Metal ion interference therapy: metal-based nanomaterial-mediated mechanisms and strategies to boost intracellular “ion overload” for cancer treatment

Abstract

Metal ion interference therapy (MIIT) has emerged as a promising approach in the field of nanomedicine for combatting cancer. With advancements in nanotechnology and tumor targeting-related strategies, sophisticated nanoplatforms have emerged to facilitate efficient MIIT in xenografted mouse models. However, the diverse range of metal ions and the intricacies of cellular metabolism have presented challenges in fully understanding this therapeutic approach, thereby impeding its progress. Thus, to address these issues, various amplification strategies focusing on ionic homeostasis and cancer cell metabolism have been devised to enhance MIIT efficacy. In this review, the remarkable progress in Fe, Cu, Ca, and Zn ion interference nanomedicines and understanding their intrinsic mechanism is summarized with particular emphasis on the types of amplification strategies employed to strengthen MIIT. The aim is to inspire an in-depth understanding of MIIT and provide guidance and ideas for the construction of more powerful nanoplatforms. Finally, the related challenges and prospects of this emerging treatment are discussed to pave the way for the next generation of cancer treatments and achieve the desired efficacy in patients.

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Wider impact

This review will significantly impact the advancement of innovative MIIT, personalized medicine, and interdisciplinary research. It has the potential to inspire researchers to develop more groundbreaking MIIT approaches, including novel treatment regimens, advanced drug delivery systems, and enhanced strategies for tailoring treatments based on patient genotypes, molecular characteristics, and pathology. Consequently, this will improve treatment accuracy and success rates. Additionally, the advancement of MIIT involves multiple fields such as biochemistry, nanotechnology, materials science and medicine, fostering interdisciplinary collaboration and cutting-edge research to tackle intricate scientific challenges.

1. Introduction

Malignant tumors pose a severe threat to human life and health as they are highly aggressive and metastatic.¹ Research indicates that tumor metastasis is accountable for approximately 90% of all cancer-related deaths.² Once a primary tumor infiltrates nearby tissues, it gains access to the lymphatic and blood systems, utilizing them as conduits for metastasis and the formation of secondary tumors.³ Over the past decade, scientists have endeavored to develop diverse cancer treatments with enhanced efficacy against malignant tumors.^4,5 Traditional cancer treatments, including surgical resection, chemotherapy, radiation, and immunotherapy, have been optimized to eradicate primary tumors.⁶ However, the clinical treatment of metastatic tumors remains extremely challenging and is associated with serious side effects such as surgical trauma, immune system damage, and neurotoxicity.⁷

With the continuous advancement of nanotechnology, there is growing interest in utilizing nanomaterials for the detection and treatment of metastatic tumors.^8–10 Compared to traditional small molecule drugs, biodegradable metal-based nanomaterials exhibit advantages, including enhanced permeability and retention (EPR) effect, diverse functionalities, and responsiveness to the tumor microenvironment (TME), making them more selective in tumor treatment while minimizing the potential adverse effects on normal tissues.^11–13 Degradation-released metal ions disrupt homeostasis in cancer cells, leading to irreversible damage or even death.^14–16 Based on this, biodegradable metal-based nanomaterials largely achieve the localized accumulation of metal ions at tumor sites, which is a difficult task to accomplish solely through the administration of metal ions. Therefore, extensive research on metal ion interference therapy (MIIT) has led to the development of a novel treatment strategy.

As research progresses, various roles of metal ions in human physiological activities are being increasingly elucidated. Certain metal ions are closely related to protein stability and biological activity. For example, Fe³⁺ and Cu²⁺ can disrupt redox homeostasis and induce ferroptosis in tumor cells by inhibiting the activity of glutathione peroxidase 4 (GPX4).^17–19 Moreover, excess Cu⁺ can trigger the aggregation of lipoylated proteins and destabilize iron–sulfur cluster proteins, resulting in mitochondrial dysfunction and cuproptosis.²⁰ Similarly, Zn²⁺, Ca²⁺, and Mg²⁺ can also act on mitochondria, resulting in cellular metabolic disorders.^21–23 Specifically, Zn²⁺ and Mg²⁺ can inhibit the activity of glycolysis-related enzymes, cut off the energy supply, and cause a starvation effect in cancer cells, thereby inducing apoptosis of cancer cells.^24,25 In the case of Ca²⁺, it can induce the release of cysteine aspartate-specific proteases (caspase) to initiate apoptosis by destroying the mitochondrial morphology. Furthermore, the released caspase 3 can activate the formation of transmembrane pores, leading to the occurrence of pyroptosis,^26,27 which is similar to the caspase 1-mediated pyroptosis initiated by Na⁺/K⁺ overload.²⁸

Additionally, metal ions have been found to serve as immune activators, stimulating the body's immune response to overcome clinical barriers associated with low tumor immunogenicity and immune-related adverse events linked to immunotherapy. For instance, Zn²⁺ and Mn²⁺ can stimulate the cyclic GMP-AMP synthase/stimulator of interferon gene (cGAS/STING) immune signaling pathway in tumor cells, thereby suppressing immune escape effects and enhancing the body's immune response to tumors.^29,30 Therefore, MIIT is a powerful therapeutic tool with strong tumor cell-killing capacity and abundant immunological effects. However, it is important to recognize that research on MIIT is still in its infancy. To date, numerous studies examining various types of MIIT have investigated their different molecular mechanisms and amplification strategies, thereby enhancing our understanding of this emerging therapeutic modality. A critical aspect in the advancement of MIIT is the design of amplification strategies that leverage interference mechanisms to optimize its therapeutic efficacy. These strategies aim to augment the treatment outcomes through synergistic treatment, disruption of ion homeostasis, and intensification of metabolic damage across diverse pathways. However, disparities exist in the research progress in different types of MIIT, necessitating a meticulous and comprehensive review to offer valuable insights for more recent advancements in the development of MIIT. By comparing differences and similarities, our perception of MIIT can be reshaped, moving beyond the limited focus on individual MIIT types to a holistic understanding of MIIT as a collective entity.

In this comprehensive review, we focus on the intrinsic mechanism of different metal ion (such as Fe³⁺/Fe²⁺, Cu²⁺/Cu⁺, Ca²⁺, Zn²⁺, Mn²⁺, Na⁺/K⁺, and Mg²⁺)-mediated interference therapies and their research progress in cancer treatment. Moreover, we shed light on the amplification strategies of MIIT, such as modulation of relevant metabolic pathways to sensitize cancer cells to MIIT, inhibition of channel protein activity to disrupt original homeostasis, and synergy with other therapeutics. Moreover, the current challenges encountered in this field and its prospects for clinical translation are also discussed. It is worth noting that this review is the first comprehensive report on the progress of emerging MIIT and its corresponding amplification strategies. We anticipate that this review will offer valuable guidance for the future development of efficient metal-based nanomaterials for effective MIIT and lay a solid foundation for the early clinical translation of MIIT.

2. Conception of metal ion interference therapy

2.1. Accurate delineation of metal ion interference therapy

Endogenous metal ions are crucial for various physiological processes within cells, such as signaling pathway regulation, enzyme catalysis, and protein synthesis.^31,32 Accordingly, to maintain these metal ions at levels within acceptable ranges, cells have established sophisticated and highly collaborative ion homeostatic regulatory systems for commonly found metal ions.^33,34 However, when the dynamic balance is severely disrupted, intracellular original metabolisms are blocked and the process of programmed cell death (e.g. apoptosis, pyroptosis, ferroptosis, and cuproptosis) is initiated.^35,36 Additionally, some nutritional metal ions can also stimulate the body's immune response to tumor cells, giving access to a novel form of immunotherapy.³⁷ Therefore, supplementation of exogenous metal ions or disruption of intracellular regulatory systems has been adopted to restrain the progression of tumors. In this case, given the huge potential of metal ions in combating tumors, the method of manipulating the level of metal ions at the tumor site has been defined as metal ion interference therapy.

2.2. Nanomaterial-facilitated metal ion interference anticancer therapy

The initial prerequisite to achieve efficient MIIT is to precisely elevate the levels of metal ions at the tumor sites. However, the short blood half-life and non-specificity of metal ions increase the risk of serious whole-body toxicity and undesirable therapeutic effects. In this case, with the rapid advancement of nanotechnology, biodegradable metal-based nanomaterials (e.g. metal oxides, metal peroxides, metal salts, metal organic frameworks (MOF), and metal complexes) have been adopted as carriers for metal ions to address the challenge in their delivery.^38,39 Most of these metal-based nanomaterials typically demonstrate TME-responsive properties, where they are particularly sensitive to the weak acidity and elevated levels of glutathione (GSH) in the TME.^40,41 Consequently, at tumor sites, these nanomaterials degrade rapidly, leading to the specific release of metal ions. This targeted degradation reduces the risk of metal ion leakage into adjacent tissues, thus minimizing the potential adverse effects of MIIT. Furthermore, the versatility of metal-based nanomaterials allows the integration of amplification strategies such as synergistic therapy and disruption of homeostatic regulatory systems, which augment the therapeutic efficacy of MIIT.⁴²

3. Progress in metal ion interference therapy

The underlying mechanisms of various types of MIIT are intricate and diverse, making it suitable for addressing a wide range of tumors. As researchers strive to optimize MIIT, several amplification strategies have been proposed and explored. These strategies are based on the disruption of the ion homeostasis regulation systems in cancer cells to induce a more pronounced ion imbalance. In this section, we provide a detailed summary of the intrinsic mechanisms of different MIIT approaches, including iron ion interference, copper ion interference, Ca²⁺ interference, Zn²⁺ interference, and some other metal ion interference therapy (Scheme 1). By virtue of their Fenton-catalytic property, iron ions generate intracellular reactive oxygen species (ROS), disrupting the redox balance, and thereby triggering ferroptosis mediated by the accumulation of toxic lipid peroxides (LPO). The aggregation of lipoylated proteins and destabilization of Fe–S cluster proteins caused by copper ions induce cuproptosis, a novel form of cell death resulting from proteotoxic stress. Ca²⁺ overload can cause mitochondrial dysfunction to initiate caspase 3-mediated apoptosis and degrade the cell skeleton to restrict the metastasis of cancer cells. Zn²⁺ interference blocks the development of tumors in multiple ways through the inhibition of glycolysis, cGAS/STING activation, and mutant p53 protein (Mutp53) degradation. However, the understanding of the effect if other metal ions on metabolic pathways by is currently rudimentary and lacks systematicity.

Scheme 1 Schematic illustration of the mechanism pathways of various metal ion interference therapies, mainly including ferroptosis, cuproptosis, Ca²⁺ interference therapy, and Zn²⁺ interference therapy. This scheme was created with Biorender.com.

3.1. Iron ion interference therapy

3.1.1. Metabolic features of iron ions and the induction mechanism of ferroptosis. As the most abundant trace element in the human body, Fe ions participate in many psychological processes, including DNA metabolism, oxygen transport,⁴³ and ATP generation.⁴⁴ However, an abnormal intracellular accumulation of Fe ions can induce a new type of cell death, which was defined as ferroptosis in 2012.⁴⁵ One important characteristic of ferroptosis is the accumulation of phospholipid hydroperoxides (PLOOHs), which are formed through the oxidation of phospholipid-containing polyunsaturated fatty acid chains (PUFA-PL) mediated by an Fe-dependent non-enzymatic Fenton reaction. Specifically, the ROS burst caused by the exacerbated Fenton reaction disrupts the original intracellular redox balance, ultimately leading to the accumulation of toxic PLOOHs. Unfortunately, the abnormal accumulation of Fe ions can be mitigated through the self-defense mechanism of cells. The cooperation between glycoprotein transferrin (TF) and its carrier protein transferrin receptor (TfR) enables the transport of extracellular Fe ions into cells.⁴⁶ Once inside cells, Fe ions are predominantly stored and transferred as ferritin, serving as the “iron pool”.⁴⁷ When intracellular free Fe ions are insufficient, ferritin is transported to autophagosomes through nuclear receptor coactivator 4 (NCOA4) and undergoes ferritinophagy for supplementation.⁴⁸ Alternatively, ferritin chelates excessive Fe ions to maintain homeostasis. Moreover, some other antioxidant defense mechanisms can also relieve the therapeutic efficacy of ferroptosis.⁴⁹ For example, to combat oxidative stress induced by excessive Fe ions, cells will overexpress GSH, a major antioxidant in mammals, to maintain the intracellular redox balance. Thus, the synthesis and metabolism of GSH significantly impact the sensitivity of cells to ferroptosis. In terms of GSH synthesis, cystine (Cys), which acts as a raw material, is transported across the cell membrane with the assistance of system Xc-. System Xc- is a cystine-glutamate exchange transporter consisting of two subunits, SLA7C2 and SLA7C11.⁵⁰ Some studies have shown that the variation in SLA7C11 plays a critical role in regulating the expression of system Xc- and determining the cellular tolerance to ferroptosis.⁵¹ Tumor suppressors, such as p53 and BRCA1-associated protein 1 (BAP1), have been found to promote ferroptosis by regulating the expression of SLA7C11.^52,53 The absence and mutations of these suppressors are conducive to improving system Xc- on the tumor cell membrane, resulting in an enhancement in ferroptosis resistance. Once inside the cell, Cys is reduced to cysteine and further utilized in the synthesis of GSH by linking with glutamate.⁵⁴ Additionally, under reducing extracellular conditions, cysteine can also enter the cell directly through the alanine-serine-cysteine (ASC) system.⁵⁵ The synthesized GSH, facilitated by the catalytic activity of selenoenzyme GPX4, plays a pivotal role in reducing toxic PLOOHs to non-toxic alcohols (PLOHs).⁵⁶ The inactivation of GPX4, which serves as a central downstream regulator of ferroptosis, is a crucial indicator of whether ferroptosis occurs. GPX4 catalytically reduces PLOOHs to their corresponding alcohols using the catalytic selenocysteine residue and two electrons provided by GSH.³⁶ Subsequently, glutathione–disulfide reductase (GSR) catalyzes the recovery of oxidized glutathione (GSSG) by utilizing two electrons provided by NADPH/H⁺.⁵⁷ Another antioxidant system that has been identified to prevent ferroptosis is the ferroptosis inhibitor protein 1 (FSP1)-ubiquinone (CoQ₁₀) pathway.⁵⁸ Acting as an oxidoreductase, FSP1 reduces CoQ₁₀ to panthenol (CoQ₁₀H₂), a lipophilic free radical scavenger capable of capturing ROS, thus restricting the accumulation of PLOOHs.⁵⁹ Furthermore, recent research has shed light on the association of ferroptosis and other metabolic pathways.⁶⁰

