A noble metal alloy nanoscale enzyme and a preparation method and application thereof

Abstract

The application discloses a noble metal alloy nano-enzyme and a preparation method and application thereof, and belongs to the technical field of nano-enzymes and tumor treatment. The loaded noble metal alloy nano-enzyme is composed of weakly oxidized porous silicon nanoparticles and noble metal alloy nano-enzymes loaded on the surface and in the pores of the weakly oxidized porous silicon nanoparticles; the weakly oxidized porous silicon nanoparticles are obtained by oxidizing porous silicon nanoparticles through a phosphate buffer solution, deionized water, an alkaline borate or phosphate solution. The loaded noble metal alloy nano-enzyme has excellent four-fold simulated enzyme activity (CAT-like, OXD-like, POD-like and GSHOx-like), good storage stability and in-vivo biodegradability, and is of great significance for tumor treatment, especially mild photothermal tumor treatment and nano-catalytic tumor treatment.

Description

Technical Field

[0001] This invention relates to the fields of nanozymes and tumor therapy, and particularly to a noble metal alloy nanozyme, its preparation method, and its application. Background Technology

[0002] Photothermal therapy (PTT) is a spatiotemporally controllable physical therapy that uses photothermal agents to convert near-infrared light energy into heat energy to ablate tumors. Compared to traditional photothermal therapy (>50°C), mild photothermal therapy (m-PTT) offers greater advantages in reducing treatment side effects and improving patient comfort. m-PTT typically raises the local tumor temperature to 38-43°C to induce cancer cell death while preventing high-temperature damage to surrounding normal tissues. However, mild photothermal irradiation often leads to the overexpression of heat shock proteins (HSPs), which promote protein refolding and repair of heat protein damage, thereby enhancing the heat resistance of tumor cells to m-PTT. To address this issue, several small-molecule inhibitors of heat shock proteins (17-AGG, quercetin, OGX-427, etc.) have been developed. However, high biotoxicity, low selectivity, and drug resistance limit the clinical translation of these HSP inhibitors. Therefore, finding effective methods to inhibit HSP expression is crucial for improving the efficacy and competitiveness of m-PTT in cancer treatment.

[0003] Nanozymes are nanoformulations with reaction kinetics and catalytic properties similar to natural enzymes, and they can inhibit HSP expression. Therefore, they provide a feasible pathway to promote m-PTT. Specifically, peroxidase-like nanozymes can decompose endogenous hydrogen peroxide (H2O2) in the tumor microenvironment (TME), generating reactive oxygen species (ROS), thereby cleaving HSPs. Furthermore, nanozymes with peroxidase (POD)-like and glutathione oxidase (GSHOx)-like activities can induce ROS generation and deplete the antioxidant glutathione (GSH) in the TME. This process ultimately leads to ferroptosis characterized by the accumulation of lipid peroxides (LPO). The large accumulation of LPO can further generate aldehyde products (such as malondialdehyde), which react with the amino acid residues of HSP molecules, causing HSP denaturation. Among these, noble metal alloy nanozymes, compared with single metal nanozymes, exhibit alloying effects, a more rational electron density distribution, and a richer catalytic site. Platinum (Pt) can be alloyed with other metals (such as ruthenium, palladium, gold, silver, and titanium) to effectively modulate electronic structure and optimize catalytic performance. Existing technology discloses a palladium-platinum alloy nanozyme that can induce tumor ferroptosis. Simultaneously, the PdPt nanozyme exhibits activities similar to catalase (CAT) and oxidase (OXD), converting H₂O₂ into oxygen (O₂), thereby reversing the hypoxic state in tumor tissue. The O₂ is then oxidized to ROS, killing tumor cells.

[0004] However, most existing alloy nanozyme synthesis methods require high-temperature conditions, complicating the preparation process. High-temperature treatment typically necessitates specialized equipment and strict temperature control procedures, which not only increases the experimental difficulty but may also lead to nanoparticle aggregation or grain growth, affecting product uniformity and catalytic performance. Chemical reduction processes rely on external reducing agents such as hydrogen, sodium borohydride, or ascorbic acid. While reducing noble metal ions, these reducing agents may generate byproducts on or around the nanoparticles, affecting the purity and activity of the final synthesized product. Furthermore, due to the high surface energy of noble metal alloy nanozymes, aggregation is highly likely, leading to a sharp decline in catalytic activity.

[0005] Therefore, we still need to develop dual-mode antitumor pathways involving ferroptosis and m-PTT using noble metal alloy nanozymes to find the optimal option for enhancing m-PTT. Furthermore, due to the high surface energy of noble metal alloy nanozymes, they are prone to aggregation, leading to a sharp decline in catalytic activity. The clearance and metabolism of noble metal alloy nanozymes larger than 5 nm in vivo should also be considered.

[0006] The use of supports not only ensures the dispersion of noble metal alloy nanozymes but also improves their bioavailability. Electrochemically etched porous silicon (PSi) is a support with abundant pore structure and surface modification potential, which can be used as a support to enhance the catalytic performance of dual nanozymes through the pore confinement effect. Furthermore, PSi supports exhibit good biodegradability, enabling the supported nanozymes to be effectively metabolized and excreted from the body. Currently, PSi-based composites have been widely applied in the biomedical field, such as sensing and monitoring, antibacterial wound treatment, and tumor therapy.

[0007] Therefore, it is of great significance to research and develop a novel noble metal alloy nanozyme for tumor treatment. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a noble metal alloy nanozyme, its preparation method and application. The noble metal alloy nanozyme is uniformly dispersed and has good quadruple nanozyme activity (CAT-like, OXD-like, POD-like, and GSHOx-like), storage stability, and excellent in vivo degradation ability and biosafety.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] This invention provides a supported noble metal alloy nanozyme, which is composed of weakly oxidized porous silicon nanoparticles and noble metal alloy nanozymes supported on their surface and in their pores.

[0011] The weakly oxidized porous silicon nanoparticles are obtained by oxidizing porous silicon nanoparticles with phosphate buffer solution, deionized water, alkaline borate or phosphate solution.

[0012] The weakly oxidized porous silica nanoparticles serve as a novel carrier and a reducing agent for nanozyme precursors. Through their pore confinement effect, they enhance the adsorption capacity of noble metal alloy nanozymes distributed within the pores for substrates, thereby promoting substrate catalysis.

[0013] Preferably, the precious metal alloy nanozyme is selected from palladium-platinum precious metal alloy nanozyme, gold-silver precious metal alloy nanozyme, or silver-palladium precious metal alloy nanozyme; more preferably, it is palladium-platinum precious metal alloy nanozyme.

[0014] In the supported noble metal alloy nanozyme of the present invention, a tight semiconductor-metal heterogeneous interface is formed between the noble metal alloy nanozyme and the Si-OH bonds on the surface of the weakly oxidized porous silicon nanoparticles, which promotes electron-hole separation and thus significantly improves the activity of the supported noble metal alloy nanozyme.

[0015] Furthermore, the unique nanoporous structure of the weakly oxidized porous silicon nanoparticles can improve the interaction between the reaction substrate and the noble metal alloy nanozyme, thereby enhancing the catalytic performance of the noble metal alloy nanozyme. Under mild photothermal stimulation, the simulated activity of the supported noble metal alloy nanozyme is further improved.

[0016] Preferably, the noble metal alloy nanozyme of this invention is selected from palladium-platinum noble metal alloy nanozymes, and the Pd content in the weakly oxidized porous silica of the supported noble metal alloy nanozyme is 80-85 μg / mg, and the Pt content is 35-50 μg / mg. In some specific embodiments of this invention, inductively coupled plasma optical emission spectrometry (ICP-OES) analysis revealed that the Pd content in the weakly oxidized porous silica was 84.13 ± 0.2117 μg / mg, and the Pt content was 39.18 ± 0.3023 μg / mg.

