A mitochondrion-targeted natural polyphenolic copper carrier overcomes tumor resistance to cisplatin by potentiating cuproptosis

A mitochondrion-targeted natural polyphenolic copper carrier overcomes tumor resistance to cisplatin by potentiating cuproptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A mitochondrion-targeted natural polyphenolic copper carrier overcomes tumor resistance to cisplatin by potentiating cuproptosis Haoyu Yang, Xiang Xiong, Xin Chen, Siqi Huang, Hongfang Dai, Liqin Yuan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7551173/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jan, 2026 Read the published version in Journal of Experimental & Clinical Cancer Research → Version 1 posted 11 You are reading this latest preprint version Abstract Platinum-based drug resistance remains a major obstacle in cancer therapy. Cuproptosis, a novel form of copper-dependent cell death regulated through mitochondrial pathways, represents a promising strategy to counteract drug resistance in tumors. However, its efficacy is constrained by several physiological barriers, including elevated intracellular glutathione (GSH) levels, inadequate copper accumulation both cytoplasmically and within mitochondria, and the overexpression of copper efflux transporters such as ATP7A/B. To overcome these limitations, we developed a mitochondrion-targeted polyphenol–copper nanocarrier (denoted bm–Cur–Cu₃@RBCm, or bCCM) by chelating Cu(II) with bisdemethylcurcumin—a tridentate ligand offering three copper-binding sites—and encapsulating the complex within red blood cell membranes (RBCm). This system enhances cuproptosis and counteracts drug resistance through three synergistic mechanisms. First, it significantly increases intracellular copper delivery via high-capacity tridentate chelation while concurrently depleting GSH to prevent the formation of inert GSH–Cu/Pt complexes, thereby increasing the bioavailability of copper and cisplatin. Second, it promotes mitochondrial copper accumulation through targeted delivery and localized GSH depletion, leading to irreversible mitochondrial damage. Third, it downregulates ATP7B expression, thereby inhibiting copper and cisplatin efflux and enhancing both cuproptosis and chemosensitivity. In vitro and in vivo evaluations demonstrated that bCCM effectively targets tumor cells and exerts potent antitumor activity against cisplatin-resistant hepatocellular carcinoma (HCC) without inducing systemic toxicity or undesirable copper accumulation. Mechanistic studies confirmed that bCCM downregulates key proteins associated with both cuproptosis and cisplatin resistance, indicating effective synergy between cuproptosis and conventional chemotherapy. This work establishes bCCM as an innovative therapeutic platform for overcoming platinum-based chemotherapy resistance, with promising potential for clinical translation in oncology. drug resistance Cuproptosis glutathione (GSH) mitochondrial copper ATPase (ATP7A/B) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Platinum-based drugs, exemplified by cisplatin (DDP), constitute a cornerstone of cancer chemotherapy. However, their clinical utility is substantially limited by the emergence of drug resistance [ 1 ] . Prolonged or repeated drug administration has been shown to induce a multimechanistic synergistic resistance phenotype characterized primarily by two interrelated processes: first, membrane transporter-mediated drug efflux—driven by the overexpression of P-glycoprotein (P-gp) and copper-transporting ATPase (ATP7B)—which significantly diminishes intracellular drug accumulation [ 2 – 4 ] ; and second, metabolic inactivation of drugs through conjugation with endogenous molecules such as glutathione (GSH) [ 5 , 6 ] . These mechanisms often coexist and reinforce one another, forming a complex resistance network. Conventional strategies such as dose escalation or the use of more cytotoxic agents not only fail to overcome this network but also may exacerbate adverse effects [ 7 , 8 ] . Consequently, the development of innovative therapeutic strategies that effectively reverse resistance while maintaining a favorable safety profile represents a critical challenge in contemporary oncology research. Cuproptosis, a novel form of regulated cell death first described by Tsvetkov et al. in 2022 [ 9 ] , is triggered by copper overload-induced mitochondrial dysfunction [ 9 – 11 ] and offers promising potential for overcoming tumor resistance to conventional therapies [ 12 – 14 ] . Specifically, enriched intracellular copper ions within cells directly bind to lipoylated components of the mitochondrial tricarboxylic acid (TCA) cycle [ 9 ] , leading to the aggregation of lipoylated proteins—particularly dihydrolipoamide S-acetyltransferase (DLAT)—and the loss of iron–sulfur (Fe–S) cluster proteins, including ferredoxin 1 (FDX1) and lipoic acid synthetase (LIAS) [ 15 , 16 ] . These events induce proteotoxic stress and ultimately result in cell death. The mechanism of copper involvement involves two key aspects: first, FDX1 and LIAS mediate the lipoylation of DLAT, enabling copper to promote its oligomerization; second, copper downregulates Fe–S cluster proteins. Together, these processes culminate in cuproptosis [ 17 , 18 ] . Notably, not all copper ions are equally effective; cuprous ions (Cu⁺) exhibit greater toxicity than cupric ions (Cu²⁺) because of their superior ability to bind lipoylated TCA cycle proteins [ 19 ] . Therefore, to induce cuproptosis, Cu²⁺ must first be reduced to Cu⁺, dissociate from ion carriers, and subsequently bind lipoic acid moieties on DLAT to trigger aggregation. Thus, enhancing both copper accumulation and its reduction to Cu⁺ represents a crucial strategy for activating cuproptosis in resistant tumors. However, the therapeutic application of cuproptosis is limited by several obstacles. Simply elevating copper levels is often insufficient to trigger effective cuproptosis within the complex tumor microenvironment. First, elevated GSH levels—common in drug-resistant cells—chelate copper ions, reducing the amount of bioavailable copper and impeding cuproptosis [ 20 – 24 ] . Second, copper efflux is mediated by copper-transporting ATPases (ATP7A/B), which expel excess copper to maintain homeostasis [ 25 , 26 ] . These same mechanisms also contribute to resistance against chemotherapeutic agents such as cisplatin via GSH-mediated inactivation and ATP7A/B-dependent efflux. Current strategies to increase the level of intracellular copper include enhancing copper uptake, minimizing copper loss, and inhibiting copper export. Several copper ionophores—such as elesclomol (ES), dithiocarbamate, bis(thiosemicarbazone) ligands, 8-hydroxyquinolines, pyrithione, and disulfiram—have been explored for their ability to promote copper accumulation and cuproptosis [ 27 – 33 ] . Among these, ES uniquely delivers copper to mitochondria, facilitating sustained copper accumulation [ 34 ] . Nonetheless, clinical trials of ES have revealed limited efficacy due to rapid systemic clearance and metabolism [ 35 ] . Other ionophores face similar translational challenges, including poor biosafety, lack of targeting, or insufficient cuproptosis induction [ 36 ] . Thus, there is an urgent need to develop safe and efficient copper delivery systems with high loading capacity, GSH depletion ability, and ATPase inhibitory activity. In this study, we designed and synthesized a novel multifunctional copper carrier, bisdemethylcurcumin (bm–Cur), which contains multiple metal-chelating groups (adjacent carbonyl and phenolic hydroxyl moieties) capable of coordinating Cu²⁺ at three distinct sites. bm–Cur also depletes GSH via Michael addition, which is mediated by its α,β-unsaturated ketone structures [ 37 – 40 ] , and inhibits the cystine/glutamate antiporter (xCT), thereby disrupting GSH synthesis [ 41 , 42 ] . Additionally, bm–Cur facilitates mitochondrial copper delivery, induces mitochondrial damage, and demonstrates selectivity toward cancer cells over normal hepatocytes [ 43 ] . It also downregulates ATP7B expression, further inhibiting copper efflux. We therefore hypothesized that bm–Cur could serve as an ideal multifunctional copper ionophore, enhancing copper delivery while simultaneously depleting GSH and suppressing ATP7B-mediated export to potentiate cuproptosis. Leveraging these properties, we complexed bm–Cur with Cu(II) and encapsulated it within red blood cell (RBCm) membranes to form a nanocomplex designated bm–Cur–Cu(II)@RBCm (bCCM) (Scheme 1 A). Upon cellular internalization, bCCM releases both copper and bm–Cur, initiating a cascade of therapeutic effects: Cu²⁺ is reduced to Cu⁺ via a Fenton-like reaction involving GSH, and the resulting Cu⁺ binds lipoylated DLAT to trigger cuproptosis. Concurrently, bm–Cur amplifies this process by depleting GSH (via Michael addition) and inhibiting xCT while also downregulating ATP7B to reduce copper export. This self-reinforcing cycle enhances copper retention and promotes cuproptosis in cisplatin-resistant hepatocellular carcinoma (HCC) cells (Scheme 1 B). Moreover, bm–Cur and bCCM specifically target mitochondria, increase mitochondrial copper levels, deplete mitochondrial GSH, and induce mitochondrial dysfunction [ 44 – 46 ] —significantly augmenting cuproptosis and reversing cisplatin resistance (Scheme 1 C). Transcriptomic analyses further supported the association between bCCM treatment and cuproptosis activation. Both in vitro and in vivo , bCCM exhibited potent anti-HCC activity without harming normal tissues, downregulating the expression of cuproptosis-related proteins (FDX1, DLAT, ATP7B, and LIAS) and resistance markers (ATP7B and P-gp). Importantly, bCCM demonstrated favorable pharmacokinetics, high tumor accumulation, minimal off-target retention, and excellent biocompatibility, underscoring its clinical translational potential. 2. Materials and methods 2.1 Materials and reagents GSH was purchased from Shanghai Yuanye Biotechnology Co. Copper perchlorate hexahydrate was purchased from Sinopharm (Beijing, China). Dimethyl sulfoxide (DMSO) and cisplatin were purchased from Shanghai McLean Biochemical Technology Co. Phosphate buffer solution (PBS), fetal bovine serum (FBS), Duchenne-modified Eagle’s medium (DMEM), trypsin, and methylthiazolium tetrazolium (MTT) were purchased from Procell Life Science and Technology Co. Ltd. (Wuhan, China). The Annexin V–FITC Apoptosis Detection Kit and BCA Protein Detection Kit were purchased from Beijing Sola Bio–technology Co. Reactive oxygen species (ROS) assay kits, GSH and GSSG assay kits, ATP assay kits, cellular mitochondrial isolation kits, 4% paraformaldehyde (PFA), Hoechst 33342, 4',6–diamidino–2–phenylindole (DAPI), and radioimmunoprecipitation (RIPA) lysates were purchased from Biyuntian (Nanjing) Bio–Technology Co. β–Actin (1:2000), FDX1 polyclonal antibodies, ATP7B polyclonal antibodies, DLAT polyclonal antibodies and LIAS polyclonal antibodies were purchased from Wuhan Protein Technology Co. xCT polyclonal antibodies were purchased from Abcam plc (Cambridge, UK). The Cellular Cuprous Fluorometric Assay Kit, Cell Copper (Cu 2+ ) Colorimetric Assay Kit (Complexing Method), Copper (Cu 2+ ) Colorimetric Assay Kit (Complexing Method), terminal deoxynucleotidyl transferase–mediated nick end labeling (Complexing Method), terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) and 3,3'–diaminobenzidine (DAB) staining were purchased from Elabscience Biotechnology Co. (1E, 6E)–1,7–Bis (3,4–dihydroxyphenyl)–1,6–heptadiene–3,5–dione) synthesized from curcumin was named bm–Cur. bm–Cur was obtained from the Laboratory of Chinese Medicine Activity Screening and Eugenics, Hunan University of Traditional Chinese Medicine. HepG2 cells were purchased from Fuheng Biological Company, Shanghai, China (RRID: CVCL_0027). BEL7402/DDP and A2780/DDP cells were purchased from Meixuan Biological Company, Shanghai, China. Hepa1–6 (RRID: CVCL_0327) and HepG2/DDP cells were purchased from Procell Life Science and Technology Co., Ltd. The BEL7402/DDP, A2780/DDP, and HepG2/DDP cell lines are all cisplatin-resistant. Cisplatin-induced cisplatin-resistant cell lines were established in vitro and verified via the CCK-8 method. 2.2 Apparatus UV spectra were measured on a UV–1800 UV spectrophotometer (Shimadzu Corporation, Tokyo, Japan). Fluorescence imaging was performed on a confocal microscope (FV 1200, objective lenses: 60× and 20×) (Olympus Corporation, Tokyo, Japan). Apoptosis analysis was performed on a flow cytometer (Shenzhen Dakovi Biotechnology Co., Ltd.). Mass spectrometry (MS) was performed with a liquid mass spectrometer (Thermo Fisher Scientific, Rockford, IL, USA). Cellular copper ion assays, tissue copper ion assays, cytosolic cuprous ion assays, MTT assays, intracellular ATP content assays, and intracellular GSH content assays were performed with a microplate detector (PerkinElmer Inc., Waltham, Massachusetts, USA). The samples for fluorescence measurements were reacted in a 37°C water bath. All plastic products used in the cell chambers were autoclaved. All the cells were cultured in a carbon dioxide incubator (HERA CELL 150i) (Thermo Fisher Scientific, Rockford, IL, USA). All the cell experiments were performed in a biosafety cabinet (Thermo Fisher Scientific, Rockford, IL, USA). 2.3 Synthesis of bCCM Five milliliters of 15 mM copper perchlorate solution was added dropwise to 5 mL of 5 mM bm–Cur solution, Tris–HCl 8.8 was added to adjust the pH to 7.4, the mixture was stirred for 2 hours, the mixture was observed to turn black, 15 mL of erythrocyte membranes were added, and the mixture was stirred for 1 hour. bCCM was obtained. 2.4 Spectroscopic Measurements The bCCM stock solution was diluted with distilled water, and GSH was prepared with distilled water. The bm–Cur solutions used for spectroscopic measurements were diluted with distilled water to the final concentration, and GSH was diluted to various concentrations. The test solutions were reacted for 120 min at room temperature, and all the spectroscopic experiments were carried out at room temperature. UV spectra were recorded after the addition of various analytes. The emission wavelengths ranged from 350 nm to 450 nm. 2.5 Cell Culture BEL7402/DDP, HepG2/DDP, HepG2 and Hepa1–6 cells were cultured in Dulbecco's modified Eagle’s medium (DMEM) (Gibco; Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (Procell Life Science and Technology Co., Ltd., China) and 100 U/mL/1% streptomycin in an incubator at 37°C with 5% humidity. A2780/DDP cells were cultured in Roswell Park Memorial Institute medium 1640 (RPMI–1640) (Gibco; Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (Procell Life Science and Technology Co., Ltd., China) and 100 U/mL/1% streptomycin in an incubator at 37°C with 5% humidity, where BEL7402/DDP was incubated with a cisplatin concentration of 200 nM, HepG2/DDP with 1 µM and A2780/DDP with 500 nM. 2.6 Cell treatment Cisplatin was dissolved in dimethyl sulfoxide at a concentration of 100 mM, copper perchlorate was dissolved in distilled water at a concentration of 100 mM, and bCCM was stored in cell culture medium and diluted to a specific concentration for cell processing. 2.7 CCK–8 assay BEL7402/DDP cells were inoculated into 96–well plates (8 × 10 3 cells/well) and incubated in cell culture medium (10% FBS) with different concentrations of bCCM (0–60 µg/mL) and cisplatin (0–20 µM) for 24 h. Then, CCK–8 solution was added to each well, and the mixture was incubated for 1 h. The cells were then subjected to a CCK–8 assay at 450 nm in a microtiter plate. 2.8 YO–PRO–1/PI apoptosis and necrosis assay BEL7402/DDP cells (5 × 10 5 cells/well) were inoculated in six–well plates and incubated with 1 mL of YO–PRO–1/PI test working solution for 20 min after 12 h of treatment with DDP (5 µM), bCCM (60 µg/mL), or bCCM (60 µg/mL) + DDP (5 µM), and red and green fluorescence was observed at the end of the incubation period via confocal laser scanning microscopy. 2.9 Cellular copper ion imaging and detection BEL7402/DDP cells (5 × 10 5 cells/well) were inoculated in six–well plates and incubated with rhodamine B hydrazide (20 µM) for 30 min after 6 h of treatment with DDP (5 µM), bCCM (60 µg/mL), or bCCM (60 µg/mL) + DDP (5 µM), and red fluorescence was observed at the end of the incubation via confocal laser scanning microscopy. For the copper ion assay, BEL7402/DDP cells were inoculated in six–well plates, and after 24 h of different treatments, the cells were collected, supplemented with 0.2 mL of lysate, lysed on ice for 10 min, and then centrifuged for 10 min at 4°C and 12,000 × g. The supernatant was collected for the assay, and some of the supernatant was retained for use in the protein concentration assay. A total of 100 µL of standard or sample to be tested was added to the bottom of each well, and 50 µL of color working solution was added. The membrane was covered, incubated at 37°C for 5 min, and the OD value of each well was detected at 580 nm. Copper ion content (µmol/gprot) = (ΔA580 – standard curve intercept) ÷ standard curve slope × sample dilution ÷ sample protein concentration. 2.10 ROS assay and live/dead staining The cells (1 × 10 6 cells/well) were cultured in 6–well plates, treated with DDP (5 µM), bCCM (60 µg/mL), or bCCM (60 µg/mL) + DDP (5 µM) for 6 h, incubated with DCFH–DA reagent for 30 min, and then immediately visualized under a confocal laser scanning microscope. Live/dead cell staining: Cells (1 × 10 6 cells/well) were cultured in 6–well plates, treated with DDP (5 µM), bCCM (60 µg/mL), or bCCM (60 µg/mL) + DDP (5 µM) for 6 h, incubated with an appropriate volume of calcein AM/PI assay working solution for 30 min, and then observed under a confocal laser scanning microscope. 2.11 Intracellular GSH assay The cells (1 × 10 6 cells/well) were cultured in 6–well plates with DDP (5 µM), bCCM (60 µg/mL), or bCCM (60 µg/mL) + DDP (5 µM) for 24 h, after which the cells were harvested. Three times the volume of the cellular precipitate was added to Protein Removal Reagent M solution (GSH and GSSG Assay Kit purchased from Beyotime Institute of Biotechnology, China). If the volume of the cellular precipitate was 10 microlitres, 30 microlitres of protein removal reagent M solution was added, and the mixture was vortexed thoroughly. The volume of the cellular precipitate can be estimated from the weight of the precipitate. The centrifuge tubes were weighed before and after collection of the cells so that the weight of the cellular precipitate could be calculated. A total of 10 mg of cellular precipitate can be roughly viewed as 10 µL. The samples were then subjected to two rapid freeze–thaw cycles via liquid nitrogen and a water bath at 37°C. The samples were left at 4°C or in an ice bath for 5 min and centrifuged at 4°C for 10 min at 10,000 × g. The supernatant was used for GSH testing. 