Metalloptosis: metal ions-induced programmed cell death based on nanomaterials for cancer therapy

3.1.1 Ferroptosis

Ferroptosis as a novel Fe-dependent PCD has been discovered in 2012,^[60] which is morphologically and mechanistically distinct from common PCDs such as apoptosis, necrosis, and autophagy. Its prominent feature is an overload of lipid peroxides on cellular membranes, mitochondria shrinkage, and reduction.^[60,61] In detail, hallmarks of ferroptosis are listed as follows. (1) Morphological features: ferroptosis is accompanied by cell membrane rupture, cytoplasmic swelling, chromosome condensation, autophagosomes increasing, and mitochondrial abnormalities.^{[60,62–63–64]} (2) Biochemical features: Fe accumulation and lipid peroxidation are the 2 most important biochemical characteristics of ferroptosis. The accumulation of Fe ions in vivo can directly induce the Fenton reaction to generate excessive ROS, ultimately leading to oxidative damage-mediated ferroptosis. In addition to the classic Fe ions-induced ferroptosis, other metal ions such as Zn ions can also achieve this process,^[65] which we will discuss in detail in the following sections. On the other hand, lipid peroxidation is mainly driven by free radical, which often occurs in the cell membrane. Its mechanism is that cell membrane lipids are oxidized and produce many metabolic products such as lipid hydroperoxides and reactive aldehydes.^[66,67] These metabolic products significantly increase during the process of ferroptosis, which can be the most important biochemical detection marker of ferroptosis. (3) Genetic features: ferroptosis is regulated by various kinds of relatively specific genes and proteins, thus, their abnormal expression is often considered a detection marker for ferroptosis. These genes and proteins are often associated with substance uptake and metabolic pathways, including cystine uptake, oxidant metabolism, lipid metabolism, energy metabolism, and Fe metabolism^[68] (Table 1). (4) Immune features: the immunological consequences of ferroptosis on the immune cells are mainly manifested as the loss of their functions. For instance, ferroptosis that occurred in T cells led to a decrease in their antiviral or antiparasitic function.^[74] More importantly, the immune system’s response is determined by ferroptosis-induced cell death. This is because various types of cell death can induce distinct immune reactions through the activation of different damage-associated molecular pattern (DAMP) signals. For instance, death cells induced by ferroptosis could activate the nuclear factor-κB pathway^[75] and target lipid metabolism-related DAMP signaling,^[76] eventually triggering the inflammatory response in peripheral macrophages.^[77] In short, the discrimination of ferroptosis is clear and specific, which emphatically includes the following points: ROS generation, lipid peroxidation, Fe abundance, glutathione peroxidase 4 (GPX4) activity, GSH depletion, release of glutamate, and cystine uptake, SLC7A11 expression and so on. These indicators are essential for detection and study in the context of ferroptosis research.

In terms of mechanisms, Fe ions-induced ferroptosis was initially discovered, and as research progressed, researchers have gradually found that the ferroptosis pathway is not solely dependent on Fe ions, some Fe-independent pathways, such as ROS metabolism (particularly lipid peroxides) and GSH metabolism, can also induce ferroptosis. Hence, the metabolic pathways of ferroptosis are mainly summarized into the following 3 pathways: Fe metabolism, ROS metabolism, and GSH metabolism (Figure 2).^[78] According to different triggering mechanisms of ferroptosis, different treatment strategies based on nanotechnology can be adopted.

3.1.1.1 Fe metabolism.

Fe ions play an essential role in inducing ferroptosis whether through enzymatic or nonenzymatic reaction. Thanks to the Fe transport systems, intracellular Fe ions can always maintain a delicate balance under normal conditions. Extracellular Fe ions can be transferred into the cytoplasm by transferrin (TF) and its corresponding transferrin receptor (TFR) and stored in the ferritin.^[61] On the other hand, intracellular Fe ions are exported outside the cell through ferroportin (FPN), which is the only known Fe ions exporter in mammal.^[79] When Fe ions accumulate in the cytoplasm, disrupting the balance of Fe ions, it can trigger oxidative stress responses and the following ferroptosis, leading to cellular damage.^[80] Interestingly, many studies have demonstrated that cancer cells have a higher demand for Fe than normal cells to maintain their rapid proliferation, specifically manifested as downregulation of FPN and upregulation of TFR1.^[70,81] Based on the high dependence of cancer cells on Fe content, cancer cells are more susceptible to Fe overload or oxidative stress compared to normal cells, thus, inducing ferroptosis by altering the intracellular Fe ion content has become one of the emerging ways to treat cancers.^[82]

At present, the majority of NMs triggering ferroptosis by changing the accumulation of Fe ions in the cytoplasm are Fe-based NMs. They can be internalized by cancer cells and degraded into ferrous (Fe²⁺) or ferric (Fe³⁺) ions in lysosomes, thereby directly boosting the intracellular Fe ions content. The excessive Fe ions can further trigger the Fenton reaction, inducing ferroptosis to kill cancer cells. For instance, the most common Fe-containing NPs are Fe₃O₄ NPs. Liang et al reported self-assembled ultrasmall Fe₃O₄ NPs with the surface modification of porphyrin-grafted lipid (PGL) to enhance ROS production and lipid peroxidation. These ultrasmall Fe₃O₄@PGL NPs with a diameter of ∼10 nm exhibited good biosafety (Figure 3A). In terms of treatment mechanism, the Fe₃O₄@PGL NPs generated abundant ROS through porphyrin-mediated photodynamic therapy (PDT), which is beneficial for the occurrence of ferroptosis. Moreover, the results indicated that the content of ROS produced was the highest only when the coincubation of NPs endocytosed by HT-29 cells and RAW264.7 cells, which suggested that Fe₃O₄@PGL NPs could also enhance the anticancer ability of ferroptosis through modulating macrophages in the TME (Figure 3B, C).^[83] To further enhance the catalytic activity of Fe-based NPs and improve their efficiency in inducing ferroptosis, Liang et al provided an effective therapy strategy to ameliorate the low ROS production efficacy of current Fe-based NPs through synthesizing body-centered cubic-ultrasmall single-crystal Fe NPs (bcc-USINPs) (Figure 3D). These bcc-USINPs remained stable in the normal physiological environment and reacted with weak acids in the TME due to their selective acidic etching of the Fe₃O₄ shell and the exposure of the Fe(0) core. The results of the catalytic activity analysis showed that bcc-USINPs exhibited superior ROS generation ability than other NPs (Figure 3E), which proved that bcc-USINPs have a stronger ability to induce ferroptosis than other NPs. More importantly, bcc-USINPs also triggered immunotherapy to enhance the synergistic effect of ferroptosis treatment. In detail, bcc-USINPs could efficiently induce immunogenetic cell death at a very low concentration (1 mg/kg) by promoting the maturation of dendritic cells (DCs) and enhancing the adaptive T cell response (Figure 3F–I). The bcc-USINPs-mediated ferroptosis treatment, when combined with programmed death-ligand 1 (PD-L1) immune checkpoint blockade immunotherapy, developed a strong immune memory, thereby potentiating the immune response. This Fe(0) delivery system based on bcc-USINPs provided an effective strategy for ferroptosis-based immunotherapy.^[84] Considering that the efficiency of ferroptosis induced by Fe-based NPs is related to the penetration depth of NPs at the tumor site, Wang et al prepared a kind of stimuli-responsive size-switchable nanocapsules with Au-Fe₂C Janus NPs as ferroptosis agents and sorafenib (SRF) as the ferroptosis enhancer. These nanocapsules exhibited superior intratumoral accumulation compared to free Au-Fe₂C NPs, subsequently penetrating deeper into tumor regions. They disrupted the intracellular redox balance, eventually leading to efficient treatment through ferroptosis.^[38]

