A noble metal-transition metal composite catalyst, its preparation method and application

Abstract

The present invention provides a noble metal-transition metal composite catalyst, a preparation method and an application thereof, belonging to the field of immunotherapy. The composite catalyst exhibits excellent peroxidase and oxidase mimicking activities and has a super strong reactive oxygen species generating ability. The composite catalyst can induce endoplasmic reticulum stress and endoplasmic reticulum stress-related apoptosis, and can significantly enhance the immunogenicity of tumors, activate the immune response, and synergistically inhibit primary and metastatic tumors with immune checkpoint blockade drugs by inducing immunogenic cell death, promoting T cell infiltration and dendritic cell maturation, and stimulating the release of inflammatory factors.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy, and particularly relates to a noble metal-transition metal composite catalyst, a preparation method thereof, and an application thereof. Background Art

[0002] Primary and metastatic malignant melanoma can maintain an immunosuppressive microenvironment by showing low immunogenicity, secreting immunosuppressive cytokines, and causing death of tolerance pathways. Tumor-associated macrophages (TAMs) are mainly immunosuppressive (M2), which is one of the key factors driving the immunosuppressive tumor microenvironment. Immunogenic cell death (ICD) is a specific form of cell death that elicits an immune response against antigens of dead or dying tumor cells. Various treatment methods, such as chemotherapy, radiotherapy, and photodynamic therapy (PDT), etc., have been used to induce ICD, which can trigger the release of damage-associated molecular patterns (DAMPs). These released or exposed signals can mediate the maturation and migration of dendritic cells (DCs), and subsequently initiate an anti-tumor adaptive immune response. It has been reported that the generation of endoplasmic reticulum stress (ER) and reactive oxygen species (ROS) is crucial for activating the intracellular signaling pathways regulating ICD.

[0003] Chemodynamic therapy (CDT) is a new and promising treatment method based on ROS under the catalysis of nanomaterials by utilizing two unique characteristics of the acidic tumor microenvironment (TME) and overexpression of hydrogen peroxide (H2O2). It is worth noting that during the treatment process, the endogenous chemical energy in the TME can directly trigger chemical reactions in tumors without external energy input, showing greater application potential in the next generation of clinical cancer treatment. ROS generated by nanomaterial catalysis can disrupt the integrity of the cell nucleus and release high-mobility group box protein B1 (HMGB1). The generation of high levels of ROS in the endoplasmic reticulum will lead to the exposure of calreticulin (CRT) and the release of adenosine triphosphate (ATP). These DAMPs related to ICD activate the host immune system against cancer by stimulating the antigen presentation of dendritic cells and the proliferation of T lymphocytes (such as CD4+ / CD8+ T cells). In addition, the generated ROS can activate macrophage reprogramming to the immunostimulatory (M1) phenotype, release anti-tumor cytokines, relieve the immunosuppressive tumor microenvironment, induce an anti-tumor innate response, form a feedback loop, and improve the immune effect.

[0004] In recent years, many types of ROS catalytic nanoagents have been found to have high catalytic activity for the CDT process, such as noble metal nanomaterials, metal oxides, and metal-organic framework materials, which can catalyze the generation of ROS from oxygen and H2O2 through existing natural pathways. So far, several platinum (Pt)-based nanomaterials have been found to have enzyme-like activities such as catalase, oxidase, and peroxidase. However, on the one hand, pure Pt catalysts are prone to agglomeration of Pt nanoparticles, which will lead to a decrease in catalytic active sites and catalyst stability; on the other hand, in order to reduce the toxicity of nanomaterials, the platinum loading of Pt-based nanomaterials still needs to be further reduced. It has been found that among various transition metal oxides, tungsten (W) oxides have multiple oxidation states. Especially when the WOx system enters the non-stoichiometric stage (i.e., the WOx form with 2 ≤ x ≤ 3), it exhibits relatively ideal oxidation and adsorption capabilities and is a good host for nanocomposites. However, the mechanism of the influence of oxygen vacancies on the surface of transition metal oxides on the catalytic performance of noble metal nanomaterials is still unclear, and there is currently no report on the preparation of ROS catalytic nanoagents by combining transition metal oxides with Pt-based nanomaterials. Summary of the Invention

[0005] The purpose of the present invention is to provide a noble metal-transition metal composite catalyst, its preparation method, and application.

[0006] The present invention provides a composite catalyst, which is a composite material prepared from a platinum salt and a transition metal oxide, and the mass ratio of the platinum salt to the transition metal oxide is (10-15):100.