2.12 Intracellular ATP assay The cells (1×10 6 cells/well) were cultured in 6–well plates containing Cisplatin (5 µM), bCCM (60 µg/mL), or bCCM (60 µg/mL) + Cisplatin (5 µM) for 24 h, and the cells were lysed by adding lysis buffer at a ratio of 200 µL of lysate per well to a 6–well plate. To sufficiently lyse the cells, a pipette can be used to repeatedly blow or shake the plate so that the lysate can fully contact and lyse the cells. The cells will be lysed immediately after contact with the lysate. After lysis, the cells were centrifuged at 12,000 × g for 5 min at 4°C, and the supernatant was removed for subsequent assays. One hundred microliters of ATP assay working solution was added to the assay wells. After leaving at room temperature for 3–5 min, 20 µL of sample or standard was added to the assay wells or tubes, which were mixed quickly with a gun (micropipette), and after an interval of at least 2 s, the RLU value or CPM was determined with a chemiluminescence meter (luminometer) or liquid flash meter. 2.13 Western blot The cells (1 × 10 6 cells/well) were cultured in 6–well plates with DDP (5 µM), bCCM (60 µg/mL), or bCCM (60 µg/mL) + DDP (5 µM) for 24 h and harvested. The protein concentration in the total extract was quantified via a BCA protein assay kit (562 nm). The expression of GST was detected strictly by western blot (WB) with different antibodies: β–actin (1:2000), xCT polyclonal (1:1000), FDX1 polyclonal (1:1000), DLAT polyclonal (1:2000), ATP7B polyclonal (1:1000), LIAS polyclonal (1:1000), and P–gp polyclonal (1:500) antibodies. Detection was carried out on a BIO–RAD ChemiDoc XRS chemiluminescence system. 2.14 Mitochondrial damage tracking Cells were seeded into 12–well plates (2 × 10 5 cells/well) with DDP (5 µM), bCCM (60 µg/mL), or bCCM (60 µg/mL) + DDP (5 µM) for 24 h. Subsequently, the cells were stained with MitoTracker Red CMXRos for 30 min, fixed with 4% PFA for 15 min and stained with DAPI (200 µL) for 15 min, followed by fluorescence imaging. 2.15 JC–1 testing The cells were seeded into 12–well plates (2 × 10 5 cells/well) with DDP (5 µM), bCCM (60 µg/mL), or bCCM (60 µg/mL) + DDP (5 µM) for 24 h. Subsequently, the cells were stained with JC–1 (2.0 µg/mL) staining solution at 37°C for 20 min, fixed with 4% PFA for 15 min and stained with DAPI (200 µL) for 15 min, followed by fluorescence imaging. 2.16 In vivo imaging BALB/C mice (5–7 weeks, 18 − 20 g) were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and were randomly divided into two groups (n = 3). BEL7402/DDP cells (5 × 10 6 cells in PBS) were injected subcutaneously into the nude mice. When the tumor volume reached approximately 80 mm 3 , the mice were injected in situ with bCCM@IR783, and small animal imaging was performed. The animal study protocol was approved by the Animal Research Ethics Committee, Hunan University of Chinese Medicine. 2.17 Nude mouse model Female BALB/c nude rats (5–7 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). The animal experimental protocol was approved by the Animal Research Ethics Committee of Hunan University of Traditional Chinese Medicine. BEL7402/DDP cells (1×10 7 cells) were suspended in PBS and then injected into the right hind abdomens of the mice. After two weeks of tumor growth, 10 mice were randomly divided into the following four groups (n = 5 in each group): the PBS group, DDP group (5 mg/kg), bCCM group (3 mg/kg) and bCCM/DDP group. The mice in the DDP group received 5 mg/kg/day Curcumin treatment (tail vein administration) once every two days. The control mice were injected with 0.9% NaCl. The mice were examined for tumor growth every 2 days. The mice died after intrahepatic injection of sodium pentobarbital. Two weeks after the administration of Curcumin, the mice were injected intraperitoneally with sodium pentobarbital (200 mg/kg). The size of each tumor was measured. Tumor tissue was collected for subsequent experiments. All the mice were euthanized at the end of the treatment. Tumors and major organs were harvested and weighed for H&E–staining (Ethics Approval Number: SLBH-202311090018). 2.18 In vivo antitumor activity assay The tumors were fixed with 4% PFA (24 h) and then sliced into paraffin sections (5 µm). H&E staining and TUNEL experiments were performed according to the manufacturer’s instructions. The expression of DLAT, FDX1, XCT and GPx4 in tumor tissues was detected by immunohistochemistry. The paraffin sections of tumor tissues subjected to different treatments were dewaxed. After acid–base repair, ovulin and d–biotin were added for blocking, and endogenous peroxidase was blocked with a 3% hydrogen peroxide solution. Then, the prepared primary antibody solutions were added to the slides and incubated for 2 h in a wet box away from light. The reaction enhancement solution was incubated for 20 min in a light–resistant environment and then incubated for 20 min in a dark environment with the secondary antibodies labeled with HRP corresponding to the primary antibodies. After DAB staining, the nucleus was restained with hematoxylin for 5 min. The sections were scanned and sealed under an inverted microscope. WB analysis was performed to detect protein expression levels in tumor tissues. The tumor tissues were disrupted with a homogenizer, and RIPA lysis buffer was added to extract total protein. Blood samples were collected for biochemical and hematological assays. Moreover, the heart, liver, spleen, lung and kidney were collected for sectioning and H&E staining. 2.19 Toxicity test of zebrafish embryos The zebrafish experiments were conducted in accordance with the Animal Ethics Committee of Hunan University of Traditional Chinese Medicine. All experiments used wild–type AB strain zebrafish. Zebrafish embryos were treated with bm–Cur or cisplatin at specified concentrations postfertilization (hpf) and analyzed at 24, 48, 72, or 96 hpf. 2.20 Analysis of Pearson's coefficient calculations Pearson's coefficient is used to measure the degree of linear correlation between two variables and has a value between − 1 and 1. A value of 1 represents a positive correlation, − 1 represents a negative correlation, and 0 represents no correlation. ImageJ software was used for Pearson coefficient analysis, ImageJ software was used to separate the color channels, pseudo–colors were added to the color channels, the Coloc2 plug–in was opened, and the algorithm was tickged for calculation. 2.21 Statistical analysis The data were analyzed with GraphPad Prism 8.4.0 software and are presented as the mean ± S.D. values; n = 3 independent experiments. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001, whereas ns represents no statistically significant difference compared with the vehicle group according to one–way ANOVA. 3. Results and discussion 3.1 Design, preparation, and characterization of the Cu–nanocarrier bCCM As a constituent unit of the Cu–nanocarrier bCCM, the bm–Cur compound was synthesized via efficient demethylation of curcumin (Fig. 1 A). The structural characterization was confirmed by 1H NMR spectrum analysis and high–resolution mass spectrometry (HR–MS) (Fig. 1 B and Fig. S1 ), which revealed a curcumin parent nucleus rich in four phenolic hydroxyl groups and two α,β-unsaturated ketones. Considering that bm–Cur with an α,β-unsaturated ketone structure can react with the sulfhydryl group of GSH to form a bm–Cur–SG complex through Michael addition, HR–MS and electrospray ionization (ESI) were performed to confirm the successful reaction between bm–Cur (C₁₉H₁₆O₆) and GSH. As expected, ESI analysis of the reaction between C₁₉H₁₆O₆ and GSH (Mw = 645.16) revealed a molecular ion peak at m/z = 646.1423 (Fig. 1 C and Fig. S2). Furthermore, the UV spectra revealed an absorbance peak at 430 nm in the sample containing bm–Cur alone. However, the absorbance intensity of bm–Cur at 430 nm decreased in a concentration-dependent manner with increasing GSH concentration (0–10 mM). Notably, the absorbance of bm–Cur progressively decreased over time at 10 mM GSH (Fig. S3), further confirming its responsiveness to GSH. Moreover, bm–Cur inhibited GSH synthesis by downregulating xCT expression (Fig. 1 D) and reducing the intracellular GSH level in a concentration-dependent manner (Fig. S4). Further tests on the cytotoxicity of bm–Cur toward tumor cells revealed a certain ability to kill both cisplatin–sensitive (HepG2 and Hepa1-6) and resistant tumor cells (BEL7402/DDP and HepG2/DDP) (Figs. S5 and S6). Moreover, bm–Cur significantly and concentration–dependently downregulated the expression of ATP7B, a copper ion and a cisplatin efflux protein (Fig. 1 E). Inspired by the remarkable properties of bm–Cur, which leverages the strong metal–chelating ability of its adjacent phenolic hydroxyl groups and ortho–diketone moieties, the supramolecular nanocomplex bCCM was prepared by directly chelating bm–Cur with Cu(II) in Tris HCl buffer supplemented with encapsulation in red blood cell membranes (RBCm) (Fig. 1 F). The simulated three–dimensional structure of bCCM clearly shows the three coordination sites of Cu(II) (Fig. 1 G). Furthermore, X–ray diffraction (XRD) analysis revealed that bCCM has almost no diffraction peaks, indicating that the bCCM has an amorphous structure with low crystallinity (Fig. 1 H). Infrared spectroscopy (IR) analysis revealed characteristic absorption peaks corresponding to hydroxyl groups at 3300–3600 cm − ¹ and carbonyl and C–O stretching vibrations at 1750–1700 cm − ¹ and 1300–1000 cm − ¹, respectively (Fig. 1 I). X–ray photoelectron spectroscopy (XPS) analysis revealed the presence of C, O, and Cu, with Cu(II) coordination peaks at 934 eV (Cu 2p3/2) and 954 eV (Cu 2p1/2) observed (Fig. 1 J and 1 K). These results confirmed the formation of bCCM containing Cu(II). TEM images revealed that the uniform sheet structure of nanocomplex bCC formed via the chelation of bm–Cur with copper ions (Fig. 1 L). After coating with RBCm, a sphere–like structure of bCCM formed (Fig. 1 L). The rapid disassembly of bCCM through a Fenton reaction between Cu(II) and GSH could deplete GSH into GSSG [ 45 ] and release single bm–Cur. The morphology of bCCM subsequently changed from flake to dispersed fragments after reacting with GSH, indicating that bCCM had been cracked by GSH, which further confirmed the response of bCCM to GSH (Fig. 1 L). We then investigated the GSH consumption capability of bCCM via UV–vis spectral analysis. UV–vis spectra revealed an absorbance peak at 430 nm in the bCCM + GSH group but not in the bCCM or GSH groups (Fig. 1 M). Moreover, the absorbance intensity increased with increasing GSH concentration (0–10 mM) in the bCCM + GSH group (Fig. 1 N) and decreased gradually over time at 10 mM GSH (Fig. 1 O). These results confirmed the response capability of bCCM to GSH and the release of bm–Cur from bCCM . We next evaluated the GSH consumption capacity of bCCM via UV–vis spectroscopy. A distinct absorbance peak at 430 nm was observed for the bCCM + GSH group, whereas neither bCCM nor GSH alone exhibited this peak (Fig. 1 M). Furthermore, the absorbance intensity at 430 nm increased in a GSH concentration-dependent manner (0–10 mM) (Fig. 1 N) and gradually decreased over time at 10 mM GSH (Fig. 1 O) in the bCCM + GSH group. These findings demonstrate that bCCM is responsive to GSH and subsequently releases bm–Cur, confirming its GSH-triggered degradation behavior. Collectively, these results demonstrate the efficient GSH consumption capability of bCCM. Moreover, the concentration of bm–Cur released from bCCM (30 µg/mL) upon GSH treatment was calculated to be 39.22 µM on the basis of changes in the absorbance at 430 nm (Fig. S7). Furthermore, the nanocomplexes exhibited excellent solubility and stability in various solutions, including normal saline (0.9% NaCl), PBS, and DMEM (Fig. S8), highlighting their suitability for in vitro and in vivo applications. 3.2 In vitro anti-resistant tumor efficacy of bCCM The successful determination of the extracellular GSH reaction activity of the bCCM nanocomplex led us to further explore its ability to consume intracellular GSH and generate ROS. First, the GSH content was measured in four liver–related cell lines: cisplatin–resistant human HCC cells (BEL7402/DDP), drug–sensitive human and mouse HCC cells (HepG2 and Hepa 1–6), and normal liver cells (HL–7702). As shown in Fig. 2 B, the GSH concentration in drug–resistant BEL7402/DDP cells was 21.42 ± 0.15 µM, which was much greater than that in normal liver cells (11.51 ± 0.10) and moderately greater than that in drug–sensitive HepG2 cells (16.04 ± 0.06) µM and Hepa 1–6 cells (15.76 ± 0.09) µM. Next, the test of the GSH consumption capacity at different concentrations of bCCM revealed that bCCM consumed GSH in a concentration–dependent manner in both BEL7402/DDP and HepG2 cells (Fig. S9). Additionally, GSH levels in BEL7402/DDP and HepG2 cells subjected to different treatments were detected, and neither bCCM nor bCCM + DDP depleted GSH significantly differently. In contrast, DDP had almost no ability to consume GSH in either BEL7402/DDP (Fig. 2 C) or HepG2 cells (Fig. S10). Moreover, the effects of various treatments on the cytoplasmic expression of xCT were examined. The results revealed significant downregulation of xCT in bCCM- and bCCM + DDP–treated BEL7402/DDP cells, which effectively inhibited GSH synthesis (Fig. 2 D and Fig. S11). Next, the overall level of intracellular ROS was evaluated via confocal laser scanning microscopy (CLSM) imaging with 2′,7′–dichlorofluorescein diacetate (DCFHDA) as an indicator, which is rapidly oxidized by ROS to generate the green fluorescence dichlorofluorescein (DCF) [ 47 ] . Notably, a sharp increase in DCF fluorescence was observed in the bCCM and bCCM + DDP groups after 6 h of incubation compared with the control (Fig. 2 E). In contrast, there was only a small increase in the fluorescence intensity of the DDP group, indicating that bCCM is a strong ROS inducer. These results demonstrated that bCCM could directly deplete intracellular GSH, increasing ROS levels. Encouraged by the superior GSH depletion effect, we further evaluated the antitumor efficacy of bCCM in vitro. First, single cells with morphological changes were observed in cisplatin–resistant tumor cells (BEL7402/DDP). As shown in Fig. 2 F, the cell underwent persistent swelling and membrane rupture until critical lysis was reached, resulting in cytoplasmic dispersion within 0–24 h after 30 µg/mL bCCM treatment. The cytotoxicity of bCCM was subsequently analyzed via an MTT assay in which DDP–resistant (BEL7402/DDP, A2780/DDP) and DDP–sensitive (HepG2) tumor cells were used as controls. As depicted in Fig. 2 G and 2 H, compared with the very weak cytotoxicity of DDP (IC 50 (47.2 ± 0.9) µM in BEL7402/DDP cells and (66.4 ± 1.7) µM in A2780/DDP cells), bCCM exhibited very strong cytotoxicity to DDP–resistant cells, with the half–maximal inhibitory concentration ( IC 50 ) of bCCM being calculated to be (65.4 ± 1.2) µg/mL in BEL7402/DDP cells and (40.2 ± 0.6) µg/mL in A2780/DDP cells after 24 h of treatment (Table S1 ). Furthermore, bCCM demonstrated excellent biological safety in normal hepatocytes (AML–12), with a cell survival rate above 95%, even at concentrations (90 µg/mL) much higher than the therapeutic level for DDP-resistant cells (7402/DDP and A2780/DDP) (Fig. S12), suggesting its selective cytotoxicity toward tumor cells. Considering the strong cytotoxicity of bCCM in DDP-resistant tumor cells, the sensitizing effect of bCCM to DDP was examined by monitoring the viability of DDP-resistant cells (BEL7402/DDP and A2780/DDP) under different conditions. Notably, the combination of bCCM with cisplatin had a significant additive antitumor effect on both DDP-resistant cell lines. In the BEL7402/DDP cells, the combined treatment (45 µg/mL bCCM + 15 µM DDP) reduced the cell viability to 58.14%, whereas the viability was 67.80% with bCCM alone and 89.52% with DDP alone. A more pronounced synergistic effect was observed in A2780/DDP cells, where the combination achieved 50.28% cell viability, versus 94.01% with bCCM alone (15 µg/mL) and 85.26% with DDP alone (15 µM). These results clearly indicate that bCCM can effectively sensitize DDP-resistant tumor cells to cisplatin treatment (Fig. 2 I). A subsequent live/dead cell costaining assay further confirmed the high cytotoxicity of bCCM in BEL7402/DDP cells. Confocal laser scanning microscopy (CLSM) images revealed abundant red fluorescence of dead cells in the bCCM- and bCCM + DDP–treated groups but abundant green fluorescence of living cells in the control and DDP groups, indicating strong cytotoxicity of bCCM and bCCM + DDP in BEL7402/DDP cells (Fig. 2 J and Fig. S13). Moreover, flow cytometry analysis revealed a significant difference in apoptosis after different treatments for 24 h. Compared with the control cells, living cells presented significant red fluorescence after bCCM treatment, indicating the strong ability of bCCM to induce apoptosis in BEL7402/DDP cells. Additionally, the bCCM combined with DDP group presented stronger red fluorescence to induce apoptosis in BEL7402/DDP cells, whereas negligible red fluorescence was observed in the single DDP group (Fig. 2 K and Fig. S14). These results clearly indicated the significant DDP sensitization effect of bCCM on the cisplatin–resistant BEL7402/DDP cells. 