3.1.2. Nanomaterial-based iron ion homeostasis interference strategies. Enhancing the effectiveness of Fe ion interference therapy in inducing ferroptosis involves disrupting the redox balance mechanism within cells. The fundamental strategies for strengthening ferroptosis include up-regulating ROS and depleting GSH. Fe-based nanomaterials, acting as classic peroxidase (POD)-like nano-enzymes, have demonstrated significant potential in amplifying the ROS levels in TME. One notable advantage is their ability to convert Fe²⁺/Fe³⁺, which facilitates the transformation of overexpressed H₂O₂ into ˙OH, while depleting GSH. This process creates a self-perpetuating cycle referred to as the “ferroptosis cycle”. For example, ultrasmall MgFe-layered double hydroxides (LDHs) loaded with artemisinin (Art) demonstrated great ability to induce ferroptosis.⁶¹ These LDHs could respond to the pH and GSH in TME to release Fe²⁺/Fe³⁺, thereby entering the “ferroptosis cycle”. Notably, the transfer of electrons between Fe²⁺ and Art led to the formation of carbon-centered radicals, which synergistically enhanced ferroptosis. Moreover, thermal therapy-induced high temperature could accelerate the “ferroptosis cycle” by increasing the non-enzymatic reaction rate, thus contributing to the ROS burst and Fe ion interference therapy.^62–64 Meanwhile, some studies have shown that ferroptosis induced by Fe ion interference can effectively inhibit the high expression of heat shock proteins (HSPs), which reverse the resistance of cancer cells to thermal therapy.⁶⁵ For example, Zeng et al.⁶⁶ coordinated croconaine with Fe³⁺ (Cro-Fe) to achieve the synergy of photothermal therapy (PTT) and ferroptosis (Fig. 1A). When exposed to the acidic TME and overexpressed GSH, Cro-Fe decomposed into Fe²⁺ and the photothermal agent croconaine (Fig. 1B). Croconaine-mediated PTT accelerated the ferroptosis cycle to generate more ROS and LPO, resulting in the down-regulation of HSP and exhibiting excellent anti-tumor therapeutic efficacy (Fig. 1C). Enhancing the H₂O₂ level in the TME is another strategy to induce efficient ferroptosis.⁵⁷ Glucose oxidase (GOx) can decompose glucose into D-glucono-δ-lactone and H₂O₂ to continuously supplement the reaction substrate and reduce the pH of TME, which is conducive to accelerating the rate of the Fenton reaction and the release of Fe ions.⁶⁷ In the current work, an Fe-based MOF (Fe-MOF) combined with GOx was utilized to enhance ferroptosis through starvation therapy (ST).⁶⁸ The metal center Fe³⁺ of Fe-MOF could be reduced by the high-level GSH at tumor sites, resulting in the collapse of its structure and the release of metal ions. Meanwhile, the good biocompatibility, easy modification, and high surface-to-volume ratio of Fe-MOF make it highly promising for the delivery of metal ions. Furthermore, the glucose depletion-mediated starvation effect could reprogram the nutritional structure of cancer cells, leading to the increased uptake of PUFAs as an alternative energy reservoir.⁶⁹ Moreover, nano-enzymes with GOx-like properties have also been developed to synergistically boost ferroptosis due to their excellent stability. For example, Hou et al.⁷⁰ synthesized a tannic acid-iron ion (Fe^IIITA) network-coated tri-metallic mesoporous nano-enzyme (PdPtAu@TF) with natural GOx-mimic properties. This nano-enzyme rapidly induced the accumulation of LPO through three distinct pathways, including ROS burst, GSH depletion, and PUFA enhancement (Fig. 1D). The PdPtAu@TF treatment significantly down-regulated the intracellular GSH content and GPX4 activity, ultimately resulting in LPO accumulation (Fig. 1E–G).

Fig. 1 (A) Mechanism diagram of the transformation between Cro-Fe and Cro.⁶⁶ (B) Iron ion release profile of Cro-Fe at different pH values.⁶⁶ (C) Photothermal conversion capacity of Cro-Fe at different pH values.⁶⁶ Reprinted with permission from ref. 66. Copyright 2021, Wiley-VCH GmbH. (D) Schematic illustration of the pathways of PdPtAu@TF to ferroptosis.⁷⁰ (E)–(G) GSH level (E), expression of GPX4 (F) and LPO level (G) in 4T1 cells treated with different concentrations of PdPtAu@TF.⁷⁰ Reprinted with permission from ref. ⁷⁰. Copyright 2023, Wiley-VCH GmbH. (H) Synthetic process and TEM image of Ce6-PEG-HNK₁₅.⁷¹ (I) Degree of co-localization of ferritin and Ce6-PEG-HNK₁₅.⁷¹ (J) Schematic diagram of Ce6-PEG-HNK₁₅ destroying ferritin.⁷¹ (K) Cytosolic iron levels in 4T1 cells treated with different formulations.⁷¹ (L) LPO levels in different groups.⁷¹ Reprinted with permission from ref. 71. Copyright 2022, Wiley-VCH GmbH. (M) Mechanism diagram of COS@MOF.⁷² (N) Bio-TEM of autophagosomes and mitochondrial changes in CT26 cell.⁷² (Yellow box: autophagosomes and red box: mitochondrial changes). (O) Autophagy indicator LC3II/LC3I ratio.⁷² (P) Expression of p62, NCOA4 and GPX4. Reprinted with permission from ref. 71. Copyright 2023, Wiley-VCH GmbH.

Ferritin, the intracellular “Fe pool”, has a powerful buffering effect on Fe ions, thus weakening exogenous ferroptosis. Additionally, ferritin has been found to be overexpressed in various malignant tumors, causing them to have greater anti-interference ability for Fe ions than normal cells.⁷³ Therefore, target-attacking ferritin can potentially be a strategy to provoke Fe ion disturbance, thus improving the therapeutic effect of ferroptosis. The ferritin-homing peptide HKN₁₅ provides a precise target to attack ferritin to achieve endogenous Fe ion interference.⁷⁴ Ferritin-hijacking Ce6-PEG-HKN₁₅ NPs prepared by self-assembly were validated to destroy ferritin under laser irradiation (Fig. 1H).⁷¹ Specifically, Ce6-PEG-HKN₁₅ NPs preferentially gathered around ferritin due to the targeting of HKN₁₅, which was supported by the higher overlap of ferritins and Ce6-PEG-HKN₁₅ (Fig. 1I). The ROS generated by laser-activated Ce6 could destroy ferritin to induce the release of Fe³⁺ (Fig. 1J). Furthermore, the disruption of the “Fe pool” effectively enhanced ferroptosis, as confirmed by the significantly elevated cytoplasmic level of Fe ions and LPO content (Fig. 1K and L). Moreover, activated autophagy is another promising pathway to induce the degradation of ferritin.⁷⁵ Du's group⁷² connected the autophagy accelerator chitosan oligosaccharides (COS) to an etched MOF to fabricate an autophagy-amplifying nanocomposite, COS@MOF (Fig. 1M). COS@MOF produced an increased number of autophagosomes, and the elevated LC3II/LC3I further confirmed the occurrence of the cellular autophagy process (Fig. 1N and O). Furthermore, the increased presence of NCOA4 and decreased levels of GPX4 suggested that autophagy facilitated the degradation of ferritin to enhance ferroptosis (Fig. 1P). Overall, specifically target-attacking ferritin or enhancing the autophagy of cells can facilitate the degradation of ferritin, thereby increasing the Fe ion interference in cancer cells.

GSH is critical in maintaining the redox balance within cells, and thus disrupting the GSH-centered antioxidant defense mechanism is an effective strategy to enhance the sensitivity of cancer cells to ferroptosis. Firstly, the content of intracellular GSH can be reduced by limiting the cellular uptake of Cys. Science reported that Cys starvation, achieved through the deletion of subunit SLC7A11, is a translatable method to induce tumor-selective ferroptosis.⁷⁶ The clinical drug sulfasalazine (SAS) can delete SLC7A11 to inhibit the synthesis of GSH, thereby strengthening the ferroptosis of cancer cells.⁷⁷ Cai's team⁷⁸ designed a hypoxia-responsive dual-drug delivery system, PAD@MS, by simply coordinating Fe³⁺ with mitoxantrone (MTO), sulfasalazine (SAS), and dopamine derivative of polyethylene glycol (Fig. 2A). Under hypoxia TME, PAD@MS obviously degraded and released SAS, which significantly down-regulated the expression of SLC7A11 to strengthen immunogenic ferroptosis therapy with ROS burst (Fig. 2B and C). The immunogenic ferroptosis could lead to the up-regulation of interferon γ (IFN-γ), which further contributed to the down-regulation of SLC7A11, ultimately resulting in a significant decrease in GSH generation (Fig. 2D). Moreover, several Cys transporter system Xc-inhibitors such as erastin and sorafenib have also been identified to strengthen ferroptosis.^45,79 In addition to small molecule drugs, the corresponding siRNA can silence the expression of system Xc- to achieve the same effect of limiting Cys uptake and GSH synthesis.⁸⁰ Besides cutting off the external supply of Cys, the Cys-depleting strategy realized based on the strong nucleophilic nature of the mercapto moiety has also received extensive attention.⁸¹ In the current work, researchers utilized the acrylate group of Aza-BDY to selectively consume intracellular Cys through Michael addition and subsequent cyclization reactions (Fig. 2E and F).⁸² Meanwhile, Aza-BDY converted into the sonosensitizer Azb-BDY, which was confirmed by the shift in its absorption peak (Fig. 2G). Consequently, there was a significant increase in the level of LPO after Aza-BDY + US treatment (Fig. 2H). In conclusion, strategically blocking the Cys supply by down-regulating SLC7A11 and inhibiting system Xc-activity can effectively inhibit intracellular GSH synthesis to enhance ferroptosis.

Fig. 2 (A) Synthetic process of PAD@MS.⁷⁸ (B) Degradation of PAD@MS under hypoxia condition.⁷⁸ (C) Release profile of SAS.⁷⁸ (D) SLC7A11 content in tumor cells with different treatments.⁷⁸ Reprinted with permission from ref. 78. Copyright 2023, Wiley-VCH GmbH. (E) Mechanism diagram of the transformation between Aza-BDY and Azb-BDY.⁸² (F) UV/vis absorption of Aza-BDY treated with different amino acids. (G) Change in UV/vis absorption of Aza-BDY before and after reaction with Cys.⁸² (H) Confocal image of LPO in tumor cells.⁸² Reprinted with permission from ref. 82. Copyright 2022, Wiley-VCH GmbH.

Some types of cancer cells can bypass system Xc-, leading to system Xc-inhibitors that cannot achieve the desired effect.⁸³ Fortunately, there are alternative strategies to promote the sensitization of tumor cells to ferroptosis. One of these strategies is the suppression of GPX4, which is the central regulator of ferroptosis. As mentioned above, by accelerating the ferroptosis cycle or introducing additional free radicals, the depletion of GSH and subsequent inactivation of GPX4 can be achieved. Nitric oxide (NO), an endogenous signaling gasotransmitter, has the ability to generate the highly reactive free radical ˙NO to induce oxidative stress and inactivate GPX4. Additionally, certain small molecule drugs, including RSL3, RSL5, and FIN56, can trigger ferroptosis by directly acting on GPX4. Among them, RSL can specifically target the nucleophilic site of GPX4 and effectively deactivate it through the alkylation of selenocysteine. Thus, nano-delivery strategies for RSL have been developed to strengthen ferroptosis.⁸⁴ Differently, FIN56 selectively enhances the lysosomal degradation of GPX4, and concurrently activates squalene synthase to deplete CoQ₁₀. The GDY-FIN56-RAP nanoplatform containing FIN56 was successfully employed to induce ferroptosis in glioblastoma, demonstrating great potential for clinical therapy.⁸⁵ In summary, the direct or indirect inactivation of GPX4 has gradually become a favored option to improve the therapeutic effect of ferroptosis.

Most ferroptosis-related amplification strategies are based on targeting the redox balance within tumor cells. Strategies such as attacking intracellular ferritin, supplying H₂O₂, and enhancing the temperature aim to increase ROS production, leading to the accumulation of toxic LPO. Simultaneously, disrupting the GSH-centered antioxidant defense system can be achieved by either blocking system Xc- or inactivating GPX4.

Iron ion interference therapy, one of the most extensively researched forms of MIIT, has shown synergistic benefits when combined with innovative therapeutic methods such as PDT/SDT and PTT. High temperature can enhance the cell membrane permeability and speed up the Fenton reaction within tumor cells, hence amplifying intracellular oxidative stress. Besides, iron ion interference alleviates the drawback of tumor thermoresistance to PTT by suppressing the expression of HSP. Subsequently, PDT and SDT function as an additional pathway to boost ROS, thus elevating the LPO levels and strengthening ferroptosis. Belonging to ICD, ferroptosis plays a significant role in modulating the immunosuppressive TME. After recognizing DAMPs such as ATP and HMGB1 released through ferroptosis, dendritic cells (DCs) undergo maturation and antigen presentation to activate cytotoxic T lymphocytes (CTLs) for tumor cell elimination. In turn, IFNγ secreted by CTLs downregulates the expression of SLC7A11 in tumor cells to accelerate ferroptosis, creating a positive feedback loop to enhance the immune response. Additionally, recent evidence indicates that the immune cells within the TME also experience ferroptosis, affecting their immune and anti-tumor characteristics. For instance, stimuli-promoting ferroptosis can modulate multiple metabolic pathways to polarize pro-tumor M2 macrophages towards anti-tumor M1 phenotypes, awakening potent phagocytic killing abilities and migration inhibition in tumor-associated macrophages.

3.2. Copper ion interference therapy

3.2.1. Metabolic features of copper ions and the induction mechanism of cuproptosis. Cu ions, as coenzyme factors, have the capability to combine with variety of proteins, forming several copper enzymes that play a crucial role in numerous life processes.⁸⁶ The Cu ion levels have also been found to be strongly associated with the appearance and progression of cancer. The reason for this is that a moderate increase in Cu ions in tumors and serum can bind to MEK1 with high affinity, thereby resulting in the activation of downstream ERK1/2 phosphorylation to promote cancer cell proliferation.⁸⁷ Fortunately, although moderately elevated Cu levels facilitate cancer development and migration, they can effectively eradicate tumors once they surpass a specific threshold, which was confirmed in a study conducted in 1989, but its mechanism remained unclear. In 2022, Tsvetkov et al. reported the mechanism of Cu-induced death, introducing a new form of cell death called cuproptosis, which is distinct from other known forms such as apoptosis, pyroptosis, and ferroptosis.³⁵

Cuproptosis, a form of proteotoxic stress-induced cell death, is closely associated with the aggregation of lipoylated proteins and the instability of iron–sulfur cluster proteins. Protein lipoylation involves the attachment of lipoic acid to specific mitochondrial proteins through amide bonds. Several key mitochondrial proteins participate in this process, including dihydrolipoamide S-acetyltransferase (DLAT), dihydrolipoamide branched chain transacylase E2 (DBT), glycine cleavage system protein H (GCSH), and dihydrolipoamide S-succinyltransferase (DLST), which are all vital for regulating carbon entry into the TCA cycle. When Cu⁺ binds to lipoylated proteins, it leads to the formation of oligomers, which severely disrupt mitochondrial respiration. Alternatively, proteins that are not lipoylated cannot bind to Cu⁺. The key genes associated with this process were identified by screening to be ferredoxin 1 (FDX1) and lipoyl synthase (LIAS). The FDX1 gene is responsible for encoding a small iron–sulfur protein that converts Cu²⁺ to the more toxic Cu⁺, while LIAS, belonging to the family of lipoic acid synthases, catalyzes the final step in the de novo synthesis of lipoic acid. After the knockdown of FDX1, the complete loss of protein lipoylation and a significant decrease in respiration were observed, confirming that FDX1 is an upstream regulator. In addition, genes that encode the component of the lipoic acid pathway, such as lipolytransferase1 (LIPT1), lipoyl synthase (LIAS), and dihydrolipoamide dehydrogenase (DLD), undoubtedly also participate in the cuproptosis process. It is important to note that tumor cells predominantly rely on glycolysis for energy metabolism, which is known as the Warburg effect, making them insensitive to cuproptosis compared to normal cells. Most Fe–S cluster proteins are important coenzymes involved in electron transfer chains (ETC) and other biochemical processes. Copper ion overload triggers a remarkable loss of Fe–S cluster proteins in an FDX1-dependent manner. However, due to its recent discovery, only the basic principles of cuproptosis are clearly understood. Therefore, the associated metabolic processes still need to be further explored to help fully recognize this new type of cell death.