It is worth noting that the induction of ferroptosis is not limited to Fe ions, and many other metal ions can also achieve ferroptosis through non-Fe ions-dependent pathways, such as Mn ions and Zn ions, which will be discussed in the following sections.

3.1.1.2 GSH metabolism.

Reduced GSH is the major intracellular antioxidant in mammals, whose biosynthesis is subject to the influence of cystine and cysteine. In the triggering mechanism of ferroptosis, GSH actually is a negative regulatory factor. Blocking cystine acquisition from the extracellular environment or inhibiting the biosynthesis of GSH can trigger ferroptosis.^[85] Many studies have demonstrated that GSH is strongly associated with cancer aggressiveness and chemoresistance, however, the existing therapeutic drugs targeting GSH cannot effectively scavenge GSH, so the relevant treatment can only serve as a supplementary strategy of cancer treatment.^[86] The emergence of nanotechnology has brought a breakthrough opportunity to solve this problem. Wang et al prepared a kind of ferroptosis-inducing agent based on arginine (Arg)-capped manganese silicate nanobubbles (AMSNs) with the efficient consumption of GSH and inactivation of GPX4. In this nanosystem, taking advantage of the argininosuccinate synthetase deficiency present in cancers, Arg as the modified surfacing ligand allowed AMSNs to have good biocompatibility and tumor homing capacity, which further dramatically enhanced the efficiency of GSH depletion and GPX4 inactivation. AMSNs were degraded to release Mn ions of higher valence (Mn³⁺ and Mn⁴⁺) in response to the weakly acidic conditions in TME. The released Mn³⁺ and Mn⁴⁺ were further reduced to Mn²⁺ in the presence of GSH, which precisely promoted the depletion of GSH. This depletion of GSH subsequently led to the upregulation of intracellular oxidative stress and the downregulation of GPX4 expression, collectively improving the treatment efficiency of ferroptosis. Meanwhile, the hollow structure also endowed AMSNs with the ability as drug delivery vehicles. Doxorubicin (Dox)-loaded AMSNs could suppress tumor growth more effectively compared with free Dox. In terms of bioimaging, Mn ions contributed to the T₁-weighted magnetic resonance imaging (MRI), ultimately achieving imaging-guided on-demand synergistic chemotherapy and ferroptosis treatment.^[87]

In addition to ameliorating the inhibitory effect of GSH on ferroptosis, targeting other antiferroptotic signaling pathways also emerges as a potent strategic approach to enhance synergistic therapeutic effects. Ferroptosis suppressor protein 1 (FSP1) can mediate detoxification of lipid peroxides through decreasing nicotinamide adenine dinucleotide phosphate (NADP⁺)/nicotinamide adenine dinucleotide phosphate (NADPH) ratio and catalyzing the recycling of coenzyme Q10 (CoQ10) to ubiquinol (CoQ10H2) for trapping lipid peroxyl radicals. The decreasing NADP⁺/NADPH ratio can further promote the conversion of cystine to cysteine and the biosynthesis of GSH and GPX4.^[88,89] Consequently, devising a treatment strategy that is capable of simultaneously inhibiting both GSH- and FSP1-mediated ferroptosis-suppressing mechanisms holds considerable therapeutic potential. Li et al developed a Cu-tetra(4-carboxyphenyl)porphyrin chloride(Fe(III)) (Cu-TCPP(Fe))-based MOF nanosystem, incorporated with Au NPs via in situ nucleation and the ferroptosis inducer Ras-selective lethal small-molecule 3 (RSL3) via π–π stacking, meanwhile modified with polyethylene glycol (PEG) and internalizing RGD peptide (iRGD) for tumor-targeted drug delivery. Mechanistically, except for the classic Fe ions-induced ferroptosis, the introduction of Au NPs caused glucose depletion because of their glucose oxidase-like activities, eventually disrupting the pentose phosphate pathway and hindering reduced GSH biosynthesis. Meanwhile, Cu ions consumed GSH by oxidizing it to glutathione (GSSG), which could suppress the GSH-mediated antiferroptotic pathway in tumor cells for amplifying ferroptotic damage. Moreover, the loaded ferroptosis inducer RSL3 could deactivate the function of GPX4 to further result in pronounced ferroptotic damage. Nonetheless, Au NPs could also increase the NADP⁺/NADPH ratio and prevent the recycling of CoQ10 to CoQ10H2, impairing the FSP1-mediated antiferroptotic pathways (Figure 4A). Specifically, Au/Cu-TCPP(Fe) nanosheet was synthetized with a typical 2-dimensional morphology and its average size was between 100 and 200 nm (Figure 4B). The functional incorporation of the Au NPs and Cu species efficiently depleted the glucose and GSH in the tumor intracellular environment, which plays critical roles in the subsequent ferroptosis sensitization (Figure 4C). The results of evaluation experiments on the GOx-like catalytic activity demonstrated that the Au/Cu-TCPP(Fe) nanosheet consistently reduced the glucose concentration over a prolonged incubation time, with the final relative levels dropped by around 51.7% after 24 h (Figure 4D). On the other hand, as shown in Figure 4E, both Cu-TCPP(Fe) and Au/Cu-TCPP(Fe) could reduce the concentration of GSH in a dose-dependent manner. After incubation with a nanosheet (100 μg/mL) for 24 h, the concentration of GSH was decreased to around 81.4% and 82.1%, evidently suggesting the GSH-consuming ability of the Cu species in the nanosheet. Furthermore, the effect of the catalytic activity of composite nanosheets on 2 independent regulatory pathways of ferroptosis (GPX4/GSH pathway and FSP1/CoQ10H2 pathway) was systematically evaluated. In terms of GPX4/GSH pathway, the results indicated that the GSH-oxidizing capability of the Cu species in the Cu-TCPP(Fe)-PEG and Au/Cu-TCPP(Fe)-PEG nanosheets caused an evident decrease of the cellular GSH (Figure 4F) and increase of the cellular GSSG levels (Figure 4G), which was consistent with the results of reduced expression of GPX4 induced by both Cu-TCPP(Fe)-PEG and Au/Cu-TCPP(Fe)-PEG (Figure 4H, I). In the terms of FSP1/CoQ10H2 pathway, Au/Cu-TCPP(Fe)-PEG could enhance the ratio of NADP⁺/NADPH (Figure 4J) and weaken the ratio of CoQ10H2 to CoQ10 (Figure 4K) in a dose-dependent manner. These changes suggested that Au NPs in the Au/Cu-TCPP(Fe)-PEG nanosheets lowered the abundance of CoQ10H2 by the consumption of glucose and the decrease of NADPH content, potentially compromising the antiferroptotic capability of FSP1 proteins. In brief, according to different signaling pathways induced by ferroptosis, this nanosystem promoted the occurrence of ferroptosis and optimized the cell lethal effect of ferroptosis, providing a strong rationale for the application of ferroptosis therapy in the clinic (Figure 4L).^[90]