[0017] Preferably, the mass ratio of palladium to platinum in the palladium-platinum alloy nanoenzyme is (2-2.5):1, more preferably (2.1-2.2):1.

[0018] This invention also provides a method for preparing the above-mentioned supported noble metal alloy nanozyme, comprising the following steps:

[0019] (1) Porous silicon nanoparticles and phosphate buffer solution were mixed and reacted to obtain weakly oxidized porous silicon nanoparticles;

[0020] (2) The palladium source, platinum source, weakly oxidized porous silicon nanoparticles and water are mixed and subjected to an in-situ reduction reaction to obtain the supported noble metal alloy nanozyme.

[0021] This invention utilizes the mild reducing activity of the Si-OH bonds abundant on the surface of weakly oxidized porous silicon nanoparticles to obtain the supported noble metal alloy nanozyme through one-step in-situ reduction deposition, thereby making the noble metal alloy nanozyme highly uniformly dispersed on the surface of the weakly oxidized porous silicon nanoparticles.

[0022] Preferably, the pH value of the phosphate buffer solution is 7-7.4.

[0023] Preferably, the ratio of the porous silicon nanoparticles to the phosphate buffer solution is 1g:(10-300)mL; more preferably, it is 1g:100mL.

[0024] Preferably, the reaction temperature in step (1) of this invention is 25°C - 50°C; more preferably, it is 37°C.

[0025] Preferably, the molar ratio of palladium in the palladium source to platinum in the platinum source is 1:(0.5-1.5); more preferably, it is 1:(0.8-1.2); and even more preferably, it is 1:1.

[0026] Preferably, the palladium source of the present invention is selected from one or more of chloropalladium acid, palladium chloride, and sodium chloropalladium; more preferably, it is chloropalladium acid.

[0027] Preferably, the platinum source is selected from one or more of chloroplatinic acid, potassium chloroplatinate, and ammonium chloroplatinate; more preferably, it is chloropalladium acid.

[0028] The present invention also provides the application of the above-mentioned supported noble metal alloy nanozyme or the supported noble metal alloy nanozyme prepared by the above-mentioned preparation method in the preparation of tumor treatment drugs.

[0029] The tumor treatment drugs include, but are not limited to, nanocatalytic tumor treatment drugs and photothermal tumor treatment drugs.

[0030] The aforementioned photothermal therapy drug for treating tumors can effectively exert the therapeutic effect of mild photothermal therapy (m-PTT) by inhibiting the expression of HSPs.

[0031] The supported noble metal alloy nanozyme of the present invention or the supported noble metal alloy nanozyme prepared by the above preparation method reverses the hypoxic state in the tumor microenvironment by converting endogenous H2O2 into oxygen.

[0032] The supported noble metal alloy nanozyme described in this invention can also generate a large amount of reactive oxygen species, consume the antioxidant glutathione, accumulate lipid peroxides, and ultimately induce iron allergy to inhibit the expression of HSPs.

[0033] Furthermore, mild photothermal stimulation can enhance the ferroptosis process of the supported noble metal alloy nanozyme, ultimately significantly inhibiting tumor development in vivo. In addition, the supported noble metal alloy nanozyme of this invention also exhibits good storage stability and in vivo biodegradability, demonstrating high biosafety.

[0034] Compared with existing technologies, the supported noble metal alloy nanozyme provided by this invention consists of weakly oxidized porous silica nanoparticles and a noble metal alloy nanozyme supported on their surface and within their pores. The weakly oxidized porous silica nanoparticles are obtained by oxidizing porous silica nanoparticles with phosphate buffer solution, deionized water, alkaline borate, or phosphate solution. The supported noble metal alloy nanozyme exhibits excellent quadruple enzyme-mimicking activities (CAT-like, OXD-like, POD-like, and GSHox-like), good storage stability, and in vivo biodegradability, which is of great significance for tumor therapy, especially mild photothermal tumor therapy and nanocatalytic tumor therapy. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the preparation of the palladium-platinum alloy nanoenzyme-supported weakly oxidized nanoporous silicon powder obtained in Example 1.

[0036] Figure 2 Attenuated total reflectance infrared spectra of different silicon materials (porous silicon, peroxide porous silicon, weakly oxidized porous silicon PSi, and silicon dioxide);

[0037] Figure 3 Transmission electron microscopy (TEM) images of porous silicon supported by palladium-platinum alloy nanozymes and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes.

[0038] Figure 4 Figure a shows the photothermal response curves of the control group, porous silicon, weakly oxidized porous silicon, porous silicon supported by palladium-platinum alloy nanozymes, and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes; Figure b shows the photothermal response curves of weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes after being irradiated by lasers with different current densities; Figure c shows the temperature change curves of weakly oxidized porous silicon supported by nanozymes after being exposed to five near-infrared light irradiation-cooling cycles.

[0039] Figure 5 Figure a shows the amount of oxygen produced after different materials react with H2O2 at 37 °C for 5 minutes; Figure b shows the double reciprocal curves of porous silicon supported by palladium-platinum alloy nanozymes and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes as CAT-like structures without near-infrared irradiation; Figure c shows the double reciprocal curves of porous silicon supported by palladium-platinum alloy nanozymes and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes as CAT-like structures with near-infrared irradiation; Figure d shows the double reciprocal curve of palladium-platinum alloy nanozymes without the weakly oxidized porous silicon support as CAT-like structures.

[0040] Figure 6 Figure a shows the amount of oxygen produced after different materials react with TMB at 37 °C for 5 minutes; Figure b shows the double reciprocal curves of porous silicon supported by palladium-platinum alloy nanozymes and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes as OXD-like under the condition of no near-infrared irradiation; Figure c shows the double reciprocal curves of porous silicon supported by palladium-platinum alloy nanozymes and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes as OXD-like under the condition of near-infrared irradiation; Figure d shows the double reciprocal curve of palladium-platinum alloy nanozymes without the weakly oxidized porous silicon support as OXD-like.

[0041] Figure 7Figure a shows the absorption spectra of different materials reacting with H2O2 and TMB at 37 °C for 5 minutes; Figure b shows the double reciprocal curves of porous silicon supported by palladium-platinum alloy nanozymes and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes as POD-like structures under conditions without near-infrared irradiation; Figure c shows the double reciprocal curves of porous silicon supported by palladium-platinum alloy nanozymes and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes as POD-like structures under conditions with near-infrared irradiation; Figure d shows the double reciprocal curves of palladium-platinum alloy nanozymes without the weakly oxidized porous silicon support as POD-like structures.

[0042] Figure 8a shows the amount of oxygen produced after different materials react with GSH and DTNB at 37 °C for 5 minutes; Figure 8b shows the double reciprocal curves of porous silicon supported by palladium-platinum alloy nanozymes and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes as GSHOx-like under no near-infrared irradiation; Figure 8c shows the double reciprocal curves of porous silicon supported by palladium-platinum alloy nanozymes and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes as GSHOx-like under near-infrared irradiation; Figure 8d shows the double reciprocal curve of palladium-platinum alloy nanozymes without the weakly oxidized porous silicon support as GSHOx-like.

[0043] Figure 9a shows the density of electronic states (DOS) of porous silicon supported by palladium-platinum alloy nanozymes; Figure b shows the density of electronic states (DOS) of weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes.

[0044] Figure 10 Cell viability of 4T1 cells after treatment with porous silica, weakly oxidized porous silica, palladium-platinum alloy nanozyme-loaded porous silica, and palladium-platinum alloy nanozyme-loaded weakly oxidized porous silica under near-infrared light irradiation.

[0045] Figure 11 The figure shows the oxygen production in 4T1 cells after different treatments, using [Ru(dpp)3]Cl2 as a fluorescent probe.

