A pH-responsive nanoplatform enhancing tumor therapy via calcium overload-induced oxidative stress to potentiate phototherapy and chemotherapy

Abstract

The specific tumor microenvironment (TME) and the ability of tumor cells to evade drug therapy pose challenges to the efficacy of single monotherapies. Herein, a multifunctional calcium carbonate-based nanoprobe (Fe₃O₄/CaCO₃-CSL/ICG) was synthesized using a simple one-step method. This nanoprobe is designed to respond specifically to the acidic TME, where the calcium carbonate shell dissolves, releasing therapeutic agents. It combines three therapeutic modalities: phototherapy, chemotherapy, and ion interference therapy. In cell experiments, it was confirmed that after entering tumor cells, the acidic intracellular environment triggered the release of calcium ions from the nanoprobe, leading to mitochondrial calcium ion overload. The loaded indocyanine green (ICG) produced photothermal and photodynamic effects under near-infrared laser irradiation. The reactive oxygen species (ROS) generated by photodynamic therapy further amplify oxidative stress caused by mitochondrial calcium overload. Additionally, celastrol (CSL) enhanced calcium ion-induced mitochondrial calcium death. Differential gene expression analysis further supported the combined therapeutic effect of Fe₃O₄/CaCO₃-CSL/ICG, indicating the regulation of genes related to calcium regulation, oxidative stress and apoptosis. In summary, we developed a responsive nanoplatform with pH-triggered degradation and controlled drug release, which enhances tumor suppression by inducing mitochondrial apoptosis through calcium overload and ROS accumulation, in combination with chemotherapy and phototherapy. This work presents a promising nanotherapeutic strategy for tumor treatment.

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1. Introduction

Cancer represents a significant global health concern and remains one of the leading causes of human mortality.¹ Consequently, cancer treatment has become the focal point for modern medical research. Currently, the primary clinical modalities for treating cancer include chemotherapy (CT), radiotherapy and thermotherapy in addition to starvation therapy and immunotherapy for malignant tumours. Some new emerging nanodrugs, nanomaterials and targeted drugs have also shown significant effects in cancer treatment. Although various therapeutic strategies are currently available for cancer treatment, their therapeutic effect remains far inferior to that expected. Therefore, researchers should aim to develop an innovative cancer therapeutic strategy aiming to mitigate the side effects of conventional treatments and efficiently eradicate cancer cells.

Calcium ion (Ca²⁺), a second messenger in cell signalling, regulates several physiological events, such as muscle contraction, neuronal excitation and cell migration, growth and death.² The crucial role of Ca²⁺ in cancer treatment has attracted the attention of researchers. ‘Calcium death’ causes cancer cell death by regulating the concentration of calcium ions inside tumour cells, resulting in intracellular calcium overload and activation of various calcium-based signalling pathways.³ Effectively transporting Ca²⁺ into cancer cells and inducing its release from the endoplasmic reticulum into the cytoplasm, with specific targeting of mitochondria, is critical for the success of calcium-induced tumor therapy. Currently, in addition to being used as drug carriers, nano-inorganic biomaterials are valued for their bioactivity, degradability and concentrated ion release in ion interference therapy for tumours.⁴ Therefore, researchers have developed various calcium interference therapeutic platforms, such as CaO₂,^5,6 CaCO₃,^7–11 CaS,¹² and CaP,^13,14 to interfere with calcium homeostasis in the mitochondria, which triggers apoptosis caused by calcium overload. Interestingly, due to the aberrant frequency of Ca²⁺ signaling in cancer cells,¹⁵ normal cells exhibit greater tolerance to the adverse effects of calcium overload compared to tumor cells. Additionally, certain naturally occurring active compounds, such as celastrol (CSL), can stimulate sustained Ca²⁺ release from the endoplasmic reticulum into the cytoplasm. This process enhances the Ca²⁺ movement into mitochondria, causing mitochondrial Ca²⁺ overload and inhibiting Ca²⁺ efflux, thereby promoting apoptosis in tumor cells.¹⁶ However, using calcium-based nanoparticles alone may have various limitations in tumour therapy. For example, as an important regulator of calcium ions, mitochondria maintains calcium homeostasis by releasing calcium ions through calcium channels,^17,18 thus weakening the effect of calcium-based nanoparticles on intracellular calcium ions in tumour cells. An increase in mitochondrial reactive oxygen species (ROS) is a typical feature of mitochondrial apoptosis,¹⁹ and thus an increase in exogenous ROS is critical for further disrupting mitochondrial calcium buffering capacity and synergistically promoting mitochondrial calcium overload.^20–22 Therefore, constructing an integrated nanoplatform based on nanomineral particles for calcium ion interference therapy synergized with exogenous reactive oxygen species to promote oxidative stress for tumor suppression has a wide range of applications.

Recently, the emphasis on cancer treatment has progressively transitioned from single therapeutic approaches to a diversified and multimodal combination therapy, which has been confirmed by basic tumour research and clinical practice.^23,24 Photothermal therapy (PTT) is a treatment method extensively explored for its application in tumor treatment. By employing photothermal agents, photothermal therapy (PTT) uses near-infrared light to generate heat, effectively ablating tumor cells. This method is noted for its high efficacy and minimal adverse effects. PTT has been widely combined with diverse anticancer treatments such as CT, chemodynamic therapy (CDT), photodynamic therapy (PDT), and immunotherapy to enhance synergistic anticancer effects through multifunctional therapeutic platforms.²⁵ The results of several studies^26–28 have demonstrated that the amalgamation of CT and PTT proves markedly superior in tumor treatment compared to any singular therapeutic approach. Indocyanine green (ICG), the only organic dye approved by the FDA for clinical use, is widely utilized in in vivo imaging and diagnostics among various photothermal conversion agents. Importantly, ICG demonstrates dual functionality in PTT and PDT. Upon exposure to near-infrared (NIR) laser irradiation, ICG exhibits robust absorption properties, converting NIR light into heat for PTT while releasing active oxygen for effective PDT. These attributes highlight ICG and its derivatives as promising candidates for dual-modality NIR fluorescence or photoacoustic imaging and synergistic PDT/PTT applications.^29,30

In this study, we selected Ca²⁺ as a therapeutic ion to induce calcium-mediated death in cells. We developed a spherical, multifunctional calcium carbonate-based nanoprobe-Fe₃O₄/CaCO₃-CSL/ICG-via a simple one-step co-precipitation method by integrating indocyanine green (ICG), celastrol (CSL) and magnetic nanoparticles (Fe₃O₄), as depicted in Scheme 1. Briefly, the nanoparticles with Fe₃O₄ as the core, encapsulated ICG and CSL through a calcium carbonate shell. The resulting Fe₃O₄/CaCO₃-CSL/ICG nanoplatform combines calcium overload therapy, photothermal/photodynamic therapy (PTT/PDT), and chemotherapy (CT), offering following advantages: (a) owing to its pH and hypersensitive properties, Fe₃O₄/CaCO₃-CSL/ICG can specifically release Ca²⁺ in the acidic tumour microenviroment. The calcium carbonate nanoparticles can not only be used as drug carriers but can also escape from lysosomes by consuming H⁺ and producing carbon dioxide, which causes lysosomal dissolution and rupture, facilitating the release of Ca²⁺ and disrupting intracellular calcium homeostasis. (b) CSL released from the nanoplatform enhances calcium-mediated cytotoxicity by promoting Ca²⁺ influx into the mitochondria from the endoplasmic reticulum.¹⁶ Moreover, CSL contributes to tumor apoptosis by modulating multiple pathways such as mitochondrial dysfunction, endoplasmic reticulum stress, and oxidative damage.^31,32 (c) ICG-loaded nanoplatforms exhibit both photodynamic and photothermal effects. PDT generates ROS, while PTT regulated calcium ion influx, amplifying ROS accumulation, inducing oxidative stress, as well as inducing mitochondrial apoptosis. (d) The spherical calcium carbonate nanoparticles synthesized via chemical precipitation improve the drug loading rate efficiency and fully encapsulate the magnetic nanoparticles and drugs, thereby minimizing premature drug leakage and reducing systemic toxicity. In conclusion, the multifunctional Fe₃O₄/CaCO₃-CSL/ICG nanoplatform represents a promising approach for combinational cancer therapy.

