Cuproptosis: an emerging domain for copper-based nanomaterials mediated cancer therapy

2.1 Copper metabolism

Copper is rich in a variety of foods, such as animal offal, seafood, cereals, vegetables, and fruits. Drinking water is also an important source of copper in the body. The currently recommended daily copper intake for adults is 2 to 3 mg.^[23] After being ingested by the human body, copper is mainly absorbed through the intestinal epithelium, with the duodenum being the main absorption site.^[24] Copper ions in food often exist in the form of divalent copper ions, but only monovalent copper ions can be stored and utilized in the body. Therefore, as shown in Figure 1, the ingested divalent copper ions are first reduced to monovalent copper ions by metalloreductases such as six-transmembrane epithelial antigen of the prostate (STEAP) on the surface of the intestinal epithelial cell membrane and then enter the intestinal epithelial cells through the copper transporter 1 (CTR1) or solute carrier family 31 member 1 (SLC31A1). Copper in the intestinal epithelial cells is transported to the other side of the intestinal epithelial cells by the copper chaperone antioxidant 1 (ATOX1) and released into the blood through the ATPase copper transporter alpha (ATP7A).^[26] Most of the copper ions in the blood are bound to ceruloplasmin, and the remaining copper ions are bound to human serum albumin and amino acids.^[27] Copper ions in the blood are transported to the liver through the portal vein system. The liver is the main storage organ for copper. The copper ions transported to hepatocytes through CRT1 are bound to metallothionein 1/2 (MT1/2). MT1 and MT2 are rich in sulfhydryl groups and therefore have a high affinity for copper ions. MT1 and MT2 in hepatocytes are the main storage sites for copper. When the copper content in the body exceeds the normal threshold, liver cells excrete excess copper into the bile and excrete it from the body through the action of ATPase copper transporter beta (ATP7B).^[28] In summary, systemic copper metabolism is mainly regulated by the small intestine and liver.

In tumor cells, copper ion homeostasis and function depend on the interaction of different types of proteins. The first group is proteins related to transmembrane copper transport, such as STEAP and CTR1, which are responsible for transporting monovalent copper ions into tumor cells. The second group is proteins that bind and store copper ions,^[29] such as MT and glutathione (GSH). MT and GSH are natural chelators of copper ions in tumor cells and play an important role in the homeostasis of intracellular copper ions. The third group is copper ion chaperones,^[30] such as superoxide dismutase copper chaperone, ATOX1, and cytochrome C oxidase copper chaperone 17. Copper ions bind to copper molecular chaperones and are then delivered to specific proteins or cell compartments to exert their effects.

2.2 Cuproptosis targets

Previous studies have shown that copper can cause cell death by inducing mitochondrial oxidative damage or destroying enzymes related to the tricarboxylic acid cycle.^[31] Tsvetkov et al^[6] identified key targets that promote cuproptosis through genome-wide CRISPR-Cas9 screening, including ferredoxin 1 (FDX1), DLAT, lipoic acid synthase (LIAS), and dihydrolipoamide dehydrogenase (DLD). The discovery of these targets helps to deepen our understanding of the molecular mechanism of cuproptosis and provides potential targets for future drug development.

3.1 Copper-ionophores hybridized nanomaterials

Ionophores are small molecules that can bind with copper ions and transport those ions into the cell via transmembrane transport.^[41,42] Therefore, the use of such special molecules to deliver copper ions is one of the most common approaches in the field of nanomedicine nowadays. Furthermore, several ionophores, such as disulfiram (DSF), diethyldithiocarbamate (DTC), and elesclomol (ES), have been found to increase the intracellular copper levels to activate cuproptosis.^[43,44] Unfortunately, the lack of tumor targeting of these ionophores, their short blood circulation period, and easy to metabolize have led to their ineffective clinical performance. The development of nanotechnology provides a new direction for the delivery of these ionophores. Wu et al synthesized a polymer micelle (PCP) using polyethylene glycol and cinnamaldehyde. The PCP is ROS-responsive and can be used to encapsulate ES-Cu (EC) compounds, resulting in ECPCP (Figure 2A–C). This formulation extends the circulation time of ES-Cu in the body and increases its concentration in tumors. Once inside cells, the PCP coating breaks down due to high ROS levels, releasing ES and copper ions to induce cell death through cuproptosis (Figure 2D–F).^[33] Similarly, Wu et al^[45] fabricated a nanocomposite of CuET and copper oxide (CuO) based on ionophore DTC (CCB) to disrupt protein homeostasis for enhanced cuproptosis-mediated tumor therapy (Figure 2G–I).

