H) The standard curve of UV-vis absorbance-concentration of Ce6 at 650 nm.(I) Ce6 release profiles from HA/CaCO_3_@Ce6 at two different pH values (5.6 and 7.4). Data are expressed as the mean ± SD (n = 3).(J) HA/CaCO_3_@Ce6 + US produces ESR spectra of ·O_2_^−^.DLS, dynamic light scattering; US, ultrasound.

To confer tumor-targeting capability, the Ce6-loaded CaCO_3_ nanoparticles were surface-functionalized with HA for CD44 receptor recognition. TEM images (Figure 1A) showed that spherical morphology was maintained throughout the synthesis. Dynamic light scattering (DLS) indicated a gradual increase in hydrodynamic diameter from 186.5 ± 1.0 nm (CaCO_3_) to 192.2 ± 8.3 nm (CaCO_3_@Ce6), and finally to 204.5 ± 0.6 nm (HA/CaCO_3_@Ce6) (Figure 1B). Zeta potential measurements (Figure 1C) showed a surface charge reversal from +9.5 mV (CaCO_3_) to −17.1 mV after HA modification, confirming successful surface coating. UV-vis and fluorescence spectroscopy (Figures 1D and 1E) verified successful Ce6 loading with preserved optical properties. Additional characterization by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA) (Figures S4–S6) confirmed the successful HA modification, showing altered CaCO_3_ characteristic peaks, appearance of HA absorption peaks, and improved thermal stability of HA/CaCO_3_@Ce6 compared with unmodified counterparts.

Characterization of HA/CaCO3@Ce6 nanoparticles

Fluorescence spectroscopy with excitation at 400 nm and emission scanning from 600 to 800 nm (Figure 1E) showed that both CaCO_3_@Ce6 and HA/CaCO_3_@Ce6 exhibited a characteristic emission peak at approximately 750 nm, matching free Ce6. BET analysis confirmed the mesoporous structure (Figures 1F and 1G). Quantitative analysis using a standard curve at 650 nm (Figure 1H) determined the drug loading rate of 12.3% and encapsulation efficiency of 73.20%. pH-responsive release studies (Figure 1I) demonstrated significantly accelerated Ce6 release at pH 5.6 relative to that at pH 7.4, confirming acid-triggered CaCO_3_.33^,^34 This degradation also consumed H^+^, potentially alleviating TME acidity. Furthermore, under pH 5.6, the faster Ce6 release coupled with ultrasound led to significantly higher ROS generation compared to that under neutral conditions (Figure S7). Electron spin resonance (ESR) analysis using DMPO spin trap confirmed ⋅O_2_^−^generation upon ultrasound irradiation (Figure 1J). Hemolysis assays showed excellent blood compatibility up to 200 μg/mL (Figure S8).

Tumor-targeted delivery and in vitro therapeutic evaluation

Tumor-targeted drug delivery represents a critical strategy to minimize systemic toxicity and maximize therapeutic efficacy via the selective accumulation of nanotherapeutic agents within tumor tissues.35^,^36 CD44, a transmembrane glycoprotein receptor, is frequently overexpressed on the surface of various cancer cell types, including human hepatocellular carcinoma (HCC) cells, making it a promising target for tumor-specific delivery.37^,^38^,^39 Given the necessity of maintaining species consistency to accurately assess immune-related therapeutic outcomes, we selected the murine Hepa1-6 hepatocellular carcinoma cell line to enable translational consistency with subsequent in vivo studies in immunocompetent C57BL/6 mice. Flow cytometry confirmed higher CD44 expression on murine Hepa1-6 cells than on human HepG2 and Hep3B cells (Figures 2A and 2B), providing the molecular basis for the selective tumor targeting capability of HA/CaCO_3_@Ce6 and enabling receptor-mediated endocytosis and intracellular accumulation of the nanoplatform.Figure 2In vitro HA-mediated targeting performance and the antitumor effects of HA/CaCO_3_@Ce6(A) Flow cytometry analysis of CD44 receptor expression on the membrane surface of the different liver cancer cells.(B) Quantitative analysis of CD44 receptor expression on the membrane surface of the different liver cancer cells. Data are expressed as the mean ± SD (n = 3), ∗∗∗p < 0.001 by Student’s t test.(C) CLSM images of Hepa1-6 cells stained with Fluo-4 after incubations with PBS, CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6 for 6 h. Scale bars, 100 μm.(D) Quantitative analysis of fluorescence intensity of Hepa1-6 cells stained with Fluo-4 after incubations with PBS, CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6 (G1–G4) for 6 h, respectively. Data are expressed as the mean ± SD (n = 3). ∗∗∗p < 0.001 by Student’s t test.(E) Cell viability of Hepa1-6 cells treated with PBS, CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6 with different concentrations for 24 h. Data are expressed as the mean ± SD (n = 3). ∗∗∗p < 0.001 by Student’s t test.(F) Cell viability of Hepa1-6 cells incubated with PBS, CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6 for 6 h and irradiated with different US intensity for 3 min. Data are expressed as the mean ± SD (n = 3), ∗∗∗p < 0.001 by Student’s t test.(G) FCM patterns of apoptotic cells in Hepa1-6 cells with different treatments.(H) Quantitative analysis of apoptotic cells in Hepa1-6 cells with different treatments. G1–G6 represent PBS, CaCO_3_, CaCO_3_@Ce6, CaCO_3_@Ce6 + US, HA/CaCO_3_@Ce6 + US, and Ce6 + US, respectively. Data are expressed as the mean ± SD (n = 3), ∗∗p ˂ 0.05; ∗∗∗p < 0.001 by Student’s t test.CLSM, confocal laser scanning microscopy; US, ultrasound.

To assess cellular uptake and intracellular Ca^2+^ dynamics, we employed Fluo-4 AM, a cell-permeable calcium indicator. This probe exhibits minimal fluorescence until cleaved by intracellular esterases to form Fluo-4, which binds to Ca^2+^ with high affinity and produces intense green fluorescence.17^,^22

Confocal laser scanning microscope (CLSM) showed significantly enhanced fluorescence in the HA/CaCO_3_@Ce6 group after 6 h incubation (Figures 2C and 2D), indicating CD44-mediated uptake and intracellular Ca^2+^ release. CCK-8 assay demonstrated excellent biocompatibility of the nanoplatform, with cell viability exceeding 80% for CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6 at concentrations below 50 μg/mL (Figure 2E). This favorable safety profile is essential for clinical translation, as it indicates minimal off-target toxicity at therapeutic doses. Importantly, ultrasound (US) irradiation triggered concentration- and intensity-dependent cytotoxicity in both CaCO_3_@Ce6 and HA/CaCO_3_@Ce6 groups, with the latter demonstrating significantly enhanced tumor cell killing efficacy than the non-targeted counterparts (Figure 2F). Flow cytometric apoptosis analysis further confirmed the highest apoptotic rate in the HA/CaCO_3_@Ce6 + US group (Figures 2G and 2H).

