Engineering manganese-based immune amplifier for chemoimmunotherapy of peritoneal metastatic colorectal cancer

Abstract

Current immunotherapies exhibit limited clinical efficacy in patients with colorectal cancer (CRC). While manganese ions (Mn) can activate the cGAS–STING pathway to potentiate innate immunity, their clinical application is limited by poor tumor accumulation and potential systemic toxicity. Alendronate (ALN), an FDA-approved agent, exerts T cell immunomodulatory activity but is hampered by low bioavailability and undesired bone targeting. To effectively potentiate antitumor immunity against CRC, we developed a manganese-alendronate (MnALN) nanomedicine via infinite coordination, leveraging Mn and ALN to synergistically eliminate tumor cells. In addition, Mn triggers reactive oxygen species (ROS)–mediated endoplasmic reticulum (ER) stress and subsequent immunogenic cell death (ICD) in tumor cells, while its combination with ALN further enhances T cell immune responses, ultimately achieving efficient tumor growth inhibition and intense anti-tumor immune response. This study presented a dual-functional MnALN nanomedicine synthesized from clinically available Mn and ALN, simultaneously activating apoptosis and inflammation-related pathways in CRC cells, which provides an effective strategy for immune tolerance CRC therapy.

Graphical abstract

Highlights

• •MnALN nanomedicine achieve tumor-specific drug release via acid-responsive degradation in the tumor microenvironment.
• •Beyond STING activation, MnALN triggers ROS-mediated ER stress to induce both apoptosis and immunogenic cell death.
• •The MnALN platform utilizes a peritoneal-confined delivery strategy to enhance tumor targeting and reduce systemic toxicity.
• •MnALN establishes a self-reinforcing chemoimmunotherapy cycle by promoting dendritic cell maturation and T-cell activation.

1. Introduction

Colorectal cancer (CRC) represents the third most common malignancy and ranks second in cancer-related mortality worldwide [1]. Metastasis occurs in approximately 50% of patients either at the time of diagnosis or during disease progression, resulting in a 5-year survival rate of less than 20% [2]. Peritoneal metastasis, the second most common site of metastasis, is associated with a particularly unfavorable prognosis [3]. Despite significant advancements in surgical methods, chemotherapy, molecular targeted therapy, and immunotherapy over the past two decades, which have improved patient outcomes with metastatic disease, the majority of metastatic CRC (mCRC) cases remain incurable [4,5]. Although cancer immunotherapy has achieved remarkable breakthroughs, its efficacy in CRC is hampered by the immunosuppressive “cold tumor” microenvironment, which limits the success of PD-1/PD-L1 blockade therapy [6]. Challenges such as the inability of traditional chemotherapies to effectively stimulate anti-tumor immunity, the toxicity and side effects of systemic treatment, and the limited bioavailability of small molecule drugs underscore the urgent need for an intelligent, efficient, and low-toxic multi-functional drug delivery system to enable the combination of chemotherapy and immunotherapy for effective tumor treatment.

ICD converts “cold tumors” into “hot tumors” by releasing DAMPs (e.g., ATP, HMGB1), which activate dendritic cells (DCs) and trigger systemic antitumor immunity, establishing itself as a vital long-term therapeutic strategy [7,8]. ICD inducers are classified into Type I and Type II based on their ability to induce endoplasmic reticulum (ER) stress. [8,9]. Among them, Type II inducers, including many metal-based agents, are particularly effective. They target the ER, disrupt cellular homeostasis through ROS-mediated stress, and induce robust DAMP release, resulting in a more potent systemic antitumor immunity than that elicited by Type I inducers [10]. As a transition metal ion, manganese (Mn) represents a promising ICD inducer. It not only generates ROS via a Fenton-like reaction to exert direct tumor cytotoxicity [11,12], but also acts as an immune adjuvant by enhancing cytotoxic T lymphocyte proliferation and DC maturation [13,14]. Furthermore, studies indicate that manganese-based compounds can sensitize tumors to radio-chemotherapy by modulating intracellular H₂O₂ levels, while also functioning as nutrient-responsive ICD inducers (e.g., MnO₂ nanoparticles) to enhance antitumor immune surveillance [15,16]. It is noteworthy, however, that chronic manganese exposure has been shown to activate the cGAS–STING pathway, induce ER stress and ROS production, and lead to neuronal apoptosis and ferroptosis, contributing to neurotoxicity [17]. Despite these advances, the spatiotemporal synergy between Mn and conventional anticancer drugs in promoting ICD remains to be fully elucidated. Moreover, the clinical translation of ICD-based therapies faces several challenges, including drug resistance, immunosuppression, and chemotherapy-related toxicity, which has prompted a growing interest in nanotechnology-based combination strategies [18].

Nanomedicine has emerged as a promising therapeutic platform in oncology, offering enhanced drug delivery efficiency and controllable release properties [19,20]. Intraperitoneal administration of nanoparticle-encapsulated chemotherapeutic agents significantly prolongs intraperitoneal drug retention compared to conventional small-molecule drugs [21]. In general, nanomedicine is associated with an extended half-life in blood circulation and improved tumor selectivity, thereby enhancing tumor accumulation and antitumor efficacy [22,23].

Recent studies have underscored the critical role of metal ions in activating antitumor immune responses [24,25]. Consequently, the development of biofunctional metal-based nanomedicines for enhanced cancer immunotherapy has emerged as a rapidly advancing research frontier [26]. However, the application of such metal-based nanomedicines in tumor immunotherapy often leads to the release of substantial amounts of toxic metal ions, which presents a major obstacle for clinical translation [27]. Therefore, refining metal-based nanomaterials and designing nanomedicines capable of co-targeting both tumor cells and immune cells holds promise for further improving the efficiency of metal-based immunotherapy. Although Mn-based nanomaterials have demonstrated considerable versatility in previous studies, their clinical potential is limited by tumor heterogeneity, complex synthesis routes, insufficient release of bioactive components, and poor bioavailability [11]. Consequently, there is a pressing need to develop readily accessible Mn-based nanomedicines for clinical metal-based cancer immunotherapy.

In recent years, carrier-free nanomedicines have attracted considerable interest due to their high drug loading capacity and absence of exogenous carrier-related toxicity [28,29]. Among these, nanomedicines formed through infinite coordination polymerization (ICP) have gained prominence in cancer therapy research, offering advantages including extremely high drug-loading capacity, synergistic multi-drug efficacy, stimuli-responsive drug release (e.g., pH-triggered mechanisms), streamlined preparation processes, and improved stability [30]. Bisphosphonates, a class of organic ligands capable of coordinating with metal ions, have drawn much attention because of their multifunctional anti-tumor properties [28,31]. Nitrogen-containing bisphosphonates (n-BPs), for instance, have received FDA approval for the treatment of osteoporosis and bone metastases in breast and prostate cancer [32,33]. They reduce skeletal-related events by inhibiting osteoclast activity and possess direct antitumor activity [31]. In addition, n-BPs can induce the accumulation of antigenic isoprenoid metabolites in target cells, thereby enhancing γδ T cell-mediated tumor killing via non-MHC restricted pathways [34]. However, the high hydrophilicity and strong negative charge of bisphosphonates limit their permeability across biological membranes, resulting in poor bioavailability [35]. Fortunately, their P-C-P backbone structure allows for enhanced bioavailability when formulated as metal-coordinated nanomedicines [36,37]. Recently, a study indicated that risedronate could coordinate with Mn to form multifunctional nanobelts that enhance oxidative stress and ultimately activate antitumor immunity [38]. Similarly, alendronate (ALN) enables the construction of cooperative nanodrugs with various anticancer agents, demonstrating potent antitumor effects [28,39]. These advances highlight the potential of bisphosphonates in innovative cancer therapies.