3.2 Metal–organic frameworks nanomaterials

Metal–organic frameworks (MOFs) were built by transition metal ions with their strongly coordinating organic ligands through self-assembly to form nanoparticles with a periodic network structure.^[46,47] Notably, the presence of a large number of coordination interactions between metal ions and organic groups in these MOF structures, which are weakened in the specific microenvironment of tumors, leads to the dissociation of the MOF structure and therefore can be used for the selective delivery of metal ions.^[48] Much effort has been devoted to developing various copper-based MOF nanomaterials for activating cuproptosis-mediated tumor therapy. Chen et al^[49] developed a system comprising bimetallic Fe-Cu MOFs as a core, which could be internalized by neutrophils to form a “cellular Trojan Horse” (Figure 3A, B). These neutrophils carried thermosensitive liposomes filled with Fe-Cu MOFs (Lip@Fe-Cu-MOFs), which released their contents at a specific temperature. The Fe-Cu MOFs exhibited robust photothermal properties (Figure 3C), whereby the generation of heat under near-infrared (NIR) light triggered drug release and enhanced cuproptosis and ferroptosis (Figure 3D). The Fenton reaction, catalyzed by Fe³⁺ or Cu²⁺, produced hydroxyl radicals that, in conjunction with Cu²⁺, depleted GSH in tumor cells. This process induced both ferroptosis and cuproptosis, attacking cancer cells on multiple fronts and enhancing treatment effectiveness while minimizing the risk of recurrence (Figure 3E).

In addition, copper-based metal-phenolic networks have likewise been extensively studied as a class of MOF materials in cuproptosis-mediated tumor therapy.^[50] Qiao et al^[50] designed metal-phenolic nanoparticles (CQG NPs) that integrated copper with quinone and glucose oxidase (GOx) to combat both dormant and recurring tumors by inducing a balanced pyroptosis and cuproptosis (Figure 3F, G). The multienzyme activity of CQG NPs activated NLRP3-mediated pyroptosis (Figure 3H, I), while cuproptosis was initiated by the release of copper ions from the degradation of nanoparticles (Figure 3J). Moreover, the susceptibility of cancer cells to cuproptosis was increased through several mechanisms: the depletion of copper chelators by a reaction with GSH, oxygen generation via a catalase-like reaction, and glucose scarcity due to starvation (Figure 3K).

Furthermore, due to the presence of pore structure on the surface of MOF materials, which can adsorb a variety of chemotherapeutic drugs, copper-based MOF nanomaterials can achieve more efficient cuproptosis-mediated tumor therapy with the assistance of chemotherapeutic drugs. Ji et al developed a multifunctional nanoparticle (M/A@MOF@CM) that was a copper-based MOF loaded with mitoxantrone and axitinib and coated with a tumor cell membrane for cuproptosis/ferroptosis/apoptosis synergistic cancer therapy (Figure 3L).^[43] This M/A@MOF@CM showed various antitumor properties, combining photothermal therapy, chemotherapy, and chemodynamic therapy to boost ICD for feasible immunotherapy (Figure 3M). This integrated strategy resulted in an 86.45% reduction in primary tumor size and a 65.61% decrease in distant tumor growth, showcasing its potential in comprehensive cancer treatment (Figure 3N).

3.3 Other copper-coordinated nanoassembly

The coordination between metal ions and organic molecules can form multifunctional nanoassemblies different from MOF structures by tuning the organic molecule species. Metal oxides or metal sulfides can form hollow mesoporous structured core-shell nanoparticles, with the hollow mesoporous structure acting as a scaffold to load hydrophobic organic compounds within the pores of the mesoporous structure, enabling controlled release of organic drugs through the porous architecture. Furthermore, the attachment and growth of nano-sized tetrairon oxide (Fe₃O₄) on the core-shell structure endow the entire nanomaterial with not only high biocompatibility but also superparamagnetism and magnetothermal response, thereby achieving a precise diagnosis of tumors and effective treatment integrating chemodynamic, chemotherapy, and other multimodal therapies. Metal ions can also coordinate with chemotherapeutic drugs, small molecule inhibitors, and other organic molecules through layer-by-layer self-assembly to form tumor microenvironment (TME)-responsive nanoparticles. These nanoparticles remain stable under normal physiological conditions but rely on their responsiveness to undergo cascade reactions that eliminate tumor cells in the TME. Liang et al^[52] introduced a novel copper-coordinated nanoassemblies (CCNAs) that integrated a photosensitizer (zinc phthalocyanine [ZnPc]), a prodrug of a chemotherapeutic (DOX) with a thioketal (TK) spacer and an IDO inhibitor (1-methyl tryptophan [1-MT]) (Figure 4A, B). These components self-assemble around Cu²⁺ ions for a combination therapy of apoptosis and cuproptosis, along with immunotherapy. Upon exposure to NIR laser irradiation, the ZnPc in CCNAs activated a photodynamic effect, producing ROS (Figure 4C, D). This led to the release of DOX, intensifying tumor cell apoptosis (Figure 4E, F). Moreover, the Cu²⁺ in CCNAs boosted the photodynamic process by catalyzing oxygen production and also induced the aggregation of harmful mitochondrial proteins, causing cell cuproptosis (Figure 4G).

Apart from photodynamic molecules that can be introduced, photothermal molecules can also be added to boost cuproptosis-mediated tumor therapy. Dai et al^[53] developed a biomimetic cuproptosis amplifier (PCD@CM) by assembling NIR-II polymer dots and DOX drug with Cu(II), and then coating them with tumor cell membranes (Figure 4H). This system targeted tumor cells, where high levels of GSH in the TME cause the release of DOX by reducing Cu(II) to Cu(I). This enabled guided photothermal and chemotherapy using NIR-II imaging (Figure 4I). The released Cu(I) caused mitochondrial protein aggregation and proteotoxic stress, inducing cuproptosis (Figure 4J). The combination of NIR-II photothermal therapy and GSH depletion makes tumor cells more susceptible to this form of cell death (Figure 4K, L).

