Cell death mediated by nanotechnology via the cuproptosis pathway: A novel horizon for cancer therapy

Cuproptosis, the current form of regulated cell death characterized by copper overload, oligomerization of lipoacylated proteins, and loss of Fe–S cluster proteins, has been proposed to function closely with human diseases, including cancer. Since its first identification in 2022, many strategies have been developed to induce cuproptosis for cancer therapy, such as small‐molecule drugs and nanomaterials. Although many reviews related to cuproptosis have been reported, they remain at a basic mechanism level, and a summary covering recent progress in the field of nanotechnologies in cuproptosis‐based cancer therapy has not yet been presented. Therefore, it is time to fill the gap and shed light on future directions for the application of this promising tool to fight against cancer. In this minireview, we first expounded the mechanism of action of cuproptosis and emphasized the feasibility of triggering cuproptosis for cancer therapy. The recent progress of cancer treatments based on nanoparticle‐induced cuproptosis was then described. Finally, the challenges and future development directions of the emerging field of cuproptosis are also discussed.

arduous and unsatisfactory in performance, as existing approaches can barely kill cancer cells while sparing surrounding normal cells. 6,7 Selectively inducing programmed cell death (PCD) in cancer cells is a promising option for cancer therapy, including apoptosis, autophagy, and necrosis. Among them, apoptosis is considered to be the preferred alternative, but the therapeutic effect is still far from satisfactory due to the intrinsic resistance induced by tumor heterogeneity. 8,9 In addition, acquired resistance results in high doses of the drug with severe side effects. Moreover, current necrosis inducers for anti-cancer applications also face technical challenges because some inflammatory signaling pathways, such as nuclear factor kappa-B (NF-κB) or mitogen-activated protein kinase (MAPK) pathways, are activated with the induction of cell necrosis, and the therapeutic effect was thus unsatisfactory. 10,11 Therefore, development of a novel cell death mode with more efficiency and fewer side effects is an urgent concern in the field of cancer therapy. Fortunately, recent studies have proposed several unidentified cell death forms with unique regulatory pathways, including ferroptosis, pyroptosis, and cuproptosis, which can circumvent the limitations of classical cell death methods and open up new opportunities for cancer treatment. [12][13][14][15] Among these non-apoptotic forms of PCD, cuproptosis has received much attention as the most emerging regulatory pathway of cell death. Interestingly, the connection between copper homeostasis and physical health was explored long before the term cuproptosis was established. The disequilibrium of copper homeostasis was repeatedly found to be associated with the development of various diseases, such as Menkes disease, Wilson's disease, neurodegenerative diseases, cardiovascular diseases, and cancer. [16][17][18][19][20] Despite the apparent importance of copper for physiology and pathology, the underlying cellular mechanisms are still largely unknown, which prompts extensive exploration of copper in the treatment of various diseases. For example, the same morphological and molecular changes were observed after treating cancer cells with disulfiram (copper ionophore), pyrazole-pyridine copper complexes, and inorganic copper, indicating that copper overload was the cause of cell death. 21 Moreover, the killing effect of elesclomol (copper ionophore) was totally lost on coadministration of MD Anderson-metastatic breast-435 (MDA-MB435) melanoma cells in the absence of serum (the source of copper), while it could be restored after adding copper instead of iron, manganese, and zinc to the serum-free medium, suggesting a potential copper ion-regulated cell death mode. 22 Afterwards, the anticancer effect of elesclomol was further corroborated by inducing a variety of cells, including melanoma cells, lung cancer cells, glioblastoma stem cells, and gynecological tumor cells, to produce reactive oxygen species (ROS). [23][24][25][26] However, the oxidative stress alone does not fully explain the mechanism of cell death, because the use of ROS scavenger N-acetylcysteine (NAC) can only partially reverse the elesclomol-induced cancer cell death. 27,28 Therefore, apart from oxidative stress, elesclomol should have additional mechanisms to regulate cancer cell death.