3.2.2. Nanomaterial-based copper ion homeostasis interference strategies. Cu-based nanomaterials as Fenton-like reagents possess a higher Fenton reaction rate and can react in a wider pH range compared to Fe-based nanomaterials. The Fenton reaction rate of traditional Fe-based nanocatalysts and Cu-based nanocatalysts is 76 m⁻¹ s⁻¹ and 1 × 10⁴ m⁻¹ s⁻¹, respectively.^88–90 Thus, based on these advantages, Cu-based nanomaterials have been increasingly utilized in ferroptosis research in recent years. With the gradual deepening of research, cuproptosis has entered our field of vision, and numerous Cu-based nanomaterials have been designed to enhance cuproptosis to achieve highly effective anti-tumor therapy. In this section, we summarize the progress in Cu-based nanomedicines in inducing cuproptosis of cancer cells and outline the key strategies to improve the therapeutic effects of cuproptosis.

Among the Cu ionophores, traditional small molecule Cu ionophores cannot effectively deliver sufficient Cu to tumor sites due to their limitations such as short blood half-life and poor specificity.⁹¹ Thus, to address these issues, intelligent nanoplatforms have been developed as Cu ionophores to induce cuproptosis, leveraging the advancements in nanotechnology. A novel ROS-responsive NP@ESCu nanoparticle was created by encapsulating the amphiphilic biodegradable polymer PHPM on the surface of elesclomol-Cu⁹² (Fig. 3A). The thioketal bonds of PHPM were broken by the intertumoral excessive ROS, leading to the release of elesclomol-Cu (Fig. 3B). Upon targeted transportation to the mitochondria, Cu²⁺ was released from the elesclomol-Cu complex and reduced to Cu⁺ by FDX1. Simultaneously, elesclomol rapidly effluxed and chelated with extracellular Cu²⁺ before being transported into tumor cells. This shuttle mechanism facilitated the continuous influx of Cu²⁺, thereby reinforcing cuproptosis. The shuttle mechanism was validated by the observed decrease in the IC50 of NP@ESCu to BIU-87, reaching 6 nM in the presence of preincubated CuCl₂ (Fig. 3C). Notably, elevated Cu⁺ concentrations consistently upregulated PD-L1 on the surface of cancer cells, thereby enhancing the efficacy of αPD-L1 (Fig. 3D).

Fig. 3 (A) Synthetic process of NP@ESCu.⁹² (B) Degradation of NP@ESCu in response to ROS.⁹² (C) Relative Cu content in tumor, expression of LIAS, and cell viability of BIU-87 cells treated with CuCl₂ and NP@ESCu.⁹² (D) Flow cytometry maps of DCs with various treatments.⁹² Reprinted with permission from ref. 92. Copyright 2023, Wiley-VCH GmbH. (E) Synthetic process of TP-M-Cu-MOF/siATP7a.⁹³ (F) mRNA level and expression of ATP7a in H69 cells with different treatments.⁹³ (G) Mitochondrial membrane potential in H69 cells treated with various formulations.⁹³ (H) Expression of Cyt C and FDX1.⁹³ All groups: I Control; II siATP7a; III TP-M-Cu-MOF; IV TP-M-Cu-MOF/siRNA^NC; V lipo-siATP7a; and VI TP-M-Cu-MOF/siATP7a. Reprinted with permission from ref. 93. Copyright 2023, Elsevier B.V.

Unfortunately, the long-term use of the Cu supplementation strategy in chronic diseases may cause tolerance to cuproptosis. Copper chaperone proteins and membrane transporters play crucial roles in maintaining intracellular Cu⁺ homeostasis. Among these proteins, the p-type ATPase copper-transporting ATPase (ATP7a/b), which can modulate the efflux of Cu⁺, is a target for disrupting intracellular Cu homeostasis.⁹⁴ Inspired by this, a pH-responsive Cu-MOF loaded with siATP7a, named TP-M-Cu-MOF/siATP7a, was designed to strengthen cuproptosis for cancer therapy⁹³ (Fig. 3E). The transcription of the ATP7a gene was significantly restricted due to the robust gene silencing, which further reduced the Cu⁺ efflux and elevated the intracellular Cu⁺ level (Fig. 3F). Furthermore, the Fenton-like catalytic property of Cu⁺ disrupted the mitochondrial metabolism to activate the related cuproptosis pathways (Fig. 3G and H). Moreover, recently, H₂S-activitated Cu₂(PO₄)(OH) NPs, which could generate ultra-small copper sulfide (Cu₉S₈) nanoparticles through an in situ sulfidation reaction, were synthesized (Fig. 4A).⁹⁵ The ultra-small Cu₉S₈ nanoparticles with an average size of 15 nm could quickly enter tumor cells through endocytosis and be degraded (Fig. 4B). Specifically, the higher endocytosis efficiency of Cu₉S₈ resulted in an intracellular ROS storm and restricted the energy supply, leading to a noticeable decrease in the activity of ATP7a (Fig. 4C). Then, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasomes were activated and the activity of FDX1 was inhibited to induce a highly effective combination of pyroptosis and cuproptosis (Fig. 4D).

Fig. 4 (A) Mechanism diagram of Cu₂(PO₄)(OH)-mediated cuproptosis and pyroptosis.⁹⁵ (B) Change in particle size and Cu²⁺ release profile of Cu₉S₈ at different pH values.⁹⁵ (C) Content of cellular copper and expression of ATP7A in HCT116 cells treated with different formulations.⁹⁵ (D) FDX1 and NLRP3 staining in HCT116 cells (scale bar = 100 μm).⁹⁵ Reprinted with permission from ref. 95. Copyright 2023, Wiley-VCH GmbH. (E) Synthetic process of BCMD NPs.⁶⁴ (F) pH-responsive-degradation of BCMD.⁶⁴ (G) Cu²⁺ and BSO release profiles of BCMD.⁶⁴ (H) Immunofluorescence image of DLAT in tumor cells with different treatments.⁶⁴ (I) Western blot analysis of POLD1, FDX1, LIAS, ACO-2 and HSP70.⁶⁴ Reprinted with permission from ref. 64. Copyright 2023, Elsevier Ltd.

With further exploration, glycolytic inhibition has been confirmed by several studies as an innovative way to strengthen cuproptosis. Improving the hypoxic microenvironment of tumors can significantly inhibit tumor glycolysis, and thus augment cuproptosis. Based on this mechanism, a Cu-based nanoreactor CCJD-FA, which is mainly composed of CaO₂, Cu-based shell, and JQ-1 (a bromodomain-containing protein 4 inhibitor), was fabricated to facilitate immunogenic cuproptosis.⁹⁶ In this nanosystem, the Cu-based shell responded to overexpressed GSH to release Cu²⁺, and subsequently the exposed CaO₂ decomposed into H₂O₂, which in turn produced O₂ through Cu²⁺-mediated catalase-like properties, thus relieving the hypoxia TME. Furthermore, JQ-1 restricted the expression of glycolysis-related enzymes. Consequently, O₂ combined with JQ-1 to effective suppress glycolysis, thereby blocking ATP generation and inactivating ATP7b. As previously stated, GOx can consume energy substrates of tumor cells and disrupt the intracellular energy metabolism. Recently, employing a GOx-engineered nonporous copper(I) 1,2,4-triazolate coordination polymer nanoplatform (GOx@[Cu(tz)]), it was confirmed that starvation therapy indeed improved cuproptosis.⁹⁷ Regrettably, the above-mentioned studies did not elaborate on the intrinsic mechanism of glycolysis inhibition-enhanced cuproptosis. Given the metabolic plasticity of cancer cells, glycolysis inhibition may trigger a metabolic shift to aerobic respiration, thus sensitizing them to cuproptosis. Furthermore, a high endogenous level of GSH can function as a thiol-containing copper chelator to prevent the occurrence of cuproptosis.⁹⁸ Thus, to address this, Huang et al.⁶⁴ devised a bidirectional amplification strategy for cuproptosis by constructing a Cu-based nanoplatform, BSO-CAT@MOF-199@DDM (BCMD) (Fig. 4E). The pH-responsive degradation of BCMD destroyed its structure, releasing Cu²⁺, buthionine-sulfoximine (BSO), and CAT (Fig. 4F and G). The goal of this strategy was to increase the O₂ concentration and decrease the GSH levels in tumors. Specifically, the secreted INF-γ combined with the released BSO to suppress the intracellular GSH content by inhibiting its synthesis. Meanwhile, the relief of hypoxia was aided by the catalytic capacity of CAT. Compared to the BMD or CMD groups, different types of lipoylated proteins and iron–sulfur cluster proteins in tumors were elevated and decreased in the BCMD group, respectively, indicating the occurrence of stronger cuproptosis (Fig. 4H and I).

Cuproptosis alone may not meet the needs of tumor treatment, and thus some work has been conducted to achieve a “1 + 1 > 2” effect by combining it with other therapies. Disulfiram (DSF) has been explored for its potential in combination with cuproptosis.⁹⁹ DSF is rapidly converted to diethyldithiocarbamate (DTC) in the cell, which can chelate with Cu²⁺ to form the more toxic and stable bis(diethyldithiocarbamate)-Cu (CuET).¹⁰⁰ Zhou et al.¹⁰¹ constructed an Au@MSN-Cu/PEG/DSF nanoplatform, as depicted in Fig. 5A and B. Under laser irradiation, Au@MSN-Cu/PEG/DSF exhibited an excellent photothermal conversion capacity (Fig. 5C). Subsequently, the Cu-doped silicone frameworks underwent temperature- and pH-dependent decomposition, leading to the release of Cu²⁺ and DSF (Fig. 5D and E). One part of Cu²⁺ was reduced to Cu⁺ by FDX1 to participate in the protein-lipid acylation process, while the other part was directly bound to DTC to form CuET (Fig. 5F). The aggregation of the NPL4 protein mediated by CuET resulted in the abnormal metabolism of ubiquitinated proteins, ultimately leading to apoptosis (Fig. 5G). The synergy of Cu²⁺ and DSF exhibited a remarkable enhancement in tumor inhibition (Fig. 5H). Another study demonstrated that CuET possessed better reduction resistance to the traditional anticancer agent cisplatin, inert reactivity with GSH, and tumor-selectivity toxicity.¹⁰² Therefore, CuET can be used as a robust and cuproptosis-dependent anticancer drug. Alternatively, Chan's group¹⁰³ synthesized a programmed responsive nanosystem, DMMA@Cu_2−xSe, to achieve the mutual promotion of thermotherapy and cuproptosis (Fig. 5I). Compared to other transition metal ions, selenium has a stronger binding capacity to copper ions, especially Cu⁺ (Fig. 5J). In an aqueous solution, Cu_2−xSe NPs gradually oxidized and released Cu²⁺/Cu⁺, exhibiting the bio-responsive release of Cu²⁺/Cu⁺ (Fig. 5K). Besides, Cu_2−xSe exhibited strong absorption and photothermal conversion efficiency in the far infrared region. Thermotherapy causes damage to cancer cells by increasing the temperature and causing mitochondrial disruption, which result in the upregulation of ROS. More ROS promoted the degradation of Cu_2−xSe NPs, which contributed to the release of Cu²⁺/Cu⁺. Finally, in synergy with thermotherapy, the degree of cuproptosis substantially increased (Fig. 5L and M).

Fig. 5 (A) Synthetic process and (B) TEM image of Au@MSN-Cu/PEG/DSF.¹⁰¹ (C) Photothermal conversion capacity of Au@MSN-Cu/PEG/DSF.¹⁰¹ Release profile of (D) Cu and (E) DSF at different temperatures and pH values.¹⁰¹ (F) Mechanism diagram of Au@MSN-Cu/PEG/DSF-mediated cuproptosis and apoptosis.¹⁰¹ (G) Expression of DLAT, LIAS and NPL4 in 4T1 cells.¹⁰¹ (H) Tumor volume change.¹⁰¹ All groups: I control; II Au@MSN-Cu/PEG/DSF; and III Au@MSN-Cu/PEG/DSF + laser. Reprinted with permission from ref. 101. Copyright 2022, Wiley-VCH GmbH. (I) Schematic illustration of DMMA@Cu2-xSe-mediated synergy of cuproptosis and thermotherapy.¹⁰³ (J) Binding capacity of selenium to different metal ions.¹⁰³ (K) Copper concentration in tumors treated with different formulations.¹⁰³ (L) Western blot analysis of FDX1, LIAS, DLST and DBT.¹⁰³ (M) Mechanism diagram of cuproptosis-mediated mitochondrial dysfunction and ROS burst.¹⁰³ Reprinted with permission from ref. 103. Copyright 2023, Wiley-VCH GmbH.

Due to the recent discovery of cuproptosis, there are limited therapeutic platforms and amplification strategies available. Among them, the most commonly utilized methods include inhibiting Cu ion efflux and enhancing the synergy with other therapeutic tools. One major challenge in copper ion interference therapy is the high resistance exhibited by cancer cells towards cuproptosis, which can be attributed to the metabolic shifts towards glycolysis and the high glutathione (GSH) content. Various approaches have been proposed in previous studies to address this issue of resistance, such as glycolysis inhibition and GSH depletion, with the intention of restoring the sensitivity of cancer cells to cuproptosis. Notably, gene p53, which is closely linked to elevated GSH levels and metabolic alterations in cancer cells, stands out as a potential target for enhancing cuproptosis. Nevertheless, the precise role of p53 in regulating cuproptosis still needs to be conclusively proven. Therefore, further investigations should be directed towards exploring this aspect in future studies.

Previous studies demonstrated the synergistic enhancement of treatments such as PTT, SDT, and PDT with copper ion-mediated Fenton-like catalysis, similar to iron ion interference. Nevertheless, although these treatments have exhibited enhanced macroscopic therapeutic effects in proteotoxic stress-induced cuproptosis, to date, research has not clearly elucidated the underlying intrinsic mechanisms. Therefore, further efforts are needed to understand these mechanisms at the molecular level. Currently, there is limited research on the immunological effects of copper ion interference, besides its induction of ICD. Extensive studies have shown that copper-mediated cell death processes can reduce the surface PD-L1 levels in cancer cells, potentially enhancing the treatment effectiveness by combining with immune checkpoint inhibitors to overcome immune evasion. However, the regulatory effects of copper ions on various immune cells remain poorly understood, necessitating further research to explore the immunological behavior influenced by copper ion interference.