[0046] Figure 12 Figure showing the ROS generation in 4T1 cells after different treatments using DCFH as a fluorescent probe.

[0047] Figure 13 The ratio of glutathione to oxidized glutathione in 4T1 cells after different treatments;

[0048] Figure 14 Malondialdehyde (MDA) levels in 4T1 cells after different treatments;

[0049] Figure 15 Western imprinted bands of HSPs in 4T1 cells after different treatments;

[0050] Figure 16To assess the viability of 4T1 cells after applying freshly prepared palladium-platinum alloy nanozymes loaded with weakly oxidized porous silica or palladium-platinum alloy nanozymes loaded with weakly oxidized porous silica stored at room temperature for one month under near-infrared light irradiation.

[0051] Figure 17 This is a graph showing the changes in tumor volume in different groups after different treatments.

[0052] Figure 18 shows the accumulation of Pd (Figure a), Pt (Figure b), and Si (Figure c) elements in major organs and tumors 1 day and 14 days after intratumoral injection of palladium-platinum alloy nanozyme-loaded weakly oxidized porous silica.

[0053] Figure 19 shows the H&E staining, Tunel staining, Ki-67 staining, HIF-1α and HSP70 staining results of tumors in each group of tumor-bearing mice after different treatments. Detailed Implementation

[0054] To further illustrate the present invention, the following detailed description of a noble metal alloy nanozyme, its preparation method, and its application, in conjunction with embodiments, is provided by the present invention.

[0055] Example 1

[0056] (1) Preparation of weakly oxidized porous silicon nanoparticles

[0057] Porous silicon (PSi) films are prepared by electrochemical etching of silicon wafers.

[0058] First, the silicon wafer is immersed in an etching solution containing hydrofluoric acid and 95% ethanol (v:v = 4:1), and then etched at 77 mA / cm. 2 Etching was performed at a current density of 600 seconds. The PSi film was then etched for 180 seconds with a 3.3% hydrofluoric acid-ethanol solution to peel it off from the silicon substrate. The PSi film was then peeled off from the silicon substrate by etching with a 3.3% hydrofluoric acid-ethanol solution for 180 seconds. Fresh porous silicon (PSi) nanoparticles were obtained after ultrasonically breaking down the PSi layer (500 W) for 30 minutes. The PSi nanoparticles were placed in PBS solution (pH=7.4), shaken at 37°C for 2 hours, and then centrifuged (10000 rpm, 10 minutes) to collect the weakly oxidized porous silicon nanoparticles.

[0059] (2) Preparation of weakly oxidized porous silica supported on palladium-platinum alloy nanoenzymes

[0060] H₂PdCl₄ (1.0 mM) and H₂PtCl₆ (1.0 mM) were mixed and added to an aqueous solution of weakly oxidized porous silica nanoparticles (2.5 mg / mL). The solution was shaken at room temperature for 10 minutes, then centrifuged at 10,000 rpm and washed three times with water to remove residual palladium(II) and platinum(IV), resulting in a palladium-platinum alloy nanozyme-loaded weakly oxidized porous silica solution. Finally, the solution was freeze-dried to obtain palladium-platinum alloy nanozyme-loaded weakly oxidized porous silica powder (wherein, by inductively coupled plasma optical emission spectrometry (ICP-OES) analysis, the palladium loading was 84.13 ± 0.2117 μg / mg, the platinum loading was 39.18 ± 0.3023 μg / mg, and the mass ratio of palladium to platinum in the palladium-platinum alloy was 2.1:1). The weakly oxidized porous silica was the weakly oxidized porous silica nanoparticles prepared in step (1) above.

[0061] Comparative Example 1

[0062] (1) First, the silicon wafer is placed in an etching solution containing hydrofluoric acid and 95% ethanol (v:v = 4:1), and then the etching is performed at 77 mA / cm. 2 Etching was performed at a current density of 600 seconds. The PSi film was then etched for 180 seconds with a 3.3% hydrofluoric acid-ethanol solution to peel it off from the silicon substrate. The PSi film was then peeled off from the silicon substrate by etching with a 3.3% hydrofluoric acid-ethanol solution for 180 seconds. Fresh porous silicon (PSi) nanoparticles were obtained after ultrasonically breaking down the PSi layer (500 W) for 30 minutes.

[0063] (2) Prepare porous silicon loaded with palladium-platinum alloy nanoenzymes using the same method as step (2) of Example 1, wherein the porous silicon is the porous silicon nanoparticles obtained in step (1).

[0064] Figure 2 The images show the attenuated total reflectance infrared spectra of different silicon materials (porous silicon, peroxide porous silicon, weakly oxidized porous silicon, and silicon dioxide). In the figures, the Si-H bonds in the porous silicon are converted to Si-OH bonds after weak oxidation with PBS, resulting in weakly oxidized porous silicon nanoparticles, as indicated by the stretching vibration peak of the Si-H bonds (2100 cm⁻¹). -1 ) and bending vibration peak (630 cm) -1 The peaks of the stretching vibrations of the Si-OH bond (3000-3700 cm⁻¹) almost completely disappeared, while the peaks of the stretching vibrations of the Si-OH bond (3000-3700 cm⁻¹) also disappeared. -1 The corresponding broad peak enhancement. In addition, the bending vibration peak of the Si-Si bond (470 cm⁻¹) is also observed. -1 The intensity also increased, possibly due to the strengthening of Si-OH bonds, which in turn increased the vibrational intensity of Si-Si bonds. In porous peroxide silicon, the peak values ​​(800 and 1100 cm⁻¹) belonging to the stretching vibrations of Si-O-Si bonds are... -1The intensity of the Si-OH bond further increases, while the peak intensity of the Si-OH bond (3000-3700 cm⁻¹) increases further. -1 The weakening of the Si-OH bonds can be explained by the high oxidation of the Si-OH bonds, resulting in a Si-O-Si cross-linked structure. Simultaneously, the Si-Si bonds (470 cm⁻¹) in the porous peroxide silicon also weaken. -1 The peak disappears at 436 cm. -1 The appearance of bending vibration peaks of Si-O-Si bonds indicates that the Si-Si framework underwent network recombination after oxidation to Si-O-Si, resulting in vibrational modes identical to those of Si-O-Si bonds in SiO2. These results suggest that the surface of weakly oxidized porous silicon nanoparticles is mainly composed of Si-OH, while the surface of peroxidized porous silicon is mainly composed of Si-O-Si-OH.

[0065] Figure 3 Transmission electron microscopy (TEM) images of porous silica loaded with palladium-platinum alloy nanozymes and porous silica loaded with palladium-platinum alloy nanozymes in weakly oxidized porous silica. The results show that both porous silica and weakly oxidized porous silica can reduce palladium(II) and platinum(IV) salts in situ, yielding two composites: porous silica loaded with palladium-platinum alloy nanozymes and porous silica loaded with palladium-platinum alloy nanozymes in weakly oxidized porous silica. However, the palladium-platinum alloy nanozymes encapsulated in weakly oxidized porous silica (average size approximately 5.2 nm) are more uniformly distributed than those encapsulated in porous silica.

[0066] Performance testing:

[0067] (1) Photothermal performance test of weakly oxidized porous silicon supported by palladium-platinum alloy nanoenzymes

[0068] Porous silica, weakly oxidized porous silica (prepared in step (1) of Example 1), porous silica supported on palladium-platinum alloy nanozymes (prepared in Comparative Example 1), and weakly oxidized porous silica supported on palladium-platinum alloy nanozymes (prepared in step (2) of Example 1) were placed in 1.5 mL centrifuge tubes and subjected to near-infrared light (808 nm, 1.0 W / cm²). 2 Irradiate for 5 minutes, and record the temperature change of the solution during the irradiation process using a thermal imager (FLIR ONE Pro, FLIR Systems, USA). Figure 4 (As shown in Figure a).

