Copper-palladium flaky nano-enzyme as well as preparation and application thereof

Abstract

The invention belongs to the technical field of nano materials, and particularly provides a copper-palladium flaky nano enzyme as well as preparation and application thereof. The preparation method of the copper-palladium flaky nano enzyme comprises the following steps: (1) uniformly mixing palladium acetylacetonate, polyvinylpyrrolidone, sodium bromide, copper acetylacetonate, DMF (Dimethyl Formamide) and water; (2) transferring the mixed solution into a reaction kettle, introducing CO into the vacuum reaction kettle for a reduction reaction, and cooling to room temperature after the reaction is finished; and (3) acetone is added into the reaction kettle for centrifugation, supernatant is discarded, a mixed solution of acetone and ethyl alcohol is continuously added for centrifugal washing of the product, the product is ultrasonically dispersed in ethyl alcohol, standing is conducted at the room temperature, and the copper-palladium flaky nano-enzyme solution is obtained. The copper-palladium flaky nano-enzyme has photo-thermal performance and multi-enzyme activity, can consume GSH and inhibit generation of GSH at the same time, further improves the ROS level in cells, induces ferroptosis of tumor cells and improves the tumor treatment effect.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials, and specifically relates to a copper-palladium sheet nanozyme and a preparation and application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by the lack of expression of estrogen (ER), progesterone receptor (PR) and human epidermal growth factor 2 (HER2). It is highly malignant, invasive and highly recurrent. Currently, chemotherapy is the main choice for TNBC patients, and its efficacy mainly depends on the induction of tumor cell apoptosis. However, tumor cells have innate or acquired resistance to apoptosis, so most TNBC patients will develop resistance shortly after chemotherapy. Therefore, an effective treatment strategy is needed to improve the treatment effect of TNBC.

[0003] Ferroptosis is a form of programmed cell death that is different from apoptosis and is characterized by the accumulation of ROS and lethal lipid peroxides. Since metastatic cancers are resistant to apoptosis and sensitive to ferroptosis, ferroptosis characterized by the accumulation of ROS and LPO has great potential for the treatment of metastatic cancer. In addition, ferroptosis can lead to damage and rupture of cell membranes, thereby releasing damage-associated molecular patterns (DAMPs) such as high-mobility group protein (HMGB1), adenosine triphosphate (ATP) and calreticulin (CRT). The released DAMPs can promote the antigen presentation function of dendritic cells (DCs), promote the activation of T cells and tumor-specific immune responses.

[0004] Studies have shown that metal-based nanomaterials that catalyze Fenton or Fenton-like reactions to produce toxic hydroxyl radicals (•OH) can directly induce cell ferroptosis, but the powerful intracellular antioxidant reduction system reduced glutathione (GSH) / glutathione peroxidase 4 (GPX4) can inhibit the generation of ROS and the accumulation of LPO by regulating the ratio of GSH and oxidized glutathione (GSSG), thereby inhibiting cell ferroptosis. However, current research has mostly focused on the reduction of GSH levels, while ignoring the issue of GSH regeneration.

[0005] Nanozymes have been widely used in ROS-mediated therapy. On the one hand, they can promote oxidative stress in tumor cells by catalyzing the decomposition of H2O2 into •OH in the tumor microenvironment by simulating peroxidase-like activity (POD enzyme-like activity); on the other hand, nanomaterials with glutathione oxidase-like activity (GSHOx enzyme-like activity) can enhance intracellular oxidative stress by consuming GSH in the tumor microenvironment. NADPH can provide electrons to glutathione reductase (GR) to promote the regeneration of reduced GSH. Therefore, nanozymes with phosphatase-like activity (NOx enzyme-like activity) can consume NADPH in tumors and inhibit the regeneration of reduced GSH, thereby further enhancing the level of internal oxidative stress in cells. However, most reported nanozymes only have POD and GSHox enzyme-like activities, while ignoring the redox regeneration system in the tumor microenvironment, making ROS-mediated tumor therapy ineffective. Summary of the invention

[0006] The purpose of the present invention is to provide a copper-palladium sheet nanozyme with photothermal performance and multi-enzyme activity, which can consume GSH while inhibiting the production of GSH, thereby further increasing the intracellular ROS level, inducing tumor cell ferroptosis, and improving the tumor treatment effect.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a method for preparing a copper-palladium sheet-like nanozyme.