3.3 bCCM-induced Cuproptosis and RNA sequencing analysis As mentioned above, GSH consumption can induce cuproptosis by decreasing chelation with copper and increasing intracellular copper ion levels, inhibiting the activity of FDX1 and DLAT and leading to enhanced therapeutic efficacy in drug–resistant tumors (Fig. 3 A). Because of the superior GSH consumption ability and copper ion-carrying ability of bCCM , we hypothesized that bCCM could significantly induce cuproptosis. The intracellular copper ion levels were subsequently monitored by a copper detection probe after different treatments. As we expected, compared with those in the control or DDP–treated groups, copper ion levels dramatically increased in bCCM – and bCCM + DDP–treated BEL7402/DDP cells, as indicated by the strong red fluorescence (Fig. 3 B and Fig. S15). Moreover, the results of the quantitative analysis of intracellular copper ions confirmed the above findings (Fig. 3 C). These results indicated that copper ions were loaded into the cells via bCCM . Moreover, Cu(II) showed no significant cytotoxicity to BEL7402/DDP cells within a certain concentration range (Fig. S16). Furthermore, considering that copper ions are further converted into Cuprous ions in the cytoplasm through GSH or FDX1 (Fig. 3 D), the intracellular Cuprous ion levels were detected. Compared with DDP alone, both bCCM and bCCM + DDP significantly increased intracellular cuprous ion levels (Fig. 3 E), which suggests that bCCM can carry copper ions into the cell and convert them to cuprous ions to trigger cuproptosis. Encouraged by the degree of copper ion influx and cuprous ion conversion achieved, the viability of bCCM combined with the cuproptosis inhibitor UK5099 (5 µM) was detected to investigate potential cuproptosis induction by bCCM . Compared with bCCM , bCCM combined with the cuproptosis inhibitor UK5099 significantly increased cell viability (up to 15 ~ 17%), whereas UK5099 alone (5 µM) had no significant effect on BEL7402/DDP cell viability (Fig. S17), indicating that the effect of bCCM (Fig. 3 F) on Cuproptosis. Western blot (WB) analysis after bCCM treatment revealed a significant decrease in the protein expression of intracellular FDX1, DLAT and LIAS, three characteristic ferroptosis suppressors, indicating effective cuproptosis induction by bCCM in BEL7402/DDP cells (Fig. 3 G and 3 H). As ATP7B is an important copper, cuprous ion and DDP transporter, the downregulation of ATP7B expression is favorable for inducing cuproptosis and reversing cisplatin resistance. Considering that ATP hydrolysis provides energy for ATP7B, intracellular ATP levels were detected after different treatments (Fig. 3 I). A dramatic decrease in ATP levels was detected in both the bCCM- and bCCM + DDP–treated groups, indicating that ATP7B activity was inhibited (Fig. 3 J). The expression of ATP7B was further investigated, and the results confirmed significant downregulation of ATP7B and P–gp expression in the bCCM- and bCCM + DDP–treated groups (Fig. 3 K and Fig. S18), suggesting that pyroptosis may weaken the resistance of BEL7402/DDP cells. As a key copper transporter responsible for Cu⁺ and DDP efflux, ATP7B plays a crucial role in regulating intracellular copper levels [ 26 ] . The downregulation of ATP7B expression promotes pyroptosis and helps overcome cisplatin resistance. Since ATP hydrolysis provides energy for ATP7B-mediated transport [ 48 ] , we measured intracellular ATP levels after different treatments (Fig. 3 I). Notably, both the bCCM and bCCM + DDP groups presented a sharp decrease in ATP levels (Fig. 3 J), suggesting impaired ATP7B activity. Furthermore, Western blot analysis confirmed a significant reduction in ATP7B and P-gp expression in these treatment groups (Fig. 3 K and Fig. S18). These findings support the hypothesis that the induction of pyroptosis may sensitize BEL7402/DDP cells by disrupting drug resistance mechanisms. To verify the above results, genome–wide high–throughput RNA sequencing (RNA–Seq) was performed on BEL7402/DDP cells treated with bCCM and PBS as a control. Gene Ontology (GO) term analysis provided insights into the enrichment of DEGs related to the cellular response to copper ions, oxidative stress, regulation of cell death, cell migration, and copper ion transport (Fig. 3 L). Notably, “copper ions” associated with Cuproptosis, which is a key inducer of Cuproptosis, were the most significantly enriched terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis further classified and annotated these DEGs, revealing that the upregulated DEGs induced by bCCM are closely correlated with the cell cycle, glutathione metabolism, ABC transporters, the citrate cycle (TCA cycle), hepatocellular carcinoma, and platinum drug resistance (Fig. 3 M). Furthermore, gene set enrichment analysis (GSEA) revealed positively and negatively regulated pathways after bCCM administration (Fig. S19A). Notably, pathways related to “glutathione metabolism”, “citrate cycle (TCA cycle)”, and “ABC transporters” were upregulated, whereas “platinum drug resistance”-associated pathways were downregulated. These findings strongly suggest that the therapeutic mechanism of bCCM against resistant HCC involves the induction of pyroptosis and the modulation of glutathione metabolic and mitochondrial pathways. Finally, the protein–protein interaction (PPI) network of 15 proteins related to platinum resistance (ABCB1, GSTA1) revealed close interactions between proteins involved in these processes (Fig. S19B). These transcriptomics analysis results confirm the role of bCCM in inducing necroptosis and further demonstrate the close relationships among pyroptosis, platinum drug resistance, and hepatocellular carcinoma (HCC) in cisplatin–resistant HCC cells (BEL7402/DDP). 3.4 bCCM induces necroptosis–mediated mitochondrial dysfunction As pivotal players in pyroptosis and targets for cancer therapy, targeting mitochondria has tremendous therapeutic potential for drug-resistant cancer [ 49 ] . Cuproptotic cells morphologically exhibit features of mitochondrial dysfunction, including disruption of the mitochondrial structure and a reduction in the mitochondrial membrane potential [ 50 ] . Increasing damage to mitochondrial membranes and inducing dysfunction are classic characteristics of pyroptosis. To confirm that bCCM induces cuproptosis in mitochondria, the mitochondrial targeting ability of bCCM was first evaluated. Confocal microscopy analysis revealed clear and rapid colocalization (0.5 h) of bCCM and bm–Cur with mitochondria in BEL7402/DDP cells (Figs. 4 A– 4 B). The ability of bCCM to induce mitochondrial dysfunction was subsequently explored. A mitochondrial REDOX imbalance, which is induced by mitochondrial GSH consumption and copper ion imbalance in response to cuproptosis, is a significant manifestation of mitochondrial dysfunction (Fig. 4 C). The mitochondrial GSH and copper ion levels in BEL7402/DDP cells were studied. First, the downregulation of xCT in mitochondria by bCCM suggested that it can also deplete mitochondrial GSH by inhibiting its synthesis (Fig. 4 D). As shown in Fig. 4 E and Fig. S20, the lowest GSH levels were detected in the mitochondria of BEL7402/DDP cells treated with bCCM compared with those treated with cisplatin alone, resulting in a 42.8% ( bCCM ) to − 3.9% (DDP) decrease in GSH levels, indicating that the greatest increase in mitochondrial GSH consumption was caused by bCCM . Notably, the decrease in GSH levels in the bCCM + DDP group was similar to that in the bCCM group and much greater than that in the DDP group (35.1% vs − 3.9%). Moreover, compared with the DDP group, both the bCCM and bCCM + DDP groups accumulated more mitochondrial copper (3.2 ~ 3.4-fold) (Fig. 4 F). Further investigation of the expression of classic mitochondrial Cuproptosis proteins (DLAT, FDX1 and LIAS) revealed that bCCM – and bCCM + DDP–treated BEL7402/DDP cells presented significantly lower protein levels than DDP-treated cells did (Fig. 4 G and Fig. S21). Together, these findings confirmed that bCCM could indeed trigger cuproptosis in mitochondria. Given that mitochondrial membrane potential (Δψm) integrity and variation are critical for mitochondrial function, we investigated the Δψm using Mito–Tracker Red CMXRos 579nm/599nm , a mitochondrial membrane potential indicator [ 51 ] . Significant red fluorescence was observed in the BEL7402/DDP cells in the control and DDP groups (indicating a normal Δψm) after 24 h of incubation, demonstrating that DDP alone had negligible effects on the Δψm. In contrast, red fluorescence was markedly diminished in cells treated with bCCM or bCCM + DDP (Fig. 4 H), reflecting severe mitochondrial membrane damage induced by bCCM . Additionally, JC–1 was used to confirm the dissipation of the mitochondrial membrane potential, which remains as a monomer in damaged mitochondria with a dissipated Δψm and aggregates in normal mitochondria with a high Δψm [ 52 ] . For BEL7402/DDP cells, in the control and DDP groups, JC–1 aggregates constituted the majority. In contrast, the number of JC–1 aggregates significantly decreased after bCCM and bCCM + DDP treatments, implying the most severe decrease in the Δψm (Fig. 4 I), which is consistent with the MitoTracker Red results. Next, morphological changes in mitochondria were directly observed via bio–TEM to evaluate mitochondrial damage in BEL7402/DDP cells. Compared with the normal morphology of the mitochondria in the PBS–treated group, the mitochondria in the bCCM -treated group presented severe destruction and swelling, increased membrane density, decreased volume, and reduced cristae (Fig. 4 J), characteristic of mitochondrial dysfunction caused by pyroptosis. Taken together, bCCM effectively targets mitochondria and induces mitochondrial cuproptosis–like dysfunction, which is characterized by changes in the expression of cuproptosis-related proteins, mitochondrial morphological damage and membrane potential dissipation. These mitochondrial effects, combined with cytoplasmic cuproptosis, synergistically enhance cuproptosis and improve therapeutic efficacy against DDP–resistant tumors. 3.5 Biosafety evaluation of bCCM in zebrafish and mouse models After confirming the in vitro necroptosis effect of bCCM , the biosafety of bCCM was evaluated in zebrafish and mouse models. Considering that zebrafish are highly useful bioassays in toxicological studies, the in vivo toxicity of bCCM was first evaluated in a zebrafish model. Zebrafish embryos were equally divided into 8 groups and coincubated with cisplatin, bCCM, bCCM + DDP, or different concentrations of bCCM . Representative images and survival curves of zebrafish embryos were acquired at different time points (Fig. 5 A, 5 B and Fig. S22). At 48 hpf, 72 hpf, and 96 hpf, the cisplatin–treated zebrafish embryos appeared dead. At 96 hpf, representative images of zebrafish larvae were captured, which revealed that both the bCCM and bCCM + DDP groups developed normally. In contrast, DDP treatment resulted in the failure of zebrafish embryos to hatch. The heart rate of zebrafish larvae at 96 hpf was measured in all groups to assess the physiological effects. No significant changes were observed in the bCCM group compared with the control and combined groups, whereas the heart rate was reduced in the DDP group. In addition, no significant changes in body length were observed in the bCCM group compared with the control and combined groups (Fig. 5 C). Moreover, there were no significant changes in the hatching rate, survival rate, body length or heart rate of zebrafish larvae in the bCCM group with different concentrations of bCCM compared with those in the control group (Fig. 5 D), indicating that bCCM has excellent biocompatibility. To further explore the antitumor effect and mechanism of therapy in vivo , the biosafety of bCCM in mice was evaluated. First, the blood physiological and biochemical parameters of the mice treated with different drugs were investigated. Prior to in vivo treatments, red blood cell (RBC) hemolysis and coagulation assays were conducted, revealing no hemolysis or coagulation phenomena within the tested concentrations (Fig. S23), indicating good blood compatibility of bCCM . Next, four groups, namely, PBS, DDP, bCCM , and bCCM + DDP , were injected into healthy mice through the tail vein four consecutive times. Thereafter, the blood, major tissues, and organs of the mice treated with different drugs were collected for further analysis on day 21. The results revealed that blood biochemical indices and parameters, including WBC, RBC, HGB, ALT, AST, PLT, CREA, and BUN, were not significantly different from those in PBS–treated mice (Fig. S24), demonstrating negligible side effects of bCCM . Additionally, H&E staining of major organs (heart, liver, spleen, lung, and kidney) revealed no morphological abnormalities (Fig. 5 E). In contrast to DDP, which significantly caused liver and spleen damage, bCCM did not affect animal growth or damage any major organs, indicating the superior biocompatibility of bCCM . 3.6 Biodistribution of bCCM and copper ions in a DDP–resistant HCC model After confirming the excellent biosafety of bCCM , a biodistribution study was performed on a DDP–resistant animal model of HCC (BEL7402/DDP). First, IR783–labeled bCCM ( bCCM @IR783) was prepared for monitoring biodistribution in the body (Fig. 6 A). The fluorescence signal of mice injected with bCCM @IR783 via the tail vein was monitored via an in vivo imaging system instrument. The results revealed a fluorescence signal at the tumor site in the mice after the injection of bCCM @IR783 for 8 h. The fluorescence intensity at the tumor site continuously increased with time and peaked at approximately 12 h. Notably, there was a strong fluorescence signal at the tumor site even at 24 h after bCCM @IR783 injection, indicating fast accumulation and long retention of bCCM @IR783 at the tumor site (Fig. 6 B). After 24 h, the mice were sacrificed, and the ex vivo biodistribution of bCCM @IR783 was studied. The results revealed that the fluorescence signal intensity was the strongest in the tumors, which was greater than that in the livers, kidneys, hearts, lungs, spleens, and intestines, indicating that bCCM @IR783 performed well at tumor targeting (Fig. 6 C). In summary, bCCM @IR783 can rapidly target and accumulate at tumor sites, which is very conducive to its further application. Furthermore, a biodistribution study of copper ions was performed. The copper ion levels in the tumor tissue and main organs (heart, liver, spleen, lung and kidney) were detected with a copper detection kit. The results revealed no significant change in copper ion levels in the main organs (Fig. 6 D) in bCCM and bCCM + DDP. Notably, the copper ion content in tumor tissues increased after bCCM and bCCM + DDP treatments. However, there was no significant difference in copper ion levels between the two groups, which revealed that copper ions accumulate at the tumor site (Fig. 6 D). These results suggest that bCCM can selectively target tumor tissue to deliver copper ions to induce cuproptosis in vivo . 3.7 In vivo anti–resistant effect of bCCM on HCC Encouraged by the excellent in vitro antidrug resistance capability and enhanced cuproptosis effect of bCCM , we next sought to explore the in vivo anticancer efficacy in a cisplatin–resistant HCC mouse model. A BEL7402/DDP tumor model (five mice per group) was established, and the drug was administered via intravenous injection (i.j.) according to the treatment schedule (Fig. 7 A). First, the body weight changes of the mice over 21 days of treatment were monitored. Negligible changes in body weight were observed in the bCCM and bCCM + DDP treatment groups compared with the control, whereas a significant decrease was noted in the DDP–treated group, indicating the good biosafety of bCCM (Fig. 7 B). As shown in Fig. 7 C, BEL7402/DDP tumor growth was effectively inhibited by bCCM and bCCM + DDP treatments, whereas DDP did not inhibit tumor growth, highlighting the superior anticancer effect of bCCM on drug–resistant HCC, as verified by tumor images (Fig. 7 D). The bCCM + DDP groups presented the lowest tumor weight and highest tumor inhibitory ratio (TIR) at the given dose (3 mg/kg + 2.5 mg/kg), in contrast to the slight reduction in tumor size and TIR caused by DDP (5 mg/kg), suggesting poor efficiency of DDP and excellent therapeutic efficiency of bCCM in resistant HCC tumors (Figs. 7 E and 7 F). Additionally, histological and immunohistochemical analyses were carried out to confirm the antitumor effect of bCCM (Fig. 7 G). Hematoxylin and eosin (H&E) staining revealed significant tumor cell destruction in the bCCM and bCCM + DDP groups, as evidenced by massive nuclear deletions and noticeable karyopyknosis, whereas the DDP groups exhibited almost normal tumor cell morphology compared with the control. Terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) staining further confirmed the H&E results, revealing negligible apoptosis in the DDP group, whereas severe apoptosis and tumor growth inhibition were observed in the bCCM and bCCM + DDP groups (Fig. 7 H and Fig. S26). These results demonstrated that bCCM administration led to excellent antitumor efficacy in resistant HCC, whereas limited efficacy was observed in the DDP group. The superior anti-resistance efficacy of bCCM in drug-resistant HCC mice may be attributed to the effects of Cuproptosis. Subsequently, immunohistochemical staining and western blotting of tumor tissues were performed to explore the mechanism underlying the resistance capability of bCCM via cuproptosis. Immunohistochemistry revealed lower expression levels of ferroptosis-related factors (FDX1, DLAT and ATP7B) and drug resistance-related factors (ATP7B and P–gp) in the bCCM and bCCM + DDP groups than in the control group (Fig. 7 I). Overall, bCCM demonstrated excellent resistance due to its ability to effectively potentiate necroptosis. 4. Conclusion In conclusion, we have developed a novel mitochondrion-targeted and GSH-responsive copper carrier, bCCM , that effectively targets cisplatin-resistant hepatocellular carcinoma. This supramolecular complex, formed by chelating bm-Cur with Cu(II) at three coordination sites and encapsulating it with RBC membranes, addresses the key limitations of cuproptosis therapy by enhancing copper delivery through the tridentate chelation architecture, which enables high-capacity copper loading and targeted delivery, comprehensive GSH depletion achieved by dual inhibition of GSH synthesis and consumption via Cu(II)-mediated Fenton reactions and Michael addition, and resistance modulation through downregulation of ATP7B to prevent copper/cisplatin efflux. In summary, this work establishes bCCM as a first-in-class therapeutic paradigm for the effective treatment of platinum-resistant malignancies, which also serves as an important example for new applications of traditional Chinese medicine. Declarations Ethics approval All the animal studies were carried out in compliance with the institutional animal care guidelines and approved protocols of the experimental animal center at Hunan University of Chinese Medicine, which is located in Hunan Province, China (Ethics Approval Number: SLBH-202311090018). Consent for publication All authors have read and approved the final manuscript, and warrant that the article is an original work and has not been published elsewhere. Data availability No datasets were generated or analysed during the current study. Competing interests Hunan University of Chinese Medicine has applied for one patent on the basis of this work. Funding This work is partially supported by the National Natural Science Foundation of China (82574571 to Y.Q.), Hunan Youth Science and Technology Innovation Talents Project (No. 2021RC3100 to Y.Q.), Open Competition Mechanism Project of Hunan University of Chinese Medicine (22JBZ023 to Y.Q.), Natural Science Foundation of Hunan Province (2024JJ5296 to Y.Q., 2025JJ50708 to X.X., 2023JJ30799 to Liqin Yuan), and Changsha Municipal Natural Science Foundation (kq2502052 to X.X.). Authors' contributions QY conceived and designed the study. YHY performed all the experiments. CX, HSQ and DHF collected the data. XX and FJL interpreted and analyzed the data. YHY, YLQ and QY wrote the manuscript. WW and QY revised the manuscript critically. Acknowledgements The authors gratefully acknowledge the assistance of the International Joint Laboratory of Traditional Chinese Medicine and Ethnic Medicine at Hunan University of Chinese Medicine, Changsha, China. References VASAN N, BASELGA J, HYMAN D. M. 