Binary mineral nanoparticles enable intravascular delivery of metal ions to tumors for metalloimmunotherapy

Domain: chemistry
Published: 2026-01-12
Editorial outcome: minor_revision

Abstract
Although disruptions in metal ion homeostasis leading to severe cellular damage and regulated cell death are a promising strategy for cancer immunotherapy, challenges in overloading these ions to tumor cells without premature release have limited their therapeutic applications. In this study, we develop binary mineral nanoparticles incorporating both Ca2+ and Na+ ions to enhance the cytotoxic effects of ion interference in cancer immunotherapy. Engineered using a microfluidics system for uniform size distribution and scalability, these nanoparticles exhibit pH-sensitive ion release. Systemically administered, they preferentially accumulate in tumors, elevating intracellular Ca2+ and Na+ levels and inducing immunogenic cell death without calcium channel activators or other small-molecule inducers. Our binary mineral nanoparticles significantly enhance antitumor immunity, especially when combined with an immune checkpoint inhibitor, leading to long-term immunity and inhibition of metastasis. This nanotechnology-enabled synergistic delivery of Ca²⁺ and Na⁺ ions represents a promising adjunct to existing metalloimmunotherapy strategies for cancer eradication.

Introduction
Metal ions are essential elements for life, playing key roles in various metabolic processes within every living cell1. They also contribute to the immune response by interacting structurally, catalytically, or regulatorily with immune sensors, ion transporters, enzymes, and downstream effector proteins2. Therefore, metal ion meticulously regulated through controlled processes of uptake, storage, and secretion1. Any disruption in the intracellular concentrations of metal ions can cause significant damage, potentially leading to cell death. Recently, the concept of manipulating intracellular ion concentrations has gained attention as a promising strategy for immunotherapy2,3. For example, the supplementation of Zn2+ or Mn2+ ions can activate cGAS-STING pathway4,5, while K+ and Na+ ions are involved in the activation of the inflammasome6,7. Additionally, an overload of Ca2+ ion is known to induce cell death8. Cytosolic Ca2+ ions are crucial in numerous intracellular signaling pathways, such as those regulating proliferation and metabolism9. They are also involved in the activation of the T cell receptor10, as well as in processes like apoptosis and pyroptosis11. In this context, Ca2+ homeostasis has emerged as a promising target to manipulate for inducing programmed cell deaths. Various delivery platforms have been developed to facilitate Ca2+ ion overload, using sources like calcium phosphate (Ca3(PO4)2), calcium carbonate (CaCO3), and calcium peroxide (CaO2)12,13. However, simply supplementing Ca2+ ions into the cytosol using calcium-based nanoparticles without the aid of a Ca2+ ion efflux inhibitor leads to only a temporary elevation of Ca2+ ion concentration, which is typically insufficient for producing antitumor effects14–17. Consequently, strategies for Ca2+ ion overload have primarily relied on the use of calcium channel activators18. Unfortunately, this approach can frequently cause undesirable side effects.

Recently, Na+ ions have garnered attention in the field of metal ion-based immunotherapy. Studies have shown that high salt intake can improve antitumor immunity in animal tumor models19,20. Since cancer cells typically contain higher levels of Na+ ions in the cytosol compared to normal cells21,22, even a small supplement of Na+ ions could potentially disrupt Na+ ion homeostasis. In fact, several nanoparticles (NPs) carrying Na+ ions demonstrated antitumor effects by increasing the osmotic stress on cancer cells, inducing caspase-1-dependent pyroptosis, and releasing damage-associated molecular patterns (DAMPs)23–25. Despite the highly promising potential of Na+ ions as inducers of immunogenic cell death (ICD), to the best of our knowledge, no NP system has been developed to deliver Na+ ions to tumors via systemic administration. This challenge is likely due to the high solubility of Na+-containing compounds in aqueous conditions, which leads to their rapid release before they can accumulate in tumor tissue. This premature release significantly limit the applicability of Na+ ions for modulating the immune system in a targeted and effective manner.

“Layer-by-layer (LbL)” assembly is a versatile technique for the controlled release of drugs, achieved by sequentially depositing alternating layers of materials with opposite charges onto a core template. This approach allows for the inclusion and controlled release of therapeutic molecules, suggesting that LbL could provide a stable and controlled means of delivering Na+ and Ca2+ ions for metalloimmunotherapy26. A significant challenge in the LbL assembly of nanoparticles is the successive nature of the layer deposition process, which necessitates multiple mixing and purification steps using methods such as sonication, vortex mixing, centrifugation, or membrane-based filtration27. These steps pose difficulties for large-scale production due to challenges in achieving homogeneous and reproducible mixing of bulk solutions, which is crucial for synthesizing LbL nanoparticles. Recent advancements in microfluidics have provided alternative methods to streamline these labor-intensive LbL assembly processes. Through fluidic layering in micro-channels, multiple layers can be assembled without the need for vortex mixers or homogenizers. In addition, a recent study demonstrated that microfluidics in LbL assembly can bypass intermediate purification steps and prevent nanoparticle aggregation, highlighting the significant advantages of applying microfluidic techniques in LbL assembly. Importantly, microfluidic-assisted LbL assembly of nanoparticles is well-suited for scalable, continuous synthesis, making it promising for clinical-scale manufacturing27,28.

In this work, we seek to leverage the distinctive antitumor properties of ion overload by developing binary mineral nanoparticles (BMNs) that incorporate both Ca2+ and Na+ ions. We develop a two-stage microfluidic system to equip BMNs with a pH-sensitive lipid layer, thereby facilitating the efficient delivery of Na+ and Ca2+ ions to the cytosol and inducing ICD in cancer cells. After systemic administration, BMNs accumulate in tumors and increase ion levels within the tumor microenvironment (TME), triggering the cancer-immunity cycle. When used in conjunction with an immune checkpoint inhibitor, the antitumor immune responses elicited by BMNs are significantly amplified, leading to effective inhibition of tumor metastasis and the establishment of long-term antitumor immunity. Given their minimal systemic toxicity, the ease of NP synthesis, effective ICD induction, and compatibility with intravenous administration, we believe the BMNs hold clinical potential for treating a range of tumors. In summary, the combined delivery of Ca2+ and Na+ ions for tumor treatment could be a promising and effective adjunct to other cancer immunotherapy approaches.

Results
Development of BMNs using a microfluidic mixing system
To synthesize BMNs consisting of both calcium (Ca2+) and sodium ions (Na+), we adapted a conventional gas-diffusion method (Supplementary Fig. 1). The modification involved using NaOH as an alkalinizing agent during the NP core preparation. To find out the optimal concentration of sodium hydroxide for preparing BMN cores containing both Ca2+ and Na+ ions, we varied the concentration of sodium hydroxide and calcium chloride during NP core preparation. With a fixed concentration of calcium chloride, the increased concentration of sodium hydroxide resulted in the larger particle size and high yields of both cations whereas it reduced the zeta potentials of the cores (Supplementary Fig. 2). In addition, high concentrations of sodium hydroxide increased the ratio of Na+ to Ca2+ (Supplementary Fig. 2). In contrast, when the concentration of sodium hydroxide was constant as 6 mM during core preparation, increasing the concentration of calcium chloride reduced the particle size and recovery efficiency of Na+ (Supplementary Fig. 3). Zeta potentials of the core and the recovery efficiency of Ca2+ increased in response to the concentration of calcium chloride, peaking at 2.70 and 4.05 mM, respectively, before declining (Supplementary Fig. 3). We selected concentrations of 2.70 mM for calcium chloride and 6 mM for sodium hydroxide as the optimal conditions for core preparation. These optimized conditions led to the formation of NP cores with a size of 120 nm, an acceptable recovery efficiency of both ions, and an appropriate Na+/Ca2+ ratio.

Next, we applied the optimized conditions to investigate the underlying formation mechanism of the BMN core. Upon adding NaOH in an aqueous solution to CaCl2 in ethanol, Ca(OH)2 precipitated due to its insolubility in ethanol, involving ~4% of the total calcium ions, as indicated by our measurements of the supernatants. Agitation during the reaction promotes CO2 gas diffusion through the gas-liquid interface29. Once dissolved, CO2 reacts with water molecules and hydroxide ions to form carbonate ions (CO32-), leading to the simultaneous precipitation of CaCO3 and Na2CO3, as they are insoluble in an ethanol-rich environment, thereby forming the BMN core29,30. This hypothesis is well-supported by our dynamic light scattering (DLS) measurements, which tracked the particles at different agitation times. Ca(OH)2 precipitated immediately after the NaOH solution was added, initially measuring ~4000 nm in size. Midway through the reaction, we observed two distinct particle size distributions: one ranging from 100–200 nm and another exceeding 600 nm. The intensity of the smaller particles increased as the reaction progressed, while the intensity of the larger particles decreased and eventually vanished after 8 h of stirring. When measuring calcium ions in the supernatant, the concentration of free calcium ions rose to 30% of the total after 2 h of stirring, then gradually fell to 0% after 24 h, suggesting the reaction had reached equilibrium. Similarly, the concentration of free sodium ions decreased between 0 and 8 h of stirring, indicating concomitant incorporation of sodium ions within the BMN core as the core grew (Supplementary Fig. 4). However, these mineral NP cores are typically unsuitable for direct in vivo applications due to their rapid precipitation in aqueous conditions, potentially leading to occlusions within the circulatory system. The LbL assembly technique enhances both the biocompatibility and colloidal stability of the NPs, making them more suitable for in vivo use15,31,32. By exploiting the strong positive charge on the BMN cores, 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), a negatively charged lipid, was coated on the NP surface via electrostatic interactions. Then, a lipid mixture consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)−2000] (DSPE-PEG2000), N-2,3-dimethylmaleic-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DMM-DOPE) was combined with the DOPA-coated NPs for the subsequent LbL assembly. DMM-DOPE, which contains an acid-cleavable amide bond (Supplementary Fig. 5), was added to give the final NPs tumor-responsive properties33.

While traditional methods for synthesizing LbL assemblies are effective to prepare BMNs with a sub-200 nm size (Supplementary Fig. 6), they often face challenges such as limited scalability, poor reproducibility, and uncontrollable NP properties. Recently, state-of-the-art microfluidic platforms have emerged in pharmaceutical development for their ability to precisely control fluid movement, reduce mixing time during synthesis, increase yield and reaction scale, and enhance heat and mass transfer34. Therefore, we developed a sequential micromixer consisting of two consecutive Tesla mixers in one device to streamline the LbL assembly (Fig. 1a and Supplementary Fig. 7). The former one was to mix the NP core and DOPA, and the latter one was to mix the DOPA-coated NPs and other lipid molecules, producing multi-layered NPs in a one-pot synthesis manner. The mixing efficiency was predicted by numerical simulation (Supplementary Fig. 8) and then experimentally investigated using rhodamine B solution (Figs. 1b, c). As two fluid streams flow parallel in a channel from ROI 1 to ROI 4, the fluorescent signals from rhodamine B became progressively diffused and homogeneous, indicating effective diffusive mixing of the two fluids, even at different total flow rates (TFR) (Fig. 1d and Supplementary Fig. 8). Mixing at a higher TFR allowed for rapid mixing of the two fluids, leading to the production of generally smaller NPs (Supplementary Fig. 8 and 9). The final BMNs exhibited a size of 183 nm with a narrow distribution (Fig. 1e) and a slightly negative zeta potential (Fig. 1f). These DLS characteristics were maintained for several days when incubated in a 5% dextrose solution at two different temperatures, 4°C and 37°C (Supplementary Fig. 10), suggesting the formation of stable BMNs in aqueous media. Transmission electron microscopy (TEM) imaging confirmed the formation of a core-shell structure: round-shaped NP cores exhibiting a bumpy surface were surrounded by enveloping lipid layers (Fig. 1g and Supplementary Fig. 11), a characteristic often observed in other metal-based NPs. Furthermore, elemental energy-dispersive spectroscopy (EDS) mapping of a single nanoparticle revealed the presence of Na+ and Ca2+ ions, as well as phosphorus (P) from phospholipids (Fig. 1h, and Supplementary Fig. 11), indicating the existence of lipid layers. An in vitro release study of BMNs demonstrated that both Ca2+ and Na+ ions were gradually released over a 48-h period (Figs. 1i, j). Additionally, the BMNs displayed higher release rates for both ions in acidic conditions compared to physiological pH conditions. This underscore the pH-responsive nature of the BMNs, which is attributable to the intrinsic properties of both CaCO3 and DMM-DOPE (Figs. 1i, j and Supplementary Fig. 12).