Herein, an engineered nanomedicine, termed MnALN, which self-assembled from ALN and Mn via infinite coordination, was designed for the treatment of peritoneal mCRC. This engineered nanomedicine achieved tumor-specific delivery and synergistically promoted tumor cell apoptosis and ICD in vivo. This dual action robustly enhanced T cell-mediated anti-tumor immune response, leading to significantly improved chemo-immunotherapeutic efficacy (Scheme 1). Furthermore, MnALN exhibited acid-responsive degradability and favorable biocompatibility. Collectively, this novel immune amplifier nanomedicine MnALN, provides new insights into the clinical treatment of patients with advanced mCRC.

Scheme 1.

Schematic illustration of the MnALN nanomedicine for potentiated chemo-immunotherapy: Upon intraperitoneal delivery, carrier-free MnALN nanomedicine selectively accumulates in the acidic tumor microenvironment. MnALN then undergoes lysosomal degradation, leading to the coordinated release of ALN (which promotes T-cell infiltration and cytotoxicity) and Mn (which induces ICD with DAMPs release). This dual action synergistically enhances dendritic cell maturation and CD8⁺ T-cell activation, ultimately eliciting systemic anti-tumor immunity and potentiating chemoimmunotherapy. The MnALN regimen effectively suppresses peritoneal metastatic colorectal cancer with minimal systemic toxicity.

2. Materials and methods

2.1. Materials

Alendronate sodium trihydrate (NaALN·3H₂O), manganese chloride tetrahydrate (MnCl₂·4H₂O), and Methylene Blue (MB) trihydrate were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). 8% hydrogen peroxide was purchased from Hunan Guangjia Bio-Chem Technology. Cell culture medium, phosphate-buffered saline (PBS), and trypsin–EDTA were purchased from Pricella (Wuhan, China). Fetal bovine serum (FBS) and penicillin-streptomycin solutions were obtained from Gibco (Guangzhou, China). Calcein/PI cell viability/cytotoxicity assay kit and DAPI dihydrochloride were purchased from Beyotime (Shanghai, China). 6-carboxyfluorescein (6-FAM) was purchased from Beijing Fluorescence Biotechnology Co., Ltd. D-Luciferin potassium salt, DiR iodide, and Cell counting kit-8 (CCK-8) assay kit were acquired from Yeasen Biotech Co., Ltd (Shanghai, China). Bcl-2, Bax, cleaved caspase-3, BiP, p-eIF2α, eIF2α, and CHOP antibodies were purchased from Cell Signaling Technology, Inc. (Shanghai, China). HMGB1, β-actin, GAPDH, secondary antibodies, enhanced chemiluminescence (ECL) kit, Reactive Oxygen Species (ROS) assay kit, N-acetyl-L-cysteine (NAC), and Enhanced ATP assay kit were purchased from Beyotime (Shanghai, China). HMGB1 ELISA kit was purchased from Ruixinbio (Quanzhou, China). Polyvinylidene fluoride (PVDF) membrane was purchased from Bio-Rad Laboratories, Inc. Pierce BCA Protein Assay kit was purchased from Thermo Fisher Scientific Inc. Total RNA extraction kit (RC112) was purchased from Vazyme (Nanjing, China). Reverse transcription kit (AG11706) and SYBR Green Premix Pro Taq HS qPCR kit (AG11701) were purchased from Accurate Biotechnology (Hunan, China). Primer sequences for IFN-β, CXCL10, and GAPDH (internal reference) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) and the primer sequences are listed in Table S1. Flow cytometry reagents were purchased from BioLegend, including: (1) Antibodies: Brilliant Violet 510™ anti-mouse CD45 (#103137), APC/Fire™ 750 anti-mouse CD3 (#100247), Alexa Fluor® 700 anti-mouse CD4 (#100536), Brilliant Violet 650™ anti-mouse CD8a (#100741), Brilliant Violet 605™ anti-mouse CD80 (#104707), PE/Cyanine7 anti-mouse CD86 (#105005), FITC anti-mouse CD11c (#117310). (2) Other reagents: 7-AAD Viability Staining Solution (#420403), Fixation Buffer (#420801), TruStain FcX™ (#101319), Brefeldin A Solution (#420601), Compensation Beads (#424602). All other reagents were consistent with those reported previously.

2.2. In vitro detection of H₂O₂-Activated hydroxyl radical (•OH) generation

The generation of hydroxyl radicals (•OH) activated by hydrogen peroxide (H₂O₂) was visualized using MB as a chromogenic indicator via UV-Vis absorption spectroscopy. Varying concentrations of MnCl₂ or MnALN were added to an 8% H₂O₂ solution containing 10 μg/mL MB. To evaluate the effect of pH on MnALN-mediated •OH generation, the pH of the MnALN-containing mixture was adjusted to 6.8 and 4.5, respectively. The reaction mixtures were incubated at designated time intervals (24, 48 h, or 72 h), and the dispersions were characterized by UV-Vis absorption spectroscopy over a wavelength range of 500–800 nm using a microplate reader. The decrease in MB absorption intensity was used to semiquantitatively assess the •OH generation capacity.

2.3. Cell lines and animals

The murine colon adenocarcinoma cell line CT26 and luciferase-labeled CT26 cell line (CT26-Luc) were obtained from Procell Life Science & Technology Co., Ltd. The human colon adenocarcinoma cell lines SW480, HT29, Caco2, and the human colon cell line NCM460 were obtained from the American Type Culture Collection. All cell lines were tested for mycoplasma contamination using a detection kit. The 6-week–old BALB/c mice (female, 16–18 g) were purchased from GemPharmatech Co., Ltd. and maintained under SPF conditions. All animal experimental protocols were approved by the Animal Care and Use Committee (TOPGM-IACUC-2024-0317)

2.4. Preparation, characterization, and in vitro degradation of MnALN

To prepare MnALN, 0.33 g of NaALN·3H₂O and 0.2 g of MnCl₂·4H₂O were dissolved in 20 mL of deionized water (ddH₂O) and adjusted to pH 6.5, respectively, and then mixed at the same rate. MnALN nanomedicine was obtained by centrifugation for freeze-drying or resuspension in alcohol for storage. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to detect the morphologies of as-synthesized ICPs. Particle size and zeta potential were measured using a Zetasizer Nano ZS90 (Malvern, Britain). The elemental composition was evaluated with energy dispersive X-ray spectroscopy (EDS) from a K-Alpha system from Thermo Scientific. X-ray diffraction (XRD) was performed using a Rigaku Ultima IV X-ray diffractometer. Fourier Transform Infrared Spectroscopy (FTIR) spectra were conducted on a Nicolet 7000-C spectrometer in the range of 400–4000 cm⁻¹. For the in vitro degradation assay, 10 mg of ICP was dispersed in 10 mL of ddH₂O, and then the dispersion was adjusted to different pH values (7.4, 6.8, 4.5, or 3.0) by using low-concentration hydrochloric acid solutions, followed by incubation at 37 °C for 24 h. Finally, the samples were transferred to glass tubes and photographed.

2.5. In vitro cellular uptake of MnALN

MnALN nanomedicine was labeled with 6-FAM for 24 h at room temperature. Briefly, CT26 cells were seeded in confocal dishes and cultured until they reached 60% density. Cells were then incubated with MnALN/6-FAM at 37 °C for 0, 1, 2, 4, and 8 h, respectively. After incubation, the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 8 min, then stained with 5 mg L⁻¹ of DAPI, and photographed by confocal laser scanning microscopy (CLSM, Zeiss LSM880, Germany). Flow cytometry (FCM) was used to quantify the uptake efficiency of the nanomedicine. CT26 cells were seeded in 6-well plates (5 × 10⁵ cells per well) and cultured until 60% density, then incubated with MnALN/6-FAM at 37 °C for 0, 1, 2, 4, 8, and 24 h, respectively. After incubation, the cells were collected for FCM, and the mean fluorescence intensity (MFI) and positive incidence uptake rate of MnALN were quantified.

