Acidic oxygen-producing nanoreactor for tumor therapy and preparation method thereof

Abstract

The present application relates to an acidic oxygen-producing nanoreactor for tumor treatment and a preparation method thereof. In the present application, Pt-AuNS exhibits excellent CAT-like catalytic activity in the range of pH 4 to 9, and doping under acidic conditions reduces the energy barrier for the decomposition of H2O2 into O2. Meanwhile, under laser irradiation at 808 nm, the plasmonic resonance generates hot electrons and photothermal effects, which significantly improve the activity of the nanomaterial CAT-like. At the same time, Pt-AuNS also has the intrinsic activity of oxidase (OXD) and the characteristics of glutathione (GSH) consumption to collapse the antioxidant stress self-preservation system of tumors. In addition, glucose oxidase (GOx) modified Pt-AuNS can cut off the nutrient supply of cancer cells, while producing H2O2, improving the treatment efficiency. In summary, the present application designs a multifunctional nanoscale enzyme system to alleviate tumor hypoxia, achieving the effect of triple therapy (starvation therapy, photothermal therapy, oxidation therapy) for synergistic treatment of tumors.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to an acidic oxygen-generating nanoreactor for tumor treatment and its preparation method. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Tumor hypoxia, caused by excessive oxygen consumption due to the overgrowth of cancer cells and insufficient oxygen supply from malformed tumor blood vessels, is one of the most representative features of the tumor microenvironment. Insufficient oxygen levels lead to the activation of hypoxia-inducible factors (HIFs), triggering a series of problems, including tumor invasion, metastasis, and immune escape. Furthermore, many strategies for improving cancer treatment efficacy are heavily reliant on oxygen, such as glutathione (GSH) depletion that disrupts redox balance and glucose oxidase (GOx)-mediated starvation therapy. Therefore, addressing tumor hypoxia is essential for achieving better therapeutic outcomes.

[0004] The discovery of nanozymes offers a novel approach to alleviating hypoxia. Nanozymes with exceptional catalase (CAT) mimicry activity have attracted considerable attention for reversing the hypoxic tumor microenvironment (TME), as they can in situ decompose excess hydrogen peroxide (H₂O₂) within the tumor, providing localized oxygen supply at the tumor site. However, the catalytic efficiency of CAT-like nanozymes is highly pH-dependent, typically exhibiting optimal activity in a weakly alkaline environment. Therefore, in the weakly acidic TME (pH ~4.5-6.5) induced by cancer cell metabolism, their oxygen-producing capacity is affected or even completely lost, significantly limiting their practical application in cancer therapy. Furthermore, while nanozymes remain stable under certain extreme conditions (such as high temperature and high pressure), their catalytic activity in the mild in vivo environment is far inferior to that of natural enzymes. Therefore, an innovative strategy is urgently needed to overcome these two key challenges: breaking the constraint of acidic pH while simultaneously enhancing the catalytic activity of nanozymes in vivo to effectively alleviate hypoxia. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide an acidic oxygen-generating nanoreactor for tumor treatment and its preparation method.

[0006] This invention ingeniously combines doping regulation and localized surface plasmon resonance (LSPR) to overcome the limitations of acidic pH and low efficiency of CAT-like nanozymes in hypoxic tumor microenvironments (TMEs). Platinum (Pt)-doped gold nanostar plasma nanozymes (Pt-AuNS) were synthesized by depositing a small number of platinum atoms into gold nanostars (AuNS). On the one hand, doping enabled Pt-AuNS to exhibit excellent CAT-like catalytic activity in a pH range of 4 to 9, and the mechanism was elucidated by density functional theory (DFT) calculations, demonstrating that doping under acidic conditions lowers the energy barrier for the decomposition of H₂O₂ into O₂. On the other hand, Pt-AuNS is also an excellent plasma with strong resonant absorption in the near-infrared (NIR) region. Under irradiation with an 808 nm laser, the hot electrons and photothermal effects generated by plasma resonance significantly improved the CAT-like activity of the nanomaterial. Simultaneously, Pt-AuNS also possesses intrinsic oxidase (OXD)-like activity and glutathione (GSH) depletion properties, thereby disrupting the tumor's antioxidant stress self-protection system. In addition, we modified Pt-AuNS with glucose oxidase (GOx) to cut off the nutrient supply to cancer cells while generating H2O2 to improve treatment efficiency. In summary, this invention designs a multifunctional nanozyme system primarily for alleviating tumor hypoxia, achieving a synergistic effect of triple therapy (starvation therapy, photothermal therapy, and oxidative therapy) in the treatment of tumors.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for preparing an acidic oxygen-generating nanoreactor, comprising the following steps:

[0009] (1) Preparation of gold nanostar (AuNS) nanoparticles: Hepes was dissolved in deionized water, then mixed with NaOH, HAuCl4 was added and mixed well, the mixture was allowed to stand in the dark, centrifuged, the supernatant was removed and the precipitate was washed, and finally dispersed in deionized water for later use.