[0007] Furthermore, the platinum salt is a tetravalent platinum salt; and / or, the transition metal oxide is a tungsten oxide; and / or, the mass ratio of the platinum salt to the transition metal oxide is (12-13):100.

[0008] Furthermore, the tetravalent platinum salt is platinum tetrachloride; and / or, the tungsten oxide is W 18 O 49 ; and / or, the mass ratio of the platinum salt to the transition metal oxide is 12.7:100.

[0009] Furthermore, the W 18 O 49 is sea cucumber-like W 18 O 49 .

[0010] Furthermore, the preparation method of the sea cucumber-like W 18 O 49 includes the following steps: Dissolve tungsten hexacarbonyl in an ethanol solution, place it in an autoclave, seal it, and heat it at 170-190 °C for 22-26 h, and then dry it to obtain sea cucumber-like W18 O 49 。

[0011] The present invention also provides a method for preparing the above composite catalyst, and the method comprises the following steps: dispersing a transition metal oxide and a platinum salt in an ethanol solution, reacting at room temperature for 10 - 14 h, centrifuging, taking the precipitate, washing, and drying to obtain the composite catalyst.

[0012] The present invention also provides the use of the above composite catalyst in the preparation of a peroxidase - like catalyst for generating reactive oxygen species.

[0013] Further, the peroxidase - like catalyst is a drug for treating primary or metastatic tumors.

[0014] Further, the tumor is melanoma.

[0015] The present invention also provides a combined drug for treating primary or metastatic tumors, which contains the above composite catalyst and an immune checkpoint blockade drug for simultaneous or separate administration in the same or different dosage unit formulations, and a pharmaceutically acceptable carrier.

[0016] Further, the immune checkpoint blockade drug is an anti - PD - L1 antibody.

[0017] W 18 O 49 is a tungsten oxide in monoclinic system.

[0018] The present invention uses 3D sea - cucumber - like W 18 O 49 and a Pt precursor as raw materials to prepare an immune - regulating preparation based on Pt nanoclusters: WOx - Pt. The Pt clusters in WOx - Pt and W 18 O 49 regulate the surface electronic structure and energy level of the Pt clusters through the strong electronic interaction of the Pt - O - W bond, and optimize the intermediate adsorption and dissociation of oxygen.

[0019] WOx - Pt exhibits excellent POD - like and OXD - like enzyme activities and has super - strong ROS - generating ability.

[0020] In vitro experiments show that the treatment with WOx - Pt can induce endoplasmic reticulum stress and ER - related apoptosis.

[0021] In vivo experiments show that WOx - Pt can significantly enhance the immunogenicity of tumors, activate the immune response, and synergistically inhibit primary and metastatic tumors with immune checkpoint blockade drugs by inducing ICD, promoting T - cell infiltration and DC maturation, and stimulating the release of inflammatory factors.

[0022] Obviously, based on the above content of the present invention, various other forms of modifications, substitutions or changes can be made without departing from the above basic technical idea of the present invention according to the common general technical knowledge and conventional means in the art.

[0023] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0024] Figure 1 .Schematic diagram of the synthesis route of WOx-Pt.

[0025] Figure 2 .SEM image (a) and TEM images (b, c) of WOx-Pt; HAADF-STEM images (d, e) of WOx-Pt; intensity profile (f) corresponding to the region in e; picture (g) of the atomic structure schematic diagram superimposed on the HAADF-STEM image. HAADF-STEM image and related element mapping images (h, i) of WOx-Pt. Scale bar, 500 nm.

[0026] Figure 3 .XRD spectra of WOx and WOx-Pt

[0027] Figure 4 .XPS characterization results of WOx-Pt: measured spectrum (a); high-resolution regions of Pt 4f (b), W 4f (c) and O 1s (d); W valence state distribution ratio (e) corresponding to d.

[0028] Figure 5 .EPR spectra of WOx and WOx-Pt.

[0029] Figure 6 .(a) TMB absorbance values at λ = 652 nm after incubation with WOx and WOx-Pt in the presence or absence of H2O2; (b) Lineweaver-Burk plot of WOx-Pt with H2O2 as the substrate; (c) V of WOx under the action of H2O2 max and K m values; (d) Comparison of TON values of WOx-Pt with other recently reported catalysts; (e) Typical Michaelis-Menten curve and Lineweaver-Burk plot of WOx-Pt with TMB as the substrate under the condition of no H2O2; (f) ·O2 - Hydroethidium (HE) probe confirms ·O2 -The existence of; (g) DPA time-dependent stimulation of catalytic oxidation; (h) Active site toxicity of WOx-Pt and KSCN.

