Copper and metformin double-doped polydopamine nanoparticles for tumor photothermal therapy

Copper and metformin double-doped polydopamine nanoparticles for tumor photothermal therapy | 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 Copper and metformin double-doped polydopamine nanoparticles for tumor photothermal therapy Yangwen Luo, Wenkai Zhang, Muge Gu, Xiangqi Zhang, Jiayu Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5223435/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The anti-tumor potentials of copper and metformin have been wildly studied. Excessive intracellular copper could lead cancer cells to cuproptosis, while metformin could activate adenosine monophosphate-activated protein kinase (AMPK) to decrease ATP and generate reactive oxygen species (ROS). This study integrated copper and metformin ably to form a polydopamine nanoparticle (CMP NPs) through coordination bonds. CMP NPs showed a high photothermal efficiency and anti-tumor efficacy both in vitro and in vivo experiments. This effective photothermal therapy (PTT) not only inhibited the growth of primary tumors but also generated an in-situ tumor vaccine-like function by immunogenic cell death to restrain the regrowth of the secondary tumors, showing a promising PTT for malignant solid tumors. nanoparticles cuproptosis anti-tumor photothermal therapy immunological memory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction According to the estimates from the International Agency for Research on Cancer (IARC), there were approximately 20 million new cases of cancer in the year 2020. Besides the frightening incidence rate of cancer, the mortality of cancer stayed at a high level, which caused 9.7 million deaths in 2020( 1 ). Metal ions have been frequently investigated in cancer therapy in recent years. Some metal ions, such as iron, calcium, copper, zinc, magnesium, and manganese( 2 ), have promising cancer therapy potential. Among these metal ions, copper has attracted researcher’s attention in recent years. The programmed cell death induced by excessive intracellular copper accumulation was termed cuproptosis. The excessive intake of copper generates intracellular reactive oxygen species (ROS) through a Fenton-like reaction, further inducing DNA damage( 3 ). More importantly, excessive intracellular copper finally induces the aggregation of dihydrolipoamide S-acetyltransferase (DLAT) and iron-sulfur cluster protein loss( 4 ). However, current applications of cuproptosis are still unable to meet the clinical needs due to some unsolved problems, which included the lack of copper transporter proteins in cancer cells( 5 , 6 ), low immunostimulatory efficacy( 7 ), and inhibition by the hypoxic tumor microenvironment( 8 ). Since copper is an essential element in tumor growth, an appropriate concentration of copper may also promote tumor growth( 9 , 10 ). Therefore, the designs of copper nanoparticles are expected to increase the copper uptake of cancer cells leveraging endocytosis( 11 ). Furthermore, combining cuproptosis with other effective drugs and safe therapies to improve immunogenetic cell death is also a hopeful therapeutic regimen. In recent years, an antihyperglycemic drug – metformin has been found to have anti-tumor efficacy by targeting mitochondria like copper. Based on current knowledge, metformin mainly inhibits the metabolism of rapidly proliferating tumors by activating adenosine monophosphate-activated protein kinase (AMPK)( 12 ). Metformin was proven to inhibit mitochondrial respiratory chain complex1, which decreases the intracellular adenosine triphosphate (ATP) and promotes ROS to damage DNA( 13 , 14 ). Anticipatedly, metformin was expected to have a synergy potential with excessive copper through its effects on mitochondria. Meanwhile, metformin has a metal-chelating site allowing the combination with copper( 15 , 16 ). To increase the copper uptake and improve immunogenetic cell death, polydopamine (PDA) was elected to act as a nanocarrier in this study. PDA has good biocompatibility( 17 , 18 ), outstanding photothermal conversion capability( 17 , 19 ), and great drug-loading capacity( 20 , 21 ). Besides, PDA-loading drugs can be released in acidic environments( 22 , 23 ) such as tumor acidic microenvironments( 24 , 25 ) and the environments of endosome and lysosome( 26 ) of cancer cells. When local photothermal therapy (PTT) is performed, the drugs are also released from the PDA( 27 , 28 ). Likewise, dopamine also has a metal-chelating site allowing the combination with copper( 29 ). Following the reasons above, we provide novel PDA nanoparticles containing copper and metformin (CMP NPs) for cancer PTT. These CMP NPs could enter the cancer cells by endocytosis and then disintegrate to produce a high level of intracellular copper. The excessive copper in cancer cells triggered cuproptosis, accompanied by depletion of GSH, decrease of ATP, and generation of ROS by a Fenton-like reaction. The influence of copper on mitochondria may also be amplified by released metformin. Moreover, CMP NPs were presented as a photothermal agent in combination with PTT. When the cancer cells were killed by cuproptosis and PTT, the cell debris continued to act as an antigen to improve immunogenetic cell death, and then generated in situ tumor vaccine-like functions triggering a systemic anti-tumor immune response. In this study, the results have represented that our CMP NPs effectively inhibit the growth and recurrence of subcutaneous tumors through cuproptosis and immunogenetic cell death combined with PTT, showing the therapeutic potential of cancer therapy. Materials and Methods Materials In the preparation of the NPs, CuCl 2 (99%) was purchased from Admas (Shanghai, China), metformin hydrochloride (99%) was purchased from Dalian Meilun Biological Technology Co., Ltd (Liaoning, China), and dopamine hydrochloride (99%) was purchased from Shanghai Macklin Reagent Co., Ltd (Shanghai, China). In cell experiments, methyl thiazolyl tetrazolium (MTT), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), 3,3'-dihexyloxycarbonocyanine iodide (JC-1), annexin V-FITC apoptosis detection kit, glutathione (GSH) peroxidase assay kit, adenosine triphosphate (ATP) assay kit, and bicinchoninic acid assay kit were all purchased from Beyotime Biotechnology (Shanghai, China). 4',6-diamidino-2-phenylindole (DAPI), indocyanine green (ICG), and rhodamine B hydrazide were purchased from Biosharp (Anhui, China). In the histopathological studies, recombinant anti-CD4 antibody (ab183685), and recombinant anti-CD8 alpha antibody (ab217344) were purchased from Abcam (Cambridge, UK). Anti-HMGB1 rabbit pAb (GB11103) was purchased from Servicebio (Hubei, China). Rabbit anti-FDX1 polyclonal antibody (abs118667) was purchased from Absin (Shanghai, China). HRP-labeled goat anti-rabbit IgG (H + L) and 3,3'-diaminobenzidine (DAB) were purchased from Beyotime Biotechnology (Shanghai, China). Female BALB/c mice (4–6 weeks old, 17–20 g) were purchased from SLAC LABORATORY ANIMAL (Shanghai, China), and maintained under specific pathogen-free conditions in all experiments. All animal experiments were performed under institutionally approved protocols (approval no. A2023207-001) by the Institutional Animal Care and Use Committee (IACUC), Shanghai Jiao Tong University. Instruments The hydrodynamic diameter, polydispersity index, and Zeta potential of the NPs were tested using a particle size analyzer (Nanobrook, Brookhaven, USA). The morphology of NPs was characterized by transmission electron microscope (Spectra 300, Thermofisher, USA). The X-ray photoelectron spectroscopy (XPS) survey was obtained using an X-ray photoelectron spectrometer (Nexsa G2, Thermo Scientific, USA). The photothermal efficacy of NPs was imaged using a thermal imager (FLIR Systems, USA). The absorbance of the experimental well was measured using a microplate reader (Biotek, USA). The Fluorescence images were obtained using a high-content imaging system (ImageXpress Micro Confocal, Molecular Devices, USA). Fluorescence intensity in cells was determined using flow cytometry (LSRFortessa X-20, BD Biosciences, USA). The histopathological images were obtained using a research slide scanner (VS200, Olympus, Japan). In the Transcriptome analysis, RNA sample concentration, quality, and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific). The library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA), and quantified using the Agilent high-sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was sequenced on NovaSeq 6000 platform (Illumina). Cell culture H22 cells were cultured in RPMI-1640 culture medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin and maintained at 37 ℃ in a humidified environment containing 5% CO 2 . Preparations of CP NPs and CMP NPs Metformin hydrochloride (2.5 mM) and dopamine hydrochloride (2.5 mM) were dissolved in 100 mL H 2 O, and then CuCl 2 (2.5 mM) was added into the solution under stirring for 0.5 h. PDA was formed in the alkaline solution, of which pH was adjusted by slowly adding NaOH solution (1 M) with continuous magnetic stirring for 12 h. During the formation of NPs, the mixture’s color changed from colorless to black. CMP NPs were obtained from the centrifugation (12000 rpm, 15 min) of the black suspension, followed by three times washing with deionized water. CP NPs or PDA were obtained by the same procedure only without metformin hydrochloride or both metformin hydrochloride and dopamine hydrochloride, respectively. The photothermal effect of the NPs In vitro photothermal effects of CP NPs and CMP NPs were studied. The NPs were dispersed in deionized water and then irradiated by a laser (808 nm). The temperature of the mixture was detected using a thermal imager. In vivo photothermal effect of CMP NPs was studied by a similar method. The female BALB/c mice were subcutaneously inoculated with H22 cells (2×10 5 cells, 0.1 mL), then CMP NPs were intratumorally injected with a single dose of 2 mg/kg after 5 days. The local temperature of the tumor site under the laser (808 nm, 1.0 W/cm 2 ) irradiation was also detected using a thermal imager. Cytotoxicity study H22 cells were seeded in 96-well plates at a density of 6×10 3 cells/well and then incubated at 37 ℃ for 12 h. The NPs were added into the wells for continued 24 h incubation. In addition, the cells from the laser-treating groups were exposed to laser irradiation (808 nm, 2 W/cm 2 ) for 5 min after 2 h of treatment. MTT reagent was added into each well with a final concentration of 0.5 mg/mL and allowed to incubate for 4 h. After the formazan formation, the supernatant was carefully removed