In vitro evaluation of ROS generation and mitochondrial dysfunction

ROS generation across treatment groups was quantitatively assessed using the dichlorofluorescein diacetate (DCFH-DA) probe, and CLSM analysis revealed a hierarchical pattern (Figures 3A and 3B): minimal ROS generation in PBS control groups; moderate enhancement in the CaCO_3_, CaCO_3_@Ce6, and Ce6 + US groups; and significantly amplified ROS production in the CaCO_3_@Ce6 + US and HA/CaCO_3_@Ce6 + US groups. Notably, the HA/CaCO_3_@Ce6 + US group exhibited the most intense green fluorescence, indicating the highest ROS levels.Figure 3In vitro evaluation of HA/CaCO_3_@Ce6 + US for ROS stimulation and mitochondrial damage(A) CLSM images of Hepa1-6 cells with different treatments for tracing ROS level. Scale bars, 100 μm.(B) Quantitative analysis of Hepa1-6 cells with different treatments for tracing ROS level. Data are expressed as the mean ± SD (n = 3), ∗∗∗p < 0.001 by Student’s t test.(C) CLSM images of Hepa1-6 cells with different treatments after JC-1 probe staining for evaluating mitochondria damages. Scale bars, 100 μm.(D) CLSM images of live/dead cells of Hepa1-6 cells with different treatments.(E) Quantitative dead cell ratio of Hepa1-6 cells with different treatments. Data are expressed as the mean ± SD (n = 3), ∗∗∗p < 0.001 by Student’s t test. G1–G6 represent PBS, CaCO_3_, CaCO_3_@Ce6, CaCO_3_@Ce6 + US, HA/CaCO_3_@Ce6 + US, and Ce6 + US, respectively.CSLM, confocal laser scanning microscopy; US, ultrasound.

To further elucidate the mitochondrial consequences of this ROS amplification, we employed JC-1 staining to assess mitochondrial membrane potential dynamics. The JC-1 probe undergoes a reversible color shift from red fluorescent J-aggregates in healthy, polarized mitochondria to green monomers upon membrane potential collapses.15 Quantitative analysis (Figures 3C and S9) showed that the HA/CaCO_3_@Ce6 + US group exhibited the most pronounced green fluorescence and minimal red fluorescence, indicating the most severe mitochondrial depolarization among all treatment groups. Cell viability assessment using calcein-AM/propidium iodide (PI) dual staining (Figures 3D and 3E) provided functional validation of the molecular observations that the HA/CaCO_3_@Ce6 + US group exhibited the highest proportion of PI-positive dead cells.

Calcium overload-induced ICD mediated by HA/CaCO3@Ce6 + US

Direct evidence of intracellular Ca^2+^ deposition was obtained using alizarin red S staining, which forms a characteristic red complex upon chelating Ca^2+^ salts.9^,^20 Staining of Hepa1-6 cells treated with various formulations (G1–G6: PBS, CaCO_3_, CaCO_3_@Ce6, CaCO_3_@Ce6 + US, HA/CaCO_3_@Ce6 + US, and Ce6 + US) revealed varying degrees of red coloration in groups containing calcium-based nanoparticles (G2–G5), indicating intracellular Ca^2^ accumulation following nanoparticle uptake (Figure 4A). Notably, the HA/CaCO_3_@Ce6 + US group displayed the most intense Ca^2^ staining, confirming maximized Ca^2^ deposition resulting from HA-mediated targeting coupled with ultrasound-enhanced nanoparticle degradation and ion release. Ultrastructural analysis by bio-transmission electron microscopy (Bio-TEM) unveiled profound morphological changes in Hepa1-6 cells following HA/CaCO_3_@Ce6 + US treatment (Figure 4B). Compared with the PBS control, the treated cells exhibited characteristic features of Ca^2+^ overload-induced damage, including pronounced mitochondrial swelling, cristae disruption, and endoplasmic reticulum (ER) vacuolization. These structural alterations correlate with the observed collapse of mitochondrial membrane potential and represent morphological hallmarks of Ca^2+^-mediated dysfunction.Figure 4. Evaluating Ca^2+^ overload and immunogenic cell death in tumor cells induced by HA/CaCO_3_@Ce6 + US(A) Alizarin red S staining showed the localize Ca^2+^ deposits forming a purplish-red complex, respectively. Scale bars, 100 μm.(B) Bio-TEM images of Hepa1-6 cells treated with HA/CaCO_3_@Ce6 + US and PBS control. Scale bars, 2 μm and 500 nm.(C) CLSM images of Hepa1-6 cells stained with CRT antibody after different treatments. Scale bars, 100 μm.(D) Quantitative immunofluorescence intensity of Hepa1-6 cells stained with CRT antibody after different treatments. Data are expressed as the mean ± SD (n = 3). ∗∗∗p < 0.001 by Student’s t test.(E) CLSM images of Hepa1-6 cells stained with HMGB1 antibody after different treatments. Scale bars, 100 μm.(F) Quantitative immunofluorescence intensity of Hepa1-6 cells stained with HMGB1 antibody after different treatments. Data are expressed as the mean ± SD (n = 3). ∗∗p < 0.05; ∗∗∗p < 0.001 by Student’s t test. G1–G6 represent PBS, CaCO_3_, CaCO_3_@Ce6, CaCO_3_@Ce6 + US, HA/CaCO_3_@Ce6 + US, and Ce6 + US, respectively.

Furthermore, we investigated the expression levels of key signaling proteins associated with the Ca^2+^–ROS cycle across different treatment groups by using western blot (WB) analysis. WB results and quantitative analysis revealed that Calpain-1 expression was highest in the HA/CaCO_3_@Ce6 + US group, whereas ATP2B4 expression was lowest in this group (Figure S10), indicating a potential inverse relationship between the two proteins under Ca^2+^ overload conditions.