[0030] Figure 7 . Results of specific endoplasmic reticulum stress tests induced by WOx-Pt in vitro. (a-c) After treating B16 / F10 cells with different concentrations of PBS, WOx, and WOx-Pt for 12 hours, Western blot analysis was used to detect the expression of caspase-3 and CHOP (C / ebp homologous protein-10) proteins. All data are expressed as mean ± standard deviation, and the statistical differences between the two groups were calculated using two-sided Student's t-test; (d) TEM images of WOx-Pt-induced endoplasmic reticulum stress in B16 / F10 cells (left: scale bar = 2 μm, right: scale bar = 500 nm), and the red arrow indicates the enlarged ER compartment.

[0031] Figure 8 . (a) Schematic diagram of the experimental design for the systemic anti-tumor immune response mechanism in B16 / f10 tumor-bearing mice; (b) Photos of tumors in each group after the entire treatment process; (c) Tumor growth curves of B16 / F10 tumor-bearing mice treated with PBS, WOx-Pt, anti-PD-L1, and WOx-Pt + anti-PD-L1; (d, g) F4 / 80 + CD80 in cells + and CD206 + Quantitative analysis; (e) Average tumor weight and (f) Average body weight of mice in each group (n = 5); (h) Tumor-infiltrating CD4 + T cells and (i) CD8 + Percentage of T cells; Quantitative analysis of TNF-α in (j) CD8 + T cells in isolated tumors.

[0032] Figure 9 . (a) Representative FCM plots of tumor-infiltrating CD4 + T cells and CD8 + T cells; FCM analysis of (b) M1 macrophages (F4 / 80 + CD80 + ) and (c) M2 macrophages (F4 / 80 + CD206 + ) in isolated tumors; Quantitative analysis of the percentage of IFN-γ in (d) CD8 + T cells; Quantitative analysis of the ratio of Treg cells to CD4 + T cells; Quantitative analysis of the percentage of mature dendritic cells in tumor-draining lymph nodes of each group on the 7th day after treatment. Detailed implementation methods

[0033] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0034] Example 1: Preparation of Spiky WOx-Pt Catalyst

[0035] The schematic diagram of the synthesis route is as Figure 1 shown.

[0036] 1. Synthesis of Spiky WOx

[0037] Spiky WOx was synthesized by a solvothermal method. 127 mg of tungsten hexacarbonyl (W(CO)6) was added to 30 mL of an ethanol solution, and it was ultrasonically dissolved for 30 min. Then it was transferred to a high-pressure sterilizer with a Teflon liner, sealed, heated at 180 °C for 24 h, and naturally cooled to room temperature. After that, it was rinsed with an ethanol solution and vacuum-dried at 80 °C overnight to obtain spiky WOx. Experiments later proved that this spiky WOx was sea urchin-like W 18 O 49 .

[0038] 2. Synthesis of Spiky WOx-Pt Catalyst

[0039] 100 mg of the spiky WOx prepared by the above solvothermal method was redispersed in 30 mL of an ethanol solution, and then 12.7 mg of platinum tetrachloride (PtCl4) was added. The mixture was vigorously stirred at room temperature for 12 h. The resulting product was centrifuged, and the precipitate was washed with an ethanol solution and freeze-dried to obtain the spiky WOx-Pt catalyst.

[0040] The beneficial effects of the present invention are demonstrated by the following experimental examples.

[0041] Experimental Example 1: Characterization and Structure Testing of Spiky WOx-Pt Catalyst

[0042] 1. Experimental Method

[0043] (1) Scanning electron microscope (SEM) images were collected using an Apreo S HiVoc (Thermo Fisher Scientific, FEI).

[0044] (2) Transmission electron microscopy (TEM), aberration-corrected high-angle annular dark-field scanning TEM (AC HAADF-STEM), and energy-dispersive spectroscopy (EDS) mapping were performed using a 200 kV Talos F200x TEM microscope from FEI Company Limited, USA, with GMS-freeanalysis.

[0045] (3) X-ray diffraction (XRD) patterns were obtained using a DX-2700BH multi-purpose X-ray diffractometer (Haoyuan Instruments) with Cu radiation at a voltage of 40 kV. The samples were scanned in the 2θ range from 5° to 80°.

[0046] (4) X-ray photoelectron spectroscopy (XPS) was performed using a K-Alpha TM + X-ray photoelectron spectrometer (Thermo Scientific), a 180° bifocal analyzer, and a 128-channel detector. During the measurement, the prepared powder sample was pressed onto a carbon wire tap and then pasted onto the sample holder for measurement. The X-ray spot size for data collection was 400 μm, with 20 survey scans and 50 scans of specific regions.