[0008] The present invention adopts the carbon monoxide reduction method to dope copper into palladium nanosheets, and synthesizes copper-palladium sheet nanozymes with surface plasmon resonance effect, which not only improves the original POD enzyme-like activity of palladium nanosheets, but also makes them have GSHOx and phosphatase-like activity (NOx enzyme-like activity). In addition, structural defects are formed by ethanol etching, which further improves the catalytic activity of multiple enzymes. Specifically, the following steps are included: (1) Palladium acetylacetonate (Pd(acac)2), polyvinyl pyrrolidone, sodium bromide (NaBr), copper acetylacetonate (Cu(acac)2), N,N-dimethylformamide (DMF) and water are mixed and uniformly mixed by stirring or ultrasonic treatment; (2) transferring the mixed solution obtained in step (1) into a reactor, introducing N2 into the reactor for vacuum replacement, introducing CO into the reactor for reduction reaction, and cooling to room temperature after the reaction is completed; (3) Add acetone to the reactor and centrifuge, discard the supernatant, continue to add a mixed solution of acetone and ethanol to centrifuge and wash the product, ultrasonically disperse the product in ethanol, and let it stand at room temperature to obtain a copper-palladium sheet nanozyme solution.

[0009] Furthermore, in step (1), the mass ratio of palladium acetylacetonate, polyvinyl pyrrolidone, sodium bromide and copper acetylacetonate is 25-27:75-85:25-26:15-25; and the volume ratio of N,N-dimethylformamide to H2O is 5:1.

[0010] Furthermore, in step (1), the volume ratio of the copper acetylacetonate to N,N-dimethylformamide is 3-5 mg:1 mL.

[0011] Furthermore, in step (2), the pressure of the introduced CO is 4-8 bar.

[0012] Furthermore, the temperature of the reduction reaction in step (2) is 100-120° C., and the reaction time is 2-4 hours.

[0013] Furthermore, in the mixed solution of acetone and ethanol in step (3), the volume ratio of acetone to ethanol is 4-5:1.

[0014] Furthermore, the mass ratio of the product in step (3) to the volume of ethanol is 2-1 mg:1 mL.

[0015] Furthermore, in step (3), the ultrasonic dispersion time is 5 to 10 minutes, and the standing time is 72 to 96 hours.

[0016] In a second aspect, the present invention provides a copper-palladium sheet nanozyme prepared by the above method.

[0017] In a third aspect, the present invention provides the use of the above-mentioned copper-palladium sheet-like nanozyme in the preparation of tumor adjuvant therapeutic drugs.

[0018] Specifically, tumor cells were co-incubated with copper-palladium sheet nanozymes and irradiated with 808nm laser.

[0019] The beneficial effects of the present invention are: The copper-palladium sheet-like nanozyme of the present invention has multi-enzyme activity and photothermal performance: On the one hand, it can simulate POD enzyme-like activity under the weak acid conditions of the tumor microenvironment, produce •OH, cause ROS accumulation, and promote lipid peroxidation. It can also simulate GSHox and NOx enzyme-like activity, while promoting GSH depletion, consuming NADPH in cells, thereby further inhibiting the generation of GSH, increasing the level of intracellular ROS, inducing ferroptosis of tumor cells, and enhancing the effect of tumor treatment (the production of a large amount of ROS and the consumption of GSH will promote GPX4 inactivation and LPO accumulation, thereby inducing ferroptosis of tumor cells. Ferroptosis further induces immunogenic death of tumor cells, triggers molecular-related damage patterns, presents signals to dendritic cells, induces systemic immunity, and enhances the therapeutic effect of immunotherapy).

[0020] On the other hand, copper-palladium sheet nanozymes can generate localized plasmon resonance (SPR) hot electrons due to their strong near-infrared light absorption. They can not only increase the temperature of the reaction system but also inject additional thermal energy into the chemical reaction, thereby further enhancing the catalytic activity of the nanozymes through photothermal therapy and synergizing with ROS, effectively inducing ferroptosis of tumor cells and promoting immunogenic cell death of tumor cells, inducing systemic immunity, and enhancing the effect of tumor treatment.