Moreover, as a central downstream regulator of ferroptosis in GSH metabolism, GPX4 combats with lipid peroxidation by transforming toxic phospholipid hydroperoxides into nontoxic phospholipid alcohols, with the generation of oxidized GSH (GSSG) as byproducts.^[91] Many chemical or biological methods can intervene in the expression of GPX4 and have also shown initial effectiveness in inducing ferroptosis.^[80,92,93] Therefore, targeting GPX4 is a powerful therapeutic strategy for ferroptosis-based cancer therapy^[94] and various kinds of nanoplatforms have been developed for suppressing GPX4.^[87,95,96] Among them, 2 common solutions are as follows: first, NMs can act as the delivery system to transport small-molecule inhibitors of GPX4 in vivo, which is capable of overcoming certain defects, such as rapid clearance from systemic circulation and poor tumor enrichment efficiency; second, NMs can reduce the expression of GPX4 by utilizing their own intrinsic physicochemical properties.^[87,96] Inspired by industrial electro-Fenton technology featured with electrochemical iron cycling, Liu et al constructed a network-like corona spontaneously consisting of Fe³⁺ ion and naturally derived tannic acid (TA) to wrap the SRF nanocores, which is a typical small-molecule GPX4 inhibitor (Figure 5A). The results showed that the formed SRF@Fe^IIITA (SFT) NPs could significantly decrease the level of GSH (Figure 5B) and GPX4 (Figure 5C) for ferroptosis initiation. Meanwhile, TA chemically reduced Fe³⁺ to Fe²⁺, offering iron redox cycling to effectively sustain the Fenton reaction and produce the lipid peroxide that was required in ferroptosis (Figure 5D). In vivo results indicated that this rational material design could advance the antitumor effect of ferroptosis through the continuous internal supply of Fe ions combined with the specific GPX4 inhibitor (Figure 5E).^[95] Moreover, the reconstituted NMs with docosahexaenoic acid were also proved to cause inactivation of lipid antioxidant GPX4 and accumulation of lipid hydroperoxides, with the consequence of significant cancer suppression.^[96]

Interestingly, as the study continues to deepen, researchers have gradually discovered some new signaling pathways of ferroptosis. The latest research indicated that supplementation with dihydroorotate or orotate, the substrate and product of dihydroorotate dehydrogenase (DHODH), could attenuate or potentiate ferroptosis induced by inhibition of GPX4, respectively, and these effects were especially pronounced in cancer cells with low expression of GPX4 (GPX4^low). Inactivation of DHODH could lead to extensive mitochondrial lipid peroxidation and ferroptosis in GPX4^low cancer cells and produce the same phenomenon in GPX4^high cancer cells with the combination of ferroptosis inducers. All these results identified a new mechanism of ferroptosis mediated by DHODH and suggested a therapeutic strategy of targeting DHODH in cancer ferroptosis treatment.^[97]

3.1.2 Cuproptosis

Copper is an essential trace element for the human body, which is primarily obtained from food. The current recommended copper intake for adults is 0.8 to 2.4 mg/d to maintain whole-body copper homeostasis.^[101] Statistically, an adult human body contains about 100 mg of copper.^[102] In one regard, copper is integral to various physiological processes in the body, acting as a catalytic and structural cofactor for a range of enzymes that facilitate critical biochemical reactions. Its role is particularly prominent in enzymes such as cytochrome c oxidase and superoxide dismutase 1, which are pivotal for cellular energy production and antioxidant defense, respectively.^[103] Beyond these functions, copper is also vital for the synthesis of neurotransmitters,^[104] iron metabolism,^[105] peptide hormone maturation, and blood clotting.^[102] While an optimal amount of copper is beneficial for the organism, excessive copper levels can precipitate a sequence of cellular metabolic disturbances that ultimately lead to cell death. Early research attributed this type of death to the accumulation of copper, which disrupts the delicate balance between the generation of ROS and the body’s antioxidative defense mechanisms.^[33] As of 2022, the notion of “cuproptosis” was formally published within the scientific community, representing a novel cellular demise pathway distinct from previously characterized mechanisms such as apoptosis, necrosis, and autophagy.^[106]

Compared to ferroptosis, cuproptosis represents a relatively novel concept in the field of metal ions-mediated cell death research. It predominantly hinges on the intracellular accumulation of copper ions, which leads to the aggregation of lipoylated proteins and subsequent loss of iron-sulfur (Fe-S) cluster proteins. This sequence of events triggers proteotoxic stress, culminating in cell death. Specifically, researchers employed the ionophore elesclomol (ES) to facilitate the transport of Cu²⁺ into cells, where an excess of Cu²⁺ was subsequently transported to mitochondria and reduced to the more toxic Cu⁺ by the mammalian ferredoxin 1 (FDX1), a critical player in cuproptosis process. Besides reducing Cu²⁺, FDX1 also lipoylates specific mitochondrial enzymes, such as dihydrolipoamide S-acetyltransferase (DLAT), crucial for maintaining normal cellular metabolism and mitochondrial functionality. When Cu⁺ binds to DLAT, it triggers the aggregation of this enzyme, subsequently impeding the tricarboxylic acid cycle. Concurrently, Cu⁺ diminishes the synthesis of Fe-S clusters. These dual actions collectively lead to cellular demise.^[106] A detailed illustration of the cuproptosis mechanism is provided in Figure 6A, offering a visual representation of how these interactions converge to induce cell death.