[0047] (5) Electron paramagnetic resonance (EPR) spectroscopy measurements were carried out using a Bruker EPR EMX Plus (Bruker Beijing Science & Technology Co., Ltd., USA) at a frequency of 9.8 GHz (microwave power: 1 mW).

[0048] 2. Experimental Results

[0049] Measurements by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed that WOx-Pt had a good sea urchin-like structure, with slightly contracted edge nanowires and a grayish-brown color instead of the dark blue of W 18 O 49 , indicating successful Pt loading ( Figure 2 a - c). Through high-resolution TEM (HRTEM) spectroscopy measurements, the diameter of the Pt nanoparticles was approximately 2 nm, and they were uniformly distributed on the surface of WOx-Pt, providing more active sites for catalysis ( Figure 2 d). The lattice fringe image of WOx-Pt clearly showed the lattice spacings produced by monoclinic W 18 O 49 and hexagonal Pt. Specifically, in addition to the lattice spacing of 0.383 nm assigned to the W 18 O 49 nanowire (010), another lattice fringe spacing of 0.221 nm could also be detected, which was exactly the Pt(111) plane ( Figure 2 e, f). In addition, Figure 2 The high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image shown in g further indicated that the Pt(111) crystal plane grew along the W 18 O 49 (010) crystal plane. The corresponding elemental mapping showed uniform distributions of Pt, W, and O elements in WOx-Pt ( Figure 2 h, i).

[0050] The present invention further studied the crystal structure of WOx-Pt by X-ray diffraction (XRD), as Figure 3 shown. WOx-Pt showed distinct monoclinic W 18 O 49Diffraction peaks: PDF#97 - 001 - 5254, but there are no diffraction peaks related to Pt nanoparticles, probably due to their small size and high dispersion. In addition, the (010) diffraction peak of WOx - Pt shows a small negative shift at 23.4°, indicating lattice expansion, which may be due to 18 O 49 interstitial doping of some Pt atoms in the monoclinic crystal structure. In addition, the peak intensity value of WOx - Pt decreases slightly, which is attributed to the uniformity of Pt loading and the 18 O 49 regularity of the crystal structure.

[0051] After confirming the crystal structure, the present invention used X - ray photoelectron spectroscopy (XPS) to study the chemical composition and valence electron states of WOx - Pt. The XPS measurement spectrum ( Figure 4 a) shows the presence of C, O, W, and Pt elements in the WOx - Pt composite. The Pt 4f XPS spectrum shows two pairs of peaks ( Figure 4 b), where the main peaks at 71.6 eV and 74.8 eV can be attributed to Pt4f of Pt0 2 / 7 and Pt4f 2 / 5 , and the remaining peaks come from PtⅡ - IV, indicating that the Pt precursor has been successfully reduced to metallic Pt. On the other hand, the high - resolution W4f spectrum of the WOx - Pt composite shows the presence of W 5+ and W 6+ species ( Figure 4 c), which is consistent with the distorted edge - sharing crystal structure of W 18 O 49 , forming low - valence W elements and metal W - W interactions in the lattice. The W 6+ / W 5+ ratio in WOx - Pt is 7.73, which is between the ratios of the original W 18 O 49 (3.35) and WO3 (19). This partial oxidation of W 5+ to W 6+ may be accompanied by an increase in related vacancies or electron transfer ( Figure 4 e). The peaks in the O1s XPS spectrum can be assigned to lattice oxygen (Olat: 530.59 eV), oxygen vacancies (Ovac: 531.89 eV), and surface - adsorbed oxygen (Osur: 533.19 eV) ( Figure 4 d). The present invention also carried out electron paramagnetic resonance (EPR) spectroscopy tests. Compared with the original W 18 O 49In contrast, the increased EPR intensity of the Pt-doped counterpart (i.e., higher unpaired electron / Ovac content) can be attributed to Pt clusters located next to charge-compensating oxygen vacancies, resulting in a lower oxidation state of Pt atoms, which is consistent with the XPS results ( Figure 5 ).