2.6. In vitro cytotoxicity study

The in vitro cytotoxicities of MnALN or a combination of NaALN and MnCl₂ were tested using the CCK-8 cell proliferation assay kit. According to the manufacturer's instructions, 5 × 10³ colorectal cancer cells (CT26, SW480, Caco2, or HT29) were seeded into 96-well plates and cultured overnight. Then, cells were incubated with various concentrations of MnALN, NaALN, or MnCl₂ for 24 or 48 h, respectively. At each time point, 10 μL of CCK-8 reagent was added to each well and incubated for an additional 1 h. Optical density (OD) at 450 nm was measured using a microplate reader (BioTek, SynergyH1, USA). Cell viability was calculated using the following formula: cell viability% = (OD_sample – OD_blank/OD_control – OD_blank) × 100%.

2.7. Assessment of cytotoxicity in co-culture experiments

MC38 tumor cells were incubated with different concentrations of NaALN with or without T cells (CTLL-2, a murine-derived cytotoxic IL-2-dependent T-cell line). Due to the adherent nature of MC38 cells and the suspension growth characteristics of CTLL-2 cells, for the co-culture system of MC38 and CTLL-2 cells, MC38 cells were first seeded into a cell culture plate and allowed to adhere overnight. After this period, the RPMI-1640 complete medium was aspirated, and CTLL-2 cells were added at a ratio of 2:1 (CTLL-2 to MC38) along with the corresponding concentrations of NaALN in fresh medium. Finally, non-adherent CTLL-2 cells were gently rinsed three times with PBS. Cytotoxicity was assessed using the CCK-8 assay according to the manufacturer's instructions.

2.8. Calcein/PI cell viability/cytotoxicity assay

Cell viability was also assessed using a Calcein/PI staining assay. Briefly, CT26 or SW480 cells were pre-seeded in 96-well plates (5000 cells per well) overnight. MnALN with varying concentrations was added to each well and incubated for 24 and/or 48 h. The cells were stained with a Calcein/PI assay kit at different time points. Image acquisition and analysis were performed using a fluorescent microscope (Leica, DMi8, Germany).

2.9. Western blot

CT26 cells were pre-seeded in the 6-well plates (5 × 10⁵ cells/well) overnight and treated with medium containing PBS, MnCl₂ (30 mg L⁻¹), NaALN (45 mg L⁻¹), MnCl₂ + NaALN, or MnALN (45 mg L⁻¹) for 24 h. In addition, to examine whether the ROS inhibitor NAC could reverse the relevant indicators, CT26 cells were treated for 24 h with PBS, NAC (5 mM), MnCl₂, MnCl₂ + NAC, MnALN, or MnALN + NAC. After different incubation periods, the total proteins of the cells were lysed and collected using RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF, 1 mM), and the concentrations of protein in the samples were measured using the bicinchoninic acid (BCA) assay. Western blotting assay was conducted according to the manufacturer's protocol, and specific bands were detected using an enhanced chemiluminescent (ECL) system (Thermo Fisher, USA). Blot bands were quantified using ImageJ software, and the band of GAPDH was considered as an internal control.

2.10. RNA sequencing

1 × 10⁶ CT26 cells were pre-seeded in a 10 cm cell culture plate overnight, followed by treatment with PBS, MnCl₂ (30 mg L⁻¹), NaALN (45 mg L⁻¹), or MnALN (45 mg L⁻¹) for 24 h. After treatments, total RNA was extracted from the cells using Qiagen RNeasy Mini kit (Germany) according to the manufacturer's protocol. RNA samples were quantified using the Qubit 2.0. The quality control of the mRNA samples was conducted using the Bioanalyzer 2100 (Agilent Technologies). RNA-seq libraries were prepared according to the standard mRNA-seq protocol and sequenced on an Illumina HiSeq 2500 platform (Illumina, PE150). Clean reads were obtained from the raw reads by removing the adaptor sequences, low-quality sequences, and reads containing poly-N with Trimmomatic software. All downstream analyses were based on high-quality clean data. RNA differential expression analysis was performed using DESeq2 software between the two different groups. Genes/transcripts with a false discovery rate (FDR) below 0.05 and an absolute fold change ≥2 were considered differentially expressed genes/transcripts (DEGs). Gene set enrichment analysis (GSEA) was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

2.11. Quantitative real-time PCR (qPCR)

CT26 cells were treated with PBS, MnCl₂ (30 mg L⁻¹), or MnALN (45 mg L⁻¹) for 24 h. Total RNA was extracted using the FastPure® RNA Kit (Vazyme) following the manufacturer's instructions, including an on-column DNase digestion step to eliminate genomic DNA. 1 μg of purified RNA was reverse-transcribed into cDNA using the Evo M-MLV RT Premix (Accurate Biotechnology). qPCR was performed on a real-time PCR system with the SYBR Green Premix Pro Taq HS Kit (Accurate Biotechnology). The 20 μL reaction mixture contained 10 μL of 2× SYBR Green Premix, 0.4 μL each of forward and reverse primers (10 μM), 1 μL of cDNA template, and 8.2 μL of nuclease-free water. Thermal cycling conditions were: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. A melting curve analysis (65–95 °C, 0.5 °C increments) was added to confirm primer specificity. GAPDH served as the internal control, and relative mRNA levels of IFN-β and CXCL10 were calculated using the 2^−ΔΔCt method.

2.12. Detection of ROS and molecules (HMGB1, ATP, CRT) related to ICD

5 × 10⁵ CT26 cells were seeded in 6-well plates and allowed to adhere overnight. Then, the cells were treated with PBS, MnCl₂ (30 mg L⁻¹), NaALN (45 mg L⁻¹), a combination of MnCl₂ and NaALN, or MnALN (45 mg L⁻¹) for 24 h. After incubation, the ROS levels in CT26 cells were stained with a DCFH-DA probe (10 μM) following the manufacturer's protocol. The corresponding images were visualized under a fluorescence microscope, and the positive rate of the cells was measured by FCM. After the treatments, the cell lysates were used to quantify high mobility group box 1 (HMGB1) by western blot as described previously. And the HMGB1 level in supernatants was also shown by the HMGB1 ELISA Kit. For ATP assessment, after different treatments, the cell supernatants were collected, and the extracellular ATP levels were measured using a microplate reader (BioTek, SynergyH1, USA) after 2 min of incubation with a standard ATP assay kit. For the detection of calreticulin (CRT) expression in vitro, after different treatments, cells were fixed with 4% formaldehyde and blocked with a 3% goat serum solution. Furthermore, the cells were co-cultured with anti-CRT antibody overnight at 4 °C, and stained with FITC-labeled anti-rabbit antibody for 1 h at room temperature. Finally, the cells were stained with DAPI for 10 min. The CRT expression in CT26 cells was captured using fluorescent microscopy (Leica, DMi8, Germany).

2.13. DC stimulation in vitro

Bone marrow-derived dendritic cells (DCs) were collected from the femurs of BALB/c mice and cultured for 7 days to generate CD11c⁺ DCs. CT26 tumor cells were cultured and treated in different groups in the upper chamber of a 24-well transwell system with 0.4 μm polycarbonate porous membranes for 24 h. The treated tumor cells were then incubated with the bone marrow-derived cells (BMDCs) seeded in the bottom well at a 2:1 ratio for another 24 h. Subsequently, the BMDCs were collected and analyzed by flow cytometry after staining with FITC anti-CD11c antibody, APC anti-CD80 antibody, and PE anti-CD86 antibody, and the maturation rate of DCs was analyzed using FlowJo software.

2.14. MnALN nanomedicine biodistributions in vivo

CT26-Luc cells were collected and resuspended in ice-cold PBS before implantation. A peritoneal tumor model was established in mice by intraperitoneal injection of 2.5 × 10⁶ cells per mouse and divided into two groups after 10 days of tumor cell incubation. MnALN nanomedicine was prelabeled with DiR dye overnight at room temperature in alcohol. To further evaluate the biodistribution of MnALN, CT26-Luc tumor-bearing BALB/c mice were intraperitoneally injected with free DiR or MnALN/DiR, and monitored using a real-time in vivo imaging system (IVIS) at 1, 4, 24, 48, and 72 h post-injection. At 72 h post-injection, the mice were sacrificed, and their tumors as well as major organs were collected for immediate, bioluminescence imaging, and fluorescence imaging were performed. Quantitative analyses of the fluorescence signals were performed using the Tanon system.