Subsequently, we assessed the cellular uptake of the BMNs in vitro by labeling them with 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide (DiR), a lipophilic near-infrared fluorescent dye. Mammalian cells maintain ionic homeostasis through various mechanisms, including ion channels, transporters, exchangers, and buffering systems. Since these mechanisms are responsible for clearing excess ions and maintaining proper ion concentrations, it is challenging to alter the cellular levels of Na+ and Ca2+ ions using Na+/Ca2+ solution35,36. Nonetheless, the uptake of DiR-labeled BMNs by 4T1 and B16F10 cells was shown to be both time-dependent (Fig. 1k) and concentration-dependent uptake (Fig. 1l), as determined by flow cytometry (Supplementary Fig. 13). Inductively coupled plasma-optical emission spectrometer (ICP-OES) analysis revealed a significant increase in intracellular calcium (Fig. 1m) and sodium (Fig. 1n) content in 4T1 cells treated with BMNs. Notably, a comparable elevation was absent in cells treated with an equivalent Na+/Ca2+ solution, indicating the effective BMN internalization. These cellular uptake of BMNs was further corroborated by confocal microscopy (Fig. 1o and Supplementary Fig. 14). In the colocalization analysis, Pearson’s correlation coefficient between the DiR and Lysotracker Green signals showed a positive correlation, suggesting that the internalized BMNs were trafficked through endolysosomal pathways. Over time, this coefficient declined, presumably due to the lysosomal disintegration of the BMNs and the subsequent diffusion of DiR dyes into cytosol (Fig. 1o and Supplementary Fig. 14). This observation suggests a time-dependent increases in the subcellular concentrations of Ca2+ and Na+ ions following BMN treatment (Supplementary Fig. 15). Flow cytometry further confirmed that BMN treatment significantly elevated the subcellular concentrations of these ions, surpassing the levels achieved with treatment of Na+/Ca2+ solution (Supplementary Fig. 15). In mammalian cells, the endoplasmic reticulum (ER) and mitochondria are the primary organelles responsible for Ca2+ homeostasis37,38. Following incubation with BMNs, the levels of Ca2+ ions in both the ER and mitochondria were dramatically increased compared to the PBS (Control) and Na+/Ca2+ solution treatments. The accumulation of Ca2+ ions in the ER rapidly reached a plateau at 2 h of incubation, while the mitochondrial Ca2+ level showed a significant increase only after 4 h of BMN treatment (Figs. 1p, q and Supplementary Fig. 16). The observed temporal difference in the Ca2+ plateau reflects the distinctive roles of the organelles. Given that the ER is the main intracellular calcium reservoir39,40, it is expected to respond rapidly to BMN-mediated Ca2+ internalization. In contrast, mitochondrial Ca2+ accumulation, in turn, might only become significant once the capacity of the ER to buffer cytosolic Ca2+ is surpassed.

Biodistribution of BMNs
After confirming the effectiveness of BMNs in increasing subcellular ion concentrations in vitro, we evaluated their tumor delivery in 4T1 tumor-bearing mice via intravenous (IV) injection of DiR-labeled BMNs. Compared to the free DiR solution, the DiR-labeled BMNs exhibited significantly higher tumor accumulation in vivo, with their accumulation increasing gradually over time (Fig. 2a). Ex vivo imaging of harvested organs showed that DiR-labeled BMNs produced significantly higher fluorescent signals in all organs except the lungs, compared to free DiR (Figs. 2b, c). Notably, the DiR-labeled BMNs demonstrated a 12-fold greater accumulation in the tumor compared to free DiR treatment. Additionally, using flow cytometry, we found pronounced elevations in mean fluorescence intensity (MFI) in most cell types studied, with the exception of granulocytic myeloid-derived suppressor cells (CD11b+Ly6G+SSChigh) and neutrophils (CD11b+Ly6G+SSClow) (Fig. 2d). The slight increase in DiR fluorescence within T cell subpopulations did not reach statistical significance (Figs. 2d, e). While such accumulations might suggest a potential risk of immune cell side effects, this concern is mitigated by previous studies demonstrating the safety of structurally similar metal compounds. For instance, NaCl nanoparticles have been shown to be safe with macrophages23. Furthermore, tumor-infiltrating T cells were demonstrated to well tolerate treatments of both calcium-based nanoparticles and high doses of NaCl, even when these interventions significantly increased the intracellular ion concentrations41,42. Consistent with these findings, BMN treatment showed negligible cytotoxicity in macrophages, DCs, and T cell subpopulations in our research (Supplementary Fig. 17), supporting the inherent tolerability of these immune cells to BMN treatment. Interestingly, the percentages of DiR-positive cells significantly increased in CD45- cells following the treatment with DiR-labeled BMNs compared to free DiR treatment (Figs. 2d, e and Supplementary Fig. 18). Next, we proceeded to measure the concentrations of Ca2+ and Na+ in the tumors. We compared BMNs to typical CaCO3 particles that lack Na+ and LbL coating. At 24 h post-administration, BMNs significantly increased Ca2+ concentration in mouse plasma (Fig. 2f) but not in mouse urine (Fig. 2g). The increase in Ca2+ levels in plasma was likely due to the extended circulation time of BMNs, which were protected by lipid layers. In the major organs, BMNs rarely altered Ca2+ concentrations, while bare CaCO3 microparticles elevated Ca2+ ion concentration in the lungs (Fig. 2h). It was due to the instability of CaCO3-based materials in aqueous solution without the lipid layers, leading to an increase in particle size and subsequent entrapment in lung tissues (Supplementary Fig. 19). Despite considerable accumulation in the reticuloendothelial system (i.e., the liver and spleen), BMNs did not cause a significant increase in Ca2+ concentrations. Consistent with the biodistribution data, BMNs significantly elevated Ca2+ concentration by ~2.3-fold in tumors compared to both the PBS and bare CaCO3 particles (Fig. 2i). Similarly, Na+ concentrations did not exhibit any significant change in tested biofluids (Figs. 2j, k) and major organs (Fig. 2l) following BMN treatment. However, there was a slight increase in Na+ content in tumor tissues, ~50% higher compared to the control group (Fig. 2m). This increase might be partly attributed to the lipid layers, which extend half-life of BMNs in the bloodstream and facilitate the release of ions when exposed to the acidic pH in the TME43.

Establishment of antitumor immunity by BMNs in multiple tumor models
In an initial study, we verified that naïve BALB/c mice tolerated a slow IV injection of Na+/Ca2+ solution containing sodium chloride and calcium chloride, with a Na+ to Ca2+ ratio of 1:40, at doses of 1 mg/kg Na+ and 40 mg/kg Ca2+. However, rapid injection or higher doses immediately caused fatal shock in the mice. Based on these results, 4T1 tumor-bearing mice were treated with PBS, Na+/Ca2+ solution (Na+: 1 mg/kg, Ca2+: 40 mg/kg), BMNs (Na+: 1.5 mg/kg, Ca2+: 60 mg/kg), or mitoxantrone (MTX)-loaded BMNs (MTX: 1.5 mg/kg, Na+: 1.5 mg/kg, Ca2+: 60 mg/kg) (Fig. 3a and Supplementary Fig. 20). Despite similar in vitro cytotoxicity, MTX-loaded BMNs significantly outperformed the MTX solution in suppressing tumor growth. The pronounced antitumor effects of MTX-loaded BMNs were likely from the NP accumulation in tumors. Interestingly, BMNs that did not contain MTX also showed significant suppression of 4T1 tumor growth compared to treatments with PBS or Na+/Ca2+ solution. Histopathological analyses showed a decrease in tumor cell volume in tumors treated with BMNs compared to those treated with PBS or Na+/Ca2+ solution (Supplementary Fig. 21 and Supplementary Table 1). Even when compared to the MTX-loaded BMNs, the reduction in 4T1 tumor growth caused by treatment with standalone BMNs was only marginally less, and this difference did not have statistical significance. In other words, BMNs containing Ca2+ and Na+ inherently exhibit antitumor effects without the need for loading anticancer small molecules. To our best knowledge, this finding has rarely been reported before. No significant differences were observed in body weight, plasma Ca2+ concentration, or clinical chemistry results. Histological analyses showed no major organ injury (Supplementary Fig. 21, Supplementary Fig. 22, and Supplementary Table 2), supporting the safety of IV BMN injection.

In a different setting, we tested various doses of BMNs based on their Na+/Ca2+ content, ranging from 0.75/30 mg/kg to 2.25/90 mg/kg, totaling 3 doses. Mouse body weight indicated good tolerance up to Na+ doses of 1.5 mg/kg and Ca2+ doses of 60 mg/kg (Fig. 3b). The antitumor efficacy of BMNs was dose-dependent in animals receiving tolerable doses (Fig. 3c and Supplementary Fig. 23). The higher dose markedly improved survival rate of the tumored animals compared to control and lower dose groups (Fig. 3d). We then compared BMNs with PEGylated CaCO3 NPs that lack Na+ ions. BMNs were again effective in suppressing 4T1 tumors, while PEGylated CaCO3 NPs barely inhibited tumor growth (Fig. 3e and Supplementary Fig. 24). Furthermore, varying the Na+/Ca2+ ratio within the BMNs showed in an in vitro cell viability assay that higher Na⁺ content increased cytotoxicity, corroborated by the annexin V and propidium iodide double staining assay (Supplementary Fig. 25). These findings clearly indicate that Na+ ions play a key role in the antitumor effects of BMNs. While previous studies have shown that CaCO3-based NPs can increase subcellular Ca2+ concentration, they typically used small molecules to disrupt Ca2+ homeostasis14,15. In contrast, our BMNs elevated ion concentrations without the use of transporter blockers. This effect is likely due to the co-delivered Na+ ion inhibiting the Na+/Ca2+ exchanger, which subsequently induces Ca2+ overload in the cytoplasm44. The effectiveness of BMNs in suppressing tumor growth was also verified in CT26 (Fig. 3f and Supplementary Fig. 26) and B16F10 tumors (Fig. 3g and Supplementary Fig. 27), with no significant changes in body weight in either model during the studies.

To uncover the mechanism behind the antitumor effects of BMNs, we analyzed the treated tumors. The immunohistochemical (IHC) analysis revealed that BMN treatment increased the levels of apoptosis markers, such as cleaved caspase-3 and cleaved PARP, while decreasing the levels of the proliferation-related protein Ki-67 and the angiogenic marker CD31 in the treated 4T1 tumors (Supplementary Fig. 28 and Supplementary Table 1). BMN treatment increased the densities of antigen-presenting cells (APCs), including F4/80+ macrophages and CD11c+ dendritic cells (DCs), in tumors compared to those treated with PBS or Na+/Ca2+ solution. Moreover, it also led to the infiltration of CD4+ and CD8+ T cells, as well as NK1.1+ natural killer cells, while inhibiting Foxp3+ regulatory T (Treg) cell recruitment. Of note, tumors treated with BMNs exhibited a reduced expression of CD206 – a canonical marker of M2-like tumor-associated macrophages (TAMs) – compared to the control (Supplementary Fig. 28 and Supplementary Table 3). This finding suggests a favorable shift toward reduced incidence of tumor resistance. Consistent with the IHC results, flow cytometric analysis of the TME demonstrated that BMN treatment increased the infiltration of CD45+CD3+ T cells in the TME, including both CD4+ and CD8+ T cells (Fig. 3h–j and Supplementary Fig. 29). Furthermore, BMNs significantly reduced the proportion of Treg cells among CD4+ T cells (Figs. 3k, l). This led to an elevated CD8+/Foxp3+ T cells ratio (Fig. 3m), which is a predictive marker for survival45. The elevated Ca2+ ion concentration in the TME, while otherwise promising, poses a risk to T cell function. Specifically, it may induce the overactivation of the calcineurin/NFAT signaling pathway in tumor-infiltrating T cells46. This pathway drives CD8+ T cell exhaustion and CD4+ T cells anergy, marked by PD-1 overexpression and severely reduced secretion of crucial cytokines such as IFN-γ, TNF-α, and IL2, ultimately hindering T cell-mediated antitumor activity47–49. However, in vitro treatment with BMNs did not elevate the nuclear translocation of NFAT1 protein in either CD8+ or CD4+ T cells, nor did it suppress the secretion of intracellular cytokines and IL2 (Supplementary Fig. 30). Consistent with these finding in vitro, BMNs also did not suppress the intracellular cytokine secretion of tumor-infiltrating T cells or increase the expression of PD-1 marker 24 h after in vivo treatment (Supplementary Fig. 31). Moreover, by day 10 of the in vivo antitumor study, T cells within the TME treated with BMNs were observed to actively produce cytokines, whereas those in the other groups showed significantly lower cytokine production (Figs. 3n, o, and Supplementary Fig. 29). Additionally, the TME of the BMN group showed an increased ratio of CD11c+MHCII+CD80+CD86+ DCs, indicating a higher rate of DC maturation (Supplementary Fig. 29). Concurrently, a lower expression of CD206 marker was observed, suggesting a desirable reduction in M2-like TAMs (Supplementary Fig. 29). These findings show that the antitumor effects of BMNs involve reprogramming immune cell populations within the TME of 4T1 tumors, as also confirmed in the CT26 tumor model (Supplementary Fig. 32). Treatment with BMNs promoted an overall immune-active state in the tumors, marked by the upregulation of type I interferon (IFN-I) genes (Ifn-α, Ifn-β), leukocyte chemokine gene (Cxcl10), and the downregulation of immunosuppressive genes (Tgf-β1, Vegf, and Il10). Notably, it significantly elevated the expression of Gzmb and Ifn-γ, genes associated with the effector functions of T cells (Fig. 3p and Supplementary Fig. 33). The BMN treatment also facilitated the maturation of DCs in the tumor-draining lymph nodes (Fig. 3q and Supplementary Fig. 29) and increased the plasma concentration of IFN-β (Supplementary Fig. 29). The treatment with BMNs led to an increase in the percentage of CD3+CD8+ T cells expressing CD62LloCD44hi in the spleen (Fig. 3r and Supplementary Fig. 29), indicating the development of an effector memory cell phenotype. These results suggest that IV administration of BMNs activates the cancer-immunity cycle in tumor animal models. To further examine the role of CD8+ T cells in antitumor effects of BMNs, we depleted CD8+ T cells using anti-mouse CD8α monoclonal antibody (aCD8) during the animal study (Fig. 3s). The depletion of CD8+ T cells completely abrogated the antitumor effects of BMNs (Fig. 3t and Supplementary Fig. 34). Taken together, these findings underscore the crucial role of immune responses mediated by CD8+ T cells in the antitumor effects exerted by BMNs.