3.4 Inorganic nanomaterials

Inorganic nanomaterials compared with organic or polymer nanomaterials have unique physicochemical properties, stronger mechanical stability while maintaining a certain biological activity, and are easier to be compounded with a variety of carriers.^[54] Metal oxides are the most common inorganic nanomaterials. The 2 main types of copper-based metal oxides are copper oxide and cuprous oxide, and both of these copper oxides have been shown to act as copper sources to activate cuproptosis. Bai et al^[55] developed MitCuOHA nanozymes using copper oxide nanorods as a base, loaded with (3-carboxypropyl) triphenylphosphonium bromide, and coated with hyaluronic acid (Figure 5A, B). These nanozymes possessed multiple enzymatic activities for combined ferroptosis and cuproptosis therapy in cancer treatment. They catalyzed the depletion of cysteine and GSH while producing H₂O₂ (Figure 5C, D), which was converted into hydroxyl radicals to induce ferroptosis in cancer cells. Additionally, MitCuOHA nanomedicine can release copper ions in the TME, and copper ions can bind to proteins in the tricarboxylic acid cycle, causing their aggregation and loss of iron–sulfur cluster proteins, leading to proteotoxic stress and cuproptosis (Figure 5E, F). Similarly, Ning et al^[56] created a biomimetic cuproptosis sensitization system by coating Cu₂O with a platelet membrane to induce multiple tumor cuproptosis (Figure 5G, H). The system incorporated an AIE photosensitizer (TBP-2), which generated hydroxyl radicals to deplete GSH and hinder copper ion efflux. Cu₂O broke down quickly under acidic and H₂O₂-rich tumor conditions, releasing copper ions (Figure 5I–K). Copper accumulation triggers the clustering of lipoylated proteins and the depletion of iron–sulfur clusters, which in turn induces protein toxicity stress, ultimately leading to cell death through cuproptosis (Figure 5L).

Currently, an increasing number of copper-based inorganic nanomaterials are being reported to be available for efficient cuproptosis activation. Man et al^[57] constructed human serum albumin-mediated defect-rich copper hydroxide nanowire (HCu nanowire) for synergetic cuproptosis and ROS-mediated apoptosis antitumor therapy (Figure 6A–C). Chan et al^[58] reported a photothermic Cu_2-xSe nanoparticle encapsulated with bioresponsive dimethyl maleic anhydride (DMMA@Cu_2-xSe) as the copper carrier to enhance the copper accumulation in tumor for achieving cuproptosis-driven enhancement of thermotherapy (Figure 6D, E). In particular, benefiting from the fact that some of the inorganic materials can trigger a change in properties due to the realization of phase transitions in tumor-specific microenvironments, some of the copper-based nanomaterials will be expected to achieve specific activation of cuproptosis in specific TMEs. Zhao et al^[59] copper hydroxyphosphate nanoparticles (Cu₂(PO₄)(OH) NPs) with hydrogen sulfide (H₂S)-responsiveness were developed to achieve an imbalance of copper homeostasis (copper overload) in tumor cells by enhancing endocytosis of copper-based nanoparticles and inhibiting exocytosis of copper ions, which further induced cellular pyroptosis and cuproptosis. Monodisperse Cu₂(PO₄)(OH) NPs synthesized by a hydrothermal method and injected intravenously into H₂S-overexpressing colon cancer microenvironments were able to be transformed into ultrasmall copper sulfide nanoparticles (Cu₉S₈ NPs) through in situ sulfation (Figure 6F, G). These ultrasmall Cu₉S₈ NPs can be endocytosed into colon cancer cells faster and release copper ions inside the cells, which dramatically increase the intracellular ROS level via a Fenton-like reaction. The increase in ROS not only activates NLRP3 inflammatory vesicles and Caspase-1 proteins, causing gasdermin D (GSDMD) cleavage and triggering cellular pyroptosis (Figure 6H), but also affects mitochondrial function, cuts off the supply of adenosine-5’-triphosphate (ATP), downregulates the expression of ATP7A, and reduces the efflux of copper ions. The combination of higher cellular uptake, copper ion release, and reduced copper ion efflux ultimately disrupts intracellular copper homeostasis and activates copper overload-mediated cuproptosis (Figure 6I, J).

4.1 Copper transporter protein regulation

Activation of cuproptosis in tumor cells is predicated on how to rapidly accumulate large amounts of copper ions within tumor cells. However, the cell has a complex network of copper-dependent transporter proteins consisting of CTR1 and ATP7A/B, to maintain its internal copper homeostasis.^[62] Jointly, these proteins coordinate copper input (CTR1) and output (ATP7A/B) to maintain intracellular copper content within a specific range, and the existence of this copper homeostatic mechanism greatly prevents the occurrence of copper overload. Typically, nanomaterials enriched in tumor sites can enter into tumor cells by endocytosis, therefore the designed and constructed copper-based nanomaterials can effectively accumulate intracellular copper content by inhibiting the expression of copper efflux proteins in tumor cells. It is worth noting that the function of copper efflux proteins (ATP7A/B) needs to be energized by ATP, and once the ATP supply is insufficient, it will lead to a decrease in expression and affect the efflux process of copper ions.^[63]

