Chemical substances and reagents
N, N-dimethylformamide, dithiodiglycolic acid, copric chloride dihydrate, triethylamine, indocyanine inexperienced (ICG), omeprazole, 3,3’,5,5’-tetramethylbenzidine (TMB), methylene blue (MB), 1,2-diaminobenzene (OPD), and triethylamine (TEA) have been purchased kind Shanghai Aladdin Biochemical Know-how Co., LTD. Ethanol was acquired from China Nationwide Pharmaceutical Group Company. Polyvinyl pyrrolidone (PVP, molecular weight = 40000) was bought from Sigma-Aldrich. DSPE-mPEG (molecular weight = 2000) was supplied by Shanghai Yare Co., LTD. Rhodamine B hydrazide (RBH) was purchased kind Biofount Know-how Co., LTD. Dulbecco’s modified eagle medium (DMEM) and phosphate buffer resolution (PBS) have been obtained from Service Biotechnology Co., LTD. Fetal bovine serum (FBS) was bought from Hyclone. Ru(dpp)3Cl2, monobromobimane, and D-luciferin potassium salt have been obtained from Macklin Biochemical Know-how Co., LTD. BBoxiProbe O26 was obtained from Bestbio Know-how Co., LTD. 5, 5’-dithio-bis (2-nitrobenzoic acid) (DTNB), 4′,6-diamidino-2-phenylindole (DAPI), and anti-HSP70 (Cat# AF1156) have been 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) have been gained from Proteintech. ATP7A (Cat# E-AB-13081) antibodies have been obtained from Elabscience Biotechnology Co., LTD. Annexin V-FITC/PI apoptosis equipment and CCK8 equipment have been obtained from Yeasen Biotechnology Co., LTD. Anti-mouse PD-1 (Cat# BE0273-100MG) was acquired from Univ Firm. India ink was purchased from Shanghai Shifeng Biotechnology Co., LTD. All chemical compounds have been used with out additional purification. All antibodies utilized in circulate cytometry are listed in Desk S1.
Synthesis of CussNV
To start, 300 mg of PVP was dissolved in 3 mL of DMF and subjected to ultrasonic agitation for five min. Subsequent, 5.2 mg of two,2’-disulfanediyldiacetic acid and 10 mg of CuCl₂·2 H₂O have been sequentially added to the answer, which was stirred for an extra 10 min. Subsequently, 0.6 mL of TEA was quickly launched below vigorous stirring, adopted by the swift addition of a combination of 6.25 mL of DMF and three.75 mL of ethanol. The ensuing resolution was transferred right into a hydrothermal synthesis reactor preheated to 150 °C and maintained at this temperature for 12 h. After cooling to room temperature, the merchandise have been collected by centrifugation at 10,000 rpm for 10 min (Sorvall ST 16R centrifuge, Thermo Fisher) and washed 3 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 in a single day to acquire CussOME. The ensuing CussOME nanoparticles have been collected by centrifugation at 10,000 rpm for 10 min and washed 3 times with ultrapure water. For PEGylation, 200 µg of DSPE-mPEG was added to the CussOME resolution and allowed to react for six h. The PEGylated nanoparticles (CussOMEp) have been then collected by centrifugation at 10,000 rpm for 10 min and washed 3 times with ultrapure water.
Characterization of Cussomep
Transmission electron microscopy (TEM) photographs have been carried out on a Hitachi HT7700 transmission electron microscope. Atomic pressure microscope (AFM) photographs have been carried out on an atomic pressure microscope (BRUKER Dimension Icon). X-ray diffraction (XRD) was carried out by a Rigaku Miniflex600 X-ray diffractometer. BET outcomes have been obtained from ASAP2020 of micromeritics (Model 4.03.). X-ray photoelectron spectroscopy (XPS) spectra have been recorded by an X-ray photoelectron spectrometer (Thermo SCIENTIFIC Nexsa, Thermo Fisher). The mapping photographs have been acquired kind a transmission electron microscope (JEM 2100 F, JEOL). The zeta potential and hydrodynamic dimension have been measured by a particle and molecular cost analyzer (Zetasizer Nano ZS, Malvern). FTIR spectra have been recorded by a Fourier rework infrared spectrometer (Thermo Nicolet IS 50, Thermo Fisher).
Degradation behaviors of cussomep
CussNV nanoparticles have been dissolved in PBS at 6.0 and seven.4 pH with or with out 10 mM GSH for 10 h, the morphology of CussNV was then characterised utilizing a TEM.