Immunogenic cell death as the antitumor mechanism of BMNs
We hypothesized that ICD would mediate the development of antitumor immunity following BMN treatment. In 4T1 cells treated with a high dose (3.75 μg Na+/mL, 150 μg Ca2+/mL) of BMNs, we observed elevated exposure of calreticulin (CRT) on the cell surface and a reduction in intracellular ATP levels 6 h after treatment. In contrast, neither a low dose (1.88 μg Na+/mL, 75 μg Ca2+/mL) of BMNs nor Na+/Ca2+ solution resulted in these outcomes (Fig. 4a–c and Supplementary Fig. 35). Notably, the BMN treatment decreased the nuclear levels of high mobility group box 1 (HMGB1) while simultaneously upregulating its level in the extracellular space (Fig. 4d and Supplementary Fig. 35). This release of HMGB1 from the nucleus is indicative of ICD, as the protein is passively secreted from dying cells during this process50,51. Consistent with this finding, the expression of ICD markers, such as CRT and HMGB1, was also increased when the tumor was treated with BMNs in vivo (Supplementary Fig. 36 and Supplementary Table 4). Additionally, BMN treatment achieved significant increases in the levels of IFN-Is (Ifn-α, Ifn-β) and lymphocyte attractant chemokines (Cxcl9, Cxcl10) (Figs. 4e, f). As a result, an appropriate dose of BMNs can lead to ICD, along with a robust release of DAMPs and IFN-Is. Then, we continued to investigate how BMNs initiate ICD and DAMP release by examining their impact on intracellular Ca2+ homeostasis. As previously demonstrated, BMNs treatment disrupted the intracellular homeostasis of Na+ and Ca2+ ions, leading to increased Ca2+ accumulation within subcellular organelles. This accumulation, in turn, can trigger the generation of reactive oxygen species (ROS), mitochondrial dysfunction23,52. Confocal imaging and flow cytometric analyses revealed that depolarization of mitochondrial membrane and generation of intracellular ROS occurred only in 4T1 cells treated with a high dose (3.75 μg Na+/mL, 150 μg Ca2+/mL) of BMNs, but not in treatment of Na+/Ca2+ solution or a low dose (1.88 μg Na+/mL, 75 μg Ca2+/mL) of BMNs (Figs. 4g, h, and Supplementary Fig. 37). The high dose of BMNs also decreased Ki-67 expression and increased γ-H2AX expression (Supplementary Fig. 38), indicating anti-proliferation and DNA damage, respectively. Western blot revealed increased expressions of proteins related to cell death, autophagy flux, and ER stress response (Supplementary Fig. 39) following BMN treatment (3.75 μg Na+/mL, 150 μg Ca2+/mL). ER stress responses marked by elevated levels of p-eIF2α, without a rise in downstream factors such as ATF4, ATF6, and XBP1s, are considered as pathognomonic indicators of ICD53. Additionally, literature has implicated autophagy as a key mechanism for ATP release from dying cells after treatment54,55. From these, we inferred that BMNs can induce ICD and DAMP release by disrupting the intracellular homeostasis of Ca2+ and Na+ ions. Recent studies have demonstrated that DAMPs and type I IFNs from cancer cells effectively bind to their corresponding pattern recognition receptors (PRRs) on APCs56,57. This binding subsequently enhances their efferocytosis capacity, maturation, and antigen cross-presentation, thereby activating the cancer-immunity cycle58. For these reasons, we investigated the downstream effects of ICD derived from BMN treatment on APCs using bone marrow-derived dendritic cells (BMDCs). When co-culturing BMDCs with fluorescently labeled 4T1 cells (Fig. 4i), we observed improved efferocytosis by BMDCs on BMN-treated 4T1 cells compared to the PBS and Na+/Ca2+ solution groups (Fig. 4j-l and Supplementary Fig. 40). Additionally, BMN treatment facilitated BMDC maturation more effectively than the other groups due to its efficient ICD induction (Fig. 4m and Supplementary Fig. 40). To study the effects of DAMP release alone, BMDCs were incubated with NP-free culture media from 4T1 cells treated under various conditions (Fig. 4n). We observed an elevation of co-stimulatory molecules (Figs. 4o, p) and the gene expression of IFN-Is (Ifn-α, Ifn-β), Cxcl10, and proinflammatory cytokines, Il6, Il12, Tnf-α (Fig. 4q and Supplementary Fig. 41), indicating the activating effect of DAMPs.

BMN-mediated ICD promotes the cancer-immunity cycle
For potent immunotherapy, induction of ICD should trigger antitumor response of both innate and adaptive immune systems to overcome the immunosuppressive TME59. Accordingly, to account for various factors present in the in vivo environment, we immunized naïve mice with dying 4T1 cells subjected to various treatments (Fig. 5a). Vaccination with the cells treated with BMNs resulted in the highest fluorescence intensity in the inguinal lymph node compared to the groups treated with PBS or Na+/Ca2+ solution (Fig. 5b, c), indicating enhanced efferocytosis due to BMN treatment. Additionally, vaccination with these cells significantly improved the maturation state of CD11c+ DCs in the lymph node (Figs. 5d, e). This suggests that BMN treatment enhanced the vaccination’s effectiveness more than Na+/Ca2+ solution treatment by eliciting stronger immunostimulatory effects. Additionally, prophylactic vaccination with BMN-treated 4T1 cells effectively prevented tumor growth (Fig. 5f-h) and enhanced animal survival compared to the control group (Fig. 5i). Notably, BMN treatment was as effective as MTX, a well-known ICD inducer, indicating that BMNs efficiently induce ICD. Antigen-specific immune recognition was also evaluated in BMN-treated, tumor-bearing mice using the B16-OVA tumor model (Fig. 5j). Consistent with results obtained in other tumor models, BMNs treatment effectively inhibited the growth of B16-OVA tumors (Fig. 5k and Supplementary Fig. 42). Moreover, the flow cytometry analysis of the tumor draining lymph nodes (TDLNs) revealed both a higher ratio of SIINFEKL-H2Kb+ DCs and an improvement of DC maturation state in the BMN group (Fig. 5l–n and Supplementary Fig. 43). This indicates that the BMN treatment effectively improved the presentation of tumor antigens by APCs in the treated mice. Additionally, the BMN group exhibited a significant increase in the frequencies of antigen-specific CD8+ T cells in both the peripheral blood (Figs. 5o, p and Supplementary Fig. 43) and secondary lymphoid organs, specifically the TDLNs and spleen (Figs. 5q, r and Supplementary Fig. 43) compared to the control and Na+/Ca2+ solution groups. Furthermore, following ex vivo restimulation with SIINFEKL peptide, the IFN-γ secretion of CD8+ T cells was significantly improved in the tumor, spleen, and TDLNs of BMN-treated mice compared to the other groups (Fig. 5s-v and Supplementary Fig. 43). This collectively demonstrates the superior effect of BMN treatment in inducing robust, tumor antigen specific immunity in the treated tumor-bearing mice. Taken together, the antitumor effects of BMNs were primarily attributed to their ion content. The overload of Na+ ion in the cytosol disrupts cellular Ca2+ homeostasis, leading to ICD and DAMP release. This, in turn, triggers antitumor responses of innate and adaptive immune systems, thus promoting of the cancer-immunity cycle.

Potent inhibition of metastasis by combining BMNs with anti-PD1 antibody
Next, we sought to evaluate whether these BMNs could potentiate their therapeutic effects when combined with anti-PD-1 antibody (aPD1) treatment in the CT26 tumor model. While BMNs or aPD1 alone had moderate effects, their combination significantly inhibited tumor growth, with two mice showing nearly complete arrest. This led to extended survival compared to other groups (Supplementary Fig. 44). None of the treated mice exhibited signs of systemic toxicity or weight loss, indicating that these treatments were well tolerated. Similar experiments were conducted in the orthotopic 4T1-luc2 tumor model (Fig. 6a). In this setting, aPD1 alone resulted in minimal tumor growth suppression; however, its antitumor effects were significantly enhanced when combined with BMNs, as observed on day 10 (Fig. 6b). Continued monitoring revealed that the BMN/aPD1 combination maintained tumor growth inhibition for three weeks after the last dose, achieving nearly complete tumor elimination in 40% of the mice (Figs. 6b, c, and Supplementary Fig. 45). Interestingly, the combination treatment also remarkably suppressed the expression of both PD1 and PD-L1 in the tumor tissues of the treated mice (Supplementary Fig. 46 and Supplementary Table 5). Bioluminescence imaging of the 4T1-luc2 orthotopic tumors on day 28 indicated that most animals developed tumor metastasis. All mice treated with either PBS or aPD1 showed luminescence in distant organs, suggesting disseminated metastasis. However, none of the mice that received the BMN/aPD1 combination showed signs of tumor metastasis, as observed by the absence of luminescence in distant organs (Fig. 6d and Supplementary Fig. 45). These findings suggest that standalone aPD1 therapy is insufficient in inhibiting spontaneous metastasis of 4T1-luc2 tumors; however, this limitation can be overcome by combining it with BMNs. The subsequent IHC revealed that the BMN/aPD1 combination significantly reduced the expression of TGF-β1 protein, a key determinant for promoting epithelial-mesenchymal transitions (EMTs) in the TME60. Subsequently, expression levels of two epithelial markers, EpCam and E-Cadherin, were markedly higher in tumors treated with the combination therapy compared to other groups. In contrast, the combination therapy led to a significant decrease in the expression of mesenchymal markers such as N-Cadherin and Vimentin (Figs. 6e, f, and Supplementary Table 6). These results support that the BMN/aPD1 combination effectively inhibited EMT processes in primary orthotopic tumors61. Inhibited EMTs are associated with reduced local invasion and subsequent intravasation of tumor cells through lymphatic and circulatory systems. This could explain the absent bioluminescence signals in the inguinal lymph nodes (Supplementary Fig. 45) and the reduced number of luciferase-positive cells in the lung tissues of mice that received the combination therapy (Figs. 6g, h, and Supplementary Table 6). No evidence of acute toxicity or tissue damage was detected in the clinical chemistry (Supplementary Fig. 47) and histology assessments (Supplementary Fig. 48 and Supplementary Table 7) of the treated animals, suggesting that the treatments were well tolerated. Lastly, we carried out another experiment using the 4T1-luc2 orthotopic model to assess the long-term effects of combining BMNs with aPD1 on animal survival (Fig. 6i). Consistent with previous results, tumor began metastasizing to distant organs on day 28 and 35 in the control group and aPD1 treatment group, respectively. BMNs delayed metastasis until day 42, while the combination therapy of BMNs with aPD1 prevented metastasis for 60 days (Fig. 6j). Also, the combination therapy significantly increased animal survival compared to other treatments (Fig. 6k). Surviving animals were then rechallenged with the same orthotopic tumors to assess whether they had developed memory immunity against tumor recurrence. Surprisingly, all mice exhibited rapid tumor elimination and survived after rechallenging, indicating the development of resistance against 4T1 tumors (Fig. 6l-n, and Supplementary Fig. 49). These results suggest a therapeutic synergy between BMNs and immune checkpoint inhibitors, effectively preventing metastasis by inhibiting EMT processes, enhancing animal survival, and preventing tumor recurrence.

Methods
Design, fabrication, and evaluation of a sequential micromixing device
Design and fabrication of a sequential micromixing device. The sequential micromixing device, consisting of two Tesla micromixers, was initially modeled using Solidworks (Dassault Systèmes SolidWorks Corp., USA), a professional 3D CAD program. The modeling process was mainly guided by previous research on Tesla micromixers, as the primary objective of this study is not to optimize geometric design. The design drawing and dimensions of the sequential micromixing system are shown in detail in Supplementary Fig. 7a, b, respectively. The sequential micromixing device was fabricated using a digital light processing (DLP) 3D printer (Max X27, Asiga, Australia) following the design process. DLP is a 3D printing method that uses an ultraviolet (UV) light source and a photo-curable resin to create solid objects. In addition, the printed micromixer device (CPX 8800H, E-Bansan, USA) was cleaned with isopropanol to eliminate any residual photo-curable resin. The conceptual design and its 3D-printing outcome for the sequential micromixing device were depicted in Supplementary Fig. 7c and Fig. 1a.

Evaluation of the mixing performance of a Tesla micromixer. As aforementioned, our sequential micromixing device comprises two Tesla mixers with identical geometry and dimensions. Consequently, the mixing performance of the single Tesla micromixer was assessed by using pure ethanol as the organic phase and an aqueous fluorescent solution (rhodamine B, 50 µM) as the aqueous phase of working fluids (Fig. 1b). The aqueous and organic phase fluids were introduced to their respective inlets at a flow rate ratio (FRR) of 3:1 using a syringe pump (Fusion 4000, Chemyx, USA). Mixing tests were performed at different total flow rates (TFRs) of 4, 20, and 36 mL/min while keeping the FRR constant to examine the effect of TFR on the mixing performance.

Fluorescence images were captured at four points (i.e., the mixing length) on the micromixers to assess the mixing performance using a fluorescence microscope (Eclipse Ci-L, Nikon) equipped with a high-speed CCD camera (Fastcam mini UX100, Photron) and a Texas red filter (excitation peak 596 nm/emission maximum of 620 nm) (Figs. 1b, c). It should be noted that fluorescence images were captured at each measurement point after the fluid flow had reached a steady state. It was also necessary to capture images while only aqueous rhodamine B and deionized water were being transported to determine the mixing index.

The mixing index was calculated using an image analysis technique with a custom MATLAB program (MATLAB R2021A, MathWorks). This image analysis program extracts pixel intensity data from collected images at four specific locations (i.e., the mixing length), indicating how the concentration of fluid species varies across a cross-section. The four locations (0.30 mm, 3.86 mm, 8.57 mm and 11.02 mm) represent the distances from the origin (0 mm) where the two inlet channels intersect (Fig. 1c). The image analysis process for calculating the mixing index involved the following steps. First, a suitable segment (measuring 80 × 220 pixels for region of interest (ROI) 1 and 80 × 100 pixels for ROI 2, 3, 4) was selected and cropped from the original images (Fig. 1d). The image type was then converted from RGB to grayscale, and the intensity of each pixel (Ii,j) was calculated. The subscripts i and j denote the coordinates for the flow direction and the direction perpendicular to the flow, respectively. Subsequently, the pixel intensity was converted into normalized pixel intensity (I'i,j) using Eq. (1). Here, Ii,jR and Ii,jW represent the pixel intensities of the images taken with only the flow of aqueous rhodamine B solution and that of DI water, respectively.