[0069] We also measured the laser power densities (0.5, 0.7, and 1.0 W / cm²) of palladium-platinum alloy nanozyme-supported weakly oxidized porous silica (400 μg / mL) in different near-infrared I regions. 2 Photothermal responsiveness under (e.g.) Figure 4(As shown in Figure b). Furthermore, to evaluate photothermal stability, the weakly oxidized porous silicon supported on palladium-platinum alloy nanozymes was irradiated with near-infrared light for 5 minutes before the light source was removed, while temperature changes during cooling were monitored. Five repeated heating and cooling cycles were performed as described above (e.g., ...). Figure 4 (As shown in Figure c).

[0070] Figure 4a shows the photothermal response curves of the control group, porous silicon, weakly oxidized porous silicon, porous silicon supported by palladium-platinum alloy nanozymes, and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes; Figure b shows the photothermal response curves of weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes after being irradiated by lasers with different current densities; Figure c shows the temperature change curves of weakly oxidized porous silicon supported by nanozymes after being exposed to five near-infrared light irradiation-cooling cycles. Figure 4 The photothermal response performance of palladium-platinum alloy nanozymes supported on porous silicon with weak oxides was investigated. As shown in Figure a, after irradiation with an 808 nm near-infrared laser for 5 minutes, the temperature of the control group solution remained almost unchanged, while the temperatures of the porous silicon and porous silicon with weak oxides increased by only about 3 °C. The temperatures of the porous silicon and porous silicon with weak oxides supported on palladium-platinum alloy nanozymes increased significantly, but the temperature of the porous silicon with weak oxides supported on palladium-platinum alloy nanozymes (59.5 °C) was slightly higher than that of the porous silicon with weak oxides supported on palladium-platinum alloy nanozymes (58 °C). Furthermore, as shown in Figure b, the porous silicon with weak oxides supported on palladium-platinum alloy nanozymes exhibited a temperature rise behavior dependent on laser power. As shown in Figure c, after five cycles of near-infrared irradiation, the temperature rise of the porous silicon with weak oxides supported on palladium-platinum alloy nanozymes remained essentially unchanged, indicating that it has good near-infrared photothermal conversion stability.

[0071] (2) Determination of catalase (CAT) activity in weakly oxidized porous silica supported on palladium-platinum alloy nanozymes

[0072] A portable dissolved oxygen analyzer was used to quantify the oxygen content produced by different materials during the catalytic process (e.g., Figure 5 (See Figure a). Palladium-platinum alloy nanozyme-loaded weakly oxidized porous silica (400 μg / mL) was mixed with different concentrations of H₂O₂ (0, 3, 6, 12, 18 mM) in PBS buffer (pH=6.5), and the real-time oxygen production was monitored over 5 minutes with or without near-infrared irradiation (e.g., [missing information]). Figure 5 Figure b (without near-infrared illumination) and figure c (with near-infrared illumination) are shown. The initial reaction temperature of the reaction system was set at 37 °C, and the near-infrared laser power was controlled at 0.5 W / cm². 2 To ensure the reaction system temperature reaches approximately 42.5℃, the initial reaction rates (V0) of various materials at different H2O2 concentrations were determined based on the real-time oxygen content variation curves. The Michaelis equation 1 / V0 = K was then used to calculate the initial reaction rate. m / Vmax +1 / V max From the double reciprocal curve, the Michaelis constant (K) can be calculated. m ) and maximum reaction rate (V max Similarly, the above method was used to analyze the CAT-like activity of the porous silicon-based nanozyme complex when heated to 42.5℃ under near-infrared light irradiation (e.g., Figure 5 (As shown in Figure d).

[0073] Figure 5 The catalase-like (CAT) activity of different materials was investigated. Materials with CAT-like activity can catalyze the decomposition of H2O2 to produce O2. As shown in Figure a, porous silica supported by palladium-platinum alloy nanozymes and porous silica supported by palladium-platinum alloy nanozymes can both catalyze the decomposition of H2O2 to produce O2. However, the amount of O2 produced by porous silica supported by palladium-platinum alloy nanozymes is significantly higher than that produced by porous silica supported by palladium-platinum alloy nanozymes.

[0074] The Michaelis constant (K) used to evaluate the performance of nanozymes was obtained through steady-state dynamics calculations. m ) and maximum reaction rate (V max K m The value represents the binding affinity of nanozymes to reaction substrates, K. m A lower value indicates that the nanozyme binds more easily to the substrate. max The value V represents the ability of nanozymes to catalyze reactions. max A higher value indicates a faster rate of nanozyme-catalyzed reaction. max / K m The Kc value reflects the catalytic efficiency of the enzyme. Figure b shows the Kc value of the weakly oxidized porous silica supported on palladium-platinum alloy nanoenzymes. m The K0 value (17.00 mM) is lower than that of porous silicon supported on palladium-platinum alloy nanoenzymes. m Value (25.62 mM), V of palladium-platinum alloy nanoenzymes supported on weakly oxidized porous silica max Value (10.71×10) -7 (M / s) is higher than that of porous silicon loaded with palladium-platinum alloy nanozymes. max Value (7.987×10) -7 M / s). Palladium-platinum alloy nanoenzymes supported on weakly oxidized porous silica (6.303 × 10⁻⁶ m / s). -5 Catalytic efficiency (V / s) max / K m ) is a porous silicon nanoenzyme supported by palladium-platinum alloy nanoparticles (3.118 × 10⁻⁶). -5 The oxygen content of the TME is twice that of the nanozyme-supported porous silica (TME). Therefore, the weakly oxidized porous silica supported by palladium-platinum alloy nanozymes is more effective in alleviating the hypoxia of the TME than the porous silica supported by palladium-platinum alloy nanozymes.

[0075] As shown in Figure c, under low-power near-infrared laser irradiation, the CAT-like activity of porous silicon supported by palladium-platinum alloy nanozymes and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes is further improved, but the catalytic efficiency of porous silicon supported by palladium-platinum alloy nanozymes (4.179×10⁻⁶) is lower. -5 The efficiency ( / s) is still significantly lower than that of weakly oxidized porous silica supported on palladium-platinum alloy nanozymes (7.825×10⁻⁶). -5 / s).

[0076] Furthermore, the effect of the pore structure of the weakly porous silica on the activity of the nanozyme loaded inside was investigated. The results in Figure d show that immersing the palladium-platinum alloy nanozyme-loaded weakly porous silica in a weakly alkaline PBS solution (pH=7.4) for 24 hours caused the pores of the weakly porous silica to collapse, resulting in a palladium-platinum alloy nanozyme with the weakly porous silica support removed. Compared to the palladium-platinum alloy nanozyme-loaded with weakly porous silica, the K0 of the palladium-platinum alloy nanozyme with the weakly porous silica support removed was significantly higher. m The value (28.29 mM) increased significantly, V max Value (7.027×10) -7 The catalytic efficiency (M / s) decreased, and the catalytic efficiency (2.484 × 10⁻⁶) also decreased. -5 The 60% decrease in / s indicates that the presence of weakly oxidized porous silica channels enhances the CAT-like activity of the internally loaded palladium-platinum alloy nanozyme.

[0077] (3) Determination of oxidase (OXD) activity in weakly oxidized porous silica supported on palladium-platinum alloy nanozymes

[0078] With or without near-infrared laser irradiation (0.5 W / cm) 2 Under the conditions described, steady-state kinetics were analyzed using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate in PBS buffer (pH=6.5) containing porous silica, weakly oxidized porous silica, palladium-platinum alloy nanozyme-supported porous silica, or palladium-platinum alloy nanozyme-supported weakly oxidized porous silica (400 μg / mL). The kinetic parameters simulating OXD (Michaelis constant (K)) were analyzed. m ) and maximum reaction rate (V max The above calculations, obtained using the Michaelis-Menten equation and double reciprocal curves, are as follows (e.g.) Figure 6 (As shown).

