Chemicals and reagents
N, N-dimethylformamide, dithiodiglycolic acid, copric chloride dihydrate, triethylamine, indocyanine green (ICG), omeprazole, 3,3’,5,5’-tetramethylbenzidine (TMB), methylene blue (MB), 1,2-diaminobenzene (OPD), and triethylamine (TEA) were bought form Shanghai Aladdin Biochemical Technology Co., LTD. Ethanol was acquired from China National Pharmaceutical Group Corporation. Polyvinyl pyrrolidone (PVP, molecular weight = 40000) was purchased from Sigma-Aldrich. DSPE-mPEG (molecular weight = 2000) was provided by Shanghai Yare Co., LTD. Rhodamine B hydrazide (RBH) was bought form Biofount Technology Co., LTD. Dulbecco’s modified eagle medium (DMEM) and phosphate buffer solution (PBS) were obtained from Service Biotechnology Co., LTD. Fetal bovine serum (FBS) was purchased from Hyclone. Ru(dpp)3Cl2, monobromobimane, and D-luciferin potassium salt were obtained from Macklin Biochemical Technology Co., LTD. BBoxiProbe O26 was obtained from Bestbio Technology Co., LTD. 5, 5’-dithio-bis (2-nitrobenzoic acid) (DTNB), 4′,6-diamidino-2-phenylindole (DAPI), and anti-HSP70 (Cat# AF1156) were obtained from Beyotime Biotechnology. HRP-conjugated affinipure goat anti-mouse IgG (H + L) (Cat# 15014), HRP-conjugated affinipure goat anti-Rabbit IgG(H + L) (Cat# 15015), anti-β-actin (Cat# 66009-1-Ig), anti-DLAT (Cat# 13426-1-AP), and anti-FDX1 (Cat# 12592-1-AP) were gained from Proteintech. ATP7A (Cat# E-AB-13081) antibodies were obtained from Elabscience Biotechnology Co., LTD. Annexin V-FITC/PI apoptosis kit and CCK8 kit were obtained from Yeasen Biotechnology Co., LTD. Anti-mouse PD-1 (Cat# BE0273-100MG) was acquired from Univ Company. India ink was bought from Shanghai Shifeng Biotechnology Co., LTD. All chemicals were used without further purification. All antibodies used in flow cytometry are listed in Table S1.
Synthesis of CussNV
To begin, 300 mg of PVP was dissolved in 3 mL of DMF and subjected to ultrasonic agitation for 5 min. Next, 5.2 mg of 2,2’-disulfanediyldiacetic acid and 10 mg of CuCl₂·2 H₂O were sequentially added to the solution, which was stirred for an additional 10 min. Subsequently, 0.6 mL of TEA was rapidly introduced under vigorous stirring, followed by the swift addition of a mixture of 6.25 mL of DMF and 3.75 mL of ethanol. The resulting solution was transferred into a hydrothermal synthesis reactor preheated to 150 °C and maintained at this temperature for 12 h. After cooling to room temperature, the products were collected by centrifugation at 10,000 rpm for 10 min (Sorvall ST 16R centrifuge, Thermo Fisher) and washed three times with ultrapure water.
Synthesis of pegylated CussOMEp
To synthesize CussOMEp, 400 µg of CussNV was dispersed in 1 mL of ultrapure water containing 0.4 µmol of OME and gently stirred overnight to obtain CussOME. The resulting CussOME nanoparticles were collected by centrifugation at 10,000 rpm for 10 min and washed three times with ultrapure water. For PEGylation, 200 µg of DSPE-mPEG was added to the CussOME solution and allowed to react for 6 h. The PEGylated nanoparticles (CussOMEp) were then collected by centrifugation at 10,000 rpm for 10 min and washed three times with ultrapure water.