[0021] The copper-palladium sheet nanozyme described in the present invention reduces the toxic side effects of traditional treatment methods by combining ferroptosis with immunotherapy and coordinating with photothermal therapy (PTT), and has important clinical application value for improving the effect of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the transmission electron microscope (TEM) imaging result of the E-CuPd NSs prepared in Example 1.

[0023] Figure 2 This is the ultraviolet-visible-near-infrared (UV-Vis-NIR) absorption spectrum of the E-CuPd NSs prepared in Example 1.

[0024] Figure 3 The corresponding temperature change curves of E-CuPd NSs solutions with different concentrations under laser irradiation.

[0025] Figure 4 This is the photothermal cycling curve of E-CuPd NSs solution with a concentration of 100 μg / mL.

[0026] Figure 5 UV absorption spectra of TMB oxidized by E-CuPd NSs group and control group.

[0027] Figure 6 UV absorption spectra of TMB oxidized by E-CuPd NSs of the present invention under different treatment conditions.

[0028] Figure 7 Figure 3 shows the UV absorption spectrum of MB degradation by E-CuPd NSs of the present invention over time.

[0029] Figure 8 The results are the test results of the GSH consumption ability of E-CuPd NSs of the present invention at different concentrations.

[0030] Fig. 9 UV absorption spectra of E-CuPd NSs of the present invention consuming NADPH at different times.

[0031] Fig.10Laser confocal images of lipid peroxidation in 4T1 cells induced by different treatments.

[0032] Fig.11 The results show that different treatment groups induced CRT externalization of 4T1 cells to the cell membrane.

[0033] Fig.12 The results show that HMGB1 of 4T1 cells induced by different treatment groups was released from the nucleus to the extranuclear space.

[0034] Fig.13 Figure 3 shows the changes in tumor size of mice during treatment under different treatment conditions.

[0035] Fig.14 The CRT eversion in the tumor site of mice in different treatment groups.

[0036] Fig.15 The release of HMGB1 in the tumor sites of mice in different treatment groups.

[0037] Fig.16 Figure 2 shows the activation of immune cells in the tumor sites of mice in different treatment groups. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0039] The raw materials of the present invention are all commercially available.

[0040] Example 1 The preparation steps of copper-palladium sheet nanozymes (E-CuPd NSs) in this embodiment are as follows: (1) 25.0 mg Pd(acac)2, 80 mg polyvinylpyrrolidone, 25.7 mg NaBr and 21.4 mg Cu(acac)2 were stirred with 5 mL N,N-dimethylformamide and 1 mL H2O in a glass container for 1 h or ultrasonically dispersed for 10 min; (2) The mixed solution of step (1) was transferred to a high-pressure reactor, and then N2 was introduced into the reactor for vacuum replacement for 3 times, and then CO was introduced to a pressure of 4 bar, and the temperature of the high-pressure reactor was raised to 120°C within 30 minutes, and the reduction reaction was carried out at 120°C for 3 hours. After the reaction was completed, the reaction was cooled to room temperature; (3) Acetone was added to the autoclave and centrifuged at 8000-10000 rpm for 5 min, and the supernatant was discarded. The product was then centrifuged and washed three times or more using a mixture of acetone and ethanol (volume ratio 5:1), each time at a speed of 8000-10000 rpm for 5 min. After the centrifugation, the product was ultrasonically dispersed in ethanol (ultrasonic dispersion for 10 minutes, the concentration of the product in ethanol was 1 mg / mL), and allowed to stand for 72 h to obtain an E-CuPd NSs solution.

[0041] The E-CuPd NSs prepared in this example were characterized and tested as follows: 1. Morphological characterization 0.2~0.5mg / mL of E-CuPd NSs was dropped onto the copper grid and its morphology was observed using a transmission electron microscope (TEM) after drying. Figure 1 This is the TEM imaging result of the E-CuPd NSs prepared in this example, which shows that the particle size is about 40-50 nm, the flaky structure, the surface is rough, the edges are irregular and the color is darker.