[0079] Figure 6 This indicates that in the TME, oxidase-like enzymes (OXD) can convert O2 into superoxide radicals (·O2). - The OXD-like activity of different materials was studied using 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic reagent. TMB can be reacted with ·O2. -Oxidation produces a blue product (oxTMB) with a characteristic UV-Vis absorption peak at 652 nm. For example... Figure 6 As shown in Figure a, both palladium-platinum alloy nanozymes-supported porous silica and palladium-platinum alloy nanozymes-supported weakly oxidized porous silica can oxidize TMB to produce color, with the palladium-platinum alloy nanozyme-supported weakly oxidized porous silica system exhibiting a higher absorbance value. Figure 6 (a) Based on the steady-state dynamics analysis in Figure 6b, the Ka of the porous silicon supported by the palladium-platinum alloy nanoenzyme was obtained. m The value is 0.4447 mM, V max The value is 1.336 × 10 -8 M / s, V max / K m The value is 3.005 × 10 -5 / s, Palladium-platinum alloy nanoenzymes supported on weakly oxidized porous silica K m The value is 0.2576 mM, V max The value is 1.713 × 10 -8 M / s, V max / K m The value is 6.649 × 10 -5 / s. Therefore, the substrate adsorption capacity, catalytic reaction rate, and catalytic efficiency of palladium-platinum alloy nanozymes supported on weakly oxidized porous silica are significantly higher than those of palladium-platinum alloy nanozymes supported on porous silica. Furthermore, low-power near-infrared irradiation can enhance the adsorption capacity of palladium-platinum alloy nanozymes supported on porous silica (K... m The value is 0.1993 mM, V max The value is 1.751 × 10 -8 M / s, V max / K m The value is 5.018 × 10 -5 / s) and palladium-platinum alloy nanozymes supported on weakly oxidized porous silica (K m The value is 0.1853 mM, V max The value is 2.139 × 10 -8 M / s, V max / K m The value is 11.54 × 10 -5 The OXD-like activity of palladium-platinum alloy nanozymes was observed, but the weakly oxidized porous silica supported by palladium-platinum alloy nanozymes still exhibited better OXD-like catalytic performance than porous silica supported by palladium-platinum alloy nanozymes. Figure 6 (As shown in Figure c). Figure 6 As shown in Figure d, the catalytic efficiency of the palladium-platinum alloy nanozyme without the weakly oxidized porous silica support is 2.523 × 10⁻⁶. -5 The pore size ( / s) was significantly lower than that of weakly oxidized porous silica loaded with palladium-platinum alloy nanozymes, which confirms that the pore structure of weakly oxidized porous silica can improve the OXD-like activity of the loaded palladium-platinum alloy nanozymes.

[0080] (4) Determination of peroxidase (POD) activity of palladium-platinum alloy nanozymes supported on weakly oxidized porous silica

[0081] Peroxidase-like activity was determined by a TMB colorimetric reaction in the presence of H₂O₂. In short, palladium-platinum alloy nanozymes loaded with weakly oxidized porous silica (400 μg / mL), TMB (0.5 mM), and H₂O₂ (3.3 mM) were reacted with or without near-infrared stimulation (0.5 W / cm²). 2 The mixture was reacted in PBS buffer (pH=6.5) for 5 minutes, and then the absorbance of the solution was recorded. The Michaelis constant (Km) at normal physiological temperature (37℃) and near-infrared irradiation-induced temperature (around 42.5℃) was determined using the Michaelis equation and double reciprocal curves. m ) and maximum reaction rate (V max )(like Figure 7 (As shown).

[0082] Figure 7 The POD-like properties of different materials were investigated. A colorimetric reaction based on 3,3',5,5'-tetramethylbenzidine (TMB) was used to study the POD-like properties of the materials. Generally, POD can catalyze the decomposition of H₂O₂ to produce ·OH, and ·OH can oxidize TMB to generate an oxidation product with characteristic absorption at 652 nm. Porous silica, weakly oxidized porous silica, porous silica supported by palladium-platinum alloy nanozymes, or weakly oxidized porous silica supported by palladium-platinum alloy nanozymes were added to a mixed solution containing H₂O₂ and TMB, and the reaction was continued for 5 minutes at a normal physiological temperature of 37 °C. Compared with the porous silica group and the weakly oxidized porous silica group, the absorbance values ​​of the porous silica group supported by palladium-platinum alloy nanozymes and the weakly oxidized porous silica group supported by palladium-platinum alloy nanozymes were significantly increased at 652 nm, with the absorbance value of the weakly oxidized porous silica group supported by palladium-platinum alloy nanozymes being higher than that of the porous silica group supported by palladium-platinum alloy nanozymes (Figure a). Steady-state kinetic analysis (Figure b) yielded the Ka of the weakly oxidized porous silica supported on palladium-platinum alloy nanozymes when H2O2 was used as the substrate. m The value is 0.5559 mM, which is much lower than the Km of porous silicon supported on palladium-platinum alloy nanozymes. m Value (1.080 mM). Palladium-platinum alloy nanoenzymes supported on weakly oxidized porous silica. max Value (5.851 × 10) -8 (M / s) is slightly higher than that of porous silicon supported on palladium-platinum alloy nanozymes. max Value (5.537 × 10) -8 M / s), while palladium-platinum alloy nanozymes supported on weakly oxidized porous silicon (10.53 × 10⁻⁶ m / s). -5The catalytic efficiency of the palladium-platinum alloy nanozyme supported on porous silicon (5.126 × 10⁻⁶ / s) was significantly better than that of the nanozyme supported on porous silicon (5.126 × 10⁻⁶ / s). -5 The catalytic efficiency was [value missing] / s. This indicates that the weakly oxidized porous silica supported by palladium-platinum alloy nanozymes exhibits superior POD-like activity compared to porous silica supported by palladium-platinum alloy nanozymes. Mild thermal stimulation induced by near-infrared irradiation can further enhance the catalytic efficiency of the porous silica system supported by palladium-platinum alloy nanozymes (K [value missing]). m =0.8194 mM, V max =6.606 × 10 -8 M / s) and palladium-platinum alloy nanozyme-supported weakly oxidized porous silica system (K m =0.4496 mM, V max =6.748 × 10 -8 The substrate adsorption capacity and catalytic reaction rate (m / s) were compared (Figure c). Notably, even with mild photothermal stimulation, the POD performance of the palladium-platinum alloy nanozyme supported on weakly oxidized porous silica was still superior to that of the palladium-platinum alloy nanozyme supported on porous silica. Furthermore, the palladium-platinum alloy nanozyme without the weakly oxidized porous silica support (3.900 × 10⁻⁶ m / s) showed better performance. -5 The catalytic efficiency of the palladium-platinum alloy nanozyme supported on weakly oxidized porous silica was only 37% of that of the palladium-platinum alloy nanozyme supported on weakly oxidized porous silica. This indicates that the pore confinement effect of weakly oxidized porous silica can promote the POD-like activity of the supported palladium-platinum alloy nanozyme (Figure d).

[0083] (5) Determination of glutathione peroxidase (GSHOx) activity in weakly oxidized porous silica supported on palladium-platinum alloy nanozymes

[0084] The GSHOx-like activity of palladium-platinum alloy nanozyme-supported weakly oxidized porous silica was measured using a colorimetric method based on 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The DTNB reaction product exhibited a characteristic absorption peak at 412 nm, and the remaining amount of GSH in the reaction system could be assessed by monitoring the absorbance at 412 nm. Steady-state kinetics were studied at normal physiological temperature (37℃) and mild photothermal temperature (~42.5℃ induced by low-power near-infrared laser). Palladium-platinum alloy nanozyme-supported weakly oxidized porous silica (400 μg / mL), GSH (0.2 mM), and DTNB (200 μg / mL) were added to PBS buffer solution (pH = 6.5), and the absorbance of the mixture (412 nm) was recorded after 5 minutes of reaction. Different concentrations of GSH were added to the above system, and the Michaelis constant (K0) of the simulated GSHOx was calculated by steady-state kinetic analysis. m ) and maximum reaction rate (V max )(like Figure 8 (As shown).