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Curcumin-glutathione interactions and the role of human glutathione S-transferase P1-1 [J]. Chem Biol Interact. 2000;128(1):19–38. PIPER J T, SINGHAL S S, SALAMEH M S, et al. Mechanisms of anticarcinogenic properties of curcumin: the effect of curcumin on glutathione linked detoxification enzymes in rat liver [J]. Int J Biochem Cell Biol. 1998;30(4):445–56. CHEN W H, CHEN Q W, CHEN Q, et al. Biomedical polymers: synthesis, properties, and applications [J]. Sci China Chem. 2022;65(6):1010–75. XU T, MA Q, ZHANG C, et al. A novel nanomedicine for osteosarcoma treatment: triggering ferroptosis through GSH depletion and inhibition for enhanced synergistic PDT/PTT therapy [J]. J Nanobiotechnol. 2025;23(1):323. SATHYABHAMA M, PRIYA DHARSHINI L C, KARTHIKEYAN A et al. The Credible Role of Curcumin in Oxidative Stress-Mediated Mitochondrial Dysfunction in Mammals [J]. Biomolecules, 2022, 12(10). ZHANG M, XU H, WU X, et al. Engineering Dual-Responsive Nanoplatform Achieves Copper Metabolism Disruption and Glutathione Consumption to Provoke Cuproptosis/Ferroptosis/Apoptosis for Cancer Therapy [J]. ACS Appl Mater Interfaces. 2025;17(14):20726–40. LIU J, YUAN Y, CHENG Y, et al. Copper-Based Metal-Organic Framework Overcomes Cancer Chemoresistance through Systemically Disrupting Dynamically Balanced Cellular Redox Homeostasis [J]. J Am Chem Soc. 2022;144(11):4799–809. ZHU J, WANG X, SU Y et al. Multifunctional nanolocks with GSH as the key for synergistic ferroptosis and anti-chemotherapeutic resistance [J]. Biomaterials, 2022, 288(121704. HANS C, SAINI R, SACHDEVA M U, S et al. 2',7'-Dichlorofluorescein (DCF) or 2',7'-dichlorodihydrofluorescein diacetate (DCFH2-DA) to measure reactive oxygen species in erythrocytes [J]. Biomed Pharmacother, 2021, 138(111512. TADINI-BUONINSEGNI F SMEAZZETTOS. Mechanisms of charge transfer in human copper ATPases ATP7A and ATP7B [J]. IUBMB Life. 2017;69(4):218–25. TANG D, CHEN X. Cuproptosis: a copper-triggered modality of mitochondrial cell death [J]. Cell Res. 2022;32(5):417–8. LI Y, LIU J. Mitochondria-Targeted Multifunctional Nanoparticles Combine Cuproptosis and Programmed Cell Death-1 Downregulation for Cancer Immunotherapy [J]. Adv Sci (Weinh). 2024;11(35):e2403520. HE W, ZHANG X-Y, GONG X, et al. Drug-Free Biomimetic Oxygen Supply Nanovehicle Promotes Ischemia-Reperfusion Therapy in Stroke [J]. Adv Funct Mater. 2023;33(21):2212919. ZHANG D, MAN D, LU J, et al. Mitochondrial TSPO Promotes Hepatocellular Carcinoma Progression through Ferroptosis Inhibition and Immune Evasion [J]. Adv Sci (Weinh). 2023;10(15):e2206669. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files image1.png Scheme 1.Schematic illustration of bCCM preparation and its mechanisms for potentiating necroptosisin drug–resistant cancer therapy. (A) Preparation of bCCM. (B) Detailed mechanism by which bCCM potentiates necroptosis. (C) Cuproptosis sensitizes BEL7402/DDP cells to cisplatin to overcome drug resistance. Supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 20 Jan, 2026 Read the published version in Journal of Experimental & Clinical Cancer Research → Version 1 posted Editorial decision: Revision requested 30 Sep, 2025 Reviews received at journal 30 Sep, 2025 Reviews received at journal 24 Sep, 2025 Reviews received at journal 22 Sep, 2025 Reviewers agreed at journal 21 Sep, 2025 Reviewers agreed at journal 13 Sep, 2025 Reviewers agreed at journal 12 Sep, 2025 Reviewers invited by journal 12 Sep, 2025 Editor assigned by journal 11 Sep, 2025 Submission checks completed at journal 11 Sep, 2025 First submitted to journal 06 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7551173","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":515345529,"identity":"7933ce25-bcc7-4150-8d52-c7a59d1b180a","order_by":0,"name":"Haoyu Yang","email":"","orcid":"","institution":"Hunan University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Haoyu","middleName":"","lastName":"Yang","suffix":""},{"id":515345533,"identity":"adef243a-aeba-4ba3-bf30-851bcc1df397","order_by":1,"name":"Xiang Xiong","email":"","orcid":"","institution":"Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Xiong","suffix":""},{"id":515345534,"identity":"042a5adb-4ae6-467b-8923-914feda8db94","order_by":2,"name":"Xin Chen","email":"","orcid":"","institution":"Hunan University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Chen","suffix":""},{"id":515345538,"identity":"faa2bb86-31c6-416f-8aab-f850101b52bb","order_by":3,"name":"Siqi Huang","email":"","orcid":"","institution":"Hunan University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Siqi","middleName":"","lastName":"Huang","suffix":""},{"id":515345539,"identity":"140ea022-3221-443b-a475-1668d538962b","order_by":4,"name":"Hongfang Dai","email":"","orcid":"","institution":"Hunan University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hongfang","middleName":"","lastName":"Dai","suffix":""},{"id":515345540,"identity":"6afcfe8a-8a15-4b2d-aa24-51df3ba13ac6","order_by":5,"name":"Liqin Yuan","email":"","orcid":"","institution":"Second Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Liqin","middleName":"","lastName":"Yuan","suffix":""},{"id":515345544,"identity":"ecafabf9-43df-4123-abd5-d5a7bb22cf20","order_by":6,"name":"Jialong Fan","email":"","orcid":"","institution":"Changsha Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jialong","middleName":"","lastName":"Fan","suffix":""},{"id":515345547,"identity":"5871d145-6079-4892-87d1-541f79881f11","order_by":7,"name":"Zhenhong Xiang","email":"","orcid":"","institution":"Hunan University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhenhong","middleName":"","lastName":"Xiang","suffix":""},{"id":515345549,"identity":"c2a3cc96-e9b9-4f42-bcfe-333a58361052","order_by":8,"name":"Wei Wang","email":"","orcid":"","institution":"Hunan University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":515345551,"identity":"391e66b6-97f5-4ab9-97d1-1a49522246b8","order_by":9,"name":"Yan Qin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYDACZgbGBx+ANL8EmCshQ4wWZsMZQFpyBgNjA1ALDzH2sElzAEmDG2AtDIS18B3nPWzM2GaXZ3y7+fijGzUWPAzsh49uwKdF8jBf4uPCtuRiszvHEptzjgEdxpOWdgOfFoPDPMbGM9uYE7fdyDFszmEDapHgMSOkxUyat60+cfMMkJZ/xGs5nLhBAqglt40ILZJAhxnOOHc8ccaNtMTZuX0SPGyE/MJ3/ozhgw9l1Yn9M5IPfM75VifHz374GF4tDAeAmJENSYANh0JULQx/CCobBaNgFIyCkQwAvIhJKAO117IAAAAASUVORK5CYII=","orcid":"","institution":"Hunan University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Qin","suffix":""}],"badges":[],"createdAt":"2025-09-06 13:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7551173/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7551173/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13046-026-03642-5","type":"published","date":"2026-01-20T15:57:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91695588,"identity":"75e0ab06-b9fd-4c5f-a02f-34d1169db316","added_by":"auto","created_at":"2025-09-19 09:30:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3731642,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the Cu nanocomplex–nanocarrier\u003cstrong\u003e bCCM \u003c/strong\u003eand its response to GSH. (A) The synthetic process of bm–Cur. (B) NMR hydrogen spectra of bm–Cur. 1H NMR (400 MHz, DMSO–d6) δ 10.06 (s, 2H), 7.52 (d, J = 16 Hz, 2H), 6.81 (d, J = 8 Hz, 2H), 6.72 (dd, J = 36, 16 Hz, 2H), 6.04 (s, 1H). (C) Reactionprocess of bm–Cur + GSH. (D–E) Expression and quantitative analysis of the xCT and ATP7B proteins in BEL7402/DDP cells after different treatments for 24 h. (n ≥ 3; error bars represent SDs, *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). (F) Preparation process of \u003cstrong\u003ebCCM\u003c/strong\u003e. (G) The simulated three–dimensional structure of the \u003cstrong\u003ebCCM\u003c/strong\u003e. (H) XRD pattern of \u003cstrong\u003ebCCM\u003c/strong\u003e. (I) FTIR spectra of \u003cstrong\u003ebCCM \u003c/strong\u003eand bm–Cur. (J) XPS spectra of \u003cstrong\u003ebCCM.\u003c/strong\u003e (K) XPS spectra for Cu 2p of \u003cstrong\u003ebCCM\u003c/strong\u003e. (L) TEM images of \u003cstrong\u003ebCC \u003c/strong\u003e(only chelatedwith copper ions),\u003cstrong\u003e bCCM \u003c/strong\u003e(coated with red blood cell membrane) and\u003cstrong\u003e bCCM\u003c/strong\u003e+ GSH (\u003cstrong\u003ebCCM\u003c/strong\u003e reactedwith GSH). Scale bar, 20 nm. (M) UV–vis spectra of \u003cstrong\u003ebCCM\u003c/strong\u003e, bm–Cur, GSH, and \u003cstrong\u003ebCCM\u003c/strong\u003e + GSH. (N) UV–vis spectra of \u003cstrong\u003ebCCM\u003c/strong\u003e (30 μg/mL) with varying GSH concentrations for 10 min. (O) UV–vis spectra of \u003cstrong\u003ebCCM\u003c/strong\u003e (30 μg/mL) with 10 mM GSH over time.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7551173/v1/5ed5cd4225a2e0771b470ac4.png"},{"id":91694724,"identity":"bbb02b2b-3a24-4b88-a881-adde922829f1","added_by":"auto","created_at":"2025-09-19 09:22:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7159044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003eGSH depletion, ROS generation, and antitumor effects of \u003cstrong\u003ebCCM\u003c/strong\u003e. (A) Proposed activation mechanism of \u003cstrong\u003ebCCM \u003c/strong\u003edepleting GSH. (B) GSH level detection in BEL7402/DDP, HepG2, Hepa1–6 and HL7702 cells. (C) GSH level detection in BEL7402/DDP cells subjected to different treatments for 24 h. (D) Western blot analysis of xCT expression in BEL7402/DDP cells. (E) Changes in ROS levels in BEL7402/DDP cells subjected to different treatments for 24 h. (F) Monitoring of single BEL7402/DDP cell morphological changes after \u003cstrong\u003ebCCM \u003c/strong\u003etreatment\u003cstrong\u003e \u003c/strong\u003eat different time points via holographic microscopy. (G, H) Cytotoxicity of different tumor cells (HepG2 cells, BEL7402/DDP and A2780/DDP) treated with different concentrations of DDP (G) or \u003cstrong\u003ebCCM\u003c/strong\u003e (H) for 24 h. (I) Cytotoxicity of bCCM combined with cisplatin in HepG2, BEL7402/DDP and A2780/DDP cells for 24 h. (J) CLSM images of live/dead staining of BEL7402/DDP cells after different treatments for 24 h. Scale bar, 100 μm. (K) Flow cytometry analysis of apoptosis in BEL7402/DDP cells after different treatments for 24 h. Data represent the mean ± standard deviation (SD) (*p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7551173/v1/9946f68d46b5e526510cde07.png"},{"id":91694722,"identity":"64c7f3c2-8dde-474a-ba5b-055bf78ec37f","added_by":"auto","created_at":"2025-09-19 09:22:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1023158,"visible":true,"origin":"","legend":"\u003cp\u003eInvestigation of pyroptosis-related characteristics and drug resistance-related protein expression and transcriptomic analysis of BEL7402/DDP cells after 24 h of various treatments. (A) Proposed mechanism by which \u003cstrong\u003ebCCM \u003c/strong\u003einduces cuproptosis to reverse resistance. (B, C) Imaging and quantitative detection of intracellular copper ion levels were performed after different treatments for 24 h. (D) The mechanism for the reduction of Cu²⁺ to cuprous Cu⁺ ions. (E) Intracellular Cu\u003csup\u003e+ \u003c/sup\u003elevel detection after different treatments. (F) Viability of \u003cstrong\u003ebCCM \u003c/strong\u003ecombined with the necroptosis inhibitor UK5099 (5 μM). (G, H) Western blot analysis of the expression of pyroptosis-related proteins (FDX1, DLAT and LIAS) in BEL7402/DDP cells. (I) Mechanistic diagram of the relationship between ATP and pyroptosis. (J) Intracellular ATP level detection after different treatments. (K) Western blot analysis of cisplatin resistance-related protein (P–gp, ATP7B) expression in BEL7402/DDP cells. (L) Gene Ontology (GO) enrichment analysis. (M) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7551173/v1/ba0dfda498c5670aa101568f.jpeg"},{"id":91695589,"identity":"e1c1f1bb-0c73-4dac-bd44-947bf0871a2f","added_by":"auto","created_at":"2025-09-19 09:30:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9500404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ebCCM \u003c/strong\u003einduces mitochondrial necroptosisand mitochondrial damage. (A–B) Colocalization of \u003cstrong\u003ebCCM \u003c/strong\u003eand bm–Cur with the MitoTracker Red probe after 0.5 h of coincubation in BEL7402/DDP cells. Scale bar, 20 μm. (C) The mechanism of cuproptosisinduced by \u003cstrong\u003ebCCM\u003c/strong\u003e in mitochondria. (D) Mito–protein expression of xCT in BEL7402/DDP cells after various treatments. (E) Mitochondrial GSH (Mito–GSH) levels are depicted in BEL7402/DDP cells. (F) Copper ion content in BEL7402/DDP cell mitochondria after various treatments. (G) Mitochondrial FDX1, DLAT, and LIAS expression in BEL7402/DDP cells subjected todifferent treatments. (I) CLSM images of mitochondrial membrane damage in BEL7402/DDP cells after 24 h of treatment by MitoTracker Red CMXRos monitoring. Scale bar, 20 μm. (J) JC–1 detection in BEL7402/DDP cells after different treatments for 24 h. (K) Bio–TEM images of BEL7402/DDP cells treated with PBS and \u003cstrong\u003ebCCM\u003c/strong\u003e. [Mito–Tracker Red CMXRos] = 200 nM, [JC–1] = 2.0 μg/mL, [bm–Cur] = 20 μM, [\u003cstrong\u003ebCCM\u003c/strong\u003e] = 30 μg/mL, [DDP] = 5 μM. The data are presented as the means ± SDs. p values were calculated via one–way ANOVA in C) and D). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7551173/v1/cd16136b9160a2e0ce4dd49b.png"},{"id":91694735,"identity":"a97d82a6-a80a-428a-84e1-fe60a376f562","added_by":"auto","created_at":"2025-09-19 09:22:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":29834688,"visible":true,"origin":"","legend":"\u003cp\u003eBiosafety assay of \u003cstrong\u003ebCCM\u003c/strong\u003e. (A) Representative images of zebrafish embryos subjected to different treatments at various time points. (B) Heart rate, hatching rate, body length and survival statistics of zebrafish under different treatments. (C) Representative images of zebrafish embryos at various time points after treatment with different concentrations of \u003cstrong\u003ebCCM\u003c/strong\u003e. (D) Copper base of the zebrafish heart rate, hatching rate, body length and survival rate after treatment with different concentrations of \u003cstrong\u003ebCCM\u003c/strong\u003e. (E)\u003cstrong\u003e \u003c/strong\u003eH\u0026amp;E staining of themain organs (heart, liver, spleen, lung, and kidney) of the mice in the different treatment groups. Scale bars, 100 μm. The dataare presented as the means ± standard deviations; n=3 independent experiments. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7551173/v1/8296c778024cadb7a71fcf9f.png"},{"id":91694728,"identity":"c97b7a98-6df1-4f27-87fe-8fe75db6a05e","added_by":"auto","created_at":"2025-09-19 09:22:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11198604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiosafety and Biodistribution of bCCM. \u003c/strong\u003e(A)\u003cstrong\u003e \u003c/strong\u003eDiagram of the preparation process of \u003cstrong\u003ebCCM@IR783\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003e(B) Fluorescence images of IR783 and \u003cstrong\u003ebCCM@IR783\u003c/strong\u003e signals in tumors. (C) Fluorescence signals of IR783 and \u003cstrong\u003ebCCM@IR783\u003c/strong\u003e in major organs and tumors. (D) Determination of the copper ioncontent in major organs and tumors.[DDP] = 5 mg/kg, [\u003cstrong\u003ebCCM\u003c/strong\u003e] = 3 mg/kg. [\u003cstrong\u003ebCCM \u003c/strong\u003e+ DDP] = 3 mg/kg + 2.5 mg/kg. The dataare shown as the means ± SDs, n = 5; p values were calculated via one–way ANOVA with Tukey’s post hoctest, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7551173/v1/242eb01a6b07f1f89ba67916.png"},{"id":91694738,"identity":"3dbefeb8-f703-4885-ab29-dd46527c381d","added_by":"auto","created_at":"2025-09-19 09:22:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":28689201,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e antidrug-resistant tumor efficacy and cuproptosis-associatedmechanism investigation in BEL7402/DDP tumor-bearing mice. (A) Therapy schedule of different treatments (n = 5 per group). (B) Images of isolated tumors. Scale bar, 1 cm. (C) Body weights of the mice during the treatments. (D) Relative tumor volume changes during therapy. (E) Weights of isolated tumors. (F) Tumor inhibition rates (TIRs) of different treatments. (G, H) H\u0026amp;E and TUNEL staining of tumors from different treatment groups. Scale bars, 100 μm and 50 μm. (I) FDX1, DLAT, ATP7B and P–gp staining images of tumor tissues after different treatments for 21 days. Scale bar, 50 µm. [DDP] = 5 mg/kg, [\u003cstrong\u003ebCCM\u003c/strong\u003e] = 3 mg/kg. [\u003cstrong\u003ebCCM +\u003c/strong\u003e DDP] = 3 mg/kg + 2.5 mg/kg. The data are shown as the means ± SDs, n = 5; p values were calculated via one–way ANOVA with Tukey’s post hoctest, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7551173/v1/6ef25d5374e8ca5212b4b586.png"},{"id":101151785,"identity":"494d7d93-e4f1-4cd7-a99b-90c8fbf121e9","added_by":"auto","created_at":"2026-01-26 16:05:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":84191083,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7551173/v1/54354daf-8946-41fc-8052-2cb44147ce4e.pdf"},{"id":91694720,"identity":"d306a5bf-98e3-4b6f-8050-3af78d3c6352","added_by":"auto","created_at":"2025-09-19 09:22:14","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1508677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003eSchematic illustration of \u003cstrong\u003ebCCM \u003c/strong\u003epreparation and its mechanisms for potentiating necroptosisin drug–resistant cancer therapy. (A) Preparation of\u003cstrong\u003e bCCM\u003c/strong\u003e. (B) Detailed mechanism by which \u003cstrong\u003ebCCM \u003c/strong\u003epotentiates necroptosis. (C) Cuproptosis sensitizes BEL7402/DDP cells to cisplatin to overcome drug resistance.