and replaced by 100 µL of DMSO. Finally, the cell plates were vibrated for 5 min and the absorbance of each well was then recorded using a microplate reader at 570 nm. In vitro cell experiments H22 cells were seeded in 6-well plates at a density of 1×10 6 cells/well and then incubated at 37 ℃ for 12 h. For other cell experiments, the cells were stimulated by three kinds of NPs or PBS for a set period, and the cells from the laser-treating group were exposed to laser irradiation (808 nm, 2.0 W/cm 2 ) for 5 min after 2 h of treatment. Before the addition of experimental probes, the cells were collected by centrifugation (2000 rpm, 5 min) and washed with PBS twice. Cellular uptake of the NPs was verified using a high-content imaging system and flow cytometry. H22 cells were stained with DAPI and washed twice with PBS. The NPs were beforehand labeled with ICG by simply mixing up and stirring for 4 h. The cellular copper produced by the NPs in H22 cells was notarized using a high-content imaging system and flow cytometry utilizing rhodamine B hydrazide as a copper probe. The H22 cells were also stained with DAPI and then incubated with rhodamine B hydrazide (10 µM in 30% DMSO) for 30 min. The apoptosis of H22 cells was studied by staining with annexin Ⅴ- FITC and propidium iodide (PI). The relative levels of reactive oxygen species and mitochondrial membrane potential were evaluated by staining with DCFH-DA and JC-1, respectively. The levels of cellular ATP and GSH were detected using commercialized assay kits following the specifications. The protein content of treated H22 cells was tested by BCA. In vivo anti-tumor efficacy of CP NPs and CMP NPs Female BALB/c were subcutaneously inoculated with H22 cells (2×10 5 cells, 0.1 mL) to establish a tumor-bearing mouse model. Upon natural growth for 5 days, the mice were intratumorally injected with the NPs at a dose of 2 mg/kg of copper once every two days. After every interventional therapy, the laser (808 nm, 1.0 W/cm 2 ) irradiation on the tumor site was performed for 5 min the next day. The body weight and tumor volume (calculated by the formula: 0.5×length×width^2) were measured and recorded once every two days. Transcriptomics analysis The total RNA of the separated tumors was isolated using the Trizol Reagent. The mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First-strand cDNA was synthesized using random oligonucleotides and Super Script II. Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400–500 bp in length, the library fragments were purified using the AMPure XP system. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15-cycle PCR reaction. Products were purified and quantified using the Agilent high-sensitivity DNA assay on a Bioanalyzer 2100 system. The sequencing library was then sequenced on the Illumina platform. Cutadapt (v1.15) software was used to filter the sequencing data to get high-quality sequences. The filtered reads were mapped to the reference genome using HISAT2 v2.0.5. HTSeq(0.9.1) statistics were used to compare the Read Count values on each gene as the original gene expression and then used FPKM to standardize the expression. Then, differential expression were analyzed by DESeq (1.30.0) with screened conditions as follows: expression difference multiple |log2FoldChange| > 1, significant P-value < 0.05. The heat map was obtained using the Euclidean and Complete Linkage methods. Study on the immunological memory effect The mice bearing subcutaneous tumors were treated with CMP NPs and photothermal therapy according to the anti-tumor efficacy study. Furthermore, the mice were deemed to recover completely if there was no macroscopic tumor for 10 days after the last treatment. The cured mice were reinoculated with H22 cells on the opposite side, whose body weight and tumor volume were also measured and recorded once every two days. The untreated mice were subcutaneously inoculated with 0.1 mL of PBS to parallel the first tumor inoculation and subsequent treatment of the above-treated mice. Once the second tumor inoculation was achieved, there was no intervention in the two groups of mice until the advent of execution, allowing the tumor to grow for 18 days naturally. Hematoxylin-eosin staining and immunohistochemistry Main organs and tumors from the experimental mice were fixed in 4% paraformaldehyde (pH 7.4) for 24 h. After fixation, the tissues were dehydrated with ethanol and embedded in paraffin. Serial cross-sections from main organs were stained with hematoxylin and eosin (H&E). Besides, Sections of the tumors were incubated with PBS with 1% TritonX-100 and 10% FBS at room temperature for 90 min and further incubated with the antibodies (CD4, 1:500; CD8, 1:500; HMGB1, 1:200; FDX1, 1:100) at 4°C overnight, following a continued incubation with universal secondary antibody at room temperature for another 60 min. Tissue coloration with 3, 3-diaminobenzidine (DAB) was observed under an inverted microscope and blocked with a neutral resin. Image analysis of immunohistochemistry was performed using Image J Analyzer software. Statistical analysis Data were expressed as mean ± S.E.M. and analyzed using GraphPad Prism V9.0 (GraphPad Software). Data were analyzed using a two-sided Student’s t-test when two groups were being compared. One-way analysis of variance (ANOVA) and Tukey’s test were used to evaluate statistical differences when more than two groups were compared. The difference was considered significant when the p-value was less than or equal to 0.05. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. Results and Discussion 1. Characterization of CMP NPs The obtained PDA, CP NPs, and CMP NPs are all black. CMP NPs contain 21.5% (w/w) metformin, and 13.8% (w/w) copper estimated by detecting the mass of unreacted metformin and ICP-MS, respectively. The shapes of CMP NPs containing main elements such as C, O, N, and Cu, were recognized as spherical particles by transmission electron microscope (Fig. 1 a). The doping of copper and metformin could increase the size of PDA and lower its Zeta potential. The effective hydrodynamic diameter of CMP NPs was about 230 nm with an -8.5 mV of Zeta potential (Fig. 1 b-d). There is an evident absorption peak (233nm) in the ultraviolet absorption spectrum of CMP NPs, which is also a characteristic peak belonging to metformin (Fig. 1 d). In the Fourier transform infrared spectroscopy (FTIR) spectra (Fig. 1 e), the different absorption peaks of CMP NPs compared to CP NPs were marked. In contrast to the chemical structure of dopamine, metformin has CN double bonds leading to the differences in the FTIR spectra at a range of ~ 1650–1700 cm − 1 between CMP NPs and CP NPs. The absorption peaks at 1190 cm − 1 and 1069 cm − 1 observed in the FTIR spectra were also deemed to belong to the stretching vibrations of C-N, while the absorption peaks at 965 cm − 1 and 882 cm − 1 may belong to the out-of-plane bending vibration of N-H like that in the benzene ring, which indicated the formation of coordination bonds between adjacent C = NH in metformin and Cu. In addition, the differences in the FTIR spectra at the range of ~ 1500–1600 cm − 1 (inferred to the stretching vibrations of C-O) and ~ 3000–3300 cm − 1 (inferred to the stretching vibrations of O-H) between PDA and CP NPs all indicated the formation of coordination bonds between C-OH in PDA and Cu. The X-ray photoelectron spectroscopy (XPS) survey of CMP NPs certified the existence of Cu, N, and O elements (Fig. 1 g), meanwhile the atomic percent of N element in CMP NPs was raised by the doping of metformin (Fig. 1 h). These results demonstrated that CMP NPs were successfully fabricated as PDA particles containing copper and metformin through a simple one-pot method. 2. Photothermal efficacy of CMP NPs CMP NPs are designed to treat solid malignant tumors combined with photothermal therapy. Hence, we evaluated its photothermal efficiency by laser illuminating and thermal imaging. In vitro experiments, both CP NPs and CMP NPs showed favorable thermal efficiency, those temperatures of the suspensions raised more than 30 ℃ after irradiating with an 808 nm laser (1.0 W/cm 2 ) for 5 min, which was proved to be repeatable without degradation. The doping of copper enhanced the photothermal property of PDA, whereas metformin almost does not influence the photothermal property of CP NPs (Fig. 2 a, b). The increased temperatures of the irradiating suspensions were determined by the concentration, laser exposure duration, and laser power, with a positive correlation (Fig. 2 c). The photothermal efficiency of CMP NPs was also tested in vivo experiment. Upon local subdermal injection of CMP NPs, the 808 nm laser irradiation (1.0 W/cm 2 ) elevated the local temperature to 55 ℃ after 3 min (Fig. 2 d, e), which has been verified to be effective for tumor photothermal therapy( 30 ). CMP NPs exerted commendable photothermal efficacy in both in vitro and in vivo studies, which is expected to be applied well in tumor PTT. 3. Pharmacological effects of CMP NPs on H22 cells To exert anti-tumor efficacy, CMP NPs should be able to enter the tumor cells and generate intracellular copper. The cellular uptake of CMP NPs in H22 cells was investigated. As the results showed in Fig. 3 a-b, the fluorescence intensity of the NPs increased as the co-incubation time went by, suggesting a time-dependent cellular uptake process. Cuproptosis is induced by excessive intracellular copper accumulation, we continued to investigate the cellular copper of the H22 cells stained with rhodamine B hydrazide after being treated with the NPs. The result of flow cytometry showed that these NPs could produce cellular copper in a time-dependent manner (Fig. 3 c, d). Since CMP NPs were designed for tumor PTT, their cytotoxicity in H22 cancer cells was tested first. As Fig. 4 a shows, the cytotoxicity of copper in H22 cells was evident, and PDA hardly affected the viability of H22 cells consistent with other studies( 31 , 32 ). When treated with CMP NPs, the cell viabilities were slightly reduced compared with CP NPs. Under the NIR laser irradiation, however, CMP NPs reduced the cell viabilities more remarkably than CP NPs. This may result from the intracellular release of metformin triggered by laser irradiation since CP NPs had shown comparable photothermal efficiency to CMP NPs. The increased cellular copper produced by these NPs was supported by the decreased GSH levels in H22 cells (Fig. 4 b), which is considered to be easy to bind to copper ions( 33 ), and reducing GSH could promote cuproptosis( 34 ). Excessive intracellular copper could promote the cellular production of ROS through dynamin-related