[0085] Figure 8 The GSH-like properties of different materials were evaluated. The reaction product of DTNB and GSH (TNB) has a typical absorption peak at 412 nm, so the remaining amount of GSH in the reaction system can be monitored by the change of the TNB absorption peak with reaction time. Different materials were added to solutions containing DTNB and TNB, and the absorbance of the mixed solution was measured after reacting at 37 °C for 5 minutes. Compared with the systems containing porous silica and weakly oxidized porous silica, the absorbance of the TNB-like material at 412 nm was significantly reduced in the systems containing palladium-platinum alloy nanozymes and systems containing palladium-platinum alloy nanozymes with weakly oxidized porous silica (Figure a). Steady-state kinetic analysis (Figure b) yielded the Kx of palladium-platinum alloy nanozyme-supported porous silica with GSH as the substrate. m The value is 0.3516 mM, V max The value is 3.283 × 10 -7 M / s, V max / K m The value is 9.337 × 10 -4 / s. Palladium-platinum alloy nanoenzymes supported on weakly oxidized porous silica K m The value is 0.3120 mM, V max The value is 5.048 × 10 -7 M / s, V max / K m The value is 16.18 × 10 -4 / s, demonstrating that the GSHOX-like activity of weakly oxidized porous silica supported by palladium-platinum alloy nanozymes is superior to that of porous silica supported by palladium-platinum alloy nanozymes. For example Figure 8 As shown in Figure c, when subjected to mild photothermal stimulation, the ability of both palladium-platinum alloy nanozymes-supported porous silicon and palladium-platinum alloy nanozymes-supported weakly oxidized porous silicon to consume GSH was improved. Among them, the ability of palladium-platinum alloy nanozymes-supported weakly oxidized porous silicon (21.93 × 10⁻⁶) to consume GSH was improved. -4 The catalytic efficiency of the palladium-platinum alloy nanozyme-supported porous silicon (14.73 × 10⁻⁶ / s) is still significantly higher than that of the palladium-platinum alloy nanozyme-supported porous silicon (14.73 × 10⁻⁶ / s). -4 / s), demonstrating that mild photothermal stimulation can effectively enhance the GSHOX-like activity of weakly oxidized porous silicon supported on palladium-platinum alloy nanozymes. For example... Figure 8 As shown in Figure d, after the pores of the weakly oxidized porous silica were destroyed, the catalytic efficiency of the palladium-platinum alloy nanozyme, with the weakly oxidized porous silica support removed, as a GSHOx-like enzyme decreased to 7.422 × 10⁻⁶. -4 / s, which further indicates that weakly oxidized porous silica has a pore confinement effect, which can enhance the GSHOx-like activity of the nanozymes loaded inside.

[0086] Figure 9The density of electronic states (DOS) plots are shown for porous silicon supported by palladium-platinum alloy nanozymes (a) and weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes (b). The results indicate that, compared to porous silicon supported by palladium-platinum alloy nanozymes, the weakly oxidized porous silicon supported by palladium-platinum alloy nanozymes exhibits higher DOS at the Fermi level (E0). f The higher DOS near the point indicates more efficient electron transfer at the interface between the weakly oxide porous silicon and the palladium-platinum alloy nanozyme. This is because the highly electronegative Si-Si-OH bonds on the surface of the weakly oxide porous silicon can form chemical bonds and electronic coupling with the noble metal, thereby enhancing the electronic interaction between the weakly oxide porous silicon and the palladium-platinum alloy nanozyme, ultimately forming a stable semiconductor-metal heterojunction. In contrast, the interaction between the Si-Si-H bonds on the porous silicon surface and the palladium-platinum alloy nanozyme is weaker, therefore the stability and electron transfer capability of the heterojunction are not as good as those of the weakly oxide porous silicon system supported by the palladium-platinum alloy nanozyme. Based on this, the d-band center of the weakly oxide porous silicon supported by the palladium-platinum alloy nanozyme (-0.39 eV) is calculated to be closer to E than the d-band center of the porous silicon supported by the palladium-platinum alloy nanozyme (-0.72 eV). f (0 eV), this is because, compared to porous silicon, palladium-platinum alloy nanoenzymes E f Electrons can be transferred more efficiently to the weakly oxidized porous silicon, increasing the unfilled degree of the d orbitals and causing the d-band center to shift upwards. This upward shift of the d-band center allows for more efficient donation of electron density to the antibonding orbitals of oxygen, reducing the strength of the OO bond and promoting the growth of ·O2. - The generation of palladium-platinum alloy nanoenzymes. Simultaneously, palladium-platinum alloy nano f The abundant holes can participate in enzyme-like catalytic reactions, thereby oxidizing different substrates.

[0087] (6) Cell culture

[0088] Mouse breast cancer cells (4T1) were purchased from Fenghui Biotechnology Co., Ltd. in China and cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin (100 units / mL) and streptomycin (100 μg / mL). The incubator conditions were set at 37°C and contained 5% CO2.

[0089] (7) Cytotoxicity

[0090] The CellTiter-Lumin kit (Beyotime Biotechnology Co., Ltd., China) was used to evaluate the cytotoxicity of different materials to 4T1 cells. 4T1 cells (8 × 10⁶ cells per well) were used. 3Cells were seeded into white 96-well plates and cultured overnight, then washed with PBS. Fresh RPMI-1640 medium containing different concentrations of the four materials (0, 80, 160, 240, 320, and 400 μg / mL) was then added to each well and cultured for 24 hours. Celltiter-Lumin reagent (100 μL) was then added to each well and shaken for 10 minutes. Chemiluminescence signals were measured using a microplate reader (Thermo Scientific 5250040, USA). Furthermore, the effects of different materials on cytotoxicity under mild photothermal stimulation were investigated. As described above, different materials were added to the seeded cells and cultured for 4 hours, then the cells were irradiated with 808 nm near-infrared light (0.5 W / cm²). 2 Irradiate for 5 minutes, continue culturing for 20 hours, and then measure cell viability using the same steps described above (e.g., ...). Figure 10 (As shown).

[0091] Figure 10 The in vitro anti-breast cancer cell (4T1) performance of different materials was investigated. For example... Figure 10 As shown, under low-power near-infrared light irradiation, the viability of 4T1 cells remained essentially unchanged with increasing concentrations of porous silica and weakly oxidized porous silica in the reaction system. However, the viability of 4T1 cells showed a significant concentration dependence between porous silica loaded with palladium-platinum alloy nanozymes and weakly oxidized porous silica loaded with palladium-platinum alloy nanozymes. At the same concentration (400 μg / mL), the survival rate of 4T1 cells treated with weakly oxidized porous silica loaded with palladium-platinum alloy nanozymes (18%) was lower than that treated with porous silica loaded with palladium-platinum alloy nanozymes (40%), demonstrating that weakly oxidized porous silica loaded with palladium-platinum alloy nanozymes is more effective than porous silica loaded with palladium-platinum alloy nanozymes in inhibiting tumor cell growth.

[0092] (8) Intracellular oxygen detection

[0093] 4T1 cells seeded in 24-well plates were cultured overnight in an anaerobic environment (1% O2 and 5% CO2). After adding different materials, the cells were cultured for another 2 hours, followed by near-infrared stimulation (0.5 W / cm²). 2 Add 2 μM tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride ([Ru(dpp)3]Cl2) probe and incubate for 6 hours. Record cell fluorescence using an inverted fluorescence microscope (e.g., 5 minutes). Figure 11 (As shown).