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7551173/v1/b1ed73538ceff172a414fbb8.png"},{"id":91694771,"identity":"a7df4740-0b5c-423d-a885-6c34e45f392c","added_by":"auto","created_at":"2025-09-19 09:22:15","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21297108,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7551173/v1/28a5a2d2ac3bf84cb257b7a3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A mitochondrion-targeted natural polyphenolic copper carrier overcomes tumor resistance to cisplatin by potentiating cuproptosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlatinum-based drugs, exemplified by cisplatin (DDP), constitute a cornerstone of cancer chemotherapy. However, their clinical utility is substantially limited by the emergence of drug resistance\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Prolonged or repeated drug administration has been shown to induce a multimechanistic synergistic resistance phenotype characterized primarily by two interrelated processes: first, membrane transporter-mediated drug efflux\u0026mdash;driven by the overexpression of P-glycoprotein (P-gp) and copper-transporting ATPase (ATP7B)\u0026mdash;which significantly diminishes intracellular drug accumulation\u003csup\u003e[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e; and second, metabolic inactivation of drugs through conjugation with endogenous molecules such as glutathione (GSH)\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. These mechanisms often coexist and reinforce one another, forming a complex resistance network. Conventional strategies such as dose escalation or the use of more cytotoxic agents not only fail to overcome this network but also may exacerbate adverse effects\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Consequently, the development of innovative therapeutic strategies that effectively reverse resistance while maintaining a favorable safety profile represents a critical challenge in contemporary oncology research.\u003c/p\u003e\u003cp\u003eCuproptosis, a novel form of regulated cell death first described by Tsvetkov et al. in 2022\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, is triggered by copper overload-induced mitochondrial dysfunction\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e and offers promising potential for overcoming tumor resistance to conventional therapies\u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Specifically, enriched intracellular copper ions within cells directly bind to lipoylated components of the mitochondrial tricarboxylic acid (TCA) cycle\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, leading to the aggregation of lipoylated proteins\u0026mdash;particularly dihydrolipoamide S-acetyltransferase (DLAT)\u0026mdash;and the loss of iron\u0026ndash;sulfur (Fe\u0026ndash;S) cluster proteins, including ferredoxin 1 (FDX1) and lipoic acid synthetase (LIAS)\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. These events induce proteotoxic stress and ultimately result in cell death. The mechanism of copper involvement involves two key aspects: first, FDX1 and LIAS mediate the lipoylation of DLAT, enabling copper to promote its oligomerization; second, copper downregulates Fe\u0026ndash;S cluster proteins. Together, these processes culminate in cuproptosis\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Notably, not all copper ions are equally effective; cuprous ions (Cu⁺) exhibit greater toxicity than cupric ions (Cu\u0026sup2;⁺) because of their superior ability to bind lipoylated TCA cycle proteins\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Therefore, to induce cuproptosis, Cu\u0026sup2;⁺ must first be reduced to Cu⁺, dissociate from ion carriers, and subsequently bind lipoic acid moieties on DLAT to trigger aggregation. Thus, enhancing both copper accumulation and its reduction to Cu⁺ represents a crucial strategy for activating cuproptosis in resistant tumors.\u003c/p\u003e\u003cp\u003eHowever, the therapeutic application of cuproptosis is limited by several obstacles. Simply elevating copper levels is often insufficient to trigger effective cuproptosis within the complex tumor microenvironment. First, elevated GSH levels\u0026mdash;common in drug-resistant cells\u0026mdash;chelate copper ions, reducing the amount of bioavailable copper and impeding cuproptosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Second, copper efflux is mediated by copper-transporting ATPases (ATP7A/B), which expel excess copper to maintain homeostasis\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. These same mechanisms also contribute to resistance against chemotherapeutic agents such as cisplatin via GSH-mediated inactivation and ATP7A/B-dependent efflux. Current strategies to increase the level of intracellular copper include enhancing copper uptake, minimizing copper loss, and inhibiting copper export. Several copper ionophores\u0026mdash;such as elesclomol (ES), dithiocarbamate, bis(thiosemicarbazone) ligands, 8-hydroxyquinolines, pyrithione, and disulfiram\u0026mdash;have been explored for their ability to promote copper accumulation and cuproptosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31 CR32\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Among these, ES uniquely delivers copper to mitochondria, facilitating sustained copper accumulation\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Nonetheless, clinical trials of ES have revealed limited efficacy due to rapid systemic clearance and metabolism\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Other ionophores face similar translational challenges, including poor biosafety, lack of targeting, or insufficient cuproptosis induction\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Thus, there is an urgent need to develop safe and efficient copper delivery systems with high loading capacity, GSH depletion ability, and ATPase inhibitory activity.\u003c/p\u003e\u003cp\u003eIn this study, we designed and synthesized a novel multifunctional copper carrier, bisdemethylcurcumin (bm\u0026ndash;Cur), which contains multiple metal-chelating groups (adjacent carbonyl and phenolic hydroxyl moieties) capable of coordinating Cu\u0026sup2;⁺ at three distinct sites. bm\u0026ndash;Cur also depletes GSH via Michael addition, which is mediated by its α,β-unsaturated ketone structures\u003csup\u003e[\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e, and inhibits the cystine/glutamate antiporter (xCT), thereby disrupting GSH synthesis\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Additionally, bm\u0026ndash;Cur facilitates mitochondrial copper delivery, induces mitochondrial damage, and demonstrates selectivity toward cancer cells over normal hepatocytes\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. It also downregulates ATP7B expression, further inhibiting copper efflux. We therefore hypothesized that bm\u0026ndash;Cur could serve as an ideal multifunctional copper ionophore, enhancing copper delivery while simultaneously depleting GSH and suppressing ATP7B-mediated export to potentiate cuproptosis.\u003c/p\u003e\u003cp\u003eLeveraging these properties, we complexed bm\u0026ndash;Cur with Cu(II) and encapsulated it within red blood cell (RBCm) membranes to form a nanocomplex designated bm\u0026ndash;Cur\u0026ndash;Cu(II)@RBCm (bCCM) (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Upon cellular internalization, bCCM releases both copper and bm\u0026ndash;Cur, initiating a cascade of therapeutic effects: Cu\u0026sup2;⁺ is reduced to Cu⁺ via a Fenton-like reaction involving GSH, and the resulting Cu⁺ binds lipoylated DLAT to trigger cuproptosis. Concurrently, bm\u0026ndash;Cur amplifies this process by depleting GSH (via Michael addition) and inhibiting xCT while also downregulating ATP7B to reduce copper export. This self-reinforcing cycle enhances copper retention and promotes cuproptosis in cisplatin-resistant hepatocellular carcinoma (HCC) cells (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Moreover, bm\u0026ndash;Cur and bCCM specifically target mitochondria, increase mitochondrial copper levels, deplete mitochondrial GSH, and induce mitochondrial dysfunction\u003csup\u003e[\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e\u0026mdash;significantly augmenting cuproptosis and reversing cisplatin resistance (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Transcriptomic analyses further supported the association between bCCM treatment and cuproptosis activation. Both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, bCCM exhibited potent anti-HCC activity without harming normal tissues, downregulating the expression of cuproptosis-related proteins (FDX1, DLAT, ATP7B, and LIAS) and resistance markers (ATP7B and P-gp). Importantly, bCCM demonstrated favorable pharmacokinetics, high tumor accumulation, minimal off-target retention, and excellent biocompatibility, underscoring its clinical translational potential.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials and reagents\u003c/h2\u003e\u003cp\u003eGSH was purchased from Shanghai Yuanye Biotechnology Co. Copper perchlorate hexahydrate was purchased from Sinopharm (Beijing, China). Dimethyl sulfoxide (DMSO) and cisplatin were purchased from Shanghai McLean Biochemical Technology Co. Phosphate buffer solution (PBS), fetal bovine serum (FBS), Duchenne-modified Eagle\u0026rsquo;s medium (DMEM), trypsin, and methylthiazolium tetrazolium (MTT) were purchased from Procell Life Science and Technology Co. Ltd. (Wuhan, China). The Annexin V\u0026ndash;FITC Apoptosis Detection Kit and BCA Protein Detection Kit were purchased from Beijing Sola Bio\u0026ndash;technology Co. Reactive oxygen species (ROS) assay kits, GSH and GSSG assay kits, ATP assay kits, cellular mitochondrial isolation kits, 4% paraformaldehyde (PFA), Hoechst 33342, 4',6\u0026ndash;diamidino\u0026ndash;2\u0026ndash;phenylindole (DAPI), and radioimmunoprecipitation (RIPA) lysates were purchased from Biyuntian (Nanjing) Bio\u0026ndash;Technology Co. β\u0026ndash;Actin (1:2000), FDX1 polyclonal antibodies, ATP7B polyclonal antibodies, DLAT polyclonal antibodies and LIAS polyclonal antibodies were purchased from Wuhan Protein Technology Co. xCT polyclonal antibodies were purchased from Abcam plc (Cambridge, UK). The Cellular Cuprous Fluorometric Assay Kit, Cell Copper (Cu\u003csup\u003e2+\u003c/sup\u003e) Colorimetric Assay Kit (Complexing Method), Copper (Cu\u003csup\u003e2+\u003c/sup\u003e) Colorimetric Assay Kit (Complexing Method), terminal deoxynucleotidyl transferase\u0026ndash;mediated nick end labeling (Complexing Method), terminal deoxynucleotidyl transferase\u0026ndash;mediated nick end labeling (TUNEL) and 3,3'\u0026ndash;diaminobenzidine (DAB) staining were purchased from Elabscience Biotechnology Co. (1E, 6E)\u0026ndash;1,7\u0026ndash;Bis (3,4\u0026ndash;dihydroxyphenyl)\u0026ndash;1,6\u0026ndash;heptadiene\u0026ndash;3,5\u0026ndash;dione) synthesized from curcumin was named bm\u0026ndash;Cur. bm\u0026ndash;Cur was obtained from the Laboratory of Chinese Medicine Activity Screening and Eugenics, Hunan University of Traditional Chinese Medicine. HepG2 cells were purchased from Fuheng Biological Company, Shanghai, China (RRID: CVCL_0027). BEL7402/DDP and A2780/DDP cells were purchased from Meixuan Biological Company, Shanghai, China. Hepa1\u0026ndash;6 (RRID: CVCL_0327) and HepG2/DDP cells were purchased from Procell Life Science and Technology Co., Ltd. The BEL7402/DDP, A2780/DDP, and HepG2/DDP cell lines are all cisplatin-resistant. Cisplatin-induced cisplatin-resistant cell lines were established \u003cem\u003ein vitro\u003c/em\u003e and verified via the CCK-8 method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Apparatus\u003c/h2\u003e\u003cp\u003eUV spectra were measured on a UV\u0026ndash;1800 UV spectrophotometer (Shimadzu Corporation, Tokyo, Japan). Fluorescence imaging was performed on a confocal microscope (FV 1200, objective lenses: 60\u0026times; and 20\u0026times;) (Olympus Corporation, Tokyo, Japan). Apoptosis analysis was performed on a flow cytometer (Shenzhen Dakovi Biotechnology Co., Ltd.). Mass spectrometry (MS) was performed with a liquid mass spectrometer (Thermo Fisher Scientific, Rockford, IL, USA). Cellular copper ion assays, tissue copper ion assays, cytosolic cuprous ion assays, MTT assays, intracellular ATP content assays, and intracellular GSH content assays were performed with a microplate detector (PerkinElmer Inc., Waltham, Massachusetts, USA). The samples for fluorescence measurements were reacted in a 37\u0026deg;C water bath. All plastic products used in the cell chambers were autoclaved. All the cells were cultured in a carbon dioxide incubator (HERA CELL 150i) (Thermo Fisher Scientific, Rockford, IL, USA). All the cell experiments were performed in a biosafety cabinet (Thermo Fisher Scientific, Rockford, IL, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Synthesis of bCCM\u003c/h2\u003e\u003cp\u003eFive milliliters of 15 mM copper perchlorate solution was added dropwise to 5 mL of 5 mM bm\u0026ndash;Cur solution, Tris\u0026ndash;HCl 8.8 was added to adjust the pH to 7.4, the mixture was stirred for 2 hours, the mixture was observed to turn black, 15 mL of erythrocyte membranes were added, and the mixture was stirred for 1 hour. bCCM was obtained.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Spectroscopic Measurements\u003c/h2\u003e\u003cp\u003eThe bCCM stock solution was diluted with distilled water, and GSH was prepared with distilled water. The bm\u0026ndash;Cur solutions used for spectroscopic measurements were diluted with distilled water to the final concentration, and GSH was diluted to various concentrations. The test solutions were reacted for 120 min at room temperature, and all the spectroscopic experiments were carried out at room temperature. UV spectra were recorded after the addition of various analytes. The emission wavelengths ranged from 350 nm to 450 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cell Culture\u003c/h2\u003e\u003cp\u003eBEL7402/DDP, HepG2/DDP, HepG2 and Hepa1\u0026ndash;6 cells were cultured in Dulbecco's modified Eagle\u0026rsquo;s medium (DMEM) (Gibco; Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (Procell Life Science and Technology Co., Ltd., China) and 100 U/mL/1% streptomycin in an incubator at 37\u0026deg;C with 5% humidity. A2780/DDP cells were cultured in Roswell Park Memorial Institute medium 1640 (RPMI\u0026ndash;1640) (Gibco; Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (Procell Life Science and Technology Co., Ltd., China) and 100 U/mL/1% streptomycin in an incubator at 37\u0026deg;C with 5% humidity, where BEL7402/DDP was incubated with a cisplatin concentration of 200 nM, HepG2/DDP with 1 \u0026micro;M and A2780/DDP with 500 nM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Cell treatment\u003c/h2\u003e\u003cp\u003eCisplatin was dissolved in dimethyl sulfoxide at a concentration of 100 mM, copper perchlorate was dissolved in distilled water at a concentration of 100 mM, and bCCM was stored in cell culture medium and diluted to a specific concentration for cell processing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 CCK\u0026ndash;8 assay\u003c/h2\u003e\u003cp\u003eBEL7402/DDP cells were inoculated into 96\u0026ndash;well plates (8 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well) and incubated in cell culture medium (10% FBS) with different concentrations of bCCM (0\u0026ndash;60 \u0026micro;g/mL) and cisplatin (0\u0026ndash;20 \u0026micro;M) for 24 h. Then, CCK\u0026ndash;8 solution was added to each well, and the mixture was incubated for 1 h. The cells were then subjected to a CCK\u0026ndash;8 assay at 450 nm in a microtiter plate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 YO\u0026ndash;PRO\u0026ndash;1/PI apoptosis and necrosis assay\u003c/h2\u003e\u003cp\u003eBEL7402/DDP cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were inoculated in six\u0026ndash;well plates and incubated with 1 mL of YO\u0026ndash;PRO\u0026ndash;1/PI test working solution for 20 min after 12 h of treatment with DDP (5 \u0026micro;M), bCCM (60 \u0026micro;g/mL), or bCCM (60 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;DDP (5 \u0026micro;M), and red and green fluorescence was observed at the end of the incubation period via confocal laser scanning microscopy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Cellular copper ion imaging and detection\u003c/h2\u003e\u003cp\u003eBEL7402/DDP cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were inoculated in six\u0026ndash;well plates and incubated with rhodamine B hydrazide (20 \u0026micro;M) for 30 min after 6 h of treatment with DDP (5 \u0026micro;M), bCCM (60 \u0026micro;g/mL), or bCCM (60 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;DDP (5 \u0026micro;M), and red fluorescence was observed at the end of the incubation via confocal laser scanning microscopy. For the copper ion assay, BEL7402/DDP cells were inoculated in six\u0026ndash;well plates, and after 24 h of different treatments, the cells were collected, supplemented with 0.2 mL of lysate, lysed on ice for 10 min, and then centrifuged for 10 min at 4\u0026deg;C and 12,000 \u0026times; g. The supernatant was collected for the assay, and some of the supernatant was retained for use in the protein concentration assay. A total of 100 \u0026micro;L of standard or sample to be tested was added to the bottom of each well, and 50 \u0026micro;L of color working solution was added. The membrane was covered, incubated at 37\u0026deg;C for 5 min, and the OD value of each well was detected at 580 nm. Copper ion content (\u0026micro;mol/gprot) = (ΔA580 \u0026ndash; standard curve intercept)\u0026thinsp;\u0026divide;\u0026thinsp;standard curve slope \u0026times; sample dilution\u0026thinsp;\u0026divide;\u0026thinsp;sample protein concentration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 ROS assay and live/dead staining\u003c/h2\u003e\u003cp\u003eThe cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) were cultured in 6\u0026ndash;well plates, treated with DDP (5 \u0026micro;M), bCCM (60 \u0026micro;g/mL), or bCCM (60 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;DDP (5 \u0026micro;M) for 6 h, incubated with DCFH\u0026ndash;DA reagent for 30 min, and then immediately visualized under a confocal laser scanning microscope. Live/dead cell staining: Cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) were cultured in 6\u0026ndash;well plates, treated with DDP (5 \u0026micro;M), bCCM (60 \u0026micro;g/mL), or bCCM (60 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;DDP (5 \u0026micro;M) for 6 h, incubated with an appropriate volume of calcein AM/PI assay working solution for 30 min, and then observed under a confocal laser scanning microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Intracellular GSH assay\u003c/h2\u003e\u003cp\u003eThe cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) were cultured in 6\u0026ndash;well plates with DDP (5 \u0026micro;M), bCCM (60 \u0026micro;g/mL), or bCCM (60 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;DDP (5 \u0026micro;M) for 24 h, after which the cells were harvested. Three times the volume of the cellular precipitate was added to Protein Removal Reagent M solution (GSH and GSSG Assay Kit purchased from Beyotime Institute of Biotechnology, China). If the volume of the cellular precipitate was 10 microlitres, 30 microlitres of protein removal reagent M solution was added, and the mixture was vortexed thoroughly. The volume of the cellular precipitate can be estimated from the weight of the precipitate. The centrifuge tubes were weighed before and after collection of the cells so that the weight of the cellular precipitate could be calculated. A total of 10 mg of cellular precipitate can be roughly viewed as 10 \u0026micro;L. The samples were then subjected to two rapid freeze\u0026ndash;thaw cycles via liquid nitrogen and a water bath at 37\u0026deg;C. The samples were left at 4\u0026deg;C or in an ice bath for 5 min and centrifuged at 4\u0026deg;C for 10 min at 10,000 \u0026times; g. The supernatant was used for GSH testing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Intracellular ATP assay\u003c/h2\u003e\u003cp\u003eThe cells (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/well) were cultured in 6\u0026ndash;well plates containing Cisplatin (5 \u0026micro;M), bCCM (60 \u0026micro;g/mL), or bCCM (60 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;Cisplatin (5 \u0026micro;M) for 24 h, and the cells were lysed by adding lysis buffer at a ratio of 200 \u0026micro;L of lysate per well to a 6\u0026ndash;well plate. To sufficiently lyse the cells, a pipette can be used to repeatedly blow or shake the plate so that the lysate can fully contact and lyse the cells. The cells will be lysed immediately after contact with the lysate. After lysis, the cells were centrifuged at 12,000 \u0026times; g for 5 min at 4\u0026deg;C, and the supernatant was removed for subsequent assays. One hundred microliters of ATP assay working solution was added to the assay wells. After leaving at room temperature for 3\u0026ndash;5 min, 20 \u0026micro;L of sample or standard was added to the assay wells or tubes, which were mixed quickly with a gun (micropipette), and after an interval of at least 2 s, the RLU value or CPM was determined with a chemiluminescence meter (luminometer) or liquid flash meter.