3.3. Calcium ion interference therapy

3.3.1. Metabolic features of calcium ion and the induction mechanism of calcium ion interference therapy. Ca²⁺ is an intracellular second messenger that regulates various vital processes, such as muscle contraction,¹⁰⁴ neuronal excitability,¹⁰⁵ and cell growth.¹⁰⁶ Besides, Ca²⁺ has already been proven to exhibit unique cytotoxicity when overloaded in tumor cells, known as Ca²⁺ interference therapy, which offers a novel approach to treat various cancers.¹⁰⁷ The homeostatic balance of Ca²⁺ depends on the sophisticated collaboration of multiple organelles including mitochondria, the endoplasmic reticulum (ER), and lysosomes.¹⁰⁸ Initially, Ca²⁺ influx is primarily mediated by three channel proteins including Ca²⁺ release-activated Ca²⁺ channel protein 1 (ORAI1),¹⁰⁹ transient receptor potential channels (TRPCs),¹¹⁰ and voltage-gated calcium channels (VGCCs).¹¹¹ Among them, ORAI1 binds to Ca²⁺ sensor protein stromal interaction molecule 1 (STIM1), and this conjugate serves as a sensor for Ca²⁺ depletion in the endoplasmic reticulum (ER), determining whether to activate or deactivate the influx of Ca²⁺. This mechanism is commonly referred to as store-operated calcium entry (SOCE).¹¹² Alternatively, the intracellular Ca²⁺ efflux relies on Ca²⁺ ATPases (PMCAs) accompanied by ATP consumption. Moreover, mitochondria play a vital role in decoding the intracellular Ca²⁺ signals. Transient Ca²⁺ oscillatory signals stimulate mitochondrial metabolism, generating pro-survival signals.¹¹³ Conversely, prolonged Ca²⁺ overload signals result in the opening of the mitochondrial permeability transition pore (mPTP), and thus trigger cell apoptosis.¹¹⁴ During this process, cytoplasmic Ca²⁺ initially enters the intermembrane space through the voltage-dependent anion-selective channel proteins (VDACs) located on the outer mitochondrial membrane (OMM).¹¹⁵ Then, the electrochemical proton gradient (ΔΨ of 150–180 mV) generated by the pH gradient and membrane potential difference alters the structure of the mitochondrial Ca²⁺ uniporter (MCU) complex on the inner mitochondrial membrane (IMM), resulting in the entry of Ca²⁺ into the mitochondrial matrix.¹¹⁶ In addition to taking up Ca²⁺ from the cytoplasm, the mitochondria around ER form close contact with it to capture the Ca²⁺ released through ryanodine receptors (RyRs) and inositol 1,4,5-triphosphate receptors (Ins (1,4,5) P3Rs) on ER.¹¹⁷ ER, the biggest intracellular Ca²⁺ store, actively pumps Ca²⁺ into it against the concentration gradient using sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPases (SERCAs).¹¹⁸ This intricate system of Ca²⁺ channels precisely regulates the intracellular distribution of Ca²⁺ and provides abundant targets for accelerating Ca²⁺ interference therapy.

Cytoplasmic Ca²⁺ overload disrupts osmotic pressure balance, making it difficult for cells to maintain normal physiological processes. Excessive Ca²⁺ combines with phosphate to form calcium phosphate (CaP), which deposits on the cell membrane and results in cell calcification.¹¹⁹ This calcification affects the binding capacity of membrane proteins and interferes with signal transduction, ultimately disrupting cellular metabolism. Besides, excessive cytoplasmic calcium activates hydrolases, which break down the cytoskeleton, repressing the migration and invasion of tumor cells. More importantly, Ca²⁺ overload can cause mitochondrial dysfunction to kill tumor cells. High levels of mitochondrial Ca²⁺ have been shown to regulate the metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis (Warburg effect). This shift typically occurs in tumor cells to support their rapid proliferation.¹²⁰ Therefore, higher Ca²⁺ uptake capacity is considered a hallmark of tumorigenesis but it also augments the tumor vulnerability to mitochondrial Ca²⁺ overload-induced apoptosis.³³ Mitochondrial Ca²⁺ overload leads to the opening of mPTP, allowing more molecules to enter the mitochondrial matrix and resulting in the remodel of IMM.¹²¹ Consequently, the second mitochondria-derived activator of caspase (SMAC) and Cyt C are released from the cristae of IMM into the cytoplasm.¹²² Subsequently, SMAC activates the conversion of pro-caspase 9 to caspase 9 by binding to the inhibitor of apoptosis proteins, while Cyt C, together with cytoplasmic protein APAF1 and caspase 9, forms apoptosomes, which can induce apoptosis.¹²³ Meanwhile, the increase in mPTP permeability depolarizes the mitochondrial membrane, leading to mitochondrial dysfunction and the release of ROS into the cytoplasm through mPTP. Overall, Ca²⁺ overload induces oxidative stress and apoptosis via mitochondrial dysfunction to kill cancer cells.

3.3.2. Nanomaterial-based calcium ion homeostasis interference strategies. Ca-based nanomaterials have emerged as promising therapeutic agents for cancer treatment.¹²⁴ The unique properties of these materials, such as high biocompatibility and controlled drug release, make them suitable for Ca²⁺ interference therapy.¹²⁵ Thus, these nanomaterials hold great potential in improving the efficiency and effectiveness of cancer treatment. With the gradual deepening of research, some effective strategies that can significantly enhance Ca²⁺ interference therapy have been proposed.

P-glycoprotein (PgP), an ATP-dependent drug pump overexpressed in tumor cells, is the major cause of the failure of clinical chemotherapy. Some studies have shown that the upregulation of HIF-1α can enhance the expression of PgP under hypoxic TME by regulating the MDR gene.¹²⁶ Ca²⁺ overload-induced mitochondrial dysfunction can decrease the oxygen (O₂) need of cancer cells, thus alleviating the hypoxia dilemma of chemotherapy. Thus, to achieve Ca²⁺ overload-augmented chemotherapy, a tumor-targeting “Ca²⁺ nanogenerator” (TCaNG) mainly containing CaP and doxorubicin (DOX) was prepared¹²⁷ (Fig. 6A). In response to the acidic TME, TCaNG specifically degraded and released Ca²⁺ and DOX in tumor cells, resulting in mitochondrial dysfunction and the significant reduction in PgP content on the cell membrane (Fig. 6B and C). Moreover, Ca²⁺ interference-mediated ATP reduction also limited the efficiency of PgP and further reduced DOX pumping out (Fig. 6D). Consequently, TCaNG effectively reversed the drug resistance in cancer cells and enhanced the treatment of the chemotherapy drug DOX. Additionally, disrupting the Ca²⁺ buffering capacity of tumor cells through the modulation of channel proteins is an effective means of achieving Ca²⁺ accumulation. The reversal of drug resistance caused by Ca²⁺ overload can prevent the efflux of modulators, facilitating their full utilization. Kaempferol-3-O-rutinoside (KAE) exhibits excellent anti-cancer properties by suppressing SERCA1 and PMCA to disrupt Ca²⁺ homeostasis and induce apoptosis.¹²⁸ Based on this, the M@CaCO₃@KAE nanocomposite, incorporating CaCO₃ and KAE, was fabricated for the successful delivery of drugs and amplification of Ca²⁺ overload¹²⁹ (Fig. 6E and F). Encapsulation of the cancer cell membrane enabled M@CaCO₃@KAE to specifically target the tumor sites through innate properties of immune escape and homologous aggregation. In this case, these two channel proteins were potently suppressed to block Ca²⁺ in the cytoplasm and mitochondria, thus amplifying the Ca²⁺ interference (Fig. 6G). Subsequently, hydrolase calpain1 activated by excessive Ca²⁺ degraded the cytoskeleton to limit the ability of tumor cells to migrate and invade (Fig. 6H). Besides, the Ca²⁺ overload-induced mitochondrial dysfunction effectively suppressed the polymerization of F-actin, which is indispensable in tumor metastasis (Fig. 6I).¹³⁰ The degradation of F-actin caused the plasma membrane to fold, resulting in vague borders and unclear silhouettes. Therefore, the metastasis of the tumor was significantly impaired (Fig. 6J).

Fig. 6 (A) Schematic illustration of TCaNG-mediated reversal of drug resistance.¹²⁷ (B) Degradation of TCaNG at different pH values.¹²⁷ (C) and (D) Expression of membrane P-gp and ATP content in tumor tissues treated with blank TCaNG for 24 h.¹²⁷ Reprinted with permission from ref. 127. Copyright 2020, the American Chemical Society. (E) Synthetic process of M@CaCO₃@KAE.¹²⁹ (F) TEM image of M@CaCO3@KAE.¹²⁹ (G) Ca²⁺ release profile of M@CaCO₃@KAE at different pH.¹²⁹ (H) Western blot analysis of Calpain1 and CAMK4.¹²⁹ (I) Intensity of p/t-coffin, G-actin and F-actin.¹²⁹ (J) Apoptosis and ki67 expression within tumor tissues after different treatments.¹²⁹ (I) Control; (II) CaCO₃ NPs; (III) KAE; (IV) CaCO₃@KAE NPs; and (V) M@CaCO₃@KAE NPs. Reprinted with permission from ref. 129. Copyright 2021, Elsevier Ltd.

Similarly, curcumin (CUR) has the same effect as KAE in enhancing Ca²⁺ interference therapy.¹³¹ For example, Zheng's group²⁶ prepared biodegradable Ca²⁺ nanomodulators (CaNMs) composed of CUR and CaCO₃ to activate pyroptosis and robust antitumor immunity (Fig. 7A). CaNMs triggered a much lower MMP compared to CUR or CaCO₃, indicating more severe mitochondria dysfunction. The activation of caspase 3 by Cyt C led to the cleavage of gasdermin E (GSDME), resulting in the formation of an N-terminal, which bind to the cell membrane phospholipids, constructing transmembrane pores (Fig. 7B). These pores facilitated the release of inflammatory molecules and cellular contents, such as lactate dehydrogenase (LDH) and ATP, thus promoting the maturation of DCs and enhancing T cell activation (Fig. 7C). Therefore, combining Ca²⁺ regulators with Ca-based nanomaterials can be expected to become a new type of immunomodulator to activate the body's immune response. However, the antigen presentation efficiency of DCs is still limited by autophagy inhibition and low viability in the complex TME.¹³² Antigens require digestion and processing through autophagy before being presented.¹³³ Thus, to address these obstacles, a simple and versatile Ca²⁺ nanogenerator (OVA@CaCO₃) was constructed.¹³⁴ This nanosystem could disrupt a series of obstacles in antigen presentation according to the following mechanisms: (i) neutralizing the acidic TME to restore the viability of DCs; (ii) alleviating autophagy inhibition to promote the processing of antigens; and (iii) triggering DAMPs to stimulate DCs (Fig. 7D). In detail, as intracellular Ca²⁺ increased, more vesicles and LC3II (an important autophagy marker) appeared in DCs, indicating a higher level of autophagy (Fig. 7E and F). Furthermore, the higher colocalization rate of antigen and autophagosome confirmed that OVA@CaCO₃ could strengthen the autophagy ability of DCs, thus enhancing Ca²⁺ interference-mediated immunotherapy (Fig. 7F).

Fig. 7 (A) Schematic illustration of mitochondrial Ca²⁺ overload-mediated pyroptosis and immunogenic cell death.²⁶ (B) Bio-TEM images of cancer cells.²⁶ (C) Released LDH content and the populations of DCs and CD8⁺ T cells in the spleen.²⁶ Reprinted with permission from ref. 26. Copyright 2022, Wiley-VCH GmbH. (D) Mechanism diagram of HOCN-mediated disruption of three barriers in antigen presentation of DCs.¹³⁴ (E) Ca²⁺ level in DCs.¹³⁴ (F) Number of vesicles, LC3-I/II protein, and colocation ratio of LC3-II and antigen in DCs.¹³⁴ Reprinted with permission from ref. 134. Copyright 2020, the American Chemical Society.

Moreover, as chemical messengers for the transfer of biological information, some gaseous molecules (e.g. H₂S and NO) actively participate in maintaining the homeostasis and physiological processes of cells.¹³⁵ H₂S plays a crucial role in regulating the channel proteins responsible for the flow of Ca²⁺.¹³⁶ Based on this, poly(acrylic acid)-stabilized CaS nanoparticles loaded with zinc protoporphyrin (ZnPP@PAA-CaS) were developed to implement H₂S gas therapy to enhance the Ca²⁺ interference and signal the cascade of Ca²⁺-induced ICD¹³⁷ (Fig. 8A). Under acidic lysosome conditions, the nano messenger broke into Ca²⁺ and H₂S, and subsequently ruptured the lysosomes via the “proton sponge effect” to release them (Fig. 8B). Fig. 8C displays that cytoplasmic Ca²⁺ stress was sharply elevated in the presence of PAA-CaS, whereas it was barely detectable with PAA-Ca. Moreover, the heme oxygenase-1 (HO-1) protection pathway for the tumor to escape death was blocked by the amplifier ZnPP, which strengthened Ca²⁺-induced apoptosis.¹³⁸ Consequently, tumor-related antigens were released and acted as an in situ vaccine to activate a powerful immune response and restrict tumor metastasis (Fig. 8D and E). Similarly, NO-induced S-nitrosylation of cysteine residues on RyRs bends its structure and keeps it open, resulting in the leakage of Ca²⁺ from the ER store.¹³⁹ Based on this mechanism, Chu's group¹⁴⁰ prepared a Ca²⁺ store-regulating system UC-ZIF/BER by loading berbamine (BER) on upconversion nanoparticles (UCNPs) coated with ZIF-82 (Fig. 8F and G). The UV emission of UCNPs triggered by NIR light isomerized the nitro–nitrite in ZIF-82, resulting in NO production and structure collapse of ZIF-82 (Fig. 8H). UC-ZIF/BER exhibited stronger cytotoxicity in 4T1 and MB-231 cells with overexpressed RyRs, confirming the key role of RyRs in NO-induced cell death (Fig. 8I and J). Meanwhile, BER was released and bound to Ca²⁺-excreted pumps to inhibit Ca²⁺ efflux. Through the synergy of BER-mediated efflux protein inhibition and NO-induced RyRs deformation, UC-ZIF/BER showed outstanding in vivo antitumor efficiency in a 4T1 subcutaneous tumor model. Additionally, Chen et al.¹⁴¹ designed a multichannel nanomodulator, Lipo CaO₂-TA-Fe³⁺/PArg, using polyarginine (PArg) as a precursor of NO to achieve a similar effect. Moreover, the high oxidative stress environment induced by ˙OH simultaneously down-regulated the Ca²⁺ efflux pump PMCA and up-regulated the Ca²⁺ influx channel on the plasma membrane.¹⁴²

Fig. 8 (A) Synthetic process of ZnPP@PAA-CaS nanomessenger.¹³⁷ (B) Release profiles of Ca²⁺ and H₂S at different pH values.¹³⁷ (C) Intracellular H₂S and Ca²⁺ content after different treatments.¹³⁷ (D) Released CRT and HMGB1. (E) Number of metastatic nodules in the lung after various treatments.¹³⁷ (C) and (D) (1) Blank; (2) PAA-Ca; (3) ZnPP; (4) PAA-CaS; and (5) ZnPP@PAA-CaS. (E) Number of metastatic nodules in the lungs after various treatments.¹³⁷ All groups: (1) blank; (2) PAA-Ca; (3) ZnPP; (4) αPD-1; (5) PAA-CaS; (6) ZnPP@PAA-CaS; (7) PAA-CaS + αPD-1; and (8) ZnPP@PAA-CaS + αPD-1. Reprinted with permission from ref. 137. Copyright 2021, the American Chemical Society. (F) Synthetic process and (G) TEM image of UC-ZIF/BER NPs.¹⁴⁰ (H) NO release capacity of UC-ZIF/BER under 980 nm NIR irradiation.¹⁴⁰ (I) Mechanism diagram of the role of NO on RyRs.¹⁴⁰ (J) Expression of RyRs in various tumor cell models.¹⁴⁰ Reprinted with permission from ref. 140. Copyright 2021, Wiley-VCH GmbH.