Scheme 1 Schematic representation of the synthesis route of Fe₃O₄/CaCO₃-CSL/ICG and the mechanism of the synergetic cancer therapy through CT, PDT/PTT and Ca²⁺ overload.

2. Materials and methods

2.1 Synthesis of Fe₃O₄/CaCO₃-CSL/ICG

Based on a study in the relevant literature³³ and previous studies conducted by our group,^28,34 the Fe₃O₄/CaCO₃-CSL/ICG composite was synthesised using a modified chemical precipitation method (Scheme 1). Briefly, CaCl₂ (1 mg) and Fe₃O₄ (60 μg) were dispersed in 5 mL of deionised water under magnetic stirring. Following an overnight (12 h) stir in darkness at room temperature (25 °C), the soultion was centrifuged (13 000 rpm, 30 min) to eliminate the supernatant and collected the precipitate. The precipitate was gently redispersed in 5 mL of deionized water containing indocyanine green (ICG, 5 μg mL⁻¹) and celastrol (CSL, 5 μg mL⁻¹), and the suspension was stirred for 12 hours in the dark to facilitate drug loading. Subsequently, 50 μg mL⁻¹ of sodium carbonate (NaCO₃) solution was added into the abovementioned mixture dropwise as a precipitant and stirred for 24 h at room temperature to form a the CaCO₃ matrix encapsulating the drugs. The Fe₃O₄/CaCO₃-CSL/ICG nanocomposites were harvested through centrifugation (8000 rpm, 30 min) and washed twice with deionized water.

2.2 Characterisation

The morphology of Fe₃O₄/CaCO₃-CSL/ICG was observed using transmission electron microscopy (TEM, Thermo Fisher Scientific, USA) at an accelerating voltage of 200 kV. The average hydrodynamic diameter and ζ potential of Fe₃O₄/CaCO₃-CSL/ICG nanoparticles were performed using dynamic light scattering (DLS, Bruker, USA) at 25 °C. The elemental composition were analyzed using X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific, USA). The optical properties of Fe₃O₄/CaCO₃-CSL/ICG were assessed with a UV-Vis spectrophotometer (Thermo Fisher Scientific, USA) over a wavelength range of 200–800 nm.

2.3 pH-Responsive behavior and drug release profile of Fe₃O₄/CaCO₃-CSL/ICG

To evaluate the pH-sensitive morphological changes of Fe₃O₄/CaCO₃-CSL/ICG nanoparticles, the nanoplatform was suspended in 1 mL PBS adjusted to pH levels of 7.4, 6.5, and 5.0 and the suspensions were incubated at 37 °C for 24 h at shaking (100 rpm). Subsequently, the solution was subjected to dilution and deposited onto a copper grid for TEM analysis. The concentration of CSL and ICG in Fe₃O₄/CaCO₃-CSL/ICG were measured using a 1260/UV HPLC system from Agilent Technologies Inc., USA. The drug loading efficiency was determined using the following formula:

To evaluate drug and ion release behavior under different pH conditions, Fe₃O₄/CaCO₃-CSL/ICG nanoparticles (1 mg mL⁻¹) were dispersed in 2 mL of PBS (pH 7.4, 6.5, and 5.0) and sealed into dialysis bags with MWCO of 3500 Da. The dialysis bags were immersed in 10 mL of PBS and kept under continuous shaking (100 rpm) at 37 °C. At specified intervals (0, 0.5, 1, 2, 4, 8, 12 and 24 h), 2.0 mL of the external PBS solution was removed and refilled with an equal volume of fresh buffer. HPLC was employed to quantify the release of ICG and CSL using the following chromatographic conditions: a 4.6 mm × 250 mm Inertex C18 column with 5 μm particle size, maintained at 35 °C, and a 20 μL injection volume. The mobile phase, consisting of 95% methanol and 5% water with 0.25% phosphoric acid (v/v), was pumped at 1.0 mL min⁻¹ flow rate for sample elution. Detection was conducted at 425 nm for CSL and 780 nm for ICG. Inductively coupled plasma mass spectrometry (ICP-MS, */i CAP Q, Thermo Fisher Scientific, Germany) was used to measure the calcium ion (Ca²⁺) release.

2.4 Singlet oxygen (¹O₂) generation detection of Fe₃O₄/CaCO₃-CSL/ICG

To assess singlet oxygen (¹O₂) generation of Fe₃O₄/CaCO₃-CSL/ICG nanoplatforms, SOSG (Thermo Fisher Scientific, USA) was employed. Fe₃O₄/CaCO₃-CSL/ICG (ICG, 10 μg mL⁻¹) platforms dissolved in PBS with different pH values (pH 7.4, 6.5 and 5.0) were mixed with SOSG (2.5 μM) and subjected to 808 nm light for different durations (0, 30, 60, 120, 150, 180, 210 and 240 s). The ¹O₂ production was assessed by measuring fluorescence intensity upon excitation (Ex: 495 nm, Em: 525 nm).

2.5 Photothermal performance of Fe₃O₄/CaCO₃-CSL/ICG

To investigate the photothermal capability of Fe₃O₄/CaCO₃-CSL/ICG under acidic conditions, Fe₃O₄/CaCO₃-CSL/ICG (1 mg mL⁻¹) was dispersed in PBS with pH levels of 7.4, 6.5, and 5.0. Each solution was subjected to laser irradiation (808 nm, 1 W cm⁻²) for different durations (0–5 min). An infrared imaging camera (Testo 865, Germany) was used to monitor the temperature variations of the samples and record the thermographic images within 5 min.

2.6 Transverse relaxation time and in vitro MR imaging

Fe₃O₄/CaCO₃-CSL/ICG was added into 1 mL of PBS with various pH values (pH 7.4, 6.5 and 5.0). T₂ images were acquired using a 3.0-T clinical MRI scanner with the following parameters: imaging frequency = 300.42 MHz, matrix = 216 × 320, field of view = 83 × 81.25 mm², slice thickness = 0.45 mm, TE = 2.42, TR = 4000 and T₂ = 1000. T₂ relaxation time was quantified on a 0.5-T micro-MRI system (Shanghai N iumag Corporation, China). Transverse relaxivity values (r₂) were determined by linear fitting of the relaxation time (T₂) against Fe molar concentrations [Fe].

2.7 Cell culture and lysosomal localization

Bel-7402 cells and normal human hepatocytes were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, P. R. China) and cultured under standard conditions (37 °C, 5% CO₂). To detect the subcellular localisation of Fe₃O₄/CaCO₃-CSL/ICG, Bel-7402 cells were incubated with Fe₃O₄/CaCO₃-CSL/ICG NPs (ICG, 5 μg mL⁻¹) for 4 or 8 h. After incubation, the cells were stained with DAPI (nucleus) and the LysoTracker green (lysosomes) probe after incubation, followed by confocal laser scanning microscopy (CLSM) imaging (Leica TCS SP8).

2.8 CCK-8 assay

The cytotoxicity of various formulations was examined using a cell counting kit-8 (CCK-8, Dojingdo, Japan). Bel-7402 cells were treated with Fe₃O₄/CaCO₃, free ICG (0.5, 1, 2, 4, 8 and 12 μg mL⁻¹), free CSL (0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 μg mL⁻¹), CSL + ICG or Fe₃O₄/CaCO₃-CSL/ICG (equivalent concentration of CSL and ICG) for 24 h. The cells were then measured for cell viability using the CCK-8 assay after being subjected or not exposed to an 808 nm laser (1 W cm⁻², 3 min).