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Western blot\u003c/h2\u003e\u003cp\u003eThe cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) were cultured in 6\u0026ndash;well plates with DDP (5 \u0026micro;M), bCCM (60 \u0026micro;g/mL), or bCCM (60 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;DDP (5 \u0026micro;M) for 24 h and harvested. The protein concentration in the total extract was quantified via a BCA protein assay kit (562 nm). The expression of GST was detected strictly by western blot (WB) with different antibodies: β\u0026ndash;actin (1:2000), xCT polyclonal (1:1000), FDX1 polyclonal (1:1000), DLAT polyclonal (1:2000), ATP7B polyclonal (1:1000), LIAS polyclonal (1:1000), and P\u0026ndash;gp polyclonal (1:500) antibodies. Detection was carried out on a BIO\u0026ndash;RAD ChemiDoc XRS chemiluminescence system.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Mitochondrial damage tracking\u003c/h2\u003e\u003cp\u003eCells were seeded into 12\u0026ndash;well plates (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) with DDP (5 \u0026micro;M), bCCM (60 \u0026micro;g/mL), or bCCM (60 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;DDP (5 \u0026micro;M) for 24 h. Subsequently, the cells were stained with MitoTracker Red CMXRos for 30 min, fixed with 4% PFA for 15 min and stained with DAPI (200 \u0026micro;L) for 15 min, followed by fluorescence imaging.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15 JC\u0026ndash;1 testing\u003c/h2\u003e\u003cp\u003eThe cells were seeded into 12\u0026ndash;well plates (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) with DDP (5 \u0026micro;M), bCCM (60 \u0026micro;g/mL), or bCCM (60 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;DDP (5 \u0026micro;M) for 24 h. Subsequently, the cells were stained with JC\u0026ndash;1 (2.0 \u0026micro;g/mL) staining solution at 37\u0026deg;C for 20 min, fixed with 4% PFA for 15 min and stained with DAPI (200 \u0026micro;L) for 15 min, followed by fluorescence imaging.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.16 In vivo imaging\u003c/h2\u003e\u003cp\u003eBALB/C mice (5\u0026ndash;7 weeks, 18\u0026thinsp;\u0026minus;\u0026thinsp;20 g) were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;3). BEL7402/DDP cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells in PBS) were injected subcutaneously into the nude mice. When the tumor volume reached approximately 80 mm\u003csup\u003e3\u003c/sup\u003e, the mice were injected \u003cem\u003ein situ\u003c/em\u003e with bCCM@IR783, and small animal imaging was performed. The animal study protocol was approved by the Animal Research Ethics Committee, Hunan University of Chinese Medicine.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.17 Nude mouse model\u003c/h2\u003e\u003cp\u003eFemale BALB/c nude rats (5\u0026ndash;7 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). The animal experimental protocol was approved by the Animal Research Ethics Committee of Hunan University of Traditional Chinese Medicine. BEL7402/DDP cells (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells) were suspended in PBS and then injected into the right hind abdomens of the mice. After two weeks of tumor growth, 10 mice were randomly divided into the following four groups (n\u0026thinsp;=\u0026thinsp;5 in each group): the PBS group, DDP group (5 mg/kg), bCCM group (3 mg/kg) and bCCM/DDP group. The mice in the DDP group received 5 mg/kg/day Curcumin treatment (tail vein administration) once every two days. The control mice were injected with 0.9% NaCl. The mice were examined for tumor growth every 2 days. The mice died after intrahepatic injection of sodium pentobarbital. Two weeks after the administration of Curcumin, the mice were injected intraperitoneally with sodium pentobarbital (200 mg/kg). The size of each tumor was measured. Tumor tissue was collected for subsequent experiments. All the mice were euthanized at the end of the treatment. Tumors and major organs were harvested and weighed for H\u0026amp;E\u0026ndash;staining (Ethics Approval Number: SLBH-202311090018).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.18 In vivo antitumor activity assay\u003c/h2\u003e\u003cp\u003eThe tumors were fixed with 4% PFA (24 h) and then sliced into paraffin sections (5 \u0026micro;m). H\u0026amp;E staining and TUNEL experiments were performed according to the manufacturer\u0026rsquo;s instructions. The expression of DLAT, FDX1, XCT and GPx4 in tumor tissues was detected by immunohistochemistry. The paraffin sections of tumor tissues subjected to different treatments were dewaxed. After acid\u0026ndash;base repair, ovulin and d\u0026ndash;biotin were added for blocking, and endogenous peroxidase was blocked with a 3% hydrogen peroxide solution. Then, the prepared primary antibody solutions were added to the slides and incubated for 2 h in a wet box away from light. The reaction enhancement solution was incubated for 20 min in a light\u0026ndash;resistant environment and then incubated for 20 min in a dark environment with the secondary antibodies labeled with HRP corresponding to the primary antibodies. After DAB staining, the nucleus was restained with hematoxylin for 5 min. The sections were scanned and sealed under an inverted microscope. WB analysis was performed to detect protein expression levels in tumor tissues. The tumor tissues were disrupted with a homogenizer, and RIPA lysis buffer was added to extract total protein. Blood samples were collected for biochemical and hematological assays. Moreover, the heart, liver, spleen, lung and kidney were collected for sectioning and H\u0026amp;E staining.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e2.19 Toxicity test of zebrafish embryos\u003c/h2\u003e\u003cp\u003e The zebrafish experiments were conducted in accordance with the Animal Ethics Committee of Hunan University of Traditional Chinese Medicine. All experiments used wild\u0026ndash;type AB strain zebrafish. Zebrafish embryos were treated with bm\u0026ndash;Cur or cisplatin at specified concentrations postfertilization (hpf) and analyzed at 24, 48, 72, or 96 hpf.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e2.20 Analysis of Pearson's coefficient calculations\u003c/h2\u003e\u003cp\u003ePearson's coefficient is used to measure the degree of linear correlation between two variables and has a value between \u0026minus;\u0026thinsp;1 and 1. A value of 1 represents a positive correlation, \u0026minus;\u0026thinsp;1 represents a negative correlation, and 0 represents no correlation. ImageJ software was used for Pearson coefficient analysis, ImageJ software was used to separate the color channels, pseudo\u0026ndash;colors were added to the color channels, the Coloc2 plug\u0026ndash;in was opened, and the algorithm was tickged for calculation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e2.21 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe data were analyzed with GraphPad Prism 8.4.0 software and are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. values; n\u0026thinsp;=\u0026thinsp;3 independent experiments. *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05; **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001; ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001, whereas ns represents no statistically significant difference compared with the vehicle group according to one\u0026ndash;way ANOVA.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Design, preparation, and characterization of the Cu\u0026ndash;nanocarrier bCCM\u003c/h2\u003e\u003cp\u003eAs a constituent unit of the Cu\u0026ndash;nanocarrier bCCM, the bm\u0026ndash;Cur compound was synthesized via efficient demethylation of curcumin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The structural characterization was confirmed by 1H NMR spectrum analysis and high\u0026ndash;resolution mass spectrometry (HR\u0026ndash;MS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which revealed a curcumin parent nucleus rich in four phenolic hydroxyl groups and two α,β-unsaturated ketones. Considering that bm\u0026ndash;Cur with an α,β-unsaturated ketone structure can react with the sulfhydryl group of GSH to form a bm\u0026ndash;Cur\u0026ndash;SG complex through Michael addition, HR\u0026ndash;MS and electrospray ionization (ESI) were performed to confirm the successful reaction between bm\u0026ndash;Cur (C₁₉H₁₆O₆) and GSH. As expected, ESI analysis of the reaction between C₁₉H₁₆O₆ and GSH (Mw\u0026thinsp;=\u0026thinsp;645.16) revealed a molecular ion peak at m/z\u0026thinsp;=\u0026thinsp;646.1423 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Fig. S2). Furthermore, the UV spectra revealed an absorbance peak at 430 nm in the sample containing bm\u0026ndash;Cur alone. However, the absorbance intensity of bm\u0026ndash;Cur at 430 nm decreased in a concentration-dependent manner with increasing GSH concentration (0\u0026ndash;10 mM). Notably, the absorbance of bm\u0026ndash;Cur progressively decreased over time at 10 mM GSH (Fig. S3), further confirming its responsiveness to GSH. Moreover, bm\u0026ndash;Cur inhibited GSH synthesis by downregulating xCT expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and reducing the intracellular GSH level in a concentration-dependent manner (Fig. S4). Further tests on the cytotoxicity of bm\u0026ndash;Cur toward tumor cells revealed a certain ability to kill both cisplatin\u0026ndash;sensitive (HepG2 and Hepa1-6) and resistant tumor cells (BEL7402/DDP and HepG2/DDP) (Figs. S5 and S6). Moreover, bm\u0026ndash;Cur significantly and concentration\u0026ndash;dependently downregulated the expression of ATP7B, a copper ion and a cisplatin efflux protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eInspired by the remarkable properties of bm\u0026ndash;Cur, which leverages the strong metal\u0026ndash;chelating ability of its adjacent phenolic hydroxyl groups and ortho\u0026ndash;diketone moieties, the supramolecular nanocomplex \u003cb\u003ebCCM\u003c/b\u003e was prepared by directly chelating bm\u0026ndash;Cur with Cu(II) in Tris HCl buffer supplemented with encapsulation in red blood cell membranes (RBCm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). The simulated three\u0026ndash;dimensional structure of \u003cb\u003ebCCM\u003c/b\u003e clearly shows the three coordination sites of Cu(II) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Furthermore, X\u0026ndash;ray diffraction (XRD) analysis revealed that \u003cb\u003ebCCM\u003c/b\u003e has almost no diffraction peaks, indicating that the bCCM has an amorphous structure with low crystallinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Infrared spectroscopy (IR) analysis revealed characteristic absorption peaks corresponding to hydroxyl groups at 3300\u0026ndash;3600 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1; and carbonyl and C\u0026ndash;O stretching vibrations at 1750\u0026ndash;1700 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1; and 1300\u0026ndash;1000 cm\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). X\u0026ndash;ray photoelectron spectroscopy (XPS) analysis revealed the presence of C, O, and Cu, with Cu(II) coordination peaks at 934 eV (Cu 2p3/2) and 954 eV (Cu 2p1/2) observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). These results confirmed the formation of \u003cb\u003ebCCM\u003c/b\u003e containing Cu(II). TEM images revealed that the uniform sheet structure of nanocomplex \u003cb\u003ebCC\u003c/b\u003e formed via the chelation of bm\u0026ndash;Cur with copper ions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). After coating with RBCm, a sphere\u0026ndash;like structure of \u003cb\u003ebCCM\u003c/b\u003e formed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). The rapid disassembly of \u003cb\u003ebCCM\u003c/b\u003e through a Fenton reaction between Cu(II) and GSH could deplete GSH into GSSG\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e and release single bm\u0026ndash;Cur. The morphology of \u003cb\u003ebCCM subsequently\u003c/b\u003e changed from flake to dispersed fragments after reacting with GSH, indicating that \u003cb\u003ebCCM\u003c/b\u003e had been cracked by GSH, which further confirmed the response of \u003cb\u003ebCCM\u003c/b\u003e to GSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). We then investigated the GSH consumption capability of \u003cb\u003ebCCM\u003c/b\u003e via UV\u0026ndash;vis spectral analysis. UV\u0026ndash;vis spectra revealed an absorbance peak at 430 nm in the \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;GSH group but not in the \u003cb\u003ebCCM\u003c/b\u003e or GSH groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM). Moreover, the absorbance intensity increased with increasing GSH concentration (0\u0026ndash;10 mM) in the \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;GSH group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN) and decreased gradually over time at 10 mM GSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO). These results confirmed the response capability of \u003cb\u003ebCCM\u003c/b\u003e to GSH and the release of bm\u0026ndash;Cur from \u003cb\u003ebCCM\u003c/b\u003e. We next evaluated the GSH consumption capacity of bCCM via UV\u0026ndash;vis spectroscopy. A distinct absorbance peak at 430 nm was observed for the \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;GSH group, whereas neither \u003cb\u003ebCCM\u003c/b\u003e nor GSH alone exhibited this peak (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM). Furthermore, the absorbance intensity at 430 nm increased in a GSH concentration-dependent manner (0\u0026ndash;10 mM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN) and gradually decreased over time at 10 mM GSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO) in the \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;GSH group. These findings demonstrate that \u003cb\u003ebCCM\u003c/b\u003e is responsive to GSH and subsequently releases bm\u0026ndash;Cur, confirming its GSH-triggered degradation behavior. Collectively, these results demonstrate the efficient GSH consumption capability of bCCM. Moreover, the concentration of bm\u0026ndash;Cur released from bCCM (30 \u0026micro;g/mL) upon GSH treatment was calculated to be 39.22 \u0026micro;M on the basis of changes in the absorbance at 430 nm (Fig. S7). Furthermore, the nanocomplexes exhibited excellent solubility and stability in various solutions, including normal saline (0.9% NaCl), PBS, and DMEM (Fig. S8), highlighting their suitability for \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e applications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.2 In vitro anti-resistant tumor efficacy of bCCM\u003c/h2\u003e\u003cp\u003eThe successful determination of the extracellular GSH reaction activity of the \u003cb\u003ebCCM\u003c/b\u003e nanocomplex led us to further explore its ability to consume intracellular GSH and generate ROS. First, the GSH content was measured in four liver\u0026ndash;related cell lines: cisplatin\u0026ndash;resistant human HCC cells (BEL7402/DDP), drug\u0026ndash;sensitive human and mouse HCC cells (HepG2 and Hepa 1\u0026ndash;6), and normal liver cells (HL\u0026ndash;7702). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the GSH concentration in drug\u0026ndash;resistant BEL7402/DDP cells was 21.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u0026micro;M, which was much greater than that in normal liver cells (11.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10) and moderately greater than that in drug\u0026ndash;sensitive HepG2 cells (16.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06) \u0026micro;M and Hepa 1\u0026ndash;6 cells (15.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09) \u0026micro;M. Next, the test of the GSH consumption capacity at different concentrations of \u003cb\u003ebCCM\u003c/b\u003e revealed that \u003cb\u003ebCCM\u003c/b\u003e consumed GSH in a concentration\u0026ndash;dependent manner in both BEL7402/DDP and HepG2 cells (Fig. S9). Additionally, GSH levels in BEL7402/DDP and HepG2 cells subjected to different treatments were detected, and neither \u003cb\u003ebCCM\u003c/b\u003e nor \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;DDP depleted GSH significantly differently. In contrast, DDP had almost no ability to consume GSH in either BEL7402/DDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) or HepG2 cells (Fig. S10). Moreover, the effects of various treatments on the cytoplasmic expression of xCT were examined. The results revealed significant downregulation of xCT in bCCM- and bCCM\u0026thinsp;+\u0026thinsp;DDP\u0026ndash;treated BEL7402/DDP cells, which effectively inhibited GSH synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and Fig. S11). Next, the overall level of intracellular ROS was evaluated via confocal laser scanning microscopy (CLSM) imaging with 2\u0026prime;,7\u0026prime;\u0026ndash;dichlorofluorescein diacetate (DCFHDA) as an indicator, which is rapidly oxidized by ROS to generate the green fluorescence dichlorofluorescein (DCF) \u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. Notably, a sharp increase in DCF fluorescence was observed in the \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;DDP groups after 6 h of incubation compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In contrast, there was only a small increase in the fluorescence intensity of the DDP group, indicating that \u003cb\u003ebCCM\u003c/b\u003e is a strong ROS inducer. These results demonstrated that \u003cb\u003ebCCM\u003c/b\u003e could directly deplete intracellular GSH, increasing ROS levels.