The cuproptosis pathway is characterized by high levels of lipoylated proteins, including glycine cleavage system protein H, dihydrolipoamide branched chain transacylase E2, dihydrolipoamide S-succinyltransferase, and aforementioned DLAT.^[107] These proteins’ lipoylation is controlled by enzymes such as lipolytransferase 1, lipoyl synthase (LIAS), and dihydrolipoamide dehydrogenase, and FDX1, with FDX1 playing a key role in initiating the lipoylation process.^[108,109]

The utilization of ionophores such as ES and disulfiram (DSF) to transport copper ions into tumor cells represents a direct and common method to induce cuproptosis.^[110] Ionophores can traverse biological membranes by forming stable complexes with ions and, upon specific stimuli, release metal ions, thereby altering intracellular and extracellular ion concentrations.^[111] However, the efficacy of existing small-molecule ionophores in inducing cuproptosis in tumor cells is somewhat limited due to their short blood half-life.^[112] The advancement of nanotechnology offers a solution to this issue by constructing nanomedicines that incorporate ionophores alongside other components, enhancing the properties of the drug delivery system. Such nanomedicines not only address the aforementioned challenges but also equip therapeutic agents with multifunctional features. They enhance cuproptosis effectiveness by promoting synergistic treatments with immunotherapy and chemo-chemodynamic therapy, or by enabling image-guided approaches for tumor therapy.^[34,113] For instance, Guo et al developed a nanomedicine, NP@ESCu, by encapsulating elesclomol-Cu with a ROS-responsive polymer (PHPM), as shown in Figure 6B. Owing to the thioketal bonds in PHPM that were sensitive to ROS, NP@ESCu released its contents when exposed to elevated ROS levels within cancer cells, leading to cuproptosis. Furthermore, NP@ESCu enhanced PD-L1 expression on cancer cells, caused maturation of DCs, and enhanced CD8⁺ T cell infiltration in tumor sites. It also triggered a shift from tumor-associated macrophages from the immunosuppressive M2 phenotype to the immunostimulatory M1 phenotype, thereby augmenting immunotherapy efficacy. The combined use of NP@ESCu and αPD-L1 in a bladder cancer mouse model demonstrated superior therapeutic outcomes, illustrating the potential of NP@ESCu as a multifaceted approach to cancer treatment.^[34] In another study, researchers used DSF/Cu-based nanomedicine to induce cuproptosis. By encapsulating DSF/Cu complexes within a tumor TME-responsive core-shell dendrimer, the resulting CSTD-Cu(II)@DSF achieved MRI-guided synergistic therapy by combining cuproptosis and chemo-chemodynamic treatment.^[113]

In addition to using ionophores for copper delivery, alternative NMs such as MOFs,^[47,49,56] inorganic NPs,^{[39,40,50,114]} and carbon nitride^[115] can also facilitate copper ions delivery into cells, effectively triggering cuproptosis. For example, Xu et al explored amorphous metal-organic frameworks (aMOF) as cuproptosis carriers due to their unique hollow structure and amorphous properties. By incorporating Cu²⁺ and Fe³⁺ during the synthesis and modifying with Dox-loaded, functionalized hyaluronan, the resultant Dox@Fe/CuTH HaMOF enhanced the intracellular copper/iron concentration in tumor cells, inducing a synergistic antitumor effect through cuproptosis, ferroptosis, and apoptosis. The inductively coupled plasma–mass spectrometry (ICP-MS) data indicated that, with prolonged incubation of the Dox@Fe/CuTH with 4T1 cells, the intracellular copper content increased 14.6 times compared to the PBS-treated control group (Figure 6C). The rise in copper levels ultimately induced the occurrence of cuproptosis in tumor cells, as evidenced by the pronounced DLAT foci in cells treated with Dox@Fe/CuTH, attributed to abnormal oligomerization of DLAT (Figure 6D). Moreover, western blot results showed a significant reduction in the levels of FDX1 and LIAS in the Dox@Fe/CuTH-treated group compared to the control group. The destabilization of these proteins further confirmed the capability of the designed nanomedicine to induce cuproptosis.^[47] Using a hydrothermal method, copper hydroxyphosphate NPs (Cu₂(PO₄)(OH) NPs) were synthesized (Figure 6E), which could respond to endogenously overexpressed H₂S to transform into ultrasmall copper sulfide NPs. This size transformation enhanced tumor cell uptake of the NPs, elevated intracellular ROS levels, and increased intracellular copper ion concentrations, ultimately leading to cuproptosis and pyroptosis. It is noteworthy that within an H₂S-enriched microenvironment, Cu₂(PO₄)(OH) NPs were able to cause mitochondrial damage and consequently decreased the expression of copper-specific efflux proteins such as ATP7A (Figure 6F). This reduction led to decreased copper excretion, further facilitating the occurrence of cuproptosis. As shown in Figure 6F, the observed reduction in FDX1, succinate dehydrogenase B (SDHB), and aconitase-2 (ACO-2) levels following the treatment with Cu₂(PO₄)(OH) NPs in an H₂S environment indicated the successful initiation of cuproptosis. After a 16-day treatment period, the tumors in H₂S-overexpressed colon cancer-bearing mice, treated with Cu₂(PO₄)(OH) NPs, showed significant suppression, as depicted in Figure 6G.^[40] By utilizing various NMs, these innovative approaches provide a range of mechanisms for “encapsulating” and “releasing” copper ions in cancer cells, thereby broadening the array of tools available for targeted cancer treatment via cuproptosis.