[0052] Experimental Example 2: POD enzyme and OXD enzyme-like activity tests of spiky WOx-Pt catalysts

[0053] 1. Experimental method

[0054] (1) Peroxidase (POD) activity was determined by colorimetry. 5 μL of the catalyst (4 mg·mL -1 ), 25 μL of 3,3,5,5-tetramethylbenzidine (TMB) (10 mg·mL -1 ), and 25 μL of H2O2 (0.1 M) were added to 2 mL of sodium acetate-acetic acid (NaOAc / HOAc) buffer [100 mM (pH 4.5 or 5.6)]. The catalytic oxidation of TMB (oxTMB) was studied by measuring the absorption change of the oxidized TMB at λmax = 652 nm (ε = 39,000 M -1 cm -1 ). Unless otherwise stated, the POD activity was carried out in air-saturated buffer. Km and Vmax were calculated using the double-reciprocal Lineweaver-Burk plot of the Michaelis-Menten equation V = Vmax×[S] / (Km + [S]), and TON = Vmax / [E], where [S] is the H2O2 concentration and [E] is the molar concentration of the metal in the nanozyme.

[0055] (2) The method for testing the oxidase (OXD) activity was similar to that for testing the peroxidase activity, except that H2O2 was absent. 15 μL of the catalyst (4 mg·mL -1 ), and 25 μL of TMB (10 mg·mL -1 ) were added to 2 mL of sodium acetate-acetic acid (NaOAc / HOAc) buffer [100 mM (pH 4.5)] for testing.

[0056] (3) Determination of ·O2 - by dihydroethidium (HE) method. HE is a specific probe that can react with ·O2 - to produce fluorescent ethidium, which is excited at 470 nm and emits at 610 nm. First, 1.5 mL of the catalyst (100 μg·mL -1 ) solution was mixed with 1.5 μL of H2O2 (0.1 M) at 37 °C for 40 min, and then 1.5 mL of the HE-ethanol solution (1 mg·mL -1)。After that, the solution was vortexed and left undisturbed for 40 minutes before fluorescence measurement (Synergy Mx).

[0057] (4) Determination by diphenylamine (DPA) method 1 O2. 25 μL of catalyst (4 mg·mL -1 ), 25 μL of H2O2 (0.1 M) and 100 μL of DPA-DMSO solution were added to 2 mL of DMSO solution to detect 1 O2, and analyzed with a UV spectrophotometer. The decomposition rate of DPA under different US irradiation times (1.0 MHz, 2.5 W cm -2 ) and laser excitation times (14 A) was recorded, and the decomposition rate was quantified by the change in relative absorbance of DPA at 378 nm. Relative absorbance = (Absorbance at λ = 378 nm in the control group - Absorbance at λ = 378 nm in the experimental group) / Absorbance at λ = 378 nm in the control group × 100%.

[0058] 2. Experimental results

[0059] In this experiment, the peroxidase-like and oxidase-like activities of the WOx-Pt catalyst for generating ROS were tested by the classical 3,3,5,5-tetramethylbenzidine (TMB) colorimetric method. In the presence of H2O2, WOx-Pt can catalyze the oxidation of TMB to generate the blue oxidant - TMB(ox-TMB), whose characteristic absorbance is at 652 nm, while the absorbance of the W 18 O 49 substrate is particularly low( Figure 6 a). Next, the maximum reaction rate (V max ), Michaelis constant (K m ), and turnover number (TON, the maximum number of converted substrates per unit active catalytic center) were further calculated. According to the Michaelis-Menten curve, when H2O2 was used as the substrate, the V max (V ma x = 6.953) of WOx-Pt was almost 26 times that of WOx (V max = 0.267). At the same time, for TMB, the V max (V max = 6.65) of WOx-Pt was also better than that of WOx (V max (V max = 0.284). The K m value of WOx-Pt (H2O2 K m = 2.477, TMB K m = 0.799) was much lower than that of WOx (H2O2 K m = 8.323, TMB K m = 8.323, TMB Km = 2.2) ( Figure 6 b, c). These excellent catalytic performances indicate that, compared with WOx, the Pt sites in WOx-Pt have stronger catalytic activity and affinity for H2O2 and TMB.

[0060] In addition, the present invention compared the catalytic activity of WOx-Pt with recently reported similar mimics (including metal oxides, metal nanoparticles, single-atom enzyme mimics, and metal-organic frameworks). The results showed that WOx-Pt exhibited extremely high max V and TON values ( Figure 6 d). Notably, WOx-Pt can also mimic the catalytic properties of OXD-like and effectively catalyze the conversion of oxygen to ROS in an O2-dependent manner. The V max value of TMB was 5.49 ( Figure 6 e).