Gratifyingly, the "cuproptosis" proposed by Tsvetkov et al. 29 provides a definitive explanation for the anticancer mechanism of elesclomol ( Figure 1). Intracellular copper ions induce a new type of regulatory cell death characterized by the aggregation of lipoacylated mitochondrial enzymes and the loss of Fe-S clusters, and inhibitors of apoptosis, necrosis, ferroptosis, and oxidative stress pathways fail to inhibit copper-induced cell death. Specifically, different metal ions, including copper, were carried by elesclomol to verify that only copper ions can mediate cancer cell death, which could be reversed by the copper ion chelating agents glutathione (GSH) and tetrathiomolybdate (TTM) but not by known inhibitors of various cell death pathways (ferrostatin-1, necrostatin-1, NAC), confirming that cell death induced by copper ions, namely cuproptosis, may be a new type of cell death mode different from traditional cell death, such as apoptosis, ferroptosis, necrosis, and autophagy. Then, mitochondrial respiration-dependent cells showed higher sensitivity to copper ions than glycolysis-dependent cells, suggesting that cuproptosis was related to mitochondrial metabolism. Further investigation revealed that the respiratory reserve capacity was significantly reduced after copper ion treatment, while basic respiration or adenosine triphosphate (ATP)-related respiration remained stable, indicating that copper acted on the components of the tricarboxylic acid (TCA) cycle rather than the electron transport chain (ETC). After that, seven TCA cycle genes were identified as relevant for the cuproptosis mechanism, including ferredoxin 1 (FDX1) (a reductase reducing Cu 2+ to Cu + ), lipoyltransferase 1 (LIPT1), lipoyl synthase (LIAS), dihydrolipoamide dehydrogenase (DLD) (three key enzymes of the lipoic acid pathway), dihydrolipoamide S-acetyltransferase (DLAT), pyruvate dehydrogenase alpha 1 (PDHA1), and pyruvate dehydrogenase beta (PDHB) (three components of the pyruvate dehydrogenase complex). Moreover, FDX1 and LIAS knockdown alleviated copper ionophore-triggered cytotoxicity, emphasizing the inherent association between the TCA cycle and cuproptosis. Furthermore, the copper ions reduced by FDX1 bound to the lipoacyl group of DLAT to promote its lipoacylation and aggregation, thereby exerting cytotoxicity. In addition, Cu + also induced the instability of the Fe-S cluster protein, which exacerbated cell death. Notably, in vivo experiments confirmed that cell death caused by copper homeostasis imbalance and cell death induced by copper ionophore belonged to the same cuproptosis mechanism. Altogether, cuproptosis is a copper-dependent form of novel cell death. The establishment of the concept not only illuminates the cytotoxic mechanism in copper ionophore, but also provides new insights for the treatment of various diseases, including cancer.
However, cuproptosis still faces several challenges before realizing effective cancer treatment, such as selectively increasing the concentration of copper ions in cancer cells, avoiding copper ion damage to normal cells, and F I G U R E 1 Cuproptosis pathway. The copper ions released by the copper ionophore bind to DLAT, causing lipoacylated DLAT oligomerization and Fe-S cluster protein instability, which ultimately triggers cuproptosis. By Figdraw. prolonging the time of cuproptosis. [30][31][32] Nanotechnology may provide an alternative to deal with such a dilemma. The past few decades have witnessed the rapid development of nanotechnology, especially in nano-drug delivery, including improved drug solubility, prolonged circulation time, preferential accumulation of drugs at lesion area and reduced systemic side effects. [33][34][35] Copper-based nanomaterials represent a novel class of cuproptosis inducers that can achieve active targeting by surface modifications and passively accumulate at the tumor site via enhanced permeability and retention (EPR) effect. 36,37 The excessive copper ion binding to DLAT in tumor cells causes aggregation of lipoacylated DLAT, and induces destabilization of Fe-S cluster proteins, which ultimately leads to cuproptosis of tumor cells, thus exerting a therapeutic effect. Although many reviews have elaborated on the close relationship between the cuproptosis mechanism and cancer, the recent progress in the field of cuproptosis nanomedicine for cancer therapy has not yet been presented.
In this minireview, we first comprehensively elaborate on the regulatory mechanism of cuproptosis in the introduction section and then summarize the representative research on the use of different nanosystems to treat cancer based on the cuproptosis mechanism ( Figure 2). Finally, the challenges and future research directions are discussed.