The above-discussed methods leverage nanoplatforms to elevate intracellular copper levels, inducing cuproptosis. As Figure 6A illustrates, there is a complex link between cuproptosis and cell metabolism. Modifying tumor metabolism to heighten copper sensitivity presents another viable strategy for inducing cuproptosis. Given that GSH, an endogenous copper chelator, can mitigate the therapeutic effect of cuproptosis by reducing free intracellular copper levels, incorporating components that deplete GSH or inhibitors blocking GSH synthesis into nanomedicines could potentially enhance the antitumor efficiency of cuproptosis. For example, researchers ingeniously utilized glucose oxidase (GOx) to respond to GSH in tumor cells, creating a nanomedicine (called GOx@[Cu(tz)]) based on nonporous copper(I) 1,2,4-triazolate ([Cu(tz)]) coordination polymer. This nanomedicine achieved dual depletion of glucose and glutathione, facilitating cuproptosis-based synergistic cancer therapy. From the intracellular GSH levels test in Figure 6H, it is evident that there was a significant decrease in GSH within tumor cells after treatment with GOx@[Cu(tz)]. Furthermore, in vivo animal experiments further confirmed that GOx@[Cu(tz)] exhibited excellent tumor suppression effects (Figure 6I).^[54] Additionally, another study incorporated buthionine-sulfoximine (BSO) to inhibit GSH synthesis and catalase (CAT) to decompose endogenous hydrogen peroxide, constructing BSO-CAT@MOF-199@DDM nanomedicine that sensitized tumor cells to cuproptosis by alleviating hypoxia and reducing GSH synthesis.^[56]

As a crucial subset of metalloptosis, research on cuproptosis is still in its infancy, with ongoing exploration into its mechanisms and the design of cuproptosis-inducing nanomedicines. Questions about the specific roles of key factors such as FDX1, specific compounds for cuproptosis, the resistance mechanisms of cuproptosis-inducing nanomedicines, the morphological features of cuproptosis, and the interaction with other cell death pathways remain open. Future designs should focus on optimizing nanomedicine transport efficiency, blood retention, tumor targeting, biocompatibility, and metabolic pathways. It is noteworthy that while the therapeutic efficacy of cuproptosis is limited when used in isolation, synergistic cancer treatments achieved through strategic NMs design can significantly enhance outcomes. Particularly, some copper-containing NMs exhibit unique properties such as exceptional photothermal conversion efficiency, catalytic activity, and MRI capabilities.^[116,117] These features hold promise for utilizing imaging techniques to achieve precise spatiotemporal delivery of copper and multimodal synergistic cancer treatment.

3.1.3 Calcium overload

Calcium ions (Ca²⁺) signaling pathway is a fundamental molecular regulation pathway that controls many cellular biological processes such as cell proliferation and apoptosis. The endoplasmic reticulum (ER) that stores the primary intracellular Ca²⁺ coordinates with other organelles and the plasma membrane to maintain the normal level of Ca²⁺. However, the imbalance of Ca²⁺ homeostasis has been considered as an important driving force in the initiation and progression of cancers.^[118] The Ca²⁺ signaling pathway is finely tuned by complex Ca²⁺ channels, transporters, pumps, exchangers, and calcium-binding proteins.^{[119–120–121–122]} Under normal circumstances, the concentration of Ca²⁺ in the cytoplasm is lower than the level of the extracellular environment.^[123,124] The significant difference between intracellular and extracellular Ca²⁺ concentrations precisely creates an opportunity for Ca-mediated cancer treatment strategies.^[119] In recent years, calcium overload as an emerging cancer therapy has been proved to disrupt the intracellular calcium homeostasis to cause irreversible cell damage.^[125,126] Hence, calcium-based nanomaterials for inducing calcium overload have received increasing attention in the interdisciplinary field of medicine and materials.^[41,127]

Due to the excellent biocompatibility, bioactivity, and biodegradability, calcium-based NPs^{[128–129–130–131–132–133]} such as calcium carbonate (CaCO₃), calcium phosphate (CaP), calcium carbonate (CaC), calcium silicate (CaSi), and calcium fluoride (CaF₂) can directly and effectively increase the level of cellular basal calcium.^[127] Their treatment mechanisms are also diverse, including calcium overload stress, ROS production, mitochondrial dysfunction, cell calcification, and abnormal immune response, which are all induced by abnormal changes in calcium concentration, especially increased intracellular Ca²⁺. Among the various calcium-based materials employed for treating calcium overload in tumors, CaCO₃ stands out as the most prevalent choice. For instance, Li et al fabricated a kind of therapeutic nanoplatform (M@CaCO₃@KAE) consisting of CaCO₃ NPs loaded with Kaempferol-3-O-rutinoside (KAE) that could effectively disrupt calcium homeostasis regulation and facilitate calcium influx. These NPs were incorporated with the cancer cell membrane (M) to ensure the targeted delivery (Figure 7A–C). Because of the response to the weak acid of TME, M@CaCO₃@KAE NPs specifically released KAE and Ca²⁺, which could break the balance of Ca²⁺ homeostasis through the role of KAE in promoting the introduction of Ca²⁺ and the continuous replenishment of Ca²⁺ by CaCO₃. The specific manifestation was the expression changes of calcium-regulated proteins (Figure 7D), indicating the serious consequences of calcium overload such as the magnified destruction of mitochondrial structure and functions, the increased cytoskeleton collapse and oxidative stress, and the ultimate cancerous cellular apoptosis (Figure 7E).^[134] Although the currently reported Ca²⁺ nanogenerators,^[137] including CaCO3^[138] and calcium peroxide (CaO₂),^[41] can impair the Ca²⁺ homeostasis of the mitochondrion to induce the calcium overload-mediated cancer therapy, there are still some limitations that need to be broken through. For example, the intramitochondrial concentration of Ca²⁺ can quickly return to a normal level owing to natural Ca²⁺ excretion via the Ca²⁺ channel, resulting in an inferior anticancer effect. Hence, designing a kind of easily-prepared Ca²⁺ nanogenerator that could continuously release Ca²⁺ is an urgent problem to be solved. Based on the above issues, Zheng et al synthesized a kind of a multichannel Ca²⁺ nanomodulator (CaNM_CUR+CDDP) encapsulated with cisplatin (CDDP) and curcumin (CUR) in hybrid CaCO₃ NPs (Figure 7F). First, CaCO₃ NPs released a mass of Ca²⁺, resulting in the excessive accumulation of Ca²⁺ in mitochondria; then, CUR further promoted the release of Ca²⁺ from the endoplasmic reticulum to the cytoplasm and inhibited Ca²⁺ efflux; finally, CDDP detrimentally affected the structural integrity and metabolic functionality of mitochondria by inducing DNA damage, leading to the mitochondrial dysfunction (Figure 7G).^[135] In addition, calcium overload caused by CaCO₃ can be effectively managed in conjunction with other treatment methods, such as immunotherapy, improving the overall efficacy of synergistic therapy. Zheng et al further fabricated acid-sensitive PEG-decorated CaCO₃ NPs incorporating CUR (^PEGCaCUR) to systematically explore their immune function (Figure 7H). In accordance with the schematic diagram for in vivo treatment (Figure 7I), the ^PEGCaCUR group, when subjected to ultrasound (US), demonstrated a preferential enhancement in the maturation of DC and activation of CD8 T cells, in comparison to the other groups. This suggested the good effect of mitochondrial Ca²⁺ overload-induced immunogenic cell death (ICD) induced by ^PEGCaCUR NPs (Figure 7J, K).^[136]