[0061] To clarify the catalytic mechanism of the WOx-Pt biocatalyst, the present invention determined the types of radical products through radical quenching experiments and fluorescence detection probe experiments. The generation of ·O2 - was demonstrated by the ·O2 - specific probe hydroethidine (HE). The WOx-Pt-HE solution showed an obvious emission peak at 610 nm ( Figure 6 f). 1 The generation of 1O2 was demonstrated by measurement with 9,10-diphenylanthracene (DPA) dye. DPA was 1 oxidized to 9,10-diphenyloxanthone (DPO2) by 1O2. As the reaction time increased, the characteristic absorption peak of DPA near 378 nm gradually decreased with the oxidation of DPO2 ( Figure 6 g). In addition, the present invention also adopted the active site poisoning method to verify the importance of Pt clusters in the WOx-Pt biocatalyst using potassium thiocyanate reagent (KSCN, which can bind to the metal center to form an inactive chelate complex). After adding KSCN, the peroxidase-like and oxidase-like activities of the WOx-Pt biocatalyst were significantly reduced, thus confirming that the Pt sites in WOx-Pt are the active centers for scavenging ROS ( Figure 6 h).

[0062] The above experimental results show that the WOx-Pt catalyst provided by the present invention exhibits excellent POD enzyme and OXD enzyme mimic activities and has a super strong ROS generation ability.

[0063] Experimental Example 3: Specific endoplasmic reticulum stress induced by spiky WOx-Pt catalyst in vitro

[0064] 1. Experimental method

[0065] (1) Western blot analysis:

[0066] After treating B16 / F10 cells with different concentrations of PBS, WOx, and WOx-Pt for 12 hours, Western blot was used to analyze the expression of caspase-3 and CHOP (C / ebp homologous protein-10) proteins. The specific operations were as follows: Total proteins were extracted from B16 / F10 cells treated with different concentrations of WOx (100 μg / mL) and WOx-Pt (20, 50, 80, or 100 μg / mL), and then separated by 6-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Beyotime, China). After blocking with TBST containing 5% skim milk powder, the PVDF membrane was incubated with primary antibodies (β-actin, CHOP, cleaved-caspase-3, pro-caspase-3, and CRT) overnight at 4°C. Then, the blots were washed three times with TBST and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 60 minutes. An automatic chemiluminescence analysis system was used to visualize the specific proteins in each group. Finally, the intensity of the Western blot was quantified using Image J software.

[0067] (2) Preparation of samples for biological electron microscopy:

[0068] B16 / F10 cells were seeded into 6-cm cell culture dishes and then treated with PBS or 50 μg / mL WOx-Pt, respectively. After 12 hours, the cells were digested and centrifuged, resuspended with 2.5% glutaraldehyde, fixed for 3 hours, and then subjected to TEM electron microscopy imaging.

[0069] 2. Experimental results

[0070] CHOP is a pro-apoptotic protein overexpressed after endoplasmic reticulum stress. The results showed that PBS treatment had little effect on CHOP activation, while different concentrations of WOx-Pt upregulated the expression of CHOP protein to varying degrees ( Figure 7 a, b). 50 μg / mL WOx-Pt significantly upregulated the expression of CHOP protein, indicating that there was severe endoplasmic reticulum stress and ER-related apoptosis in cells after WOx-Pt treatment. In addition, all treatment groups activated the mitochondrial apoptotic protein Caspase-3, and 50 μg / mL WOx-Pt induced the highest level of cleaved Caspase-3 and the lowest level of pro-caspase-3 (as the precursor of cleaved Caspase-3, pro-caspase-3 needs to be cleaved into cleaved Caspase-3 to have biological activity) ( Figure 7a, c). The above results indicate that there is crosstalk between endoplasmic reticulum stress and mitochondria, and endoplasmic reticulum stress can induce mitochondria-related cell death.

[0071] Meanwhile, through transmission electron microscopy observation of cell samples with different treatment methods in this experiment, it was found that the treatment with 50 μg / mL WOx-Pt significantly enlarged the endoplasmic reticulum lumen of B16 / F10 cells (indicated by arrows), further demonstrating the most obvious endoplasmic reticulum stress state ( Figure 7 d).

[0072] The above experimental results indicate that the treatment with WOx-Pt can induce endoplasmic reticulum stress and ER-related apoptosis.