2.9 Apoptosis analysis

The Annexin V-FITC/PI assay was performed to analyse the apoptotic of Bel-7402 cells treated with PBS (as control), Fe₃O₄/CaCO₃ (10 μg mL⁻¹), free CSL, free ICG, CSL + ICG or Fe₃O₄/CaCO₃-CSL/ICG (CSL 0.8 μg mL⁻¹; ICG, 8 μg mL⁻¹) for 24 h. After exposure to 808 nm laser irradiation (1 W cm⁻², 3 min) or without it, cells were further incubated for an additional 4 h. Subsequently, the cells were collected, stained with Annexin V-FITC/PI, and analyzed using flow cytometry (FCM).

2.10 Intracellular distribution of Ca²⁺ released by Fe₃O₄/CaCO₃-CSL/ICG

Bel-7402 cells were treated with Fe₃O₄/CaCO₃ (10 μg mL⁻¹), Fe₃O₄/CaCO₃-CSL/ICG (10 μg mL⁻¹), or PBS for 4 h and stained with DAPI and Fluo-3 AM probe. CLSM (Leica TCS SP8) was employed for visualizing the intracellular Ca²⁺ distribution. For quantitative analysis, cells were digested, resuspended in PBS, and analyzed via FCM to determine fluorescence intensity from Fluo-3 AM.

2.11 Release of Ca²⁺ by Fe₃O₄/CaCO₃-CSL/ICG in mitochondria

Bel-7402 cells were treated with Fe₃O₄/CaCO₃-CSL/ICG (equivalent to 1 mM Ca²⁺) for 2 h or 4 h and stained with DAPI, Fluo-3 AM (Ca²⁺) and MitTracker red probes. The distribution of Ca²⁺ within the mitochondria were observed using CLSM (Leica TCS SP8), and merged images were used to evaluate colocalization of Ca²⁺ and mitochondria.

2.12 Intracellular reactive oxygen species (ROS) generation

Intracelluar ROS levels were measured using DCFH-DA probe. Bel-7402 cells were treated with free ICG (8 μg mL⁻¹), free CSL (0.8 μg mL⁻¹) and Fe₃O₄/CaCO₃-CSL/ICG (CSL 0.8 μg mL⁻¹; ICG, 8 μg mL⁻¹) for 6 h, followed by incubation with DCFH-DA for 20 min. Subsequently, the cells treated with Fe₃O₄/CaCO₃-CSL/ICG and ICG were irradiated with an 808 nm laser (1 W cm⁻², 3 min) and analysed using FCM. Moreover, cells were imaged using CLSM to visualize ROS production.

2.13 Mitochondrial morphology and function analysis

Changes in intracellular mitochondrial membrane potential were assessed using JC-1 staining. Bel-7402 cells were cultured with PBS (as control), Fe₃O₄/CaCO₃, free CSL, free ICG, CSL + ICG or Fe₃O₄/CaCO₃-CSL/ICG (CSL 0.8 μg mL⁻¹; ICG, 8 μg mL⁻¹) for 6 h, followed by staining with JC-1 for 20 min and washing with PBS three times. Mitochondrial membrane potential changes were visualized using CLSM (Leica TCS SP8). To observe mitochondrial morphology ultrastructural changes, Bel-7402 cells were collected, fixed with glutaraldehyde, dehydrated, embedded in paraffin and sliced and observed using biological TEM (Bio-TEM). For intracellular ATP quantification, Bel-7402 cells were treatment with ATP lysate and subsequent centrifugation at 12 000 rpm to gather the supernatant to quantify intracellular ATP content using an ATP testing kit.

2.14 RNA isolation and bioinformatic analysis

Total RNA was extracted using the TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's protocol. RNA purity and integrity were assessed via NanoDrop 2000 spectrophotometer (Thermo Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies), respectively. Libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep kit. Differential expression analysis was performed using the DESeq2,³⁵ with thresholds set as p-value < 0.01, |fold change| > 2, and VIP > 1. Based on the hypergeometric distribution, GO³⁶ enrichment analysis of DEGs were performed to screen significant enriched terms using R (v 3.2.0). Significant enrichment terms were visualized through column diagram, the chord diagram and bubble diagram generated in R (v 3.2.0).

2.15 Cell cycle analysis

Bel-7402 cells were exposed to various treatments including PBS, Fe₃O₄/CaCO₃, free ICG, free CSL, CSL + ICG, or Fe₃O₄/CaCO₃-CSL/ICG for 12 h. Cells treated with ICG-containing formulations were then subjected to NIR laser irradiation at 808 nm (1 W cm⁻², 3 min). After treatment, cells were collected and stained with a cell cycle staining kit at room temperature for 30 min in the dark. Cell cycle analysis was conducted using a FACSCalibur flow cytometer.

2.16 Xenograft tumour mouse model and in vivo MRI of Fe₃O₄/CaCO₃-CSL/ICG

BALB/c nude mice (5 weeks old, both genders) were acquired from GemPharmatech Co., Ltd (Jiangsu, China). All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Shanghai Jiao Tong University and approved by the Animal Ethics Committee of Shanghai Jiao Tong University (approval no. 202401264). Tumor models were established by subcutaneously injecting Bel-7402 cells (2 × 10⁶ cells in 0.1 mL of PBS) into the right flanks of the mice. Fe₃O₄/CaCO₃-CSL/ICG was injected into the tumor-bearing mice through the tail vein, and MRI scans were conducted at 0 and 12 h post-injection using a 3.0-T clinical MRI instrument to evaluate the tumor targeting ability of nanoparticles in vivo.

2.17 In vivo photothermal effect of Fe₃O₄/CaCO₃-CSL/ICG and antitumour effects

Tumor-bearing mice received tail vein injections of normal saline, free ICG (8 μg mL⁻¹), or Fe₃O₄/CaCO₃-CSL/ICG (ICG 8 μg mL⁻¹). After 12 h of injection, tumors underwent irradiation with an 808 nm laser (1 W cm⁻²) for 5 min. The temperature variations of the tumor tissues were monitored using an infrared imaging camera (Testo-865). To evaluate the anticancer efficacy, mice-bearing tumors were randomly divided into six groups (n = 5 per group) and treated with various drug formulations as follows: (1) PBS; (2) Fe₃O₄/CaCO₃; (3) free CSL; (4) free ICG + laser; (5) free CSL + ICG + laser and (6) Fe₃O₄/CaCO₃-CSL/ICG + laser. ICG and CSL concentrations were 10 and 5 mg kg⁻¹, respectively. Treatments were administered via tail vein injection every other day for a total of three sessions. For groups (4)–(6), tumors were irradiated with laser (808 nm, 1 W cm⁻² for 10 min) for 12 h after injection. The tumour size was measured with a caliper and the volumes were calculated using the formula: volume = width² × length/2. Body weights and tumour volumes were recorded every 2 days for 14 days. Upon study completion, mice were euthanized and their major organs (heart, liver, spleen, lungs, kidneys) and tumors were harvested. Tissue samples were subjected to hematoxylin and eosin (H&E) staining. To further investigate oxidative stress levels within tumor tissues following treatment, frozen tumor sections were stained with dihydroethidium (DHE) and the red fluorescence indicating ROS generation was visualized using a fluorescence microscope. Additional immunohistochemistry staining with anti-Ki67 antibody and TUNEL staining were performed to analyse cell proliferation and apoptosis at the tumour site. To assess the biosafety of the nanoplatform in vivo, healthy BALB/c mice were intravenously injected with saltine (as control) or Fe₃O₄/CaCO₃-CSL/ICG (5 mg kg⁻¹). At 14 days post-injection, blood samples were collected for complete blood count (CBC) and serum biochemistry analysis, including liver (ALT, AST) and kidney function markers (BUN, CRE). For biodistribution analysis, tumor-bearing mice were sacrificed at 12 h following tail vein injection of Fe₃O₄/CaCO₃-CSL/ICG (5 mg kg⁻¹). The major organs and tumors were excised, washed, weighted and decomposed by concentrated nitric acid. The final tissue solutions were used to measure the concentration of Fe by ICP-MS.