2.15. ICP-MS analysis of manganese concentration in serum and tumor tissues

To quantitatively evaluate manganese accumulation in serum and tumor tissues, mice bearing peritoneal disseminated CT26 tumors were intraperitoneally administered MnCl₂ (13 mg/kg) or MnALN (20 mg/kg) nanoparticles (four doses, 24 h after the final injection). Tumor tissues were excised and weighed (100–200 mg per sample). For tumor tissue digestion, each sample was first minced into small pieces using sterile scissors and then transferred into the inner vessel of a Sineo microwave digestion system (Model MDS-6G, Shanghai Sineo Microwave Chemistry Technology Co., Ltd., China). Subsequently, 4 mL of concentrated sulfuric acid (H₂SO₄) and 2 mL of hydrogen peroxide (H₂O₂, 30% v/v) were added to each vessel. The digestion program was set as follows: stage 1, 130 °C for 5 min at power level 4; stage 2, 160 °C for 20 min at power level 4. After completion, an aliquot of 100 μL of the digested solution was then mixed with 900 μL of 1 M sodium hydroxide (NaOH) solution, followed by centrifugation at 12,000 rpm for 5 min. The supernatant was collected and subjected to inductively coupled plasma mass spectrometry (ICP-MS) to determine manganese concentration. Serum samples were diluted appropriately with 1% (v/v) H₂SO₄ and directly analyzed by ICP-MS. All measurements were performed in triplicate, and manganese concentrations were calculated against standard curves prepared from certified reference standards.

2.16. In vivo antitumor performance of MnALN

The BALB/c mice were randomly divided into four groups (n = 8) at 6 days after tumor cell implantation. The mice in the three experimental groups were intraperitoneally injected with 0.4 mL of PBS containing MnCl₂ (13 mg/kg), NaALN (20 mg/kg), or MnALN (20 mg/kg) every 5 days, and the control group was injected with the same amount of PBS. Body weights and abdominal circumference of the mice were measured every 4 or 8 days. Changes in the size of abdominal tumors were marked by injecting luciferin and monitored by using the IVIS. Twenty days after drug injection, three-eighths of the mice in each group were sacrificed under sterile conditions. Tumor tissues from each group were removed and fixed with 10% formalin. H&E and TUNEL staining were performed to evaluate the morphology and apoptosis of tumor cells, immunohistochemistry (IHC) was used to detect the expression of CRT and HMGB1, and immunofluorescence (IF) was used to detect the infiltration of CD8⁺ T cells in tumor tissue.

2.17. Detection of T cell infiltration in tumor and ascites by flow cytometry

On day 20 post-treatment, mice were euthanized by CO₂ inhalation. Fresh tumor tissues were harvested and dissociated using collagenase IV and DNase at 37 °C for 40 min, while ascites was collected by lavaging the abdominal cavity with ice-cold Cell Staining Buffer. Single-cell suspensions from both sources were filtered through 40 μm mesh, treated with red blood cell lysis buffer (0.83% NH₄Cl) on ice for 5 min, and resuspended in Cell Staining Buffer at 1 × 10⁶ cells/mL. Cells were blocked with TruStain FcX™ on ice for 15 min, then stained with antibodies against CD45, CD3, CD4, and CD8a (BioLegend), followed by 7-AAD viability staining. Stained samples were acquired on a flow cytometer, and data were analyzed using FlowJo software (v10.8). T-cell subsets were defined as CD45⁺CD3⁺CD4⁺ (CD4⁺ T) and CD45⁺CD3⁺CD8a⁺ (CD8⁺ T).

2.18. Biocompatibility assay

After different treatments for 20 days, the mice were sacrificed, and their major organs were harvested and fixed with formalin. H&E staining was performed to observe the tissue morphology under an optical microscope. In addition, the normal colorectal cells NCM460 were seeded in 96-well plates overnight. Then, MnALN at different concentrations was added, and the plates were incubated at 37 °C for 24 and 48 h, respectively. Finally, the cell viability was assessed by the CCK8 assay.

2.19. Hemolysis assay

To further evaluate the biocompatibility of MnALN nanomedicine, whole blood was collected in an anticoagulant tube and diluted with normal saline into a 2% red blood cell (RBC) suspension. Different concentrations of 2 mL MnALN or MnCl₂ solutions were prepared with normal saline and mixed with 2 mL of the diluted RBC suspension in each group. Normal saline alone incubated with blood was regarded as the negative control, while deionized water incubated with blood was regarded as the positive control. All samples were incubated at 37 °C for 3 h and then centrifuged at 1500 rpm for 5 min. Then 100 μL of supernatant from each group was measured at an optical density of 570 nm using a microplate reader. The hemolysis rate (HR) was calculated using the following formula: HR % = (A_sample - A_negative)/(A_positive - A_negative) × 100%.

2.20. Statistical analysis

Data are shown as the average (±SD) from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 8.0. A t-test was applied to compare the two groups. ∗, ∗∗, and ∗∗∗ represented p-value <0.05, <0.01, and <0.001, respectively, and they were increasingly regarded as having statistical significance.

3. Results and discussion

3.1. Preparation and characterization of MnALN nanomedicine

The MnALN nanomedicine was synthesized via coordination between the oxygen atom of the phosphonate groups in alendronate and Mn. As shown in Fig. 1A, alendronate and Mn formed an infinite coordination structure in an aqueous solution at pH 6.5 after stirring for 30 min. The obtained MnALN NPs were amorphous and spherical, with an average diameter of approximately 190 nm and a Zeta potential of 37.9 mV (Fig. 1B and C, and Figure S1). Elemental mapping confirmed the existence of Mn- and N - containing phosphonate in infinite coordination polymerizations (ICPs) using an Energy Dispersive X-ray (EDX) spectrometer (Fig. 1D). The amorphous nature of MnALN ICPs was further corroborated by X-ray diffraction (XRD) (Fig. 1E). The broad absorption band centered at 1060 cm⁻¹ (metal–O–P stretching vibration, dotted line) in the Fourier transform infrared (FTIR) spectra confirmed the covalent binding of Mn to phosphate (Fig. 1F) [40]. Furthermore, the pH-responsive degradation of MnALN was demonstrated, as shown by its disintegration in solutions at pH 7.4, 6.8, 4.5, and 3.0 for 24 h (Fig. 1G).

Fig. 1.

Preparation and characterization of MnALN NPs. (A) Schematic diagram of the synthetic procedure of MnALN. (B) Size and zeta potential of MnALN NPs. (C) SEM and TEM images at varied magnifications of MnALN NPs. (Scale bar: 100 nm). (D) Electron energy spectrum analysis (EDS) of MnALN NPs. (E) XRD patterns of MnALN NPs. (F) FTIR spectra of NaALN and MnALN NPs. (G) Photographs of 5 mg mL⁻¹ MnALN NPs in deionized water at pH values of 7.4, 6.8, 4.5, and 3.0 for 24 h. (H-J) Time-dependent degradation curves of methylene blue (MB) measured at 24 h, 48 h, or 72 h. (H) Effect of varying concentrations of MnCl₂ on MB degradation. (I, J) Effect of varying concentrations of MnALN on MB degradation at pH 6.8 (I) and pH 4.5 (J).

