An S / N co-doped carbon-based nanoenzyme@DOX composite material, its preparation method and application

Abstract

This invention discloses an S / N co-doped carbon-based nanozyme@DOX composite material, its preparation method, and its applications. Using silica as a hard template, zinc chloride as a pore-forming agent, glucosamine hydrochloride as both the N and C sources, and thiourea as the S source, S and N are simultaneously doped into the carbon framework via a one-pot high-temperature pyrolysis annealing process. Finally, hydrofluoric acid is used to remove the hard template, resulting in a layered porous S / N co-doped carbon-based nanozyme (SNC). After PEGylation modification, the layered porous structure facilitates the loading of the anticancer drug doxorubicin (DOX), yielding the S / N co-doped carbon-based nanozyme@DOX composite material. This composite material exhibits significant photothermal effects and enzyme-like catalytic activity responsive to the tumor microenvironment. It can accumulate and sustain-release drugs at the tumor site, achieving synergistic multimodal therapy of tumors using chemo / catalytic / photothermal methods, thereby improving the efficacy of tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of nanoenzyme technology, specifically relating to an S / N co-doped carbon-based nanoenzyme@DOX composite material, its preparation method, and its application. Background Technology

[0002] Cancer is one of the leading causes of death worldwide, posing a serious threat to human life and health. Currently, the main treatments for cancer include radiotherapy, chemotherapy, and surgical resection, but these methods all come with varying degrees of side effects. Therefore, finding safe and effective cancer treatment strategies is crucial. Excessive reactive oxygen species (ROS) can damage biological macromolecules such as DNA and proteins in tumor cells, ultimately leading to tumor cell apoptosis. Enzyme therapy, as an in vivo catalytic therapy modality for tumors, can regulate ROS through catalytic reactions, thereby inducing tumor cell apoptosis, and has attracted widespread attention from researchers. Furthermore, enzyme therapy can activate the body's immunity and enhance immune system function. However, natural enzymes face challenges in practical applications, such as high cost, poor stability, and difficulty in preservation, which significantly limit their application in clinical treatment.

[0003] With the development of nanoscience, an increasing number of nanomaterials have been found to possess enzyme-like catalytic activity, including carbon-based nanomaterials, metal nanomaterials, and metal oxide nanostructures. These nanomaterials with catalytic activity similar to natural enzymes are called nanozymes. Compared with natural enzymes, nanozymes have many significant advantages, such as low cost, high stability, ease of preparation, and tunable catalytic activity. Furthermore, in the tumor microenvironment, nanozymes can utilize the weak acidity of the tumor as a reaction condition and intratumoral H2O2 as a reactant to mimic peroxidase (POD) activity, triggering the Fenton / Fenton-like reaction and catalyzing the production of a large amount of ·OH from H2O2, thereby inducing tumor cell apoptosis and achieving chemodynamic therapy (CDT) in response to the tumor microenvironment. Among them, carbon-based nanozymes, due to their good biocompatibility, can significantly reduce toxic reactions to the body and are widely used in tumor CDT. In addition, carbon-based nanozymes also have strong near-infrared absorption and can be used for tumor photothermal therapy. In photothermal therapy, they can rapidly increase local temperature and enhance the anti-tumor effect. However, further improving the catalytic activity of carbon-based nanozymes and enhancing the efficacy of CDT therapy remains a challenge. Summary of the Invention

[0004] The purpose of this invention is to provide an S / N co-doped carbon-based nanozyme@DOX composite material, its preparation method, and its applications. This composite material is a drug-loaded nanozyme composite material with heteroatom doping and a layered porous structure. It exhibits significant photothermal effects and enzyme-like catalytic activity, and can accumulate and sustain drug release at tumor sites, achieving synergistic anti-tumor treatment through chemotherapy, enzyme-like catalytic therapy, and photothermal effects, resulting in highly efficient anti-tumor efficacy.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A method for preparing S / N co-doped carbon-based nanozyme@DOX composite material is provided, comprising the following steps:

[0007] 1) Synthesis of layered porous S / N co-doped carbon-based nanozymes: Glucosamine hydrochloride (C6H) was used to synthesize nanozymes. 13 NO5·HCl), thiourea (CH4N2S) and zinc chloride are dissolved together in SiO2 suspension. After stirring, the solution is freeze-dried. Then, the freeze-dried powder is pyrolyzed under an inert atmosphere. Finally, the template is removed with hydrofluoric acid, which yields a layered porous S / N co-doped carbon-based nanozyme, denoted as SNC nanozyme.