NANOTHERANOSTICS FOR CUPROPTOSIS-BASED CANCER THERAPY
Since the concept was proposed, increasing evidence has demonstrated the anti-cancer promise of cuproptosis induction, and much effort has been devoted to the design and development of various cuproptosis-based nanomaterials for the eradication of malignancies. These nanomaterials upregulate the local copper ion concentration by delivering copper ions to the tumor lesion site. Subsequently, if the DLAT oligomerization and loss of Fe-S clusters are detected, cuproptosis is believed to be achieved. For example, He and coworkers 38 reported a copper-based nanomedicine (CuET NP) including the copper (II) bis(diethyldithiocarbamate) (CuET) encapsulated by the bovine serum albumin shell to replace drug-resistant cisplatin for the treatment of non-small-cell lung cancer. Cisplatin resistance was attributed to the high concentration of GSH, and CuET could be a candidate for alternative therapy because of its GSH-resistant performance endowed by the chelating geometry of CuET and the strong bonding of Cu-S. After intravenous injection, the CuET was found to accumulate obviously in tumor cells due to the EPR effect. CuET not only reduced the expression of FDX1 to induce cuproptosis but also bound to the P97 segregase adaptor NPL4 and induced cytotoxicity, thus demonstrating excellent tumor inhibition ability (tumor inhibition rate: 56%). These results illustrated that nanosystem-induced cuproptosis of tumor cells might be a promising cancer treatment strategy.
Due to the heterogeneity of tumor cells, chemotherapy alone may be less efficient and less comfortable in the treatment of some cancers, so it is necessary to deliver combination therapies in a number of ways for improving the treatment effect. 39 Figure 3E). These results illustrated that cuproptosis via consuming intracellular glucose and GSH concentrations is a promising cancer therapeutic strategy.
Cu + plays a major role in copper ion-mediated cancer treatment, as it promotes DLAT oligomerization and loss of Fe-S clusters to induce cuproptosis in tumor cells. Although Cu + represents a critical regulatory factor in the cuproptosis pathway and delivery of Cu + can contribute to inducing cuproptosis, direct delivery of Cu + is difficult due to its instability, which has prompted a motion toward the delivery of Cu 2+ , and Cu 2+ is reduced to Cu + in vivo for inducing cell cuproptosis. For example, Xu and coworkers 42 developed a photothermally triggered copperdoped nanosystem (Au@mesoporous silica nanoparticle (MSN)-Cu/polyethylene glycol (PEG)/DSF) including the mesoporous silica-coated Au nanorod (NR) (Au@MSN) as a photothermal agent with the excellent Cu 2+ and anti-cancer drug disulfiram (DSF) loading ability for synergistic cuproptosis/apoptosis/photothermal therapy (PTT) ( Figure 4A). The synthesized Au@MSN-Cu/PEG/DSF had a good photothermal-triggered degradation perfor-mance because the heat could accelerate the breakage of the Cu-O bond and promote the release of Cu 2+ and DSF. As shown in Figure 4B, the Cu 2+ release from the light irradiated Au@MSN-Cu/PEG/DSF group showed a significant increase with time compared to the non-irradiated group, confirming the excellent photothermal-triggered Cu 2+ release performance of Au@MSN-Cu/PEG/DSF. The released Cu 2+ reacted with the DSF in situ to form the cytotoxic CuET, which converted Cu 2+ to Cu + and caused a significant reduction in cell viability; the reduction in cell viability was reversed by treatment with the copper chelating agent TTM, indicating that Cu 2+ -mediated cell death was associated with cuproptosis ( Figure 4C). Moreover, the light-irradiated Au@MSN-Cu/PEG/DSF group significantly downregulated the expression of DLAT and LIAS, further confirming that Cu 2+ -mediated cell death was cuproptosis ( Figure 4D). Apart from Cu 2+ -mediated cuproptosis, the released DSF-mediated apoptosis and Au@MSN-mediated PTT also played a synergistic therapeutic role. Benefiting from the synergistic therapeutic effect of cuproptosis/apoptosis/PTT, after injecting the Au@MSN-Cu/PEG/DSF in vivo, the tumor growth of tumor-bearing mice was significantly suppressed (80.9% inhibition rate average), indicating the excellent anticancer effect of the Au@MSN-Cu/PEG/DSF ( Figure 4E,F). This study provides a promising strategy for cancer treatment via cuproptosis/apoptosis/PTT synergistic therapy.