Characterization of Cussomep
Transmission electron microscopy (TEM) images were carried out on a Hitachi HT7700 transmission electron microscope. Atomic force microscope (AFM) images were conducted on an atomic force microscope (BRUKER Dimension Icon). X-ray diffraction (XRD) was conducted by a Rigaku Miniflex600 X-ray diffractometer. BET results were obtained from ASAP2020 of micromeritics (Version 4.03.). X-ray photoelectron spectroscopy (XPS) spectra were recorded by an X-ray photoelectron spectrometer (Thermo SCIENTIFIC Nexsa, Thermo Fisher). The mapping images were acquired form a transmission electron microscope (JEM 2100 F, JEOL). The zeta potential and hydrodynamic size were measured by a particle and molecular charge analyzer (Zetasizer Nano ZS, Malvern). FTIR spectra were recorded by a Fourier transform infrared spectrometer (Thermo Nicolet IS 50, Thermo Fisher).
Degradation behaviors of cussomep
CussNV nanoparticles were dissolved in PBS at 6.0 and 7.4 pH with or without 10 mM GSH for 10 h, the morphology of CussNV was then characterized using a TEM.
Release behaviors of OME and copper ions from CussOMEp
10 mg of CussOMEp were dispersed in PBS solutions at pH 7.4 and pH 6.0 with or without 10 mM GSH. At specified time points, the released OME and cupric in the supernatant were collected by centrifugation at 10,000 rpm for 20 min and the copper contents and OME were quantified using atomic absorption spectrometry and UV-visible spectrophotometer (UV-8000 S, Shanghai Metash Instruments Co., Ltd.), respectively. The absorption peak intensity at 278 nm of OME was used to determine the standard curve.
Dissolved oxygen determination
A solution containing 50 µg/mL CussNV and 10 mM H₂O₂ was prepared, and its oxygen content was measured using a dissolved oxygen analyzer (JPB607A, Leici).
•OH generation ability of CussNV
To evaluate the •OH generation capacity of CussNV, a 50 µg/mL solution of CussNV was dispersed in 1 mL of PBS and treated with 0.5 mM TMB, 50 µM MB, or 20 mM OPD, followed by the addition of 100 µM H₂O₂. The mixture was incubated for 30 min, after which the absorbance of the resulting oxidation products was measured using a UV-visible spectrophotometer and a microplate reader (1510, Thermo Fisher). Additionally, electron spin resonance (ESR) spectroscopy was performed to verify the generation of hydroxyl radicals (•OH). Briefly, 50 mM H₂O₂ and 100 µg/mL CussNV were mixed in a buffer at pH 6.0 for 15 min. Subsequently, 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added, and the ESR spectra were recorded to detect •OH production. A sample without CussNV served as the control group.
For the analysis of POD-like activity, the 250 µg/mL CussNV were thoroughly combined with TMB solution, followed by the addition of H2O2 at concentrations of 2.5 mM, 5 mM, 10 mM, 15 mM, and 20 mM. The absorbance at 655 nm was measured within 30 min to determine the kinetic parameters of the activity of the POD-like enzyme.
GSH depletion ability of CussNV
The GSH depletion capacity of CussNV was assessed using the DTNB reagent. A solution of PBS containing 10 mM GSH was incubated with varying concentrations of CussOMEp at 37 °C for 12 h. Following incubation, the mixture was reacted with DTNB for 4 h. The ultraviolet absorbance of the above solution at 412 nm was then measured using a UV-visible spectrophotometer.
For assessment the GSHox-like activity, CussNV at a concentration of 250 µg/mL was incubated with GSH at concentrations of 0.1 mM, 0.75 mM, 1.25 mM, 2 mM, and 2.5 mM. Following incubation, the mixture was centrifuged, and the resulting supernatant underwent a colorimetric reaction with DTNB. The absorbance at 412 nm was subsequently measured within 1 h to determine the kinetic parameters of nanozyme.
Cell culture
Luc-4T1 cells were generated by lentiviral transfection of the luciferase reporter gene into 4T1 cells. Both 4T1 and Luc-4T1 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, maintained at 37 °C in a 5% CO₂ atmosphere, and routinely sub-cultured.
Cell uptake
The FITC labeled CussNVp (CussNVp@FITC) was synthesized for cellular uptake investigation. Briefly, 50 µg 4T1 cells were incubated with 50 µg FITC for 12 h, then the mixture was centrifuge for several times to obtain CussNVp@FITC. After that, 4T1 cells were seeded into a 6-well plate at a density of 1.0 × 105 cells per well and cultured for 24 h. Subsequently, the cells were treated with CussNVp@FITC at a concentration of 50 µg/mL for various time intervals. After treatment, the presence of green fluorescence signals within the cells were assessed using flow cytometry (Novocyte 3130, ACEA).