To further evaluate the Fenton-like catalytic activity of the MnALN nanomedicine, methylene blue (MB) degradation assays were performed under different pH conditions to monitor •OH generation. MB, a common blue dye, is effectively decolorized upon oxidation by •OH produced in Fenton reactions. The experiments were carried out with 8% H₂O₂, and the degradation percentages were quantified after 48 h of reaction. As shown in the UV-Vis spectra (Fig. 1H–J), free MnCl₂ under neutral conditions exhibited a pronounced Fenton-like reaction, achieving 30.67% MB degradation. In contrast, MnALN induced only a weak Fenton-like reaction at pH 6.8, with 14.4% MB degradation. However, when the pH was adjusted to 4.5—mimicking the acidic lysosomal environment—substantially enhanced degradation of MB (20.58%) was observed, indicating a marked increase in Fenton reaction activity. The control group, containing only MB and H₂O₂, showed only weak MB degradation under the same conditions. These results provide direct evidence that MnALN undergoes acid-responsive degradation and activates its Fenton-like activity predominantly under acidic conditions, such as those found in lysosomes (pH ∼4.5).

Collectively, these results confirm the successful preparation of a novel pH-sensitive nanomedicine with a nanostructure and positive surface charge, which could effectively release Mn and ALN in the acidic tumor microenvironment. The formation of the metal-O-P coordination bond yielded nanoparticles with a positive surface charge, enhancing cellular interaction. Most critically, the demonstrated pH-responsive degradation is the key point of this design. MnALN remains stable at physiological pH but disintegrates rapidly in acidic environments, mirroring the extracellular tumor microenvironment (TME) and, more acutely, the lysosomal compartments after endocytosis. This property ensures precise, spatially controlled release of the therapeutic payload specifically within tumor cells, minimizing off-target effects.

3.2. Cellular uptake and cytotoxicity of MnALN NPs in CRC cells

The cellular uptakes of MnALN NPs were first investigated using CLSM. As shown in Fig. 2A, green fluorescence from 6-FAM-labeled MnALN NPs was detected in the cytoplasm after 1 h of incubation, and the signal intensity increased in a time-dependent manner. Consistent with these observations, flow cytometry (FCM) analysis revealed that both the mean fluorescence intensity (MFI) and positive cellular uptake rate of MnALN in CT26 cells increased in a time-dependent manner (Fig. 2B–D).

Fig. 2.

In vitro cellular uptake and anti-tumor efficiency of MnALN NPs in different tumor cells. (A) Detection of cellular uptake of 20 mg L⁻¹ MnALN in CT26 cells at 0, 2, 4, and 8 h from CLSM. Scale bar: 50 μm. (B) FCM analysis and (C–D) quantification mean fluorescence intensity (MFI) and positive incidence uptake rate of MnALN in CT26 cells at 0, 1, 2, 4, 8, and 24h (n = 3). (E–F) Viabilities of different cells treated with different concentrations of MnALN NPs for 24 h and 48 h. (G) Calcein/PI staining of CT26 cells after incubating with MnALN NPs at different concentrations for 24 h. Scale bar: 100 μm. (H) Bubble plots of the 10 remarkable enrichment pathways of DEGs by KEGG analysis. The critical signaling pathways were related to oxidative stress, apoptosis, and immune regulation. (I–L) GSEA enrichment of DEGs centralized in (I) NF-κB, (J) TNF signaling pathway, (K) Cell cycle, and (L) DNA replication in control versus MnALN group. (M) Heat map of DEGs related to oxidative stress, antioxidant response, inflammation, and immune regulation in the control versus MnCl₂ versus NaALN versus MnALN group.

The antitumor efficacy of MnALN was assessed against a panel of colorectal cancer cell lines using CCK-8 assays. After 24 h of treatment, MnALN exhibited varying cytotoxicities, with significantly lower inhibitory concentrations (IC₅₀) values in SW480 (1.676 mg L⁻¹) and CT26 cells (9.082 mg L⁻¹) compared to Caco-2 (41.67 mg L⁻¹) and HT29 (34.4 mg L⁻¹) cells (Fig. 2E). This differential sensitivity may be linked to variations in lysosomal abundance among cell lines. As shown in Fig. S2, analysis of the HUMAN PROTEIN ATLAS database revealed higher expression levels of lysosome-related proteins (LAMP2, LAMP3, and CD63) in SW480 cells than in Caco-2 and HT29 cells, suggesting a high lysosomal content that may facilitate more efficient endocytic degradation of MnALN. Notably, after 48 h of treatment, the IC₅₀ values for most cell lines decreased below 10 mg L⁻¹, indicating potent tumor-killing activity at low concentrations (Fig. 2F). The high sensitivity of CT26 supported their selection for subsequent in vivo tumor modeling. The time- and concentration-dependent cytotoxic effects were further validated by Calcein-AM/PI staining (Fig. 2G and Fig. S3).

To elucidate the underlying mechanisms of MnALN-induced cell death, we performed RNA sequencing. As shown in Fig. 2H, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes (DEGs) revealed significant enrichment of 10 pathways related to oxidative stress, apoptosis, and immune regulation. Gene Set Enrichment Analysis (GSEA) further indicated strong enrichment in the NF-κB, TNF-α, and HIF-1 signaling pathways (Fig. 2I, J, and Fig. S4A), which are implicated in cellular stress, inflammatory responses, and antitumor immunity. Conversely, pathways related to cell cycle, DNA replication, and peroxisome function were suppressed (Fig. 2K, L, and Fig. S4B), suggesting the inhibition of tumor proliferation. A heatmap of the selected DEGs related to oxidative stress, antioxidant response, and immune regulation is presented in Fig. 2M. Specifically, genes associated with cellular stress response (Hsp90aa1, etc.), lysosomal function and pH regulation (Atf6v0d1, Atf6v1b2, etc.), apoptosis regulation (Trp53inp2, Trp53bp2, Nfkb1, etc.), and immune activation (Csf2, Ccl27a, Ccl5, Cxcl2, etc.) were significantly upregulated in the MnALN group. Conversely, genes involved in antioxidant defense (Sod3, etc.) and mitochondrial respiration (Cox10, Cox18, etc.) were downregulated substantially. These transcriptional alterations collectively suggest that MnALN triggers a complex stress and inflammatory response, which may contribute to its biological effects.

3.3. Synergism of Mn and NaALN in tumor cell killing

To further explore the anti-tumor mechanisms of Mn and ALN, both individually and in combination, we first assessed their cytotoxic efficacy against tumor cells using the CCK-8 assay. As shown in Fig. 3A, MnCl₂ at concentrations below 0.5 μM did not significantly induce cell death, whereas the addition of a low concentration of NaALN (40 μM) markedly reduced the viability of tumor cells. Similarly, ALN alone at concentrations below 40 μM exhibited minimal cytotoxicity, but the subsequent addition of a low concentration of MnCl₂ (0.25 μM) markedly reduced the viability of tumor cells (Fig. 3B). These results demonstrated a pronounced synergistic effect between Mn and ALN in eliminating tumor cells.

Fig. 3.

In vitro evaluation of the combination of MnCl₂ and NaALN in apoptosis and immune response activation. (A) Viabilities of SW480 cells treated with different concentrations of MnCl₂ under 40 μM NaALN for 24 h. (B) Viabilities of SW480 cells treated with different concentrations of NaALN under 0.5 μM MnCl₂ for 24 h (n = 5). (C) Fluorescence images of CT26 cells at 24 h after different treatments and stained with DCFH-DA. Scale bar: 200 μm. (D) FCM analysis on the level of intracellular ROS in CT26 cells after different treatments for 24 h and stained with DCFH-DA. (E) Western blotting analysis of endoplasmic reticulum stress-related (Bip, p-eIF2α, CHOP) proteins in CT26 cells after different treatments for 24 h. (F) Immunofluorescence images of calreticulin (CRT) expression on CT26 cells treated with different therapies. Scale bar: 50 μm. (G) ELISA for HMGB1 in the supernatant of CT26 cells after different treatments. (H) Extracellular ATP level of tumor cells after different treatments. (I–J) Flow cytometry plot and quantification of DC cells incubated with CT26 cells after different treatments in transwell chambers. (K) Western blotting analysis of apoptosis-related proteins in CT26 cells after different treatments for 24 h. (L) Schematic diagram of the anti-tumor mechanism of MnALN in tumor cells. (n = 3) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

To delineate the specific mechanisms underlying Mn- and ALN- synergistically induced cell death, RNA-seq was performed on the cells. In MnCl₂-treated tumor cells, 4038 DEGs were identified under the threshold criteria (fold change ≥2, P-value <0.05), including 1535 upregulated and 2503 downregulated genes (Fig. S5A). KEGG enrichment analysis revealed 10 significant enriched pathways associated with oxidative stress, apoptosis, and tumor immunity (Fig. S5B). Among these, the NF-κB and p53 signaling pathways were markedly upregulated, whereas DNA replication and cell cycle pathways were suppressed (Fig. S5C–F). Key genes (Sod2, Cox6a2, Hsp90ab1, Ndufa13, Ndufb4, Ndufv2, Ndufaf3, etc.) involved in oxidative stress and antioxidant responses were significantly altered in the MnCl₂ group (Fig. S5G).