2.18 Statistical analysis

The data are presented as the mean ± standard deviation (SD) from at least three independent experiments. Group comparisons were conducted using one-way ANOVA and Student's t-test. P-Values less than 0.05 were considered statistically significant. GraphPad Prism 7.0 software was employed for all statistical calculations. *p < 0.05 was considered significant, and **p < 0.01 and ***p < 0.001 were considered highly significant.

3. Results and discussion

3.1 Synthesis and characterisation of Fe₃O₄/CaCO₃-CSL/ICG

The structural characteristics of the synthesized nanoparticles were examined through transmission electron microscopy (TEM). Illustrated in Fig. 1a, the Fe₃O₄/CaCO₃-CSL/ICG displayed a well-dispersed spherical morphology with an approximate particle size of 100 nm. Dynamic light scattering (DLS) analysis (Fig. 1b) indicated a hydrated particle size of 120 nm for Fe₃O₄/CaCO₃-CSL/ICG, aligning with the TEM in observations. These results indicated that the CaCO₃ nanoparticles loaded with drugs maintained good morphology and dispersibility. The Ca 2p peak and Fe 2p peak in the X-ray photoelectron spectroscopy (XPS) spectrum (Fig. 1c) confirmed the presence of Ca and Fe elements in Fe₃O₄/CaCO₃-CSL/ICG, indicating that the synthesised product was calcium carbonate and that it was successfully coated by Fe₃O₄ nanoparticles.

Fig. 1 (a) TEM images and (b) size distribution of Fe₃O₄/CaCO₃-CSL/ICG. (c) XPS characterisation of Fe₃O₄/CaCO₃-CSL/ICG. (d) UV-Vis absorption spectra of Fe₃O₄, CSL, ICG and Fe₃O₄/CaCO₃-CSL/ICG. (e) HPLC determination of Fe₃O₄/CaCO₃-CSL/ICG. (f) Generation of ¹O₂ measured using SOSG reagent for Fe₃O₄/CaCO₃-CSL/ICG at different pH values under 808 nm laser irradiation. (g) Fe₃O₄/CaCO₃-CSL/ICG precipitation by centrifugation after 12 h at different pH values. (h) TEM images of Fe₃O₄/CaCO₃-CSL/ICG incubated in PBS at pH 5.0, 6.5, and 7.4 for 24 h.

To verify the successful loading of ICG and CSL into Fe₃O₄/CaCO₃-CSL/ICG, ultraviolet-visible (UV-Vis) absorption spectroscopy and high-performance liquid chromatography (HPLC) were performed. As shown in Fig. 1d, compared with free ICG, the Fe₃O₄/CaCO₃-CSL/ICG showed distinct absorption peaks between 700 and 900 nm, with the main peak of ICG red-shifted 117 nm (from 778 to 895 nm). Additionally, a characteristic absorption peak of CSL appeared near 410 nm, confirming the successful co-loading of ICG and CSL within the calcium carbonate carrier. HPLC analysis (Fig. 1e and Fig. S1†) showed that the retention times of the Fe₃O₄/CaCO₃-CSL/ICG loaded peaks of ICG and CSL were 2.58 min and 1.24 min, respectively, and no mutual interference was observed in the retention times of CSL and ICG, providing confirmation of the successful loading of both compounds onto the carrier. According to the results of HPLC (Table S1†), drug loading efficiencies was significantly improved by up to 89.87% ± 1.84% and 55.10% ± 13.84% for CSL and ICG, respectively, compared with electrostatic adsorption methods. In summary, the abovementioned results confirmed the successful synthesis and efficient dual-drug loading of the Fe₃O₄/CaCO₃-CSL/ICG nanoplatform.

To further assess the stability of the synthesized nanoprobes, we measured the zeta potential of Fe₃O₄, Fe₃O₄/CaCO₃, and Fe₃O₄/CaCO₃-CSL/ICG nanoparticles, as well as the hydrodynamic diameter of Fe₃O₄/CaCO₃-CSL/ICG in various media over time. As shown in Fig. S2,† the Fe₃O₄ nanoparticles exhibited a zeta potential of −19.14 ± 0.71 mV, while the potential shifted to 30.75 ± 3.07 mV and 29.45 ± 2.94 mV after the coating with CaCO₃ and subsequent loading of CSL and ICG, respectively, indicating successful surface modification and enhanced colloidal stability. Moreover, the hydrodynamic size (Fig. S3a†) and polydispersity index (PDI) (Fig. S3b†) of Fe₃O₄/CaCO₃-CSL/ICG remained relatively stable over 24 hours in physiological saline, phosphate-buffered saline (PBS), and complete culture medium, with no apparent aggregation observed. Collectively, these results collectively suggest that Fe₃O₄/CaCO₃-CSL/ICG nanoparticles possess good physicochemical stability under physiological conditions.

3.2 pH-Responsive degradation and drug release of Fe₃O₄/CaCO₃-CSL/ICG

To evaluate the pH-responsive degradation behavior of the synthesised nanoplatform, Fe₃O₄/CaCO₃-CSL/ICG nanoplatform was incubated in PBS with various pH values (5.0, 6.5, 7.4) for 24 h. After centrifugation, the colour of the supernatant deepened as the pH decreased, indicating that the nanoplatforms gradually decomposed and released of the encapsulated drugs under acidic environments (Fig. 1g). The morphological changes of the nanoplatforms after incubation were observed using TEM. Fig. 1h shows that the nanoplatforms could maintain a spherical structure in pH 7.4 PBS, partially degraded at pH 6.5 and disintegrated into smaller fragments at pH 5.0, confirming their acid-sensitive structural instability. Subsequently, the pH-responsive release of ICG and CSL was quantified using HPLC. In neutral pH (7.4), the nanoprobe released minimal cumulative release of ICG and CSL, maintaining structural integrity as observed in Fig. 2a and Fig. S4,† which is in agreement with the findings of TEM. In contrast, at pH 6.5, there was a significant increase in drug release. At pH 5.0, the cumulative release reached 70.78% for ICG and 79.60% for CSL within 4 hours. Additionally, inductively coupled plasma mass spectrometry (ICP-MS) was utilized to characterize the release of calcium ions and the release profiles of calcium ions (Fig. 2b) showed a similar pH-responsive trend and correlated well with the drug release data. Collectively, these results demonstrated that the synthesized Fe₃O₄/CaCO₃-CSL/ICG nanoplatform possesses robust acid-responsive degradation properties, enabling efficient drug and calcium ion release in acidic tumor microenvironments.

Fig. 2 (a) Cumulative release of CSL and ICG. (b) Ca²⁺ released from Fe₃O₄/CaCO₃-CSL/ICG at different pH values (n = 3). (c) Photothermic heating curves and (d) thermal images of Fe₃O₄/CaCO₃-CSL/ICG at various pH values with 808 nm laser irradiation (1 W cm⁻²) for different times. (e) T₂-Weighted MR images and (f) relaxation rates (r₂) of Fe₃O₄/CaCO₃-CSL/ICG with various concentrations at three different pH values.