Cytotoxicity evaluation
4T1 cells were seeded into 96-well plates at a density of 1.0 × 10⁴ cells per well and cultured for 24 h. Cells were then treated with varying concentrations of OME (0, 1, 2, 5, 10, 20, 50, and 100 µM), CussNVp (0, 20, 40, 60, 80, 100, 150, and 200 µg/mL), or CussOMEp (0, 20, 40, 60, 80, 100, 150, and 200 µg/mL) for an additional 24 h. After treatment, the media were replaced with fresh medium containing 10 µL of CCK-8 solution per well. Cell viability was determined by measuring absorbance at 450 nm.
Intracellular Cu2+ detection
4T1 cells (1 × 10⁶) were seeded into confocal dishes and allowed to adhere. After 24 h of treatment with various probes, the cells were incubated with a Cu²⁺-specific fluorescence probe (RBH, 1 µM) at 37 °C for 30 min, followed by fixation with 4% paraformaldehyde for 15 min. The cells were then stained with DAPI for 10 min. Fluorescence imaging was conducted using a confocal laser scanning microscopy (CLSM, FV3000, Olympus), with excitation at 510 nm and emission at 578 nm for the Cu²⁺ probe, and excitation at 360 nm and emission at 460 nm for DAPI. In addition, the intracellular Cu2+ concentration was quantified by using the Cu²⁺ fluorescence probe and recording using a fluorescence spectrophotometer (F-7100, Hitachi) with excitation at 510 nm and emission at 578 nm. The statistical results of fluorescent images were counted using the Image J software.
Intracellular distribution of ATP7A
4T1 cells (1 × 106) were seeded into confocal dishes and incubated with OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL) for 24 h. The cells were then fixed in 4% paraformaldehyde for 15 min and blocked with 5% BSA for 2 h. Next, the cells were incubated overnight with ATP7A antibody (1: 200), followed by a 1-hour incubation with goat anti-rabbit IgG(H + L) conjugated to AF488 at 4 °C. After staining DAPI for 10 min, the fluorescence intensity of the cell samples was imaged using a CLSM. Fluorescence excitation and emission wavelengths were set at 488 nm and emission at 520 nm for the AF488-conjugated antibody, and at 360 nm and 460 nm for DAPI, respectively.
Intracellular oxygen, •OH and liperfluo evaluation
4T1 cells (1 × 10⁶) were seeded into confocal dishes and incubated for 24 h to allow for adherence. After that, cell samples were incubated with OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL) for 24 h. Then the cells were then treated with either 50 µM of Ru(ddp)₃Cl₂, a fluorescent oxygen probe, at 37 °C for 2 h, or a 1: 1000 dilution of BBoxiProbe O26, a •OH fluorescent probe, at 37 °C for 30 min or 10 µg/mL liperfluo, a ferroptosis fluorescent probe, at 37 ℃ for 30 min. After treatments, cells were fixed with 4% paraformaldehyde at 4 °C for 15 min. Following fixation, the cells were stained with DAPI for 10 min and imaged using a CLSM. The imaging parameters were set as follows: excitation at 488 nm and emission at 510 nm for both Ru(ddp)₃Cl₂, BBoxiProbe O26, and liperfluo, and excitation at 360 nm and emission at 460 nm for DAPI.
Intracellular GSH detection
Nonfluorescent bromobimane can be converted into fluorescent compounds in the presence of small thiols, such as GSH. To assess intracellular GSH levels, 4T1 cells were received with indicated treatments and stained with 100 µM bromobimane at 37 °C for 30 min. Blue fluorescence was detected using a CLSM, with excitation at 392 nm and emission at 478 nm, to visualize GSH levels.