In contrast, ALN did not directly induce oxidative stress but primarily modulated anti-tumor immune pathways (Fig. S6). Under the same threshold criteria, 1894 DEGs were identified in NaALN-treated cells, comprising 646 upregulated and 1248 downregulated genes (Fig. S6A). KEGG enrichment analysis highlighted 10 pathways significantly associated with apoptosis and immune regulation (Fig. S6B). GSEA indicated that DEGs in NaALN-treated cells were enriched in apoptotic DNA fragmentation, p53-mediated intrinsic apoptotic signaling, TNF signaling, and T cell-mediated immune responses to tumor cells (Fig. S6C–F). Additionally, several genes encoding pro-inflammatory chemokines and immune cell attractants (e.g., Ccl2, Ccl7, Cxcl5, Nfkbie, Cxcl1, Cxcl2, and Cxcl10) were significantly modulated, indicating the potential of NaALN to enhance anti-tumor immunity (Fig. S6G). Based on these findings, we validated whether NaALN enhances T cell-mediated tumor cell killing using a co-culture system of tumor cells and T cells. Consistent with RNA-seq results, the tumor cell survival rate remained above 80% in the T cell-only group. In contrast, the addition of a low concentration of NaALN significantly enhanced the cytotoxicity of T cells against tumor cells, reducing tumor cell survival rate to below 50% (Fig. S7). These results suggested that the core therapeutic power of MnALN stems from the potent synergy between its constituent ions, Mn²⁺ and ALN. Neither component alone at low concentrations is highly cytotoxic, but their combination elicits dramatic cell death. And the RNA-seq analyses reveal their complementary roles: Mn²⁺ acts primarily as a chemotherapeutic sensitizer, inducing profound oxidative stress (via Fenton-like reactions), disrupting mitochondrial function, and activating pro-apoptotic pathways. Conversely, ALN functions as an immunomodulator, priming the tumor cells for immune recognition by upregulating pro-inflammatory chemokines (e.g., CCL2, CXCL10) and sensitizing them to T-cell-mediated killing. This division of labor is elegantly unified within the single MnALN nanoparticle.

3.4. In vitro anti-tumor mechanism of MnALN

It has been reported that several non-ferrous metal ions, including Mn, copper (Cu), iron (Fe), and cobalt (Co), can catalyse Fenton-like reactions to generate ROS [41]. To investigate the role of oxidative stress in MnALN-induced cytotoxicity, we measured intracellular ROS levels using a DCFH-DA fluorescent probe. As shown in Fig. 3C, minimal ROS fluorescence was detected in cells treated with PBS or NaALN alone, whereas a modest signal was observed in the MnCl₂ group. In contrast, co-treatment of MnCl₂ and NaALN, as well as the MnALN NPs, elicited the most intense fluorescence. Consistently, FCM analysis (Fig. 3D and Fig. S8) further confirmed that both the MnCl₂ + NaALN and MnALN NPs induced a higher mean fluorescence intensity and a greater proportion of ROS-positive cells than monotherapies or controls, underscoring the capability of MnALN to provoke ROS-mediated tumor cell death.

Given that ROS can trigger ER stress [42] and subsequent ICD [43,44], we evaluated key ER stress markers by western blot analysis. As expected, MnALN treatment significantly up-regulated the expression of ER stress markers, including BiP and phosphorylated eIF2α (p-eIF2α), in CT26 cells after 24 h (Fig. 3E). Notably, the pro-apoptotic transcription factor CHOP, another ER stress marker, was markedly elevated in the MnALN group, suggesting that ROS-induced ER stress promotes apoptotic cell death. Moreover, the expression of calreticulin (CRT), a hallmark of ICD, was significantly enhanced in the MnALN-treated group (Fig. 3F). Additionally, the release of HMGB1 and ATP into the tumor microenvironment was substantially increased in both the MnCl₂–NaALN combination group and the MnALN NPs group compared with the other groups (Fig. 3G and H; Fig. S9), implying the release of DAMPs that facilitate DCs maturation and adaptive immune activation.

To further substantiate the role of ROS in MnALN-induced ER stress and ICD, we employed the ROS scavenger NAC. As shown in Fig. S10A and B, NAC co-treatment significantly attenuated the MnALN-induced upregulation of ER stress markers, including BiP, p-eIF2α, and CHOP. Consistently, the increased extracellular ATP level, a hallmark of ICD, was also reversed by NAC. Similar results were observed in the MnCl₂ group, whereas NAC alone had no noticeable effect. These findings collectively demonstrate that ROS generation is an upstream trigger of ER stress and subsequent ICD in MnALN-treated tumor cells, reinforcing the mechanistic linkage between oxidative stress and immunogenic cell death.

In addition to ER stress, manganese is known to trigger the cGAS-STING pathway, a central axis for innate immune activation. To determine whether MnALN also engages this pathway, we examined key STING signaling effectors. As shown in Fig. S11A, treatment with either MnCl₂ or MnALN notably increased the phosphorylation of TBK1, IRF3, and STING, whereas total TBK1, IRF3, and STING levels remained largely unchanged. Moreover, qPCR analysis (Fig. S11B) demonstrated that MnALN significantly upregulated the mRNA expression of interferon-β (IFN-β) and CXCL10, two representative type I interferon-related immune response genes downstream of STING activation. These results indicate that MnALN activates STING-associated innate immune signaling in CT26 cells. Given that STING signaling can promote type I interferon production and enhance cross-priming of T cells, this pathway may cooperate with ROS-induced ER stress and ICD to potentiate antitumor immunity, thereby further supporting the role of MnALN as an immune amplifier.

To verify whether MnALN-induced ICD activates DCs, we co-cultured differently treated tumor cells with bone marrow-derived cells (BMDCs) and assessed DC maturation via flow cytometry using antibodies against CD80 and CD86. As shown in Fig. 3I and J, the MnALN NPs group exhibited a significant increase in mature DCs, indicating effective antigen presentation and immune activation.

ER stress plays a dual role in cell fate determination, governed by its intensity, duration, and specificity. While intense yet specific ER stress represents an effective strategy for eliciting ICD, sustained and unmitigated ER stress leads to classical apoptosis [45,46]. Intriguingly, MnALN induced caspase-dependent apoptosis in CT26 cells, as evidenced by upregulation of Bax and cleaved caspase-3 expression, and downregulation of Bcl-2 levels (Fig. 3K). Collectively, these results demonstrate that Mn and ALN synergistically eliminate tumor cells through ROS generation, ER stress-induced apoptosis, and ICD activation (Fig. 3L).

3.5. Distribution of MnALN NPs in vivo

To investigate the biodistribution and antitumor efficacy of MnALN in a murine model of peritoneal disseminated CRC, we intraperitoneally inoculated luciferase-expressing CT26 (CT26-Luc) cells into BALB/c mice. Tumor progression was monitored non-invasively using an in vivo imaging system (IVIS). Consistent with our previous findings, solid tumors and scattered micro-nodules were observed in the mesentery 6 days post-inoculation, confirming successful model establishment.