To quantify ICD induction, we performed immunofluorescence staining for CRT and HMGB1 in Hepa1-6 cells. The results demonstrated significantly enhanced CRT surface exposure in the HA/CaCO_3_@Ce6 + US group compared with all control groups (Figures 4C and 4D), with quantitative analysis confirming the highest fluorescence intensity in this treatment group. HMGB1 staining revealed markedly reduced intracellular HMGB1 levels in the HA/CaCO_3_@Ce6 + US group (Figures 4E and 4F), indicating active release of this nuclear damage- associated molecular pattern (DAMP). Furthermore, we investigated another typical characteristic indicator of ICD, the intracellular ATP content. The results demonstrated that following various treatments, the intracellular ATP level in the HA/CaCO_3_@Ce6 + US group decreased remarkably and was the lowest among all groups (Figure S11).

In vivo antitumor efficacy evaluation

Tumor formation was initially confirmed using ^68^Ga-FAPI PET/CT imaging, which demonstrated specific contrast agent accumulation in tumor tissues with no uptake in surrounding muscle (Figure 5A). This imaging modality provided superior spatial resolution and quantitative accuracy compared with fluorescence imaging, enabling precise assessment of nanoparticle distribution and therapeutic efficacy. For therapeutic evaluation, the consistent Ce6 equivalent dose of 3.5 mg/kg was applied across all nanoparticle treatment groups in animal experiments, followed by ultrasound (3 W/cm^2^, 5 min) 6 h post-injection. Tumor growth and body weight were monitored over a 13-day treatment period. As shown in Figures 5B and 5C, the HA/CaCO_3_@Ce6 + US group displayed the most potent antitumor efficacy, showing the strongest suppression of tumor growth among all groups. Consistently, endpoint tumor weights were lowest in the HA/CaCO_3_@Ce6 + US group (Figure 5D). Notably, all groups maintained stable body weights throughout the study (Figure 5E), indicating excellent systemic safety. Furthermore, the HA/CaCO_3_@Ce6 + US treatment significantly prolonged survival, with 80% of mice surviving to the study endpoint (Figure 5F).Figure 5In vivo assessment of antitumor properties of HA/CaCO_3_@Ce6 + US(A) ^68^Ga-FAPI PET/CT imaging of small animals indicated the success of Hepa1-6 tumor-bearing formation.(B) Tumor growth curves of implanted C57BL/6 mice with different treatments. Data are expressed as the mean ± SD (n = 5). ∗∗p < 0.05; ∗∗∗p < 0.001 by Student’s t test.(C) Representative photographs of tumors with different treatments of implanted C57BL/6 mice.(D) The weights of Hepa1-6 tumors harvested from the C57BL/6 mice. Data are expressed as the mean ± SD (n = 5). ∗∗p < 0.05; ∗∗∗p < 0.001 by Student’s t test.(E) Body weight growth curves of mice in different treatment groups. Data are expressed as the mean ± SD (n = 5).(F) Survival curves of the tumor-bearing mice after different treatments.(G) Tumors in different treatment groups were stained for H&E, TUNEL, Ki 67, CRT, and HMGB1. Scale bars, 100 μm. G1–G6 represent PBS, CaCO_3_, CaCO_3_@Ce6, CaCO_3_@Ce6 + US, HA/CaCO_3_@Ce6 + US, and Ce6 + US, respectively. The consistent Ce6 equivalent dose of 3.5 mg/kg was applied across all nanoparticle treatment groups in the animal experiment.

Hematoxylin and eosin staining (H&E) and dUTP nick-end labeling (TUNEL) staining demonstrated the most extensive necrosis and apoptosis in the HA/CaCO_3_@Ce6 + US group (Figure 5G), indicating the greatest histopathological damage among all treatments. Consistently, immunohistochemistry revealed the lowest Ki67 expression in the HA/CaCO_3_@Ce6 + US group (Figures 5G and S12), further corroborating its potent antiproliferative activity. Alizarin red staining of tumor tissues subjected to different treatments showed that the HA/CaCO_3_@Ce6 group (group 5, G5) exhibited the most prominent calcium salt deposition (Figure S13), indicating that HA/CaCO_3_@Ce6 can effectively accumulate in tumor tissues. Simultaneously, the results of dihydroethidium (DHE) fluorescence staining of frozen tumor tissue sections also indicated that this group displayed the highest intensity of ROS signals (Figure S14).

Furthermore, by detecting the translocation of CRT to the cell membrane surface (Figures 5G and S15) and the release of HMGB1 into the extracellular space (Figures 5G and S16),10^,^40 it was confirmed that the nanoplatform can effectively initiate a series of DAMP cascade reactions, which are pivotal for subsequently activating the adaptive immune response against tumor antigens. Furthermore, the robust induction of ICD, as evidenced by CRT exposure and HMGB1 release, suggests a critical involvement of dendritic cell (DC) maturation in the observed antitumor immunity. We, thus, conducted an analysis of the maturation state of DCs. The results indicated that in the HA/CaCO_3_@Ce6 treatment group, the proportion of CD80^+^CD86^+^ mature DCs was the highest among all groups examined (Figure S17). This finding further corroborates the notion that this treatment can effectively enhance antitumor immune activation. Additionally, this treatment reduced HIF-1α expression (Figure S18) and tumor vascular density (Figure S19).

Biosafety evaluation of HA/CaCO3@Ce6 nanoplatform

The biocompatibility of HA/CaCO_3_@Ce6 was comprehensively evaluated in healthy C57BL/6 mice following the intravenous administration of PBS, free Ce6, CaCO_3_, CaCO_3_@Ce6, or HA/CaCO_3_@Ce6 (n = 3 per group). After 14 days, major organs including heart, liver, spleen, lungs, kidneys, and brain were harvested for H&E staining. Microscopic analysis revealed no significant histopathological abnormalities or tissue damage in any of the examined organs across all the groups (Figure 6A). Additionally, serum biochemical analysis was performed to evaluate potential hepatotoxicity and nephrotoxicity. Liver function markers including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-BIL), albumin (ALB), and alkaline phosphatase (ALP), as well as renal function markers comprising creatinine (CREA), blood urea nitrogen (BUN), and uric acid (UA), were measured. Statistical analysis demonstrated no significant differences in any of these parameters between the HA/CaCO_3_@Ce6 group and control groups (Figure 6B).Figure 6. Blood and histological biological safety of HA/CaCO_3_@Ce6(A) Microscopic images of H&E-stained tissue slices of different organs harvested from mice after caudal intravenous injections of different nanoparticles. Scale bars, 100 μm.(B) The liver function (ALT, AST, TBIL, ALB, and ALP) and renal function (BUN, CREA, and UA) indices of mice in each group after caudal intravenous injections of different nanoparticles. Data are expressed as the mean ± SD (n = 3). G1–G5 represent PBS, Ce6, CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6, respectively. The consistent Ce6 equivalent dose of 3.5 mg/kg was applied across all nanoparticle treatment groups in the animal experiment.ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin; ALB, albumin; ALP, alkaline phosphatase; BUN, blood urea nitrogen; CREA, creatinine; UA, uric acid.