Western blot analysis
4T1 cells were seeded into a 6-well plate at a density of 1.0 × 10⁵ cells per well and cultured for 24 h. The cells were then treated with OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL) for 24 h. After treatment, the cells were harvested, washed with ice-cold PBS, and lysed. β-actin served as the loading control, detected using anti-actin antibodies, while the expression levels of GPX4, HSP70, DLAT, and FDX1 were assessed using their respective antibodies (dilution 1: 1000). Membranes were then incubated with secondary antibodies against rabbit IgG or mouse IgG (dilution 1: 5000) and imaged using a multifunctional fluorescent and luminescent gel imager (Cheml XRQ, Gene Company Limited).
Apoptosis analysis
4T1 cells (1.0 × 10⁶) were seeded into 6-well plates and incubated for 24 h in the presence of OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL). After treatment, the cells were stained with 5 µL Annexin V-FITC and 5 µL propidium iodide (PI), followed by fluorescence signal analysis using a FCM.
Hemolysis assay
All animal experiments were conducted in accordance with protocols approved by the Animal Experimental Ethics Committee of Fujian Normal University (Approval No. IACUC-20230036). Female Balb/c mice (4–6 weeks old) were obtained from Shanghai Slack Laboratory Animal Co., LTD. Blood samples were collected via orbital puncture and placed in anticoagulant tubes. Red blood cells were isolated by centrifugation at 3000 rpm for 5 min and diluted to a final concentration of 2%. Various concentrations of CussNVp and CussOMEp were incubated with the red blood cells for 2 h at 37 °C. Phosphate-buffered saline (PBS) and 0.5% Triton X-100 were used as negative and positive controls, respectively. Hemolysis was quantified by measuring the absorbance at 576 nm, and hemolysis rates were calculated using the following formula:
Hemolysis (%) = (OD576sample – OD576N) / (OD576P – OD576N) × 100%.
Biosafety evaluation
Balb/c mice (4–6 weeks old) were randomly assigned to two groups (n = 6 per group) and intravenously administered either PBS or 300 µg of CussOMEp per mouse. Body weights were measured every five days for 90 days. Blood and serum samples were collected to assess key physiological parameters. Major organs were harvested, weighed, and subjected to H&E staining for morphological analysis.
In vivo biodistribution assay
ICG-labeled CussNVp (denoted as CussICGp) was synthesized for in vivo imaging. In brief, 100 µg of ICG was mixed with 100 µg of CussNVp and incubated overnight. The unbound ICG was then removed by centrifuge, and the loading rate of ICG was determined using UV-visible spectrophotometry. Six 4T1 tumor-bearing mice were randomly divided into two groups: (1) Free ICG, and (2) CussICGp. Each group of mice received a 100 µg dosage of ICG via tail vein injection. Fluorescence imaging was conducted using an in vivo imaging system (IVIS) at various times (0, 0.5, 2, 4, 8, 12, 24, and 48 h). At 48 h post-injection, the mice were euthanized, and their tumor tissues and major organs (heart, liver, spleen, lung, and kidney) were collected for ex vivo imaging. Furthermore, to quantify the tumor-targeting capacity of CussNVp, twelve 4T1 tumor-bearing mice were administered either PBS, 0.054 µmol of OME, 300 µg of CussNVp, or 300 µg of CussOMEp intravenously injection, and the mice were euthanasia at 48 h post-injection. The tumor tissues were collected, and the copper contents were assessed using the fluorescent probe.
In vivo synergistic antitumor efficacy
The 4T1 tumor model was established by subcutaneously injecting 1 × 10⁶ 4T1 cells into the right flank of healthy female Balb/c mice. Once tumors reached a volume of 100 mm³, the mice were randomly assigned to four groups (n = 4) and intravenously administered one of the following treatments: PBS, 0.054 µmol OME per mouse, 300 µg CussNVp per mouse, or 300 µg CussOMEp per mouse. Tumor volumes and body weights were monitored every two days.