I'i,j = (Ii,j - Ii,jW) / (Ii,jR - Ii,jW) (1)

After determining the value of I'i,j for each pixel, its mean (μ) and standard deviation (σ) were calculated using Eqs. (2) and (3).

μ = (1/n:m) * Σi=1n Σj=1m I'i,j (2)

σ = sqrt((1/n:m) * Σi=1n Σj=1m (I'i,j - μ)2) (3)

Finally, the mixing index (η) was determined using Eq. (4) based on the calculated standard deviation, where σmax, which was set to 0.5, denotes the maximum standard deviation for the case where the two fluids were completely unmixed.

η = (1 - σ/σmax) × 100% (4)

As a result, when the standard deviation of the measurement points lowers in relation to the maximum standard deviation, the mixing efficiency increases. Put simply, when the intensity of all pixels is similar, the mixing efficiency is higher.

Numerical simulation for prediction of the mixing performance. To predict the mixing performance of the Tesla micromixer used in this study, numerical simulations were performed using the commercial finite element-based software COMSOL Multiphysics® (COMSOL Inc., Sweden). The geometry of the numerical model closely resembled that of the physical system, as shown in Supplementary Fig. 7d. The mesh was created using a hybrid grid consisting of free tetrahedral, prisms, triangles, and quads, totaling 5,353,197 elements.

To determine the flow fields in the Tesla micromixer, the continuity Eq. (5) and Navier-Stokes Eq. (6) were solved numerically, assuming Newtonian and incompressible fluids and steady state flow conditions.

∇·u = 0 (5)

ρ(u·∇u) = -∇p + μ∇2u (6)

The density and dynamic viscosity of the fluid are represented by ρ and μ, respectively. The numerical simulation employs the fluid properties of DI water (i.e., ρ of 998.2 kg/m3 and μ of 1.009 mPa∙s), ignoring the effects of the diluted rhodamine B concentration. u and p represent the velocity vector and pressure in the micromixer, respectively. The injection flow rates and open boundary conditions were imposed boundary conditions at the channel inlets and outlet, respectively. The boundary conditions of all channel walls were set to no-slip.

The concentration field for mass transport was obtained by numerically solving the convection-diffusion Eq. (7). This calculation utilized the previously determined velocity fields and incorporated CR and DR, which represent the concentration and mass diffusivity of the aqueous rhodamine B solution and ethanol, respectively.

u·∇CR = DR∇2CR (7)

The numerical simulation used D values of 4.27×10−10 m2/s for aqueous rhodamine B solution and 2.0×10−9 m2/s for ethanol. The boundary conditions for the concentrations of aqueous rhodamine solutions entering the two inlets were 1.0 mol/m3 and 0 mol/m3. The boundary conditions for the channel walls and outlets were set to non-flux and mass flux continuity conditions, respectively.

The initial calculation of the area mean (μx) and standard deviation (σx) of the normalized concentration (C'R) of rhodamine B at each location, determined by using the following formulas:

μx = (1/Ax) * ∫∫ CR' dydz (8)

σx = sqrt((1/Ax) * ∫∫ (CR' - μx)2 dydz) (9)

Here, C'R is defined as the concentration of rhodamine B divided by its initial concentration. The cross-sectional area parallel to the xy-plane is represented by Ax.

The mixing index (ηx) can then be determined from the calculated standard deviation using Eq. (10).

ηx = (1 - σx/σx,max) × 100% (10)

Here, σx,max represents the maximum standard deviation for BMNs completely unmixed fluids.

Recovery efficiency (%) = (ion in collected samples / total ion inputs) × 100% (11)

In vitro release of the ions from BMNs was characterized in two different pH conditions (Ca2+- and Na+-free Tris-HCl buffer pH 6.5 and 7.4) at 37°C with shaking at 100 rpm. At predetermined time points, BMNs samples were centrifuged at 16,000g for 30 min, and the ion contents were measured in the supernatants.

Cell culture
4T1 (CRL-2539), CT26 (CRL-2638) cells from the American Type Culture Collection (ATCC, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (HyClone, USA) containing 10% fetal bovine serum (FBS) (HyClone, USA) and 1% penicillin/streptomycin (HyClone, USA). B16F10 (CRL-6475) cells (ATCC, VA, USA) were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, HyClone) supplemented with 10% FBS and 1% PS. 4T1-luc2 cells were prepared as previously described58 and maintained in RPMI 1640 containing 10% FBS and 0.3 mg/mL Geneticin (G418 Sulfate, Thermo Fisher Scientific, USA). The cell lines are authenticated by ATCC. No additional authentication was performed by the authors. All cell lines were tested negative for mycoplasma.

Bone marrow-derived dendritic cells (BMDCs) and bone-marrow-derived macrophages (BMDMs) were differentiated as previously described63. Briefly, bone marrow cells were collected from femurs and tibias of 6-week-old BALB/c mice and cultured in the complete RPMI 1640 medium containing 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF, BioLegend) for BMDCs or macrophage colony-stimulating factor (M-CSF, BioLegend) for BMDMs. On day 8, completely differentiated BMDCs were collected with a ratio of CD11c+ cells of 90% or above, meanwhile BMDMs were collected on day 7.

For isolation and activation of splenic T cells, 96-well plates were first coated with 5 μg/mL solution of anti-mouse CD3ε (BioLegend) in PBS for at least 2 h. Splenic CD8+ and CD4+ T cells were isolated from spleens of BALB/c mice using the EasySep™ Mouse CD8+ T Cell Isolation Kit and EasySep™ Mouse CD4+ T Cell Isolation Kit (STEMCELL Technologies), respectively. The isolated cells were then activated for 48 h in complete RPMI 1640 medium supplemented with 20 ng/mL recombinant mouse IL-2 (BioLegend), 50 μM β-mercaptoethanol (Sigma-Aldrich), and 5 μg/mL anti-mouse CD28 antibody (BioLegend).

Characterization of in vitro cellular uptake of nanoparticles and cytosolic ion concentrations
In vitro cellular uptake of DiR-labeled BMNs was studied in 4T1 and B16F10 cells by flow cytometry and confocal microscopy. Briefly, for flow cytometric analysis, cells were seeded in 12-well plates at a density of 1×105 cells/well overnight. The cultured cells were then incubated with DiR-labeled BMNs at various concentrations (0, 10, 20, 40, or 80 µg/mL) of Ca2+ ion for 1 h or at a Ca2+ concentration of 20 µg/mL for various duration of time (0, 30, 60, or 120 min). The DiR signal was observed by flow cytometry (FACS Verse, BD Biosciences). For confocal imaging, the cells were seeded on 18 mm coverslips. After incubation with DiR-labeled BMNs, the cells were washed three times with PBS and co-stained with LysoTracker Green DND-26 (50 nM) (Invitrogen) and Hoechst 33342 (2 µg/mL) (Thermo Fisher Scientific) for 30 min at 37°C. Subsequently, the cells were mounted on microscope slides and imaged using a K1-Fluo confocal laser scanning microscope (Nanoscope Systems Inc., South Korea). The colocalization analysis, Pearson’s correlation coefficient was calculated using a JaCoP Plugin in ImageJ. The levels of total intracellular ions were also analyzed by flow cytometry and confocal microscopy. After the cells were treated with DiR-labeled BMNs (Ca2+: 80 μg/mL), they were incubated with sodium-binding benzofuran isophthalate acetoxymethyl ester (SBFI-AM, Invitrogen) or Fluo-3 AM (Sigma-Aldrich) to detect Na+ ions or Ca2+ ions, respectively, according to the manufacturers’ protocols. For organelle-specific Ca2+ ion analysis, cancer cells were treated with BMNs or Na+/Ca2+ solution (Ca2+: 80 μg/mL; Na+: 2 μg/mL) for the specified time intervals. Thereafter, treated cells were stained to detect Ca2+ in target organelles. To detect Ca2+ ion in the ER, cells were incubated with Mag-Fluo-4 AM (AAT Bioquest) and LumiTracker® ER Red (Lumiprobe). To detect mitochondrial Ca2+ ion, cells were incubated with Rhod-2 AM (MedChemExpress) and MitoTracker™ Deep Red FM. All staining procedures followed the manufacturers’ instructions. The fluorescence intensities of ion indicators were measured using FlowJo V10 and ImageJ for flow cytometry and confocal imaging, respectively.

In vitro evaluation of the anticancer effects and mechanisms of BMNs
In vitro cytotoxicity assay of BMNs was conducted using the MTS cell proliferation assay. Briefly, 4T1 cells, BMDCs, BMDMs, CD4+ T cells, or CD8+ T cells were cultured in 96-well plates at a density of 5×103 cells/well for cancer cells, BMDCs, or BMDMs and 105 cells/well for T cell subpopulations. The cells were then treated with various NPs for 24 h at 37°C. Cytotoxicity of BMNs was assayed using CellTiter 96® AQueous MTS reagent (G1111, Promega) according to the manufacturer’s instructions. Absorbance (Abs) was measured at the wavelength of 490 nm using a multimode microplate reader (Tecan Infinite F200, Switzerland). Cell viability was calculated using the following formula:

Viability (%) = (Abs sample - Abs blank) / (Abs control - Abs blank) × 100 (12)

For Annexin V and Propidium Iodide (PI) double staining a apoptosis assay, the cells treated with various NPs were incubated with FITC Annexin V for 15 min after washing with 1X Annexin V binding buffer. PI was added to the stained cells just before flow cytometric analysis.

To assess BMN-triggered release of DAMPs from cells, cells were treated with PBS, Na+/Ca2+ solution, or BMNs at various ion concentrations. At 6 h post-treatments, the cells were collected and washed with cold 2% FBS in PBS, followed by incubation with FITC anti-mouse calreticulin antibody (NBP1-47518F, Novus Biologicals) for 30 min. PI was added to the stained cells immediately before analyzing in flow cytometry. For confocal imaging, the cells treated with BMNs were fixed with 0.25% paraformaldehyde for 20 min at room temperature, incubated with rabbit anti-mouse calreticulin antibody (Ab2907, Abcam) for 30 min on ice, followed by incubation with goat anti-rabbit IgG secondary antibody conjugated to DyLight 488 (Thermal Fisher Scientific) for 1 h in the dark. Thereafter, the stained cells were mounted on slide glasses and observed using a confocal microscope. The intracellular levels of ATP were measured using either the luminescent ATP detection assay kit (Ab113849, Abcam) according to the manufacturer’s instruction or by confocal imaging following incubation of the cells with quinacrine solution (5 µM) for 30 min57.

To study the generation of reactive oxygen species (ROS) and the changes in mitochondrial membrane potential (ΔΨm), the 4T1 cells were treated with PBS, Na+/Ca2+ solution, or BMNs at various ion concentrations for 6 h. Levels of ROS and mitochondrial membrane potential were measured by using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich) and JC-1 reagent (MedChemExpress), respectively. Then, the stained cells were examined using confocal microscopy or flow cytometry with slight modifications.

For immunocytochemistry, 4T1 cells seeded on coverslips were treated with PBS, Na+/Ca2+ solution, or BMNs at concentrations of Na+ of 3.75 µg/mL and Ca2+ of 150 µg/mL. The cells were fixed with 0.25% paraformaldehyde for 20 min, and permeabilized using 0.2% Triton X-100 (Sigma-Aldrich) for 15 min at room temperature. Subsequently, the cells were incubated with anti-HMGB1 (3935S, Cell Signaling Technology), Ki-67 (Ab16667, Abcam), or γ-H2AX (2577S, Cell Signaling Technology) primary antibodies at 4°C overnight, followed by incubation with a goat anti-rabbit IgG secondary antibody conjugated to DyLight 488 (Thermal Fisher Scientific) for 1 h. The stained cells were mounted on slide glasses and examined in confocal microscopy.

For western blot, 4T1 cells variously treated were washed and lysed in RIPA buffer (89901, Thermo Fisher Scientific) supplemented with Halt™ Protease Inhibitor Cocktail (87786, Thermo Fisher Scientific) and EDTA (5 µM) and stored at -80°C for further analysis. Protein concentrations were determined using Pierce™ BCA Protein Assay Kits (23225, Thermo Fisher Scientific). Cell lysates were then denatured at 95°C for 15 min. Lysate proteins were separated by SDS-PAGE and transferred onto PVDF membranes (IPVH00010, Millipore Sigma). The membranes were blocked using 3% BSA (A7030, Sigma-Aldrich) in tris-buffered saline plus tween 20 (0.05%) for 1 h at room temperature, followed by incubation with a specific primary antibody at 4°C overnight. Thereafter, the membranes were washed and incubated with HRP-conjugated secondary antibody with shaking at room temperature for 1 h. Proteins were detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (34580, Thermo Fisher Scientific). The antibodies used for western blot are listed in Supplementary Table 8.

For assessments of type I interferon responses, 4T1 cells were seeded in 6-well plates at a density of 2×105 cells/well. The cells were treated with PBS, Na+/Ca2+ solution, or BMNs at ion concentrations of Na+: 3.75 µg/mL and Ca2+: 150 µg/mL for 24 h. The treated cells and cell-cultured media were then collected and subjected to RT-qPCR and ELISA, respectively.