Launch behaviors of OME and copper ions from CussOMEp
10 mg of CussOMEp have been dispersed in PBS options at pH 7.4 and pH 6.0 with or with out 10 mM GSH. At specified time factors, the launched OME and cupric within the supernatant have been collected by centrifugation at 10,000 rpm for 20 min and the copper contents and OME have been quantified utilizing atomic absorption spectrometry and UV-visible spectrophotometer (UV-8000 S, Shanghai Metash Devices Co., Ltd.), respectively. The absorption peak depth at 278 nm of OME was used to find out the usual curve.
Dissolved oxygen willpower
An answer containing 50 µg/mL CussNV and 10 mM H₂O₂ was ready, and its oxygen content material was measured utilizing a dissolved oxygen analyzer (JPB607A, Leici).
•OH era means of CussNV
To judge the •OH era capability of CussNV, a 50 µg/mL resolution of CussNV was dispersed in 1 mL of PBS and handled with 0.5 mM TMB, 50 µM MB, or 20 mM OPD, adopted by the addition of 100 µM H₂O₂. The combination was incubated for 30 min, after which the absorbance of the ensuing oxidation merchandise was measured utilizing a UV-visible spectrophotometer and a microplate reader (1510, Thermo Fisher). Moreover, electron spin resonance (ESR) spectroscopy was carried out to confirm the era of hydroxyl radicals (•OH). Briefly, 50 mM H₂O₂ and 100 µg/mL CussNV have been blended 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 have been recorded to detect •OH manufacturing. A pattern with out CussNV served because the management group.
For the evaluation of POD-like exercise, the 250 µg/mL CussNV have been totally mixed with TMB resolution, adopted by the addition of H2O2 at concentrations of two.5 mM, 5 mM, 10 mM, 15 mM, and 20 mM. The absorbance at 655 nm was measured inside 30 min to find out the kinetic parameters of the exercise of the POD-like enzyme.
GSH depletion means of CussNV
The GSH depletion capability of CussNV was assessed utilizing the DTNB reagent. An answer of PBS containing 10 mM GSH was incubated with various concentrations of CussOMEp at 37 °C for 12 h. Following incubation, the combination was reacted with DTNB for 4 h. The ultraviolet absorbance of the above resolution at 412 nm was then measured utilizing a UV-visible spectrophotometer.
For evaluation the GSHox-like exercise, CussNV at a focus of 250 µg/mL was incubated with GSH at concentrations of 0.1 mM, 0.75 mM, 1.25 mM, 2 mM, and a couple of.5 mM. Following incubation, the combination was centrifuged, and the ensuing supernatant underwent a colorimetric response with DTNB. The absorbance at 412 nm was subsequently measured inside 1 h to find out the kinetic parameters of nanozyme.
Cell tradition
Luc-4T1 cells have been generated by lentiviral transfection of the luciferase reporter gene into 4T1 cells. Each 4T1 and Luc-4T1 cells have been cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, maintained at 37 °C in a 5% CO₂ ambiance, and routinely sub-cultured.
Cell uptake
The FITC labeled CussNVp (CussNVp@FITC) was synthesized for mobile uptake investigation. Briefly, 50 µg 4T1 cells have been incubated with 50 µg FITC for 12 h, then the combination was centrifuge for a number of occasions to acquire CussNVp@FITC. After that, 4T1 cells have been seeded right into a 6-well plate at a density of 1.0 × 105 cells per effectively and cultured for twenty-four h. Subsequently, the cells have been handled with CussNVp@FITC at a focus of fifty µg/mL for varied time intervals. After therapy, the presence of inexperienced fluorescence indicators inside the cells have been assessed utilizing circulate cytometry (Novocyte 3130, ACEA).
Cytotoxicity analysis
4T1 cells have been seeded into 96-well plates at a density of 1.0 × 10⁴ cells per effectively and cultured for twenty-four h. Cells have been then handled with various 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 extra 24 h. After therapy, the media have been changed with recent medium containing 10 µL of CCK-8 resolution per effectively. Cell viability was decided by measuring absorbance at 450 nm.
Intracellular Cu2+ detection
4T1 cells (1 × 10⁶) have been seeded into confocal dishes and allowed to stick. After 24 h of therapy with varied probes, the cells have been incubated with a Cu²⁺-specific fluorescence probe (RBH, 1 µM) at 37 °C for 30 min, adopted by fixation with 4% paraformaldehyde for 15 min. The cells have been then stained with DAPI for 10 min. Fluorescence imaging was carried out utilizing 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. As well as, the intracellular Cu2+ focus was quantified through the use of the Cu²⁺ fluorescence probe and recording utilizing a fluorescence spectrophotometer (F-7100, Hitachi) with excitation at 510 nm and emission at 578 nm. The statistical outcomes of fluorescent photographs have been counted utilizing the Picture J software program.