[0010] (2) Preparation of platinum-gold nanostars (Pt-AuNS) nanoparticles: AuNSs and H2PtCl6 were stirred and preheated at 80℃, and then AA was added to the mixed solution to react. After the reaction was completed, the precipitate was centrifuged and washed to obtain Pt-AuNS nanoparticles. The Pt-AuNS nanoparticles were resuspended in deionized water to obtain an aqueous solution of Pt-AuNS nanoparticles.

[0011] (3) Preparation and modification of Pt-AuNS-GOx nanoparticles: DHLA was added to the aqueous solution of Pt-AuNS nanoparticles and stirred at room temperature; centrifuged, the supernatant was removed and the precipitate was collected and resuspended in deionized water to obtain Pt-AuNS nanoparticles modified with carboxyl groups; NHS and EDC were dissolved in H2O2, and 100 μL of each were gradually added to the carboxyl-modified Pt-AuNS solution; the carboxyl groups were activated by stirring at room temperature; GOx aqueous solution was added dropwise to the activated Pt-AuNS solution, the reaction was stopped by stirring at room temperature for a period of time, centrifuged, and the precipitate was washed and resuspended in deionized water; finally, the precipitate was passivated with HS-PEG overnight and washed twice with DPBS to obtain the final product Pt-AuNS-GOx nanoparticles.

[0012] Furthermore, in step (1), 1g of Hepes was dissolved in 28.4mL of deionized water, then mixed with 1.6mL of NaOH (1M), and 150μL of HAuCl4 (40mM) was added.

[0013] Furthermore, in step (1), the time for keeping the container in the dark is 0.5-3 hours, preferably 1 hour.

[0014] Furthermore, in step (2), the concentration of AuNSs is 60 mg / L, the concentration of H2PtCl6 is 1 mM, and the concentration of AA is 1 mM; the volume ratio of Pt-AuNS, H2PtCl6, and AA is 6:4:5.

[0015] Furthermore, in step (2), the mixture is preheated at 700 rpm for 10 min.

[0016] Furthermore, in step (2), after adding AA, the reaction is carried out for 30 minutes.

[0017] Further, in step (3), 100 μL LDHLA (15 mM) was added to 10 mL of Pt-AuNS nanoparticle aqueous solution (60 mg / L), and the mixture was stirred at 750 rpm for 2 h at room temperature. After centrifugation, the supernatant was removed and the precipitate was collected. The precipitate was resuspended in 4 mL of deionized water to obtain carboxyl-modified Pt-AuNS nanoparticles. Then, 2 mg NHS and 3 mg EDC were weighed and dissolved in 1 mL of H2O2. 100 μL of each solution was gradually added to 4 mL of carboxyl-modified Pt-AuNS solution. The carboxyl groups were activated by stirring at room temperature. A 1 mg / mL GOx aqueous solution was prepared. 1 mL of GOx aqueous solution was added dropwise to the activated Pt-AuNS solution. After stirring at 750 rpm for 2 h at room temperature, the reaction was stopped. The mixture was centrifuged, and the precipitate was washed three times with deionized water and resuspended in 10 mL of deionized water. Finally, the precipitate was treated with 0.1 mM... The nanoparticles were passivated overnight with HS-PEG (Mw = 5000 Da) and washed twice with DPBS to obtain the final product, Pt-AuNS-GOx nanoparticles.

[0018] Furthermore, the centrifugation speed in steps (1), (2), and (3) is 10,000-13,000 rpm, and the centrifugation time is 10-20 min.

[0019] In a second aspect, the present invention provides an acidic oxygen-generating nanoreactor prepared by the above-described preparation method.

[0020] A third aspect of the present invention provides the application of the above-described acidic oxygen-generating nanoreactor in the preparation of drugs for treating tumors.