protein 1 (Drp1)-mediated mitochondrial fission( 35 , 36 ) and can restrict the ATP energy supply( 37 ). Meanwhile, metformin also influences the mitochondria and induces ROS ( 13 , 14 ). Here, we generally studied the abilities to produce ROS and ATP reduction of the NPs in H22 cells. The co-incubation with the NPs for 6 h notably increased the intracellular ROS levels (Fig. 4 d, e). The ATP levels of H22 cells were decreased by the NPs (Fig. 4 c). These results were consistent with another experiment showing the changes in mitochondrial membrane potential caused by the NPs (Fig. 5 ). It was obvious that there were more cells stained with green fluorescence and fewer cells stained with red fluorescence in the NP groups than in the PBS group, indicating mitochondrial degeneration and follow-up necrosis. The above results implied an apoptosis or necrosis outcome of H22 cells caused by the co-incubation with the NPs. Hence, we utilized annexin V-FITC combined with PI to investigate the cell’s apoptosis or necrosis rate after treatment with the NPs for 3 h and 12 h. As the results shown by flow cytometry (Fig. 6 ), the annexin V-FITC positive percent of the treated groups was significantly higher than that of the PBS group after 3h and 12 h, demonstrating that the NPs could remarkably damage cancer cells to death. Noteworthily, both in the 3 h and 12 h period, the percent of classical apoptosis cells (stained positively by annexin V-FITC meanwhile negatively by propidium iodide, shown as Q3 quadrant in the plot) had no evident changes, which might result from the cell membrane destruction induced by excessive copper( 38 ), remaindering that cuproptosis is different from apoptosis and other programmed cell deaths( 4 ). Above all, among most of the above results, CMP NPs performed better than CP NPs, which was further transcended by the addition of near-infrared laser irradiation. 4. In vivo anti-tumor efficacy of CMP NPs in H22 tumor-bearing mice model After verifying the remarkable killing effect of CMP NPs on H22 Cells, its anti-tumor efficacy in BALB/c mice bearing H22 cells was integrally studied. The experimental mice were divided into six groups and treated with different NPs as illustrated in Fig. 7 a. Firstly, there was no significant body weight loss of mice in the groups treated with the NPs, except for the group treated with CMP NPs plus laser irradiation, which was supposed to result from their experienced pain caused by the photothermal effect. There was also no evident change in the major organs across all groups revealed by histopathological analysis (Fig. 10 a). The results manifested that CMP NPs inhibited the tumor growth compared with saline and saline plus laser irradiation. Encouragingly, treatment with CMP NPs plus laser irradiation could further significantly inhibit tumor growth (Fig. 7 b-f), representing a successful therapeutic regimen combining cuproptosis with photothermal therapy. The TUNEL staining of the treated tumor tissues echoed the previous results (Fig. 8 a). The immunohistochemistry staining results showed an increased expression of ferredoxin-1 (FDX1) and decreased expression of high-mobility group box 1 (HMGB1) in the tumors from the CMP NP group (Fig. 8 b-d). FDX1 is the key regulator of copper ionophore–induced cell death, which reduces Cu ( Ⅱ ) to more toxic Cu (Ⅰ). Knockout FDX1 could rescue cells from cuproptosis( 4 , 39 ). Therefore, increased expression of FDX1 may help CMP NPs exert their cytotoxicity in cancer cells. Besides, HMGB1 is associated with the hallmarks of cancer( 40 ) and is considered to contribute to tumorigenesis( 41 , 42 ). The decreased expression of HMGB1 by CMP NPs in this study suggested an outcome of immunogenic cell death. Moreover, the anti-tumor effect of CMP NPs in the H22 cells-bearing mice model was further verified by transcriptomics analysis. We found 676 differential expression genes of the tumors between the saline group and the CMP NPs group, where 666 differential expression genes were upregulated, and 10 differential expression genes were downregulated in the CMP NPs group (Fig. 9 a, b). The differential expression genes were enriched into some sets of genes from Gene ontology (GO) (Fig. 9 c) and pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Fig. 9 d) using Gene Set Enrichment Analysis (GSEA) analysis. In GO, the expression of metabolism-related and immune-related genes was altered by CMP NPs. The expression genes involved in ribosome and mitochondrial were downregulated, and those engaged in pyroptosis and immune-related sets of genes, such as activation of the innate immune response, mast cell activation, positive regulation of interferon-alpha and interleukin-1 beta production, and so on, were upregulated. This result reemerged in KEGG pathways. More importantly, the pathways of the citrate cycle and oxidative phosphorylation were found to be downregulated in the CMP NPs group. In contrast, the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway, and protein digestion and absorption( 43 ) were upregulated. The alteration of the citrate cycle pathway confirmed the cuproptosis caused by CMP NPs( 4 ). Oxidative phosphorylation was regarded as a therapeutic target for cancer therapy( 44 , 45 ), which may be inhibited in this study by the released metformin from CMP NPs( 46 ). The NOD-like receptor is the key component of the host innate immune system( 47 , 48 ), its up-regulation suggested an immune activation induced by CMP NPs. In brief, CMP NPs had successfully exerted anti-tumor efficacy through cuproptosis and antimetabolic effect, and consequently induced immunogenic cell death to activate immune responses. 5. In-situ tumor vaccine-like function generates immune memory effect in the tumor-reinoculated mice mode CMP NPs combined with laser irradiation exerted an outstanding anti-tumor efficacy in our preceding study, which even cured several tumor-bearing mice, we continued to probe the in-situ tumor vaccine-like function induced by the immunogenic cell death, which was triggered by CMP NPs plus laser irradiation and further generated immune memory effect in those cured mice through a tumor rechallenging model illustrated as Fig. 10 a. The results shown in Fig. 9 b demonstrated a body weight recovery in the mice cured by CMP NPs plus laser irradiation. More importantly, the growth of reinoculated tumors in the mice recovered from earlier combined therapy was significantly inhibited. By contrast, the tumors in the mice without any treatment had grown unrestrainedly (Fig. 10 c-e). The more infiltrated CD4 + T and CD8 + T lymphocytes in the tumors from treated mice were observed in the immunohistochemistry staining results (Fig. 11 f-h). The organ index (expressed by organ weight/body weight) of the main organs from the mice showed an in vivo safety of CMP NPs plus laser irradiation. In summary, CMP NPs combined with PTT could inhibit tumor growth, lead the cancer cells to immunogenic cell death, followed by in-situ tumor vaccine-like function, and eventually generate an immune memory effect to restrain the tumor regrowth. Conclusion In conclusion, we have provided polydopamine nanoparticles – CMP NPs doped with copper and metformin for tumor photothermal therapy. CMP NPs could act as a nanocarrier for copper uptake to induce cuproptosis and for metformin to restrain the tumor metabolism and perform as a photothermal agent applied to PTT. The carried metformin and copper affect the mitochondria of cancer cells and produce cellular ROS, along with the reduction of ATP and GSH. In the animal model, CMP NPs effectively inhibited tumor growth combined with PTT. Moreover, CMP NPs plus NIR laser irradiation could induce an in-situ tumor vaccine-like function by immunogenic cell death to restrain tumor regrowth. Declarations Ethics approval and consent to participate All animal experiments were performed under institutionally approved protocols (approval no. A2023207-001) by the Institutional Animal Care and Use Committee (IACUC), Shanghai Jiao Tong University. Consent for publication All authors have approved the manuscript and agree with the submission and publication. Availability of data and materials Readers who need data or materials from this article could contact the corresponding authors by e-mail. Competing interests All authors declare no competing interests. Funding This study was supported by the National Natural Science Foundation of China (22071148) and Fundamental Research Funds for the Central Universities (2232023A-07) to Jiefeng Shen. Authors' contributions Yangwen Luo prepared the nanoparticles, performed in vitro and in vivo experiments, analyzed the data, and wrote the manuscript; Wenkai Zhang, Muge Gu, Xiangqi Zhang, Wei Yu, and Jiayu Wang offered advice and assistance in the experiments; Weien Yuan, Jiefeng Shen, Guoqiang Yang, and Hua Yang designed this project, commented on the experiments and draft writing, and supervised the study. Acknowledgements We acknowledge Shanghai Bioprofile for assistance in the transcriptomics analysis. Authors' information Authors and Affiliations Department of Hepatic Oncology, Shanghai Geriatric Medical Center, Shanghai 201104, P. R. China; Department of Liver Surgery and Transplantation, Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai 200032, P. R. China Hua Yang Frontiers Science Center for Transformative Molecules, School of Chemistry and Chemical Engineering, Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, P. R. China Yangwen Luo & Guoqiang Yang College of Chemistry and Chemical Engineering, Donghua University, Shanghai 201620, P. R. China Jiefeng Shen Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, P. R. 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Polydopamine surface-modified nanocarriers for improved anticancer activity: Current progress and future prospects. OpenNano. 2022. Yu B, Liu J, Liu S, Zhou F. Pdop layer exhibiting zwitterionicity: a simple electrochemical interface for governing ion permeability. Chem Commun. 2010. Feng H, Zhu X, Chen R, Liao Q, Ye D, Zhang B et al. pH response of zwitterionic polydopamine layer to palladium deposition in the microchannel. Chem Eng J. 2018. Wojtkowiak JW, Verduzco D, Schramm KJ, Gillies RJ. Drug Resistance and Cellular Adaptation to Tumor Acidic pH Microenvironment. Mol Pharm. 2011. Zahn LM. Effects of the tumor microenvironment. Science. 2017. Mellman I, Fuchs R, Helenius A. Acidification of the endocytic and exocytic pathways. Annu Rev Biochem. 