[0042] 2. Ultraviolet-visible-near infrared (UV-Vis-NIR) absorption spectrum test and analysis The E-CuPd NSs solution with appropriate concentration was tested by UV-Vis-NIR absorption spectrum. Figure 2 This is the UV-Vis-NIR absorption spectrum of the E-CuPd NSs prepared in this example. It can be seen that it has a significant absorption peak in the near-infrared region, indicating that the E-CuPd NSs of the present invention has excellent optical properties in this band.

[0043] 3. Photothermal performance evaluation of E-CuPd E-CuPd NSs solutions with different concentrations were irradiated with 808 nm laser at a power of 1.5 W / cm 2 The temperature change of the E-CuPd solution was recorded by an infrared thermal imager.

[0044] like Figure 3 The corresponding temperature change curves of E-CuPd NSs solutions with different concentrations under laser irradiation are shown. The results show that the E-CuPd NSs of the present invention has a strong light-to-heat conversion ability. In addition, the laser on-off irradiation of 100μg / mL E-CuPd NSs solution was performed, and its temperature change was recorded, such as Figure 4 As shown, after multiple laser switching cycles, E-CuPdNSs still maintains strong photothermal performance, indicating that it has excellent photothermal stability.

[0045] Example 2 In this example, an ultraviolet spectrophotometer was used to further measure the enzyme-like catalytic activity of the E-CuPd NSs prepared in Example 1. The specific steps are as follows: 1. Determination of POD enzyme-like activity of E-CuPd by 3,3',5,5'-tetramethylbenzidine (TMB) TMB can be oxidized to oxTMB by substances such as ROS. E-CuPd NSs are co-incubated with H2O2 to generate ROS, and then TMB is added. TMB will be oxidized to oxTMB. oxTMB has a characteristic absorption peak at 652nm. Therefore, whether E-CuPd NSs has POD enzyme-like activity can be evaluated by observing the absorption peak at 652nm.

[0046] TMB (0.16 mM) was dissolved in substrate buffer solution (pH=5), and H2O2 (10 mM) was added to form a substrate solution. 3µL E-CuPd NSs (1 mg / mL) (denoted as E-CuPd NSs group) and 3µL PBS (denoted as control group) were added to 3mL of substrate solution, respectively. After 5 minutes, the absorbance of TMB at 652 nm was measured using a UV-visible spectrophotometer.

[0047] like Figure 5 As shown, compared with the control group added with PBS, the E-CuPd NSs group has a maximum absorption peak at 652 nm, indicating that the E-CuPd NSs of the present invention has good POD enzyme-like activity.

[0048] To further determine whether the addition of H2O2 and light have an effect on the POD enzyme activity of E-CuPd NSs, three groups were set up: E-CuPd NSs group: 3 µL of E-CuPd NSs (1 mg / mL) was added to the substrate solution containing only TMB (0.16 mM). After 5 min, the absorbance of TMB at 652 nm was measured using a UV-visible spectrophotometer.

[0049] E-CuPd NSs+H2O2 group: 3µL E-CuPd NSs (1mg / mL) was added to the substrate solution formed by TMB (0.16 mM) and H2O2 (10mM). After 5 minutes, the absorbance of TMB at 652 nm was measured using a UV-visible spectrophotometer.

[0050] E-CuPd NSs+H2O2+L 808Group: 3 µL of E-CuPd NSs (1 mg / mL) was added to the substrate solution formed by TMB (0.16 mM) and H2O2 (10 mM), and irradiated under 808 nm laser. After 5 min, the absorbance of TMB at 652 nm was measured using a UV-visible spectrophotometer.

[0051] like Figure 6 As shown, E-CuPd NSs can catalyze the production of ROS only in the presence of H2O2, and under the irradiation of 808nm laser, the absorption of E-CuPd NSs is maximum at 652nm, which indicates that photothermal can promote the production of ROS by E-CuPd NSs.

[0052] 2. Methylene blue (MB) measurement of •OH generation ability of E-CuPd NSs MB will degrade after reacting with •OH, which will reduce the absorption peak of MB solution at 660 nm. Therefore, MB was co-incubated with E-CuPd NSs, and the ability of E-CuPd NSs to produce •OH was detected by measuring the absorbance of MB solution at 660 nm.