[0021] The present invention has the following beneficial effects:

[0022] This invention successfully synthesized an acidic oxygen-generating nanoreactor, which experimentally demonstrated excellent CAT-like catalytic activity within a pH range of 4 to 9. Metal doping lowered the energy barrier for the decomposition of H₂O₂ into O₂ under acidic conditions. Furthermore, the plasma properties of Pt-AuNS significantly enhanced the CAT-like activity of the nanomaterial. Simultaneously, Pt-AuNS also exhibits intrinsic oxidase-like (OXD) activity and glutathione (GSH) depletion properties, thereby disrupting the tumor's antioxidant stress self-protection system. The acidic oxygen-generating nanoreactor of this invention possesses significant anti-cancer efficacy. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are provided to further illustrate the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention.

[0024] Figure 1 a) is a schematic diagram of the synthesis of Pt-AuNS-GOx and b) is a schematic diagram of the underlying mechanism.

[0025] Figure 2 a) is a large-field TEM image of Pt-AuNS nanoparticles, and b) is an HR-TEM image of Pt-AuNS nanoparticles and a selected area electron diffraction (SAED) pattern of the selected area.

[0026] Figure 3 Pt-AuNS-like CAT activity synthesized with different H2PtCl6 concentrations.

[0027] Figure 4 This is an analysis of the metal content in different nanoparticles, where ND represents undetected.

[0028] Figure 5 a) shows the protein content of BCA-quantified nanoparticles, and b) shows the protein analysis image by SDS-PAGE.

[0029] Figure 6The hydration particle size (a) and zeta potential (b) of AuNS, Pt-AuNS, and Pt-AuNS-GOx nanoparticles are given.

[0030] Figure 7 To evaluate the catalase-like activity of Pt-AuNS-GOx nanoparticles at different pH values.

[0031] Figure 8 The effect of light on the CAT-like activity of Pt-AuNS-GOx.

[0032] Figure 9 The effect of light irradiation on the OXD-like activity of Pt-AuNS-GOx.

[0033] Figure 10 The glucose consumption of Pt-AuNS-GOx nanoparticles of different concentrations in a weakly acidic environment (pH=6.5).

[0034] Figure 11 To investigate the cytotoxicity of different concentrations of nanoparticles on mouse breast cancer (4T1) cells under no light (-) or light (+) conditions.

[0035] Figure 12 Figure a) shows digital photographs of tumors removed from mice in each treatment group after euthanasia at 20 days; Figure b) shows the tumor mass of each treatment group as shown in Figure a). Detailed Implementation

[0036] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] Materials: Tetrachloroauric acid tetrahydrate (HAuCl4·4H2O), platinum hexachlorohydrate (H2PtCl6·6H2O), sodium hydroxide (NaOH), L-ascorbic acid (AA), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer, dihydrolipoic acid (DHLA), tetramethylammonium hydroxide 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (NHS), glucose oxidase (GOx), mercaptopolyethylene glycol (HS-PEG, Mw = 5000 Da), phosphate-buffered saline (PBS), 3,3′,5,5′-tetramethylbenzidine (TMB), Cell Counting Kit-8 (CCK-8) cell counting reagent and 2′,7′-dichlorodifluorofluorescein diacetate (DCFH-DA; ≥94%) were purchased from Sigma-Aldrich. Malondialdehyde (MDA) assay kit, hydrogen peroxide (H2O2) assay kit, and reduced glutathione (GSH) kit were purchased from Nanjing Jiancheng Biotechnology Institute. Calcein-AM / PI live / dead cell double staining kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. All other chemicals were obtained from Adamas beta and were ready for use without further purification. All experiments used deionized (DI) water (Millipore Milli-Q grade, 18.2 MΩ).

[0038] Example 1

[0039] The preparation method of the acidic oxygen-generating nanoreactor is as follows (synthesis schematic diagram and mechanism are shown in the figure). Figure 1 As shown):

[0040] (1) Preparation of gold nanostars (AuNS) nanoparticles: First, weigh 1g of Hepes and dissolve it in 28.4mL of deionized water, then mix it with 1.6mL of NaOH (1M). Add 150μL of HAuCl4 (40mM) and mix quickly with a pipette. Let it stand in the dark for 1h. Centrifuge at 13000rpm for 15min, discard the supernatant and take the precipitate, wash it 3 times with deionized water, and finally disperse it in deionized water for later use.