[0008] 2) Disperse and dissolve the SNC nanozyme obtained in step 1) in ultrapure water, and then add phosphatidylethanolamine-polyethylene glycol 2000-amine (DSPE-PEG). 2K -NH2), stir overnight, centrifuge to obtain precipitate, the precipitate is recorded as S-PEG, disperse the precipitate in ultrapure water to obtain S-PEG dispersion;

[0009] 3) Mix the S-PEG dispersion obtained in step 2) with doxorubicin hydrochloride (DOX) solution in the dark, stir overnight, and separate the precipitate to obtain the S / N co-doped carbon-based nanozyme@DOX composite material.

[0010] According to the above scheme, in step 1), the mass ratio of glucosamine hydrochloride, thiourea, zinc chloride and SiO2 is 2:0.3-0.4:0.1-0.2:1.5-2.5.

[0011] According to the above scheme, in step 1), the inert atmosphere is a flowing nitrogen atmosphere.

[0012] According to the above scheme, in step 1), the pyrolysis conditions are: calcination at 800-1000℃ for 1.5-2.5 hours. Preferably, the heating rate is 4-6℃ / min.

[0013] According to the above scheme, in step 1), the concentration of hydrofluoric acid is 8-15 wt%.

[0014] According to the above scheme, in step 2), SNC nanozyme and DSPE-PEG 2K The mass ratio of -NH2 is 4:12 to 18.

[0015] According to the above scheme, in step 2), the SNC nanozyme is dispersed and dissolved in ultrapure water, and the concentration of the resulting SNC nanozyme dispersion is 0.8 to 1.2 mg / mL.

[0016] According to the above scheme, in step 2), the centrifugation conditions are 10000-12000 rpm for 8-12 minutes.

[0017] According to the above scheme, in step 2), the concentration of the S-PEG dispersion is 0.8 to 1.2 mg / mL.

[0018] According to the above scheme, in step 3), the mass ratio of S-PEG to doxorubicin hydrochloride is 3:3 to 5.

[0019] According to the above scheme, in step 3), the concentration of doxorubicin hydrochloride solution is 0.8–1.2 mg / mL.

[0020] According to the above scheme, in step 3), the precipitate is separated by centrifugation; preferably, the centrifugation conditions are a speed of 10000-12000 rpm for 8-12 minutes.

[0021] This invention provides an application of the S / N co-doped carbon-based nanozyme@DOX composite material prepared by the above method in the preparation of antitumor drugs.

[0022] This invention provides a method for preparing S / N co-doped carbon-based nanoenzymes@DOX composite materials. Using silica as a hard template, zinc chloride as a pore-forming agent, glucosamine hydrochloride as both the N and C sources, and thiourea as the S source, S and N are simultaneously doped into the carbon framework via a one-pot high-temperature pyrolysis annealing process. Finally, hydrofluoric acid is used to remove the hard template, resulting in a layered porous S / N co-doped carbon-based nanoenzyme (SNC).

[0023] The SNC nanozyme prepared in this invention, through heteroatom doping, leverages the synergistic effect between heteroatoms to regulate the charge distribution of the carbon support, thereby influencing the adsorption energy of the intermediate and enhancing the catalytic activity of the active sites. Simultaneously, its layered porous structure, with its highly exposed number of active sites and abundant edge defects, results in even higher catalytic activity and highly selective peroxidase (POD)-like activity. This allows it to effectively catalyze H2O2 in the tumor microenvironment, generating ·OH and causing damage to cancer cells. Furthermore, the layered porous structure provides a high density of catalytic active sites while also containing numerous drug-loading sites. After PEGylation modification, it can load the anticancer drug doxorubicin, accumulating in the tumor area through the enhanced permeability and retention effect (EPR) to promote cell apoptosis. Finally, the photothermal effect of the carbon-based nanomaterials themselves can also increase the temperature of the tumor region, further promoting cell apoptosis.