Theoretical investigation of ⋅O2−production

Theoretical calculations examined Ce6’s electrostatic potential, orbital distributions, oxygen interaction, and superoxide generation. The electrostatic potential map (Figure 7A) highlighted electronegative regions on carboxyl oxygens and the porphyrin ring. Projected density of states (PDOS) analysis (Figure 7B) indicated dominant p and d orbital contributions near the Fermi level. Time-dependent density functional theory (TD-DFT) calculations identified strong absorption bands (Figure 7C). Molecular orbital distributions showed lowest unoccupied molecular orbital (LUMO) (Figure 7D) and highest occupied molecular orbital (HOMO) (Figure 7E) localized on the porphyrin ring, with a HOMO-LUMO gap of 1.647 eV. Ce6 exhibited strong binding to O_2_ (−18.6 kcal mol^−1^, Figure S20), with the LUMO and LUMO+1 localized on O_2_’s π∗ orbitals upon binding. The energy gap between Ce6’s HOMO and O_2_’s π∗ orbitals (0.972 eV) was smaller than Ce6’s intrinsic π→π∗ gap (Figures 7F and 7G), favoring electron transfer. For Ce6@⋅O_2_^−^, the HOMO-LUMO gap narrowed to 0.365 eV, with HOMO-1 and HOMO-2 localized on ⋅O_2_^−^, indicating preferential electron transfer to oxygen. Mulliken charge analysis suggested an electron transfer tendency of 0.732 eV from Ce6 to ⋅O_2_^−^.Figure 7. Theoretical investigation of ·O_2_^−^ production(A) Electrostatic potential map of the Ce6 molecule.(B) PDOS of the Ce6 molecule.(C) TD-DFT plot of the Ce6 molecule.(D) LUMO distribution of the Ce6 molecule.(E) HOMO distribution of the Ce6 molecule.(F) HOMO/LUMO distributions of Ce6@O_2_ molecules.(G) HOMO/LUMO distributions of Ce6@·O_2_^−^ molecules.PDOS, projected density of states; TD-DFT, time-dependent density functional theory; HOMO, highest occupied molecular orbital; LUMO, lowest unoccupied molecular orbital.

Discussion

This study successfully developed a multifunctional HA/CaCO_3_@Ce6 nanoplatform that synergistically combines Ca^2+^ overload with SDT for enhanced treatment of deep-seated tumors. The results demonstrate a coordinated mechanism of action that operates at multiple levels to induce potent antitumor effects both in vitro and in vivo.

The core innovation lies in the strategic design of the nanocarrier itself. The mesoporous CaCO_3_ structure serves a dual purpose: as a high-capacity reservoir for the sonosensitizer Ce6 and as a source of Ca^2+^. Its pH-responsive degradation is a critical feature, triggered selectively in the acidic TME.33^,^34^,^41 This not only enables a controlled release of Ce6 but also initiates the Ca^2+^ overload process. The released Ca^2+^ ions preferentially accumulate in mitochondria, disrupting the membrane potential and electron transport, thereby inducing intrinsic oxidative stress. Concurrently, CO_2_ generated from CaCO_3_ degradation can undergo inertial cavitation under ultrasound, providing a mechanical component to the therapy that enhances cell membrane permeability and sonodynamic efficacy.42^,^43

The surface modification with HA transforms nanoparticles from a passively accumulating agent into an active targeting system. The significant enhancement in cellular uptake and therapeutic outcome observed in CD44-high Hepa1-6 cells, compared to non-targeted controls, underscores the importance of receptor-mediated endocytosis in maximizing intracellular nanoparticle concentration. This active targeting is crucial for deep-seated tumors where the enhanced permeability and retention (EPR) effect alone may be insufficient for adequate drug delivery.37^,^38^,^39 The CCK8 and apoptosis assays verified the powerful cytotoxic effect of the HA-targeting group. This synergistic effect can be attributed to multiple mechanisms: (1) HA-mediated active targeting enhances tumor-specific accumulation; (2) Acid-triggered degradation of CaCO_3_ in the TME generates Ca^2+^, inducing mitochondrial Ca^2+^ overload; and (3) Ultrasound activation of Ce6 produces abundant ROS, Furthermore, Ca^2+^ overload and ROS generation establish a self-amplifying cycle that exacerbates cellular damage.

The synergy between Ca^2+^ overload and SDT creates a self-amplifying cycle of cellular damage. Ca^2+^ influx into mitochondria generates a baseline level of ROS through disruption of the electron transport chain.9 This oxidative stress is dramatically amplified by the ultrasound-activated Ce6, which produces copious amounts of ⋅O_2_^−^. The generated ROS, in turn, further disrupts cellular Ca^2+^ homeostasis by oxidizing thiol groups in Ca^2+^-handling proteins. As suggested by our WB data, the observed degradation of ATP2B4 and activation of Calpain-1 indicate a breakdown of Ca^2+^ efflux mechanisms, leading to further intracellular Ca^2+^ accumulation. This mitochondrial Ca^2+^ accumulation triggers a cascade of detrimental effects: (1) disruption of mitochondrial membrane potential; (2) impairment of electron transport chain function; and (3) induction of oxidative stress imbalance.44 Additionally, CO_2_ released during degradation can undergo inertial cavitation under ultrasound, mechanically disrupting cellular structures and thereby augmenting SDT efficacy.32 This establishes a vicious positive feedback loop— Ca^2+^ overload begets more ROS, and ROS beget more Ca^2+^ dysregulation—culminating in catastrophic mitochondrial failure.