Based on this, Xu et al^[64] developed a novel copper/iron hybrid hollow amorphous MOF with doxorubicin and hyaluronan (DOX@Fe/CuTH HaMOF) as an oxidative stress amplifier and copper/iron metabolic disrupter for anticancer therapy. In this nanomaterials system, the content of intracellular H₂O₂ could be promoted by the effect of DOX (Figure 7A), and combined with its Fenton catalytic properties, it can dramatically increase the intracellular level of ROS (Figure 7B), which in turn leads to mitochondrial damage and affects the production of ATP (Figure 7C), ultimately resulting in a decrease in the expression of ATP7A protein (Figure 7D). More copper ions will be locked up inside the tumor cell, enabling more effective activation of cuproptosis (Figure 7E–G). Furthermore, besides utilizing oxidative stress-induced mitochondrial damage to regulate the copper transporter proteins, it is also possible to inhibit the expression of copper efflux proteins by inhibiting glycolysis to cut off ATP supply. In addition, Yan et al^[65] built an OPDEA-coated copper-based MOF system (OMP) to deliver siRNA targeting PDK1 (siPDK) for achieving antitumor effect via cuproptosis. The delivered siPDK could reduce ATP production by suppressing intracellular glycolysis (Figure 7H), leading to the blockage of the Cu efflux protein ATP7B (Figure 7I); consequently, this enhances the sensitivity of cancer cells to the cuproptotic effects triggered by OMPs (Figure 7J, K). In addition, it is also feasible to increase the activation efficiency of cuproptosis by reducing ATP7A protein expression through directly targeted delivery of siRNA targeting ATP7A.^[66]

Copper transporter protein modulation strategy based on copper-based nanomaterials is focused on increasing intracellular copper levels by inhibiting the expression of copper efflux proteins (ATP7A/B). Three types of approaches are currently available: (1) increasing intracellular ROS levels through copper-based nanomaterials, which in turn causes mitochondrial dysfunction and reduces ATP production capacity, can effectively reduce ATP7A/B protein expression; (2) inhibition of intracellular glycolysis, which subsequently reduces ATP supply, similarly inhibits protein expression; and (3) delivery of siRNA for the corresponding protein directly inhibits protein expression.

4.2 GSH depletion

GSH, a naturally biological tripeptide, plays a crucial role in regulating intracellular redox balance. As the tumor cells show an overall oxidative stress state, reduced GSH is highly expressed within the tumor cells.^[67] These excess reduced GSH will chelate with free intracellular copper ions, which restricts the binding of copper to the lipoylated components in the TCA, inhibiting the efficacy of cuproptosis. Thus, depletion of intracellular GSH can increase DLAT oligomerization and sensitize tumor cells to cuproptosis.

Utilizing the strong reducing property of GSH, it will be promising to deplete GSH through redox by constructing material systems containing multivalent metal ions. Chen et al^[68] constructed a Cu₂O@Mn₃Cu₃O₈ (CMCO) nanozyme with a core-shell structure that served as the TME-activated copper deliverer for safe and efficient cuproptosis (Figure 8A). The redox activity of Cu⁺/Cu²⁺ and Mn²⁺/Mn³⁺/Mn⁴⁺ in CMCO reacted with the GSH in the TME, converting it to oxidized glutathione (GSSG) through a GSHOx-like mechanism (Figure 8B). This reaction reduced the ability of GSH to chelate copper ions (Figure 8C), leading to increased copper toxicity and the induction of cuproptosis in cancer cells (Figure 8D, E). Similarly, Zhang et al^[69] reported CuMoO₄ nanodots with multienzyme-like activities that respond to the TME for multimodal cancer treatment. The CuMoO₄ nanodots, containing variable-valent metals (Cu⁺/Cu²⁺ and Mo⁵⁺/Mo⁶⁺), effectively depleted intracellular GSH through a GSHOx-like activity, sensitizing tumor cells to cuproptosis (Figure 8F).

Although relying on the variable metal ion reduction strategy can efficiently consume GSH, the overall GSH depletion effect is still not satisfactory. Therefore, several strategies to assist GSH depletion have been developed for more efficient realization of intracellular GSH depletion. Zhao et al^[70] utilized gallic acid (GA) with GSH-depleting properties to construct copper-based metal-phenolic network nanoparticles (Cu-GA NPs) for polyphenol drug-assisted dysregulation of intracellular redox homeostasis. The Cu-GA NPs enabled the rapid release of copper ions and GA under the conditions of the TME overexpressed GSH (Figure 8G). The released GA and Cu²⁺ continuously consume the remaining GSH to achieve GSH depletion and simultaneously Cu²⁺ will be reduced to Cu⁺ with higher Fenton catalytic activity (Figure 8H, I). Subsequently, ROS levels within the tumor cells are significantly increased by the Fenton-like reaction of released Cu⁺ (Figure 8J). Furthermore, an excessive buildup of Cu⁺ within tumor cells leads to the clustering of DLAT and a decrease in the expression of iron–sulfur cluster proteins, ultimately realizing the dual-wheel-driven tumor therapy of apoptosis and cuproptosis under the regulation of redox homeostasis (Figure 8K). The depletion of intracellular GSH is likewise effectively assisted by the delivery of GSH generation inhibitors. Huang et al developed a copper-based nanoplatform, MOF-199@DDM, designed to deliver buthionine-sulfoximine (BSO). BSO is an inhibitor of intracellular GSH synthesis, which makes tumor cells more susceptible to cuproptosis-inducing effects.^[71]