[0041] (2) Preparation of platinum-gold nanostars (Pt-AuNS) nanoparticles: 6 mL of AuNSs (60 mg / L) and 4 mL of H2PtCl6 (1 mM) were preheated at 80 °C with stirring at 700 rpm for 10 min. Then, 5 mL of AA (1 mM) was added to the mixed solution and the reaction was carried out for 30 min. After the reaction was completed, the mixture was centrifuged at 13000 rpm for 15 min, and the precipitate was washed three times with deionized water to obtain Pt-AuNS nanoparticles.

[0042] (3) Preparation and modification of Pt-AuNS-GOx nanoparticles: 100 μL LDHLA (15 mM) was added to 10 mL of Pt-AuNS (60 mg / L), and the mixture was stirred at 750 rpm for 2 h at room temperature. After centrifugation at 13000 rpm for 15 min, the supernatant was removed and the precipitate was resuspended in 4 mL of deionized water to obtain carboxyl-modified Pt-AuNS nanoparticles. Then, 2 mg of NHS and 3 mg of EDC were weighed and dissolved in 1 mL of H2O2, and 100 μL of each were gradually added to 4 mL of carboxyl-modified Pt-AuNS solution. The carboxyl groups were activated by stirring at 600 rpm for 30 min at room temperature. A 1 mg / mL GOx aqueous solution was prepared. 1 mL of GOx aqueous solution was added dropwise to the activated Pt-AuNS solution, and the reaction was stopped after stirring at 750 rpm for 2 h at room temperature. The mixture was centrifuged at 10000 rpm for 15 min, and the precipitate was washed three times with deionized water and resuspended in 10 mL of deionized water. Finally, the nanoparticles were passivated overnight with 0.1 mM HS-PEG (Mw = 5000 Da) and washed twice with DPBS to obtain the final product, Pt-AuNS-GOx nanoparticles.

[0043] Morphology of Pt-AuNS nanoparticles: 10 μL of Pt-AuNS nanoparticles were diluted to 1 mL with deionized water. 10 μL of the diluted nanoparticles were then dropped onto a copper grid and dried overnight in an electronic desiccant oven. The morphology, lattice spacing, and selected area electron diffraction (SAED) of the nanoparticles were observed using a high-resolution transmission electron microscope (HR-TEM). Morphological characterization of Pt-AuNS: TEM images are shown below. Figure 2 As shown in a), the vast majority of Pt-AuNS nanoparticles are multi-branched star-shaped structures, with a size of approximately 40 nm, proving the successful synthesis of Pt-AuNS nanoparticles. HR-TEM lattice imaging is as follows. Figure 2 b) The lattice spacing is approximately 0.231 nm, which is between the interplanar spacing of pure gold (0.235 nm) and platinum (0.228 nm), so the lattice spacing is the alloy (111) plane; the fast Fourier transform of the selected region shows the diffraction light of the five characteristic diffraction peaks of the face-centered cubic (fcc) lattice.

[0044] Metal content in AuNS and Pt-AuNS nanoparticles: 10 μL of AuNS and Pt-AuNS nanoparticles were each digested overnight with aqua regia, then diluted to 10 mL by acid removal. The Au and Pt content was quantified by ICP-MS to obtain the proportion of each element in the Pt-AuNS nanoparticles. CAT activity of Pt-AuNS nanoparticles with different feed ratios: (e.g.) Figure 3As shown, the oxygen (O2) generation performance of Pt-AuNS improves with increasing Pt precursor (H2PtCl6). However, once the concentration of H2PtCl6 exceeds 1 mM, the catalytic activity begins to decrease, proving that more Pt does not necessarily lead to better CAT-like activity; the optimal catalytic rate is achieved when the Pt to Au ratio is appropriate. The metal content in Pt-AuNS synthesized from 1 mM Pt precursor (H2PtCl6) is as follows: Figure 4 As shown, inductively coupled plasma mass spectrometry (ICP-MS) determined the Pt / Au ratio to be approximately 12.8%.

[0045] SDS-PAGE and BCA protein detection experiments on nanomaterials: Individual GOx, AuNS, Pt-AuNS, and Pt-AuNS-GOx nanoparticles were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to verify successful GOx coupling, and the specific GOx loading was detected by BCA protein detection. BCA protein quantification and SDS-PAGE analysis of Pt-AuNS-GOx nanoparticles were also performed. Figure 5 As shown in a), the concentration of GOx protein added during the synthesis of the material was 148.79 μg / mL, and the protein concentration on the Pt-AuNS-GOx nanoparticles obtained by centrifugation was 77.4 μg / mL, resulting in a calculated coupling rate of 52%. SDS-PAGE protein analysis showed... Figure 5 As shown in b), only channel 5 (Pt-AuNS-GOx nanoparticles) showed the same imprint as channel 1, proving that the protein on the nanoparticles is GOx.