[0024] The S / N co-doped carbon-based nanozyme@DOX composite material obtained in this invention passively accumulates in tumor tissue through high permeability and retention effect (EPR effect), releasing DOX and effectively reducing pharmacokinetic losses. It also interferes with tumor cell proliferation by releasing the drug under the low pH conditions of the tumor microenvironment (TME). Simultaneously, its POD-like activity exhibits high selectivity, utilizing the hydroxyl radicals generated by high H2O2 catalysis in the TME environment to induce cellular oxidative stress, thereby damaging cancer cells without harming normal tissues. Furthermore, combined with the material's photothermal effect, it promotes cell apoptosis by increasing the temperature in the tumor region, thus enhancing the therapeutic effect. This method aims to achieve a comprehensive anti-tumor therapy, inhibiting tumor growth and spread through the combined action of multiple mechanisms including chemo / photothermal / CDT.

[0025] The beneficial effects of this invention are:

[0026] 1. This invention employs a one-pot high-temperature pyrolysis method. By introducing S and N sources, heteroatom doping and pore-forming processes involving silica and zinc chloride are achieved to form a layered porous structure, which improves the catalytic activity of the material and provides numerous drug-carrying sites. The layered porous structure then facilitates the introduction of the anticancer drug doxorubicin hydrochloride, resulting in an S / N co-doped carbon nanozyme (SNC) loaded with the anticancer drug DOX. The resulting composite material exhibits a certain photothermal effect and highly selective, excellent enzyme-like catalytic activity. It can accumulate and release the drug in large quantities in the tumor lesion area through the EPR effect, achieving synergistic antitumor therapy through chemotherapy, enzyme-like catalytic therapy, and photothermal effects. This multi-mechanism approach inhibits tumor growth and spread while effectively reducing the possibility of chemotherapy damaging normal tissues, achieving highly efficient antitumor effects and providing new ideas for developing novel and highly effective combined treatment strategies for malignant tumors.

[0027] 2. This invention uses a one-pot high-temperature pyrolysis method, which has a simple preparation process, short production cycle, and inexpensive and readily available raw materials, thus saving production costs. Furthermore, the production process does not use highly toxic raw materials or volatile chemical reagents, making it safe and environmentally friendly, and suitable for large-scale production. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] Figure 1 The image shows a transmission electron microscope (TEM) image of the SNC nanozyme prepared in Example 1.

[0030] Figure 2 This is a high-resolution transmission electron microscope image of the SNC nanozyme prepared in Example 1.

[0031] Figure 3 The image shows a high-angle annular dark-field image (left) and an elemental analysis diagram obtained by energy dispersive spectroscopy for the SNC nanozyme prepared in Example 1, where red represents C, green represents N, blue represents O, and yellow represents S.

[0032] Figure 4 The UV-Vis spectra of the SNC nanozyme prepared in Example 1 under different conditions for POD activity testing are shown.

[0033] Figure 5 The UV-Vis spectrum is the result of quantitative testing of the POD activity of the SNC nanozyme prepared in Example 1.

[0034] Figure 6 The temperature changes of the SNC nanozyme prepared in Example 1 at different concentrations are shown.

[0035] Figure 7 The temperature change of the SNC nanozyme photothermal effect under different powers is shown in Example 1.

[0036] Figure 8 The UV-Vis spectrum of the SPD composite material prepared in Example 1 is shown.

[0037] Figure 9 The cell survival rate of the SNC nanozyme prepared in Example 1 under different treatments.

[0038] Figure 10 The cell survival rate of the SPD composite material prepared in Example 1 under different treatments.

[0039] Figure 11 The cell survival rate of the SNC nanozyme and SPD composite material prepared in Example 1 under different treatment conditions.

[0040] Figure 12 The images show the fluorescence of live and dead cells under different conditions for SNC nanozymes and SPD composite materials prepared in Example 1.

[0041] Figure 13 The distribution of the SNC nanozyme prepared in Example 1 in mice.