Seven days after tumor implantation, the tumor-targeting capability of MnALN was evaluated using IVIS following intraperitoneal administration of either free DiR or DiR-labeled MnALN nanomedicine. As shown in Fig. 4A, strong fluorescence signals colocalized with bioluminescent tumor regions in mice treated with MnALN/DiR, indicating a specific tumor accumulation. In contrast, free DiR diffused widely throughout the peritoneal cavity within 48 h and failed to concentrate at the tumor sites. Ex vivo imaging of excised tumors and major organs 72 h post-injection further validated these findings (Fig. 4A). Quantitative analysis revealed a significantly higher fluorescence intensity in tumors from the MnALN/DiR group than in the free DiR group, whereas hepatic fluorescence was slightly elevated in mice receiving free DiR (Fig. 4B). These results demonstrate that MnALN/DiR was rapidly distributed throughout the abdominal cavity within 1 h and subsequently accumulated preferentially at tumor sites, maintaining high local concentrations for up to 72 h. In contrast, free DiR was rapidly cleared, primarily through hepatic metabolism. In summary, MnALN nanomedicine exhibited prolonged retention and enhanced tumor-targeting capacity within the peritoneal cavity.

Fig. 4.

In vivo biodistribution of MnALN/DiR and quantitative ICP-MS analysis of manganese accumulation. (A) Images of bioluminescence (Luc) for the tumor and fluorescence (red) for DiR in CT26-Luc tumor-bearing mice at 1, 4, 24, 48, and 72 h, and the ex vivo images for tumor tissues and major organs at 72 h after intraperitoneal injection of free DiR (up), and MnALN/DiR (down). (B) Fluorescence intensities of tumors and major organs in these two groups of mice. P-values of different tissues of these two groups were compared. ∗∗p < 0.01. (C) ICP-MS quantification of manganese concentrations in serum of mice treated with MnCl₂ or MnALN (four intraperitoneal injections, samples collected 24 h after last injection; ns, not significant). (D) ICP-MS quantification of manganese concentrations in tumor tissues under the same treatment regimen. Data are shown as mean ± SD (n = 3). ∗p < 0.05 versus MnCl₂ group.

To quantitatively validate the tumor-selective retention of MnALN, we measured manganese levels in serum and tumor tissues by ICP-MS. CT26 peritoneal disseminated tumor-bearing mice were intraperitoneally administered MnCl₂ or MnALN nanoparticles four times, and samples were collected 24 h after the final injection. As shown in Fig. 4C, serum manganese levels were comparable between the MnCl₂ and MnALN groups, indicating similar systemic absorption after intraperitoneal administration. In contrast, the manganese accumulation in tumor tissues was significantly higher in the MnALN-treated group than in the free MnCl₂ group (Fig. 4D). These quantitative ICP-MS data are consistent with the fluorescence imaging results and further support that MnALN nanoparticles achieve superior tumor accumulation.

The enhanced tumor targeting of MnALN/DiR compared to small-molecule agents may be attributed to the peritoneum-plasma barrier, which restricts nanoparticle efflux and maintains high intraperitoneal drug concentrations. In essence, our approach leverages the anatomical features of the peritoneal cavity to extend the drug half-life, introducing what we term the “peritoneal confinement” concept. Since the nanoparticles are largely retained within the peritoneal space and are engineered to release their payload specifically in response to the acidic tumor microenvironment and endolysosomal uptake, the system achieves exceptional tumor-selective accumulation. This targeting strategy is further facilitated by the nanoscale size and positive charge of MnALN/DiR NPs, which promote direct contact with tumor cells and preferential accumulation in the tumor tissue. In contrast, other intraperitoneal organs are enveloped by the peritoneum, which consists of tightly packed mesothelial cells and is less permeable to NPs [47]. Consequently, this strategy not only significantly improves therapeutic efficacy and bioavailability but also offers a potent and safe treatment modality for peritoneal tumors.

3.6. Antitumor effects of MnALN NPs in vivo

Encouraged by the in vitro antitumor activity and favorable biodistribution profile, we evaluated the in vivo therapeutic efficacy of MnALN in a CT26-Luc peritoneal mouse model. Six days after intraperitoneal injection of tumor cells, the tumor-bearing mice were randomly divided into 4 groups (8 mice in each group) and treated with PBS, MnCl₂ (13 mg/kg), NaALN (20 mg/kg), and MnALN nanomedicine (20 mg/kg) via intraperitoneal injection every five days for a total of 45 days. On day 20, three mice in each group were sacrificed to evaluate physiological indicators and tumor parameters, and the remaining mice were monitored until the experimental endpoint on day 45, when all mice were euthanized (Fig. 5A). Bioluminescence images and quantitative analysis of the luminescence intensity of abdominal tumors revealed that MnALN treatment most effectively suppressed tumor growth, whereas NaALN alone showed a relatively modest inhibitory effect (Fig. 5B and C). The MnCl₂ group exhibited only a slight reduction in bioluminescence compared to the control group, consistent with the poor bioavailability and short half-life of MnCl₂ in vivo. The abdominal girth was recorded over 25 days as an indicator of ascites development and tumor progression. The PBS control group showed a significant increase in abdominal circumference, whereas the MnCl₂ and NaALN groups exhibited moderate increases. Notably, only the MnALN group maintained normal abdominal girth throughout this study period (Fig. 5D). Survival analysis over 45 days revealed that the PBS group had a median survival of approximately 25 days, whereas 80% of the mice treated with MnALN survived until the end of the experiment, with the longest survival among all groups (Fig. 5E).

Fig. 5.

In vivo anti-tumor efficacy assessments of MnALN NPs. (A) Schematic illustration of in vivo therapy design. (B) Bioluminescence images of CT26-Luc tumor-bearing mice individually treated with PBS, MnCl₂ (13 mg/kg), NaALN (20 mg/kg), or MnALN (20 mg/kg) at day 0, 4, 8, 16, and 30. (C) Bioluminescence intensities were measured from the images of (B) and compared with the control at day 0, 4, 8, and 16: ∗p < 0.05, ∗∗p < 0.01, n = 5. (D) Measurement of the abdominal girth of mice in the individual group (n = 5). (E) Survival proportions of PBS, MnCl₂, NaALN, or MnALN groups (n = 5). (F) TUNEL and H&E staining images of tumor tissue sections from different groups. Scale bar: 50 μm.

Furthermore, H&E and TUNEL staining of tumor tissues revealed that, compared with the PBS control, both MnCl₂ and NaALN treatment induced moderate levels of necrosis and apoptosis. Notably, the MnALN group exhibited the most extensive tumor cell death, characterized by pronounced nuclear fragmentation and positive TUNEL signals (Fig. 5F). Taken together, these results demonstrated that MnALN nanomedicine effectively suppresses tumor growth, controls abdominal circumference expansion, and prolongs survival in a murine model of peritoneal metastatic CRC, exhibiting superior therapeutic efficacy compared to MnCl₂ or NaALN alone.

3.7. In vivo immune activation and antitumor mechanism of MnALN

Given the superior antitumor efficacy of MnALN in the peritoneal metastatic CRC model, as evidenced by the visibly reduced tumor burden in the MnCl₂ and NaALN groups and the most pronounced tumor suppression in the MnALN group (Fig. 6A and B), we next investigated the underlying antitumor immune mechanisms of MnALN in vivo. To determine whether MnALN induces immunogenic cell death (ICD) within tumor tissues, tumors collected on day 20 after treatment were subjected to immunohistochemical (IHC) analysis. IHC staining revealed markedly stronger expression of calreticulin (CRT) and high mobility group box 1 (HMGB1) in the MnALN-treated group compared to controls, supporting the induction of ICD-associated immunogenic signaling in vivo (Fig. 6E).

Fig. 6.