[0046] Characterization of hydrated particle size and zeta potential of nanomaterials: 100 μL of AuNS, Pt-AuNS, and Pt-AuNS-GOx nanoparticles were diluted to 2 mL with deionized water. The particle size and zeta potential of the nanoparticles in an aqueous environment were detected using a dynamic light scattering instrument. The experiment was repeated three times, and the data were recorded. Characterization of the particle size and potential of nanoparticles: as follows... Figure 6 As shown in a), through gradual modification, the hydrated particle size of AuNS and Pt-AuNS did not change significantly. The addition of Pt did not alter the shape and size of AuNS. After coupling GOx to modify PEG, the measured hydrated particle size was slightly larger due to some agglomeration of nanoparticles. Meanwhile, it can be seen that... Figure 6 As shown in b), the zeta potentials of different nanoparticles are different, proving that the final material was successfully prepared.

[0047] The decomposition path of H₂O₂ on the metal surface was calculated using density functional theory (DFT). The software used was the Vienna Ab-initio Simulation Package (VASP). The calculation method involved first-principles density functional theory calculations using VASP, employing projected fused wave (PAW) to describe the interactions between electrons and ions. The generalized gradient approximation and PBE exchange-related functionals were used in the calculations. All structural optimizations and energy calculations were performed at a plane cutoff energy of 500 eV. To avoid interactions between slab structures in the periodic direction, interactions were selected in the vertical direction. The vacuum layer thickness was determined. The computational module used was a 3×3 supercell, with different models built for different materials. For Brillouin zone sampling, a 3×3×1 Monkhorst-Pack grid was used for geometric optimization. Long-range dispersion force correction employed Grimme's DFT-D3 correction. Calculation results show that oxygen production energy consumption on Au-Pt under acidic conditions is less than that on pure Au and pure Pt, demonstrating the superiority of doping.

[0048] UV-Vis absorption spectra of nanomaterials: AuNS, Pt-AuNS, and Pt-AuNS-GOx nanoparticles were diluted to the same concentration with deionized water. The solutions were placed in quartz cuvettes, and the UV-Vis absorption spectra of each solution were detected using a UV-Vis spectrophotometer in the wavelength range of 280 nm to 1000 nm. Comparison was performed to demonstrate that Pt-AuNS-GOx nanomaterials exhibit excellent absorption in the near-infrared region.

[0049] Photothermal performance testing of nanomaterials: 1.5 mL of different nanomaterials (1. PBS; 2. AuNS (20 μg / mL); 3. Pt-AuNS (20 μg / mL); 4. Pt-AuNS-GOx (20 μg / mL)) were placed in an EP tube and irradiated with an 808 nm laser for 10 minutes. Temperature changes were recorded using a thermal infrared imager, and heating curves were plotted. The photothermal conversion capability of each nanomaterial under near-infrared laser was evaluated, demonstrating that the excellent plasmonic properties of Pt-AuNS-GOx nanomaterials convert light energy into heat energy.

[0050] Assay for catalase-like activity of nanomaterials: 100 μL of different materials (1. PBS; 2. AuNS (60 μg / mL); 3. Pt-AuNS (60 μg / mL); 4. Pt-AuNS-GOx (60 μg / mL)) were added to 10 mM H2O2. An oxygen analyzer was immediately inserted into the solution to monitor O2 generation. The effects of pH and temperature on enzyme activity were then investigated. Buffer solutions with different pH values ​​were prepared, and Pt-AuNS-GOx and 10 mM H2O2 were added. Oxygen generation was monitored using an oxygen analyzer. 100 μL of Pt-AuNS-GOx was added to 2.6 mL of buffer (Ph = 7) + 300 μL of (0.1 M) H2O2. After reacting in water baths at different temperatures for 10 min, 300 μL of ammonium molybdate was immediately added, and the absorbance at 405 nm was measured. Finally, the effect of light on enzyme activity was investigated. The oxygen production rate was measured under different concentrations of H₂O₂, both under light and without light, to determine the influence of light. The catalase-like activity of Pt-AuNS-GOx nanoparticles at different pH values ​​was also examined. Figure 7 As shown, within a pH range of 4-9, Pt-AuNS-GOx nanoparticles can catalytically decompose H2O2 to generate O2. The highest O2 production occurs at pH 7, while the production is almost identical at pH 6 and pH 9. At pH levels as low as 2 or 3, O2 production is virtually negligible. This demonstrates that the synthesized nanomaterials can produce oxygen under acidic conditions. Light-enhanced CAT enzyme activity of Pt-AuNS-GOx nanoparticles was investigated: the same concentration of nanomaterials was added to different concentrations of H2O2, and the initial reaction rate was determined by measuring the initial rate of O2 production. Figure 8 As shown, the reaction rate in the illuminated group was much higher than that in the non-illuminated group, proving that illumination can indeed enhance the CAT-like enzyme activity of Pt-AuNS-GOx nanoparticles.