[0053] Specifically, MB (5 µg / mL) was dissolved in a buffer solution with pH = 5.0, H2O2 was added to form a substrate solution, and then 20 µL of E-CuPd NSs (1 mg / mL) was added to the substrate solution, and the UV-visible absorption spectra of MB in different segments were measured using a UV spectrophotometer.

[0054] like Figure 7 As shown, after the MB solution was co-incubated with E-CuPd NSs, the absorption peak of the MB solution decreased significantly over time, indicating that the E-CuPd NSs of the present invention have a good ability to generate •OH.

[0055] 3. GSHOx enzyme-like activity assay of E-CuPd NSs (GSH consumption capacity assay) GSH reacts with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) to generate yellow 2-nitro-5-mercaptobenzoic acid and glutathione disulfide (GSSG). 2-nitro-5-mercaptobenzoic acid has a characteristic absorption peak at 412 nm. E-CuPd NSs are co-incubated with GSH, and then DTNB is added and co-incubated for 15 minutes. The content of reduced glutathione can be quantitatively detected by the change in absorbance. Therefore, DTNB can be used to evaluate the ability of E-CuPd NSs to consume GSH.

[0056] The depletion of GSH was determined by a colorimetric reaction with DTNB. Specifically, GSH (1 mM) was dissolved in a buffer solution at pH 6.5 to form a substrate buffer solution, and then different concentrations of E-CuPd NSs were added to the GSH substrate buffer system. After 1 h, 50 µL DTNB (10 mg / mL) was added, and after 30 min, the UV-visible absorption spectrum of 2-nitro-5-mercaptobenzoic acid was measured by a UV spectrophotometer.

[0057] like Figure 8 As shown, with the increase of the concentration of E-CuPd NSs, the absorption peak of 2-nitro-5-mercaptobenzoic acid at 412 nm gradually decreased, which indicates that the E-CuPd NSs of the present invention can consume GSH in the solution and has GSHox enzyme-like activity.

[0058] 4. NOx Enzyme-like Activity Assay of E-CuPd NSs Since oxidized GSSG can be reduced to GSH with the participation of NADPH, inhibiting NADPH can inhibit the regeneration of GSH. By observing the change of the characteristic absorption peak of NADPH at 340nm, it can be judged whether E-CuPd NSs has the ability to consume NADPH.

[0059] NADPH (100 µg / mL) was dissolved in a buffer solution of pH=6.5, and 50 µg / mL E-CuPd NSs was added. The changes in the ultraviolet absorbance of the supernatant were monitored at different time periods.

[0060] like Fig. 9 It can be seen that as the mixing time of E-CuPd NSs and NADPH increases, the absorption peak of NADPH at 340 nm gradually decreases, that is, NADPH is gradually consumed, which indicates that the E-CuPd NSs of the present invention has NOx enzyme-like activity.

[0061] Example 3 In this example, 4T1 (mouse breast cancer) cells were used to verify the effects of the E-CuPd NSs of the present invention on inducing ferroptosis and immunogenic cell death of tumor cells.

[0062] 1. Verification of the effect of inducing ferroptosis in tumor cells 4T1 cells in the logarithmic growth phase were cultured at 1×10 5 After the cells adhered to the wall, different treatment groups were set up, namely: control group (control): serum-free culture medium; L 808Group: 4T1 cells were irradiated with 808 nm laser; E-CuPd NSs group: 4T1 cells were co-incubated with E-CuPd NSs; E-CuPd NSs+L 808 Group: 4T1 cells were co-incubated with E-CuPd NSs and irradiated with 808 nm laser after 4 h. 808 The concentration of E-CuPd NSs in the groups was the same, 20 μg / mL. After 24 h of culture, the serum-free culture medium of the experimental groups and the control group was aspirated, washed twice with PBS, and the lipid peroxidation fluorescent probe (C11 BODIPY 581 / 591 ) dye was used to stain 4T1 cells at 37°C for 20 minutes, then the excess dye was washed away with PBS, and diluted serum-free culture medium containing Hoechst 33342 was added. The cells were incubated at 37°C in the dark for 5 minutes, and finally the serum-free culture medium containing Hoechst 33342 was removed. The cells were washed twice with PBS, and confocal photography of 4T1 cells was performed using a laser.