As previously stated, the combination of Fenton agents with Ca-based nanomaterials can effectively enhance the level of cellular oxidative stress, thereby promoting Ca²⁺ inflow and causing Ca²⁺ disorder in cancer cells. Interestingly, Ca-based nanomaterials can be effectively endowed with Fenton-like properties by changing their crystal structure, synthesizing nano-reagents with high efficiency in inducing intracellular oxidative stress and Ca²⁺ disturbance through a one-step method. For the first time, a recent study reported that a type of valency-invariable CaF₂ crystal obtained by the direct precipitation method possesses peroxidase (POD)-like activity¹⁴³ (Fig. 9A). The activity of CaF₂, unlike the valence change-dependent POD-like activity of transition metal ions, was derived from the CaF₂(110) facet. Specifically, H₂O₂ was absorbed on the (110) facet with an energy drop of 0.92 eV, and then decomposed into OH*, which formed a bond with CaF₂via Ca–O binds. In addition, free ˙OH was generated with an energy of 1.67 eV. Subsequently, OH* combined with nearby H⁺ to produce H₂O, which desorbed from the facet, accompanied with a low endothermic energy of 0.78 eV (Fig. 9B). The cavitation effect of US irradiation could accelerate the rate and promote the degree of this process (Fig. 9C). Consequently, the generated ˙OH inhibited the expression of Ca²⁺ pump PMCA4 ATPase (ATP2B4) (Fig. 9D). Moreover, the mitochondrial dysfunction-induced decrease in ATP content further impaired the efficiency of ATP2B4, leading to an intensified Ca²⁺ overload and creating a “vicious cycle” (Fig. 9E). Therefore, the powerful Ca²⁺ interference capacity of CaF₂ could effectively inhibit the growth of tumors.

Fig. 9 (A) Schematic illustration of CaF₂-mediated ultrasound-augmented calcium interference therapy.¹⁴³ (B) POD-like catalytic pathway and the free energy variation of CaF₂(110) facet.¹⁴³ (C) ESR spectra of ˙OH trapped by DMPO in different groups.¹⁴³ (D) Expression of Calpain-1 and ATP2B4 in 4T1 cells.¹⁴³ (E) Relative ATP content in 4T1 cells with various treatment.¹⁴³ Reprinted with permission from ref. 143. Copyright 2022, Wiley-VCH GmbH. (F) Schematic illustration of plasma membrane damage mediated amplification of calcium interference therapy.¹⁴⁴ (G) Cell membrane-anchoring capacity of CMA-nPS.¹⁴⁴ (H) H1299 cell killing capacity of CMA-nPS.¹⁴⁴ (I) Digital photos of tumors with different treatments.¹⁴⁴ Reprinted with permission from ref. 144. Copyright 2022, Wiley-VCH GmbH.

Different from supplementing exogenous Ca²⁺ and regulating channel proteins to disrupt Ca²⁺ homeostasis, inducing cell membrane damage is a novel approach to trigger endogenous Ca²⁺ overload. Inspired by this, Gao et al.¹⁴⁴ proposed a strategy for achieving endogenous Ca²⁺ interference therapy by damaging tumor plasma membranes through the photodamage caused by photocatalysis. They constructed a cell-membrane-anchoring nano-photosensitizer (CMA-nPS) by embedding a dibenzocyclooctyne (DBCO)-decorated photosensitizer into a nanovesicle (Fig. 9F). In the acidic TME, the fusion of CMA-nPS and the tumor plasma membrane was triggered due to the transformation of the VSV-G structure into an unfolded fusion state. With an increase in the co-incubation time, CMA-nPS was observed to colocalize well with the plasma membrane (Fig. 9G). Subsequently, under NIR laser irradiation, the ROS in situ generated by the anchored photosensitizer significantly oxidized the phospholipids and increased the membrane permeability. The integrity of the plasma membrane was assessed using propidium iodide, and the results showed that CMA-nPS + L caused the most severe damage to the cell membrane, enabling extracellular Ca²⁺ influx and leading to tumor cell death, exhibiting an outstanding antitumor effect (Fig. 9H and I).

One of the most remarkable features of Ca²⁺ interference therapy is its ability to limit malignant tumor metastasis and invasion through cell calcification and cytoskeleton degradation. Given the clear understanding of the homeostatic regulatory system of Ca²⁺, the inhibition of channel proteins to control intracellular Ca²⁺ distribution has become the mainstream strategy for amplifying Ca²⁺ interference therapy. With the help of various channel protein inhibitors (e.g. small molecule drugs, ROS, and gas molecules), Ca²⁺ interference therapy has demonstrated powerful anti-tumor effects and considerable clinical translational potential. Moving forward, it is crucial to focus on the modulation of relevant immune cells by Ca²⁺ as a second messenger. This will allow the utilization of Ca²⁺ interference as a means of enhancing immunotherapy.

The effects of synergistic therapeutics can disrupt intracellular Ca²⁺ homeostasis by altering the state of channel proteins and amplifying the interference of Ca²⁺ on metabolism. The photothermal effect activates TRPV channels, leading to the promotion of Ca²⁺ influx and exacerbation of ion overload in cancer cells. In addition, ROS generated by PDT and SDT regulate the endogenous Ca²⁺ distribution by changing the conformation of channel proteins, resulting in localized Ca²⁺ overload. Besides, Ca²⁺ overload-mediated mitochondrial dysfunction further exacerbates the intracellular oxidative stress, thereby intensifying the therapeutic effects of ROS-dependent treatment. Ca²⁺ interference therapy can also trigger different forms of ICD, such as apoptosis and pyroptosis, to evoke the body's immune response to tumor cells. Besides elevating the abundance of tumor-associated antigens and DAMPs, enhancing the antigen processing and presentation efficiency of DCs is also an effective way to improve the cytotoxic T cell-mediated killing of tumor cells. High Ca²⁺ levels can enhance autophagy in DCs to accelerate antigen processing and promote immunological synapse formation to facilitate antigen presentation from DCs to CTLs. The latest research has shown that high calcium levels can polarize macrophages towards the M1 phenotype, thereby triggering the innate immune system's recognition and elimination of cancer cells. Furthermore, the NF-kB/IRF3 pathway activated by Ca²⁺ within tumor cells can also reprogram macrophage polarization to the M1 phenotype via the upregulation of IFN-1 expression. Therefore, Ca²⁺ interference can comprehensively coordinate the innate and adaptive immune systems for efficient immunotherapy.

3.4. Zinc ion interference therapy

3.4.1. Metabolic features of Zn²⁺ and the induction mechanism of Zn²⁺ interference therapy. Zn²⁺ plays a vital role as an essential auxiliary component in key metabolic regions of various enzyme molecules engaged in protein synthesis, such as RNA polymerase.¹⁴⁵ However, an overabundance of Zn²⁺ can prove detrimental to tumor cells given that it enhances the generation of endogenous ROS and disrupts MMP, leading to oxidative stress, a starvation effect, and an immune response. These effects effectively impede tumor proliferation and metastasis. The ETC are comprised of a series of complexes situated on the IMM, which work together to help organisms acquire energy. It has been confirmed through various studies that Zn²⁺ has the ability to suppress the ETC and increase the leakage of electrons to O₂ at complexes I and III. Consequently, this promotes the production of superoxide anion radicals (˙O₂⁻) and H₂O₂ in both normal and cancer cells.¹⁴⁶ Subsequently, severe oxidative stress can further trigger various cell death patterns such as pyroptosis and ICD. Moreover, abnormal electron transfer on the IMM can also disrupt ATP production by depolarizing the MMP during aerobic respiration. However, cancer cells prefer glycolysis over aerobic respiration for rapid proliferation. In this case, Zn²⁺ can impair two key glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphofructokinase (PFK), leading to the interruption of glycolysis.¹⁴⁷ Furthermore, Zn²⁺ can also degrade nicotinamide adenine dinucleotide (NAD⁺), an essential redox factor and substrate in ATP production.¹⁴⁸ A decrease in the intracellular NAD⁺ content significantly affects energy availability. Alternatively, a high level of Zn²⁺ exhibits the capacity to selectively degrade the mutant p53 proteins (Mutp53). The wild-type p53 protein (WTp53), a tumor suppressor, can elevate the expression of several apoptotic genes such as Bak and Bax, causing the programmed death of cells.¹⁴⁹ Unfortunately, WTp53 usually mutates to Mutp53 in cancer cells.¹⁵⁰ Compared to WTp53, Mutp53 not only loses the tumor-suppressing characteristic but also obtains new oncogenic functions, which is well known as Mutp53 gain-of-function (GOF).¹⁵¹ Due to the unique GOF, an important feature of Mutp53 is its tumor-promoting activity, including tumor growth, metastasis, and therapeutic resistance.^152,153 Besides, it can even eliminate the effects of other tumor suppressors such as p63 and p73. Over the past decade, many studies have been devoted to degrading Mutp53 by triggering autophagy or utilization of proteases.^154,155 Through the ubiquitination-mediated proteasomal (UPS) pathway, Zn²⁺ overload-mediated degradation of Mutp53 is a novel strategy to inhibit the development of tumors.¹⁵⁶ Additionally, Zn²⁺ can also trigger the body's immune response to cancer cells via the cGAS/STING signaling pathway.¹⁵⁷ cGAS senses intracellularly damaged DNA, and then activates STING, which stimulates two key transcription factors, NF-Kb and IRF3 in the cytoplasm.¹⁵⁸ Afterward, they enter the nucleus to exercise transcriptional functions to activate the expression of type I interferons (IFNB1) and inflammatory factors (ISG56).¹⁵⁹ Mitochondrial damage due to Zn²⁺ overload will release small amounts of DNA, which allows cGAS to work. More importantly, Zn²⁺ can enhance the enzymatic activity of cGAS proteins by inducing phase separation.¹⁶⁰ As a result of cGAS/STING activation, IFNB1 is expressed to stimulate multiple immune cells, thus enhancing the body's immune response to tumors.¹⁶¹

3.4.2. Nanomaterial-based zinc ion homeostasis interference strategies. Due to the diversity of mechanisms of Zn²⁺ in inducing cell disturbances, a variety of Zn-based nanosystems have been developed for Zn²⁺ interference in the treatment of cancer. In terms of glycolytic inhibition, an intelligent LND@HMPB-Zn nanosystem with dual glycolysis inhibition ability was constructed.¹⁶² Lonidamine (LND), an indazole-3-carboxylic acidic derivative, could bind to hexokinase (HK), which catalyzed one of the three rate-limiting irreversible enzymatic reactions of glycolysis. Therefore, this nanosystem enabled dual inhibition of glycolysis at different sites based on the combination of LND and excessive Zn²⁺. However, when the glycolytic pathway is blocked, cancer cells up-regulate glycolytic flux by virtue of their own adaptive capacity, leading to the intracellular up-regulation of glucose transporter (GLUT1), which will relieve the glycolytic inhibition of Zn²⁺ on cancer cells. Thus, to solve this problem, a dual gate-controlled “nano-enabled energy interrupter” (HZ@GD) was designed (Fig. 10A).²⁵ HZ@GD could specifically degrade at the tumor sites due to its dual response to low pH and hyaluronidase, as manifested by the change in its particle size and the release of Zn²⁺ and DNAzyme (Fig. 10B and C). Activated by Zn²⁺, GLUT1 mRNA-cleaving DNAzyme could cleave specific mRNAs multiple times to silence the GLUT1 gene, resulting in a significant decrease in the amount of GLUT1 in cancer cells (Fig. 10D). Furthermore, the intertumoral LA and ATP levels significantly decreased by 62.5% and 83.1% in the HZ@GD group, respectively, reflecting the superior energy depletion capacity of the “nano-enabled energy interrupter” (Fig. 10E). Unfortunately, cancer cells exhibit metabolic plasticity, enabling them to switch from the glycolytic pathway to the OXPHOS (aerobic respiratory) pathway for energy supply and proliferation when the glycolysis is inhibited. Metabolic inhibitor Zn-carnosine metallodrug network nanorods (Zn-Car NM) were constructed to completely block the energy supply by simultaneously shutting down both OXPHOS and glycolysis¹⁶³ (Fig. 10F). By undergoing “ion exchange” with Zn²⁺, the mitochondrial Cu²⁺ was depleted and Zn²⁺ and carnosine (Car) were released from Zn-Car NM (Fig. 10G and H). The depletion of Cu²⁺ inactivated mitochondrial complex IV, and subsequently disrupted MMP to limit the process of OXPHOS. In another aspect, Car removed the glycolytic intermediate products and excessive Zn²⁺ disrupted the metabolism of NAD⁺ to suppress the NAD⁺-dependent enzymes (Fig. 10I). Therefore, the combination of Zn²⁺ and Car achieved dual-gate control of glycolysis. Consequently, intracellular ATP was depleted to starve cancer cells (Fig. 10I and J). Based on the glycolysis blockade pathway, Zn²⁺-interference therapy can be an alternative to GOx-based ST. Besides, Zn²⁺-based nanomaterials are more stable for delivery and the function of Zn²⁺ is not limited by the TME.

Fig. 10 (A) Mechanism diagram of HZ@GD-activated gene silencing therapy.²⁵ (B) Degradability of HZ@GD treated with HAase at different pH values.²⁵ (C) Zn²⁺ and DNAzyme release profile of HZ@GD with different treatments.²⁵ (D) Immunofluorescence image of GLUT1 protein.²⁵ (Blue: DAPI; red: GLUT1; scale bar: 100 μm.) (E) Expression of GLUT1 mRNA and relative LA and ATP content in tumor tissues with different treatments.²⁵ Reprinted with permission from ref. 25. Copyright 2021, The Authors. (F) Synthetic process of Zn-Car NMs.¹⁶³ (G) Mechanism diagram and release profile of Zn²⁺ from Zn-Car.¹⁶³ (H) Confocal fluorescent images of free Cu in 4T1 cells treated with different formulations.¹⁶³ (I) COX activity, ratio of JCagg/JCmono, ratio of NAD+/NADH and ATP content in 4T1 cells.¹⁶³ (J) Relative 4T1 tumor volume with i.t. injection.¹⁶³ Reprinted with permission from ref. 163. Copyright 2023, the American Chemical Society.