3.3 pH responsiveness produces ¹O₂ and enhances the photothermal properties of Fe₃O₄/CaCO₃-CSL/ICG

ICG, with its near-infrared (NIR) absorption and fluorescence capabilities, converts light energy into thermal energy, widely applicable in both PTT and PDT. Consequently, the optical properties of Fe₃O₄/CaCO₃-CSL/ICG were investigated. Acting as an effective photosensitizer, ICG promotes the production of singlet oxygen (¹O₂), a reactive oxygen species critical to PDT.³⁷ The generation of ¹O₂ was assessed by monitoring the fluorescence emitted by the singlet oxygen sensor green (SOSG) under 808 nm laser irradiation for Fe₃O₄/CaCO₃-CSL/ICG. As shown in Fig. 1f and Fig. S5,† the fluorescence intensity increased markedly with decreasing pH, indicating that ¹O₂ generation by Fe₃O₄/CaCO₃-CSL/ICG is enhanced under acidic conditions.

Subsequently, we investigated the pH-responsive enhanced photothermal properties of Fe₃O₄/CaCO₃-CSL/ICG under NIR laser irradiation. As illustrated in Fig. 2c and d, the temperature of Fe₃O₄/CaCO₃-CSL/ICG solutions increased significantly over time compared to PBS controls, confirming the photothermal conversion capability of the nanoparticles. A further increase in temperature and heating rate was observed at lower pH values, likely due to the accelerated release of ICG caused by CaCO₃ decomposition in acidic environments. The abovementioned experimental results confirm that Fe₃O₄/CaCO₃-CSL/ICG exhibits strong pH-responsive behavior, enhancing both ¹O₂ production and photothermal efficacy in mildly acidic environments typical of the tumor microenvironment.

3.4 pH-Responsive MR imaging of Fe₃O₄/CaCO₃-CSL/ICG

Superparamagnetic Fe₃O₄ nanoparticle possess strong magnetic properties and function as T₂-weighted MRI contrast agents by shortening the T₂ relaxation time of water protons in tissues. To assess the impact of pH-responsive degradation on magnetic resonance imaging (MRI), Fe₃O₄/CaCO₃-CSL/ICG nanoparticles were incubated in PBS solutions with varying pH values (7.4, 6.5, 5.0) for 24 hours, followed by T₂-weighted MRI analysis. As shown in Fig. 2e, a concentration-dependent darkening of the MRI signal was observed: as the nanoparticle concentration increased, the brightness of the images decreased. Moreover, at the same concentration, the brightness of the images gradually diminished as the pH value decreased. The transverse relaxation rate (r₂) of each group was examined further to demonstrate the effect of pH on the T₂ signal (Fig. 2f). The r₂ value increased from 4.058 (pH 7.4) to 8.209 mM⁻¹ s⁻¹ (pH 5.0). These results revealed that the T₂-weighted imaging effect of Fe₃O₄/CaCO₃-CSL/ICG can be effectively enhanced with the decomposition of CaCO₃ and the liberation of Fe₃O₄ in an acidic environment, which is favourable for MRI.

3.5 In vitro cytotoxicity of Fe₃O₄/CaCO₃-CSL/ICG

Inspired by the excellent properties of Fe₃O₄/CaCO₃-CSL/ICG, in vitro therapeutic efficacy was evaluated at a cellular level. The cytotoxicity and cytocompatibility of Fe₃O₄/CaCO₃-CSL/ICG were investigated through the CCK-8. In Fig. 3a, Fe₃O₄/CaCO₃ demonstrated minimal cytotoxicity towards Bel-7402 cells, even at high concentrations, with cell viability maintained above 90%. This highlights the excellent biocompatibility of the synthesized nanoplatforms. The cellular activity of all other treatment groups was dose-dependent. At the same treatment concentration, the cellular activity of the Fe₃O₄/CaCO₃-CSL/ICG group was the lowest compared with the CSL and ICG groups, and all groups showed statistically significant cellular activity, indicating that the combination of CT, phototherapy (PDT/PTT) and mitochondrial calcium overload in the Fe₃O₄/CaCO₃-CSL/ICG nanoparticles contributed to enhanced inhibition of Bel-7402 cell proliferation.

Fig. 3 (a) Cell viability assay for various treatments on Bel-7402 cells over 24 h. Data are presented as mean ± SD (n = 3). All comparisons were made vs. Fe₃O₄/CaCO₃-CSL/ICG + laser group. ***p < 0.001, **p < 0.01 (vs. Fe₃O₄/CaCO₃ group); ^###p < 0.001, ^##p < 0.01 (vs. CSL group); ^⋇⋇⋇p < 0.001, ^⋇⋇p < 0.01, ^⋇p < 0.05 (vs. ICG + laser group); ^▲p < 0.05 (vs. CSL + ICG + laser group); ns: not significant. (b) Cell viability assay in human liver cancer cells (Bel-7402) or normal cells (L02) incubated with Fe₃O₄/CaCO₃-CSL/ICG NPs. Data are presented as mean ± SD (n = 3). ***p < 0.001, **p < 0.01, *p < 0.05. (c) Flow cytometric analysis of apoptosis in Bel-7402 cells following various treatments. (d) Co-incubation ratio analysis and (e) CLSM images of the Fe₃O₄/CaCO₃-CSL/ICG and ICG biodistribution in Bel-7402 cell lysosomes following varying treatment times.

Because of differences in calcium signalling pathways in normal and tumour cells¹⁵ and the fact that CSL exhibited relatively specific cytotoxic effects towards cancer cells,³⁸ the cytotoxicity of Fe₃O₄/CaCO₃-CSL/ICG to L02 normal human hepatocytes was also examined. As shown in Fig. 3b, Fe₃O₄/CaCO₃-CSL/ICG demonstrated no significant cytotoxicity towards L02 cells across the tested concentration range (0.5–12 μg mL⁻¹). At each corresponding concentration, the viability of L02 cells was significantly higher than that of Bel-7402 cells (p < 0.001 at 0.5–4 μg mL⁻¹, p < 0.05 at 8 μg mL⁻¹, and p < 0.01 at 12 μg mL⁻¹). These results indicates that Fe₃O₄/CaCO₃-CSL/ICG is specifically cytotoxic to tumor cells. In contrast, normal cells exhibited greater tolerance to the adverse effects of the nanoplatform.

The apoptotic effect of Fe₃O₄/CaCO₃-CSL/ICG on Bel-7402 cells was examined using flow cytometry (FCM) with Annexin V-FITC/PI double staining (Fig. 3c). The analysis showed that the early and late apoptotic rates increased significantly to 63.5% in cells treated with Fe₃O₄/CaCO₃-CSL/ICG. Compared to the monotherapy groups (CSL, ICG + laser) and controls, this increase in Fe₃O₄/CaCO₃-CSL/ICG group was statistically significant (p < 0.001) (Fig. S6†). These results were consistent with the CCK-8 assay, suggesting that the developed nanoplatforms effectively induced apoptosis in Bel-7402 cells. Therefore, the Fe₃O₄/CaCO₃-CSL/ICG nanoplatforms demonstrated strong cytotoxicty against Bel-7402 cells in vivo, attributed to the combined effects of phototherapy, chemotherapy and calcium ion therapy for cancer treatment in vitro.