1986. Yuan Z, Lin C, He Y, Tao B, Chen M, Zhang J et al. Near-Infrared Light-Triggered Nitric-Oxide-Enhanced Photodynamic Therapy and Low-Temperature Photothermal Therapy for Biofilm Elimination. ACS Nano. 2020. Gao G, Jiang Y-W, Jia H-R, Wu F-G. Near-infrared light-controllable on-demand antibiotics release using thermo-sensitive hydrogel-based drug reservoir for combating bacterial infection. Biomaterials. 2018. Bacchella C, Dell’Acqua S, Nicolis S, Monzani E, Casella L. A Cu-bis(imidazole) Substrate Intermediate Is the Catalytically Competent Center for Catechol Oxidase Activity of Copper Amyloid-β. Inorg Chem. 2021. Chen Q, Xu L, Liang C, Wang C, Peng R, Liu Z. Photothermal therapy with immune-adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapy. Nat Commun. 2016. Ruan S, Yin W, Chang J, Yang Y, Sun J, Ma X et al. Acidic and hypoxic tumor microenvironment regulation by CaO2-loaded polydopamine nanoparticles. J Nanobiotechnol. 2022. Zhang Y, Ding X, Xie F, Gao M, Qiu J, Wang Z, et al. Targeted Recruitment and Degradation of Estrogen Receptor α by Photothermal Polydopamine Nanoparticles for Breast Tumor Ablation. Advanced Healthcare Materials; 2022. Liu H-J, Chen W, Wu G, Zhou J, Liu C, Tang Z et al. Glutathione-Scavenging Nanoparticle-Mediated PROTACs Delivery for Targeted Protein Degradation and Amplified Antitumor Effects. Adv Sci. 2023. Lu S, Li Y, Yu Y. Glutathione-Scavenging Celastrol-Cu Nanoparticles Induce Self-Amplified Cuproptosis for Augmented Cancer Immunotherapy. Adv Mater. 2024. Xu J, Zheng B, Zhang S, Liao X, Tong Q, Wei G et al. Copper Sulfide Nanoparticle-Redirected Macrophages for Adoptive Transfer Therapy of Melanoma. Adv Funct Mater. 2021. Shimada K, Reznik E, Stokes ME, Krishnamoorthy L, Bos PH, Song Y, et al. Copper-Binding Small Molecule Induces Oxidative Stress and Cell-Cycle Arrest in Glioblastoma-Patient-Derived Cells. Cell Chemical Biology; 2018. Chang J, Yin W, Zhi H, Chen S, Sun J, Zhao Y et al. Copper Deposition in Polydopamine Nanostructure to Promote Cuproptosis by Catalytically Inhibiting Copper Exporters of Tumor Cells for Cancer Immunotherapy. Small. 2024. Zhao G, Sun H, Zhang T, Liu J-X. Copper induce zebrafish retinal developmental defects via triggering stresses and apoptosis. Cell Communication Signal. 2020. Tsvetkov P, Detappe A, Cai K, Keys HR, Brune Z, Ying W et al. Mitochondrial metabolism promotes adaptation to proteotoxic stress. Nat Chem Biol. 2019. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011. Pusterla T, Nèmeth J, Stein I, Wiechert L, Knigin D, Marhenke S et al. Receptor for advanced glycation endproducts (RAGE) is a key regulator of oval cell activation and inflammation-associated liver carcinogenesis in mice. Hepatology. 2013. Wang S, Zhang Y. HMGB1 in inflammation and cancer. J Hematol Oncol. 2020. Zu H, Wu Y, Meng H, Cheng X, Wang Y, Zhang LW et al. Tumor Metabolism Aiming Cu2–xS Nanoagents Mediate Photothermal-Derived Cuproptosis and Immune Activation. ACS Nano. 2024. Nayak AP, Kapur A, Barroilhet L, Patankar MS. Oxidative Phosphorylation: A Target for Novel Therapeutic Strategies Against Ovarian Cancer. Cancers. 2018. Rao S, Mondragón L, Pranjic B, Hanada T, Stoll G, Köcher T et al. AIF-regulated oxidative phosphorylation supports lung cancer development. Cell Res. 2019. Lee M, Hirpara JL, Eu J-Q, Sethi G, Wang L, Goh B-C et al. Targeting STAT3 and oxidative phosphorylation in oncogene-addicted tumors. Redox Biol. 2018. Chen G, Shaw MH, Kim Y-G, Nuñez G. NOD-like receptors: role in innate immunity and inflammatory disease. Annu Rev Pathol. 2008. Zheng C. The emerging roles of NOD-like receptors in antiviral innate immune signaling pathways. Int J Biol Macromol. 2020. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-5223435","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":365920209,"identity":"15b444cf-0e0b-442b-a088-9c533c15d049","order_by":0,"name":"Yangwen Luo","email":"","orcid":"","institution":"Frontiers Science Center for Transformative Molecules, School of Chemistry and Chemical Engineering, Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yangwen","middleName":"","lastName":"Luo","suffix":""},{"id":365920210,"identity":"1cdb8cc8-4b46-48b2-be9c-aa3db58a0096","order_by":1,"name":"Wenkai Zhang","email":"","orcid":"","institution":"Engineering Research Center of Cell \u0026 Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenkai","middleName":"","lastName":"Zhang","suffix":""},{"id":365920211,"identity":"cafd3414-f925-42de-ade5-f195c7fc612e","order_by":2,"name":"Muge Gu","email":"","orcid":"","institution":"Engineering Research Center of Cell \u0026 Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Muge","middleName":"","lastName":"Gu","suffix":""},{"id":365920212,"identity":"d7207f3a-a670-4a26-84b9-42bbd956fa7a","order_by":3,"name":"Xiangqi Zhang","email":"","orcid":"","institution":"Engineering Research Center of Cell \u0026 Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiangqi","middleName":"","lastName":"Zhang","suffix":""},{"id":365920213,"identity":"4aa1f14b-560f-4d8d-a8b4-14fef56f33a7","order_by":4,"name":"Jiayu Wang","email":"","orcid":"","institution":"Engineering Research Center of Cell \u0026 Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong 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University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weien","middleName":"","lastName":"Yuan","suffix":""},{"id":365920216,"identity":"e0a6252b-7ecb-46b2-abf8-811ce8839d44","order_by":7,"name":"Jiefeng Shen","email":"","orcid":"","institution":"College of Chemistry and Chemical Engineering, Donghua University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiefeng","middleName":"","lastName":"Shen","suffix":""},{"id":365920217,"identity":"c8a48b83-6d2a-4e3b-a4de-12e520e97061","order_by":8,"name":"Guoqiang Yang","email":"","orcid":"","institution":"Frontiers Science Center for Transformative Molecules, School of Chemistry and Chemical Engineering, Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guoqiang","middleName":"","lastName":"Yang","suffix":""},{"id":365920218,"identity":"83ec4dcd-b100-4ccb-9647-fdfba4709be9","order_by":9,"name":"Hua Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYBACAygtx94AYTM2EKvFmOcAqVoSe4jWYi6R/Ezi547a9B6J5I2fCxhsZDccYH72AJ8WyxlpZpK9Z47n9kikFUvPYEgz3nCAzdwAnxaDGwlmErxtx3L3S+QYSPMwHE7ccICHTQK/lvRvkn/bjqXzSOQY/+Zh+E+Mlhwzad62mgSgFjOgLQeI0HLmTbG1bNsBwx6eZ2XWPAbJxjMPs5nh13I8fePNt2118jzsyZtv81TYyfYdb36GVwsQsAAVHIaZAMTMBNSDlHxgYKgjrGwUjIJRMApGLgAA/I1IUdSRTUEAAAAASUVORK5CYII=","orcid":"","institution":"Department of Hepatic Oncology, Shanghai Geriatric Medical Center","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-10-08 08:38:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5223435/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5223435/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66772576,"identity":"e0a7e833-a831-4724-8830-87ea84c9983f","added_by":"auto","created_at":"2024-10-16 10:36:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":271621,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of CMP NPs. a) TEM images and elements mapping of CMP NPs. b) Hydrodynamic diameters of PDA, CP NPs, and CMP NPs (n = 3). c) Zeta potentials of PDA, CP NPs, and CMP NPs (n = 3). d) Hydrodynamic diameter distributions of PDA, CP NPs, and CMP NPs. e) Ultraviolet (UV) absorption spectra of dopamine, metformin, PDA, CP NPs, and CMP NPs. f) Fourier transform infrared spectroscopy (FTIR) spectra of dopamine, metformin, CP NPs, and CMP NPs. g) X-ray photoelectron spectroscopy (XPS) survey of CMP NPs. h) Atomic percent of three elements in CP NPs and CMP NPs measured by XPS. Data are represented as mean ± S.E.M.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/02c74f161ef6ef6c8cd441eb.png"},{"id":66772357,"identity":"5d6ce1a3-52b4-48b5-975c-b3778685e072","added_by":"auto","created_at":"2024-10-16 10:28:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":352566,"visible":true,"origin":"","legend":"\u003cp\u003eThe photothermal efficacy of CMP NPs. a) Temperature variation curves and b) thermal images of PDA, CP NPs, and CMP NPs excited by NIR laser (808 nm, 1.0 W/cm\u003csup\u003e2\u003c/sup\u003e). c) Temperature variation curves of CMP NPs in different concentrations under different power of NIR laser. In vivo d) temperature variation curve and e) thermal images of the subcutaneous tumor site treated by CMP NPs and excited by NIR laser (808 nm, 1.0 W/cm\u003csup\u003e2\u003c/sup\u003e) (n = 5). Data are represented as mean ± S.E.M.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/bcdc0cd5b1b757e5612cbe20.png"},{"id":66774189,"identity":"5534031a-5087-433a-bd84-761719db5ad0","added_by":"auto","created_at":"2024-10-16 10:44:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":499992,"visible":true,"origin":"","legend":"\u003cp\u003eCellular uptake of CP NPs and CMP NPs in H22 cells. a) Representative fluorescent images and b) flow cytometric profiles of H22 cells treated with the NPs labeled with indocyanine green. c) Representative fluorescent images and d) flow cytometric profiles of H22 cells incubated with rhodamine B hydrazide after the treatment of the NPs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/84d54a350a0fb4c95dacdb09.png"},{"id":66772575,"identity":"2472aa7f-5dbd-4b3f-9d60-ad1c762092dc","added_by":"auto","created_at":"2024-10-16 10:36:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":203427,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of CP NPs and CMP NPs on the cell viability, GSH, ATP, and ROS of H22 cells. a) Cell viabilities of H22 cells and NIH 3T3 cells after a series of treatments for 24 h, with or without laser irradiation (5 min, 808 nm, 2 W/cm\u003csup\u003e2\u003c/sup\u003e).\u0026nbsp; b) Cellular GSH relative level of H22 cells incubated with the NPs for 24 h. c) Cellular ATP level of H22 cells incubated with the NPs for 24 h. d) Representative fluorescent images and e) flow cytometric profiles of intracellular ROS of H22 cells with treatments for 6 h. Data are represented as mean ± S.E.M. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/be231bb4761ae4653c95b046.png"},{"id":66772573,"identity":"7096f3de-b480-4f92-9a52-111c63084cb8","added_by":"auto","created_at":"2024-10-16 10:36:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":653153,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative fluorescent images and flow cytometric plots demonstrating the difference in mitochondrial membrane potential between the H22 cells with different treatments for 12 h.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/1136f354874c0f9d7d119159.png"},{"id":66772367,"identity":"caf2a578-0421-4af9-b4c9-413cd338c791","added_by":"auto","created_at":"2024-10-16 10:28:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":323894,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometric plots and quantifications of the H22 cells stained with annexin Ⅴ- FITC and PI after various treatments (n = 3). Data are represented as mean ± S.E.M. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/dfc635e105f07b88c8d7b8ea.png"},{"id":66772358,"identity":"1c853b08-6001-44f8-b726-ef777e573193","added_by":"auto","created_at":"2024-10-16 10:28:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":235260,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo anti-tumor efficacy of the NPs in the H22 tumor-bearing mice model. a) Schematic showing the experimental schedule for exploring the anti-tumor efficacy of the NPs in the BALB/c mice subcutaneously bearing H22 cells tumor. b) Body weight changes of the mice (n = 5). Tumor volume changes of c) each group (n = 5) and d) each mouse throughout the period of the experiment. e) Photograph showing the separated tumors from the mice at the end of the treatment course. f) Weight of tumors separated from the mice at the end of the treatment course (n = 5). Data are represented as mean ± S.E.M. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, ns, not significant.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/745270599bff96c315e7c62e.png"},{"id":66772361,"identity":"20bd52f5-dcc4-41e9-8b58-302263ccdcf3","added_by":"auto","created_at":"2024-10-16 10:28:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":616508,"visible":true,"origin":"","legend":"\u003cp\u003eThe staining results of the tumors separated from the BALB/c mice. a) Representative images of the TUNEL staining. b) Immunohistochemistry staining and c), d) quantification of FDX1 and HMGB1 in the tumors separated from the BALB/c mice at the end of the treatment course. Data are represented as mean ± S.E.M. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/3028dce693fd0f3f3a0dd081.png"},{"id":66772577,"identity":"374d1067-6331-4653-84f9-96ed790674fe","added_by":"auto","created_at":"2024-10-16 10:36:50","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":385524,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomics analysis of separated tumors from saline/CMP NPs-treated BALB/c mice. a) Cluster heat map showing the differential expressions between the saline and CMP NPs groups. b) Volcano plot displaying the differences in gene expression between the saline group and CMP NPs group. Representative Gene Set Enrichment Analysis (GSEA) analysis results of the gene sets from c) Gene ontology (GO) and d) Kyoto Encyclopedia of Genes and Genomes (KEGG) in the transcriptomics analysis results.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/ea3e3ef25f7a4327cfe46a5e.png"},{"id":66772362,"identity":"21ec60cf-325e-45ce-ae57-f7678d058154","added_by":"auto","created_at":"2024-10-16 10:28:49","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":625130,"visible":true,"origin":"","legend":"\u003cp\u003eThe immune memory effect in the BALB/C mice recovered from CMP NPs plus laser irradiation. a) Schematic showing the experimental schedule. b) Body weight changes of the mice after the previous treatment cycle (n = 4). c) Tumor volume changes and e) tumor weight of the mice after the previous treatment cycle (n = 4). d) Photograph showing the separated tumors from the mice having experienced twice inoculations of H22 cells (n = 4). h) Immunohistochemistry staining and f), g) quantifications of CD4 and CD8 in the tumor tissues separated from the mice at the end of the experiment (n = 3). Data are represented as mean ± S.E.M. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/208369533a3602a0c9b62205.png"},{"id":66772364,"identity":"4792ffa2-9f82-406f-8a34-fd670b88ed6d","added_by":"auto","created_at":"2024-10-16 10:28:50","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":625039,"visible":true,"origin":"","legend":"\u003cp\u003eTissue staining and organ index of the main organs from the mice indicate the biological safety of CMP NPs. a) Representative H\u0026amp;E staining images and b) organ indexes (n = 5) of the main organs from the H22 tumor-bearing BALB/c mice in the anti-tumor efficacy study. c) Organ indexes of the main organs from the BALB/c mice having experienced twice inoculations of H22 cells (n = 4). Data are represented as mean ± S.E.M. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/4c376da80e7efd3bdcec5993.png"},{"id":71041123,"identity":"a1f6a788-0812-46fa-81b5-3c35c71cbbae","added_by":"auto","created_at":"2024-12-10 13:47:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4934594,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5223435/v1/d015d0f3-0ee5-4d7c-bee7-911ed0663a0b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Copper and metformin double-doped polydopamine nanoparticles for tumor photothermal therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAccording to the estimates from the International Agency for Research on Cancer (IARC), there were approximately 20\u0026nbsp;million new cases of cancer in the year 2020. Besides the frightening incidence rate of cancer, the mortality of cancer stayed at a high level, which caused 9.7\u0026nbsp;million deaths in 2020(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Metal ions have been frequently investigated in cancer therapy in recent years. Some metal ions, such as iron, calcium, copper, zinc, magnesium, and manganese(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), have promising cancer therapy potential. Among these metal ions, copper has attracted researcher\u0026rsquo;s attention in recent years. The programmed cell death induced by excessive intracellular copper accumulation was termed cuproptosis. The excessive intake of copper generates intracellular reactive oxygen species (ROS) through a Fenton-like reaction, further inducing DNA damage(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). More importantly, excessive intracellular copper finally induces the aggregation of dihydrolipoamide S-acetyltransferase (DLAT) and iron-sulfur cluster protein loss(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). However, current applications of cuproptosis are still unable to meet the clinical needs due to some unsolved problems, which included the lack of copper transporter proteins in cancer cells(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), low immunostimulatory efficacy(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), and inhibition by the hypoxic tumor microenvironment(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Since copper is an essential element in tumor growth, an appropriate concentration of copper may also promote tumor growth(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Therefore, the designs of copper nanoparticles are expected to increase the copper uptake of cancer cells leveraging endocytosis(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Furthermore, combining cuproptosis with other effective drugs and safe therapies to improve immunogenetic cell death is also a hopeful therapeutic regimen.\u003c/p\u003e \u003cp\u003eIn recent years, an antihyperglycemic drug \u0026ndash; metformin has been found to have anti-tumor efficacy by targeting mitochondria like copper. Based on current knowledge, metformin mainly inhibits the metabolism of rapidly proliferating tumors by activating adenosine monophosphate-activated protein kinase (AMPK)(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Metformin was proven to inhibit mitochondrial respiratory chain complex1, which decreases the intracellular adenosine triphosphate (ATP) and promotes ROS to damage DNA(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Anticipatedly, metformin was expected to have a synergy potential with excessive copper through its effects on mitochondria. Meanwhile, metformin has a metal-chelating site allowing the combination with copper(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo increase the copper uptake and improve immunogenetic cell death, polydopamine (PDA) was elected to act as a nanocarrier in this study. PDA has good biocompatibility(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), outstanding photothermal conversion capability(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), and great drug-loading capacity(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Besides, PDA-loading drugs can be released in acidic environments(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) such as tumor acidic microenvironments(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) and the environments of endosome and lysosome(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) of cancer cells. When local photothermal therapy (PTT) is performed, the drugs are also released from the PDA(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Likewise, dopamine also has a metal-chelating site allowing the combination with copper(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFollowing the reasons above, we provide novel PDA nanoparticles containing copper and metformin (CMP NPs) for cancer PTT. These CMP NPs could enter the cancer cells by endocytosis and then disintegrate to produce a high level of intracellular copper. The excessive copper in cancer cells triggered cuproptosis, accompanied by depletion of GSH, decrease of ATP, and generation of ROS by a Fenton-like reaction. The influence of copper on mitochondria may also be amplified by released metformin. Moreover, CMP NPs were presented as a photothermal agent in combination with PTT. When the cancer cells were killed by cuproptosis and PTT, the cell debris continued to act as an antigen to improve immunogenetic cell death, and then generated in situ tumor vaccine-like functions triggering a systemic anti-tumor immune response. In this study, the results have represented that our CMP NPs effectively inhibit the growth and recurrence of subcutaneous tumors through cuproptosis and immunogenetic cell death combined with PTT, showing the therapeutic potential of cancer therapy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eIn the preparation of the NPs, CuCl\u003csub\u003e2\u003c/sub\u003e (99%) was purchased from Admas (Shanghai, China), metformin hydrochloride (99%) was purchased from Dalian Meilun Biological Technology Co., Ltd (Liaoning, China), and dopamine hydrochloride (99%) was purchased from Shanghai Macklin Reagent Co., Ltd (Shanghai, China).\u003c/p\u003e \u003cp\u003eIn cell experiments, methyl thiazolyl tetrazolium (MTT), 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), 3,3'-dihexyloxycarbonocyanine iodide (JC-1), annexin V-FITC apoptosis detection kit, glutathione (GSH) peroxidase assay kit, adenosine triphosphate (ATP) assay kit, and bicinchoninic acid assay kit were all purchased from Beyotime Biotechnology (Shanghai, China). 