MnALN reduces tumor burden and active anti-tumor immunity in tumor tissue and ascites. (A) Representative photograph of abdominal dissection from a treated mouse, showing mesenteric tumor nodules (yellow arrows). (B) White-light image of tumors collected from the mesentery. (C) Representative flow cytometry plots of CD4⁺ and CD8⁺ T cells in tumor tissue. Numbers indicate the percentage of CD8⁺ T cells among total T cells. (D) Quantification of the proportion of CD8⁺ T cells in tumor tissue. ∗∗p < 0.01, (n = 3). (E) Immunohistochemistry images of tumor tissue stained with CRT and HMGB1. Scale bar: 50 μm. (F) Immunofluorescent images of tumor tissue stained with CD3 and CD8. Scale bar: 100 μm. (G) Representative flow cytometry plots of CD4⁺ T cells and CD8⁺ T cells from ascites. (H) Quantification of the proportion of CD8⁺ T cells in ascites. (I and J) ELISA quantification of TNF-α and IFN-γ levels in mouse ascites (ng/mL). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 versus CTL (PBS) group.

As ICD is known to promote the activation and recruitment of cytotoxic T lymphocytes, we further assess T cell infiltration in tumor tissues. Flow cytometric analysis (Fig. 6C and D) showed that the average proportion of tumor-infiltrating CD8⁺ T cells among CD45⁺ CD3⁺ T cells was 7.15% in the PBS group, increased to 9.10% and 12.88% with MnCl₂ or NaALN alone, and reached 18.70% in the MnALN group. Consistently, immunofluorescence staining for CD3 and CD8 further confirmed the substantially increased T cell accumulation in MnALN-treated tumors, whereas MnCl₂ or NaALN monotherapies induced only moderate increases (Fig. 6F and Fig. S12A). Additionally, IHC analysis revealed a concentration-dependent increase in T cell density in MnALN-treated tumors (Fig. S12B).

Beyond solid tumor tissues, we also examined the immune status in ascites, which represents a key pathological feature of advanced peritoneal dissemination. As shown in Fig. S12C, ascites accumulation was notably reduced in the MnALN group compared to controls. Flow cytometric analysis of ascites-derived cells (Fig. 6G and H) showed a similar immune activation trend: the average proportions of CD8⁺ T cells among CD45⁺ CD3⁺ T cells were 2.58% in the PBS group, 3.73% in the MnCl₂ group, 4.84% in the NaALN group, and 17.8% in the MnALN group. To further evaluate functional immune responses elicited by MnALN, pro-inflammatory cytokines in ascites were quantified by ELISA (Fig. 6I and J). MnALN treatment significantly elevated tumor necrosis factor-α (TNF-α) from 0.468 ng/mL (PBS) to 1.051 ng/mL, and interferon-γ (IFN-γ) from 0.379 ng/mL to 0.471 ng/mL. Although MnCl₂ and NaALN also induced moderate increases in these cytokines, the MnALN group consistently exhibited the highest levels of inflammatory cytokines.

Collectively, these results indicate that MnALN suppresses peritoneal tumor growth, reduces ascites formation, promotes CD8⁺ T cell accumulation, and enhances Th1-type cytokine production within the peritoneal tumor microenvironment. This coordinated immune activation contributes to the exceptional in vivo therapeutic efficacy of MnALN against aggressive peritoneal disseminated CRC. Given that malignant ascites is a hallmark of advanced peritoneal metastasis and is commonly associated with poor prognosis, the robust CD8⁺ T cell infiltration in ascites following MnALN treatment highlights its promising translational potential for patients with late-stage peritoneal disseminated CRC.

3.8. Biocompatibility of MnALN nanomedicine

The biocompatibility of MnALN was systematically evaluated using a series of in vitro and in vivo assays. First, the toxicity of MnALN on human normal colonic epithelial cells NCM460 was assessed using the CCK-8 assay. As shown in Fig. S13A, the cytotoxic effect of MnALN on NCM460 cells was significantly less than that on tumor cells. Hemocompatibility was further examined using a hemolysis assay, where a hemolysis rate exceeding 5% is generally indicative of erythrocyte damage. As illustrated in Fig. 7A and B, all tested concentrations of MnALN and MnCl₂ resulted in hemolysis rates below 5%, with no visible hemolysis observed even at the highest concentrations (400 mg L⁻¹ for MnALN and 2 mM for MnCl₂). Moreover, no significant body weight loss was observed in any groups, suggesting minimal systemic toxicity of the treatments (Fig. S13B). Finally, the histocompatibility of different treatments was evaluated by H&E staining. After 20 days of treatment, the tumor-bearing mice were sacrificed, and the major organs were extracted. As shown in Fig. 7C, no obvious histological abnormalities were detected in the heart, liver, spleen, lung, or kidney of mice treated with MnCl₂, NaALN, or MnALN, suggesting the absence of significant organ-level toxicity. These results suggested that MnALN showed minimal toxicity to normal colon epithelial cells, no significant hemolytic activity, and caused no observable histological damage to major organs, underscoring its favorable therapeutic window attributed to the localized intraperitoneal delivery and tumor-specific activation. However, the potential impact of MnALN treatment on bone metabolic homeostasis, such as bone mineral density, was not evaluated in the present study. Therefore, systematic assessment is warranted in future investigations, particularly given the well-established clinical activity of alendronate in bone metabolism. Importantly, during the observation period, MnALN-treated mice showed no evidence of impaired mobility, body weight loss, or treatment-related systemic toxicity. Collectively, these results demonstrated that the as-synthesized MnALN nanomedicine possesses excellent biocompatibility, supporting its potential for further translational development.

Fig. 7.

In vitro and in vivo biosafety evaluation of MnALN NPs. (A–B) Hemolysis test of MnALN and MnCl₂ at different concentrations. (C) H&E staining image of tissue slice of main organs in PBS, MnCl₂, NaALN, and MnALN NPs groups. Scale bar: 100 μm.

4. Conclusions

In summary, an engineered manganese-based immune amplifier MnALN nanomedicine was designed and successfully synthesized via coordination polymerization and self-assembly of alendronate sodium with manganese. A key innovation of this platform is its tumor-microenvironment-responsive degradation, which enables the tumor-specific release of both active components. Beyond the known role of Mn in activating the cGAS–STING pathway, we discovered that nanoformulated Mn also acts as a Type II ICD inducer via ER stress. Together with alendronate-mediated T-cell activation, this dual mechanism establishes a synergistic chemo-immunotherapeutic cycle that robustly promotes dendritic cell maturation and antitumor immunity. The antitumor efficacy and biocompatibility of MnALN were systematically validated via multiple in vitro assays. In a murine model of CRC peritoneal metastasis, intraperitoneal injection of MnALN demonstrated efficient tumor-targeting and markedly suppressed tumor proliferation and ascites formation. Collectively, this work established a versatile nanomedicine with favorable therapeutic effects and biosafety for advanced CRC treatment.

Compliance with ethics requirements

All animal procedures were performed in strict adherence to the protocol approved by the Animal Welfare and Ethics Committee (TOPGM-IACUC-2024-0317).

CRediT authorship contribution statement

Miaojuan Qiu: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Software, Writing – original draft. Yingfei Wen: Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation. Sufen Fang: Data curation, Investigation, Methodology, Resources, Software, Validation. Tian Hao: Data curation, Formal analysis, Investigation, Methodology, Software. Huihui Xu: Formal analysis, Investigation, Methodology, Resources, Software. Xinyi Deng: Formal analysis, Methodology, Software. Takeshi Yamakawa: Formal analysis, Validation, Visualization, Writing – review & editing. Binbin Li: Data curation, Formal analysis, Funding acquisition, Supervision. Shiqiang Zhang: Data curation, Funding acquisition, Resources. Yingying Lu: Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing. Changhua Zhang: Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing. Jing Zhao: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia

component 1

Data availability

No data was used for the research described in the article.