GSH overexpressed in tumor cells has a strong ligand-chelating effect, which can effectively chelate the accumulated copper ions in the cell and then cut off the effect of copper ions and cuproptosis-related proteins, reducing the possibility of the emergence of cuproptosis. Two potential modalities have been developed to deplete GSH in order to increase the sensitivity of tumor cells to cuproptosis: (1) construction of copper-based nanomaterials with variable metal ions to regulate intracellular GSH levels by redox reaction. (2) Delivery of GSH-depleting chemotherapeutic molecules or GSH synthesis inhibitors with the assistance of copper-based nanomaterials.

4.3 Increasing intracellular oxygen content

Mitochondrial respiration involving cuproptosis necessitates the engagement of oxygen (O₂), whereas the TME is typified by hypoxia, which will also restrict the impact of cuproptosis. Alleviating the hypoxic environment of the tumor (increasing intracellular oxygen content) would be expected to enhance the sensitivity of tumor cells to cuproptosis.

Peroxides are a class of inorganic materials containing peroxygen bonds (–O–O–), which can produce H₂O₂ stably under acidic conditions and release O₂ under certain conditions, being considered as potential carriers of O₂.^[72] On the basis of calcium peroxide nanoparticles (CaO₂), Jin et al have engineered and synthesized a nanoreactor for immunotherapy that triggers cuproptosis, designated as CCJD-FA. This nanoreactor encapsulates copper-based shell-coated CaO₂ and the bromodomain-containing protein 4 inhibitor JQ-1, along with DSPE-PEG-FA (Figure 9A).^[63] Within tumor cells, CCJD-FA disintegrates under the influence of GSH and an acidic milieu, exposing CaO₂, which then produces H₂O₂ by interacting with H⁺ (Figure 9B–D). This H₂O₂, in conjunction with the liberated Cu²⁺, undergoes a Fenton-like reaction, releasing O₂ to alleviate hypoxia and instigate cuproptosis (Figure 9E–G).

Catalase (CAT), a kind of enzyme widely existing in organism, is capable of efficiently catalyzing the release of O₂ from H₂O₂, reversing the hypoxic TME,^[74] which enhances mitochondrial respiration and promotes cuproptosis. The researchers used a technique called rolling circle amplification to combine long-chain DNA with catalase, and then obtained the enzyme-core spherical nucleic acid nanoplatform (CAT-ecSNA-Cu) through the chelation of copper ions (Figure 9H).^[73] CAT can maintain its activity under the protection of long-chain DNA. In the TME, CTA catalyzes hydrogen peroxide to produce oxygen, which enhances the aerobic respiration of tumor cells and increases the sensitivity of tumor cells to copper death. Notably, some copper-based nanomaterials with CAT-like enzyme activity can likewise catalyze the production of O₂ to alleviate hypoxia in the TME (Figure 9I, J), thereby promoting the therapeutic efficiency of cuproptosis (Figure 9K, L).^[68]

Tumor hypoxia microenvironment can inhibit cell cuproptosis. There are 2 approaches that have been confirmed to increase intracellular oxygen content for the enhancement of cuproptosis. (1) Peroxide-based oxygen generation: design nanoreactors incorporating peroxides to release oxygen within the TME, mitigating hypoxia and boosting cuproptosis. (2) Catalase-enhanced mitochondrial respiration: utilize catalase-functionalized nanoplatforms to catalyze the conversion of H₂O₂ to O₂, thereby sensitizing tumor cells to cuproptosis by enhancing mitochondrial respiration.

5.1 Cuproptosis combined chemotherapy

Chemotherapy is still the first choice for the treatment of cancers in clinical practice, and correspondingly developed chemotherapeutic drugs are mainly effective in combating cancer by activating apoptosis.^[76] However, tumor cells derive multiple drug-resistant resistances to counteract the function of these chemotherapeutic drugs, resulting in unsatisfactory efficacy.^[77] In addition, conventional chemotherapeutic agents without tumor selectivity require high-dose injections to achieve anticancer effects that are frequently accompanied by severe toxic side effects. These unavoidable obstacles urgently seek to develop multimechanism combination strategies to alleviate the above dilemma.

Fortunately, some studies have found that the introduction of cuproptosis can solve some of the difficulties in the application of chemotherapeutic drugs. Drug resistance, especially for drug efflux transporter-mediated chemoresistance, stands out as one of the predominant factors leading to the suboptimal outcomes in the field of contemporary cancer chemotherapy. Lu et al^[78] developed a copper-based nanoplatform by coordinating with ellagic acid (EA-Cu), which effectively combated resistance to cancer chemotherapy through cuproptosis (Figure 10A). Within this system, EA curbed the self-sustaining properties of tumor cells by inhibiting the hedgehog signaling pathway. Concurrently, Cu²⁺ diminished the stem-like characteristics of cells by interfering with their mitochondrial function, which significantly boosted the effectiveness of chemotherapy induced by DOX. Furthermore, EA worked in tandem with copper ions to trigger mitochondrial dysfunction and cuproptosis, leading to a significant reduction in ATP levels. This reduction consequently inhibited the function of P-glycoprotein, a key player in drug resistance by facilitating the expulsion of chemotherapeutic agents, thereby increasing the sensitivity of tumor cells to chemotherapy mediated by DOX.