[0042] Figure 14 This diagram illustrates the changes in tumor volume in mice after treatment with the control group, Example 1 group, and Example 1 combined with near-infrared therapy.

[0043] Figure 15 This diagram illustrates the changes in body weight of mice after treatment in the control group, Example 1 group, and Example 1 combined with near-infrared therapy group.

[0044] Figure 16 Histological analysis of hematoxylin-eosin (H&E) stained tissue sections of tumors obtained after treatment in the control group, Example 1 group, and Example 1 combined with near-infrared group, as well as Ki-67 antigen and TUNEL immunostaining. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0046] Example 1

[0047] A method for preparing S / N co-doped carbon-based nanozyme@DOX composite material is provided, comprising the following steps:

[0048] The first step is the synthesis of layered porous S / N co-doped carbon nanozymes.

[0049] 2g C6H 13 NO5·HCl, 334 mg CH4N2S, and 150 mg ZnCl2 were dissolved together in 20 mL of suspension containing 2 g SiO2. The solution was then stirred and lyophilized. Subsequently, the resulting powder was pyrolyzed in a flowing nitrogen atmosphere, heated to a target temperature of 900 °C at a rate of 5 °C / min, and held at this temperature for 2 h. Finally, the template was removed using 10 wt% hydrofluoric acid to obtain the SNC nanozyme.

[0050] Figure 1-2 The images show transmission electron microscopy (TEM) and high-resolution TEM images of the SNC nanozyme, respectively. The images reveal that the nanozyme has a layered porous structure containing numerous spherical pores, which facilitates the exposure of catalytic active sites and the loading of anticancer drugs.

[0051] Figure 3The high-angle annular dark-field image and energy dispersive spectroscopy (EDS) elemental analysis of the SNC nanozyme show the uniform distribution of C, O, N, and S elements, proving S / N co-doping. The layered porous structure endows the SNC nanozyme with a high number of exposed active sites, abundant edge defects, and a large specific surface area, which is beneficial for accelerating charge transport and enhancing the POD activity of SNC nanozymes.

[0052] The second step is the modification of SNC nanozymes.

[0053] The SNC nanozyme obtained in the first step was ultrasonically disrupted and dispersed in ultrapure water to obtain a 1 mg / mL SNC nanozyme dispersion. Then, 4 mL of the SNC nanozyme dispersion (1 mg / mL) was mixed with 15 mg of DSPE-PEG. 2K The mixture of -NH2 was stirred overnight, and then centrifuged at 11000 rpm for 10 min to obtain S-PEG precipitate. 4 mL of ultrapure water (with a resistivity of 18.2 MΩ·cm) was added to redissolve the precipitate, resulting in an S-PEG dispersion of 1 mg / mL.

[0054] The third step is the synthesis of S / N co-doped carbon-based nanozyme@DOX composite materials.

[0055] The 3 mL L-PEG dispersion (1 mg / mL) obtained in the second step was mixed with 4 mL of doxorubicin hydrochloride solution (1 mg / mL) in the dark and stirred overnight. Then, the mixture was centrifuged at 11,000 rpm for 10 min to obtain a precipitate, which is the S / N co-doped carbon-based nanoenzyme@DOX composite material, denoted as SPD. 3 mL of ultrapure water was added to redissolve the precipitate to obtain the SPD dispersion.

[0056] The S / N co-doped carbon-based nanozyme@DOX composite material prepared in this embodiment was used to test enzyme activity and in vitro and in vivo antitumor efficacy. The method is as follows:

[0057] (1) POD-like activity test

[0058] The POD-like activity of Example 1 was determined colorimetrically using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate in 0.1 M acetate-sodium acetate (HAc-NaAc) buffer (pH = 4.0). TMB exhibited a characteristic absorption peak at 652 nm after oxidation by reactive oxygen species such as ·OH. Nanozyme solution (10 μL, 500 μg / mL), hydrogen peroxide (100 μL, 1 mM aqueous solution of H2O2), and TMB solution (50 μL, 10 mM) were added sequentially to 0.20 mL of HAc-NaAc buffer and mixed thoroughly. The mixture was incubated in the dark at 37°C for 1 minute. The final concentrations of nanozyme, hydrogen peroxide, and TMB were fixed at 500 μg / mL, 100 mM, and 1 mM, respectively. The UV-Vis absorption spectra of the test solutions were recorded using a multi-functional microplate reader. The results are shown below. Figure 4 As shown, no significant color change was observed in the TMB or TMB+H2O2 groups. However, upon addition of SNC nanozymes, the solution color changed from colorless to blue, exhibiting a high characteristic absorption at 652 nm. This indicates that SNC nanozymes possess good POD-like activity and can effectively oxidize TMB for color development. In contrast, the SNC+Laser group showed higher absorbance, demonstrating that the Laser can further enhance reactive oxygen species (ROS) generation.

[0059] To further investigate the relationship between concentration and catalytic efficiency, we varied the concentration of H2O2, and the results were as follows: Figure 5 It can be observed that in the presence of SNC nanozymes, the absorbance value increases with the increase of H2O2 concentration. These test results all demonstrate that Example 1 has significant POD-like activity.

[0060] (2) Photothermal performance test

[0061] The temperature change of SNC nanozymes under near-infrared irradiation (Laser) was measured using a thermometer.

[0062] SNC nanozymes were dispersed into solutions of different concentrations (50, 75, 100, and 150 μg / mL). Then, 1 mL of each concentration solution was taken and subjected to a power of 1.5 W / cm². 2 Irradiate with near-infrared light for ten minutes. Figure 6 The temperature changes of solutions at different concentrations were shown over a ten-minute period. The results indicated a positive correlation between the concentration of the dispersion and the increase in solution temperature. To further investigate the effect of the near-infrared irradiation power on the photothermal efficiency of SNC nanozymes, we analyzed the temperature change curves of a 100 μg / mL dispersion at different powers. Figure 7 As shown, the rate at which the solution temperature rises also increases with increasing power.

[0063] (3) Drug release detection

[0064] The bonding status of materials can be determined by the characteristic absorption peaks of DOX ultraviolet light.

[0065] We used a multi-functional microplate reader to analyze the UV-Vis absorption spectra of the drug doxorubicin, as well as SNC and SPD solutions, such as... Figure 8 As shown, compared to the SNC solution, the SPD solution exhibits the characteristic absorption peak of doxorubicin at 490 nm. This phenomenon indicates that the SPD composite material successfully carries doxorubicin.

[0066] (4) In vitro efficacy testing

[0067] The antitumor effect of the material was evaluated using the MTT cytotoxicity assay.

[0068] To evaluate the effects of different concentrations of SNC, SPD, and DOX on 4T1 cells, 4T1 cells were seeded in 96-well plates and cultured overnight (1 × 10⁶ cells per well). 5 (Number of cells). After the cells stabilized, they were treated with different materials for 24 hours. After incubation, the cells were washed with PBS, and 20 μL of MTT solution was added to each well, followed by incubation for another 4 hours. Then, the supernatant was removed, and 150 μL of dimethyl sulfoxide (DMSO) solution was added. The cells were then placed on a light-protected shaker and shaken to dissolve any crystals. Finally, the UV-Vis absorption spectrum at 490 nm was recorded using a multi-mode microplate reader. Figure 9 As shown, even at SNC concentrations as high as 100 μg / mL, cell viability remained above 90%. However, in the group treated with both SNC and H2O2, cell viability decreased with increasing SNC nanozyme concentration. This is because the enzyme-like catalytic activity of SNC can convert H2O2 into hydroxyl radicals (·OH), inducing tumor cell apoptosis. Figure 10 The efficacy of SPD combined with PTT was demonstrated. Cell viability decreased with increasing DOX concentration, as SPD effectively releases the drug, thereby inhibiting cell growth. The therapeutic effect was further enhanced when used in combination with PTT. Compared to monotherapy, SPD combined with laser therapy exhibited more significant anti-tumor proliferative activity. We evaluated the effects of different interventions on cell growth inhibition under TME conditions, and the results are as follows: Figure 11 As shown, compared with other intervention groups, SPD combined with laser therapy exhibited the most significant inhibitory effect on cell growth under TME conditions. This indicates that in TME, the dual intervention of SPD's enzyme-catalyzed therapy (CDT) and photothermal therapy (PTT) can effectively inhibit tumor cell growth.