3.6 Intracellular uptake and distribution of Fe₃O₄/CaCO₃-CSL/ICG

The previous results confirmed the promising in vitro therapeutic effects of Fe₃O₄/CaCO₃-CSL/ICG. To further investigate the mechanism underlying Fe₃O₄/CaCO₃-CSL/ICG-induced apoptosis, we focused on the intracellular behavior of the nanoparticles, particularly their pH-responsive characteristics. Typically, nanoparticles are internalized by tumor cells through endocytosis and accumulate in lysosomes, where the acidic environment (pH 4.0–6.0) can trigger the degradation of pH-sensitive carriers and promote drug release. As previously mentioned, drug delivery and calcium ion release from Fe₃O₄/CaCO₃-CSL/ICG nanoparticles required an acidic environment. Therefore, we verified whether the nanoparticles were localized within lysosomes after cellular uptake. CLSM was used to determine the colocalisation of Fe₃O₄/CaCO₃-CSL/ICG and lysosomes. Red fluorescence from ICG was employed to trace the Fe₃O₄/CaCO₃-CSL/ICG, LysoGreen (green fluorescence) labeled lysosomes, and DAPI with blue fluorescence marked the cellular nucleus. In Fig. 3d and e, the colocalisation coefficient of Fe₃O₄/CaCO₃-CSL/ICG with lysosomes, measured by Pearson's localization and overlap coefficient, was 92.9% after 4 h of incubation. However, after 8 h, this value decreased to 65.42% for Fe₃O₄/CaCO₃-CSL/ICG, whereas it remained high for ICG. These findings revealed that Fe₃O₄/CaCO₃-CSL/ICG could effectively escape from the lysosomes after initial uptake, facilitating intracellular drug and calcium ion release under acidic conditions.

3.7 Evaluation of intracellular calcium ions distribution

Based on the previous findings, we hypothesize that CaCO₃ in Fe₃O₄/CaCO₃-CSL/ICG nanoparticles reacts with protons inside the lysosome, inducing a proton sponge effect. This process increases lysosomal osmotic pressure, eventually leading to lysosomal rupture and release of both calcium ions along with encapsulated drugs into the cytoplasm. To examine the effect of Fe₃O₄/CaCO₃-CSL/ICG on intracellular calcium levels, Bel-7402 cells were incubated with Fe₃O₄/CaCO₃ or Fe₃O₄/CaCO₃-CSL/ICG for 4 h. Intracellular Ca²⁺ was detected using the calcium-sensitive probe Fluo-3 AM, followed by analysis with confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) (Fig. 4a and Fig. S7†). Cells co-incubated with either Fe₃O₄/CaCO₃ or Fe₃O₄/CaCO₃-CSL/ICG showed increased fluorescence intensity compared to the control cells, indicating a rise in free intracellular Ca²⁺ levels, consistent with the FCM results. The cells incubated with Fe₃O₄/CaCO₃-CSL/ICG showed the highest fluorescence intensity, indicating an elevated concentration of intracellular free Ca²⁺, likely due to CaCO₃ degradation and CSL mediated continuously promoted Ca²⁺ release from the endoplasmic reticulum. These findings confirm that Fe₃O₄/CaCO₃-CSL/ICG effectively increases intracellular calcium concentrations.

Fig. 4 (a) CLSM images of intracellular Ca²⁺ generation following Fe₃O₄/CaCO₃ and Fe₃O₄/CaCO₃-CSL/ICG treatment. (b–d) CLSM images and colocalization ratio analysis of Ca²⁺ biodistribution released from Fe₃O₄/CaCO₃-CSL/ICG in mitochondria of Bel-7402 cells over time (n = 3). (e) CLSM images of Bel-7402 cells following several treatments, including both irradiation with an 808 nm laser and no irradiation.

The elevated intracellular Ca²⁺ levels can disrupt calcium homeostasis. This could result in an influx of Ca²⁺ into the mitochondria, triggering oxidative stress induced by calcium overload and subsequently causing cell apoptosis. To confirm the intracellular release of Ca²⁺ from Fe₃O₄/CaCO₃-CSL/ICG into the mitochondria, CLSM was utilized to visualize Ca²⁺ localization within mitochondria. The colocalization was analyzed using ImageJ (Fig. 4b–d). MitoRed (red fluorescence) was used to label mitochondria, Fluo-3 AM (green) to trace Ca²⁺, and DAPI (blue) to stain nuclei. As depicted in Fig. 4b, mitochondrial Ca²⁺ levels increased progressively with longer incubation times of Fe₃O₄/CaCO₃-CSL/ICG. The colocalization coefficient between Ca²⁺ and mitochondria rose from 83.5% to 96.1% (Fig. 4c and d), confirming that nanoparticle-derived calcium effectively accumulates in mitochondria.

3.8 Fe₃O₄/CaCO₃-CSL/ICG induces oxidative stress

The abovementioned experiments demonstrated that Fe₃O₄/CaCO₃-CSL/ICG can degrade within the cytoplasm, leading to the release of ICG and CSL. Upon laser irradiation, the released ICG act as a photosensitizer to generate singlet oxygen for PDT. The increase of ROS is a typical manifestation of mitochondrial apoptosis.¹⁹ Thus, the intracellular ROS level were assessed using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. As singlet oxygen oxidizes non-fluorescent DCFH-DA into fluorescent DCF (green), intracellular ROS generation was evaluated via confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) by measuring green fluorescence intensity. Control cells exhibited almost no green fluorescence, whereas cells treated with Fe₃O₄/CaCO₃ or CSL alone showed weak signals, likely due to effects on cellular calcium homeostasis. In contrast, ICG and Fe₃O₄/CaCO₃-CSL/ICG-treated cells showed progressively enhanced green fluorescence after 808 nm laser irradiation. Notably the strongest signal was observed in the Fe₃O₄/CaCO₃-CSL/ICG group (Fig. 4e), which was consistent with FCM findings (Fig. S8†). This consistency suggests that upon intracellular release, ICG has the capacity to generate a substantial quantity of ROS under laser irradiation, which may further amplify cellular oxidative stress levels.

Studies have shown that oxidative stress affects the cell cycle through multiple pathways, leading to cell cycle arrest.^39–42 We evaluated the impact of Fe₃O₄/CaCO₃-CSL/ICG nanoparticles on the cell cycle using FCM (Fig. S9†). The Fe₃O₄/CaCO₃-CSL/ICG + laser group exhibited a notably elevated proportion of cells in the G₂/M phase. This cell arrest may be attributed to the combined effects of CSL-induced chemotherapy, ICG-mediated phototherapy, and oxidative stress triggered by Ca²⁺ overload, which together contributed to inhibiting the proliferation of Bel-7402 cells.

3.9 Mitochondrial damage induces apoptosis

The combination of excessive mitochondrial Ca²⁺ influx and abundant singlet oxygen (¹O₂) generation upon laser activation of ICG can result in mitochondrial dysfunction. Mitochondrial membrane integrity is critical in apoptosis, and calcium overload can destabilize the membranes, releasing pro-apoptotic factors. To assess mitochondrial function and structural integrity, we investigated changes in mitochondrial membrane potential and morphology to determine whether apoptosis was induced via a mitochondrial pathway. Excess Ca²⁺ accumulation in mitochondria can cause mitochondrial depolarization and fragmentation. Therefore, we utilized the JC-1 probe as a fluorescent indicator to observe the mitochondrial membrane potential in cells subjected to various materials, evaluating mitochondrial integrity. The JC-1 aggregates within mitochondria and emits red fluorescence inhealthy cells. In apoptotic cells, loss of membrane potential causes JC-1 to exist in its monomeric form, emitting green fluorescence. Therefore, alterations in mitochondrial membrane potential can be identified through changes in green and red fluorescence. As shown in Fig. 5a, cells treated with PBS exhibited strong red fluorescence, while those exposed to ICG, CSL, or Fe₃O₄/CaCO₃-CSL/ICG showed a gradual increase in green fluorescence. Quantification of the red-to-green (R/G) fluorescence ratio (Fig. 5b) revealed a notable decrease in the Fe₃O₄/CaCO₃-CSL/ICG group, suggesting marked depolarization of the mitochondrial membrane and significant mitochondrial damage, likely due to the combined effects of ICG, CSL, and Ca²⁺ release.