Nanoparticle-mediated activation of bone marrow-derived dendritic cells in vitro
To investigate the efferocytosis of BMDC using flow cytometry, 4T1 cells were stained with CellTrace™ CFSE Cell Proliferation Kit (C34554, Thermo Fisher Scientific) according to the manufacturer’s protocol and cultured in 12-well plates at a density of 5×104 cells/well overnight. The cultured cells were then treated with PBS, Na+/Ca2+ solution, or BMNs. At 24 h post-treatment, 5×104 completely differentiated BMDCs were added into the wells containing treated 4T1 cells and co-cultured for additional 4 h at 37°C. The co-cultured cells were harvested using a cell scraper. After washing with cold PBS, the cell suspensions were incubated with CD16/32 Fc blocker (101302, BioLegend) for 30 min on ice, followed by 1 h incubation with APC anti-mouse CD11c, APC/Cy7 anti-mouse CD86, and PerCP/Cy5.5 anti-mouse I-A/I-E antibodies in the dark. The intensities of CFSE and the ratio of mature BMDCs were analyzed using flow cytometry. For confocal microscopy-based analysis of BMDC efferocytosis, cancer cells were subjected to the similar protocol, while BMDCs were stained with 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide (DiR) (D12731, Thermo Fisher Scientific) using a manufacturer’s instruction prior to co-culture for 4 h with treated cancer cells. After scrapping, cell suspensions were transferred to a new 12-well plate containing coverslips pretreated with poly-L-lysine, followed by incubation at 37°C for 1 h to allow cell attachment. The coverslips were then mounted on the slide glasses and imaged by confocal microscopy.

To assess if DAMP release from the treated cancer cells stimulates BMDCs, 4T1 cells were treated with PBS, Na+/Ca2+ solution, or BMNs for 24 h. Then, cell-free culture media were collected by centrifugation at 16,000g for 30 min. The cell-free media were added to completely differentiated BMDCs for 24 h at 37°C. The BMDCs were then treated with CD16/32 Fc blocker for 30 min on ice, followed by incubation with APC anti-mouse CD11c, PerCP/Cy5.5 anti-mouse I-A/I-E, FITC anti-mouse CD80, and APC/Cy7 anti-mouse CD86 antibodies for 1 h in the dark. The stained cells were analyzed by flow cytometry. Similarly, independent experiments were conducted to analyze the expression of type I interferons (Ifn-α, Ifn-β, and Cxcl10) and pro-inflammatory cytokines (Il6, Il12, and Tnf-α) in the treated BMDCs using RT-qPCR.

In vivo biodistribution of BMNs
To examine the biodistribution of BMNs, 5×105 4T1 cells were subcutaneously injected into the right flanks of 6-week-old BALB/c mice. When tumor size was about 200 mm3, tumor-bearing mice received IV injection of either free DiR solution or DiR-labeled BMNs at a DiR dose of 0.5 mg/kg. The fluorescence images were taken using a PerkinElmer IVIS Lumine III System at predetermined timepoints. At 24 h after injection, the mice were euthanized, and tumors and major organs were collected for ex vivo fluorescence imaging. The collected tumors were further treated with a digestive solution containing collagenase D (1 mg/mL) and DNase I (30 IU) to obtain single-cell suspensions. The single-cell suspensions were stained with various antibodies for flow cytometric characterization of BMN distribution across various cell types within the TME.

To investigate the effect of the lipid layering on the tumor accumulation of BMNs, we conducted another independent study in which tumor-bearing mice received PBS, bare CaCO3 particles, or BMNs at a dose of Ca2+ of 60 mg/kg. Bare CaCO3 particles were prepared by simple mixing of CaCl2 and Na2CO3 solutions in distilled water. At 24 h post-treatment, plasma, urine, tumors, and major organs were collected from the treated mice. Ca2+ ion concentrations in biofluids, tumors, and organs were measured using an inductively coupled plasma-optical emission spectrometer (ICP-OES) method.

Inductively coupled plasma-optical emission spectrometer (ICP-OES)
To detect the Ca2+ and Na+ contents in the cultured cells or mouse tissues were collected and washed 3 times with cold PBS followed by 3 times of washing with Ca2+- and Na+-free 5% dextrose solution. The collected cancer cells (~1−2×105 cells in 50 μL), biofluids (20 μL) or tissue samples (20-30 mg) were digested using 1 mL of the aqua regia solution (HNO3:HCl molar ratio 1:3) at 100oC for 2 h. Thereafter, the solutions were diluted up to 20 mL with deionized water in volumetric flasks. The concentrations of Ca2+ and Na+ ions in the solutions were measured using the Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES, Perkin Elmer OPTIMA8300, NFEC-2019-03-254855) at Core Research Support Center for Natural Products and Medical Materials at Yeungnam University. Na+ and Ca2+ ions were detected and measured at 589.592 nm for Na+ ions; and 317.933 nm for Ca2+ ions.

Antitumor effects of BMNs
In vivo prophylactic vaccination. 1×106 4T1 cells were cultured in T-25 flask (Corning, NY, USA) at 37°C with 5% CO2. The cells were treated with mitoxantrone (1.5 µg/mL), or BMNs (Na+: 3.75 µg/mL, Ca2+: 150 µg/mL) for 24 h. Thereafter, the treated cells in each flask were collected, washed briefly three times with 0.1 N lactic acid and then cold PBS, followed by resuspension in 100 µL cold PBS. The collected cells were used to immunize 6-week-old BALB/c mice by subcutaneous injection at tail bases. Each BALB/c mouse was two vaccinations with a 7-day interval between them. Seven days after the second vaccination, the treated mice were challenged with a subcutaneous injection of 3×105 4T1 living cells on their right flank. Tumor size was measured every other day using a digital caliper (CD-15CPX, Mitutoyo, Tokyo, Japan), and tumor volume was calculated using the following formula: volume = long×width2×0.5. To study the lymphatic transport of the dying 4T1 cells upon vaccination, cancer cells were stained with eBioscience™ Cell Proliferation Dye eFluor™ 670 (Thermo Fisher Scientific) following the manufacturer’s instruction. The fluorescently labeled, dying 4T1 cells were then subjected to the same procedure for vaccination as described above. At 24 h after injection, inguinal lymph nodes were collected from the animals. Fluorescence imaging was conducted with the harvested lymph nodes using the PerkinElmer IVIS Lumine III System, and the maturation state of dendritic cells in inguinal lymph nodes was analyzed using flow cytometry.

Assessment of murine tolerability of BMNs. 5×105 4T1 cells in 100 µL PBS were subcutaneously inoculated into the right flanks of 6-week-old BALB/c mice. When tumor volume reached a size of 70 mm3, tumor-bearing mice were randomly divided into 4 groups and received different doses (Na+: 0, 0.75, 1.5, 2.25 mg/kg) of BMNs on days 0, 3, and 6 of the study. The body weight and tumor volume of the animals were monitored every other day.

In vivo mouse tumor models and treatment with BMNs. 4T1 and CT26 tumors were established in BALB/c mice, while B16F10 tumors were developed in C57BL/6 mice as previously described. Once the tumors reached a volume of ~70 mm3 with 4T1 tumors (n=6) or 50 mm3 with CT26 (n=6) and B16F10 (n=5) tumors, the mice were treated with PBS, Na+/Ca2+ solution at a dose of Na+ of 1 mg/kg + Ca2+ of 40 mg/kg, or BMNs (Na+: 1.5 mg/kg + Ca2+: 60 mg/kg) on days 0, 3, 6 of the studies. On day 10, tumors and major organs were isolated from the treated mice for further analysis.

T cell blocking experiments. The 4T1 tumors were established in BALB/c mice as described above. Treatments started when tumors reached a volume of ~70 mm3 (n=6). 1 day before starting treatment with BMNs (marked as day -1), the mice received an IP injection of 100 µg anti-mouse CD8α antibody (Clone 2.43, BioXCell) for depletion of CD8+ T cell or 100 µg IgG (LTF-2, Bio X Cell) as an isotype control. The antibody treatments were given every 3 d, on days -1, 2, 5, and 8, while BMNs were intravenously administered on day 0, 3, and 6 (Na+: 1.5 mg/kg + Ca2+: 60 mg/kg) of the study. On day 10, tumor-bearing mice were euthanized to collect tumors. T cell depletion was confirmed in peripheral blood on day 7 and in the TME on day 10 of treatments.

Tumor antigen-specific responses. B16-OVA tumors were established by subcutaneously injecting 5×105 cells to the right flanks of the mice. When tumor volume reached ~50 mm3, tumor-bearing mice were randomly divided into three groups (n=8 per group) and administered treatment via IV injection on days 0, 4, and 7. The treatment groups were: PBS (Control), Na+/Ca2+ solution (Na+: 1.0 mg/kg + Ca2+: 40 mg/kg), and BMNs (Na+: 1.5 mg/kg + Ca2+: 60 mg/kg). On day 10, the treated mice were euthanized, and tumors, spleens, TDLNs, and peripheral blood were collected. To detect the OVA-specific CD8+ T cells, the single cells suspensions isolated from spleen, TDLNs, and blood were stained with PE-Labeled Mouse H-2Kb-OVA (SIINFEKL) Tetramer Protein (AcroBiosystem), followed by staining with appropriate surface marker antibodies. Cells isolated from OTI mice were used as positive controls for this assay. To examine tumor antigen-specific response of CD8+ T cells, cells isolated from tumor, TDLNs, and spleens were restimulated ex vivo with SIINFEKL (2 μg/mL, Vac-sin, Invitrogen) for 6 h in the presence of 1× Protein Transport Inhibitor Cocktail (004980, Invitrogen), followed by intracellular cytokine staining assays.

Assessment of calcineurin/NFAT signaling pathway
For in vitro treatments, activated splenic CD8+ or CD4+ T cells were treated with Na+/Ca2+ solution or BMNs at a dose of Ca2+: 150 μg/mL and Na+: 3.75 μg/mL for 24 h. The proteins from treated cells were fractionated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo Fisher Scientific). Cytoplasmic and nuclear proteins were then separated via SDS-PAGE, transferred to a PVDF membrane, and detected as described above. For intracellular cytokine staining, Stimulation Cocktail (00-4970, Invitrogen) and Protein Transport Inhibitor Cocktail (004980, Invitrogen) were added to each well at 1× working concentrations 6 h prior to cell collection. The cells were then stained with fluorophore-conjugated antibodies and analyzed using flow cytometry. For in vivo assessment, 4T1 tumors were established in BALB/c mice as previously described. When tumor volume reached 150-200 mm3, tumor-bearing mice were administered PBS (Control), Na+/Ca2+ solution (Ca2+: 40 mg/kg; Na+: 1 mg/kg), or BMNs (Ca2+: 60 mg/kg; Na+: 1.5 mg/kg). At 24 h post injections, tumors were collected to prepare single cell suspensions. Tumor-infiltrating CD8+ and CD4+ T cells were isolated using positive selection kits (18953 and 18952, STEMCELL Technologies). Isolated T cells were then restimulated, followed by intracellular cytokine staining for flow cytometry analysis.

Antitumor efficacy of combining BMNs with anti-PD1 antibody
In subcutaneous CT26 tumor-bearing mice. CT26 cells were inoculated to the right flanks of 6-week-old BALB/c mice. On day 6, mice were divided into 4 groups (n=6), and they received IV injections of PBS or BMNs (Na+: 1.5 mg/kg, Ca2+: 60 mg/kg) through on days 6, 9, and 12 and anti-mouse PD-1 antibody (aPD1) (BE0033-2, Bio X Cell) (10 mg/kg) through IP injections on days 7, 10, and 13. Tumor size was monitored every 2 d.

In orthotopic 4T1-luc tumor-bearing mice. 2×106 4T1-luc cells suspended in 50 µL fresh RPMI 1640 and Matrigel (354262, Corning) were inoculated into the 4th mammalian fat pad on the right side of 6-week-old BALB/c mice. On day 14, the mice were divided into four groups (n=5) and treated with BMNs alone or in combination with aPD1, with a total of three doses administered as previously described. Tumor volumes were measured every 2 d. On day 28 of the study, the mice were administered D-luciferin potassium salt (FL08608, Biosynth) at a dose of 150 mg/kg via IP injection. Fifteen min later, the mice were anesthetized using 2.5% isoflurane, and bioluminescence images were taken using a PerkinElmer IVIS Lumina III System. Tumors and major organs were then immediately isolated and subjected to ex vivo imaging. The images were analyzed using Living Image v. 4.7.3 software (Perkin Elmer, Waltham, MA, USA). The collected tumors and lungs were fixed in 10% neutral buffered formalin in PBS solution for further analyses. To assess the anti-metastasis effects of the combinatorial therapy on the spontaneous metastasis progression in the tumor-bearing mice, an independent study was conducted. On days 21, 28, 35, 42, 49, and 60, the mice were administered with D-luciferin at a dose of 150 mg/kg via IP injection for in vivo bioluminescence imaging. The survival rate of the mice was monitored until day 90 of the study. On day 180, the mice completely cured from the tumors (n=4) were rechallenged with 2×106 4T1-luc2 cells into the 4th mammalian fat pad on the left side to evaluate the development of memorial antitumor immunity. The naïve BALB/c mice (n=6) were used as control.

Enzyme-linked immunosorbent assay (ELISA)
Concentrations of various proteins in cell culture media or mouse plasma were assayed using ELISA according to the manufacturers’ protocols: IFN−β (Mouse IFN-β DuoSet ELISA kit, R&D Systems), CXCL10 (Mouse CXCL10 DuoSet ELISA kit, R&D Systems), HMGB1 (HMGB1 ELISA Kit, ABclonal).

Histopathology, immunohistochemistry, and immunofluorescence
For hematoxylin and eosin staining, tumor tissues and major organs were processed by an automated tissue processor (Shandon Citadel 2000, Thermo Fisher Scientific). Then, 4 µm sections were prepared using a microtome (RM2255, Leica Biosystems) and stained with hematoxylin and eosin solution.

For immunohistochemistry, 4 µm sections were embedded in MeOH and 0.03% H2O2 solution for 30 min to eliminate any endogenous peroxidase activity. Next, epitope retrieval was conducted before non-specific IgG binding was blocked using normal horse serum. The sections were then incubated with primary antibodies at 4°C overnight in humidity chambers, followed by incubating with biotinylated universal secondary antibody and VECTASTAIN® ELITE® ABC Kit (PK-6200, Vector Lab, CA, USA) for 1 h. Finally, the stained sections were incubated with a peroxidase substrate (SK-4100, Vector Lab, CA, USA) and observed under a light microscope (Nikon Eclipse 80i, Japan). An automated analyzer was used to process the images. Information of the antibodies is provided in Supplementary Table 9.