The inefficient cytotoxic selectivity of chemotherapeutic agents has likewise been an obstacle to the further development of chemotherapy. It is worth noting that one class of chemotherapeutic drugs is of the metal ion potentiating type.^[82] And one of the characteristics of cuproptosis is an overload of intracellular copper ions. Therefore, copper-based chemotherapy drugs hold great potential to achieve cuproptosis synergetic with chemotherapy. DSF, approved by the Food and Drug Administration for treating alcoholism, was found to possess antitumor activity via inducing apoptosis of tumor cells.^[83] Interestingly, some recent studies suggested that the antitumor capacity of DSF could be strongly amplified once it was combined with endogenous copper ions (Cu²⁺), while DSF by itself exhibited low toxicity.^[84] Therefore, DSF is a potential “pro-drug” with Cu²⁺ as an enhancer to realize in situ chemotherapy. Ni et al^[79] engineered a TME-activated core-shell structured tecto dendrimer, which was laden with copper ions and DSF, designated as CSTD-Cu(II)@DSF. This formulation was intended for a dual therapeutic strategy that leverages cuproptosis to enhance chemo-chemodynamic therapy (Figure 10B). Upon systemic circulation, the CSTD-Cu(II)@DSF accumulated at the tumor site, where it released its therapeutic payload in response to the tumor's mildly acidic and elevated levels of ROS microenvironment. Once inside the cells, the elevated levels of intracellular Cu(II) ions initiated the oligomerization of lipoylated proteins, precipitating proteotoxic stress that led to cuproptosis. Additionally, these ions catalyzed the process of lipid peroxidation, a critical component of chemodynamic therapy. The CSTD-Cu(II)@DSF also induced mitochondrial dysfunction and cell cycle arrest at the G2/M phase, which, combined with DSF, amplified the apoptotic effect on the cancer cells. By combining the cytotoxic effects of chemotherapy, the selective stress induced by cuproptosis, and the oxidative stress generated through chemodynamic therapy, it offered a multifaceted approach to overcoming cancer. Likewise, researchers led by Zhong utilized Cu₂O@CuBTC-DSF@HA nanocomposites, which were capable of responding to acidic conditions by dissociating to catalyze the conversion of DSF into the toxic dithiocarbamate-copper complexes (CuET). This process was designed to occur in the TME and it worked in conjunction with cuproptosis to amplify the therapeutic effects of treatment (Figure 10C).^[80]

The compound artemisinin (ART), known for its antimalarial properties, has uncovered its potential in the fight against cancer.^[85] Its molecular structure, featuring a sesquiterpene with an endoperoxide bridge, is susceptible to cleavage by transition metal ions, resulting in the formation of a carbon-centered radical (·C). This process significantly boosts oxidative stress within cells. Xu et al^[81] introduced a novel hollow nanoplatform (HNP) that incorporated CuET and loaded with ART to enhance oxidative stress in cancer therapy (Figure 10D). This HNP was designed to be responsive to both the acidic pH and the high levels of GSH within TME. Upon exposure to these conditions, the ART@CuT/ETH HNPs disassemble, releasing Cu²⁺ ions. The released copper ions played a dual role: They catalyzed the formation of ·C and hydroxyl radicals (·OH) through reactions with ART and endogenous H₂O₂, respectively, thereby intensifying oxidative stress. Additionally, copper ions bind to the DLAT, leading to its aggregation and the induction of cuproptosis. The platform also incorporated the ligand TPH, which features disulfide bonds and is adept at depleting intracellular GSH. This GSH depletion further magnified oxidative stress and supported the induction of cuproptosis. The approach of amplifying oxidative stress through 3 distinct pathways showed promise in the realm of copper-based cancer therapies.

5.2 Cuproptosis combined chemodynamic therapy

Chemodynamic therapy (CDT) is an emerging field in cancer treatment that leverages the cytotoxic potential of ROS, which are produced from the conversion of endogenous H₂O₂ through Fenton or Fenton-like reactions, to provoke apoptosis in tumor cells.^[86] Recognized for its precise targeting of cancer cells and minimally invasive characteristics, CDT has gained attention in the search for novel cancer therapies. Copper ions are a kind of metal ions with Fenton-like catalytic activity and have higher Fenton catalytic activity compared with conventional iron ion catalysts.^[87] Once copper overload is induced to form in tumor cells, oxidative stress is also generated. When the oxidative stress exceeds the cellular threshold, it is expected to enable chemodynamic therapy in combination with cuproptosis.