In addition to CaCO₃, calcium oxide (eg, CaO₂) is also an available Ca²⁺ nanogenerator. Zhang et al prepared a kind of pH-sensitive sodium-hyaluronate-modified calcium peroxide nanoparticles (SH-CaO₂ NPs) to induce an abnormal cytoplasm accumulation of free Ca²⁺. Interestingly, these NPs created an artificial calcium-overloading stress in tumor cells, while normal cells were more tolerant of the adverse influence of NPs than tumor cells. Meanwhile, the enrichment of Ca²⁺ showed the potential to cause tumor calcification, which may further inhibit the growth of tumor tissue.^[41]

3.2.1 Zinc ions-induced cell death

Zinc (Zn) is a necessary trace element for the daily activities of the body, ranking second in abundance. It is closely associated with various biological functions, including cell cycle progression, signal transduction, gene expression, immune functions, meiosis, and numerous other physiological processes.^[139] Statistical data indicate that the activity of around 300 enzymes and the structural integrity of approximately 2000 transcription factors are zinc dependent.^[140] Women typically have around 1.5 g of zinc in their bodies, while men have about 2.5 g, predominantly located in the bone and skeletal muscle.^[141] Zinc homeostasis dysregulation is closely linked to cancer, with zinc ions not only induce ferroptosis or calcium overload but also directly cause apoptosis, necrosis, etc. Although zinc ions-mediated cell death has not yet been classified as a new type of PCD, it possesses distinct characteristics and will be the focus of discussion in this section.

Recent research increasingly highlights the significant role of zinc in ferroptosis. Zinc oxide NPs (ZnO NPs), approved as a safe substance by the United States Food and Drug Administration, exhibit good biocompatibility and cost-effectiveness, offering broad application prospects in the biomedical field.^[142] A study by Zhang et al in 2020 demonstrated that ZnO NPs could trigger ferroptosis in human umbilical vein endothelial cells (HUVECs) by increasing ROS, lipid peroxidation, and depleting glutathione (Figure 8A–D).^[143] Further investigations revealed that the zinc chelator N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) could protect cells from erastin-induced cell death; supplementation with zinc alone (ZnCl₂) or together with the iron chelator deferoxamine significantly heightened the sensitivity of MDA-MB-231 (Figure 8E) and HT-1080 cells to ferroptosis.^[144] Additional studies identified that ZIP7 plays a crucial role by regulating zinc transport from the ER to the cytosol, essential for ferroptosis. Blocking ZIP7’s function genetically or chemically safeguarded cells against ferroptosis, a protection that was negated with the addition of zinc (Figure 8F).^[144] These findings confirmed that Zn²⁺ and ZnO NPs are capable of inducing ferroptosis, suggesting that zinc administration could serve as a promising approach to promote the death of cancer cells.

Moreover, zinc overload can impair mitochondrial function and promote organelle death, coordinating with Ca²⁺-induced damage to disrupt intracellular calcium homeostasis, ultimately leading to cell death.^[146,147] While Zn²⁺ and Ca²⁺ are known to share many transport pathways, the precise nature of their interplay in the context of PCD remains to be clearly defined.

Lysosomes are key organelles in cells, fundamental for metabolism and protein breakdown, and crucial for cell growth, division, and differentiation.^[148] Unstable Zn²⁺ within cells is partly stored in lysosomes. Just as with iron, Zn²⁺ can also be released from its unstable reserve, influencing mitochondrial and lysosomal functions, thereby inducing zinc ions-mediated cancer cell death.^[149] Mucolipin TRP channel 1 (TRPML1/MCOLN1; ML1) is a cation channel that allows the passage of Ca²⁺ and Zn²⁺/Fe²⁺, mainly found on the membranes of late endosomes and lysosomes in all mammalian cells.^[150] It is essential for several lysosomal activities, such as transport, exocytosis, biogenesis, and heavy metal homeostasis. In metastatic melanoma cells with maladaptive upregulation of the ML1 lysosomal channel, Du et al discovered a kind of lysosomal Zn²⁺ release triggering nonapoptotic cell death, which they termed as lysozincrosis.^[145] Upon treating these cells with TRPML-specific synthetic agonist (ML-SA), lysosomal Zn²⁺ release was triggered, leading to mitochondrial swelling/damage and rapid depletion of cellular ATP. This process induced necrosis, as illustrated in Figure 8G. As expected, administering ML-SA5, a specific type of ML-SA, significantly reduced tumor volume in MeWo-FmC-bearing mice over a 42-day treatment period without any relapse, demonstrating a more pronounced antitumor effect compared to the control group (Figure 8H, I).^[145]

It is worth noting that the influence of zinc ions on cancer is notably varied, depending on the specific type of cancer involved.^[140] Besides the aforementioned lysozincrosis in metastatic melanoma, studies indicate that ZnO NPs particularly induce cancer cell death through the apoptotic pathway in certain cancer cell lines, such as cervical carcinoma, lung carcinoma, head and neck squamous cell carcinoma, urinary bladder carcinoma, and head and neck squamous cell carcinoma.^[151,152] Additionally, reports indicate that ZnFe₂O₄-based nanosystems could programmatically initiate and amplify the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)/stimulator of interferon genes (STING) signaling pathway in tumor cells, thereby inhibiting tumor progression and postoperative recurrence.^[153]

In summary, current studies on zinc ions-mediated cell death mainly align with established PCD pathways, with no specific cell death mechanisms attributed to zinc ions induction yet identified. Research into the anticancer mechanisms of zinc ions-based nanomedicine, especially focusing on ZnO, remains relatively underexplored. Moreover, given the varying dependence of different cancer cells on zinc ions, establishing a universal principle for zinc ions-mediated cell death requires further investigation. However, it is undeniable that disrupting the zinc homeostasis in cancer cells represents a promising anticancer strategy.