Recently, a variety of endogenous stimulus-responsive nanomaterials have been designed to respond to certain unique features in the tumor microenvironment, such as hypoxia, acidic pH, high ROS, overexpressed enzymes, and enriched GSH, for improving the selectivity and specificity of cancer treatment. 43 For example, Zhang and coworkers 44 developed a pH-responsive nano-delivery system (HFn-Cu-regorafenib (REGO) NPs) consisting of human H-ferritin (HFn), chemotherapeutic agent regorafenib and Cu 2+ to induce autophagy and cuproptosis for glioblastoma treatment ( Figure 5A). Benefiting from the modification of HFn, HFn-Cu-REGO NPs showed good blood-brain barrier permeation, tumor-site accumulation, and pH-responsive disassembly capability. Upon treatment with HFn-Cu-REGO NPs, the pH-responsive nano-delivery system was responsive, disassembled, and released regorafenib and Cu 2+ in response to the acidic pH, causing concentrations of regorafenib and Cu 2+ was locally elevated in the tumor region. On the one hand, excessive intracellular Cu 2+ activated the copper homeostasis system, leading to the upregulation of copper efflux receptors and downregulation of copper uptake receptors ( Figure 5B); on the other hand, Cu 2+ promoted the aggregation of lipoacylated DLAT ( Figure 5C) and triggered cuproptosis. In addition, the released regorafenib induced lethal autophagy arrest to exert the therapeutic effect by preventing autophagy lysosomal fusion. Based on the Cu 2+ -induced cuproptosis and regorafenib-mediated lethal autophagy arrest, HFn-Cu-REGO NP-treated tumorbearing mice showed delayed tumor growth and the lowest bioluminescence among all groups, indicating the optimal anti-cancer effect of HFn-Cu-REGO NPs ( Figure 5D). This study provides new insights into the treatment of cancer via targeting the delivery of Cu 2+ in response to endogenous stimulation to induce cuproptosis in cancer cells.
Because complicated pathophysiology involves multiple mechanisms, cancer exhibits similar acidic environments compared with other diseases, such as inflammation and cardiovascular disease, leading to nonspecific accumulation of single-pH-responsive nanomaterials. 45,46 Multi-stimuli responsive nanomaterials have been explored to achieve responsiveness to multiple pathological environmental variations for improved specificity and targeting. 47,48 For example, Suo and coworkers 49 designed a GSH/pH-responsive hollow amorphous metal organic framework (HaMOF) (DOX@Fe/CuTH) consisting of Cu 2+ , disulfide bond (S-S)-bearing 3,3′dithiobis(propionohydrazide) and Fe 3+ encapsulated by hyaluronan shell with good doxorubicin (DOX) loading ability for cuproptosis/ferroptosis/apoptosis synergistic cancer therapy ( Figure 6A). Because of the high affinity between hyaluronan and CD44 receptor overexpression on the surface of cancer cells, hyaluronan endowed the DOX@Fe/CuTH with cancer site-specific targeting capability, resulting in enhanced tumor-site accumulation and preferential internalization within cancer cells. After cell internalization, DOX@Fe/CuTH showed GSH/pH-responsive cargo (Cu 2+ , Fe 3+ , and DOX) release due to the GSH-triggered fracture of disulfide bonds and acidic pH-triggered weakening of ion coordination. The Cu 2+ released from DOX@Fe/CuTH induced abnormal oligomerization of DLAT, with more obvious DLAT foci than PBS-treated cells. In addition, the expression levels of FDX1 and LIAS in the DOX@Fe/CuTH-treated  Figure 6B). Meanwhile, the poor cell vitality after DOX@Fe/CuTH treatment was significantly restored by the copper chelating agent ( Figure 6C). These results collectively confirmed that Cu 2+ induced cancer cell death through the cuproptosis pathway. Apart from Cu 2+ -mediated cuproptosis, Fe 3+ -mediated ferroptosis and DOX-mediated apoptosis also exhibited synergistic therapeutic effects. Therefore, after intravenous injection, the tumor volume after nano-drug treatment was 0.7-fold that before treatment, remarkably smaller than those of control (4.1-fold), DOX (3.0-fold), CuTH (2.4-fold), and Fe/CuTH (1.4-fold) groups. The results demonstrated that DOX@Fe/CuTH had a potent anti-cancer effect via cuproptosis/ferroptosis/apoptosis synergistic therapy ( Figure 6D).