[0073] Experimental Example 4: Application of Spiky WOx-Pt Catalyst in Immunotherapy against Tumors

[0074] 1. Experimental Method

[0075] (1) Verification of the immunotherapy effect on tumor-bearing mice (B16 / F10 cells):

[0076] B16 / F10 cells were subcutaneously injected into the right hind limb of C57BL / 6 mice at a concentration of 1×10 6 cells per 100 μL. When the tumor volume reached approximately 50 - 100 mm 3 , the mice were randomly divided into 4 groups (n = 5 in each group): Control group, anti-PD-L1 group, WOx-Pt group, and WOx-Pt + anti-PD-L1 group. The treatment methods for each group were as follows:

[0077] Control group: Tumor-bearing mice were injected intratumorally with PBS, once every 2 days, for a total of 3 injections;

[0078] anti-PD-L1 group: Tumor-bearing mice were intraperitoneally injected with anti-PD-L1 (100 μg / mouse), once every 2 days, for a total of 3 injections;

[0079] WOx-Pt group: Tumor-bearing mice were injected intratumorally with WOx-Pt (10 mg / kg), once every 2 days, for a total of 3 injections;

[0080] WOx-Pt + anti-PD-L1 group: Tumor-bearing mice were injected intratumorally with WOx-Pt (10 mg / kg), and anti-PD-L1 (100 μg / mouse) was intraperitoneally injected into the mice at the intervals of WOx-Pt treatment, once every 2 days, for a total of 3 injections.

[0081] The tumor size and body weight were monitored daily. The tumor size was measured using calipers, and the tumor volume was calculated and recorded according to the formula of length × width 2 × 0.5.

[0082] (2) Tumor-infiltrating lymphocyte analysis:

[0083] After the treatment was completed, the tumors of each mouse were removed and cut into small pieces, and then ground into tissue homogenates. After lysing red blood cells and centrifuging, the obtained single cells were labeled with fluorescently labeled flow antibodies. Then, the situation of tumor-infiltrating lymphocytes was analyzed by flow cytometry. Antibodies such as anti-CD3-PE, anti-CD4-APC / Cy7, anti-CD8-FITC, anti-F4 / 80-APC, anti-CD80-FITC, anti-CD206-PerCP / Cy5.5, anti-CD45-PE / Cy7, anti-CD62L-PerCP / Cy5.5, anti-CD44-APC were all diluted according to the instructions before use.

[0084] 2. Experimental results

[0085] This experiment evaluated the synergistic effect of WOx-Pt and immune checkpoint blockade drugs (anti-PD-L1 antibody, also known as anti-PD-L1) in systemic anti-tumor immunotherapy ( Figure 8 a). As Figure 8 shown in b and c, WOx-Pt treatment successfully inhibited the primary tumor in the initial stage, which was attributed to sufficient ICD induction and activation of anti-tumor immunity. However, as the disease course prolonged, single WOx-Pt treatment could not achieve the ideal therapeutic effect (the tumor volume was 451.78 mm 3 ) after the treatment was completed. However, after synergistic action with the anti-PD-L1 antibody, compared with the control group (the tumor volume was 1162 mm 3 ) after the treatment was completed, the subcutaneous tumor in the WOx-Pt + anti-PD-L1 group was significantly inhibited (the tumor volume was 328 mm 3 ) ( Figure 8 c). All mice were sacrificed on the 7th day after tumor treatment, and the weight of the excised tumor tissue ( Figure 8 e) further confirmed the synergistic treatment effect. There was no significant difference in the body weight of mice in each group ( Figure 8 f), indicating that WOx-Pt has potential therapeutic effects and minimal side effects. These results show that compared with single use of anti-PD-L1 antibody or WOx-Pt treatment, the combined use of anti-PD-L1 antibody and WOx-Pt treatment has significantly enhanced anti-tumor efficacy, confirming that WOx-Pt can enhance the anti-tumor efficacy of PD-L1 blockade treatment. It shows that WOx-Pt synergizes with immune checkpoint blockade drugs and can effectively stimulate the systemic anti-tumor immune response.

[0086] Table 1. Figure 8Average volume data of tumor tissues in each group after treatment in c

[0087]

[0088]

[0089] Table 2. Figure 8 Average weight data of tumor tissues excised on the 7th day after treatment in each group in e

[0090] Grouping Average weight of tumor tissue (g) Control group 1.81 anti-PD-L1 group 0.98 WOx-Pt group 0.6 WOx-Pt + anti-PD-L1 group 0.17

[0091] Next, in this experiment, the mechanism of WOx-Pt-induced systemic anti-tumor immunity was investigated by analyzing the infiltration of white blood cells. As is well known, CD8 + T cells (cytotoxic T lymphocytes, CTLs, CD3 + CD8 + T cells) can release perforin, granzyme, and granulysin to kill tumor cells, and CD4 + T cells (CD3 + CD4 + T cells) play a crucial role in regulating adaptive immunity. Therefore, in this experiment, CD8 + and CD4 + T cells in B16 / F10 subcutaneous tumors were evaluated on the 7th day after treatment. The results showed that although the treatment of mice with anti-PD-L1 or WOx-Pt alone led to a moderate increase in CD8 + and CD4 + T cells, the treatment of mice with WOx-Pt + anti-PD-L1 had the highest promoting effect on the intratumoral infiltration of CD8 + and CD4 + T cells, which were 1.46 times and 1.67 times that of the control group, respectively ([[]] Figure 8 h, 8i and Figure 9 a).