[0063] like Fig.10 As shown, there are significant differences in the induction of lipid peroxidation in tumor cells by different treatment groups. E-CuPd NSs alone can induce lipid peroxidation in tumor cells, but the effect is limited. When irradiated with 808nm laser, the effect of E-CuPd NSs inducing lipid peroxidation in tumor cells is significantly enhanced, which will provide a new idea for inducing ferroptosis of tumor cells.

[0064] 2. Verification of the effect of inducing immunogenic cell death in tumor cells 4T1 cells in the logarithmic growth phase were inoculated into confocal microplates. After the cells adhered to the wall, different treatment groups were set up, namely: control group: serum-free culture medium; L 808 Group: 4T1 cells were irradiated with 808 nm laser; E-CuPd NSs group: 4T1 cells were co-incubated with E-CuPd NSs; E-CuPd NSs+L 808 Group: 4T1 cells were co-incubated with E-CuPdNSs and irradiated with 808 nm laser after 4 h.

[0065] After 24 h, the E-CuPd NSs group and the E-CuPd NSs+L 808The same concentration of E-CuPd NSs was added to the two groups and cultured for 24 h. The cells were then fixed with 4% paraformaldehyde, blocked with bovine serum albumin, permeabilized, and incubated with primary antibodies (Anti-CRT and Anti-HMGB1) overnight. After incubation with secondary antibodies at room temperature, laser confocal microscopy was used for observation.

[0066] like Fig.11 As shown in the figure, the fluorescence labeling of 4T1 cells treated with E-CuPd NSs alone was stronger than that of 4T1 cells not treated with E-CuPd NSs, while E-CuPd NSs+L 808 The treated 4T1 cells had the strongest fluorescence labeling, indicating that the E-CuPd NSs+L 808 The treated 4T1 cells had more CRT externalization to the cell membrane.

[0067] The release of HMGB1 in 4T1 cells was further characterized. Fig.12 As shown, the HMGB1 fluorescence signal in the nucleus of 4T1 cells treated with E-CuPd NSs was significantly weakened, indicating that E-CuPd NSs can promote the release of HMGB1 in the nucleus.

[0068] The above results indicate that E-CuPd NSs can deplete GSH by producing abundant ROS and consuming NADPH to block the regeneration of GSH, thereby effectively cascading oxidative stress in tumor cells, inducing ferroptosis of tumor cells and further stimulating immunogenic cell death.

[0069] Example 4 In this example, Balb / c mice were used to construct a breast cancer model to verify the effect of the E-CuPd NSs of the present invention in inducing ferroptosis and immunogenic death of tumor cells.

[0070] First, the experiment set up different groups and measured tumor size and immune infiltration in the tumor site to evaluate the effects of different treatments on inhibiting tumor growth.

[0071] In this example, mice were randomly divided into 4 groups: control group: PBS was injected into the tail vein; 808 Group: PBS injected into tail vein followed by laser irradiation; E-CuPd NSs group: E-CuPd NSs injected into tail vein; E-CuPd NSs+L 808 Group: tail vein injection of E-CuPd NSs followed by laser irradiation.

[0072] When the mice were 5 weeks old, tumors were inoculated. The right mammary gland of the mice was depilated one day before inoculation. 4T1 cells in the logarithmic growth phase were resuspended in serum-free medium and the cell suspension was mixed with matrix gel at a ratio of 1:1 to make the final density of 4T1 cells 5×10 6 50 μL of cell suspension was subcutaneously inoculated into the right mammary gland of mice and the tumor volume was increased to 100 mm. 3 The drug was administered every three days, with each group of mice receiving a dose of 2.5 mg / mL. The light-exposed group was treated with laser (1.5 W·cm for 5 min) 8 h after administration. -2 ).

[0073] Fig.13 The changes in tumor size of mice during treatment are shown. 808 The group was able to significantly inhibit the metastasis of tumor cells and showed excellent anti-tumor effects. This may be due to the synergistic effect of local hyperthermia triggered by NIR and the enhanced catalytic effect of multi-enzyme mimic activity, which activated the immune response combined with ferroptosis and photothermal therapy.