Yu et al^[88] designed a Cu⁺ and DNAzyme-driven nanocascade system (ZIF-8-Cu₂O-DNA) that enhanced both cuproptosis and CDT (Figure 11A). Within the weak acidic environment of the tumor, the system was designed to release DNA, Zn²⁺ ions, and Cu⁺ ions. The Cu⁺ ions, which excel in mediating Fenton-like reactions and cuproptosis, were released to efficiently trigger these processes while coupled with the generation of Cu²⁺ ions. These Cu²⁺ ions were then partially recycled back into Cu⁺ by GSH, forming a supply loop that ensured a sustained synergistic effect. The consumption of GSH in this cycle also boosted cuproptosis and CDT. Simultaneously, the DNA and Zn²⁺ form DNAzymes that degrade catalase-related RNA, causing an accumulation of H₂O₂ and further amplifying the therapeutic effect. The GSH-depleting properties, integrated into copper-based nanomaterials that can induce CDT, open up the possibility of combining cuproptosis with ferroptosis, offering a novel and multipronged approach to cancer treatment.

The efficacy of CDT agents can be substantially heightened by inducing localized hyperthermia within the tumor.^[91] Photothermal therapy (PTT) is an extensively utilized technique that employs photothermal agents to convert NIR light into heat, damaging tumor cells.^[92] By incorporating PTT into CDT, the therapeutic outcome is further enhanced, creating a complementary strategy that leverages the thermal effects of PTT with the oxidative mechanisms of CDT for a more potent cancer treatment. Song et al^[89] developed nanomotors (CuSiO₃@Au-Pd NMs) that utilized NIR light to enhance cellular uptake and tumor penetration for achieving a cuproptosis-assisted synergistic therapeutic approach combining PTT and CDT (Figure 11B). These nanomotors were capable of self-propulsion upon NIR irradiation, facilitating their entry into cells and their ability to reach deep within tumor tissues. Once internalized, the Cu²⁺ ions released from the nanomotors initiate the aggregation of DLAT in the TCA cycle, leading to proteotoxic stress and the induction of cuproptosis in cells. Additionally, the Cu⁺ ions, produced by the reduction of Cu²⁺ by GSH, triggered Fenton-like reactions that generated amounts of toxic ROS. The gold-palladium nanoparticles (Au-Pd NAs) within the CuSiO₃@Au-Pd NMs contributed to their exceptional photothermal capabilities, further boosting the effectiveness of the cancer treatment. The CuSiO₃@Au-Pd NMs-mediated synergistic therapy, which combined PTT with CDT and cuproptosis, presented a promising strategy for efficient and multifaceted cancer treatment (Figure 11C).

Recent research has verified that an increase in intracellular ROS can initiate pyroptosis, a novel form of PCD characterized by the upregulation of gasdermin family proteins. In this regard, it is expected that with the addition of CDT, simultaneous activation of cellular cuproptosis and pyroptosis to improve the efficiency of tumor treatment. Wang et al^[90] constructed an integrated nanoplatform (hCZAG), which used the zeolitic imidazolate framework-8 (ZIF-8) as its basement. This platform featured Cu²⁺ and Zn²⁺ as active sites and incorporated glucose oxidase (GOx) to simultaneously induce pyroptosis and cuproptosis (Figure 11D). The GOx within the hCZAG efficiently increased intracellular H₂O₂ content. Meanwhile, the Cu²⁺ ions could be reduced to Cu⁺ by the overexpressed endogenous GSH, and both Cu²⁺ and Cu⁺ ions could engage in Fenton-like reactions, thereby enhancing the generation of ROS and amplifying oxidative stress. Significantly, the surge in ROS caused by the hCZAG nanoplatform activated Caspase-1 proteins, leading to the cleavage of GSDMD, and ultimately inducing pyroptosis. Furthermore, the accumulation of Cu⁺ ions could cause the aggregation of DLAT, which triggered cuproptosis. This dual-modality approach to cancer therapy, activating both pyroptosis and cuproptosis pathways, presented a promising strategy for enhancing the effectiveness of tumor treatments.