Mutp53 is another star target for Zn²⁺ interference therapy.¹⁶⁴ For instance, Zhang's group¹⁵⁶ synthesized ZnFe NPs, which exhibited the strongest Mutp53 degradation ability with a Zn : Fe ratio of 1 : 2 (denoted as ZnFe-4) (Fig. 11A and B). ZnFe-4 selectively degraded several types of translated Mutp53 rather than disrupting their transcription and translation processes (Fig. 11C and D). Furthermore, the degradation of Mutp53 can up-regulate the expression of p21, another tumor suppressor. The treatment with ZnFe-NPs significantly induced cell cycle arrest, decreased migratory and invasive capacity, and reduced colony formation (Fig. 11E). In another study, Mn-ZnO₂ NPs were found to exhibit the ability to promote the degradation of Mutp53 and accumulation of WTp53. This co-regulation was achieved through the synergistic effects among Mn²⁺, Zn²⁺, and ROS¹⁶⁵ (Fig. 11F and G). Specifically, a high concentration of Mn²⁺ and ROS activated the ataxia-telangiectasia mutated (ATM)-p53 signaling pathway to stabilize and accumulate WTp53.¹⁶⁶ The activated WTp53 induced the transcription of proapoptotic genes such as Bax, thereby suppressing tumor growth (Fig. 11H). Zn²⁺-mediated Mutp53 degradation and Mn²⁺-mediated WTp53 accumulation effectively impaired GOF and initiated the death process in tumor cells. Consequently, Mn-ZnO₂ exhibited better tumor inhibition than ZnO₂ alone (Fig. 11I).

Fig. 11 (A) Preparation and treatment mechanism of ZnFe-4 nanoparticles.¹⁵⁶ (B) Morphological change in ZnFe-4 at different pH values.¹⁵⁶ (C) Capacity of ZnFe-4 to degrade different types of Mutp53.¹⁵⁶ (D) Relative level of p53 mRNA in H1299 cells.¹⁵⁶ (E) Cell cycle arrest and clonal formation of ES-2 cells treated with PBS or ZnFe-4 for 12h.¹⁵⁶ Reprinted with permission from ref. 156. Copyright 2020, Wiley-VCH GmbH. (F) Schematic illustration of the preparation and treatment mechanism of Mn-ZnO₂ NP.¹⁶⁵ (G) Zn²⁺ release profile of Mn-ZnO₂.¹⁶⁵ (H) Expression of Mutp53, ATM, p-ATM, p-WTp53, WTp53 and Bax in MDA-MB-231 cells.¹⁶⁵ (I) TUNEL staining analysis to detect the apoptotic MDA-MB-231 cells.¹⁶⁵ Reprinted with permission from ref. 165. Copyright 2022, The Authors.

Zn-based nanomaterials also play a crucial role in immune activation and regulation, which is helpful in achieving a synergistic enhancement in interference therapy with metal ion immunotherapy. Zhang's team synthesized a Zn²⁺-doped LDH (Zn-LDH)-based immunomodulating adjuvant¹⁶⁷ (Fig. 12A). In the acidic TME, Zn–OH is susceptible to hydrolytic fracture to elevate the pH of TME and release a certain amount of Zn²⁺, thereby effectively disrupting endo-/lysosomes to block autophagy and triggering mitochondrial damage. Moreover, Zn-LDHs can activate the cGAS/STING pathway, as confirmed by the significant up-regulation of IFNB1 and ISG56 (Fig. 12B). Consequently, the treatment of Zn-LDHs allowed up-regulation of the “eat me” signal (CRT and p53) and down-regulation of the “do not eat me” signal (PD-L1 and CD47) (Fig. 12C and D). Meanwhile, these variations promoted the conversion of M2-type tumor-associated macrophages (TAMs) to M1-type TAMs and the maturation of DCs, which can highly increase the efficiency of antigen presentation (Fig. 12E). In general, Zn-LDHs are simple but robust immunomodulating adjuvants that not only induce ICD but also reduces immune escape. In addition to enhancing the infiltration of immune cells to tumor by inducing ICD, Zn-based nanomaterials can also directly act on DCs and activate the matrix metalloproteinase pathway to stimulate the immune response of the body and alleviate the tumor suppressive microenvironment. Matrix metalloproteinase is an endogenous Zn²⁺-dependent proteolytic enzyme, which is responsible for the degradation of the tumor extracellular matrix (ECM) and matrix membranes.¹⁶⁸ The dense structure of the tumor ECM is a physical barrier that prevents the deep penetration of T cells and antibodies, impairing their killing ability. Also, an increase in the stiffness of the tumor ECM hampers the ability of DCs to re-recognize and internalize tumor antigens.¹⁶⁹ Thus, to overcome these tissues, Ding's group designed a Zn-MOF vaccine that directly targets DCs to enhance antigen uptake and presentation.¹⁷⁰ This vaccine, when taken up by DCs, enhanced antigen cross-presentation through lysosomal swelling-released OVA and cGAS/STING pathway-activated expression of interferon. Furthermore, in the acidic TME, the vaccine released Zn²⁺ to activate matrix metalloproteinase-2, thereby enhancing the T cell invasion and tumor-killing ability. However, despite the successful immune response elicited by these immune adjuvants, the low enrichment efficiency at the tumor site remains a significant obstacle in achieving the desired therapeutic effect. Recently, it has been shown that disruption of epithelial cells at the tumor vasculature can effectively enhance the EPR effect. Yang et al.¹⁵⁷ constructed a STING agonist, ZnCDA, which significantly reduced the blood vessel density at the tumor. Knocking out the STING gene in epithelial cells significantly attenuated this effect. This study implied that the targeting of nanomedicines can be enhanced through the STING pathway, and thus greatly increase their potential for clinical application.

Fig. 12 (A) Schematic illustration of the preparation of Zn-LDHs and mechanism of Zn-LDH-mediated robust and safe metalloimmunotherapy.¹⁶⁷ (B)–(D) Relative levels of ISG56 and IFNB1 mRNA (B), CRT (C), and PD-L1(D) in B16F10 cells.¹⁶⁷ (E) Phagocytosis of dying B16F10 cells by DCs.¹⁶⁷ Reprinted with permission from ref. 167. Copyright 2022, Wiley-VCH GmbH. (F) Synthetic process and (G) TEM image of UHSsPZH NPs.¹⁷¹ (H) Zn²⁺ release profile of UHSsPZH NPs at different pH values. (I) Mechanism diagram of Zn²⁺-mediated Ca²⁺ channel protein destruction in vivo.¹⁷¹ (J) Two pathways of Zn²⁺-induced apoptosis.¹⁷¹ (H) Expression of Oria1 and ZnT1.¹⁷¹ Reprinted with permission from ref. 171. Copyright 2023, The Authors.

Most of the above-mentioned studies focused on the use of Zn-based nanomaterials to supplement exogenous Zn²⁺ and achieve Zn²⁺ interference therapy. To maintain the intracellular Zn²⁺ levels, the body strictly regulates the activities of the Zrt/Irt-related protein (ZIP) and Zn transporter (ZnT1).¹⁷² ZIP is responsible for the influx of Zn²⁺ from extracellular organelles into the cytoplasm, while ZnT1 plays the opposite role.¹⁷³ Jiang et al.¹⁷¹ proposed a “block and attack” strategy, which is achieved by combining gene silencing using siRNA with Zn²⁺ supplementation. To implement this strategy, they created a nanocomposite called UHSsPZH, which stands for UCNPs-Hy@mSiO₂@PEI-siRNA@ZnO₂@HA (Fig. 12F and G). “Attack” is the typical Zn²⁺ supplementation strategy, whereas “block” is to limit the Zn²⁺ efflux via gene silencing of ZnT1. Compared to “block” or “attack” alone, “block and attack” triggered a higher intracellular Zn²⁺ concentration (Fig. 12H and I). Furthermore, Zn²⁺ can chelate with the abundant cysteine residues on the Ca²⁺ channel protein ORAI1 to form a Zn²⁺-cysteine complex, which distorts the conformational structure of ORAI1 (Fig. 12J). In vivo studies showed that USsPZH could inhibit both the ZnT1 and ORAI1 channels, and thus trigger dual-ion disorder (Fig. 12K). Therefore, the normal physiological function of ORAI1 was compromised, resulting in the down-regulation of the intracellular Ca²⁺ concentration. Ultimately, the oxidative stress generated by Zn²⁺-interference worked synergistically with the disturbances in Ca²⁺ homeostasis to accelerate apoptosis.

Zn²⁺ interference with the multidimensional containment of tumor cell development can be summarized as Mutp53 degradation, cGAS/STING activation, and glycolysis inhibition. Among these mechanisms, Zn²⁺ uniquely possesses the ability to degrade Mutp53 compared to other metal ions. Mutp53 is a crucial regulator of tumor cell metabolic remodeling, rapid multiplication, and apoptosis inhibition. Therefore, Zn²⁺ interference can reverse the GOF properties of cancer cells, thereby enhancing the effectiveness of other therapeutic treatments. Current research on Zn²⁺ interference therapy has primarily focused on understanding the underlying mechanisms and has only explored the inhibition of Zn²⁺ efflux proteins in isolation. Moving forward, it is necessary to devote greater attention to regulating the metabolism of Zn²⁺ and improving the efficiency of Zn²⁺-activated antitumor pathways.

Zn²⁺ interference therapy in combination with other therapeutic modalities, specifically ROS-dependent treatments such as PDT and SDT, has not been extensively studied. These therapies can induce mitochondrial damage and subsequent release of mitochondrial DNA, which amplifies Zn²⁺-mediated cGAS/STING activation. Additionally, photothermal effects have been found to enhance the glycolysis-inhibiting properties of Zn²⁺. More importantly, Zn²⁺ interference has the potential to improve the immunosuppressive TME and reverse the immune evasion of tumor cells from multiple dimensions. Basically, Zn²⁺ activates the cGAS/STING pathway by promoting the separation of the cGAS-DNA phase to restore the body's immune surveillance of the tumors. Recently, the physical nature of the TME has gained significant attention as a crucial factor affecting the effectiveness of immunotherapy, complementing the traditional focus on its biochemical aspects. At the tumor site, the ECM is notably stiffer than in normal tissue, creating a formidable physical barrier that hinders the antigen presentation capacity of DCs and the infiltration of T cells. Matrix metalloproteinases, when activated by Zn²⁺ acting as cofactors, play a key role in ECM modification by promoting its softening, thereby facilitating the functional activities of immune cells within the TME. Notably, Zn²⁺ exhibits a further significant impact by selectively degrading Mutp53, disrupting the immune evasive strategies employed by tumor cells. This mechanism involves the down-regulation of PD-L1 and the release of inflammatory cytokines, ultimately enhancing the immune response against tumor cells.

3.5. Other metal ion interference therapies

3.5.1. Mechanisms and strategies of nanotechnology-mediated Mn²⁺ interference therapy. Manganese is an important nutrient trace element needed for various physiological processes such as development, antioxidant defense, reproduction, and neuronal function. Furthermore, it is incorporated in many metalloenzymes and participates in numerous important enzymatic reactions.¹⁷⁴ The valence-variable manganese ion displays a diverse range of enzymatic activities crucial for maintaining redox homeostasis. Among these activities, manganese superoxide dismutase functions as a significant mitochondrial antioxidant, utilizing Mn³⁺ at its catalytic site to convert superoxide anions within the mitochondria into O₂ and H₂O₂.¹⁷⁵ Notably, nearly all Mn-based oxides demonstrate substantial acid responsiveness and catalase (CATase) activity, facilitating the breakdown of H₂O₂ to generate Mn²⁺ and O₂. Subsequently, Mn²⁺ exhibits Fenton-like catalytic features by further catalyzing the decomposition of H₂O₂ to produce ˙OH, thereby intensifying oxidative stress. Excessive Mn²⁺ causes oxidative stress, genotoxicity, membrane perturbations, and protein dysfunction in cancer cells due to its Fenton-like catalytic property. One of the major reasons for the popularity of manganese ion interference therapy is its great potential in tumor immunology. Mn²⁺ can effectively activate the cGAS/STING pathway and NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, inducing ICD and modulating the immunosuppressive TME. Research suggests that Mn²⁺ exhibits stronger activation ability in the cGAS/STING pathway compared to other metal ions.¹⁷⁶ This can significantly enhance the sensitivity of cGAS, enabling it to produce cGAMP in response to lower dsDNA stimulation. Furthermore, the affinity between cGAMP and STING at the ER surface is also heightened, boosting IFN-1 expression.¹⁷⁷ Consequently, the enhanced antigen presentation of macrophages and DCs activates CTLs and NKs to eliminate cancer cells, effectively boosting anti-tumor immunotherapy via the dual activation of innate and adaptive immunity strategies. Additionally, ROS burst mediated by Mn²⁺ activates the NLRP3 inflammatory pathway, and subsequently initiates caspase 1-mediated pyroptosis, triggering a powerful inflammatory and immune response at the tumor site.¹⁷⁸ Manganese ions help counteract the reducing environment by engaging in redox reactions and facilitating the transformation of TAMs from the M2 phenotype to the M1 phenotype.¹⁷⁹ In conclusion, manganese ion interference therapy shows great potential in disrupting cellular metabolism and enhancing the body's immune response.

Recently, a pH-responsive MRI theranostic nanoplatform was constructed, which was comprised of Se nanoparticles, manganese carbonyl (MnCO), and Fe₃O₄.¹⁸⁰ The Se nanoparticles facilitated O₂⁻ production and rapidly activated superoxide dismutase, triggering cascade reactions that produced H₂O₂. The presence of Mn²⁺ facilitated the Fenton-like reaction, leading to the generation of ˙OH, which induced quick protein degradation and oxidation. Additionally, Mn²⁺ played a role in inhibiting the generation of ATP, thereby effectively starving cancer cells. Moreover, similar to Zn²⁺, Mn²⁺ also participates in activating the cGAS/STING pathway in humans to stimulate the body's immune response and facilitate Mn²⁺ interference-mediated immunotherapy.^181,182 To enhance the Mn²⁺ activation of the cGAS/STING pathway, a gas nano-adjuvant based on gas-induced mitochondria dysfunction was designed (Fig. 13A). Once inside cancer cells, the gas nano-adjuvant disassembled in response to the overexpressed GSH and low pH, leading to the release of MnCO and H₂S (Fig. 13B). Under NIR-irradiated AIEgen-based PDT/PTT, the prodrug MnCO in situ decomposed to Mn²⁺ and CO. Consequently, the MMP was depolarized due to the induction of H₂S and CO, leading to the subsequent release of mitochondrial DNA (Fig. 13C). Moreover, mitochondrial DNA and Mn²⁺ can trigger the activation of the cGAS/STING pathway to promote the synthesis of related cytokines through phosphorylation of TBK1 and IRF3, thereby arousing the immune response to tumors (Fig. 13C–E). It should be noted that Mn²⁺ exhibits a distinct feature from Zn²⁺ given that it can bypass STING to directly or indirectly enhance the phosphorylation of two downstream transcription factors, p65 and IRF3.³⁰ Notably, recent studies have established the relationship between IFN gene transcription and the formation of enhancesomes, which include phosphorylated IRF3 and p65.¹⁸³ In this context, the research by Sun's group's showed that Mn²⁺ has the ability to trigger the STING-independent phosphorylation of p65 and TBK1³⁰ (Fig. 13F). Through thermal shift tests, it was found that Mn²⁺ could not enhance the affinity between STING and STING agonists (Fig. 13G). After knocking out STING, Mn²⁺ could still initiate the phosphorylation process of TBK1 and p65, but not IRF3. Therefore, Mn²⁺ exhibited robust immune activation potential by strengthening the phosphorylation of IRF3 in the presence of STING agonists. Inspired by this, Mn²⁺ was combined with cyclic dinucleotide (CDN), a conventional STING agonist, to achieve the amplification of the STING signaling cascade and up-regulation of IFN-β (Fig. 13H). Moreover, Mn²⁺ promotes the survival and proliferation of memory T cells and significantly enhances the killing effect of natural killer cells (NKs), thereby increasing the host's immune surveillance of the tumor. In summary, Mn²⁺-mediated immunotherapy has shown great potential for clinical application, and thus received widespread attention.