CONCLUSION AND PERSPECTIVES
Cancer is already the second most lethal disease after cardiovascular disease, and this grave situation has led to the search for effective control and intervention techniques. [50][51][52] Cuproptosis is a new form of PCD proposed in 2022 in which copper ions play an important role. 29 Since then, the cellular regulatory mechanisms and signaling pathways involving cuproptosis have been investigated extensively, which propelled the movement toward cuproptosis inducers represented by copper-based nanomaterials for the eradication of malignant tumors. As a novel model of cell death regulation, cuproptosis not only addresses the problem of resistance introduced by apoptosis but also circumvents the inflammatory risk of necrosis. 53,54 In this minireview, we first comprehensively expound the regulatory mechanism of cuproptosis in the introduction section and then introduce the application of the cuproptosis mechanism in the field of nanomedicine, which may provide new insights into designing copperbased nanomaterials for cancer therapy. Despite the rapid development of studies on cuproptosis-based cancer therapy, challenges remain to be addressed, along with tremendous opportunities. Firstly, the understanding of the complicated molecular mechanism and associated regulatory pathways of cuproptosis is still in its infancy, and more studies are required to understand the exact regulatory mechanism. Knowledge of the mechanism of cuproptosis may help in designing a more effective anti-cancer nanomaterial. 15,55 Secondly, cuproptosis is a process closely related to cellular copper metabolism, so it is imperative to reasonably control intracellular copper ion concentrations in both in vitro and in vivo studies. Regarding cancer, cuproptosis is a double-edged sword because it can not only kill cancer cells but also induce copper toxicosis to destroy normal cells. 56,57 Therefore, particular attention should be paid to the parameters such as type, dosage, and timing to obtain an optimal relationship between efficacy and side effects in practical applications. 58,59 Thirdly, the mechanism of copper ion-mediated cancer treatment may include Fenton reaction and cuproptosis on the basis of the present study. Fenton reaction refers to the reaction of copper ions with H 2 O 2 to generate •OH, showing an increase in ROS, 60 while cuproptosis is characterized by copper ion-induced DLAT oligomerization and Fe-S cluster loss. Thus, a clear understanding of the mechanism of action could aid the rational design of highly selective and specific copper-based nanosystems. Meanwhile, this also suggests that combining crosstalk between different cell death phenotypes may be an effective therapeutic regimen and possess potential clinical application in cancer treatment. 61,62 Fourthly, the existing nanomaterials are weak in the responsive release of copper ions because of the small difference in enzyme activity or acidity between the tumor and normal tissue resulting from the diversity and heterogeneity of the tumor. 7 Hence, there is a pressing demand for designing ultra-sensitive nanomaterials that can be activated within a very narrow threshold. 63,64 Fifthly, during the circulation process, the dissolution of copper in copper-based nanomaterials seriously affects the therapeutic efficacy and cycle stability. Several nanoparticles mentioned in this review specifically deliver Cu + /Cu 2+ to tumor region through coordination encapsulation and surface modification, and Cu 2+ is reduced to Cu + in vivo, resulting in excessive Cu + in tumor cells. After that, Cu + promotes DLAT oligomerization and Fe-S cluster loss, finally achieving effective cuproptosis. Therefore, in order to better apply cuproptosis to cancer therapy, future research should focus on how to improve the specificity, stability, and biocompatibility of nanocarriers and intracellular Cu + concentration. For example, supramolecular nanomaterials exhibit strong stability due to their dynamic characteristics and high correlation constant between matching groups, and may be a promising cuproptosis inducer. 65,66 Lastly, it is highly anticipated that cuproptosis can be extended to the treatment of other diseases. In addition to chemotherapy, nano-drug-based cell cuproptosis therapies are also expected to be widely combined with other treatments, which can further improve the treatment efficiency of tumor therapies based on cell cuproptosis.
In conclusion, cuproptosis-based nanomedicine is a promising cancer treatment strategy. We believe that with the deepening of our understanding of cuproptosis and its relationship with nanomaterials, cancer therapeutic outcomes will be continuously improved through the design and development of copper-based nanomaterials.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.