For immunofluorescence, 4 µm sections of lung tissues were processed as described above. These sections were then subjected to heat-induced antigen retrieval followed by permeabilization in MeOH for 10 min. After blocking non-specific binding, the sections were stained with an anti-luciferase antibody (L0519, Sigma-Aldrich) at 4°C overnight. Subsequently, the stained sections were incubated with Alexa Fluor 555-conjugated anti-rabbit IgG for 1 h at room temperature and counterstained with 4’,6-diamidino-2-phenylindole (DAPI). The sections were then imaged by fluorescent microscopy and analyzed using an automated imaging analyzer.

Flow cytometry
Single-cell suspensions were prepared from tumors, spleens, tumor-draining lymph nodes, and mouse blood by appropriate protocols. For blood samples, 50 µL blood was placed in 1.5 mL microcentrifuge tubes, and red blood cells (RBCs) were removed by adding 950 µL 1X RBC lysis buffer (420301, BioLegend) for 15 min at room temperature. For spleens and lymph nodes, the samples were ground using plungers of 1 mL syringe and subjected to the RBC removal protocol. For tumors, collected specimens were cut into small pieces, ground, and suspended in a digestive solution containing 1 mg/mL collagenase D (11088866001, Roche) and 30 IU DNase I (11284932001, Roche) for 1 h at 37°C with shaking. The digested tissues were passed through a 70 µm cell strainer (352350, Corning Life Sciences), and RBCs were removed. The prepared single-cell suspensions were incubated with CD16/32 Fc blocker for 30 min on ice, followed by staining with fluorophore-conjugated antibodies for 1 h in the dark. For staining intracellular proteins, such as Foxp3, IFN-γ, and TNF-α, the cells were fixed and permeabilized using the Foxp3 Transcription Factor Staining Buffer set (00-5523-00, Thermo Fisher Scientific) according to the manufacturer’s protocol before staining. For staining intracellular cytokines, the cells were stimulated for 6 h with Cell Stimulation Cocktail (00-4970, Invitrogen) and Protein Transport Inhibitor Cocktail (004980, Invitrogen) before staining the intracellular proteins. The cells were then analyzed using FACS Verse (BD Bioscience, San Jose, CA, USA) or Cytek Aurora flow cytometer (Cytek Biosciences, NFEC-2025-09-308501), and the results were analyzed using FlowJo v. 10 software (Supplementary Fig. 50). Information about the antibodies is provided in Supplementary Table 10.

Reverse transcription quantitative PCR
Total RNA from cultured cells or tumor cells was extracted using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Complementary DNAs (cDNAs) were synthesized from the extracted RNA using the GoScript™ Reverse Transcription kit (Promega). Expressions of the specific genes were evaluated using TOPreal™ SYBR Green PreMIX (Enzynomics, South Korea). The reactions were run on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, CA, USA). The gene expressions were normalized to the gene expression of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) by a ΔΔCt method. Information about the primer sequences is provided in Supplementary Table 11.

Statistics and reproducibility
Unless otherwise specified, all experiments were repeated at least three times to ensure that similar results were reproducible. Similar results were obtained from at least three experiments for Figs. 1g, h; and 4g, h. Data are presented as the mean±SD with the indicated sample size. Data were analyzed using one- or two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. The statistical comparisons between 2 groups were analyzed by unpaired two-tailed Student’s t-test. Survival rates between groups were compared using the log-rank (Mantel-Cox) test. All statistical analyses were conducted by GraphPad Prism v. 8.4 software. Differences were considered statistically significant when p value was less than 0.05. Error bars, p values, n numbers and statistical tests used are reported in the figures or figure legends. No statistical method was used to predetermine sample size. Samples were randomly allocated into experimental groups. The investigators were not blinded to allocation during experiments and outcome assessment.