Intracellular distribution of ATP7A
4T1 cells (1 × 106) have been seeded into confocal dishes and incubated with OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL) for twenty-four h. The cells have been then mounted in 4% paraformaldehyde for 15 min and blocked with 5% BSA for two h. Subsequent, the cells have been incubated in a single day with ATP7A antibody (1: 200), adopted 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 depth of the cell samples was imaged utilizing a CLSM. Fluorescence excitation and emission wavelengths have been 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 analysis
4T1 cells (1 × 10⁶) have been seeded into confocal dishes and incubated for twenty-four h to permit for adherence. After that, cell samples have been incubated with OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL) for twenty-four h. Then the cells have been then handled with both 50 µM of Ru(ddp)₃Cl₂, a fluorescent oxygen probe, at 37 °C for two 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 therapies, cells have been mounted with 4% paraformaldehyde at 4 °C for 15 min. Following fixation, the cells have been stained with DAPI for 10 min and imaged utilizing a CLSM. The imaging parameters have been set as follows: excitation at 488 nm and emission at 510 nm for each Ru(ddp)₃Cl₂, BBoxiProbe O26, and liperfluo, and excitation at 360 nm and emission at 460 nm for DAPI.
Intracellular GSH detection
Nonfluorescent bromobimane will be transformed into fluorescent compounds within the presence of small thiols, akin to GSH. To evaluate intracellular GSH ranges, 4T1 cells have been obtained with indicated therapies and stained with 100 µM bromobimane at 37 °C for 30 min. Blue fluorescence was detected utilizing a CLSM, with excitation at 392 nm and emission at 478 nm, to visualise GSH ranges.
Western blot evaluation
4T1 cells have been seeded right into a 6-well plate at a density of 1.0 × 10⁵ cells per effectively and cultured for twenty-four h. The cells have been then handled with OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL) for twenty-four h. After therapy, the cells have been harvested, washed with ice-cold PBS, and lysed. β-actin served because the loading management, detected utilizing anti-actin antibodies, whereas the expression ranges of GPX4, HSP70, DLAT, and FDX1 have been assessed utilizing their respective antibodies (dilution 1: 1000). Membranes have been then incubated with secondary antibodies towards rabbit IgG or mouse IgG (dilution 1: 5000) and imaged utilizing a multifunctional fluorescent and luminescent gel imager (Cheml XRQ, Gene Firm Restricted).
Apoptosis evaluation
4T1 cells (1.0 × 10⁶) have been seeded into 6-well plates and incubated for twenty-four h within the presence of OME (10.8 µM), CussNVp (60 µg/mL), or CussOMEp (60 µg/mL). After therapy, the cells have been stained with 5 µL Annexin V-FITC and 5 µL propidium iodide (PI), adopted by fluorescence sign evaluation utilizing a FCM.
Hemolysis assay
All animal experiments have been carried out in accordance with protocols accredited by the Animal Experimental Ethics Committee of Fujian Regular College (Approval No. IACUC-20230036). Feminine Balb/c mice (4–6 weeks outdated) have been obtained from Shanghai Slack Laboratory Animal Co., LTD. Blood samples have been collected by way of orbital puncture and positioned in anticoagulant tubes. Crimson blood cells have been remoted by centrifugation at 3000 rpm for five min and diluted to a ultimate focus of two%. Numerous concentrations of CussNVp and CussOMEp have been incubated with the pink blood cells for two h at 37 °C. Phosphate-buffered saline (PBS) and 0.5% Triton X-100 have been used as detrimental and optimistic controls, respectively. Hemolysis was quantified by measuring the absorbance at 576 nm, and hemolysis charges have been calculated utilizing the next system:
Hemolysis (%) = (OD576pattern – OD576N) / (OD576P – OD576N) × 100%.
Biosafety analysis
Balb/c mice (4–6 weeks outdated) have been randomly assigned to 2 teams (n = 6 per group) and intravenously administered both PBS or 300 µg of CussOMEp per mouse. Physique weights have been measured each 5 days for 90 days. Blood and serum samples have been collected to evaluate key physiological parameters. Main organs have been harvested, weighed, and subjected to H&E staining for morphological evaluation.
In vivo biodistribution assay
ICG-labeled CussNVp (denoted as CussICGp) was synthesized for in vivo imaging. In short, 100 µg of ICG was blended with 100 µg of CussNVp and incubated in a single day. The unbound ICG was then eliminated by centrifuge, and the loading fee of ICG was decided utilizing UV-visible spectrophotometry. Six 4T1 tumor-bearing mice have been randomly divided into two teams: (1) Free ICG, and (2) CussICGp. Every group of mice obtained a 100 µg dosage of ICG by way of tail vein injection. Fluorescence imaging was carried out utilizing an in vivo imaging system (IVIS) at varied occasions (0, 0.5, 2, 4, 8, 12, 24, and 48 h). At 48 h post-injection, the mice have been euthanized, and their tumor tissues and main organs (coronary heart, liver, spleen, lung, and kidney) have been collected for ex vivo imaging. Moreover, to quantify the tumor-targeting capability of CussNVp, twelve 4T1 tumor-bearing mice have been administered both PBS, 0.054 µmol of OME, 300 µg of CussNVp, or 300 µg of CussOMEp intravenously injection, and the mice have been euthanasia at 48 h post-injection. The tumor tissues have been collected, and the copper contents have been assessed utilizing the fluorescent probe.