5.3 Cuproptosis combined immunotherapy

Cancer immunotherapy, which involves the strategic use of the immune system to identify and eliminate malignant cells, has become a pivotal and innovative method in oncological care.^[93] Nevertheless, the typically weak immune reaction following immunotherapy often leads to the development of adverse effects associated with the immune system. Recently, several studies found that activation of ICD can further promote the liberation of antigens from tumors and the maturation process of dendritic cells (DCs). Hence, it amplifies the immunogenicity of the tumor and invigorates the immune system’s response.^[94,95] Cuproptosis has been reported to trigger the ICD, releasing damage-associated molecular patterns (DAMPs) to promote the recruitment of T cells.^[96,97] The mechanism of ICD induced by copper ions involves multiple aspects, such as copper ions and mitochondrial function, oxidative stress, DAMPs release, and activation of immune response. These mechanisms work together to cause tumor cell death and activate antitumor immune response.^[98] Under normal physiological conditions, cells maintain a balance between oxidation and antioxidant defense. Copper ions can destroy the cellular antioxidant system and lead to tumor cell death.^[99] On the one hand, copper ions undergo a Fenton-like reaction, catalyzing hydrogen peroxide to produce a large amount of ROS, which cause multiple damages to cells, including DNA damage, interference with mitochondrial function, and destruction of cell membrane integrity.^[100] On the other hand, copper can oxidize reduced GSH to oxidized glutathione disulfide (GSSG), leading to the depletion of the antioxidant GSH, thereby interfering with the GSH-related antioxidant defense system and leading to tumor cell apoptosis.^[101] In addition, the copper ion carrier elesclomol can bind extracellular divalent copper ions and transport them to intracellular compartments. FDX1 reduces divalent copper ions to monovalent copper ions, inhibiting the synthesis of Fe–S cluster proteins. LIAS is an upstream regulator of protein lipoylation and can regulate the lipoylation of mitochondrial enzymes such as DLAT. Monovalent copper ions can bind to lipoylated DLAT in the TCA cycle and induce the aggregation of lipoylated proteins.^[6] These abnormal processes together lead to proteotoxic stress and mitochondrial dysfunction, ultimately leading to cell death. Hence, the immunotherapeutic effect of tumors could be enhanced by regulating the cell cuproptosis. Du et al^[102] prepared a bismuth copper oxide nanomaterial enriched with copper vacancies (BCO-V_Cu), which possessed superior piezoelectric and ferroelectric characteristics. The incorporation of copper vacancies (V_Cu) led to an enhancement in the piezoelectric properties and residual ferroelectric polarization. The optimized BCO-V_Cu exhibited outstanding ferroelectric catalytic capabilities and a strong initial polarization, thereby efficiently generating ROS through a ferroelectric catalytic mechanism. This advanced ferroelectric nanomaterial was capable of being precisely controlled, allowing it to facilitate penetration into cells by initially increasing the permeability of the cell membrane and then inhibiting the efflux of copper ions via specific proteins. This process intensified the activation of cuproptosis, inducing ICD and provoking a robust immune response. In turn, the occurrence of ICD stimulated the maturation of DCs and the polarization of macrophages toward the M1 phenotype, promoting the differentiation of cytotoxic T cells for immunotherapy (Figure 12A). Likewise, Liu et al^[105] synthesized morphology-transforming multifunctional nanotransformers (CTMF NPs) to deliver copper ions and induce cuproptosis by disrupting the TCA cycle, which yielded notable ICD and synergistic treatment by multiple anticancer mechanisms in a single therapeutic entity.

In addition, tumor cells tend to express programmed death ligand 1 (PD-L1) on their surface and achieve immune escape by binding to programmed death receptor 1 (PD-1) on T cells (immune cells), inhibiting the function of T cells, thereby leading to immune escape.^[106] Hence, utilizing immune checkpoint inhibitors (ICIs), particularly those targeting the PD-1/PD-L1 axis, has demonstrated unprecedented clinical outcomes in various malignancies.^[107] Moreover, on the basis of cuproptosis-mediated ICD, the combination of ICIs will be able to further improve the immunotherapeutic efficiency of tumors. Lu et al^[103] developed an innovative cuproptosis-inducing nanosystem (ES@CuO) involving CuO nanoparticles with ionophore ES. Upon internalization by tumor cells, ES@CuO underwent degradation, liberating Cu²⁺ ions and ES to initiate cuproptosis, effectively suppressing the proliferation of murine B16 melanoma cells. Furthermore, the ES@CuO could enhance cuproptosis-mediated immune responses and alter the typically immunosuppressive TME by increasing the infiltration of lymphocytes into the tumor and promoting the secretion of inflammatory cytokines, resulting in an effective immune response against cancer. In addition, the integration of ES@CuO with PD-1 immunotherapy could significantly boost the antitumor efficacy in a murine melanoma model (Figure 12B). In a related study, Xu et al^[104] constructed a spiky copper-based MOF (S@Cu-MOF) and combined it with αPD-1 for cancer therapy. The spiky structure could promote tumor cell uptake of S@Cu-MOF. Moreover, this synergistic approach has been shown to further enhance cuproptosis-mediated cancer immunotherapy. By combining the cuproptosis activation property of the S@Cu-MOF with the immune checkpoint inhibition offered by αPD-1, it could overcome the limitations of individual treatments and provide a more potent therapeutic effect against cancer (Figure 12C).

Furthermore, recent studies indicated that intratumoral Cu levels in tumors could affect PD-L1 expression, consequently affecting the responsiveness of tissue to immune therapy.^[108,109] Guo et al^[98] built a ROS-sensitive polymer for coencapsulating ES and Cu to create nanoparticles (NP@ESCu). Once these nanoparticles penetrate cancer cells, the high levels of ROS within the cells trigger the release of ES and Cu. The released ES would be quickly effluxed and act as a chelator, facilitating the uptake of additional extracellular Cu²⁺ ions into the cancer cells. This procedure established a shuttling mechanism that promoted the continuous accumulation of Cu within the mitochondria of the cancer cells, leading to the occurrence of cuproptosis. Significantly, the sustained increase in copper levels within the cells can induce the upregulation of PD-L1 expression on the cancer cell surface. This upregulation is a key factor in enhancing the effectiveness of αPD-L1 for immune therapy. In a bladder cancer mouse model, the combined antitumor efficacy of NP@ESCu and αPD-L1 was evaluated. This study marked the first instance of integrating a nanomaterial capable of inducing cuproptosis with αPD-L1 to augment the therapeutic impact on cancer (Figure 12D).