A particularly significant finding is the nanoplatform’s ability to induce ICD. Ca^2+^ overload-induced mitochondrial dysfunction activates ER stress through the PERK-eIF2α-ATF4 signaling axis, which drives the translocation of CRT from the ER lumen to the cell surface.10^,^40^,^45^,^46 This process effectively translates mitochondrial damage into immunogenic “eat-me” signals that facilitate phagocytic recognition by dendritic cells. Concurrently, the massive ROS production promotes CRT exposure and the concomitant release of HMGB1 and ATP efflux.47^,^48^,^49 The detection of surface-exposed CRT, released HMGB1, and extracellular ATP confirms the activation of a complete DAMP cascade.45 This transforms the therapy from a locally cytotoxic treatment into a systemic immunotherapeutic strategy. The induction of ICD is likely a direct consequence of the severe ER stress and mitochondrial damage triggered by the Ca^2+^-ROS cycle.17^,^40 The subsequent maturation of DCs, as evidenced by increased CD80^+^CD86^+^ populations, validates the initiation of an adaptive immune response. This immunomodulatory dimension is a major advantage, as it can potentially address tumor recurrence and metastasis by establishing a systemic, antigen-specific immune memory.23

The in vivo results robustly translate the mechanistic findings into therapeutic efficacy. The superior tumor growth suppression, extended survival, and histological evidence of extensive apoptosis and necrosis in the HA/CaCO_3_@Ce6 + US group confirm the platform’s potential. Importantly, the treatment also modulated the TME by alleviating hypoxia (reduced HIF-1α) and inhibiting angiogenesis. The reduction in tumor hypoxia is multifactorial, potentially resulting from improved perfusion due to vascular normalization effects of Ca^2+^, CO_2_-induced cavitation that enhances oxygenation, and the consumption of H^+^ ions during CaCO_3_ degradation that mitigates acidosis.18^,^43 A less hypoxic and acidic TME is inherently less aggressive and more susceptible to therapy.

The theoretical calculations provide a fundamental understanding of the sonodynamic process at the molecular level. The small energy gap between Ce6’s HOMO and O_2_’s π∗ orbitals, and the clear electron transfer tendency to form ⋅O_2_^−^, offer a quantum mechanical rationale for Ce6’s high ROS generation efficiency under ultrasound. This complements the experimental ESR data and strengthens the mechanistic foundation of the SDT component.

In conclusion, this work presents a rationally designed nanotheranostic platform that successfully integrates tumor targeting, pH-responsive drug release, Ca^2+^ overload, and SDT. It moves beyond a simple combination by exploiting the inherent biochemical and biophysical links between Ca^2+^ signaling and oxidative stress to create a self-reinforcing therapeutic cycle. While the current subcutaneous model provides proof-of-concept, the platform’s design—particularly its active targeting and ability to modulate the TME—addresses key challenges in treating deep-seated tumors, such as poor penetration and immunosuppression. Future studies employing orthotopic models will be valuable to further validate its efficacy against anatomically challenging internal malignancies. This multifaceted approach, combining direct cytotoxicity with immune activation, represents a promising strategy for improving outcomes in solid tumor therapy.

Limitations of the study

While our results demonstrate the platform’s strong preclinical potential, key steps remain for clinical translation. Future work will focus on: (1) conducting advanced safety and efficacy studies in more clinically relevant orthotopic models; and (2) exploring its modular design for combination therapies with other anticancer agents. Addressing these points will be crucial for advancing this ion-interference SDT toward clinical application.

Resource availability

Lead contact

Further information and material requests should be directed to and will be fulfilled by the lead contact, Bao Li (libao@hust.edu.cn).

Materials availability

Nanomaterials described in this study are available upon request following the completion of a materials transfer agreement from the lead contact.

Data and code availability

• •Data reported in this paper will be shared by the lead contact upon request.
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This work was supported by The 10.13039/501100001809National Science Foundation of China (no. 82400119), the Science and Technology Bureau Dawning project of Wuhan (no. 2023020201020536 and 2023020201020543), the 10.13039/501100003819Natural Science Foundation of Hubei Province, China (no. 2023AFB454), and the Collaborative Academic Innovation Project of Shandong Cancer Hospital (no. TS006).

Author contributions

M.C. and D.L. designed the study; M.C., Y.W., and H.S. performed the experiments; Y.N. and C.L. analyzed the data and organized the figures; M.C., Z.W., L.X., and J.Z. wrote the manuscript; M.C., X.C., and B.L. revised the manuscript.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCESOURCEIDENTIFIERAntibodiesAnti-Mouse CD44 Rabbit Recombinant AntibodySanying BiotechnologyCat No. 15675-1-AP; RRID: AB_2076198High Mobility Group Box 1 (HMGB1) Polyclonal antibodySanying BiotechnologyCat No. 10829-1-AP; RRID: AB_2232989Calreticulin (CRT) Polyclonal antibodySanying BiotechnologyCat No. 10292-1-AP; RRID: AB_2069615CoraLite488-conjugated Goat Anti-Rabbit IgGSanying BiotechnologyCat No. SA00013-2; RRID: AB_2797132CD31 Polyclonal antibodySanying BiotechnologyCat No. 23083-1-AP; RRID: AB_2881055Hif-1 alpha Polyclonal antibodSanying BiotechnologyCat No. 20960-1-AP; RRID: AB_10732601Ki-67 Polyclonal antibodySanying BiotechnologyCat No. 27309-1-AP; RRID: AB_2756525Calpain 1 Rabbit mAbABclonal BiotechnologyCat No. A8710; RRID: AB_2863593ATP2B4 Rabbit mAbABclonal BiotechnologyCat No. A10105; RRID: AB_2757628β-Actin Rabbit mAbABclonal BiotechnologyCat No. AC026; RRID: AB_2768234CD80 Monoclonal AntibodyThermo Fisher ScientificCat No. A14723; RRID: AB_2534239CD86 Monoclonal AntibodyThermo Fisher ScientificCat No. 12-0862-82; RRID: AB_465768Chemicals, peptides, and recombinant proteinsCaCl_2_·2H_2_OAladdin scientificCat No. 22691-02-7NH_4_HCO_3_Aladdin scientificCat No. 1066-33-7Hyaluronic Acid (HA)Maclean Biochemical TechnologyCat No. 9004-61-9Chlorin e6 (Ce6)Yuanye biotechnologyCat No. 19660-77-6Critical commercial assaysROS assay kitBeyotime biotechnologyCat No.Y061804Cell Counting Kit-8 (CCK8) assay kitBeyotime biotechnologyCat No.C0039Calcein-AM/PI assay kitBeyotime biotechnologyCat No.C2015LAnnexin V-FITC/PI assay kiBeyotime biotechnologyCat No.C1062LAlizarin Red S staining solutionBeyotime biotechnologyCat No.C0140Fluo-4 AM calcium ion fluorescent probe kitBeyotime biotechnologyCat No.S1061SEnhanced mitochondrial membrane potential assaykitBeyotime biotechnologyCat No.C2003SATP Assay KitBeyotime biotechnologyCat No.S0026

Experimental model and study participant details

The Hepa 1-6 cell line, Hep3B cell line and HepG2 cell line were derived from the Key Laboratory of Molecular Imaging of Hubei Province. All cells were cultured in DMEM medium containing 1% penicillin-streptomycin solution and 10% fetal bovine serum under the culture conditions of 37°C and 5% CO_2_ humidification environment.