In vivo synergistic antitumor efficacy
The 4T1 tumor mannequin was established by subcutaneously injecting 1 × 10⁶ 4T1 cells into the appropriate flank of wholesome feminine Balb/c mice. As soon as tumors reached a quantity of 100 mm³, the mice have been randomly assigned to 4 teams (n = 4) and intravenously administered one of many following therapies: PBS, 0.054 µmol OME per mouse, 300 µg CussNVp per mouse, or 300 µg CussOMEp per mouse. Tumor volumes and physique weights have been monitored each two days.
On day 14, mice have been euthanasia, the tumor tissues and tumor-draining lymph nodes have been collected. Tumor tissues have been subjected to H&E staining, in addition to IHC evaluation utilizing anti-HIF-1α, anti-GPX4, and anti-FDX1 antibodies. In addition to, single-cell suspension of tumor tissues was ready for immune profiling. Macrophage polarization was evaluated utilizing 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 have been 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, whereas B cell infiltration was measured utilizing anti-B220-FITC. As well as, the single-cell suspension of tumor-draining lymph nodes have been ready for DC maturation evaluation. Single-cell suspensions have been ready, and cells have been stained with anti-CD11c-BV421, anti-CD80-FITC, and anti-CD86-PE antibodies at 4 °C for 30 min, adopted by circulate cytometric evaluation.
In vivo anti-abscopal exercise
Major tumors have been established by subcutaneously injecting 1 × 106 4T1 cells into the appropriate flank of wholesome feminine Balb/c mice. The tumor-bearing mice have been then randomly divided into 4 teams (n = 8) and administered both PBS, 0.054 µmol of OME, 300 µg of CussNVp, or 300 µg of CussOMEp intravenously on day 0. Moreover, mice obtained αPD-1 (1 mg/kg) therapy on days 2, 5, and eight. On day 9, 2 × 105 4T1 cells have been injected subcutaneously into the left flank to ascertain abscopal tumors. Tumor volumes and physique weights have been recorded each different day for as much as 34 days.
On day 34, mice have been euthanasia, and the lymph nodes, tumors, and spleens have been collected to make single-cell suspensions for immune response evaluation. For DC maturation evaluation, lymph node single-cell suspensions have been stained with anti-CD11c-BV421, anti-CD80-FITC, and anti-CD86-PE at 4 °C for 30 min. For tumor single-cell suspensions evaluation, 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 have been examined by staining with anti-CD3-PE/Cy7, anti-CD4-BV421, and anti-CD8a-APC/Cy7. Tumor cells have been mounted and permeabilized utilizing the BD Pharmingen™ Transcription Issue Buffer Set for 45 min previous to further staining. After treating with brefeldin A for six h earlier than staining with anti-IFNγ-APC. Regulatory T cell inhibition was evaluated utilizing anti-Foxp3-PE following centrifugation and analyzed by circulate cytometry. Reminiscence T cells have been 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 exercise
To determine main tumors, 1 × 106 4T1 cells have been subcutaneously injected into the appropriate flank of Balb/c mice. As soon as the tumor quantity reached roughly 100 mm³, the mice have been 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 eight. On day 9, the mice obtained an intravenous injection of two × 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 carried out utilizing an IVIS Spectrum (PerkinElmer) below anesthesia 10 min after the D-luciferin injection. Tumor volumes and physique weights have been recorded bi-daily for as much as 38 days. To evaluate lung metastasis, lung tissues have been full of India ink and subsequently stained with H&E.
Density purposeful principle simulation
The preliminary construction of the nanocluster was obtained by conformational search with the CREST program. After acquiring the wavefunctions primarily based on B3LYP purposeful and def2-SVP foundation within the Gaussian 16 program (Rev A.03), the weak interplay evaluation was carried out utilizing the interplay area indicator (IRI) within the Multiwfn program [48, 49].
Statistical evaluation
Quantitative knowledge are expressed as imply ± normal deviation. Statistical analyses have been carried out utilizing one-way ANOVA with GraphPad Prism 8.0 software program. Statistical significance is indicated by an asterisk, with the next designations: ns (not important), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. A P worth of 0.05 or much less was thought of statistically important.