Fig. 5 (a and b) Potential of mitochondrial membrane and fluorescence ratio between red and green of Bel-7402 cells following various treatments. (c) Bio-TEM microimages of Bel-7402 cells before and following various treatments for 12 h. (d) Intracellular relative ATP content analysis. ***p < 0.001, **p < 0.01, *p < 0.05.

Mitochondrion is central to calcium overload-induced mitochondrial dysfunction and apoptosis. To further visualise the extent of mitochondrial damage, we observed the mitochondrial morphology of Bel-7402 cells in different treatment groups by biological TEM (Fig. 5c). As illustrated in Fig. 5c, cells in the PBS group maintained normal morphology, whereas the mitochondria of cells treated with CSL, ICG and Fe₃O₄/CaCO₃ showed slight damage, displayed pronounced mitochondrial swelling, vacuolization, loss of cristae, and membrane rupture (Fig. 5c, red arrows), indicating extensive mitochondrial injury.

Given the critical role of mitochondrial membrane potential in ATP synthesis, we further quantified intracellular ATP levels using an ATP detection kit (Fig. 5d). In contrast to control group, all treatment groups showed a significant decrease in ATP content (p < 0.05), with the Fe₃O₄/CaCO₃-CSL/ICG treatment group exhibiting the most pronounced reduction (as low as 17.47%). The results demonstrated that Fe₃O₄/CaCO₃-CSL/ICG induces mitochondrial damage and dysfunction through calcium overload. In summary, depolarization of the mitochondrial membrane and impaired ATP synthesis serve as key markers of early apoptosis. We propose that these effect result from the combined actions of CT, PDT, PTT, and oxidative stress induced by mitochondrial calcium overload.

3.10 RNA sequencing and bioinformatic analysis

To gain a comprehensive understanding of the molecular mechanism underlying the combined therapeutic effect of Fe₃O₄/CaCO₃-CSL/ICG on liver cancer tumors, we performed RNA sequencing analysis to compare gene expression changes in Bel-7402 cells with and without Fe₃O₄/CaCO₃-CSL/ICG treatment.

Principal component analysis (PCA) was performed using SIMCA 14 software, revealing that the data points representing the treatment and control groups were distributed in distinct three-dimensional spaces (Fig. 6a). The internal validation parameters were R²X = 0.963 and Q² = 0.946. This indicates that the model was not overfitted. Based on the PCA results, we further incorporated grouping information for partial least squares discriminant analysis (PLS-DA) (R²X = 0.963, R²Y = 0.999, Q² = 0.999). The results demonstrated significant genetic differences between the treatment and control groups (Fig. 6b), consistent with PCA and cluster analysis findings (Fig. 6c). To identify differentially expressed genes (DEGs) between the treatment and control groups, we generated a Volcano plot (Fig. 6d) and applied a threshold of fold change > 2 and p < 0.01, identifying 3309 DEGs. A heatmap was then constructed based on these DEGs (Fig. 6e). To refine the selection and identify key drug intervention pathways, we further screened the DEGs using the variable importance in projection (VIP) scores (VIP > 1) obtained from PLS-DA analysis. This yielded 89 DEGs (Fig. 6f and Table S2†), of which 36 were upregulated and 53 were downregulated following Fe₃O₄/CaCO₃-CSL/ICG treatment. A comparison of Fig. 6e and f revealed that the refined DEGs exhibited more pronounced differential expression between the two groups. We conducted Gene Ontology (GO) enrichment analysis on 89 DEGs following Fe₃O₄/CaCO₃-CSL/ICG treatment, identifying multiple regulated pathways (Fig. 6g–i and Table S3†). Fig. 6g illustrates significant GO enrichment related to oxidative stress response, mitochondrial dysfunction, and calcium binding. These findings align with experimental results, indicating that mitochondrial calcium imbalance plays a crucial role in apoptosis. Accumulation of ROS and mitochondrial membrane depolarization exacerbate cellular damage, thereby activating apoptotic pathways. Fig. 6h and i provide a detailed enrichment analysis of DEGs after Fe₃O₄/CaCO₃-CSL/ICG treatment, highlighting their roles in specific biological processes, molecular functions, and cellular components.

Fig. 6 RNA sequencing analysis of control and Fe₃O₄/CaCO₃-CSL/ICG treatment on Bel-7402 cells. (a) PCA analysis, (b) PLS-DA analysis, (c) sample cluster analysis, (d) Volcano map, (e) Heat map (p < 0.01, |fold change| > 2), (f) Heat map (p < 0.01, |fold change| > 2, VIP > 1), (g) GO analysis for DEGs, (h) GO chord diagram of differentially expressed genes, (i) GO enrichment Circos plot for DEGs.

The GO enrichment results reflect a cascade of cellular events initiated by calcium overload, which contributes to mitochondrial dysfunction and apoptosis. Specifically, calcium overload leads to excessive ROS accumulation, thereby triggering oxidative stress. This results in mitochondrial damage (e.g., loss of membrane potential and respiratory chain dysfunction), activation of apoptosis signaling, and eventually energy metabolism disorders (such as impaired ATP synthesis), thereby accelerating cell death. In conclusion, the analysis confirms that Fe₃O₄/CaCO₃-CSL/ICG affects cell survival and promotes apoptosis by regulating oxidative stress, mitochondrial function, and calcium signaling, thereby exerting anti-tumor effects.

3.11 Imaging and distribution of Fe₃O₄/CaCO₃-CSL/ICG in vivo

Fe₃O₄ nanoparticles are widely used in MRI due to their superparamagnetic properties, good biosafety, and excellent surface modifiability. Therefore, we performed T₂-weighted MRI on tumour-bearing BALB/c mice before and after tail vein injection of Fe₃O₄/CaCO₃-CSL/ICG to verify the intratumoral accumulation of the synthesized nanoplatforms at the tumour site (Fig. 7b). A pronounced T₂ signal darkening was observed at the tumor site post-injection (Fig. 7b), confirming that Fe₃O₄/CaCO₃-CSL/ICG exhibits excellent MRI contrast enhancement and effective tumor-targeting capability in vivo.

Fig. 7 (a) Treatment scheme. (b) T₂-Weighted MR images of the tumour-bearing mouse obtained before injection (left) and 12 h post intravenous injection (right) with Fe₃O₄/CaCO₃-CSL/ICG. (c) The corresponding tumour temperature changes of mice treated with Fe₃O₄/CaCO₃-CSL/ICG for various times. NIR: 808 nm laser with 1 W cm⁻² power density. (d) Thermal images of mice treated with Fe₃O₄/CaCO₃-CSL/ICG for different times. (e) Tumour growth curves and (f) body weight changes of Bel-7402 tumour-bearing mice following various treatments (n = 5, *p < 0.05, ***p < 0.001) (after nude mice with tumors died, no more data were recorded).

3.12 PTT effect of Fe₃O₄/CaCO₃-CSL/ICG in vivo

Encouraged by the strong photothermal conversion observed in vitro, we evaluated the in vivo PTT performance of Fe₃O₄/CaCO₃-CSL/ICG. Tumor-bearing BALB/C mice were injected with PBS, free ICG or Fe₃O₄/CaCO₃-CSL/ICG. Following a 12-hour interval post-injection, the tumors were irradiated with an 808 nm laser (1 W cm⁻²) for 5 min and tumour temperature was recorded using an infrared thermal imaging system. In Fig. 7c and d, tumors in the PBS group showed negligible temperature increase. In contrast, mice injected with Fe₃O₄/CaCO₃-CSL/ICG exhibited a rapid temperature rise, reaching up to 46 °C under laser irradiation significantly higher than that observed in the free ICG group. These results demonstrate that Fe₃O₄/CaCO₃-CSL/ICG possesses potent photothermal performance in vivo, effectively accumulating at the tumor site and enabling localized thermal ablation.