Female C57BL/6 mice (4-8 weeks old) were purchased from Hubei Bainet Biotech Company (NO.422023100014309) and raised in SPF-level specific pathogen-free facilities. All animal experiments were conducted in accordance with the relevant policies of the National Health Commission and were approved by the Laboratory Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IACUC NO:2024-4692).

Method details

Synthesis of HA/CaCO3@Ce6 nanoparticles

Synthesis of mesoporous nano-CaCO_3_: Mesoporous calcium carbonate nanoparticles were prepared via a co-precipitation method. Briefly, 150 mg of CaCl_2_·2H_2_O was dissolved in 100 mL of anhydrous ethanol, followed by the addition of 5 g NH_4_HCO_3_. The reaction proceeded under continuous stirring at 40°C for 72 h. The resulting white precipitate was collected by centrifugation at 12,000 rpm for 10 min to obtain a white CaCO_3_ precipitate. Then the precipitate was washed 2–3 times with anhydrous ethanol to yield mesoporous CaCO_3_.

Synthesis of CaCO_3_@Ce6: The sonosensitizer Ce6 was loaded onto the mesoporous CaCO_3_nanoparticles via an electrostatic adsorption and physical trapping method. Briefly, 10 mg of the as-synthesized CaCO_3_ nanoparticles were dispersed in 1 mL of deionized water (10 mg/mL) and mixed with 1 mL of a Ce6 solution (2 mg/mL in deionized water). The mixture was subjected to sonication in a bath sonicator for 30 minutes to facilitate the diffusion of Ce6 molecules into the mesopores of the carrier and incubated in the dark at room temperature for 2 hours under gentle agitation. Finally, free Ce6 was removed by centrifugation (10,000 rpm, 10 min) to obtain the desired product CaCO_3_@Ce6.

Synthesis of HA/CaCO_3_@Ce6: The CaCO3@Ce6 nanoparticles were functionalized with HA for CD44 targeting. Specifically, 2 mL of CaCO_3_@Ce6 (10 mg/mL) was mixed with an HA solution (4 mg/mL) and sonicated for 30 min. The mixture was then stirred vigorously for 4 h to complete the coating process. After centrifugation (10,000 rpm, 10 min), the precipitate was washed 1–2 times with PBS to obtain the final product HA/CaCO_3_@Ce6.

Characterization of HA/CaCO3@Ce6 nanoparticles

The morphology of CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6 was observed and imaged using transmission electron microscopy (TEM, JEM-1200EX, JEOL). The hydrodynamic diameter and zeta potential of the nanoparticles were measured via dynamic light scattering (DLS, Nano ZS90, Malvern Instruments Ltd.). Ultraviolet-Visible Spectroscopy (UV-Vis) absorption spectra were detected using the spectrophotometer (UV-2450, Shimadzu), while the fluorescence spectra of Ce6, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6 were recorded using a fluorescence spectrophotometer (EDX-720, Shimadzu). To conduct a more in - depth analysis of the composition of HA/CaCO_3_@Ce6, we further verified its elemental composition, Ce6 loading capacity, and the surface modification of HA using techniques such as elemental mapping analysis, X - ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo Fishe), X - ray diffraction (XRD, Japan RIKEN), Fourier - transform infrared spectroscopy (FTIR, Nicolet Nexus 6700, Thermo Fishe), and thermogravimetric analysis (TGA, Perkin Elmer).

The mesoporous structure and loading capacity of CaCO_3_ were analyzed using a surface area and porosity analyzer (ASAP 2460, Micromeritics). The encapsulation efficiency and drug loading capacity of Ce6 were calculated based on a standard calibration curve. To evaluate the release profile of Ce6 from HA/CaCO_3_@Ce6, 1 mL of the nanoparticle solution was loaded into a dialysis bag in fifferent pH of PBS (pH 7.4 / pH 5.6) under gentle agitation. The absorbance of released Ce6 was measured at different time intervals. Additionally, free radical generation by the nanocomposites was detected using electron spin resonance spectroscopy (ESR, Bruker A300-10/12). To simultaneously determine the release rate of Ce6 under different pH conditions (pH 7.4 and pH 5.6) and analyze its correlation with the generation efficiency of reactive oxygen species (ROS), we employed the DCFH-DA probe method. At various time points, 500 μL of the released Ce6 solution was mixed with 2500 μL of freshly prepared DCFH solution (obtained by dissolving DCFH-DA in PBS and incubating it in the dark for activation). After ultrasonic treatment (1 W, 3 minutes) of the reaction system, the fluorescence intensity of DCF was immediately measured using a UV-Vis spectrophotometer at an excitation wavelength of 488 nm and an emission wavelength of 530 nm to assess the ROS generation level.

Hemolysis assay

Hemocompatibility was evaluated using a standard hemolysis assay. Whole blood was collected from healthy BALB/c mice via the orbital venous plexus and centrifuged at 3,000 rpm for 15 min to separate red blood cells (RBCs). Preparing 1mL of HA/CaCO_3_@Ce6 nanoparticles at different concentrations (0, 20, 50, 100, and 200μg/mL) compared to ultrapure water (positive control) and PBS (negative control). Then, 20 μL of RBC suspension was added to each well, incubated at 37°C for 4 h. After centrifugation (3,000 rpm, 15 min), the samples were photographed, and the absorbance (OD) at 542 nm was measured. Hemolysis (%) = OD (ultrapure water control)−OD (PBS control)/OD (sample)−OD (PBS control) ×100%.

Detection of CD44 expression

To assess the CD44 expression level on murine hepatocellular carcinoma cells (Hepa1-6), HepG2, Hep3B (CD44-positive controls), the different cells were seeded in 6-well plates and cultured overnight. According to the manufacturer’s protocol for the Anti-Human/Mouse CD44 antibody and the CoraLite488-conjugated secondary antibody manual, the cells were stained and subsequently analyzed by flow cytometry (BD LSRFortessa™, FCM).