4',6-diamidino-2-phenylindole (DAPI), indocyanine green (ICG), and rhodamine B hydrazide were purchased from Biosharp (Anhui, China).\u003c/p\u003e \u003cp\u003eIn the histopathological studies, recombinant anti-CD4 antibody (ab183685), and recombinant anti-CD8 alpha antibody (ab217344) were purchased from Abcam (Cambridge, UK). Anti-HMGB1 rabbit pAb (GB11103) was purchased from Servicebio (Hubei, China). Rabbit anti-FDX1 polyclonal antibody (abs118667) was purchased from Absin (Shanghai, China). HRP-labeled goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) and 3,3'-diaminobenzidine (DAB) were purchased from Beyotime Biotechnology (Shanghai, China).\u003c/p\u003e \u003cp\u003eFemale BALB/c mice (4\u0026ndash;6 weeks old, 17\u0026ndash;20 g) were purchased from SLAC LABORATORY ANIMAL (Shanghai, China), and maintained under specific pathogen-free conditions in all experiments. All animal experiments were performed under institutionally approved protocols (approval no. A2023207-001) by the Institutional Animal Care and Use Committee (IACUC), Shanghai Jiao Tong University.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInstruments\u003c/h3\u003e\n\u003cp\u003eThe hydrodynamic diameter, polydispersity index, and Zeta potential of the NPs were tested using a particle size analyzer (Nanobrook, Brookhaven, USA). The morphology of NPs was characterized by transmission electron microscope (Spectra 300, Thermofisher, USA). The X-ray photoelectron spectroscopy (XPS) survey was obtained using an X-ray photoelectron spectrometer (Nexsa G2, Thermo Scientific, USA). The photothermal efficacy of NPs was imaged using a thermal imager (FLIR Systems, USA). The absorbance of the experimental well was measured using a microplate reader (Biotek, USA). The Fluorescence images were obtained using a high-content imaging system (ImageXpress Micro Confocal, Molecular Devices, USA). Fluorescence intensity in cells was determined using flow cytometry (LSRFortessa X-20, BD Biosciences, USA). The histopathological images were obtained using a research slide scanner (VS200, Olympus, Japan). In the Transcriptome analysis, RNA sample concentration, quality, and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific). The library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA), and quantified using the Agilent high-sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was sequenced on NovaSeq 6000 platform (Illumina).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eH22 cells were cultured in RPMI-1640 culture medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin and maintained at 37 ℃ in a humidified environment containing 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003ch3\u003ePreparations of CP NPs and CMP NPs\u003c/h3\u003e\n\u003cp\u003eMetformin hydrochloride (2.5 mM) and dopamine hydrochloride (2.5 mM) were dissolved in 100 mL H\u003csub\u003e2\u003c/sub\u003eO, and then CuCl\u003csub\u003e2\u003c/sub\u003e (2.5 mM) was added into the solution under stirring for 0.5 h. PDA was formed in the alkaline solution, of which pH was adjusted by slowly adding NaOH solution (1 M) with continuous magnetic stirring for 12 h. During the formation of NPs, the mixture\u0026rsquo;s color changed from colorless to black. CMP NPs were obtained from the centrifugation (12000 rpm, 15 min) of the black suspension, followed by three times washing with deionized water. CP NPs or PDA were obtained by the same procedure only without metformin hydrochloride or both metformin hydrochloride and dopamine hydrochloride, respectively.\u003c/p\u003e\n\u003ch3\u003eThe photothermal effect of the NPs\u003c/h3\u003e\n\u003cp\u003eIn vitro photothermal effects of CP NPs and CMP NPs were studied. The NPs were dispersed in deionized water and then irradiated by a laser (808 nm). The temperature of the mixture was detected using a thermal imager.\u003c/p\u003e \u003cp\u003eIn vivo photothermal effect of CMP NPs was studied by a similar method. The female BALB/c mice were subcutaneously inoculated with H22 cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells, 0.1 mL), then CMP NPs were intratumorally injected with a single dose of 2 mg/kg after 5 days. The local temperature of the tumor site under the laser (808 nm, 1.0 W/cm\u003csup\u003e2\u003c/sup\u003e) irradiation was also detected using a thermal imager.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity study\u003c/h2\u003e \u003cp\u003eH22 cells were seeded in 96-well plates at a density of 6\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well and then incubated at 37 ℃ for 12 h. The NPs were added into the wells for continued 24 h incubation. In addition, the cells from the laser-treating groups were exposed to laser irradiation (808 nm, 2 W/cm\u003csup\u003e2\u003c/sup\u003e) for 5 min after 2 h of treatment. MTT reagent was added into each well with a final concentration of 0.5 mg/mL and allowed to incubate for 4 h. After the formazan formation, the supernatant was carefully removed and replaced by 100 \u0026micro;L of DMSO. Finally, the cell plates were vibrated for 5 min and the absorbance of each well was then recorded using a microplate reader at 570 nm.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vitro cell experiments\u003c/h3\u003e\n\u003cp\u003eH22 cells were seeded in 6-well plates at a density of 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/well and then incubated at 37 ℃ for 12 h. For other cell experiments, the cells were stimulated by three kinds of NPs or PBS for a set period, and the cells from the laser-treating group were exposed to laser irradiation (808 nm, 2.0 W/cm\u003csup\u003e2\u003c/sup\u003e) for 5 min after 2 h of treatment. Before the addition of experimental probes, the cells were collected by centrifugation (2000 rpm, 5 min) and washed with PBS twice.\u003c/p\u003e \u003cp\u003eCellular uptake of the NPs was verified using a high-content imaging system and flow cytometry. H22 cells were stained with DAPI and washed twice with PBS. The NPs were beforehand labeled with ICG by simply mixing up and stirring for 4 h.\u003c/p\u003e \u003cp\u003eThe cellular copper produced by the NPs in H22 cells was notarized using a high-content imaging system and flow cytometry utilizing rhodamine B hydrazide as a copper probe. The H22 cells were also stained with DAPI and then incubated with rhodamine B hydrazide (10 \u0026micro;M in 30% DMSO) for 30 min.\u003c/p\u003e \u003cp\u003eThe apoptosis of H22 cells was studied by staining with annexin Ⅴ- FITC and propidium iodide (PI). The relative levels of reactive oxygen species and mitochondrial membrane potential were evaluated by staining with DCFH-DA and JC-1, respectively.\u003c/p\u003e \u003cp\u003eThe levels of cellular ATP and GSH were detected using commercialized assay kits following the specifications. The protein content of treated H22 cells was tested by BCA.\u003c/p\u003e\n\u003ch3\u003eIn vivo anti-tumor efficacy of CP NPs and CMP NPs\u003c/h3\u003e\n\u003cp\u003eFemale BALB/c were subcutaneously inoculated with H22 cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells, 0.1 mL) to establish a tumor-bearing mouse model. Upon natural growth for 5 days, the mice were intratumorally injected with the NPs at a dose of 2 mg/kg of copper once every two days. After every interventional therapy, the laser (808 nm, 1.0 W/cm\u003csup\u003e2\u003c/sup\u003e) irradiation on the tumor site was performed for 5 min the next day. The body weight and tumor volume (calculated by the formula: 0.5\u0026times;length\u0026times;width^2) were measured and recorded once every two days.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomics analysis\u003c/h2\u003e \u003cp\u003eThe total RNA of the separated tumors was isolated using the Trizol Reagent. The mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First-strand cDNA was synthesized using random oligonucleotides and Super Script II. Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3\u0026prime; ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400\u0026ndash;500 bp in length, the library fragments were purified using the AMPure XP system. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15-cycle PCR reaction. Products were purified and quantified using the Agilent high-sensitivity DNA assay on a Bioanalyzer 2100 system. The sequencing library was then sequenced on the Illumina platform.\u003c/p\u003e \u003cp\u003eCutadapt (v1.15) software was used to filter the sequencing data to get high-quality sequences. The filtered reads were mapped to the reference genome using HISAT2 v2.0.5. HTSeq(0.9.1) statistics were used to compare the Read Count values on each gene as the original gene expression and then used FPKM to standardize the expression. Then, differential expression were analyzed by DESeq (1.30.0) with screened conditions as follows: expression difference multiple |log2FoldChange| \u0026gt; 1, significant P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The heat map was obtained using the Euclidean and Complete Linkage methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStudy on the immunological memory effect\u003c/h2\u003e \u003cp\u003eThe mice bearing subcutaneous tumors were treated with CMP NPs and photothermal therapy according to the anti-tumor efficacy study. Furthermore, the mice were deemed to recover completely if there was no macroscopic tumor for 10 days after the last treatment. The cured mice were reinoculated with H22 cells on the opposite side, whose body weight and tumor volume were also measured and recorded once every two days. The untreated mice were subcutaneously inoculated with 0.1 mL of PBS to parallel the first tumor inoculation and subsequent treatment of the above-treated mice. Once the second tumor inoculation was achieved, there was no intervention in the two groups of mice until the advent of execution, allowing the tumor to grow for 18 days naturally.