3.2.2 Sodium ions-induced cell death

Sodium ions (Na⁺), a kind of common physiological ion, are crucial for normal cell function, typically existing at concentrations an order of magnitude higher extracellularly (145 mM) than intracellularly (12 mM).^[154] Conversely, intracellular potassium (K⁺) levels are maintained at a higher concentration. This asymmetry in Na⁺/K⁺ concentration gradients is vital for amino acid transport, pH maintenance, and cell volume regulation.^[155,156] Disrupting sodium homeostasis in tumor cells can induce significant osmotic changes, leading to apoptosis or necrosis. However, targeting tumor cells with excess Na⁺ is challenging due to the strict cellular ion transport regulation of live cells and the short circulation lifetime of single-ion delivery. Ingeniously designed sodium-based NMs can bypass these hurdles, as they enter cells via endocytosis and release Na⁺ internally, increasing osmotic pressure and causing selective tumor cell death while sparing normal cells due to their lower sodium levels.^[157]

However, ionic compounds such as sodium chloride (NaCl) and sodium bicarbonate (NaHCO₃), selected for their availability and excellent biocompatibility, are challenging to encapsulate in NMs due to their high solubility in water.^[158] This makes the delivery of sodium ions to tumor cells using NMs a complex task. It is able to overcome these challenges through kinds of preparation strategies, ensuring stable encapsulation and effective delivery within the TME. Jiang et al used sodium oleate and molybdenum chloride as precursors, with oleylamine as a surfactant, to synthesize pure-phase NaCl nanoparticles (SCNPs) via a microemulsion reaction in a hexane/ethanol mixed solvent.^[157] Subsequently, the obtained SCNPs surface was modified with a layer of PEGylated phospholipid to enhance its water solubility, resulting in the phospholipid-coated PSCNPs (Figure 9A). PSCNPs could enter tumor cells via endocytosis, releasing a significant amount of Na⁺/Cl⁻, leading to increased osmolarity and rapid cell lysis. Interestingly, PSCNPs showed minimal toxicity toward normal cells like human prostate epithelial cell line (HPrECs) and mouse spermatogonial stem cells (C18-4). This selective cytotoxicity was attributed to the higher uptake by rapidly proliferating tumor cells and their inherently higher intracellular sodium concentrations ([Na⁺]_int), making them more susceptible to PSCNPs-induced osmotic shock. Figure 9B illustrates the varying cytotoxic effects on different cells and their correlation with cellular [Na⁺]_int, indicating that [Na⁺]_int plays a vital role in determining the susceptibility of cancer cells to PSCNPs. In vivo experiments demonstrated that PSCNPs suppressed tumor growth (Figure 9C) and induced apoptosis and necroptosis in cancer cells, with an increase in survival (Figure 9D). Moreover, the study also revealed that PSCNPs could induce ICD, simultaneously killing cancer cells and enhancing anticancer immunity. To further enhance the blood circulation time and sodium ions responsive release capability of NaCl-based nanomedicines, Li et al designed and synthesized a kind of GSH-responsive nanomedicines, termed NaCl@ssss-VHMS (Figure 9E), which comprised NaCl nanocrystals internally and a virus-mimicking hollow mesoporous tetrasulfide-organosilica (ssss-VHMS) externally.^[158] The morphological advancements in NaCl@ssss-VHMS facilitated its rapid internalization into tumor cells via spike surface-assisted adhesion and invasion. Once inside, it degraded under the action of excessively produced GSH, releasing substantial amounts of Na⁺/Cl⁻, disrupting the ionic homeostasis of tumor cells, and inducing apoptosis, ferroptosis, as well as pyroptosis. The intracellular Na⁺ and Cl⁻ ion levels were monitored using Na⁺ (sodium-binding benzofuran isophthalate acetoxymethyl ester [SBFI-AM]) and Cl⁻ indicator probe (MQAE), respectively. The enhanced fluorescence signals of SBFI-AM and MQAE in NaCl@ssss-VHMS-treated HepG2 cells, compared to control groups, underscored its superior ion delivery performance (Figure 9F). In vivo imaging experiments also demonstrated the efficient accumulation of NaCl@ssss-VHMS within tumors (Figure 9G). In addition to NaCl, sodium citrate NPs have also been employed for sodium ion delivery, resulting in increased intracellular osmotic pressure and mediating tumor cell death.^[160]

As another type of sodium-containing inorganic salt, sodium bicarbonate (NaHCO₃) NPs are also employed in sodium ions-mediated cell death. Distinct from the aforementioned NaCl NPs, NaHCO₃ NPs possess weak alkalinity, which allows them to release a substantial amount of Na⁺ inside tumor cells while concurrently rectifying the acidosis of the TME.^[161] In a recent study, Ding et al synthesized NaHCO₃ NPs using sodium oleate and ammonium hydrogen carbonate as precursors through a rapid microemulsion method (Figure 9H).^[159] NaHCO₃ NPs were subsequently modified with distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG) for enhanced biocompatibility. The resultant weakly alkaline NaHCO₃ NPs could modulate lactic acid metabolism through acid-base neutralization. Treatment with NaHCO₃ NPs significantly increased intracellular pH levels (Figure 9I) and markedly reduced the lactic acid concentration in the culture medium (Figure 9J), demonstrating the substantial potential of NaHCO₃ NPs in regulating lactic acid metabolism and maintaining cellular pH stability. Moreover, like the aforementioned NaCl NPs, NaHCO₃ NPs could also release significant quantities of Na⁺ within tumor cells, leading to an increase in intracellular osmolarity. This process could trigger the pyroptosis pathway and ICD, thereby enhancing immune responses.