[0051] OXD Activity Assay of Nanomaterials: The absorption intensity of TMB at 652 nm was determined by UV-Vis absorption spectroscopy, and the oxidase catalytic performance of Pt-AuNS-GOx was analyzed based on this. 2 mL of acetate buffer solution (0.1 M, pH = 4.0) was added to a cuvette, followed by a certain amount of Pt-AuNS-GOx. After mixing thoroughly, the mixture was placed in a UV-Vis-NIR spectrophotometer to scan the baseline. Then, 20 μL of TMB (50 mM) was added, and the mixture was quickly mixed. The absorption curve was measured every five minutes for 20 min. Argon gas was passed through the buffer solution (to completely remove dissolved oxygen), and other conditions remained unchanged to measure the oxidation of TMB under hypoxic conditions. Effect of Light on OXD Enzyme Activity of Pt-AuNS-GOx Nanoparticles: The same concentration of nanomaterials was added to the same concentration of TMB, and after different treatments (1. no light at room temperature, 2. light, 3. light but water cooling (to eliminate temperature effects)) and reacted for the same time, their absorption spectra were measured. Figure 9 As shown, the characteristic peak (652nm) of TMB-OX in the light-illuminated group was the highest. After water cooling treatment, the absorbance decreased by 84%, proving that light irradiation can enhance the activity of its OXD-like enzyme. Among them, the increase in temperature is the main reason for the enhancement of enzyme activity, while the effect of hot electrons is relatively small.

[0052] GSH consumption experiment: DTNB working reagent was prepared by dissolving DTNB in ​​phosphate buffer (pH=8.0) containing 1 mm EDTA. Pt-AuNS-GOx (concentration range: 0-40 μg / ml) was dispersed in 1 ml of 0.2 mg / ml GSH and reacted in the dark for 5 h. Then, 400 μl of DTNB was added, and the mixture was incubated for another 30 min. Finally, the absorbance at 412 nm was measured, and the absorbance of the supernatant was obtained. It was found that the absorbance at 412 nm decreased in a concentration-dependent manner. When the concentration of the nanomaterial reached 40 μg / ml, the GSH content was only 42.53%, proving that Pt-AuNS-GOx-like OXD enzyme activity can consume the reducing substance GSH.

[0053] Glucose Consumption Experiment: Glucose consumption was analyzed using DNS reagent. 36.4 g KaC4O6·4H2O was added to 100 ml of deionized water and heated to dissolve. Then, 4.25 g NaOH, 1.26 g C7H4N2O7, and 1 g phenol were added sequentially. After cooling, the solution was diluted to 200 ml with deionized water and stored in a brown bottle at room temperature. The nanoparticles were added to a buffer solution containing 1 mg / ml glucose and reacted at 37°C for 4 h. The supernatant (1 ml) was collected and reacted with 3 ml of DNS reagent in a 100°C oil bath for 5 minutes. After dilution by the same factor, the absorbance at 532 nm was measured. Test of the ability of Pt-AuNS-GOx nanoparticles to consume glucose in a weakly acidic environment: GOx can reduce glucose to gluconic acid and hydrogen peroxide using O2. The ability of Pt-AuNS nanoparticles to reduce glucose was evaluated after coupling GOx to them. Figure 10 As shown, Pt-AuNS-GOx nanoparticles of different concentrations were added to a glucose solution of the same concentration and reacted for the same amount of time. The remaining glucose content in the solution was then measured. It was found that under weakly acidic conditions, glucose consumption was concentration-dependent on the nanoparticles. In the reaction group with added H2O2, because Pt-AuNS-GOx can catalyze the decomposition of H2O2 into O2, providing reactants for glucose consumption, the glucose was consumed more thoroughly. Therefore, the glucose content in the group with added H2O2 was lower than that in the group without added H2O2. This demonstrates that Pt-AuNS-GOx nanoparticles have the ability to consume glucose and also possess cascade catalytic capabilities, decomposing H2O2 into O2 to utilize it for consuming more glucose.