Fig. 13 (A) Synthetic process of gas nanoadjuvant.¹⁸⁴ (B) Degradability and gas release profiles of gas nanoadjuvants.¹⁸⁴ (C) Cytosolic mitochondrial DNA and released cytokines IFN-β, CXCL10, and IL-6.¹⁸⁴ (D) Expression of STING, TBK1, IRF3 and their phosphorylation in tumors.¹⁸⁴ (E) Flow cytometric assay of mature DCs and tumor-infiltrating CD8⁺ T cells.¹⁸⁴ Reprinted with permission from ref. 184. Copyright 2023, The Author(s). (F) Mechanism diagram of Mn²⁺-mediated elevation of IFN-β expression, phosphorylation of p65, TBK1, and IRF3 in STING knock-out cells, and inhibition of Mn²⁺-mediated IFN-β by p65 nucleus translocation.³⁰ (G) Affinity between STING and STING agonists.³⁰ (H) Synthetic process of CDN-Mn nanoparticles.³⁰ Reprinted with permission from ref. 30. Copyright 2021, The Author(s).

3.5.2. Mechanisms and strategies of nanomaterial-based sodium/potassium ion interference therapy. Mammalian cells maintain a low ratio of intracellular to extracellular Na⁺ and high ratios of K⁺. The asymmetric concentration gradients are mainly maintained by the sodium-potassium pump and provide driving forces for several physiological processes, such as amino acid transport, pH stabilization, and osmotic pressure homeostasis.¹⁸⁵ A low osmotic pressure outside or high osmotic pressure inside the cell leads to cell swelling and cytoskeletal damage. When the change exceeds the tolerance limit of the cell, the cell membrane ruptures and releases its content. Thus, to effectively disrupt the osmotic pressure balance in tumor cells, it is necessary to increase the concentration of Na⁺/K⁺ at the tumor site. In this case, Li et al.¹⁸⁶ constructed a virus-mimicking GSH-responsive NaCl@ssss-VHMS to achieve the systematic delivery of NaCl crystals (Fig. 14A). NaCl@ssss-VHMS rapidly entered tumor cells through spike surface-assisted adhesion and invasion, surpassing the efficiency of conventional spherical nanomaterials. Upon entering the cells, ssss-VHMS underwent rapid degradation via thiol-tetrasulfide exchange with intracellular GSH, resulting in the explosive release of Na⁺/Cl⁻. Fig. 14B shows that the NaCl@ssss-VHMS group showed a higher level of Na⁺ and Cl⁻ compared to the NaCl group, indicating that this nanoplatform has superior Na⁺ delivery capabilities to enhance the accumulation of Na⁺ in tumor cells. The surge in osmolarity caused by the burst of Na⁺ triggered cell swelling, plasma membrane destruction, and the observation of cell debris through bio-TEM analysis (Fig. 14C). Meanwhile, the disruption of the endolysosome induced the formation of NLRP3 inflammasomes, and subsequently activated caspase 1 (Fig. 14D). Consequently, the GSDMD-formed pores released the cell contents, resulting in pyroptosis (Fig. 14E). By increasing the osmolarity and triggering cellular pyroptosis, Na⁺ interference therapy displays powerful antitumor effects and holds great potential for future clinical application. Similarly, Ding et al.¹⁸⁷ synthesized biodegradable K₃ZrF₇:Yb/Er upconversion nanoparticles (ZrNPs) as pyroptosis inducers for tumor immunotherapy (Fig. 14E). Degraded by acidic lysosomes, ZrNPs released a large amount of K⁺ and [ZrF₇]³⁻ (Fig. 14F). Osmolarity-activated caspase 1 could promote the maturation of the inflammatory molecule IL-1β, which was released from the pyroptosis-formed pores to activate ICD (Fig. 14G). Additionally, it has been verified that intertumoral high K⁺ suppressed the anti-tumor capacity of TAMs. TAMs are mainly categorized into two main distinct inflammatory (M1) and immunosuppressive (M2) phenotypes.¹⁸⁸ M1-type TAMs are tumor-killing cells responsible for immune promotion, whereas M2-type TAMs suppress immunity and contribute to tumor development.¹⁸⁹ Numerous studies have shown that the tissue microenvironment and extrinsic factors such as cytokines released in inflammation can control the polarization of TAMs. Besides, due to the plasticity of TAMs, they can repolarize to another state.¹⁹⁰ The inwardly rectifying K⁺ channel Kir2.1 on TAMs is activated by intertumoral high K⁺ and is a central regulator of the functional polarization of TAMs.¹⁹¹ Kir2.1 deficiency reprograms tumor metabolism and upregulates a series of immune activation gene sets, leading to the repolarization of TAMs to the M1-type.¹⁹² Thus, decreasing K⁺ in the TME is a potential pathway to activate the tumor immune response.

Fig. 14 (A) Synthetic process and corresponding TEM images of NaCl@ssss-VHMS.¹⁸⁶ (B) CLSM images of Na⁺ and Cl⁻ level.¹⁸⁶ (C) Morphology changes in HepG2 cells treated with NaCl@ssss-VHMS.¹⁸⁶ (D) Caspase-1 activation and Capase-3 activation in HepG2 cells with different formulations.¹⁸⁶ Reprinted with permission from ref. 186. Copyright 2022, the American Chemical Society. (E) Schematic illustration of Zr NP-mediated pyroptosis-enhanced immunotherapy.¹⁸⁷ (F) Level of K⁺ in 4T1 cells.¹⁸⁷ (G) Relative c-GSDMD content and profiles of released IL-1β in 4T1 cells, mature rate of DCs, population of CD8⁺ T cells, and CD8⁺/CD4⁺ ratio in the lymph node.¹⁸⁷ Reprinted with permission from ref. 187. Copyright 2021, the American Chemical Society.

3.5.3. Mechanisms and strategies of nanotechnology-mediated magnesium ion interference therapy. Magnesium, the second most abundant cation in cells after potassium, serves as a vital cofactor in various biological processes, including oxidative phosphorylation, energy generation, and the synthesis of proteins and nucleic acids.¹⁹³ Intracellular oxidative stress and antioxidant defense mechanisms are intricately linked to the levels of intracellular Mg²⁺. A deficiency in Mg²⁺ can lead to the disruption of ETC on the IMM, resulting in the down-regulation of ATP synthase and subsequent mitochondrial dysfunction and the generation of ROS.¹⁹⁴ Mg²⁺ deficiency promotes apoptosis by elevating cytochrome C discharge through Bax or the VDAC, while inhibiting anti-apoptotic proteins such as the Bcl-2 family. Moreover, Mg²⁺ also acts as an immunomodulator by regulating cytotoxic T-cell activation by altering the conformation of LFA-1. When stimulated by extracellular Mg²⁺, the co-stimulatory cell-surface molecule LFA-1 on CD8⁺ T cells adopts its active conformation, resulting in increased Ca²⁺ flux, signal transduction, metabolic reprogramming, immune synapse formation, and enhanced T cell toxicity.¹⁹⁵ Therefore, sufficient Mg²⁺ is significant for improving the efficiency of immunotherapy. However, magnesium ion-disrupting therapy is still in its early stages, with only a limited number of nanomaterials being used in this area.

Mg-based nanomaterials have various applications in clinical settings, such as being used as screws and pins in orthopedics and as biodegradable scaffolds in the cardiovascular field.^196,197 Additionally, Mg-based nanomaterials exhibit increased cytotoxicity towards cancer cells. The biocompatibility and degradability of magnesium-based nanomaterials lead to the generation of surface effects, including increase in pH, increase in osmotic pressure, and hydrogen evolution, which have the potential to directly target cancer cells and reduce tumor growth.¹⁹⁸ Moreover, the Mg²⁺ released by biodegradable Mg-based nanomaterials can serve as a coenzyme factor, which effectively collaborates with DNA enzymes to inhibit the expression of MDR mRNA and further reverse the drug resistance of tumors to chemotherapy. Inspired by this, a versatile DNA nanodrug (MCD@TMPyP4@DOX) was fabricated to achieve effective MDR silencing via Mg²⁺-activated DNAzymes.²³ Triggered by photodynamic-induced ROS burst, the nanodrug self-disassembled to release DNAzymes and Mg²⁺. Subsequently, DNAzymes cleaved the MDR mRNA under the action of cofactor Mg²⁺, resulting in a significant decrease in PgP expression. Meanwhile, ROS-mediated mitochondrial dysfunction impaired ATP production, thus limiting the DOX pumping efficiency of PgP. This research presented a new way that Mg²⁺ effectively improves the effect of gene therapy as a cofactor.

Mg²⁺ can also actively participate in cellular energy metabolism. Mg²⁺-dependent enzymes such as HK,¹⁹⁹ PFK,²⁰⁰ and pyruvate kinase (PK),²⁰¹ which are the three key enzymes involved in glycolysis, make Mg²⁺ deficiency an effective strategy for blocking glycolysis in tumor cells. Recently, a GSH-responsive nanoplatform MSN/EDTA&rotenone@PAASH was subtly designed to achieve Mg²⁺ interference-mediated glycolysis inhibition.²⁰² Within this nanoplatform, EDTA chelated high intracellular concentrations of Mg²⁺ to form EDTA-Mg complexes, inactivating HK, PFK, and PK. Simultaneously, rotenone diffused into the intermembrane space and blocked the electron transfer during OXPHOS, thereby disrupting ATP synthesis. In addition to participating in energy generation, Mg²⁺ has a close relationship with DNA replication and stability, which participates in various DNA repair patterns, such as nucleotide excision repair, base excision repair, and mismatch repair, and stabilizes the natural DNA conformation through hydrogen bonding. Therefore, when there is an intracellular deficiency of Mg²⁺, DNA becomes more vulnerable to damage from ROS. Several studies have shown that a moderate Mg²⁺ level is strongly associated with mitochondrial health and intracellular redox homeostasis. Insufficient Mg²⁺ disrupts the ETC, which leads to a burst of ROS, subsequently disrupting the existing antioxidant defense mechanisms of cells. This process includes the down-regulation of intracellular GSH and vitamin E levels, which accelerates the oxidation of lipids.

Metal-based nanomaterials, including various types such as metal oxides, metal peroxides, metal salts, MOFs, and complexes, have been extensively utilized for targeted metal ion interference therapy in the context of cancer treatment. Here, we comparatively analyze the types, sizes, and responsive properties of the different metal-based nanomaterials that have been developed thus far and the treated tumor models (Table 1). Most of these nanomaterials demonstrated a propensity to actively interact with the acidic tumor microenvironment and exploit overexpressed GSH to modulate the metal ion levels within the ECM. Consequently, the use of degradable metal-based nanomaterials offers heightened tumor selectivity, while minimizing the adverse impacts on healthy tissues. Concomitantly, nano-sized drugs are capable of being internalized by cancer cells through endocytosis, subsequently undergoing degradation within lysosomes, which in turn leads to elevated intracellular metal ion concentrations. Additionally, we summarize the biosafety, injection modalities, dosages, and corresponding therapeutic effects of various nanomaterial-mediated MIIT in Table 2. Furthermore, the use of complex functionalized nanomedicines for achieving tumor ablation through multimodal synergistic therapy adds another layer of complexity, making it difficult to compare the therapeutic effects of metal ion interference therapy alone. Fortunately, the current nanomedicine being studied has demonstrated excellent biocompatibility and therapeutic outcomes in xenograft mice. By incorporating diverse amplification strategies and metal-based nanomaterials through a rational design approach, the interference of metal ions can be maximized for enhanced therapeutic efficacy. Consequently, it is evident that the utilization of nanotechnology-mediated metal ion interference therapy holds promise as a potent strategy for advancing cancer treatment methodologies.

Table 1 Nanomaterials for metal ion interference therapy

Type	Formulation	Metal-based nanomaterials	Metal ion content (%)	Particle size	Responsive degradation	Tumor model	Ref.
Iron ion interference	LDHP	Fe/Al-LDH	38.52	433	pH	4T1 cells	203
	Cro-Fe@BSA	Cro-Fe complex	17.0 ± 3.6	91.3 ± 10.4	pH	4T1 cells	66
	sSFP	FePt	7.57	325	pH	4T1 cells	204
	Fe-MOF-RP	Fe-TCPP	1.03	∼150	GSH	B16F10 cells	205
	PFP@Au-Fe2C-SRF	Fe2C	22.7	320.2	pH and laser	4T1 cells	206
	PAD@MS	Fe complex	10.2	∼130	Hypoxia and pH	CT26 and MC38 cells	78
	PCSFG	Fe-GA coordinate	13.5	∼45	Temperature and pH	4T1 cells	207
Calcium ion interference	M@CaCO3@KAE	CaCO3	—	∼100	pH	A549 cells	129
	CSSG	CaS	—	80	pH	4T1 cells	208
	ZnPP@PAA-CaS	CaS	51	122	pH	4T1 cells	137
	CaO2-N770@MSNs	CaO2	8.1	464.1	pH	CT26 cells	140
	Lipo CaO2-TA-Fe^3+/PArg	CaO2	4.11	43.8	pH	4T1 cells	141
	CCMH	CaO2	20.21	180	pH	4T1 cells	209
	CEL/ACC-LP NPs	CaCO3	17.61 ± 0.41	∼120	pH	CT26 cells	210
Copper ion interference	NP@ESCu	Elescomol-Cu	—	87.5	ROS	MB49 cells	92
	RBC-Zr@APC/C	CuO/Cu-MOF	8.61	184 ± 10	—	4T1 cells	211
	CuO2-MSN@TA-Cu^2+	CuO2/TA-Cu^2+	2.46	167 ± 0.344	pH	4T1 cells	212
	T-HCN@CuMS	Cu^2+	0.44	—	pH	143B cells	213
	Cu-GA	Cu-gallic acid	20	120	pH and GSH	4T1 cells	214
	CAT-ecSNA-Cu	Cu^2+	80.8	415	—	CT26 cells	215
	DMMA@Cu2−xSe	Cu2−xSe	—	130	H2O2 and pH	A375 cells	103
	Cu-MCGH	Cu-MCN	0.47	∼250	—	4T1 cells
	BSO-CAT@MOF-199 @DDM	MOF-199	—	∼50	pH	GL261 cells	64
	Au@MSN-Cu/PEG/DSF	Cu-MSN	11.5	221.81	Temperature and pH	4T1 cells	101
Zinc ion interference	Zn-LDHs	Zn-LDH	6.7	∼100	pH	B16F10 cells	167
	ZnS@BSA	ZnS	—	∼100	pH	HCC cells	161
	ALA&Dz@ZIF-PEG	ZIF-8	15.5	∼78	pH	4T1 cells	216
	HZ@GD	ZIF-8	—	117	HAase and pH	B16F10 cells	25
	DSF@Zn-DMSNs	Zn-DMSNs	26.4	230.10 ± 0.40	pH	CT26 cells	217
	UHSsPZH NPs	ZnO2	—	60.67 ± 8.57	pH	4T1 cells	171
	ZnFe-4 NPs	Zn oleate	25	32	pH	ES-2 cells	156
Manganese ion interference	IFNγ/uMn-LDHs	Mn-LDHs	—	58 ± 1	GSH and Ph	4T1 cells	218
	CDN-Mn particle	Mn complex	—	117.9 ± 41.42	—	CT26 and B16F10 cells	30
	BPNS@Mn^2+/CpG	BPNS@Mn^2+	—	∼310	Temperature and pH	4T1 cells	219
	MTHMS	MnCO	—	128.1 ± 5.9	Temperature and pH	4T1 cells	184
Sodium/potassium ion interference	K3ZrF7:Yb/Er UCNPs	K3ZrF7	—	∼20	pH	4T1 cells	187
	NaCl@ssss-VHMS	NaCl	∼39.5	∼150	GSH and pH	HepG2 cells	186