[0094] Figure 11Oxygen production in 4T1 cells after treatment with different materials was evaluated. Among the five treatment groups, the fluorescent oxygen probe showed the weakest red fluorescence in the palladium-platinum alloy nanozyme-loaded porous silica group, indicating that palladium-platinum alloy nanozyme-loaded porous silica can most effectively reverse the hypoxic environment of the tumor microenvironment (TME). Furthermore, the quenching of the red fluorescence signal in 4T1 cells treated with palladium-platinum alloy nanozyme-loaded porous silica was more pronounced under mild photothermal stimulation, demonstrating the generation of more O2. These results indicate that palladium-platinum alloy nanozyme-loaded porous silica exhibits mildly photothermally enhanced CAT-like activity in tumor cells.

[0095] (9) Intracellular ROS detection

[0096] 4T1 cells were seeded in 24-well plates and treated as described above. Reactive oxygen species (DCFH-DA) probes were added and incubated for 30 minutes. Cells were then washed three times with serum-free RPMI-1640 medium to thoroughly remove residual DCFH-DA before recording fluorescence images (e.g., ...). Figure 12 (As shown).

[0097] Figure 12 The changes in ROS levels in 4T1 cells after treatment with different materials were tested to evaluate the POD-like properties of different materials at the cellular level. First, the production of ·OH in 4T1 cells after the addition of different materials was examined under conditions without near-infrared light irradiation. Figure 12 As shown, compared with the porous silicon group, the weakly oxidized porous silicon group, and the porous silicon group loaded with palladium-platinum alloy nanozymes, the weakly oxidized porous silicon group loaded with palladium-platinum alloy nanozymes exhibited the strongest green fluorescence, demonstrating that the weakly oxidized porous silicon group loaded with palladium-platinum alloy nanozymes can also be used at the cellular level as a POD-like nanozyme to catalyze the decomposition of H2O2 to generate a large amount of ·OH for killing tumor cells. Furthermore, after near-infrared light irradiation, the green fluorescence of the weakly oxidized porous silicon group loaded with palladium-platinum alloy nanozymes was further enhanced, indicating that mild photothermal stimulation can enhance the POD-like performance of the weakly oxidized porous silicon group loaded with palladium-platinum alloy nanozymes in 4T1 cells.

[0098] (10) Detection of intracellular glutathione levels

[0099] 4T1 cells (1×10⁴ cells per well) 5After seeding (number of cells) into 6-well plates and culturing for 12 h, porous silica, weakly oxidized porous silica, palladium-platinum alloy nanozyme-loaded porous silica, and palladium-platinum alloy nanozyme-loaded weakly oxidized porous silica were added, respectively. The cells were cultured for another 8 h, and the GSH and GSSG levels in the tumor cells after different treatments were measured using the GSH / GSSG kit (Beyotime Biotechnology Co., Ltd.). Furthermore, after culturing the palladium-platinum alloy nanozyme-loaded porous silica group and the palladium-platinum alloy nanozyme-loaded weakly oxidized porous silica group for 2 h, they were irradiated with a near-infrared laser for 5 min, and cultured for another 6 h before detection (e.g.,...). Figure 13 (As shown).

[0100] Figure 13 The GSHOx-like properties of different materials in 4T1 cells were evaluated. Compared with the control group, the ratio of glutathione to oxidized glutathione in 4T1 cells of porous silica and weakly oxidized porous silica remained essentially unchanged. However, in 4T1 cells with palladium-platinum alloy nanozymes-loaded porous silica and palladium-platinum alloy nanozymes-loaded weakly oxidized porous silica, the ratio of glutathione to oxidized glutathione decreased, with a more significant decrease in the palladium-platinum alloy nanozyme-loaded weakly oxidized porous silica group. This demonstrates that palladium-platinum alloy nanozymes-loaded weakly oxidized porous silica can more effectively oxidize intracellular glutathione, exhibiting GSHOx-like properties.

[0101] (11) Detection of intracellular malondialdehyde (MDA)

[0102] 4T1 cells were seeded into 6-well plates and subjected to different treatments before complete lysis using Western Lysis Buffer at 4°C. The supernatant was then collected by centrifugation at 10000 g for 10 minutes. The MDA content in different samples was determined using an MDA detection kit (Beyotime). Figure 14 (As shown).

[0103] Figure 14The changes in malondialdehyde (MDA) levels in tumor cells after treatment with different materials were investigated. Without near-infrared light stimulation, the lowest MDA expression level was observed in 4T1 cells treated with palladium-platinum alloy nanozymes loaded with porous silica (PSA). This demonstrates that PSA induced the accumulation of large amounts of lipid peroxides (LPO) through ROS accumulation and GSH consumption, indicating that PSA can induce ferroptosis. After low-power light stimulation, the MDA content in 4T1 cells from both the PSA-loaded porous silica group and the PSA-loaded PSA group decreased further. However, the MDA level in the PSA-loaded PSA group was still significantly lower than that in the PSA group, demonstrating that mild photothermal stimulation can promote a more efficient ferroptosis response in PSA-loaded PSA.

[0104] (12) Detection of intracellular heat shock protein (HSP) levels

[0105] 4T1 cells (1 × 10⁵ cells per well) were placed in 6-well plates. The HSP70 assay was performed in two groups. The first group was cultured for 8 hours with different materials but without near-infrared irradiation. The second group was cultured for 2 hours with different materials, followed by near-infrared laser irradiation (0.5 W / cm²). 2 (5 minutes) Continue culturing for 6 hours, then digest with trypsin and collect cells. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% milk, the membrane was incubated with primary antibody at 4°C for 12 hours, followed by incubation with secondary antibody for 1 hour. Finally, the membrane was exposed to an ECLplus imaging system to observe protein bands (e.g., ...). Figure 15 (As shown).

[0106] Figure 15 The study investigated the changes in HSP levels in tumor cells after treatment with different materials. It was observed that under mild photothermal stimulation, the HSP70 level in the palladium-platinum alloy nanozyme-loaded weakly oxidized porous silica group significantly decreased. This is because the palladium-platinum alloy nanozyme-loaded weakly oxidized porous silica, with its quadruple mimicry enzyme activity, generates a large amount of ROS and LPO during the induced ferroptosis response, thereby inhibiting HSP70 expression and preventing HSP70 self-repair, thus making 4T1 tumor cells more sensitive to mild photothermal therapy.

[0107] (13) Stability Study of Palladium-Platinum Alloy Nanoenzymes Supported in Weakly Oxide Porous Silica

[0108] After being stored at room temperature and under dry conditions for one month, freshly prepared palladium-platinum alloy nanozymes loaded with weakly oxidized porous silica, or palladium-platinum alloy nanozymes loaded with weakly oxidized porous silica stored for one month, were applied to 4T1 cells. After culturing for four hours, 808 nm light stimulation (1.0 W / cm2, 5 min) was applied, and then cell viability was measured as described above (e.g., Figure 16 (As shown).

[0109] Figure 16 The storage stability of palladium-platinum alloy nanozymes supported on porous silica was investigated. Under near-infrared light stimulation, compared with freshly prepared palladium-platinum alloy nanozymes supported on porous silica, the growth inhibition rate of palladium-platinum alloy nanozymes supported on porous silica remained above 99.7% after one month of storage. These results demonstrate that palladium-platinum alloy nanozymes supported on porous silica possesses good storage stability and potential for practical applications.