Detection of intracellular calcium ion concentration

Hepa1-6 cells (5×10^4^ cells/well) were plated in 12-well plates and allowed to adhere overnight. The cells were then treated with PBS, CaCO_3_, CaCO_3_@Ce6, HA/CaCO_3_@Ce6 for 6 hours (The concentration of all nanomaterials was 50 μg/mL). After incubation, the spent medium was carefully aspirated, followed by three consecutive PBS washes to remove residual components. According to the protocol of the Fluo-4 AM calcium ion fluorescent probe kit, the cells were stained with Fluo-4 AM fluorescent probe and incubated for 30 minutes, then fluorescence images were captured using a Confocal Laser Scanning Microscope (CLSM 710, Zeiss, Germany), and the fluorescence intensity of each group was quantified for analysis.

Nanoparticle cytotoxicity analysis

Hepa1-6 cells were seeded in 96-well plates at a density of 5×10^3^ cells per well and cultured for 24 h. Subsequently, the cells were treated with culture media containing different concentrations (0, 5, 10, 20, 50, and 100 μg/mL) of nanomaterials (PBS, CaCO_3_, CaCO_3_@Ce6, HA/CaCO_3_@Ce6) for an additional 24 h. Following incubation, we carefully aspirated the supernatant and replaced it with a 1:10 dilution of CCK-8 reagent in basal medium (100 μL/well). After 1 h of dark incubation at 37 °C, we quantified cell viability by measuring optical density at 450 nm using a SpectraMax microplate reader.

In vitro tumor cell proliferation detection

Hepa1-6 cells were plated in 96-well culture plates (5×10^3^ cells/well) and maintained in complete medium for 24 hours prior to treatment. The cells were then treated with PBS, CaCO_3_, CaCO_3_@Ce6, HA/CaCO_3_@Ce6 (50 μg/mL) for 6 h. Following incubation, the cells were exposed to ultrasound irradiation at varying intensities (0, 1, and 3 W/cm^2^) for 3 min using a calibrated ultrasonic transducer. Continue to cultivate for 24 hours, the cell viability was detected using the CCK-8 assay kit.

In vitro tumor cell ROS production

The generation of intracellular ROS in treated Hepa1-6 cells was detected with a fluorescent ROS Assay Kit. The experiment was conducted with six experimental groups: PBS, CaCO_3_, CaCO_3_@Ce6, CaCO_3_@Ce6+US, HA/CaCO_3_@Ce6+US and Ce6+US (50 μg/mL, with ultrasound irradiation at 1.0 W/cm^2^ for 3 minutes). Following treatments, cells were incubated with DCFH-DA probe (diluted 1:1000 in culture medium) for 30 minutes at 37 °C under light-protected conditions. We removed unbound probes by washing cells with PBS, then quantified ROS production via Confocal Laser Scanning Microscope (CLSM 710, Zeiss, Germany). Fluorescence intensity data were normalized to control groups before statistical analysis.

In vitro tumor cell apoptosis and live/dead situation detection

Hepa 1-6 cells were seeded in 12-well plates and divided into six experimental groups. After 24 hours of incubation, the cells were subjected to ultrasound irradiation (1.0 W/cm^2^, 3 minutes). Following treatment, the cell monolayers were digested using EDTA-free trypsin, washed twice with ice-cold PBS, and resuspended in binding buffer. Cell apoptosis was assessed by FITC-Annexin V/PI co-staining per kit instructions, followed by quantitative assessment using FCM (BD LSRFortessa™).

Live (Calcein-AM+) and dead (PI+) cell populations were distinguished by Confocal Laser Scanning Microscope (CLSM 710, Zeiss, Germany).

In vitro tumor cell mitochondrial membrane potential detection

Changes in mitochondrial membrane potential (ΔΨm) were evaluated using a JC-1 assay kit. After 24 hours of seeding in 12-well plates, Hepa 1-6 cells were treated with PBS, CaCO_3_, CaCO_3_@Ce6, CaCO_3_@Ce6+US, HA/CaCO_3_@Ce6+US and Ce6+ US( 50 μg/mL, 1.0 W/cm^2^, 3 minutes). Following treatment, cells were rinsed with PBS and incubated with JC-1 working solution at 37 °C for 30 min in the dark. After removal of the staining solution and multiple washes with PBS, the fluorescence was imaged using a confocal laser scanning microscope (CLSM 710, Zeiss). JC-1 aggregates (red fluorescence, high ΔΨm) and monomeric forms (green fluorescence, low ΔΨm) were detected, and the red/green fluorescence ratio was quantified to assess ΔΨm collapse.

In vitro detection of tumor cell calcium overload and Bio-TEM imaging

After treatment, the Hepa 1-6 tumor cells incubated with 1 mL of Alizarin Red S staining solution at 37 °C for 30 min. The cells were then thoroughly washed with PBS, and calcium deposition in each group was observed and recorded under a microscope, with a purplish-red complex was formed.

For Bio-TEM imaging, Hepa 1-6 tumor cells were rapidly washed twice with PBS after different treatments, followed by fixation with 2.5% glutaraldehyde at 4°C for 12 hours. The cell slides were then rinsed three times with phosphate buffer and post-fixed in 1% osmium tetroxide at 4°C for 2 hours. Subsequently, the samples were washed by double-distilled water, dehydrated through an ethanol gradient, and subjected to solvent replacement using isoamyl acetate. Critical point drying was performed, followed by coating with an ion sputter coater. After processing, the samples were imaged using a scanning electron microscope (HITACHI Regulus 8100, Hitachi, Japan).

Western blot of cells in vitro

To verify the role of key signaling pathways in the calcium-ROS cycle, we treated the cells in 6-well plates with the following different conditions: PBS, CaCO_3_, CaCO_3_@Ce6, CaCO_3_@Ce6 + US, HA/CaCO_3_@Ce6 + US and Ce6 + US (50 μg/mL, with ultrasound irradiation at 1.0 W/cm^2^ for 3 minutes). Subsequently, the proteins of the cells were extracted to detect the expression levels of the proteins related to the calcium-ROS cycle, ATP2B4 R and Calpain 1. In brief, after different treatments, total intracellular proteins were extracted using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using a micro UV-Vis spectrophotometer (Thermo Scientific™ NanoDrop™ One, USA). Equal amounts of protein samples (40–60 μg) from each group were subjected to electrophoresis separation on a 10% SDS-PAGE gel (200 V, 36 min), and then transferred to a PVDF membrane by constant current electrotransfer (400 mA, 80 min or 150 min, adjusted according to the molecular weight of the target protein). The membrane was blocked with QuickBlock™ blocking buffer at room temperature for 30 min to reduce non-specific binding. Subsequently, the membrane was incubated with the corresponding primary antibodies at 4 °C overnight. After three washes with TBST, the membrane was incubated with Alexa Fluor® 680-labeled fluorescent secondary antibody working solution at room temperature in the dark for 1 h. After another three washes with TBST, the signals were detected using an Odyssey DLx near-infrared dual-color laser imaging system (Gene Company Limited, China), and the band intensities were quantified using Image Studio software.