On day 14, mice were euthanasia, the tumor tissues and tumor-draining lymph nodes were collected. Tumor tissues were subjected to H&E staining, as well as IHC analysis using anti-HIF-1α, anti-GPX4, and anti-FDX1 antibodies. Besides, single-cell suspension of tumor tissues was prepared for immune profiling. Macrophage polarization was evaluated using anti-CD45-PE/Cy5, anti-CD11b-APC/Cy7, anti-F4/80-PE/CF594, and anti-CD206-Alexa Fluor 647. Cytotoxic T lymphocytes and helper T cells were analyzed by staining with anti-CD45-PE, anti-CD3-PE/Cy7, anti-CD4-APC, and anti-CD8a-PerCPCy5.5. Neutrophil infiltration was assessed with anti-CD11b-APC/Cy7, anti-F4/80-PE/CF594, and anti-Ly6G-PE, while B cell infiltration was measured using anti-B220-FITC. In addition, the single-cell suspension of tumor-draining lymph nodes were prepared for DC maturation analysis. Single-cell suspensions were prepared, and cells were stained with anti-CD11c-BV421, anti-CD80-FITC, and anti-CD86-PE antibodies at 4 °C for 30 min, followed by flow cytometric analysis.
In vivo anti-abscopal activity
Primary tumors were established by subcutaneously injecting 1 × 106 4T1 cells into the right flank of healthy female Balb/c mice. The tumor-bearing mice were then randomly divided into four groups (n = 8) and administered either PBS, 0.054 µmol of OME, 300 µg of CussNVp, or 300 µg of CussOMEp intravenously on day 0. Additionally, mice received αPD-1 (1 mg/kg) treatment on days 2, 5, and 8. On day 9, 2 × 105 4T1 cells were injected subcutaneously into the left flank to establish abscopal tumors. Tumor volumes and body weights were recorded every other day for up to 34 days.
On day 34, mice were euthanasia, and the lymph nodes, tumors, and spleens were collected to make single-cell suspensions for immune response analysis. For DC maturation assessment, lymph node single-cell suspensions were stained with anti-CD11c-BV421, anti-CD80-FITC, and anti-CD86-PE at 4 °C for 30 min. For tumor single-cell suspensions analysis, macrophage polarization was evaluated by staining with anti-CD45-PE/Cy5, anti-CD11b-APC/Cy7, anti-F4/80-PE/CF594, anti-CD86-APC, and anti-CD206-FITC. Cytotoxic T lymphocyte and helper T cell populations were examined by staining with anti-CD3-PE/Cy7, anti-CD4-BV421, and anti-CD8a-APC/Cy7. Tumor cells were fixed and permeabilized using the BD Pharmingen™ Transcription Factor Buffer Set for 45 min prior to additional staining. After treating with brefeldin A for 6 h before staining with anti-IFNγ-APC. Regulatory T cell inhibition was evaluated using anti-Foxp3-PE following centrifugation and analyzed by flow cytometry. Memory T cells were analyzed by staining spleen cell suspensions with anti-CD3-APC, anti-CD8a-PerCP/Cy5.5, anti-CD44-FITC, and anti-CD62L-BV650.
In vivo anti-metastatic activity
To establish primary tumors, 1 × 106 4T1 cells were subcutaneously injected into the right flank of Balb/c mice. Once the tumor volume reached approximately 100 mm³, the mice were intravenously administered PBS, 0.054 µmol of OME, 300 µg of CussNVp, or 300 µg of CussOMEp (n = 8). Subsequently, αPD-1 antibody (1 mg/kg) was administered intravenously on days 2, 5, and 8. On day 9, the mice received an intravenous injection of 2 × 105 Luc-4T1 cells to induce lung metastasis. For bioluminescence imaging, D-luciferin potassium salt (150 mg/kg) was intraperitoneally injected on days 21, 25, and 30. Imaging was performed using an IVIS Spectrum (PerkinElmer) under anesthesia 10 min after the D-luciferin injection. Tumor volumes and body weights were recorded bi-daily for up to 38 days. To assess lung metastasis, lung tissues were filled with India ink and subsequently stained with H&E.
Density functional theory simulation
The initial structure of the nanocluster was obtained through conformational search with the CREST program. After obtaining the wavefunctions based on B3LYP functional and def2-SVP basis in the Gaussian 16 program (Rev A.03), the weak interaction analysis was performed using the interaction region indicator (IRI) in the Multiwfn program [48, 49].
Statistical analysis
Quantitative data are expressed as mean ± standard deviation. Statistical analyses were performed using one-way ANOVA with GraphPad Prism 8.0 software. Statistical significance is indicated by an asterisk, with the following designations: ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. A P value of 0.05 or less was considered statistically significant.