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin-eosin staining and immunohistochemistry\u003c/h2\u003e \u003cp\u003eMain organs and tumors from the experimental mice were fixed in 4% paraformaldehyde (pH 7.4) for 24 h. After fixation, the tissues were dehydrated with ethanol and embedded in paraffin. Serial cross-sections from main organs were stained with hematoxylin and eosin (H\u0026amp;E). Besides, Sections of the tumors were incubated with PBS with 1% TritonX-100 and 10% FBS at room temperature for 90 min and further incubated with the antibodies (CD4, 1:500; CD8, 1:500; HMGB1, 1:200; FDX1, 1:100) at 4\u0026deg;C overnight, following a continued incubation with universal secondary antibody at room temperature for another 60 min. Tissue coloration with 3, 3-diaminobenzidine (DAB) was observed under an inverted microscope and blocked with a neutral resin. Image analysis of immunohistochemistry was performed using Image J Analyzer software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. and analyzed using GraphPad Prism V9.0 (GraphPad Software). Data were analyzed using a two-sided Student\u0026rsquo;s t-test when two groups were being compared. One-way analysis of variance (ANOVA) and Tukey\u0026rsquo;s test were used to evaluate statistical differences when more than two groups were compared. The difference was considered significant when the p-value was less than or equal to 0.05. * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ns, not significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e1. Characterization of CMP NPs\u003c/h2\u003e \u003cp\u003eThe obtained PDA, CP NPs, and CMP NPs are all black. CMP NPs contain 21.5% (w/w) metformin, and 13.8% (w/w) copper estimated by detecting the mass of unreacted metformin and ICP-MS, respectively. The shapes of CMP NPs containing main elements such as C, O, N, and Cu, were recognized as spherical particles by transmission electron microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The doping of copper and metformin could increase the size of PDA and lower its Zeta potential. The effective hydrodynamic diameter of CMP NPs was about 230 nm with an -8.5 mV of Zeta potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-d). There is an evident absorption peak (233nm) in the ultraviolet absorption spectrum of CMP NPs, which is also a characteristic peak belonging to metformin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). In the Fourier transform infrared spectroscopy (FTIR) spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), the different absorption peaks of CMP NPs compared to CP NPs were marked. In contrast to the chemical structure of dopamine, metformin has CN double bonds leading to the differences in the FTIR spectra at a range of ~\u0026thinsp;1650\u0026ndash;1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e between CMP NPs and CP NPs. The absorption peaks at 1190 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1069 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e observed in the FTIR spectra were also deemed to belong to the stretching vibrations of C-N, while the absorption peaks at 965 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 882 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e may belong to the out-of-plane bending vibration of N-H like that in the benzene ring, which indicated the formation of coordination bonds between adjacent C\u0026thinsp;=\u0026thinsp;NH in metformin and Cu. In addition, the differences in the FTIR spectra at the range of ~\u0026thinsp;1500\u0026ndash;1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (inferred to the stretching vibrations of C-O) and ~\u0026thinsp;3000\u0026ndash;3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (inferred to the stretching vibrations of O-H) between PDA and CP NPs all indicated the formation of coordination bonds between C-OH in PDA and Cu. The X-ray photoelectron spectroscopy (XPS) survey of CMP NPs certified the existence of Cu, N, and O elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg), meanwhile the atomic percent of N element in CMP NPs was raised by the doping of metformin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). These results demonstrated that CMP NPs were successfully fabricated as PDA particles containing copper and metformin through a simple one-pot method.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2. Photothermal efficacy of CMP NPs\u003c/h2\u003e \u003cp\u003eCMP NPs are designed to treat solid malignant tumors combined with photothermal therapy. Hence, we evaluated its photothermal efficiency by laser illuminating and thermal imaging. In vitro experiments, both CP NPs and CMP NPs showed favorable thermal efficiency, those temperatures of the suspensions raised more than 30 ℃ after irradiating with an 808 nm laser (1.0 W/cm\u003csup\u003e2\u003c/sup\u003e) for 5 min, which was proved to be repeatable without degradation. The doping of copper enhanced the photothermal property of PDA, whereas metformin almost does not influence the photothermal property of CP NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). The increased temperatures of the irradiating suspensions were determined by the concentration, laser exposure duration, and laser power, with a positive correlation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The photothermal efficiency of CMP NPs was also tested in vivo experiment. Upon local subdermal injection of CMP NPs, the 808 nm laser irradiation (1.0 W/cm\u003csup\u003e2\u003c/sup\u003e) elevated the local temperature to 55 ℃ after 3 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e), which has been verified to be effective for tumor photothermal therapy(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). CMP NPs exerted commendable photothermal efficacy in both in vitro and in vivo studies, which is expected to be applied well in tumor PTT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3. Pharmacological effects of CMP NPs on H22 cells\u003c/h2\u003e \u003cp\u003eTo exert anti-tumor efficacy, CMP NPs should be able to enter the tumor cells and generate intracellular copper. The cellular uptake of CMP NPs in H22 cells was investigated. As the results showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b, the fluorescence intensity of the NPs increased as the co-incubation time went by, suggesting a time-dependent cellular uptake process. Cuproptosis is induced by excessive intracellular copper accumulation, we continued to investigate the cellular copper of the H22 cells stained with rhodamine B hydrazide after being treated with the NPs. The result of flow cytometry showed that these NPs could produce cellular copper in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince CMP NPs were designed for tumor PTT, their cytotoxicity in H22 cancer cells was tested first. As Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea shows, the cytotoxicity of copper in H22 cells was evident, and PDA hardly affected the viability of H22 cells consistent with other studies(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). When treated with CMP NPs, the cell viabilities were slightly reduced compared with CP NPs. Under the NIR laser irradiation, however, CMP NPs reduced the cell viabilities more remarkably than CP NPs. This may result from the intracellular release of metformin triggered by laser irradiation since CP NPs had shown comparable photothermal efficiency to CMP NPs. The increased cellular copper produced by these NPs was supported by the decreased GSH levels in H22 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), which is considered to be easy to bind to copper ions(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), and reducing GSH could promote cuproptosis(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExcessive intracellular copper could promote the cellular production of ROS through dynamin-related protein 1 (Drp1)-mediated mitochondrial fission(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and can restrict the ATP energy supply(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Meanwhile, metformin also influences the mitochondria and induces ROS (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Here, we generally studied the abilities to produce ROS and ATP reduction of the NPs in H22 cells. The co-incubation with the NPs for 6 h notably increased the intracellular ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, e). The ATP levels of H22 cells were decreased by the NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). These results were consistent with another experiment showing the changes in mitochondrial membrane potential caused by the NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). It was obvious that there were more cells stained with green fluorescence and fewer cells stained with red fluorescence in the NP groups than in the PBS group, indicating mitochondrial degeneration and follow-up necrosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe above results implied an apoptosis or necrosis outcome of H22 cells caused by the co-incubation with the NPs. Hence, we utilized annexin V-FITC combined with PI to investigate the cell\u0026rsquo;s apoptosis or necrosis rate after treatment with the NPs for 3 h and 12 h. As the results shown by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), the annexin V-FITC positive percent of the treated groups was significantly higher than that of the PBS group after 3h and 12 h, demonstrating that the NPs could remarkably damage cancer cells to death. Noteworthily, both in the 3 h and 12 h period, the percent of classical apoptosis cells (stained positively by annexin V-FITC meanwhile negatively by propidium iodide, shown as Q3 quadrant in the plot) had no evident changes, which might result from the cell membrane destruction induced by excessive copper(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), remaindering that cuproptosis is different from apoptosis and other programmed cell deaths(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Above all, among most of the above results, CMP NPs performed better than CP NPs, which was further transcended by the addition of near-infrared laser irradiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4. In vivo anti-tumor efficacy of CMP NPs in H22 tumor-bearing mice model\u003c/h2\u003e \u003cp\u003eAfter verifying the remarkable killing effect of CMP NPs on H22 Cells, its anti-tumor efficacy in BALB/c mice bearing H22 cells was integrally studied. The experimental mice were divided into six groups and treated with different NPs as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea. Firstly, there was no significant body weight loss of mice in the groups treated with the NPs, except for the group treated with CMP NPs plus laser irradiation, which was supposed to result from their experienced pain caused by the photothermal effect. There was also no evident change in the major organs across all groups revealed by histopathological analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003ea). The results manifested that CMP NPs inhibited the tumor growth compared with saline and saline plus laser irradiation. Encouragingly, treatment with CMP NPs plus laser irradiation could further significantly