\u003c/p\u003e\u003cp\u003eEncouraged by the superior GSH depletion effect, we further evaluated the antitumor efficacy of \u003cb\u003ebCCM\u003c/b\u003e \u003cem\u003ein vitro.\u003c/em\u003e First, single cells with morphological changes were observed in cisplatin\u0026ndash;resistant tumor cells (BEL7402/DDP). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, the cell underwent persistent swelling and membrane rupture until critical lysis was reached, resulting in cytoplasmic dispersion within 0\u0026ndash;24 h after 30 \u0026micro;g/mL bCCM treatment. The cytotoxicity of \u003cb\u003ebCCM\u003c/b\u003e was subsequently analyzed via an MTT assay in which DDP\u0026ndash;resistant (BEL7402/DDP, A2780/DDP) and DDP\u0026ndash;sensitive (HepG2) tumor cells were used as controls. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, compared with the very weak cytotoxicity of DDP (IC\u003csub\u003e50\u003c/sub\u003e (47.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9) \u0026micro;M in BEL7402/DDP cells and (66.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7) \u0026micro;M in A2780/DDP cells), \u003cb\u003ebCCM\u003c/b\u003e exhibited very strong cytotoxicity to DDP\u0026ndash;resistant cells, with the half\u0026ndash;maximal inhibitory concentration (\u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e) of \u003cb\u003ebCCM\u003c/b\u003e being calculated to be (65.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2) \u0026micro;g/mL in BEL7402/DDP cells and (40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6) \u0026micro;g/mL in A2780/DDP cells after 24 h of treatment (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Furthermore, \u003cb\u003ebCCM\u003c/b\u003e demonstrated excellent biological safety in normal hepatocytes (AML\u0026ndash;12), with a cell survival rate above 95%, even at concentrations (90 \u0026micro;g/mL) much higher than the therapeutic level for DDP-resistant cells (7402/DDP and A2780/DDP) (Fig. S12), suggesting its selective cytotoxicity toward tumor cells. Considering the strong cytotoxicity of \u003cb\u003ebCCM\u003c/b\u003e in DDP-resistant tumor cells, the sensitizing effect of \u003cb\u003ebCCM\u003c/b\u003e to DDP was examined by monitoring the viability of DDP-resistant cells (BEL7402/DDP and A2780/DDP) under different conditions. Notably, the combination of \u003cb\u003ebCCM\u003c/b\u003e with cisplatin had a significant additive antitumor effect on both DDP-resistant cell lines. In the BEL7402/DDP cells, the combined treatment (45 \u0026micro;g/mL \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;15 \u0026micro;M DDP) reduced the cell viability to 58.14%, whereas the viability was 67.80% with \u003cb\u003ebCCM\u003c/b\u003e alone and 89.52% with DDP alone. A more pronounced synergistic effect was observed in A2780/DDP cells, where the combination achieved 50.28% cell viability, versus 94.01% with \u003cb\u003ebCCM\u003c/b\u003e alone (15 \u0026micro;g/mL) and 85.26% with DDP alone (15 \u0026micro;M). These results clearly indicate that \u003cb\u003ebCCM\u003c/b\u003e can effectively sensitize DDP-resistant tumor cells to cisplatin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003eA subsequent live/dead cell costaining assay further confirmed the high cytotoxicity of \u003cb\u003ebCCM\u003c/b\u003e in BEL7402/DDP cells. Confocal laser scanning microscopy (CLSM) images revealed abundant red fluorescence of dead cells in the \u003cb\u003ebCCM-\u003c/b\u003e and \u003cb\u003ebCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP\u0026ndash;treated groups but abundant green fluorescence of living cells in the control and DDP groups, indicating strong cytotoxicity of \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP in BEL7402/DDP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ and Fig. S13). Moreover, flow cytometry analysis revealed a significant difference in apoptosis after different treatments for 24 h. Compared with the control cells, living cells presented significant red fluorescence after \u003cb\u003ebCCM\u003c/b\u003e treatment, indicating the strong ability of bCCM to induce apoptosis in BEL7402/DDP cells. Additionally, the \u003cb\u003ebCCM\u003c/b\u003e combined with DDP group presented stronger red fluorescence to induce apoptosis in BEL7402/DDP cells, whereas negligible red fluorescence was observed in the single DDP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK and Fig. S14). These results clearly indicated the significant DDP sensitization effect of \u003cb\u003ebCCM\u003c/b\u003e on the cisplatin\u0026ndash;resistant BEL7402/DDP cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.3 bCCM-induced Cuproptosis and RNA sequencing analysis\u003c/h2\u003e\u003cp\u003eAs mentioned above, GSH consumption can induce cuproptosis by decreasing chelation with copper and increasing intracellular copper ion levels, inhibiting the activity of FDX1 and DLAT and leading to enhanced therapeutic efficacy in drug\u0026ndash;resistant tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Because of the superior GSH consumption ability and copper ion-carrying ability of \u003cb\u003ebCCM\u003c/b\u003e, we hypothesized that \u003cb\u003ebCCM\u003c/b\u003e could significantly induce cuproptosis. The intracellular copper ion levels were subsequently monitored by a copper detection probe after different treatments. As we expected, compared with those in the control or DDP\u0026ndash;treated groups, copper ion levels dramatically increased in \u003cb\u003ebCCM\u003c/b\u003e\u0026ndash; \u003cb\u003eand bCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP\u0026ndash;treated BEL7402/DDP cells, as indicated by the strong red fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Fig. S15). Moreover, the results of the quantitative analysis of intracellular copper ions confirmed the above findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results indicated that copper ions were loaded into the cells via \u003cb\u003ebCCM\u003c/b\u003e. Moreover, Cu(II) showed no significant cytotoxicity to BEL7402/DDP cells within a certain concentration range (Fig. S16). Furthermore, considering that copper ions are further converted into Cuprous ions in the cytoplasm through GSH or FDX1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), the intracellular Cuprous ion levels were detected. Compared with DDP alone, both \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;DDP significantly increased intracellular cuprous ion levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), which suggests that \u003cb\u003ebCCM\u003c/b\u003e can carry copper ions into the cell and convert them to cuprous ions to trigger cuproptosis.\u003c/p\u003e\u003cp\u003eEncouraged by the degree of copper ion influx and cuprous ion conversion achieved, the viability of \u003cb\u003ebCCM\u003c/b\u003e combined with the cuproptosis inhibitor UK5099 (5 \u0026micro;M) was detected to investigate potential cuproptosis induction by \u003cb\u003ebCCM\u003c/b\u003e. Compared with \u003cb\u003ebCCM\u003c/b\u003e, bCCM combined with the cuproptosis inhibitor UK5099 significantly increased cell viability (up to 15\u0026thinsp;~\u0026thinsp;17%), whereas UK5099 alone (5 \u0026micro;M) had no significant effect on BEL7402/DDP cell viability (Fig. S17), indicating that the effect of bCCM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) on Cuproptosis. Western blot (WB) analysis after bCCM treatment revealed a significant decrease in the protein expression of intracellular FDX1, DLAT and LIAS, three characteristic ferroptosis suppressors, indicating effective cuproptosis induction by bCCM in BEL7402/DDP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). As ATP7B is an important copper, cuprous ion and DDP transporter, the downregulation of ATP7B expression is favorable for inducing cuproptosis and reversing cisplatin resistance. Considering that ATP hydrolysis provides energy for ATP7B, intracellular ATP levels were detected after different treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). A dramatic decrease in ATP levels was detected in both the bCCM- and bCCM\u0026thinsp;+\u0026thinsp;DDP\u0026ndash;treated groups, indicating that ATP7B activity was inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). The expression of ATP7B was further investigated, and the results confirmed significant downregulation of ATP7B and P\u0026ndash;gp expression in the bCCM- and bCCM\u0026thinsp;+\u0026thinsp;DDP\u0026ndash;treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK and Fig. S18), suggesting that pyroptosis may weaken the resistance of BEL7402/DDP cells.\u003c/p\u003e\u003cp\u003eAs a key copper transporter responsible for Cu⁺ and DDP efflux, ATP7B plays a crucial role in regulating intracellular copper levels\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. The downregulation of ATP7B expression promotes pyroptosis and helps overcome cisplatin resistance. Since ATP hydrolysis provides energy for ATP7B-mediated transport\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e, we measured intracellular ATP levels after different treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Notably, both the \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;DDP groups presented a sharp decrease in ATP levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ), suggesting impaired ATP7B activity. Furthermore, Western blot analysis confirmed a significant reduction in ATP7B and P-gp expression in these treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK and Fig. S18). These findings support the hypothesis that the induction of pyroptosis may sensitize BEL7402/DDP cells by disrupting drug resistance mechanisms.\u003c/p\u003e\u003cp\u003eTo verify the above results, genome\u0026ndash;wide high\u0026ndash;throughput RNA sequencing (RNA\u0026ndash;Seq) was performed on BEL7402/DDP cells treated with \u003cb\u003ebCCM\u003c/b\u003e and PBS as a control. Gene Ontology (GO) term analysis provided insights into the enrichment of DEGs related to the cellular response to copper ions, oxidative stress, regulation of cell death, cell migration, and copper ion transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Notably, \u0026ldquo;copper ions\u0026rdquo; associated with Cuproptosis, which is a key inducer of Cuproptosis, were the most significantly enriched terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis further classified and annotated these DEGs, revealing that the upregulated DEGs induced by \u003cb\u003ebCCM\u003c/b\u003e are closely correlated with the cell cycle, glutathione metabolism, ABC transporters, the citrate cycle (TCA cycle), hepatocellular carcinoma, and platinum drug resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). Furthermore, gene set enrichment analysis (GSEA) revealed positively and negatively regulated pathways after \u003cb\u003ebCCM\u003c/b\u003e administration (Fig. S19A). Notably, pathways related to \u0026ldquo;glutathione metabolism\u0026rdquo;, \u0026ldquo;citrate cycle (TCA cycle)\u0026rdquo;, and \u0026ldquo;ABC transporters\u0026rdquo; were upregulated, whereas \u0026ldquo;platinum drug resistance\u0026rdquo;-associated pathways were downregulated. These findings strongly suggest that the therapeutic mechanism of bCCM against resistant HCC involves the induction of pyroptosis and the modulation of glutathione metabolic and mitochondrial pathways. Finally, the protein\u0026ndash;protein interaction (PPI) network of 15 proteins related to platinum resistance (ABCB1, GSTA1) revealed close interactions between proteins involved in these processes (Fig. S19B). These transcriptomics analysis results confirm the role of \u003cb\u003ebCCM\u003c/b\u003e in inducing necroptosis and further demonstrate the close relationships among pyroptosis, platinum drug resistance, and hepatocellular carcinoma (HCC) in cisplatin\u0026ndash;resistant HCC cells (BEL7402/DDP).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.4 bCCM induces necroptosis\u0026ndash;mediated mitochondrial dysfunction\u003c/h2\u003e\u003cp\u003eAs pivotal players in pyroptosis and targets for cancer therapy, targeting mitochondria has tremendous therapeutic potential for drug-resistant cancer\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Cuproptotic cells morphologically exhibit features of mitochondrial dysfunction, including disruption of the mitochondrial structure and a reduction in the mitochondrial membrane potential\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. Increasing damage to mitochondrial membranes and inducing dysfunction are classic characteristics of pyroptosis. To confirm that \u003cb\u003ebCCM\u003c/b\u003e induces cuproptosis in mitochondria, the mitochondrial targeting ability of \u003cb\u003ebCCM\u003c/b\u003e was first evaluated. Confocal microscopy analysis revealed clear and rapid colocalization (0.5 h) of \u003cb\u003ebCCM\u003c/b\u003e and bm\u0026ndash;Cur with mitochondria in BEL7402/DDP cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The ability of \u003cb\u003ebCCM\u003c/b\u003e to induce mitochondrial dysfunction was subsequently explored. A mitochondrial REDOX imbalance, which is induced by mitochondrial GSH consumption and copper ion imbalance in response to cuproptosis, is a significant manifestation of mitochondrial dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The mitochondrial GSH and copper ion levels in BEL7402/DDP cells were studied. First, the downregulation of xCT in mitochondria by \u003cb\u003ebCCM\u003c/b\u003e suggested that it can also deplete mitochondrial GSH by inhibiting its synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and Fig. S20, the lowest GSH levels were detected in the mitochondria of BEL7402/DDP cells treated with \u003cb\u003ebCCM\u003c/b\u003e compared with those treated with cisplatin alone, resulting in a 42.8% (\u003cb\u003ebCCM\u003c/b\u003e) to \u0026minus;\u0026thinsp;3.9% (DDP) decrease in GSH levels, indicating that the greatest increase in mitochondrial GSH consumption was caused by \u003cb\u003ebCCM\u003c/b\u003e. Notably, the decrease in GSH levels in the \u003cb\u003ebCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP group was similar to that in the \u003cb\u003ebCCM\u003c/b\u003e group and much greater than that in the DDP group (35.1% vs \u0026minus;\u0026thinsp;3.9%). Moreover, compared with the DDP group, both the \u003cb\u003ebCCM and bCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP groups accumulated more mitochondrial copper (3.2\u0026thinsp;~\u0026thinsp;3.4-fold) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Further investigation of the expression of classic mitochondrial Cuproptosis proteins (DLAT, FDX1 and LIAS) revealed that \u003cb\u003ebCCM\u003c/b\u003e\u0026ndash; and \u003cb\u003ebCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP\u0026ndash;treated BEL7402/DDP cells presented significantly lower protein levels than DDP-treated cells did (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and Fig. S21). Together, these findings confirmed that \u003cb\u003ebCCM\u003c/b\u003e could indeed trigger cuproptosis in mitochondria.\u003c/p\u003e\u003cp\u003eGiven that mitochondrial membrane potential (Δψm) integrity and variation are critical for mitochondrial function, we investigated the Δψm using Mito\u0026ndash;Tracker Red CMXRos\u003csup\u003e579nm/599nm\u003c/sup\u003e, a mitochondrial membrane potential indicator\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Significant red fluorescence was observed in the BEL7402/DDP cells in the control and DDP groups (indicating a normal Δψm) after 24 h of incubation, demonstrating that DDP alone had negligible effects on the Δψm. In contrast, red fluorescence was markedly diminished in cells treated with \u003cb\u003ebCCM\u003c/b\u003e or \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;DDP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), reflecting severe mitochondrial membrane damage induced by \u003cb\u003ebCCM\u003c/b\u003e. Additionally, JC\u0026ndash;1 was used to confirm the dissipation of the mitochondrial membrane potential, which remains as a monomer in damaged mitochondria with a dissipated Δψm and aggregates in normal mitochondria with a high Δψm \u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. For BEL7402/DDP cells, in the control and DDP groups, JC\u0026ndash;1 aggregates constituted the majority. In contrast, the number of JC\u0026ndash;1 aggregates significantly decreased after \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP treatments, implying the most severe decrease in the Δψm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI), which is consistent with the MitoTracker Red results. Next, morphological changes in mitochondria were directly observed via bio\u0026ndash;TEM to evaluate mitochondrial damage in BEL7402/DDP cells. Compared with the normal morphology of the mitochondria in the PBS\u0026ndash;treated group, the mitochondria in the \u003cb\u003ebCCM\u003c/b\u003e-treated group presented severe destruction and swelling, increased membrane density, decreased volume, and reduced cristae (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ), characteristic of mitochondrial dysfunction caused by pyroptosis. Taken together, \u003cb\u003ebCCM\u003c/b\u003e effectively targets mitochondria and induces mitochondrial cuproptosis\u0026ndash;like dysfunction, which is characterized by changes in the expression of cuproptosis-related proteins, mitochondrial morphological damage and membrane potential dissipation. These mitochondrial effects, combined with cytoplasmic cuproptosis, synergistically enhance cuproptosis and improve therapeutic efficacy against DDP\u0026ndash;resistant tumors.