CRT and HMGB1 immunofluorescence

Hepa 1-6 cells were divided into six experimental groups and treated with PBS, CaCO_3_, CaCO_3_@Ce6, CaCO_3_@Ce6 + US, HA/CaCO_3_@Ce6 + US and Ce6 + US, (50 μg/mL, 1.0 W/cm^2^, 3 minutes), respectively. After treatment, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed with PBS, non-specific binding was blocked using 5% BSA in PBST for 20 min. Primary antibody incubation was performed with anti-CRT/HMGB1 (Proteintech, 1:500) in blocking buffer at 4°C for 16 h. Following extensive PBS washes, secondary staining was conducted with CoraLite 488-conjugated goat anti-rabbit IgG (Proteintech, 1:200) for 2 h at room temperature in the dark. Nuclei were counterstained with DAPI (1:1000). The expression patterns of CRT and HMGB1 in each treatment group were visualized and documented using a confocal laser scanning microscope (CLSM 710, Zeiss, Germany).

Detection of intracellular ATP content

To detect the changes in intracellular ATP levels, Hepa1-6 cells were seeded in 6-well plates at a density of 1.0×10^5^ cells per well and cultured overnight to allow them to adhere. Subsequently, according to the experimental groups, the corresponding drugs were added and ultrasonic treatment was performed. After incubation, the cells from each group were collected and lysed to extract intracellular ATP. 60 μL of the cell lysate was added to the detection wells of a specially coated ATP detection plate, followed by the addition of 100 μL of ATP detection reagent working solution. The mixture was incubated at room temperature in the dark for 3–5 minutes to eliminate background interference. Then, the luminescence value was immediately measured using a chemiluminescence detector (Thermo, USA), and the ATP content was quantified based on the standard curve.

In vivo therapeutic experiment

C57BL/6 mice bearing Hepa 1-6 tumors were randomly divided into six groups (n=5 per group) when tumor volumes reached 80–100 mm^3^. Tumor formation was confirmed in three randomly selected mice using ^68^Ga-FAPI small-animal PET/CT imaging (1 μCi/mg; InliView-3000B, Novel Medical). Mice received tail vein injections of the formulations every other day for a total of seven treatments (days 0, 2, 4, 6, 8, 10, and 12): PBS, CaCO_3_, CaCO_3_@Ce6, CaCO_3_@Ce6 + US, HA/CaCO_3_@Ce6 + US, Ce6 + US. The US irradiation parameters: 3.0 W /cm^2^, 5 min and the consistent Ce6 equivalent dose of 3.5 mg/kg was applied across all nanoparticle treatment groups in animal experiment. Body weight and tumor size were monitored throughout the treatment period. Tumor volume was calculated as: Volume = Length × Width× Width /2. On day 12, all the mice were euthanized and tumors were harvested for gross examination and weighing, and photographed for gross morphological analysis. Tumor sections were subjected to: H&E staining for assessment of necrosis, TUNEL assay for apoptosis quantification, Expression profiling of Ki67, CD31, Hif-1α, CRT, and HMGB1 were performed by immunohistochemistry (IHC). To further assess the levels of calcium deposition and ROS generation within tumor tissues, we subjected the tumor tissues to frozen - section staining for ROS detection and paraffin embedding, followed by alizarin red staining to detect calcium deposition. Additionally, For survival analysis, an additional cohort of mice (n=5/group) was monitored for 60 days post-treatment to evaluate long-term therapeutic efficacy.

The detection of DC maturation

C57BL/6 mice inoculated with Hepa 1 - 6 tumors were randomly allocated into six groups (n = 3). Starting from day 0, corresponding formulations were administered via tail - vein injection every other day, with a total of seven treatments carried out on days 0, 2, 4, 6, 8, 10, and 12. The treatment regimens were as follows: PBS, CaCO_3_, CaCO_3_@Ce6, CaCO_3_@Ce6 + US, HA/CaCO_3_@Ce6 + US, and Ce6 + US. The parameters of ultrasound were set at 3.0 W/cm^2^ for a duration of 5 min. After the completion of the treatment, tumor tissues from each group of mice were harvested. Single - cell suspensions were prepared through mechanical grinding and enzymatic digestion methods. The cell suspensions were blocked with 5% bovine serum albumin (BSA) at 4°C for 30 min. Then, surface - marker antibodies used for detecting the maturation of dendritic cells (DCs) were added for staining according to the coloring scheme of CD11c - APC , MHC II-PerCP-Cy5.5, CD86 - FITC, and CD80 - PE. Subsequently, flow cytometry was employed to analyze the degree of DC maturation within the tumor microenvironment of each group.

Biocompatibility and histological safety analysis of blood and tissues

Fifteen 6-week-old female C57BL/6 mice were randomly divided into five groups (n=3) and recieved intravenous injections of PBS, Ce6, CaCO_3_, CaCO_3_@Ce6, and HA/CaCO_3_@Ce6, the consistent Ce6 equivalent dose of 3.5 mg/kg was applied across all nanoparticle treatment groups in animal experiment. After two weeks, Following retro-orbital blood collection, samples were centrifuged (12,000 rpm, 10 min) to isolate serum. Biochemical assays were performed to assess liver and kidney function. The biochemical parameters assessed included ALT, AST, T-BIL, ALB, ALP, BUN, CREA, and UA. Additionally, major organs (heart, liver, spleen, lungs, kidneys, and brain) were harvested from all mice for H&E staining to evaluate potential pathological damage.

Quantification and statistical analysis

All statistical analyses in this study were performed using SPSS 22.0 software (Chicago, IL, USA). Data are presented as mean ± standard deviation (SD) from at least three independent experiments (n≥3). Group comparisons were conducted using student’s t test. Statistical methods for each analysis are fully described in the figure legends. A two-sided P value˂0.05 was considered statistically significant, with ∗∗P˂0.05 and ∗∗∗P˂0.001 denoting significance levels. All the graphs were generated using GraphPad Prism 5 software (La Jolla, CA, USA).

Chen et al. develop a pH-responsive calcium carbonate nanoplatform that targets tumors and degrades in acidic microenvironments. This induces mitochondrial calcium overload and creates a self-amplifying reactive oxygen species loop under ultrasound, effectively enhancing sonodynamic therapy and antitumor immunity against deep-seated tumors.