inhibit tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-f), representing a successful therapeutic regimen combining cuproptosis with photothermal therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe TUNEL staining of the treated tumor tissues echoed the previous results (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). The immunohistochemistry staining results showed an increased expression of ferredoxin-1 (FDX1) and decreased expression of high-mobility group box 1 (HMGB1) in the tumors from the CMP NP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eb-d). FDX1 is the key regulator of copper ionophore\u0026ndash;induced cell death, which reduces Cu \u003csup\u003e(\u003c/sup\u003eⅡ\u003csup\u003e)\u003c/sup\u003e to more toxic Cu (Ⅰ). Knockout FDX1 could rescue cells from cuproptosis(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Therefore, increased expression of FDX1 may help CMP NPs exert their cytotoxicity in cancer cells. Besides, HMGB1 is associated with the hallmarks of cancer(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) and is considered to contribute to tumorigenesis(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). The decreased expression of HMGB1 by CMP NPs in this study suggested an outcome of immunogenic cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, the anti-tumor effect of CMP NPs in the H22 cells-bearing mice model was further verified by transcriptomics analysis. We found 676 differential expression genes of the tumors between the saline group and the CMP NPs group, where 666 differential expression genes were upregulated, and 10 differential expression genes were downregulated in the CMP NPs group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003ea, b). The differential expression genes were enriched into some sets of genes from Gene ontology (GO) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003ec) and pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003ed) using Gene Set Enrichment Analysis (GSEA) analysis. In GO, the expression of metabolism-related and immune-related genes was altered by CMP NPs. The expression genes involved in ribosome and mitochondrial were downregulated, and those engaged in pyroptosis and immune-related sets of genes, such as activation of the innate immune response, mast cell activation, positive regulation of interferon-alpha and interleukin-1 beta production, and so on, were upregulated. This result reemerged in KEGG pathways. More importantly, the pathways of the citrate cycle and oxidative phosphorylation were found to be downregulated in the CMP NPs group. In contrast, the nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway, and protein digestion and absorption(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) were upregulated. The alteration of the citrate cycle pathway confirmed the cuproptosis caused by CMP NPs(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Oxidative phosphorylation was regarded as a therapeutic target for cancer therapy(\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), which may be inhibited in this study by the released metformin from CMP NPs(\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). The NOD-like receptor is the key component of the host innate immune system(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), its up-regulation suggested an immune activation induced by CMP NPs. In brief, CMP NPs had successfully exerted anti-tumor efficacy through cuproptosis and antimetabolic effect, and consequently induced immunogenic cell death to activate immune responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e5. In-situ tumor vaccine-like function generates immune memory effect in the tumor-reinoculated mice mode\u003c/h2\u003e \u003cp\u003eCMP NPs combined with laser irradiation exerted an outstanding anti-tumor efficacy in our preceding study, which even cured several tumor-bearing mice, we continued to probe the in-situ tumor vaccine-like function induced by the immunogenic cell death, which was triggered by CMP NPs plus laser irradiation and further generated immune memory effect in those cured mice through a tumor rechallenging model illustrated as Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003ea. The results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003eb demonstrated a body weight recovery in the mice cured by CMP NPs plus laser irradiation. More importantly, the growth of reinoculated tumors in the mice recovered from earlier combined therapy was significantly inhibited. By contrast, the tumors in the mice without any treatment had grown unrestrainedly (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003ec-e). The more infiltrated CD4\u003csup\u003e+\u003c/sup\u003e T and CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes in the tumors from treated mice were observed in the immunohistochemistry staining results (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ef-h). The organ index (expressed by organ weight/body weight) of the main organs from the mice showed an in vivo safety of CMP NPs plus laser irradiation. In summary, CMP NPs combined with PTT could inhibit tumor growth, lead the cancer cells to immunogenic cell death, followed by in-situ tumor vaccine-like function, and eventually generate an immune memory effect to restrain the tumor regrowth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we have provided polydopamine nanoparticles \u0026ndash; CMP NPs doped with copper and metformin for tumor photothermal therapy. CMP NPs could act as a nanocarrier for copper uptake to induce cuproptosis and for metformin to restrain the tumor metabolism and perform as a photothermal agent applied to PTT. The carried metformin and copper affect the mitochondria of cancer cells and produce cellular ROS, along with the reduction of ATP and GSH. In the animal model, CMP NPs effectively inhibited tumor growth combined with PTT. Moreover, CMP NPs plus NIR laser irradiation could induce an in-situ tumor vaccine-like function by immunogenic cell death to restrain tumor regrowth.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed under institutionally approved protocols (approval no. A2023207-001) by the Institutional Animal Care and Use Committee (IACUC), Shanghai Jiao Tong University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have approved the manuscript and agree with the submission and publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReaders who need data or materials from this article could contact the corresponding authors by e-mail.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (22071148) and Fundamental Research Funds for the Central Universities (2232023A-07) to Jiefeng Shen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYangwen Luo prepared the nanoparticles, performed in vitro and in vivo experiments, analyzed the data, and wrote the manuscript; Wenkai Zhang, Muge Gu, Xiangqi Zhang, Wei Yu, and Jiayu Wang offered advice and assistance in the experiments; Weien Yuan, Jiefeng Shen, Guoqiang Yang, and Hua Yang\u003csup\u003e\u0026nbsp;\u003c/sup\u003edesigned this project, commented on the experiments and draft writing, and supervised the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Shanghai Bioprofile for assistance in the transcriptomics analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003eDepartment of Hepatic Oncology, Shanghai Geriatric Medical Center, Shanghai 201104, P. R. China; Department of Liver Surgery and Transplantation, Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai 200032, P. R. China\u003c/p\u003e\n\u003cp\u003eHua Yang\u003c/p\u003e\n\u003cp\u003eFrontiers Science Center for Transformative Molecules, School of Chemistry and Chemical Engineering, Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, P. R. China\u003c/p\u003e\n\u003cp\u003eYangwen Luo \u0026amp; Guoqiang Yang\u003c/p\u003e\n\u003cp\u003eCollege of Chemistry and Chemical Engineering, Donghua University, Shanghai 201620, P. R. China\u003c/p\u003e\n\u003cp\u003eJiefeng Shen\u003c/p\u003e\n\u003cp\u003eEngineering Research Center of Cell \u0026amp; Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, P. R. China\u003c/p\u003e\n\u003cp\u003eWenkai Zhang, Muge Gu, Xiangqi Zhang, Jiayu Wang, Wei Yu \u0026amp; Weien Yuan\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians; 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Wang Y, Zhao L, Stenzel MH, Jiang Y. Metal ion interference therapy: metal-based nanomaterial-mediated mechanisms and strategies to boost intracellular ion overload for cancer treatment. Mater Horiz. 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYadav AA, Patel D, Wu X, Hasinoff BB. Molecular mechanisms of the biological activity of the anticancer drug elesclomol and its complexes with Cu(II), Ni(II) and Pt(II). 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Int J Biol Macromol. 2020.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"nanoparticles, cuproptosis, anti-tumor, photothermal therapy, immunological memory ","lastPublishedDoi":"10.21203/rs.3.rs-5223435/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5223435/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe anti-tumor potentials of copper and metformin have been wildly studied. Excessive intracellular copper could lead cancer cells to cuproptosis, while metformin could activate adenosine monophosphate-activated protein kinase (AMPK) to decrease ATP and generate reactive oxygen species (ROS). This study integrated copper and metformin ably to form a polydopamine nanoparticle (CMP NPs) through coordination bonds. CMP NPs showed a high photothermal efficiency and anti-tumor efficacy both in vitro and in vivo experiments. This effective photothermal therapy (PTT) not only inhibited the growth of primary tumors but also generated an in-situ tumor vaccine-like function by immunogenic cell death to restrain the regrowth of the secondary tumors, showing a promising PTT for malignant solid tumors.\u003c/p\u003e","manuscriptTitle":"Copper and metformin double-doped polydopamine nanoparticles for tumor photothermal therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-16 10:28:44","doi":"10.21203/rs.3.rs-5223435/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"edeaba61-07aa-4d00-88db-8e8d8de2185f","owner":[],"postedDate":"October 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-10T13:39:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-16 10:28:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5223435","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5223435","identity":"rs-5223435","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}