[0110] (14) Study on the in vivo antitumor properties of palladium-platinum alloy nanozymes supported on weakly oxidized porous silica

[0111] BALB / c mice (5-6 weeks old, female) were purchased from SLAC Laboratory Animal Co., Ltd. (China). All animal experiments were approved by the Laboratory Animal Center of Zhejiang University and supervised by the Animal Ethics Committee of Zhejiang University (ZJU20240391). 4T1 cells (1×10⁻⁶) were subcutaneously injected into the right hind leg of BALB / c mice. 6 (Number of tumors), establishing a 4T1 tumor model. The tumor volume grew to approximately 100 mm. 3 Mice were randomly divided into three groups (n=4 per group): (i) saline + near-infrared light irradiation group; (ii) porous silica supported by palladium-platinum alloy nanozyme + near-infrared light irradiation group; (iii) porous silica supported by palladium-platinum alloy nanozyme + near-infrared light irradiation group. PBS, porous silica supported by palladium-platinum alloy nanozyme, or porous silica supported by palladium-platinum alloy nanozyme were injected intratumorally into the tumors of mice, respectively. Two hours after injection, the tumors were treated with an 808nm laser (0.5W / cm²). 2 The tumor area was irradiated for 5 minutes. Mouse weight and tumor volume were measured and recorded every two days. Tumor volume = 0.5 × tumor length × (tumor width)^2. After 14 days of different treatments, mice were sacrificed, and tumor tissue was removed for photography and weighing. The tumor growth inhibition rate (TGI) was calculated as follows: TGI = (1 - tumor weight in treatment group / tumor weight in control group) × 100% (e.g., 5 minutes). Figure 17 (As shown).

[0112] Figure 17The efficacy of palladium-platinum alloy nanozymes-loaded porous silica in treating breast cancer in vivo was investigated. In vivo experiments showed that after 14 days of treatment, tumors in mice treated only with saline and near-infrared light stimulation grew uncontrollably. In mice treated with both palladium-platinum alloy nanozymes-loaded porous silica and near-infrared light stimulation, tumor volume decreased, and tumor growth was controlled to some extent, with a tumor growth inhibition rate (TGI) of 70.3%. Notably, the tumor volume in mice treated with both palladium-platinum alloy nanozymes-loaded porous silica and near-infrared light stimulation was significantly reduced, with a TGI value as high as 90.0%, demonstrating that palladium-platinum alloy nanozymes-loaded porous silica exhibits excellent anti-breast cancer effects in vivo under low-power near-infrared light assistance.

[0113] (15) Study on the distribution of organisms in vivo

[0114] To assess the accumulation and metabolism of weakly porous silica loaded with palladium-platinum alloy nanozymes in vivo, a biodistribution study was conducted. Tumor-bearing mice were sacrificed 1 day and 14 days after treatment. Major organs and tumors were collected and nitrated in nitric acid for 24 hours. The pH of the solution was then adjusted to 6.5, and the solution was filtered through a 0.45 μm membrane. The contents of silicon, palladium, and platinum in each organ were calculated using ICP-OES analysis (e.g.,...). Figure 18 (As shown).

[0115] Figure 18 shows that, based on biodistribution analysis, palladium, platinum, and silicon were present in the liver, spleen, lungs, kidneys, and tumors of mice one day after administration, demonstrating that the weakly porous silica loaded with palladium-platinum alloy nanozymes can enter the in vivo circulation. After 14 days, the levels of palladium, platinum, and silicon in tumors and major organs were significantly reduced, indicating that the biodegradable weakly porous silica carrier allows the internally loaded palladium-platinum alloy nanozymes to be metabolized into orthosilicic acid and effectively excreted. These experimental results demonstrate that the weakly porous silica loaded with palladium-platinum alloy nanozymes possesses excellent biodegradability.

[0116] (16) Histological and pathological analysis

[0117] Tumor tissues and major organs from sacrificed mice were immersed in 4% paraformaldehyde for 24 hours, followed by paraffin embedding and sectioning for H&E, TUNEL, Ki-67, HIF-1α, and HSP70 staining analysis (e.g., Figure 19 (As shown).

[0118] Figure 19 illustrates the antitumor pathway of palladium-platinum alloy nanozymes loaded with weakly porous silica through histopathological examination. H&E staining of tumor sections from different treatment groups of palladium-platinum alloy nanozymes revealed the most significant tumor tissue damage in the group treated with palladium-platinum alloy nanozymes loaded with weakly porous silica + near-infrared light irradiation. Simultaneously, terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) and Ki-67 assays showed that under low-power near-infrared light irradiation, palladium-platinum alloy nanozymes loaded with weakly porous silica were more effective than palladium-platinum alloy nanozymes in killing tumor cells and inhibiting their proliferation. HIF-1α immunofluorescence staining showed that the oxygenation level in the group treated with palladium-platinum alloy nanozymes loaded with weakly porous silica + near-infrared light irradiation was the highest among the three groups, characterized by a sharp decay of red fluorescence. This is attributed to the excellent CAT-like activity of palladium-platinum alloy nanozymes loaded with weakly porous silica, which can reverse the hypoxic state of the tumor microenvironment (TME). Furthermore, the expression of HSP70 in tumors treated with both palladium-platinum alloy nanozymes-loaded porous silica and near-infrared light irradiation was maximally inhibited, indicating that palladium-platinum alloy nanozymes-loaded porous silica can induce ferroptosis, thereby enhancing the effect of mild photothermal therapy. In conclusion, in in vivo experiments, palladium-platinum alloy nanozymes-loaded porous silica demonstrated a stronger ability to inhibit tumor growth than palladium-platinum alloy nanozymes-loaded porous silica.

[0119] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A supported noble metal alloy nanozyme, characterized in that, It consists of weakly oxidized porous silicon nanoparticles and noble metal alloy nanozymes loaded on their surface and in their pores. The weakly oxidized porous silicon nanoparticles were prepared by the following method: The porous silicon nanoparticles were placed in a PBS solution with a pH of 7.4, shaken at 37°C for 2 hours, and then centrifuged at 10,000 rpm for 10 minutes to collect the weakly oxidized porous silicon nanoparticles. The precious metal alloy nanozyme is selected from palladium-platinum precious metal alloy nanozymes.

2. The supported noble metal alloy nanozyme according to claim 1, characterized in that, The Pd content in the weakly oxidized porous silicon of the supported noble metal alloy nanozyme is 80-85 μg / mg, and the Pt content is 35-50 μg / mg.

3. The supported noble metal alloy nanozyme according to claim 1, characterized in that, The mass ratio of palladium to platinum in the palladium-platinum noble metal alloy nanozyme is (2-2.5):

1.

4. The method for preparing the supported noble metal alloy nanozyme according to any one of claims 1-3, characterized in that, Includes the following steps: (1) The porous silicon nanoparticles were placed in a PBS solution with a pH of 7.4, shaken at 37°C for 2 hours, and then centrifuged at 10,000 rpm for 10 minutes to collect the weakly oxidized porous silicon nanoparticles. (2) The palladium source, platinum source, weakly oxidized porous silicon nanoparticles and water are mixed and subjected to an in-situ reduction reaction to obtain the supported noble metal alloy nanozyme.

5. The method according to claim 4, characterized in that, The ratio of the porous silicon nanoparticles to the PBS solution is 1 g: (10-300) mL.

6. The method according to claim 4, characterized in that, The molar ratio of palladium in the palladium source to platinum in the platinum source is 1:(0.5-1.5).

7. The method according to claim 4, characterized in that, The palladium source is selected from one or more of chloropalladic acid, palladium chloride, and sodium chloropalladate. The platinum source is selected from one or more of chloroplatinic acid, potassium chloroplatinate, and ammonium chloroplatinate.

8. The application of the supported noble metal alloy nanozyme according to any one of claims 1-3 or the supported noble metal alloy nanozyme prepared by the preparation method according to any one of claims 4-7 in the preparation of drugs for treating tumors; The tumor is breast cancer.

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