[0054] Cytotoxicity assay of Pt-AuNS-GOx nanoparticles to normal cells: The biocompatibility of Pt-AuNS-GOx nanoparticles was assessed by analyzing their cytotoxicity to human umbilical vein endothelial cells (HUVECs) (ATCC) using the CCK8 assay. In short, cells were seeded into 96-well plates (8000-10000 cells / well) and cultured overnight. Cells were then treated with different Au concentrations (0, 10, 20, 30, 40 μg / mL) of Pt-AuNS-GOx. After 48 hours of culture, CCK8 was added, and culture was continued for another 2 hours. Cell viability was assessed using a microplate reader. Even at a high concentration of 40 μg / mL, normal cell viability remained above 90%, indicating that the Pt-AuNS-GOx nanomaterials are safe and non-toxic to normal cells.

[0055] Cytotoxicity assay of nanomaterials against mouse breast cancer (4T1) cells: To investigate the anticancer mechanism of Pt-AuNS-GOx nanoparticles, we co-incubated different nanomaterials with the same Au concentration with 4T1 cells for 12 hours. The light-illuminated group was irradiated at 808 nm, 1.0 W for 10 min, followed by another 12 hours of incubation. Cells were then washed twice with PBS, and CCK8 was added for a further 2 hours of incubation. The cytotoxicity to 4T1 cells was assessed using a microplate reader. Figure 11 As shown, the enzyme-like activities (OXD-like and CAT-like) generated by doping disrupt the original redox balance of cancer cells, resulting in Pt-AuNS exhibiting significantly higher cytotoxicity against 4T1 cells than AuNS. By cutting off the energy supply to cancer cells and consuming glucose within them, Pt-AuNS-GOx inhibits their proliferation, thus further enhancing the therapeutic effect of GOx-modified cells. Furthermore, near-infrared irradiation significantly promotes apoptosis in cancer cells, thanks to the excellent plasma properties of Pt-AuNS-GOx. The LSPR-excited hot electrons and photothermal activity greatly improve catalytic activity. These results confirm the significant anticancer efficacy of Pt-AuNS-GOx nanoparticles.

[0056] Establishment of a mouse breast cancer model: Mice (Balb / c, 6 weeks old, approximately 40g) were purchased from Nanjing Scoray Biotechnology Co., Ltd., and acclimatized in the laboratory for 1 week. All animal experiments were conducted according to the procedures approved by the Experimental Animal Center of Shandong University. 4T1 cells were digested with trypsin, resuspended in sterile PBS, and placed on ice. Hair was removed from the backs of the mice, and 1×10⁷ cells were injected into the upper thigh. The tumor size was measured to be approximately 100 mm using calipers. 3 It can be used to conduct in vivo experiments.

[0057] Therapeutic efficacy of nanomaterials against tumors in mice: Mice with successfully established tumor models were divided into 6 groups (mice treated with PBS and PBS+NIR as positive controls, GOx, Pt-AuNS, Pt-AuNS-GOx, and Pt-AuNS-GOx+NIR) for treatment to assess the therapeutic efficacy of Pt-AuNS-GOx nanoparticles against breast cancer in mice. In short, PBS and each nanoparticle (Au concentration 16 mg / kg) were intravenously injected into each group of mice. After 24 hours, the light-treated group was irradiated for 5 minutes (808 nm, 1.5 W), and the nanomaterials were injected again after 72 hours. This process was repeated. Mouse weight and tumor volume were recorded every two days. On day 20, the mice were euthanized, and the tumors were removed, weighed, and photographed.

[0058] Evaluation of the therapeutic effect of Pt-AuNS-GOx nanoparticles on breast cancer model mice: Successfully modeled breast cancer mice were divided into 6 groups, with at least 4 mice in each group. Mice treated with PBS and PBS+NIR served as positive controls. Treatments included Pt-AuNS, Pt-AuNS-GOx, and Pt-AuNS-GOx+NIR. Tumors were removed, photographed, and weighed on day 20. Figure 12 As shown, two mice in the Pt-AuNS-GOx+NIR treatment group were cured, while the tumors in the other two mice were smaller than those at the time of successful modeling, indicating that Pt-AuNS-GOx+NIR has a significant tumor-inhibiting effect and a good therapeutic effect.