Ethics statement
Female BALB/c mice and C57BL/6 mice, aged 5-6 weeks, were obtained from Yeungnam Bio Inc. (South Korea) and used for all in vivo experiments. All animal experiments were carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) and the Institutional Authority for Laboratory Animal Care and Handling at Yeungnam University (Approval numbers: 2022-017, 2023-023, 2023-032, 2023-033). A tumor volume of 2000 mm3 was defined as the humane endpoint euthanasia. Animals were euthanized immediately if they exhibited any signs of distress, including but not limited to severe weight loss, ulceration, or a moribund state. In selected cases, and only when animals maintained normal body weight, activity, and showed no clinical signs of distress, tumor volumes were allowed to temporarily exceed 2000 mm3 under prior IACUC approval, solely for the purpose of evaluating the durability of antitumor efficacy and long-term therapeutic responses (Fig. 3g, Supplementary Fig. 27 and 44). Throughout the study, animal welfare was continuously monitored, and clinical condition-based humane endpoints were strictly prioritized over tumor size alone.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This interesting and novel study introduces binary mineral nanoparticles (BMNs) engineered via microfluidics to co-deliver\ncalcium and sodium ions to tumors, hence inducing immunogenic cell death to promote antitumor immunity without the need\nfor cytotoxic drugs. The most notable innovation lies in the scalable, pH-sensitive nanoparticle design, which enables stable\nsystemic delivery of dual ions, and effective disruption of ion homeostasis in cancer cells to synergize with immune\ncheckpoint inhibitors to suppress tumor growth and metastasis. The authors provided a thorough and well-structured point-\nby-point response to all reviewer comments, which is deemed a commendable effort. The authors responded positively to\nsuggestions such as adding microfluidic platform context, improving imaging resolution, and quantifying fluorescence\nintensities. Some minor points are noted, as detailed below:\n1. A more elaborated limitation section should be in place to reflect these points. In addition, the authors openly\nacknowledged limitations (e.g., lack of in vivo ICD marker validation, absence of tumor antigen specificity data) and\nproposed future experiments to address them.\n2. While the responses were detailed, some explanations, particularly those involving complex nanoparticle formation\nmechanisms and ion release kinetics, could benefit from clearer visual aids or simplified summaries.\n3. Several critical experiments (e.g., immune cell-specific cytotoxicity, PD-1/PD-L1 expression, tumor antigen-specific T cell\nresponses) were deferred to future revisions. Including at least one piece of data on these fronts would have strengthened\nthe manuscript’s impact.", "reviewer_2": "The authors have addressed the previous reviews.\nI recommend completing the remaining studies during the revision process, as the authors stated in the Authors' Response\npages.", "reviewer_3": "The authors have inadequately addressed the concerns, many of which could be nailed by some easy experiments, but\nauthors did not. Therefore, I am not entirely convinced that the current manuscript could be accepted by Nat. Commun.\nJust name a few..\nCytotoxicity of various immune cells (at least in vitro) induced by BMNs were not investigated, which should be easy to\nsupplement.\nThe metal ion concentrations throughout the text should be quantified by ICP. The quantification using fluorescent probes for\nthe minor variation in the specific context here is not convinced by the reviewer.\nThe current data cannot strongly support that BMNs are layer-by-layer assembled (Fig. 1h and Supplementary Fig. 11)."}, "author_response": "A Response Letter for the Manuscript titled:\nBinary Mineral Nanoparticles Enable Intravascular Delivery of Metal Ions to\nTumors for Metalloimmunotherapy\nWe sincerely appreciate the Editor for giving us an opportunity to submit our revised\nmanuscript to the journal. We have made efforts to address all points raised by the\nreviewers in both the previous and current reviews. In this letter, to respond to the\ncomments from each Reviewer, we present the answers for the questions in this\nrevision followed by the additional experiments which were deferred in the previous\nrevision. The changes we have made are highlighted in the revised manuscript. Our\npoint-by-point responses are provided below along with the line number corresponding\nto each response.\nReviewer #1 (Remarks to the Author):\nThis interesting and novel study introduces binary mineral nanoparticles (BMNs)\nengineered via microfluidics to co-deliver calcium and sodium ions to tumors, hence\ninducing immunogenic cell death to promote antitumor immunity without the need for\ncytotoxic drugs. The most notable innovation lies in the scalable, pH-sensitive\nnanoparticle design, which enables stable systemic delivery of dual ions, and effective\ndisruption of ion homeostasis in cancer cells to synergize with immune checkpoint\ninhibitors to suppress tumor growth and metastasis. The authors provided a thorough\nand well-structured point-by-point response to all reviewer comments, which is\ndeemed a commendable effort. The authors responded positively to suggestions such\nas adding microfluidic platform context, improving imaging resolution, and quantifying\nfluorescence intensities. Some minor points are noted, as detailed below:\nWe thank the Reviewer for your comments on our manuscript. We have\nprovided point-to-point response to your minor concerns below.\nR.1.1. A more elaborated limitation section should be in place to reflect these points.\nIn addition, the authors openly acknowledged limitations (e.g., lack of in vivo ICD\nmarker validation, absence of tumor antigen specificity data) and proposed future\nexperiments to address them.\nWe thank the Reviewer for bringing these important aspects to our attention.\nWe have conducted additional experiments and revised the manuscript to thoroughly\naddress these points.\nFirst, we conducted additional experiments to examine tumor antigen-\nspecificity in the tumor-bearing mice by utilizing the B16-OVA melanoma model (Fig.\n5j). We found that BMN treatment effectively promoted the cross-presentation of the\nantigen (SIINFEKL) by DCs in the TDLNs (Fig. 5l, m). This finding is further\ncorroborated by the observation that tetramer+CD8+ T cells were detected in the\nperipheral blood and lymphoid organs (spleen and TDLNs) of BMN-treated mice,\nclearly demonstrating an improved recognition of the tumor antigen. Additionally, the\nantigen-specific cell functional response was confirmed using ex vivo restimulation\nwith the SIINFEKL peptide. As expected, CD8+ T cells isolated from the tumors and\nsecondary lymphoid organs of BMN-treated mice showed a significant enhancement\nin IFN-γ secretion (Fig. 5s-v and Supplementary Fig. 43). Collectively, these results\nstrongly support our hypothesis that BMNs effectively induce ICD in cancer cells,\nthereby leading to the reinitiation of the cancer-immunity cycle in the treated animal\nmodel. We have presented these tumor antigen specificity data in Figure 5j-v and\nSupplementary Figures 42 and 43, with the corresponding text incorporated into the\nResults section as follows:\nLine 397:\n‘…Antigen-specific immune recognition was also evaluated in BMN-treated, tumor-\nbearing mice using the B16-OVA tumor model (Fig. 5j). Consistent with results\nobtained in other tumor models, BMNs treatment effectively inhibited the growth of\nB16-OVA tumors (Fig. 5k and Supplementary Fig. 42). Moreover, the flow cytometry\nanalysis of the tumor draining lymph nodes (TDLNs) revealed both a higher ratio of\nSIINFEKL-H Kb+ DCs and an improvement of DC maturation state in the BMN group\n2\n(Fig. 5l-m and Supplementary Fig. 43). This indicates that the BMN treatment\neffectively improved the presentation of tumor antigens by APCs in the treated mice.\nAdditionally, the BMN group exhibited a significant increase in the frequencies of\nantigen-specific CD8+ T cells in both the peripheral blood (Fig. 5o, 5p and\nSupplementary Fig. 43) and secondary lymphoid organs, specifically the TDLNs and\nspleen (Fig. 5q, 5r and Supplementary Fig. 43) compared to the control and Na+/Ca2+\nsolution groups. Furthermore, following ex vivo restimulation with SIINFEKL peptide,\nthe INF-γ secretion of CD8+ T cells was significantly improved in the tumor, spleen,\nand TDLNs of BMN-treated mice compared to the other groups (Fig. 5s-v and\nSupplementary Fig. 43). This collectively demonstrates the superior effect of BMN\ntreatment in inducing robust, tumor antigen specific immunity in the treated tumor-\nbearing mice…’\nSecond, we validated the expression levels of ICD markers, such as CRT and\nHMGB1, in the in vivo study using IHC. The results are presented in Supplementary\nFig. 36 and Table 4, and the corresponding text has been revised in the manuscript as\nfollows:\nLine 341:\n‘…Consistent with this finding, the expression of ICD markers, such as CRT and\nHMGB1, was also increased when the tumor was treated with BMNs in vivo\n(Supplementary Fig. 36 and Supplementary Table 4)…’\nR.1.2. While the responses were detailed, some explanations, particularly those\ninvolving complex nanoparticle formation mechanisms and ion release kinetics, could\nbenefit from clearer visual aids or simplified summaries.\nWe thank the Reviewer for this suggestion. As per the Reviewer’s suggestion,\nwe have added an illustration and chemical reactions depicting the NP core formation\nin Supplementary Fig. 4. We also have expanded our discussions about the\nincorporation of Na+ ions in the BMN cores. However, due to the word count\nconstraints required by the Journal, we have placed this detailed information, along\nwith the supporting data, in Supplementary Information (Supplementary Discussion\n1,2).\nLine 98:\n‘…To synthesize BMNs consisting of both calcium (Ca2+) and sodium ions (Na+), we\nadapted a conventional gas-diffusion method (Supplementary Fig. S1). The\nmodification involved using NaOH as an alkalinizing agent during the NP core\npreparation. Optimization processes are detailed in Supplementary Information\n(Supplementary Discussion 1)…’\nLine 105:\n‘…Analyzing the concentrations of free ions in the reaction mixture, in combination\nwith dynamic light scattering (DLS) size measurement, revealed that Na+ ions were\nincorporated concomitantly with the growth of the NP core (Supplementary Fig. 4).\nMoreover, the stirring conditions significantly influenced both the growth of the NP core\nand the rate of ion incorporation. A detailed explanation of the proposed BMN core\nformation mechanism is provided in Supplementary Information (Supplementary\nDiscussion 2)…’\nR.1.3. Several critical experiments (e.g., immune cell-specific cytotoxicity, PD-1/PD-\nL1 expression, tumor antigen-specific T cell responses) were deferred to future\nrevisions. Including at least one piece of data on these fronts would have strengthened\nthe manuscript’s impact.\nWe are pleased to provide additional data as recommended by the Reviewers.\nTo examine BMNs’ cytotoxicity against immune cells, we isolated various immune cell\npopulations from mouse, including BMDMs – BMDCs, CD4+ T cells, and CD8+T cells\n– and conducted MTS assays following BMN treatment. As shown in Supplementary\nFig. 17, most cell types tolerated to BMN treatment within its working concentration\nrange. BMNs induced significant toxicity on these immune cells only when treated at\nan extremely high concentration (i.e., Ca+: 2,000 µg/mL, Na+: 50 µg/mL)\n(Supplementary Fig. 17), a dose substantially higher than the IC observed in 4T1\n50\ncells. This suggests a relative safety profile for BMNs across various immune cell types.\nWe have added a brief discussion detailing these findings as follows:\nLine 198\n‘…Additionally, using flow cytometry, we found pronounced elevations in mean\nfluorescence intensity (MFI) in most cell types studied, with the exception of\ngranulocytic myeloid-derived suppressor cells (CD11b+Ly6G+SSChigh) and neutrophils\n(CD11b+Ly6G+SSClow) (Fig. 2d). The slight increase in DiR fluorescence within T cell\nsubpopulations did not reach statistical significance (Fig. 2d, and 2e). While such\naccumulations might suggest a potential risk of immune cell side effects, this concern\nis mitigated by previous studies demonstrating the safety of structurally similar metal\ncompounds. For instance, NaCl nanoparticles have been shown to be safe with\nmacrophages.23 Furthermore, tumor-infiltrating T cells were demonstrated to well\ntolerate treatments of both calcium-based nanoparticles and high doses of NaCl, even\nwhen these interventions significantly increased the intracellular ion\nconcentrations.39,40 Consistent with these findings, BMN treatment showed negligible\ncytotoxicity in macrophages, DCs, and T cell subpopulations in our research\n(Supplementary Fig. 17), supporting the inherent tolerability of these immune cells to\nBMN treatment…’\nWe also analyzed the expression of PD1 and PD-L1 in the tumors collected\nfrom the in vivo study of the BMN/aPD1 combination therapy. As shown in the\nSupplementary Fig. 46 and Table 5, BMN treatment alone significantly reduced the\nexpression of both PD1 and PD-L1 in the tumor tissues. Notably, this reduction was\neven more significant in the BMN/aPD1 combination group.\nLine 430\n‘…Interestingly, the combination treatment also remarkably suppressed the\nexpression of both PD1 and PD-L1 in the tumor tissues of the treated mice\n(Supplementary Fig. 46 and Supplementary Table 5)…’\nFor the tumor antigen-specific T cell responses, please refer to our answer to\nR1.1.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed the previous reviews.\nI recommend completing the remaining studies during the revision process, as the\nauthors stated in the Authors’ Response pages.\nWe thank the Reviewer for giving us the opportunity to further improve our\nmanuscript. We are pleased to provide the additional data required for the remaining\nstudies that we committed to in the previous round of peer review. For clarity in this\nrevision, we have included the relevant comments from the previous round, followed\nby our corresponding detailed responses and data updates.\nR.2.1. (From R.2.1. of the previous review) The authors incorporated phospholipids\ncontaining an acid-cleavable bond to impart tumor-responsive properties to their NPs.\nTo validate the tumor responsiveness of BMNs, an additional experiment should be\nconducted by constructing control NPs incorporating phospholipids without the acid-\ncleavable bond and comparing tumor-specific ion accumulation between the control\nNPs and BMNs.\nWe thank the Reviewer for their comment. We conducted an additional in vitro\nrelease experiment to investigate the role of DMM-DOPE, the phospholipid containing\nan acid-cleavable amide bond. While CaCO -based NPs are well-established in the\n3\nliterature to possess an inherent pH-responsive release nature, our NPs also exhibited\nthis property, even without DMM-DOPE. Notably, the incorporation of DMM-DOPE into\nthe lipid layers significantly enhanced this pH-dependent release pattern\n(Supplementary Fig. 12). A brief discussion addressing this point was added to the\nmanuscript as follows:\nLine 152\n‘…Additionally, the BMNs displayed higher release rates for both ions in acidic\nconditions compared to physiological pH conditions. This underscores the pH-\nresponsive nature of the BMNs, which is attributable to the intrinsic properties of both\nCaCO and DMM-DOPE (Fig. 1i, 1j and Supplementary Fig. 12)…’\nto be an overall increase in T cell, NK cell, and APC populations within the TME, while\napoptosis markers are also elevated. Could this indicate that apoptosis is being\ninduced in immune cells? As mentioned earlier, the results in Fig. 2e show that NP\nuptake in cancer cells is minimal compared to other cell types. Can the authors\nexperimentally demonstrate that BMNs induce apoptosis specifically in cancer cells,\nas hypothesized, rather than in other cells within the TME?\nAs previous mentioned, we conducted additional experiments to address this\nimportant point. For the detailed results and analysis regarding the cytotoxicity of\nBMNs in different immune cells, please refer to our answer to R.1.3.\nR.2.3. (From R.2.8. of the previous review) In Figs. 3i and j, as well as Supplementary\nFig. 25, what are the percentages of CD8 and CD4 T cells within the CD45+ cell\npopulation? Additionally, how does the overall percentage of CD3+ T cells within the\nCD45+ cell population change?\nWe thank the Reviewer for this valuable suggestion. To investigate the ratio of\nT cell subpopulations within CD45+ cells, we repeated an in vivo study of 4T1 tumor\nmodel. Flow cytometry analysis showed that BMN treatment enhanced the ratios of\nCD45+CD3+ T cells in the TME (Supplementary Fig. 29a-b). Moreover, the ratios of\nboth CD4+ and CD8+ T cells within the CD45+ population were also increased,\ndemonstrating the potential of BMNs to favorably reprogram the immune\nmicroenvironment of treated tumors (Supplementary Fig. 29c-e).\nBesides these immunological findings, we also collected data on tumor volume\nand tumor weight (Fig. R2.1). The results obtained were completely similar to those of\nour initial experiment, effectively demonstrating the reproducibility of our findings.\nHowever, to avoid confusing readers, we have decided to keep these data from the\nnew experiment solely within this response letter.\nFigure R2.1. The antitumor effect of BMNs in 4T1 tumor model. (a) Individual\ntumor growth in the groups treated with indicated formulations. (b) Average tumor\ngrowth curves of different groups. (c) Photograph and (d) weight of tumors collected\nfrom treated mice on day 10 of the study. (e) Body weight of tumor-bearing mice over\ntime of the study. (1) Control (PBS), (2) Na+/Ca2+ solution, (3) BMNs. Data are\npresented as mean ± SD with n=8. (b) Two-way ANOVA with Tukey’s multiple\ncomparison tests. (d) One-way ANOVA with Tukey’s multiple comparison tests.\nR.2.4. (From R.2.11. of the previous review) Please confirm the ICD marker\nmeasurements performed in cancer cells in Fig. 4 and Supplementary Fig. 28 in tumor\ntissue as well. Specifically, to support the authors’ hypothesis, could you examine ICD\nmarkers in “cancer cells” within the TME?\nWe thank the Reviewer for your suggestion. As previously noted, we are\npleased to provide the data of the ICD marker expression in the tumor tissues, which\nwe assessed via IHC. For detailed information, please refer to our answer to R1.1.\nAdditionally, investigating the cancer cell-specific expression of ICD markers\nwithin tumor sections typically requires multiplex IHC. We regret to inform you that we\nwere unable to conduct the multiplex IHC experiments for this revision due to the\ncombined constraints of time and the technical challenge of building a reliable antibody\npanel.\nR.2.5. (From R.2.12. of the previous review) In the TME analysis presented in Fig. 3,\nthe authors only show changes in T cell populations. However, the results in Fig. 2e\nsuggest that BMNs may also affect other cell types. Please conduct a more detailed\nTME immune profiling to show how the immune cell populations change. In particular,\nbased on the results in Fig. 2e, there may be changes in the tumor-associated\nmacrophage (TAM) population and phenotype.