[0074] Next, immunofluorescence was used to determine whether tumor cells underwent ICD, such as CRT turnover and HMGB1 release. These DAMPs can promote the maturation and activation of DC cells. Fig.14 As shown, compared with the control group, the NIR group (L 808 The fluorescence of CRT was almost unobservable in the tumor cells of the PBS group, indicating that NIR-II laser irradiation alone could not effectively induce the translocation of CRT. After the introduction of E-CuPd NSs, CRT fluorescence was clearly observed in the E-CuPd NSs group due to its ability to consume GSH to amplify intracellular oxidative stress, and E-CuPd NSs further increased CRT fluorescence exposure by combining with 808nm laser. Compared with the PBS group, E-CuPd NSs+L 808 The secretion of extracellular HMGB was significantly increased in the group ( Fig.15 ), which indicates that E-CuPd NSs can effectively induce ICD in tumor cells.

[0075] Afterwards, tumors were collected from tumor-bearing mice for flow cytometry analysis to explore the effect of E-CuPd NSs on DC cell maturation. Fig.16 As shown in A, compared with the light-only group (L 808 Compared with the E-CuPd NSs group, the proportion of mature DCs cells increased significantly.

[0076] Since mature DCs are potent stimulators of T cell immune priming, we continued to investigate the potential of E-CuPd NSs to enhance the infiltration of effector T cells within tumors. + CD4 in T cells + and CD8 + Subgroup.

[0077] like Fig.16 As shown in B, CD3 + +CD8 + The proportion of double-positive cell populations increased significantly, and E-CuPd NSs treatment in vivo increased intratumoral CD8 + T cells (cytotoxic T lymphocytes) transform the tumor microenvironment from "cold" to "hot".

[0078] The above results indicate that E-CuPd NSs can induce ICD in TNBC, enhance tumor immunogenicity, promote the maturation of antigen-presenting dendritic cells, and ultimately lead to the activation of CD8 + T cells kill cancer cells and mediate anti-tumor immunity.

Claims

1. A method for preparing a copper-palladium sheet-like nanozyme, characterized in that: The following steps are involved: (1) mixing palladium acetylacetonate, polyvinyl pyrrolidone, sodium bromide, copper acetylacetonate, N,N-dimethylformamide and water, and mixing them uniformly by stirring or ultrasonic treatment; (2) transferring the mixed solution obtained in step (1) into a reactor, introducing N2 into the reactor for vacuum replacement, introducing CO into the reactor for reduction reaction, and cooling to room temperature after the reaction is completed; (3) Add acetone to the reactor and centrifuge, discard the supernatant, continue to add a mixed solution of acetone and ethanol to centrifuge and wash the product, ultrasonically disperse the product in ethanol, and let it stand at room temperature to obtain a copper-palladium sheet nanozyme solution.

2. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of palladium acetylacetonate, polyvinyl pyrrolidone, sodium bromide and copper acetylacetonate is 25-27:75-85:25-26:15-25; and the volume ratio of N,N-dimethylformamide to H2O is 5:

1.

3. The preparation method according to claim 1, characterized in that: In step (1), the volume ratio of the copper acetylacetonate to N,N-dimethylformamide is 3-5 mg:1 mL.

4. The preparation method according to claim 1, characterized in that: In step (2), the pressure of the introduced CO is 4-8 bar.

5. The preparation method according to claim 1, characterized in that: The temperature of the reduction reaction in step (2) is 100-120° C., and the reaction time is 2-4 hours.

6. The preparation method according to claim 1, characterized in that: In the mixed solution of acetone and ethanol in step (3), the volume ratio of acetone to ethanol is 4-5:

1.

7. The preparation method according to claim 1, characterized in that: The mass ratio of the product in step (3) to the volume of ethanol is 2-1 mg:1 mL.

8. The preparation method according to claim 1, characterized in that: The ultrasonic dispersion time in step (3) is 5 to 10 minutes, and the standing time is 72 to 96 hours.

9. The copper-palladium sheet-like nanozyme prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the copper-palladium sheet-like nanozyme according to claim 9 in the preparation of tumor adjuvant therapeutic drugs.

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