[0059] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an acidic oxygen-generating nanoreactor, characterized in that, Includes the following steps: (1) Preparation of AuNS nanoparticles: Hepes was dissolved in deionized water, then mixed with NaOH, HAuCl4 was added and mixed well, the mixture was allowed to stand in the dark, centrifuged, the supernatant was removed and the precipitate was washed, and finally dispersed in deionized water for later use. (2) Preparation of Pt-AuNS nanoparticles: AuNSs and H2PtCl6 were stirred and preheated at 80 °C. Then, L-ascorbic acid was added to the mixed solution for reaction. After the reaction was completed, the mixture was centrifuged and the precipitate was washed to obtain Pt-AuNS nanoparticles. The Pt-AuNS nanoparticles were resuspended in deionized water to obtain an aqueous solution of Pt-AuNS nanoparticles. The concentration of AuNSs was 60 mg / L, the concentration of H2PtCl6 was 1 mM, and the concentration of L-ascorbic acid was 1 mM. The volume ratio of Pt-AuNS, H2PtCl6 and L-ascorbic acid was 6:4:

5. (3) Preparation and modification of Pt-AuNS-GOx nanoparticles: Dihydrolipoic acid was added to the aqueous solution of Pt-AuNS nanoparticles and stirred at room temperature; centrifuged, the supernatant was removed and the precipitate was taken, and resuspended in deionized water to obtain Pt-AuNS nanoparticles modified with carboxyl groups; NHS and EDC were dissolved in H2O2, and 100 μL of each were gradually added to the Pt-AuNS solution modified with carboxyl groups; the carboxyl groups were activated by stirring at room temperature; GOx aqueous solution was added dropwise to the activated Pt-AuNS solution, and the reaction was stopped by stirring at room temperature for a period of time, centrifuged, and the precipitate was washed and resuspended in deionized water; finally, passivation was performed overnight with HS-PEG, and washing was performed twice with DPBS to obtain the final product Pt-AuNS-GOx nanoparticles, which is the acidic oxygen-generating nanoreactor.

2. The preparation method according to claim 1, characterized in that, In step (1), 1 g of Hepes was dissolved in 28.4 mL of deionized water, then mixed with 1.6 mL of 1 M NaOH, and 150 μL of 40 mM HAuCl4 was added.

3. The preparation method according to claim 1, characterized in that, In step (1), the time for keeping the plant in the dark is 0.5-3 hours.

4. The preparation method according to claim 3, characterized in that, The light-protected standing time is 1 hour.

5. The preparation method according to claim 1, characterized in that, In step (2), the mixture is preheated at 700 rpm for 10 minutes.

6. The preparation method according to claim 1, characterized in that, In step (2), L-ascorbic acid is added and the reaction is carried out for 30 minutes.

7. The preparation method according to claim 1, characterized in that, In step (3), 100 μL of 15 mM dihydrolipoic acid was added to 10 mL of 60 mg / L Pt-AuNS, and the mixture was stirred at 750 rpm for 2 h at room temperature. After centrifugation, the supernatant was removed and the precipitate was collected. The precipitate was resuspended in 4 mL of deionized water to obtain carboxyl-modified Pt-AuNS nanoparticles. Then, 2 mg of NHS and 3 mg of EDC were weighed and dissolved in 1 mL of H2O2. 100 μL of each solution was gradually added to 4 mL of carboxyl-modified Pt-AuNS solution. The carboxyl groups were activated by stirring at room temperature. A 1 mg / mL GOx aqueous solution was prepared. 1 mL of GOx aqueous solution was added dropwise to the activated Pt-AuNS solution. The reaction was stopped after stirring at 750 rpm for 2 h at room temperature. The mixture was centrifuged, and the precipitate was washed three times with deionized water and resuspended in 10 mL of deionized water. Finally, the precipitate was treated with 0.1 mM Mw=5000... Da was passivated with HS-PEG overnight and washed twice with DPBS to obtain the final product, Pt-AuNS-GOx nanoparticles.

8. The preparation method according to claim 1, characterized in that, The centrifugation speed in steps (1), (2), and (3) is 10,000-13,000 rpm, and the centrifugation time is 10-20 min.

9. The preparation method according to claim 1, characterized in that, The acidic oxygen-generating nanoreactor prepared according to the preparation method described in claim 1.

10. The application of the acidic oxygen-generating nanoreactor prepared by the method according to claim 9 in the preparation of drugs for treating tumors.

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