\nWe thank the Reviewer for this insightful suggestion. As requested, we\nconducted additional experiments to analyze the expression of the CD206 protein, a\ncanonical marker for M -like macrophages, using different analytical methods. Both\n2\nflow cytometry and IHC analysis confirmed a reduction of CD206 expression in tumors\ntreated with BMNs compared to the control and Na+/Ca2+ solution groups\n(Supplementary Fig. 28, 29, and Table 3). This result supports the effect of BMNs in\nfavorably reprogramming the immune conditions of TME of treated mice. We have\nincluded a detailed discussion of this result in the revised manuscript as follows:\nLine 284\n‘…Of note, tumors treated with BMNs exhibited a reduced expression of CD206 – a\ncanonical marker of M -like tumor-associated macrophages (TAMs) – compared to the\n2\ncontrol (Supplementary Fig. 28 and Supplementary Table 3). This finding suggests a\nfavorable shift toward reduced incidence of tumor resistance. Consistent with the IHC\nresults, flow cytometric analysis of the TME demonstrated that BMN treatment\nincreased the infiltration of CD45+CD3+ T cells in the TME, including both CD4+\nand CD8+ T cells (Fig. 3h-j and Supplementary Fig. 29)…’\nLine 307\n‘…Additionally, the TME of the BMN group showed an increased ratio of\nCD11c+MHCII+CD80+CD86+ DCs, indicating a higher rate of DC maturation\n(Supplementary Fig. 29). Concurrently, a lower expression of CD206 marker was\nobserved, suggesting a desirable reduction in M -like TAMs (Supplementary Fig. 29)…’\nadditional experiments, such as ELISA, to quantify the amount secreted, rather than\nrelying solely on confocal microscopy imaging.\nWe thank the Reviewer for the suggestion to enhance the rigor of our data. We\nare pleased to provide the additional ELISA assay results quantifying the extracellular\nconcentration of HMGB1 secreted by 4T1 cells following different regimens. In\nagreement with the results of other ICD markers, the Na+/Ca2+ solution or low dose of\nBMNs did not induce significant HMGB1 secretion to the extracellular space.\nConversely, the high dose of BMNs superiorly triggered the release of the HMGB1\nprotein (Fig. 4d). The discussion of this result has been added to the revised\nmanuscript as follows:\nLine 336\n‘…Notably, the BMN treatment decreased the nuclear levels of high mobility group box\n1 (HMGB1) while simultaneously upregulating its level in the extracellular space (Fig.\n4d and Supplementary Fig. 35). This release of HMGB1 from the nucleus is indicative\nof ICD, as the protein is passively secreted from dying cells during this process.48,49\nConsistent with this finding, the expression of ICD markers, such as CRT and HMGB1,\nwas also increased when the tumor was treated with BMNs in vivo (Supplementary\nFig. 36 and Supplementary Table 4).…’\nR.2.7. (From R.2.17. of the previous review) The BMN and aPD-1 combination\nappears to significantly inhibit tumor growth. To explain this antitumor efficacy, have\nthe authors assessed the expression of PD-1 or PD-L1?\nWe thank the Reviewer for the insightful comment. Please refer to our answer\nto R.1.3.\nReviewer #3 (Remarks to the Author):\nThe authors have inadequately addressed the concerns, many of which could be\nnailed by some easy experiments, but authors did not. Therefore, I am not entirely\nconvinced that the current manuscript could be accepted by Nat. Commun. Just name\na few..\nWe sincerely appreciate the Reviewer’s continued commitment to ensuring the\nrigor and quality of our manuscript. This time, we believe the substantial new data,\nalongside our revised interpretations, have significantly strengthened the paper. We\nearnestly hope the Reviewer will re-evaluate the manuscript in light of this new\nevidence and concur that the manuscript has substantially improved.\n3.1. Cytotoxicity of various immune cells (at least in vitro) induced by BMNs were not\ninvestigated, which should be easy to supplement.\nThis important point regarding the potential cytotoxicity of BMNs against\nimmune cells has now been addressed with the inclusion of new data. For detailed\ninformation, please refer to our answer to R.1.3.\n3.2. The metal ion concentrations throughout the text should be quantified by ICP. The\nquantification using fluorescent probes for the minor variation in the specific context\nhere is not convinced by the reviewer.\nWe thank the Reviewer for the recommendation to use ICP for metal ion assay.\nIn this revision, we used an inductively coupled plasma – optical emission\nspectrometer (ICP-OES) to measure the ion concentrations in the biological samples.\nFor cultured 4T1 cells, as shown in Fig. 1m and 1n, Na+/Ca2+solution treatment did\nnot change the levels of ions inside the treated cells. In contrast, BMNs dramatically\nelevated the content of both ions inside the cells by 30-folds of Ca2+ ion and 5-folds of\nNa+ ion.\nFurthermore, the biodistribution assays were repeated and the ion contents in\ndifferent organs of treated mice were measured via ICP-OES. Similarly with the\nprevious data, we obtained higher levels of calcium and sodium ions in the tumor\ntissues of BMN-treated mice compared to other treatment groups. In contrast, Ca2+\nand Na+ contents in other organs rarely changed, except in the case of lung of mouse\ntreated with the bare CaCO particle (Fig. 4f-m).\nfor the ICP-OES method was included in the Method as follows:\nLine 232, Supplementary Information\n‘…To detect the Ca2+ and Na+ contents in the cultured cells or mouse tissues were\ncollected and washed 3 times with cold PBS followed by 3 times of washing with Ca2+-\nand Na+-free 5% dextrose solution. The collected cancer cells (~ 1-2 ´ 105 cells in 50\nµL), biofluids (20 µL) or tissue samples (20-30 mg) were digested using 1 mL of the\naqua regia solution (HNO :HCl molar ratio 1:3) at 100oC for 2 h. Thereafter, the\n3\nsolutions were diluted up to 20 mL with deionized water in volumetric flasks. The\nconcentrations of Ca2+ and Na+ ions in the solutions were measured using the\nInductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES, Perkin Elmer\nOPTIMA8300, NFEC-2019-03-254855) at Core Research Support Center for Natural\nProducts and Medical Materials at Yeungnam University. Na+ and Ca2+ ions were\ndetected and measured at 589.592 nm for Na+ ions; and 317.933 nm for Ca2+ ions…’\n3.3. The current data cannot strongly support that BMNs are layer-by-layer assembled\n(Fig. 1h and Supplementary Fig. 11).\nWe appreciate the Reviewer for the sharp distinction regarding the assembly\nmethodology.\nThe LbL assembly during the BMN synthesis is supported by DLS analysis\n(Supplementary Fig. 6). As the NP core underwent the initial DOPA layering, we\nobserved a slight size increase accompanied by a small attenuation of the positive\nzeta potential. Subsequent complexation of the initial DOPA layer with other lipid\ncomponents resulted in the neutralization, and even anionization, of the zeta potential,\nwhich strongly suggests the successful formation of the lipid layer on the BMN surface.\nWe recognize that our process is a modified LbL assembly, which utilizes the principle\nof sequential coating. Specifically, the second step involves the complexation of the\ninitial DOPA anchoring layer with a bulk lipid solution. While this is not a strict,\nalternating LbL deposition, the process is sequential, relying on the DOPA layer to\npromote the subsequent self-assembly of the remaining lipid components onto the\ncore.\nIn this revision, we have further strengthened this point using imaging data.\nWe provided the higher resolution TEM images of BMNs, which allow for the detection\nof the lipid layer covering the NP surface (Fig. 1g). We also included supplementary\nTEM image with a broader field of view (Supplementary Fig. 11b) to give a clearer\nobservation of BMN morphology. For comparative context, we added the TEM image\nof uncoated NP cores (Supplementary Fig. 11a), which resembles published TEM\nimages of CaCO NPs. By comparing these images, we conclude that the low-contrast\n3\nlining observed around the NPs is highly likely to represent the lipid layers assembled\nvia the LbL process. The corresponding information was added to Method section.\nLine 56, Supplementary Information\n‘…Morphology and elemental mapping of BMNs were characterized by using a\ntransmission electron microscopy (TEM) (H-7600, Hitachi) and field emission\ntransmission electron microscope (FE-TEM) equipped with elemental energy-\ndispersive spectroscopy (EDS) (Titan G2 80-200, ChemiSTEM Cs Probe, Fei\nCompany), respectively…’\n3.4. (From R.3.4. of the previous review) Since BMNs were found to accumulate in\nvarious immune cells, as shown in Figures 4d and 4e, measuring the subsequent Ca²\n⁺ and Na⁺ levels in affected cells/tissue is essential to assess the direct impact of\nthese nanoparticles on immune cell function. Additionally, it would be helpful to\nanalyze Na⁺ level changes in tumor cells treated with BMNs to further understand\ntheir influence on tumor cell physiology. One caveat about Ca2+ is that overactivated\nCa/NFAT signaling in T cells tend to cause CD4 T cell anergy and CD8 T cell\nexhaustion, which could sabotage anti-tumor immunity. These caveats should be\naddressed experimentally.\nWe sincerely appreciate the Reviewer for raising this critical point regarding\nthe potential effect for BMN treatment to disrupt Ca2+ homeostasis in tumor infiltrating\nimmune cells.\nWe first want to confirm that the general cytotoxicity profile of BMNs against\nvarious immune cell populations is presented. For detailed information, please refer to\nour answer to R.1.3.\nAs per the Reviewer’s suggestion, we conducted additional, targeted\nexperiments to directly address the risk of T cell exhaustion. We collected and\nactivated splenic CD8+ and CD4+ T cells and exposed them with different treatment\nfor 24 h (Supplementary Fig. 30a). Western blot analysis showed no significant change\nin the nuclear or cytoplasmic expression of the NFAT1 protein either CD8+ or CD4+ T\ncells (Supplementary Fig. 30b-c and 30i-j). This strongly indicates that BMN treatment\ndoes not affect the nuclear translocation of NFAT1 protein. Intracellular cytokine\nstaining revealed that T cells in the BMN-treated group showed an improved secretion\nof IL-2, IFN-γ, and TNF-α (Supplementary Fig. 30d-g and 30k-n). Furthermore, BMN\ndid not elevate PD1 expression on the T cell surface (Supplementary Fig. 30h and\n30o).\nWe also characterized the function of tumor-infiltrating T cells following BMN\ntreatment in the in vivo tumor model (Supplementary Fig. 31a). Similarly, we found no\nsignificant change in PD1 expression, but observed significantly elevated levels of\nintracellular cytokines (Supplementary Fig. 31b-k). (We not that due to limited sample\nsize, Western blot analysis was not conducted using in vivo samples). We believe that\nthese newly added results decisively demonstrate that BMN treatment is unlikely to\noveractivate the Ca2+/NFAT signaling pathway in a way that induces CD8+ T cell\nexhaustion or CD4+ T cell anergy.\nIn addition, BMN treatment was found to facilitate the maturation of DCs and\nconcurrently reduce the M -like macrophage populations within the TME.\nmanuscript as follows:\nLine 294\n‘…The elevated Ca2+ ion concentration in the TME, while otherwise promising, poses\na risk to T cell function. Specifically, it may induce the overactivation of the\ncalcineurin/NFAT signaling pathway in tumor-infiltrating T cells.44 This pathway drives\nCD8+ T cell exhaustion and CD4+ T cells anergy, marked by PD-1 overexpression and\nseverely reduced secretion of crucial cytokines such as IFN-γ, TNF-α, and IL2,\nultimately hindering T cell-mediated antitumor activity.45–47 However, in vitro treatment\nwith BMNs did not elevate the nuclear translocation of NFAT1 protein in either CD8+\nor CD4+ T cells, nor did it suppress the secretion of intracellular cytokines and IL2\n(Supplementary Fig. 30). Consistent with these finding in vitro, BMNs also did not\nsuppress the intracellular cytokine secretion of tumor-infiltrating T cells or increase the\nexpression of PD-1 marker 24 h after in vivo treatment (Supplementary Fig. 31)…’\nLine 307\n‘…Additionally, the TME of the BMN group showed an increased ratio of\nCD11c+MHCII+CD80+CD86+ DCs, indicating a higher rate of DC maturation\n(Supplementary Fig. 29). Concurrently, a lower expression of CD206 marker was\nobserved, suggesting a desirable reduction in M -like TAMs (Supplementary Fig. 29)…’\n2\n3.5. (From R.3.7. of the previous review) Figure 4 shows that only BMNs with a high\ndose of Na⁺ can initiate ICD and DAMP release. The authors should clarify the role of\nCa2+ in BMNs regarding these effects and whether a sodium-only nanoparticle could\nachieve similar results. This would provide insight into the necessity of Ca2+ in the\nformulation.\nWe thank the Reviewer for their comment. We acknowledge that the\npreparation of a stable, sodium-only nanoparticle is technically challenging due to the\nvery high water solubility of sodium salts. Therefore, to clarify the individual effect of\nNa+ ions, we attempted to use an NaCl solution at an extremely high concentration as\na proxy for a Na+-only treatment.\nWe first determined the IC value of the NaCl solution in 4T1 cells using an\nNaCl solutions at various concentrations (ranging from 25% to 200% of the IC value).\n(Fig. R3.1b-c). NaCl solutions significantly decreased the intracellular ATP level once\n4T1 cells were treated at a concentration of 200% IC (Fig. R3.1d). Interestingly, NaCl\n50\ntreatments did not upregulate the levels of extracellular HMGB1 protein (Fig. R3.1e).\nWe interpret the effect of the NaCl solution on releasing intracellular molecules (such\nas ATP and HMGB1) as primarily resulting from a cell lysis effect due to the extremely\nhigh concentration (Fig. R3.1f). Based on these in vitro results, we concluded that the\nNaCl solution treatment did not induce true ICD effects, but rather lysed the cancer\ncells once the concentration was sufficiently high.\nConsequently, we did not pursue an in vivo model for the solution, as the\nconcentration required to show an in vitro effect is not biocompatible for safe\nintravenous administration.\nFigure R3.1. NaCl solution treatment fails to induce ICD in 4T1 cells. (a) In vitro\ncytotoxicity of NaCl solution in 4T1 cells. (b-e) Investigation of effect of NaCl solution\nin inducing the expression of ICD markers such as calreticulin (b-c), ATP (d), and\nHMGB1 (e) in 4T1 cells treated with various NaCl concentrations around the IC value.\n50\n(f) Representative images of NaCl-treated 4T1 cells indicated the lysing effect of the\nhigh concentration salt solution. (a,b,d,e) Data are presented as mean ± SD with n=6\nin a, n=4 in b,d, and n=3 in e. (b,d,e) One-way ANOVA with Tukey’s multiple\ncomparison tests.\n3.6. (From R.3.8. of the previous review) For figure 4g-h, the authors are\nrecommended to provide more details regarding organelle-specific Ca2+ levels\n(maybe by using organelle-specific Ca2+ indicator) as well as membrane potential at\nmore time points. For ROS levels, what specific ROS species are elevated?\nWe thank the Reviewer for affirming the value of providing organelle-specific\nCa2+ levels; we agree that this data strongly supports the clarification of the BMN’s\nmechanism of action.\nTo track organelle-specific Ca2+ level changes, we investigated the\nconcentrations in the ER and mitochondria using Mag-Fluo-4 AM and Rhod-2 AM,\nrespectively. We focused on these two organelles because: (1) The ER and\nmitochondria are the main organelles that serve as Ca2+ ion storage; (2) we\nhypothesize the primary mechanism of BMN is the Ca2+ overload-mediated\ndysfunction of mitochondria and ER stress, leading to ICD in treated cancer cells.\nOur new experiments revealed that BMN treatment successfully induced an increase\nin Ca2+ ion levels in both the ER and mitochondria in a time-dependent manner.\nInterestingly, the Ca2+ ion levels reached a plateau at 2 h in the ER but at 4 h in the\nmitochondria (Fig. 1p-q and Supplementary Fig. 16a-b). Furthermore, we\ndemonstrated the more potent effect of BMNs in elevating the concentration of Ca2+\nions in both organelles compared to the Na+/Ca2+ solution (Supplementary Fig. 16c-\nd).\nFinally, regarding the investigation of mitochondrial membrane potential, we\nare pleased to provide additional data assessing the membrane potential at various\ntime points (Supplementary Fig. 37).\nFor the comment related to ROS, we had addressed this point in the previous\nround of revision.\nLine 178\n‘…In mammalian cells, the endoplasmic reticulum (ER) and mitochondria are the\nprimary organelles responsible for Ca2+ homeostasis.35,36 Following incubation with\nBMNs, the levels of Ca2+ ions in both the ER and mitochondria were dramatically\nincreased compared to the PBS (Control) and Na+/Ca2+ solution treatments. The\naccumulation of Ca2+ ions in the ER rapidly reached a plateau at 2 h of incubation,\nwhile the mitochondrial Ca2+ level showed a significant increase only after 4 h of BMN\ntreatment (Fig. 1p, 1q and Supplementary Fig. 15). The observed temporal difference\nin the Ca2+ plateau reflects the distinctive roles of the organelles. Given that the ER is\nthe main intracellular calcium reservoir,37,38 it is expected to respond rapidly to BMN-\nmediated Ca2+ internalization. In contrast, mitochondrial Ca2+ accumulation, in turn,\nmight only become significant once the capacity of the ER to buffer cytosolic Ca2+ is\nsurpassed…’"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have made substantial revisions and responded appropriately to this reviewer's suggestions. The updated analyses and clarifications adequately resolve my prior major concerns.\nOne minor concern: it remains unclear what the following incomplete sentence means in response to Comment 3.4 — “(We note that due to the limited sample size, Western blot analysis was not conducted using in vivo samples).”", "reviewer_3": "The authors have addressed all my concerns, I have no other questions and the paper should be ready to go."}, "author_response": "A Response Letter for the Manuscript titled:\nBinary Mineral Nanoparticles Enable Intravascular Delivery of Metal Ions to\nTumors for Metalloimmunotherapy\nWe earnestly thank the Editor and Reviewers for your consideration on our revised\nmanuscript. Here, we are pleased to provide point-by-point responses to the remaining\ncomments from Reviewers.\nReviewer #1 (Remarks to the Author):\nThe authors have made substantial revisions and responded appropriately to this\nreviewer's suggestions. The updated analyses and clarifications adequately resolve\nmy prior major concerns.\nWe appreciate the Reviewer for your comments and suggestions, which\nsubstantially improved our manuscript.\nR1.1. One minor concern: it remains unclear what the following incomplete sentence\nmeans in response to Comment 3.4 — “(We note that due to the limited sample size,\nWestern blot analysis was not conducted using in vivo samples).”\nWe apologize for the incomplete and unclear sentence in our previous\nresponse. Our intention was to clarify the methodology used for analyzing PD-1\nexpression in the in vivo samples. When analyzing the in vitro samples, we employed\nboth Western blot and flow cytometry, as ample cell material was available. However,\ndue to the inherent limitation in sample size obtained from the in vivo studies, we\nrestricted our analysis of PD-1 expression to flow cytometry alone. We hope this\nexplanation resolves this minor concern regarding our analytical methods.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed all my concerns, I have no other questions and the paper\nshould be ready to go.\nWe are grateful to the Reviewer for their thorough and constructive feedback."}]

Decision letter