Currently, NMs employed in sodium ions-mediated cell death are known for their good biocompatibility and simple raw material components. They induce tumor cell death by disrupting the ionic homeostasis of tumor cells, leading to a sharp increase in osmotic pressure. Additionally, some of them can also regulate lactic acid metabolism through acid-base neutralization, thereby reversing the weakly acidic, immunosuppressive TME, offering broad application prospects. In this therapeutic strategy, the controllable preparation of NMs is crucial, ensuring prolonged blood circulation while preventing the off-target leakage and degradation of water-soluble sodium-containing compounds. Future developments could involve designing more comprehensive theranostic nanoplatforms, using imaging techniques to investigate the specific mechanisms and metabolic pathways in sodium ions-mediated cell death, such as fluorescent Na⁺ reporter SBFI-AM or ²³Na MRI. Finally, sodium ions-mediated cell death holds significant research value in improving immune responses and achieving tumor immunotherapy, with current studies making beneficial explorations in this area.^{[157,159,160]}

3.2.4 Other ions-induced cell death

Other essential metal ions, including magnesium (Mg), cobalt (Co), molybdenum (Mo), manganese (Mn), and potassium (K) ions, are also crucial for the growth and development of humans,^[22] showing potential in inducing metalloptosis.

Magnesium ion (Mg²⁺) as an essential trace element for the human body, participates in various physiological activities in cardiovascular, neurological systems, and muscles.^[162] More importantly, Mg²⁺ can act as an activator involved in the synthesis of at least 200 enzymes^[163] and regulate other momentous ion channels such as K⁺ and Ca²⁺.^[164] Hence, abnormal levels of Mg²⁺ in the body can trigger various diseases, and some nanotheranostics based on Mg²⁺ have shown initial effectiveness in treating cancer. For instance, MgO NPs exerted a strong inhibitory effect on cancer cells^[165] through the production of ROS because of the release of Mg²⁺ or that of ligands as well as byproducts.^{[166–167–168]} The generated ROS further caused oxidative damage to DNA, protein denaturation, and lipid peroxidation, leading to the eventual cell death.^[169] However, the detailed molecular mechanism of Mg²⁺-mediated cell death still needs to be clarified.^[170,171] Meanwhile, the biocompatibility and cytotoxicity of MgO NPs is also a key issue that potentially hinders the translation of MgO NPs in the field of cancer therapy,^[172] which may be solved through the green synthesis routes of MgO NPs.^[171,173] For instance, compared with Co oxide NPs, MgO NPs synthesized by the green method showed less toxicity and great bioavailability.^[174] Moreover, the researchers have also found that introducing Mg²⁺ into NMs can promote the rolling circle amplification of DNA, ultimately achieving the antitumor effect of the functionalized DNA.^[175]

Co and Mo have been reported to induce ferroptosis. Studies indicate that Co NPs significantly downregulated the expression of GSH, GPX4, and SLC7A11 proteins in Balb/3T3 mouse fibroblast cells, while leading to a notable increase in intracellular levels of ROS, MDA, cobalt, and iron.^[176] However, ferrostatin-1 showed rescue effects on cells after treatment of Co NPs, suggesting a close association between Co NPs-induced cytotoxicity and ferroptosis.^[176] In another study, cobaltous oxide nanodots were encapsulated within dendritic mesoporous silica NPs, followed by further surface modifications to yield iCoDMSNs. iCoDMSNs treatment increased transferrin receptor levels and reduced solute carrier family 40 member 1 (SLC40A1), leading to Fe²⁺ accumulation and subsequent ferroptosis.^[177] Similarly, molybdenum sulfide NPs were able to generate ROS, thereby triggering lipid peroxidation and ferroptosis in cancer cells.^[178] Additionally, the simultaneous integration of Co and Mo into NMs could enhance the induction of ferroptosis more effectively. Wu et al introduced a strategy of nonferrous ferroptosis, utilizing a CoMoO₄-phosphomolybdic acid nanosheet hybrid. This approach leveraged accelerated Mo(V)-Mo(VI) conversion to increase lipid peroxide levels, enhanced GSH consumption for GPX4 enzyme deactivation, and initiated a ROS surge, effectively inducing ferroptosis.^[57]

Manganese-based NPs can induce ferroptosis in tumors through mechanisms such as GSH depletion, increased oxygen and ROS production, and enhanced lipid peroxidation. For example, when oral squamous cell carcinoma cells (Cal-27) were treated with MnO₂ nanoflowers under near-infrared radiation, there was an increase in cellular Fe²⁺, an accumulation of LPO, and a downregulation of GPX4, effectively inducing ferroptosis.^[179] Similarly, manganese silicate nanobubbles demonstrated a high capacity for depleting GSH, inducing ferroptosis through the inactivation of GPX4.^[180] Furthermore, Mn can influence iron homeostasis by altering iron uptake and the expression of circulating transport and regulatory proteins; alterations in iron levels can, in turn, impact Mn balance and its biological roles.^[181] Mn²⁺ may also contend with essential ions such as Ca²⁺ and Mg²⁺ for cellular entry via different transport mechanisms, suggesting its potential to trigger PCD by unbalancing ion equilibrium.^[182] Moreover, Mn-containing NPs can stimulate innate and adaptive immunity through the cGAS-STING pathway, playing an important role in the immunotherapy of tumors.^[183,184] As an illustration, hollow mesoporous silica-coated MnO NPs have been utilized in MRI-guided immune-chemodynamic therapy, where MnO NPs served in activating the cGAS-STING pathway for immunotherapy, upregulating ROS through Fenton-like reactions, and enhancing T1-weighted MRI.^[184] This research effectively leveraged the unique properties of the Mn element for antitumor therapy.

As a vital physiological ion, potassium ions are integral to numerous crucial cellular functions, including metabolism, growth, and apoptosis.^[185] Analogous to NaCl, KCl NPs are also applicable in cancer therapy. The intracellular release of a substantial amount of K⁺ and Cl⁻ could alter the osmotic pressure within cancer cells, subjecting them to a hypertonic state. This change induced the rupture and subsequent death of the cancer cells.^[186] Furthermore, potassium ionophores have demonstrated anticancer capability. For example, valinomycin, a natural ionophore, is able to pump intracellular potassium ions out of the cytosol, leading to mitochondrial damage and inducing apoptosis.^[187] Additionally, a helical polypeptide-based potassium ionophore was shown to induce ER stress-mediated apoptosis in vivo.^[188] However, it is important to note that the research on the antitumor applications of NPs constructed based on potassium ionophores remains comparatively limited.

Although the discussed metal elements in this section can mediate cell death, the exact pathways of their cancer-killing effects need more research. There is a current gap in definitive studies confirming these pathways. Future work must elucidate how these metals exert specific molecular interactions within cancer cells to maximize their therapeutic value. Unraveling these mechanisms will illuminate the details of metalloptosis and guide the development of targeted cancer treatments leveraging these metals’ unique attributes.