[0069] Cell status was observed and determined using the Calcein-AM / PI live / dead cell double staining kit.

[0070] 4T1 cells were seeded in 12-well plates fitted with slides and cultured overnight. After cell growth stabilized, cells were incubated with different materials (SNC, SNC+H2O2, SPD, SPD+laser, SPD+H2O2+laser) for 6 hours. After incubation, cells were rinsed twice with 1x AssayBuffer (1 mL each time). After rinsing, 500 μL of 1x AssayBuffer and 0.3 μL of Calcein-AM (stock solution) were added, mixed, and incubated at 37°C in the dark for 20-25 minutes. 1.7 μL of the PI stock solution provided in the kit was added to the stained cells, and staining was carried out at room temperature in the dark for 5 minutes. After fluorescence incubation, cells were washed with 1x PBS, fixed with 1 mL of paraformaldehyde for 10 minutes, and finally mounted with 15-20 μL of anti-fluorescence quencher. Live cells (yellow-green fluorescence) and dead cells (red fluorescence) were simultaneously detected under a fluorescence microscope using a 490±10 nm excitation filter. Additionally, only dead cells can be observed using a 545nm emission filter. Alternatively, detection can be performed directly using a suitable filter under a fluorescence microplate reader. It is recommended to avoid light throughout the entire process of fluorescent staining cells. Results are as follows... Figure 12 As shown, under TME conditions, the SPD and PTT combined treatment group exhibited the strongest red fluorescence, indicating the highest number of dead cells, while almost no green fluorescence was observed, indicating a very low number of live cells. This result is consistent with the findings of the MTT cytotoxicity assay.

[0071] (4) In vivo efficacy testing

[0072] Female BALB / c mice (6 weeks old, weighing approximately 20g) were selected as the animal model. Animal experiments in this study were strictly conducted according to the protocol approved by the Ethics Committee of Wuhan University of Science and Technology School of Medicine (Approval No.: 2024123). Mouse breast cancer 4T1 cells (8 × 10⁻⁶) were subcutaneously injected into the right back. 5 Tumor modeling was performed.

[0073] Before in vivo efficacy testing, 100 μL of SNC nanozyme carrying cy7 fluorescent agent was injected into the tail vein of a tumor-bearing mouse. The dose of cy7 was 0.05 mg / kg. Subsequently, the fluorescence distribution in the mouse was observed using small animal imaging technology. The results are as follows: Figure 13 As shown, obvious fluorescence signals can be detected in the tumor area 12 hours after injection.

[0074] 4T1 tumor-bearing mice were randomly divided into three groups (n=4 per group): a control group, the Example 1 group, and the Example 1 combined with near-infrared laser group. Each experimental group received 7 mg / kg once daily. The Example 1 combined with near-infrared laser group received near-infrared laser at 1 W / cm² one day after administration. 2 The tumor area was irradiated with a high power for 5 minutes. During the treatment, body weight and tumor size were measured and recorded every other day for 16 days.

[0075] After 16 days of observation, the results were as follows: Figure 14 As shown, in stark contrast to the control group, tumor growth was effectively inhibited after injection of the SPD nanocomposite material synthesized in Example 1. The inhibition rate in Example 1 reached 64%. Combined with nanozyme activity testing, we can conclude that the excellent therapeutic effect of Example 1 stems from the synergistic effect of drug chemotherapy and CDT. It is worth mentioning that the tumor inhibition rate in the combination of Example 1 and near-infrared spectroscopy was further improved, reaching 85%, which can be attributed to the increased catalytic efficiency of near-infrared spectroscopy and its role in tumor ablation. Figure 15 As shown, the body weight of mice in each group remained basically unchanged over 16 days, which further illustrates that the toxicity and side effects of these synthetic nanozymes on mice are negligible.

[0076] Next, histopathological analysis was performed on the obtained tumor tissue. The degree of damage to the tumor tissue by nanozymes was evaluated by hematoxylin and eosin (H&E) staining; the proliferation of tumor cells was detected by Ki-67 antigen; and the apoptosis of tumor cells was detected by TUNEL antigen.