3.13 Antitumour effect of Fe₃O₄/CaCO₃-CSL/ICG in vivo

Encouraged by the MR imaging capability, tumo-targeting accumulation and photothermal performance of Fe₃O₄/CaCO₃-CSL/ICG, we established a Bel-7402 tumour-bearing nude mouse model to evaluate the in vivo antitumor efficacy of Fe₃O₄/CaCO₃-CSL/ICG. BALB/c nude mice with Bel-7402 tumors were randomly divided into six groups (n = 5 per group): (a) PBS, (b) Fe₃O₄/CaCO₃, (c) CSL, (d) ICG + laser, (e) CSL + ICG + laser and (f) Fe₃O₄/CaCO₃-CSL/ICG + laser. All drug formulations were administered via tail vein injection on days 1, 4, and 7 (Fig. 7a). The doses administered were 5 mg kg⁻¹ for CSL and 10 mg kg⁻¹ for ICG. After 12 h of administration, mice in groups (d)–(f) were exposed to an 808 nm laser with a power density of 1 W cm⁻² for 5 min and repeated irradiation once every 5 min. Changes in mice group-wise tumor volumes and body weights are illustrated in Fig. 7e and f. In Fig. 7e and Fig. S10,† the tumor growth was suppressed to varying degrees in all treatment groups except for the PBS (control) group, indicating the therapeutic potential of each formulation. In particular, tumour growth in the Fe₃O₄/CaCO₃-CSL/ICG group was completely inhibited, showing statistically significant differences compared to all other groups (p < 0.001). The outcomes shown above suggested that monotherapy alone is insufficient for effectively tumor suppression, whereas Fe₃O₄/CaCO₃-CSL/ICG, through multimodal therapy, achieves significant tumor inhibition. Moreover, Fe₃O₄/CaCO₃-CSL/ICG exhibited excellent biosafety with minimal systemic toxicity, likely due to its enhanced tumor retention, increased vascular permeability, and selective pH-responsive intracellular drug release.

To assess the therapeutic efficacy of Fe₃O₄/CaCO₃-CSL/ICG in tumor-bearing mice, tumor tissues were analyzed using hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and Ki67 immunostaining. As shown in Fig. 8, H&E staining of tumor tissues in the control group revealed nuclei morphology and intact tissue structural. In contrast, varying degrees of cellular damage, including nuclear pyknosis, structural disorganization, and vacuolization, were observed in other treatment groups. The TUNEL assay yielded consistent results, showing markedly stronger red fluorescence (indicative of apoptotic cells) in the Fe₃O₄/CaCO₃-CSL/ICG group compared to other groups, suggesting significant induction of tumor cell apoptosis. Intratumoral ROS levels are closely linked to tumor cell apoptosis. Therefore, dihydroethidium (DHE) staining was used to detect ROS levels in tumor tissues. Red fluorescence intensity from DHE staining reflects the degree of ROS accumulation, it is usually positively correlated with elevated levels of oxidative stress. As shown in Fig. S11,† the Fe₃O₄/CaCO₃-CSL/ICG group exhibited the most intense red fluorescence, indicating elevated ROS production likely resulting from efficient intratumoral nanoparticle accumulation. In contrast, the CSL, ICG + laser, and CSL + ICG + laser groups showed relatively weak fluorescence signals, which can be attributed to the poor tumor-targeting capability of free CSL and ICG. Minimal fluorescence was observed in the control group, while the Fe₃O₄/CaCO₃ group exhibited only faint fluorescence, potentially due to the calcium homeostasis maintained by tumor cells. These results align with the TUNEL results and collectively suggested that the nanocarrier-mediated targeted delivery enables effective intratumoral drug accumulation. Upon laser irradiation, this system generates substantial ROS, triggering oxidative stress and promoting apoptosis in tumor cells. Ki-67 staining further confirmed reduced cell proliferation in the Fe₃O₄/CaCO₃-CSL/ICG group, as evidenced by weak green fluorescence, consistent with its observed antitumor efficacy in vivo. Moreover, no significant body weight loss was observed in any group throughout the experimental period (Fig. 7f), indicating minimal systemic toxicity.

Fig. 8 Tumor sections from mice receiving various treatments stained with TUNEL, H&E, and Ki-67. Scale bar: 100 μm.

In addition, blood routine and biochemical analyses were performed to assess systemic toxicity. As shown in Fig. S12,† the hematological parameters (WBC, RBC, and PLT) and liver and kidney function indicators (ALT, AST, TBIL, BUN, and CRE) in the Fe₃O₄/CaCO₃-CSL/ICG group showed no significant differences compared to the control group, indicating good biocompatibility and minimal systemic toxicity of the synthetic Fe₃O₄/CaCO₃-CSL/ICG nanoprobes in vivo. Additionally, H&E staining from all groups revealed no observable histopathological abnormalities, confirming that the synthesized nanoparticles were non-toxic to normal tissues and demonstrated favorable biosafety (Fig. S13†). Furthermore, to investigate the in vivo behavior of the nanoprobes, we conducted comprehensive pharmacokinetic and biodistribution studies of the Fe₃O₄/CaCO₃-CSL/ICG nanoprobe following tail vein injection. The circulation half-life (t_1/2) of Fe₃O₄/CaCO₃-CSL/ICG was calculated to be 5.79 ± 0.9 hours (Fig. S14a†), indicating a prolonged blood retention time that may enhance tumor accumulation. ICP-MS elemental analysis of Fe content at 12 hours post-injection revealed that the nanoprobes exhibited favorable biodistribution (Fig. S14b†), with notable accumulation in tumor tissues (9.64% ID g⁻¹), suggesting an effective passive targeting capability. Collectively, these findings indicated that Fe₃O₄/CaCO₃-CSL/ICG nanoprobes possess favorable pharmacokinetic properties and in vivo tumor-targeting performance, laying a solid foundation for future therapeutic applications. In summary, the Fe₃O₄/CaCO₃-CSL/ICG multifunctional nanoprobe effectively suppresses tumor growth through the integration of phototherapy, chemotherapy, and calcium ion therapy, offering a promising strategy for tumor treatment.

4. Conclusions

We designed and prepared a multifunctional nanotherapeutic probe with pH-responsive degradation properties for MR imaging-guided PDT/PTT/CT and mitochondrial calcium overload-based tumour treatment. Drug loading in the synthesised nanoplatform was achieved by incorporating the therapeutic agents during the formation of calcium carbonate nanoparticles. This strategy improved drug encapsulation efficiency and conferred an excellent pH-responsive degradation ability that enabled targeted vertex release of drugs and calcium ions at tumour sites. The resulting increase in intracellular calcium ion concentration led to calcium overload and activation of mitochondria-related apoptosis. Meanwhile, the therapeutic effect of phototherapy and CT further enhanced tumor cell inhibition by Fe₃O₄/CaCO₃-CSL/ICG. Both in vitro and in vivo results revealed that this MR-guided nanoplatform could effectively restrain tumour growth with favorable biocompatibility through laser-activated, multi-modal therapy. These findings suggested that Fe₃O₄/CaCO₃-CSL/ICG holds promising potential for future cancer treatment applications.

Author contributions

Shanshan Fan: investigation, data curation, writing – original draft. Shengsheng Cui: investigation, formal analysis, writing – review & editing. Xinni Pan: investigation. Haisong Tan: investigation. Cheng Cao: investigation. Yueqi Zhu: funding acquisition, project administration. Yanlei Liu: funding acquisition, project administration, writing – review & editing.

Conflicts of 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.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgements

This research was financially supported by the National Natural Science Foundation of China (grant no. 82073380, 81921002 and 8202010801), the Interdisciplinary Program of Shanghai Jiao Tong University (YG2022ZD011 and YG2021QN129) and the China Postdoctoral Science Foundation (no. 2020TQ0191). All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao University.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5bm00599j

‡ These authors contributed equally to this work.

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