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

doi:10.21203/rs.3.rs-5223435/v1

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

https://doi.org/10.21203/rs.3.rs-5223435/v1

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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

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

In the preparation of the NPs, CuCl₂ (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% CO₂.

Preparations of CP NPs and CMP NPs

Metformin hydrochloride (2.5 mM) and dopamine hydrochloride (2.5 mM) were dissolved in 100 mL H₂O, and then CuCl₂ (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 (2×10⁵ 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/cm²) irradiation was also detected using a thermal imager.

Cytotoxicity study

H22 cells were seeded in 96-well plates at a density of 6×10³ 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/cm²) 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 1×10⁶ 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/cm²) 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 (2×10⁵ 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/cm²) 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.

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. 1a). 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. 1b-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. 1d). In the Fourier transform infrared spectroscopy (FTIR) spectra (Fig. 1e), 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. 1g), meanwhile the atomic percent of N element in CMP NPs was raised by the doping of metformin (Fig. 1h). 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/cm²) 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. 2a, b). The increased temperatures of the irradiating suspensions were determined by the concentration, laser exposure duration, and laser power, with a positive correlation (Fig. 2c). 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/cm²) elevated the local temperature to 55 ℃ after 3 min (Fig. 2d, 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. 3a-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. 3c, d).

Since CMP NPs were designed for tumor PTT, their cytotoxicity in H22 cancer cells was tested first. As Fig. 4a 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. 4b), 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. 4d, e). The ATP levels of H22 cells were decreased by the NPs (Fig. 4c). 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. 7a. 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. 10a). 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. 7b-f), representing a successful therapeutic regimen combining cuproptosis with photothermal therapy.

The TUNEL staining of the treated tumor tissues echoed the previous results (Fig. 8a). 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. 8b-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. 9a, b). The differential expression genes were enriched into some sets of genes from Gene ontology (GO) (Fig. 9c) and pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Fig. 9d) 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. 10a. The results shown in Fig. 9b 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. 10c-e). The more infiltrated CD4⁺ T and CD8⁺ T lymphocytes in the tumors from treated mice were observed in the immunohistochemistry staining results (Fig. 11f-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.

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.

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 Yangdesigned 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. China

Wenkai Zhang, Muge Gu, Xiangqi Zhang, Jiayu Wang, Wei Yu & Weien Yuan

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No competing interests reported.

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

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Materials

Instruments

Cell culture

Preparations of CP NPs and CMP NPs

The photothermal effect of the NPs

Cytotoxicity study

In vitro cell experiments

In vivo anti-tumor efficacy of CP NPs and CMP NPs

Transcriptomics analysis

Study on the immunological memory effect

Hematoxylin-eosin staining and immunohistochemistry

Statistical analysis

Results and Discussion

1. Characterization of CMP NPs

2. Photothermal efficacy of CMP NPs

3. Pharmacological effects of CMP NPs on H22 cells

4. In vivo anti-tumor efficacy of CMP NPs in H22 tumor-bearing mice model

5. In-situ tumor vaccine-like function generates immune memory effect in the tumor-reinoculated mice mode

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1