[0077] The results are as follows Figure 16 As shown, after treatment in Example 1, the tumors in mice exhibited microscopic damage to the nuclei and cytoplasm of tumor cells, while macroscopically they showed loose tissue with many gaps, and significant apoptosis and tissue destruction. Figure 16 (Leftmost column) The phenomenon was more pronounced after Example 1 combined with near-infrared spectroscopy treatment. As shown by Ki-67 antigen immunohistochemical staining, Ki-67 expression was significantly reduced after Example 1 combined with near-infrared spectroscopy treatment, and the predicted tumor proliferation trend was weakened. Figure 16 (Middle column) Only after treatment in Example 1 did TUNEL maintain a certain expression level. Immunohistochemical analysis of TUNEL revealed cell apoptosis; the expression level of TUNEL in mouse tumors treated with the combination of Example 1 and near-infrared spectroscopy was significantly increased, indicating that apoptosis had occurred. Figure 15 (Rightmost column) The phenomenon was more pronounced after treatment with near-infrared spectroscopy in Example 1. In summary, the mouse tumors treated with near-infrared spectroscopy in Example 1 showed high levels of ROS accumulation, indicating strong apoptosis. It exhibited good anti-tumor properties in vivo.

[0078] The above embodiments are the result of continuous experimentation with raw material dosages to achieve high catalytic efficiency of the obtained nanozymes within the minimum raw material dosage range. Through strict control of each process step and multiple trials, a layered porous nanomedicine carrier system has been developed that can be used in the field of tumor treatment for chemotherapy, enzyme-like catalytic therapy, and photothermal effect-induced apoptosis.

[0079] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing S / N co-doped carbon-based nanoszyme@DOX composite material, characterized in that, The method comprises the following steps: 1) synthesis of hierarchical porous S / N co-doped carbon-based nanosensor: dissolve glucose hydrochloride, thiourea and zinc chloride in SiO2 suspension, stir the solution and freeze-dry, then pyrolyze the freeze-dried powder under inert atmosphere, and finally remove the template with hydrofluoric acid to obtain hierarchical porous S / N co-doped carbon-based nanosensor, denoted as SNC nanosensor; wherein the mass ratio of glucose hydrochloride, thiourea, zinc chloride and SiO2 is 2:0.334:0.15:2; 2) The SNC nanoszyme prepared in step 1) is dispersed in ultrapure water, and then DSPE-PEG is added 2K -NH2, stirring overnight, centrifuging to obtain a precipitate, which is denoted as S-PEG, and dispersing the precipitate in ultrapure water to obtain an S-PEG dispersion; 3) mix the S-PEG dispersion solution obtained in step 2) with a doxorubicin hydrochloride solution in the dark, stir overnight, separate the precipitate to obtain S / N co-doped carbon-based nanosensor@DOX composite material.

2. The production method according to claim 1, characterized by, In step 1), the pyrolysis conditions are: calcination at 800-1000℃ for 1.5-2.5 h.

3. The preparation method according to claim 1, characterized in that, In step 1), the concentration of hydrofluoric acid is 8-15wt%.

4. The method of claim 1, wherein, In the step 2), the mass ratio of SNC nanoszyme and DSPE-PEG 2K -NH2 is 4:12~18.

5. The preparation method according to claim 1, characterized in that, In step 2), the SNC nanosensor is dispersed in ultrapure water, and the concentration of the obtained SNC nanosensor dispersion solution is 0.8-1.2 mg / mL.

6. The method of claim 1, wherein, In step 3), the concentration of the S-PEG dispersion solution is 0.8-1.2 mg / mL; the concentration of the doxorubicin hydrochloride solution is 0.8-1.2 mg / mL.

7. The preparation method according to claim 1, characterized in that, In step 3), the mass ratio of S-PEG to doxorubicin hydrochloride is 3:3-5.

8. S / N co-doped carbon-based nanosensor@DOX composite material prepared by the preparation method of any one of claims 1-7.

9. Use of the S / N co-doped carbon-based nanosensor@DOX composite material of claim 8 in the preparation of an anti-breast cancer drug.

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