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Table 2 Biosafety and therapeutic efficacy of nanomaterials for various MIIT

Formulation	Dose	Synergy therapy	Injection modality	Tumor inhibition (%)	Biosafety	Ref.
A@P/uLDHs	5 mg kg^−1	—	i.v. injected at day 0, 4, 8, 12, 16	95	Good	220
LDHP	10 mg kg^−1	—	i.t. injected at day 0, 3, 6	∼76	Good	203
Fe-RA	12 mg kg^−1	—	i.t. injected at day 0, 2, 4, 6, 8	∼63	Good	221
AuSi@FePB	5 mg	PTT	i.v. injected at day −1, 1, 3	∼87	Good	222
PDA-DTC/Cu	5 mg kg^−1	—	i.v. injected every 2 days	66.3	Good	223
CuP/Er	Cu: 3.5 mg kg^−1	—	i.v. injection every 3 days	86.5	Good	224
PTC	5 mg kg^−1	PDT	i.v. injection at day 0, 3, 6, 9, 12, 15, 18	90	Good	225
ECPCP	ES concentration: 5 mg kg^−1	—	i.v. injected at day 0, 2, 4, 6	74.8	Good	226
CD@CaCO3P	CaCO3 dose at 15 mg kg^−1	—	i.v. injected at day 1, 4, 7	85	Good	227
PEG/CaO2@p-780	CaO2 dose at 2 mg kg^−1	PTT	i.v. injected at day 0, 2, 4, 6, 8, 10, 12	80	Good	228
CaGlu NPs	62.5 mg kg^−1	—	i.v. injected at day 0, 2, 4, 6, 8, 10, 12, 14	71.4	Good	229
ZPM@OVA-CpG	OVA: 50 μg	—	i.v. injected at day 7, 14, 21	76.2	Good	230
ZMCH	10 mg kg^−1	PDT	i.v. injected every two days	90	Good	231
Zn-LDH	1mg	—	i.t. injected at day 0, 6	88.2	Good	232
PZnO@DOX	1 mg	SDT and chemotherapy	i.v. injected at day 0, 2, 4	70.6	Good	233
MnPt	Mn: 2.5 mg kg^−1	—	i.v. injected at day 0, 2, 4, 6, 8, 10, 12	72.1	Good	234
MMP	20 mg kg^−1	—	i.v. injected at day 0, 2, 4	∼88	Good	235

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4. Summary and outlook

4.1. Summary

In this review, we systematically summarized the intrinsic mechanisms of different types of MIIT and provided an overview of the recent advancements in the corresponding amplification strategies. Based on the concept of MIIT, the most basic amplification strategy is to disrupt the ion homeostatic regulatory mechanisms in cancer cells, thus exacerbating the abnormal accumulation of metal ions. With a deeper understanding of metal ion-related metabolism, the modulation of these metabolic pathways, in conjunction with metal ion overloading, can sensitize cancer cells to MIIT. Additionally, the pursuit of further therapeutic efficacy has led to the exploration and utilization of various amplification strategies. Metal-based nanoplatforms with sono/photo-catalytic properties or photothermal conversion capacity have been also investigated to combine SDT/PDT/PTT with MIIT, resulting in enhanced therapeutic outcomes compared to single treatments. Furthermore, taking advantage of TME-responsive nanoplatforms, the spatial-temporal precision and efficacy of MIIT have been highly improved. Next, we discuss the challenges encountered by MIIT in the clinical treatment of cancers and some perspectives on its future development.

4.2. Challenges

(i) Currently, some understanding of mechanisms is still in its infancy and lacks systematicity, often attributing the therapeutic effects to a single pathway and ignoring the impact of metal ions on full cellular metabolism. In fact, tumor cell metabolism encompasses a complex interplay among various pathways, wherein perturbations in one pathway can cascade into others.

(ii) The uncertain fate of nanoparticles in the body following systemic or topical administration is a hurdle in clinical translation. Without proper surface engineering, nanomedicines are rapidly captured by the mononuclear phagocytic system (MPS), and subsequently cleared from the circulation. Also, despite evading MPS clearance, traversing the blood–brain barrier remains a formidable challenge for efficient nanoparticle accumulation at tumor sites.

(iii) Assessing the in vivo safety of degradable metal-based nanomedicines also poses a significant challenge. Given their status as trace elements in the human body, metal ions have the potential to disrupt homeostatic equilibrium. Consequently, the inevitable leakage of these ions into the body's fluid circulation poses potential risks, affecting the body's homeostatic regulatory system. For instance, the excessive leakage of calcium ions may precipitate blood clot formation in blood vessels. Hence, the judicious assessment of the introduced metal ion burden on the body represents a critical challenge that cannot be overlooked.

4.3. Perspectives

(i) The current focus of MIIT research predominantly focuses on single metal ion interference, overlooking the potential of bi-metal ion interference therapy. However, understanding the underlying mechanism of MIIT suggests the possibility of a synergistic effect from multi-metal ion interference. Compared to single MIIT, bi-MIIT exhibits mutual reinforcement in disrupting intracellular ion homeostasis, amplifying metabolic damage, and modulating the immunosuppressive TME, which is expected to improve the therapeutic efficiency of MIIT. For instance, the mitochondrial disorder induced by Ca²⁺ overload could impair ATP synthesis, and then block the copper ion efflux mediated by Cu-ATP, resulting in more severe copper ion overload and aggregation of lipoylated proteins.²³⁶ Moreover, the challenge of inefficient immunotherapy caused by the immunosuppressive TME can be overcome by the synergistic enhancement of the immune response through multi-MIIT. ICD induced by ferroptosis and the cGAS/STING pathway activated by Mn²⁺ can collectively promote DC maturation, which addresses the issue of poor therapeutic efficacy due to insufficient antigen presentation resulting from single stimulation.²³⁷ The interference of genes or proteins crossing over in metabolic pathways by different metal ions shows the potential feasibility of amplified metabolic disruption by multi-metal ion interference therapy. The latest research has shown that the degradation of Mutp53 by Zn²⁺ has the potential to reverse the high GSH content in tumor cells, consequently sensitizing them to cuproptosis and ferroptosis.²³⁸ Hence, it is imperative to recognize the potential of bi-metal ion interference in MIIT and prioritize its exploration in future developments, given that it exhibits potential to achieve more significant therapeutic effects with greater efficiency.

(ii) Emerging research has demonstrated that metal ions play an important role in immune modulation, pioneering a new form of immunotherapy known as metalloimmunotherapy. However, the current studies on metalloimmunotherapy are mainly focused on the activation and regulation of antigen presenting cells (APCs) and CTLs, while neglecting the effect NKs and B lymphocyte cells. Additionally, metal ions have the potential to down-regulate the expression of immunosuppressive cytokines and remodel the TME to reverse tumor immune escape. Therefore, future research efforts should aim to elucidate the underlying mechanisms of metalloimmunotherapy to achieve the desired anti-tumor effects comprehensively.

(iii) The surface modification of nanomaterials is essential to enhance the precision of metal ion delivery given that it can improve their internalization and targeting capabilities. Encapsulating nanomedicines with erythrocyte membranes and homologous cancer cell membranes enables them to effectively evade the immune system, leading to increased accumulation at the tumor site. Additionally, incorporating targeting molecules such as hyaluronic acid and folic acid facilitates the binding of nanomedicines to specific receptors on the membrane of cancer cells, thereby enhancing their cellular uptake. Moreover, the internalization efficiency of nanomedicines is also influenced by factors such as their size, shape, and surface charge. Thus, considering a combination of these factors can optimize the delivery of metal ions.

(iv) In xenograft mouse models, numerous metal-based nanomaterials have been designed and confirmed to possess exceptional therapeutic effects. However, the in vivo fate and ultimate therapeutic efficacy of nanomedicines are significantly influenced by critical properties such as their size, shape, and surface properties. Therefore, relying on incomplete or one-sided data from individual cases may result in overlooking certain elusive biological phenomena. By integrating detailed information from these cases, the creation of a field known as nanomedomics can enhance our comprehension of nanomedicine.²³⁹ Within nanomedomics, examining crucial factors affecting barriers such as the blood circulation, accumulation at the tumor site, intratumor penetration, and endocytosis can substantially optimize the design of nanomedicines, thereby increasing the likelihood of their successful clinical translation. Establishing a comprehensive database opens up opportunities for employing artificial intelligence and machine learning tools to design nanomedicines from diverse perspectives, challenging the conventional therapeutic effect-centered design approach. In addition to adopting a more rational design of nanomedicines, innovative techniques, strategies, and tools have been employed to address the limitations of xenograft mouse models in nanomedicine research. Researchers have turned to microfluidic tumor microarrays and 3D in vitro models to simulate the human TME and vasculature system.²⁴⁰ These models aim to provide a more comprehensive understanding of the nanoparticle enrichment at the tumor site by addressing the shortcomings of EPR-mediated passive or active targeting in xenograft models. It is acknowledged that the EPR effect in xenograft models does not accurately represent real tumors found in the human body due to the inherent tumor heterogeneity and individual factors, leading to uncertainty. As a response, patients are now being graded based on the strength of the EPR effect, increasing the likelihood of benefiting from nanomedicine therapies.

In summary, an overview of the development and strategies of MIIT can expedite the clinical application of nanomedicine. However, as a nascent therapeutic approach, MIIT still faces several challenges and opportunities for improvement. Addressing the aforementioned issues is crucial for realizing the full potential of MIIT and facilitating a qualitative leap in nanomedicine development.

Abbreviations

EPR	Enhanced permeability and retention
TME	Tumor microenvironment
GPX4	Glutathione peroxidase 4
Caspase	Cysteine aspartate-specific proteases
cGAS	Cyclic GMP-AMP synthase
STING	Stimulator of interferon genes
MIIT	Metal ion interference therapy
TCA	Tricarboxylic acid
RSL	RAS-selective lethal
ROS	Reactive oxygen species
PLOOHs	Phospholipid hydroperoxides
PUFA-PL	Phospholipid-containing polyunsaturated fatty acid chain
ACSL4	Acyl-CoA synthetase long-chain family member 4
PUFA	Polyunsaturated fatty acyl
LPCAT3	Lysophosphatidylcholine acyltransferase 3
TF	Transferrin
TfR	Transferrin receptor
NCOA4	Nuclear receptor coactivator 4
GSH	Glutathione
Cys	Cystine
BAP1	BRCA1-associated protein 1
ASC	Alanine-serine-cysteine
GSR	Glutathione–disulfide reductase
GSSG	Oxidized glutathione
FSP1	Ferroptosis inhibitor protein 1
CoQ10	Ubiquinone
CoQ10H2	Panthenol
PgP	P-glycoprotein
MDR	Multi drug resistance
POD	Peroxidase
LDHs	Layered double hydroxides
Art	Artemisinin
HSP	Heat shock protein
PTT	Photothermal therapy
LPO	Lipid hydroperoxides
GOx	Glucose oxidase
ST	Starvation therapy
CAT	Catalase
Ce6	Chlorin e6
COS	Chitosan oligosaccharides
MOF	Metal organic frame
SAS	Sulfasalazine
MTO	Mitoxantrone
IFN	Interferon
NO	Nitric oxide
FGF	Fibroblast growth factor
FDX1	Ferredoxin 1
IL	Interleukin
ETC	Electron transport chain
LIAS	Lipoyl synthase
DLAT	Dihydrolipoamide S-acetyltransferase
DBT	Dihydrolipoamide branched chain transacylase E2
GCSH	Glycine cleavage system protein H
DLST	Dihydrolipoamide S-succinyltransferase
PDH	Pyruvate dehydrogenase
α-KDH	α-Ketoglutarate dehydrogenase
ATP7a/b	Copper-transporting ATPase
Cyt C	Cytochrome C
NLRP3	Nucleotide-binding oligomerization domain-like receptor protein 3
BSO	Butythione sulfoxideimine
DDM	Dodecyl-beta-D-maltoside
DSF	Disulfiram
DTC	Diethyldithiocarbamate
CuET	Bis(diethyldithiocarbamate)–copper
NIR	Near infrared
ORAI1	Ca^2+ release-activated Ca^2+ channel protein 1
TRPCs	Transient receptor potential channels
VGCCs	Voltage-gated calcium channels
STIM1	Ca^2+ sensor protein stromal interaction molecule 1
ER	Endoplasmic reticulum
SOCE	Store-operated calcium entry
PMCAs	Ca^2+ ATPases
mPTP	Mitochondrial permeability transition pore
VDACs	Voltage-dependent anion-selective channel proteins
OMM	Outer mitochondrial membrane
MCU	Mitochondrial Ca^2+ uniporter
RyRs	Ryanodine receptors
Ins (1,4,5) P3Rs	Inositol 1,4,5-triphosphate receptors
SERCAs	Sarcoplasmic/endoplasmic reticulum Ca^2+ ATPases
CaP	Calcium phosphate
OXPHOS	Oxidative phosphorylation
SMAC	Second mitochondria-derived activator of caspase
HIF-1α	Hypoxia-inducible factor-1α
DOX	Doxorubicin
KAE	Kaempferol-3-O-rutinoside
CUR	Curcumin
GSDME	Gasdermin E
LDH	Lactate dehydrogenase
BER	Berbamine
UCNPs	Upconversion nanoparticles
PArg	Polyarginin
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase
PFK	Phosphofructokinase
NAD^+	Nicotinamide adenine dinucleotide
Mutp53	Mutant p53 proteins
WTp53	Wild-type p53 protein
GOF	Gain-of-function
UPS	Ubiquitination-mediated proteasomal
LND	Lonidamine
HK	Hexokinase
GLUT1	Glucose transporter
Car	Carnosine
ATM	Ataxia telangiectasia mutated
ECM	Extracellular matrix
ZIP	Zrt/Irt-related protein
ZnT1	Zn transporter
NKs	Natural killer cells
PK	Pyruvate kinase
APCs	Antigen presenting cells
CTLs	Cytotoxic T lymphocytes
MPS	Mononuclear phagocytic system

Data availability

Data availability is not applicable to this article as no new data were created or analyzed in this study.

Conflicts of interest

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

Acknowledgements

The authors gratefully acknowledge the financial support from National Natural Science Foundation of China (Grant No. 52101287).

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