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Biosafety evaluation of bCCM in zebrafish and mouse models\u003c/h2\u003e\u003cp\u003eAfter confirming the \u003cem\u003ein vitro\u003c/em\u003e necroptosis effect of \u003cb\u003ebCCM\u003c/b\u003e, the biosafety of bCCM was evaluated in zebrafish and mouse models. Considering that zebrafish are highly useful bioassays in toxicological studies, the \u003cem\u003ein vivo\u003c/em\u003e toxicity of \u003cb\u003ebCCM\u003c/b\u003e was first evaluated in a zebrafish model. Zebrafish embryos were equally divided into 8 groups and coincubated with cisplatin, \u003cb\u003ebCCM, bCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;DDP, or different concentrations of \u003cb\u003ebCCM\u003c/b\u003e. Representative images and survival curves of zebrafish embryos were acquired at different time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and Fig. S22). At 48 hpf, 72 hpf, and 96 hpf, the cisplatin\u0026ndash;treated zebrafish embryos appeared dead. At 96 hpf, representative images of zebrafish larvae were captured, which revealed that both the \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;DDP groups developed normally. In contrast, DDP treatment resulted in the failure of zebrafish embryos to hatch. The heart rate of zebrafish larvae at 96 hpf was measured in all groups to assess the physiological effects. No significant changes were observed in the \u003cb\u003ebCCM\u003c/b\u003e group compared with the control and combined groups, whereas the heart rate was reduced in the DDP group. In addition, no significant changes in body length were observed in the \u003cb\u003ebCCM\u003c/b\u003e group compared with the control and combined groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Moreover, there were no significant changes in the hatching rate, survival rate, body length or heart rate of zebrafish larvae in the \u003cb\u003ebCCM\u003c/b\u003e group with different concentrations of \u003cb\u003ebCCM\u003c/b\u003e compared with those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), indicating that \u003cb\u003ebCCM\u003c/b\u003e has excellent biocompatibility.\u003c/p\u003e\u003cp\u003eTo further explore the antitumor effect and mechanism of therapy \u003cem\u003ein vivo\u003c/em\u003e, the biosafety of \u003cb\u003ebCCM\u003c/b\u003e in mice was evaluated. First, the blood physiological and biochemical parameters of the mice treated with different drugs were investigated. Prior to \u003cem\u003ein vivo\u003c/em\u003e treatments, red blood cell (RBC) hemolysis and coagulation assays were conducted, revealing no hemolysis or coagulation phenomena within the tested concentrations (Fig. S23), indicating good blood compatibility of \u003cb\u003ebCCM\u003c/b\u003e. Next, four groups, namely, PBS, DDP, \u003cb\u003ebCCM\u003c/b\u003e, and \u003cb\u003ebCCM\u0026thinsp;+\u0026thinsp;DDP\u003c/b\u003e, were injected into healthy mice through the tail vein four consecutive times. Thereafter, the blood, major tissues, and organs of the mice treated with different drugs were collected for further analysis on day 21. The results revealed that blood biochemical indices and parameters, including WBC, RBC, HGB, ALT, AST, PLT, CREA, and BUN, were not significantly different from those in PBS\u0026ndash;treated mice (Fig. S24), demonstrating negligible side effects of \u003cb\u003ebCCM\u003c/b\u003e. Additionally, H\u0026amp;E staining of major organs (heart, liver, spleen, lung, and kidney) revealed no morphological abnormalities (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In contrast to DDP, which significantly caused liver and spleen damage, \u003cb\u003ebCCM\u003c/b\u003e did not affect animal growth or damage any major organs, indicating the superior biocompatibility of \u003cb\u003ebCCM\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Biodistribution of bCCM and copper ions in a DDP\u0026ndash;resistant HCC model\u003c/h2\u003e\u003cp\u003eAfter confirming the excellent biosafety of \u003cb\u003ebCCM\u003c/b\u003e, a biodistribution study was performed on a DDP\u0026ndash;resistant animal model of HCC (BEL7402/DDP). First, IR783\u0026ndash;labeled \u003cb\u003ebCCM\u003c/b\u003e (\u003cb\u003ebCCM\u003c/b\u003e@IR783) was prepared for monitoring biodistribution in the body (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The fluorescence signal of mice injected with \u003cb\u003ebCCM\u003c/b\u003e@IR783 via the tail vein was monitored via an \u003cem\u003ein vivo\u003c/em\u003e imaging system instrument. The results revealed a fluorescence signal at the tumor site in the mice after the injection of \u003cb\u003ebCCM\u003c/b\u003e@IR783 for 8 h. The fluorescence intensity at the tumor site continuously increased with time and peaked at approximately 12 h. Notably, there was a strong fluorescence signal at the tumor site even at 24 h after \u003cb\u003ebCCM\u003c/b\u003e@IR783 injection, indicating fast accumulation and long retention of \u003cb\u003ebCCM\u003c/b\u003e@IR783 at the tumor site (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). After 24 h, the mice were sacrificed, and the ex vivo biodistribution of \u003cb\u003ebCCM\u003c/b\u003e@IR783 was studied. The results revealed that the fluorescence signal intensity was the strongest in the tumors, which was greater than that in the livers, kidneys, hearts, lungs, spleens, and intestines, indicating that \u003cb\u003ebCCM\u003c/b\u003e@IR783 performed well at tumor targeting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In summary, \u003cb\u003ebCCM\u003c/b\u003e@IR783 can rapidly target and accumulate at tumor sites, which is very conducive to its further application.\u003c/p\u003e\u003cp\u003eFurthermore, a biodistribution study of copper ions was performed. The copper ion levels in the tumor tissue and main organs (heart, liver, spleen, lung and kidney) were detected with a copper detection kit. The results revealed no significant change in copper ion levels in the main organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) in \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP. Notably, the copper ion content in tumor tissues increased after \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP treatments. However, there was no significant difference in copper ion levels between the two groups, which revealed that copper ions accumulate at the tumor site (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These results suggest that \u003cb\u003ebCCM\u003c/b\u003e can selectively target tumor tissue to deliver copper ions to induce cuproptosis \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.7 In vivo anti\u0026ndash;resistant effect of bCCM on HCC\u003c/h2\u003e\u003cp\u003eEncouraged by the excellent \u003cem\u003ein vitro\u003c/em\u003e antidrug resistance capability and enhanced cuproptosis effect of \u003cb\u003ebCCM\u003c/b\u003e, we next sought to explore the \u003cem\u003ein vivo\u003c/em\u003e anticancer efficacy in a cisplatin\u0026ndash;resistant HCC mouse model. A BEL7402/DDP tumor model (five mice per group) was established, and the drug was administered via intravenous injection (i.j.) according to the treatment schedule (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). First, the body weight changes of the mice over 21 days of treatment were monitored. Negligible changes in body weight were observed in the \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP treatment groups compared with the control, whereas a significant decrease was noted in the DDP\u0026ndash;treated group, indicating the good biosafety of \u003cb\u003ebCCM\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, BEL7402/DDP tumor growth was effectively inhibited by \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP treatments, whereas DDP did not inhibit tumor growth, highlighting the superior anticancer effect of \u003cb\u003ebCCM\u003c/b\u003e on drug\u0026ndash;resistant HCC, as verified by tumor images (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The \u003cb\u003ebCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP groups presented the lowest tumor weight and highest tumor inhibitory ratio (TIR) at the given dose (3 mg/kg\u0026thinsp;+\u0026thinsp;2.5 mg/kg), in contrast to the slight reduction in tumor size and TIR caused by DDP (5 mg/kg), suggesting poor efficiency of DDP and excellent therapeutic efficiency of \u003cb\u003ebCCM\u003c/b\u003e in resistant HCC tumors (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eAdditionally, histological and immunohistochemical analyses were carried out to confirm the antitumor effect of \u003cb\u003ebCCM\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Hematoxylin and eosin (H\u0026amp;E) staining revealed significant tumor cell destruction in the \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;DDP groups, as evidenced by massive nuclear deletions and noticeable karyopyknosis, whereas the DDP groups exhibited almost normal tumor cell morphology compared with the control. Terminal deoxynucleotidyl transferase\u0026ndash;mediated nick end labeling (TUNEL) staining further confirmed the H\u0026amp;E results, revealing negligible apoptosis in the DDP group, whereas severe apoptosis and tumor growth inhibition were observed in the \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u003c/b\u003e\u0026thinsp;+\u0026thinsp;DDP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH and Fig. S26). These results demonstrated that \u003cb\u003ebCCM\u003c/b\u003e administration led to excellent antitumor efficacy in resistant HCC, whereas limited efficacy was observed in the DDP group. The superior anti-resistance efficacy of \u003cb\u003ebCCM\u003c/b\u003e in drug-resistant HCC mice may be attributed to the effects of Cuproptosis. Subsequently, immunohistochemical staining and western blotting of tumor tissues were performed to explore the mechanism underlying the resistance capability of \u003cb\u003ebCCM\u003c/b\u003e via cuproptosis. Immunohistochemistry revealed lower expression levels of ferroptosis-related factors (FDX1, DLAT and ATP7B) and drug resistance-related factors (ATP7B and P\u0026ndash;gp) in the \u003cb\u003ebCCM\u003c/b\u003e and \u003cb\u003ebCCM\u0026thinsp;+\u003c/b\u003e\u0026thinsp;DDP groups than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). Overall, \u003cb\u003ebCCM\u003c/b\u003e demonstrated excellent resistance due to its ability to effectively potentiate necroptosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn conclusion, we have developed a novel mitochondrion-targeted and GSH-responsive copper carrier, \u003cb\u003ebCCM\u003c/b\u003e, that effectively targets cisplatin-resistant hepatocellular carcinoma. This supramolecular complex, formed by chelating bm-Cur with Cu(II) at three coordination sites and encapsulating it with RBC membranes, addresses the key limitations of cuproptosis therapy by enhancing copper delivery through the tridentate chelation architecture, which enables high-capacity copper loading and targeted delivery, comprehensive GSH depletion achieved by dual inhibition of GSH synthesis and consumption via Cu(II)-mediated Fenton reactions and Michael addition, and resistance modulation through downregulation of ATP7B to prevent copper/cisplatin efflux. In summary, this work establishes \u003cb\u003ebCCM\u003c/b\u003e as a first-in-class therapeutic paradigm for the effective treatment of platinum-resistant malignancies, which also serves as an important example for new applications of traditional Chinese medicine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the animal studies were carried out in compliance with the institutional animal care guidelines and approved protocols of the experimental animal center at Hunan University of Chinese Medicine, which is located in Hunan Province, China (Ethics Approval Number: SLBH-202311090018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final manuscript, and warrant that the article is an original work and has not been published elsewhere.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHunan University of Chinese Medicine has applied for one patent on the basis of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is partially supported by the National Natural Science Foundation of China (82574571 to Y.Q.), Hunan Youth Science and Technology Innovation Talents Project (No. 2021RC3100 to Y.Q.), Open Competition Mechanism Project of Hunan University of Chinese Medicine (22JBZ023 to Y.Q.), Natural Science Foundation of Hunan Province (2024JJ5296 to Y.Q., 2025JJ50708 to X.X., 2023JJ30799 to Liqin Yuan), and Changsha Municipal Natural Science Foundation (kq2502052 to X.X.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQY conceived and designed the study. YHY performed all the experiments. CX, HSQ and DHF collected the data. XX and FJL interpreted and analyzed the data. YHY, YLQ and QY wrote the manuscript. WW and QY revised the manuscript critically.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the assistance of the International Joint Laboratory of Traditional Chinese Medicine and Ethnic Medicine at Hunan University of Chinese Medicine, Changsha, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVASAN N, BASELGA J, HYMAN D. M. A view on drug resistance in cancer [J]. Nature. 2019;575(7782):299\u0026ndash;309.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTADINI-BUONINSEGNI F, BARTOLOMMEI G, MONCELLI M R, et al. Translocation of platinum anticancer drugs by human copper ATPases ATP7A and ATP7B [J]. 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IUBMB Life. 2017;69(4):218\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTANG D, CHEN X. Cuproptosis: a copper-triggered modality of mitochondrial cell death [J]. Cell Res. 2022;32(5):417\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLI Y, LIU J. Mitochondria-Targeted Multifunctional Nanoparticles Combine Cuproptosis and Programmed Cell Death-1 Downregulation for Cancer Immunotherapy [J]. Adv Sci (Weinh). 2024;11(35):e2403520.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHE W, ZHANG X-Y, GONG X, et al. Drug-Free Biomimetic Oxygen Supply Nanovehicle Promotes Ischemia-Reperfusion Therapy in Stroke [J]. Adv Funct Mater. 2023;33(21):2212919.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHANG D, MAN D, LU J, et al. Mitochondrial TSPO Promotes Hepatocellular Carcinoma Progression through Ferroptosis Inhibition and Immune Evasion [J]. Adv Sci (Weinh). 2023;10(15):e2206669.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"drug resistance, Cuproptosis, glutathione (GSH), mitochondrial copper, ATPase (ATP7A/B)","lastPublishedDoi":"10.21203/rs.3.rs-7551173/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7551173/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlatinum-based drug resistance remains a major obstacle in cancer therapy. Cuproptosis, a novel form of copper-dependent cell death regulated through mitochondrial pathways, represents a promising strategy to counteract drug resistance in tumors. However, its efficacy is constrained by several physiological barriers, including elevated intracellular glutathione (GSH) levels, inadequate copper accumulation both cytoplasmically and within mitochondria, and the overexpression of copper efflux transporters such as ATP7A/B. To overcome these limitations, we developed a mitochondrion-targeted polyphenol\u0026ndash;copper nanocarrier (denoted bm\u0026ndash;Cur\u0026ndash;Cu₃@RBCm, or bCCM) by chelating Cu(II) with bisdemethylcurcumin\u0026mdash;a tridentate ligand offering three copper-binding sites\u0026mdash;and encapsulating the complex within red blood cell membranes (RBCm). This system enhances cuproptosis and counteracts drug resistance through three synergistic mechanisms. First, it significantly increases intracellular copper delivery via high-capacity tridentate chelation while concurrently depleting GSH to prevent the formation of inert GSH\u0026ndash;Cu/Pt complexes, thereby increasing the bioavailability of copper and cisplatin. Second, it promotes mitochondrial copper accumulation through targeted delivery and localized GSH depletion, leading to irreversible mitochondrial damage. Third, it downregulates ATP7B expression, thereby inhibiting copper and cisplatin efflux and enhancing both cuproptosis and chemosensitivity. \u003cem\u003eIn vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e evaluations demonstrated that bCCM effectively targets tumor cells and exerts potent antitumor activity against cisplatin-resistant hepatocellular carcinoma (HCC) without inducing systemic toxicity or undesirable copper accumulation. Mechanistic studies confirmed that bCCM downregulates key proteins associated with both cuproptosis and cisplatin resistance, indicating effective synergy between cuproptosis and conventional chemotherapy. This work establishes bCCM as an innovative therapeutic platform for overcoming platinum-based chemotherapy resistance, with promising potential for clinical translation in oncology.\u003c/p\u003e","manuscriptTitle":"A mitochondrion-targeted natural polyphenolic copper carrier overcomes tumor resistance to cisplatin by potentiating cuproptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-19 09:22:09","doi":"10.21203/rs.3.rs-7551173/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-30T06:12:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T04:11:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T15:28:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T22:00:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"335627652767792691301468529600816312112","date":"2025-09-22T02:28:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124955462276514007430675464727511825646","date":"2025-09-13T06:38:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120127885159549601263903004145353138666","date":"2025-09-12T14:45:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-12T06:19:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-11T15:45:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-11T15:30:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Experimental \u0026 Clinical Cancer Research","date":"2025-09-06T13:07:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"346fbd1c-7e56-41ff-a597-fa769478fc43","owner":[],"postedDate":"September 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T16:01:28+00:00","versionOfRecord":{"articleIdentity":"rs-7551173","link":"https://doi.org/10.1186/s13046-026-03642-5","journal":{"identity":"journal-of-experimental-and-clinical-cancer-research","isVorOnly":false,"title":"Journal of Experimental \u0026 Clinical Cancer Research"},"publishedOn":"2026-01-20 15:57:02","publishedOnDateReadable":"January 20th, 2026"},"versionCreatedAt":"2025-09-19 09:22:09","video":"","vorDoi":"10.1186/s13046-026-03642-5","vorDoiUrl":"https://doi.org/10.1186/s13046-026-03642-5","workflowStages":[]},"version":"v1","identity":"rs-7551173","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7551173","identity":"rs-7551173","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}