[0092] To further verify the advantages of using WOx-Pt, this experiment evaluated the effect of WOx-Pt on reprogramming tumor-associated macrophages (TAMs) from the tumor-promoting M2 phenotype to the tumor-suppressing M1 phenotype. The results showed that the proportion of M1 macrophages increased after treatment with WOx-Pt alone (17.2% in the control group and 31.5% in the WOx-Pt group), and the proportion of M2 macrophages decreased (52.1% in the control group and 45.6% in the WOx-Pt group), with no significant difference compared with the group treated with anti-PD-L1 alone, which may be due to the role of ROS production. However, compared with the control group, the percentage of M1 macrophages in the WOx-Pt + anti-PD-L1 group increased significantly (up to 67.7%), and the percentage of M2 macrophages decreased significantly (down to 6.81%) ( Figure 8d and Figure 9 b, 9c). These results are attributed to the role of WOx-Pt in generating ROS and inducing ICD, as well as the synergistic effect of immune checkpoint blockade, which integrated to improve the immune microenvironment, reprogrammed TAMs towards the tumor-killing M1 phenotype, and thus enhanced the anti-tumor effect.

[0093] As is well known in the art, cytotoxic T lymphocytes can release interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) to promote the immune microenvironment. In this experiment, the CD3 + CD8 + TNF-α + T cells and IFN-γ-positive CD3 + CD8 + IFN-γ + T cells in B16 / F10 subcutaneous tumors were then studied. Notably, after WOx-Pt + anti-PD-L1 treatment, the levels of CD3 + CD8 + IFN-γ + T cells and CD3 + CD8 + TNF-α + T cells in the tumor increased significantly ( Figure 8 j and Figure 9 d). In addition, the number of Tregs (CD3 + CD4 + Foxp3 + ) in the WOx-Pt + anti-PD-L1 group was reduced compared with the control group ( Figure 9 e). These results indicate that the combination of WOx-Pt and anti-PD-L1 can significantly promote the tumor infiltration of anti-tumor lymphocytes.

[0094] Flow cytometry was also used in this experiment to detect the induction of dendritic cell maturation in tumor-draining lymph nodes. Mature dendritic cells (DCs) were represented by CD11c + CD80 + CD86 + cells. The results showed that the treatment with WOx-Pt + anti-PD-L1 had the best effect on promoting DC maturation, and the expressions of CD80 and CD86 were significantly upregulated compared with other treatment groups ( Figure 9 f), indicating the best effect on DC maturation and activation, and having great potential to stimulate subsequent tumor-specific immune responses.

[0095] The above results indicate that, based on its excellent multi-enzyme mimicking catalytic behavior, WOx-Pt can significantly enhance tumor immunogenicity, activate the immune response, and synergistically inhibit primary and metastatic tumors with immune checkpoint blockade drugs by inducing ICD, promoting T cell infiltration and DC maturation, and stimulating the release of inflammatory factors.

Claims

1. A combined drug for treating primary or metastatic tumors, characterized in that: The tumor is melanoma, which contains a composite catalyst and an anti-PD-L1 antibody for simultaneous or separate administration in the same or different dosage unit formulations, as well as a pharmaceutically acceptable carrier; The composite catalyst is a composite material prepared from platinum tetrachloride and W 18 O 49 as raw materials. The mass ratio of the platinum tetrachloride to W 18 O 49 is (12 - 13):100; The preparation method of the said W 18 O 49 comprises the following steps: Dissolve tungsten hexacarbonyl in an ethanol solution, place it in an autoclave, seal it, and heat it at 170-190 °C for 22-26 h, then dry it to obtain W 18 O 49 ; The preparation method of the composite catalyst comprises the following steps: dispersing a transition metal oxide and a platinum salt in an ethanol solution, reacting at room temperature for 10-14 h, centrifuging, taking the precipitate, washing, and drying to obtain the composite catalyst; 2. The combined medicament according to claim 1, wherein: The platinum tetrachloride and W 18 O 49 have a mass ratio of 12.7:100.

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