Multifunctional nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a multifunctional nano-enzyme as well as a preparation method and application thereof, and belongs to the technical field of nano-enzyme synthesis. Aiming at the problems that a traditional nano-enzyme is single in activity, limited in treatment effect, poor in tumor active targeting specificity and the like, the multifunctional nano-enzyme provided by the invention is simple and efficient in preparation method, and is prepared by taking N and P double-doped hollow carbon nanospheres as a carrier, loading L-arginine and glucose oxidase and taking an aptamer as a targeting molecule. The multifunctional nano-enzyme is small in particle size, high in dispersion degree and good in biological safety. The multifunctional nano-enzyme not only has the activities of peroxidase, nitric oxide synthase and glucose oxidase, but also has excellent photo-thermal conversion capacity in tumor targeted therapy. The multifunctional nano-enzyme provided by the invention has a tumor focus specific recognition capability, can be highly enriched at a tumor part, finally realizes the purpose of photothermal / catalytic treatment combined tumor treatment, and provides a new thought and platform for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanozyme synthesis, and specifically relates to a multifunctional nanozyme and a preparation method and application thereof. Background Art

[0002] Since enzyme-driven catalytic reactions occur continuously in organisms and can achieve efficient and selective biotransformations, enzyme-based catalytic therapy has attracted much attention in recent years. However, natural enzymes are subject to many limitations in practical applications due to their single catalytic ability, high manufacturing cost, and high complexity. Nanozymes combine the functions of nanomaterials and enzymes, have the advantages of strong catalytic activity, high stability, and simple preparation, and have attracted widespread attention from scientists. Since the peroxidase-like (POD) activity of Fe3O4 nanoparticles was reported in 2007, researchers have developed a variety of nanomaterials with enzyme catalytic activity, which can be widely used in the biomedical field, including tumor treatment. For example, catalase-like (CAT) can decompose H2O2 into O2 to alleviate hypoxia in tumor tissues; glucose oxidase-like (GOx) can oxidize glucose into gluconic acid and H2O2, consume glucose, and be used for catalytic therapy; POD-like enzymes can decompose H2O2 into hydroxyl radicals (•OH), inducing oxidative stress and triggering cell apoptosis.

[0003] In the prior art, patent CN115569190A provides a multifunctional nanozyme immune adjuvant and its preparation method and application, the preparation method includes reacting the solution of porous ternary metal nanoparticles with an acid solution having a carboxyl group to obtain COOH-ternary metal; reacting the solution of bovine serum albumin with a manganese salt, then adjusting the pH of the system to 10-12, and continuing the reaction to obtain a BSA-Mn complex; loading the BSA-Mn complex on the surface and pores of the COOH-ternary metal to obtain a multifunctional nanozyme. The nanozyme inhibits tumor growth by immunotherapy, however, the nanozyme lacks active targeting ability and only relies on the EPR effect to enter the tumor lesion. Less nanozyme accumulates in the tumor site, which ultimately leads to limited therapeutic effect.

[0004] The paper Glucose-Responsive Sequential Generation of Hydrogen Peroxide and Nitric Oxide for Synergistic Cancer Starving-Like / Gas Therapy provides a new type of intelligent nano-therapeutic agent, which uses hollow mesoporous silica nanoparticles to simultaneously load GOx and L-arginine (L-Arg), thereby achieving green and non-invasive synergistic treatment for malignant tumors. However, the hollow mesoporous silica nanoparticles used as the carrier of the nano-therapeutic agent do not have the ability to convert light into heat, and the treatment that relies solely on the NO and •OH produced by the nano-therapeutic agent cannot achieve satisfactory therapeutic effects.

[0005] Although nanozymes have made some research progress, they still have great potential for development in terms of integrating multiple enzyme activities, improving enzyme activity, active targeted therapy, and therapeutic mechanisms. Therefore, it is necessary to develop a more excellent multifunctional nanozyme that integrates multiple favorable factors into a single nanozyme, increases its enrichment in tumor tissues, improves treatment efficiency, and thus achieves targeted and precise tumor treatment. Summary of the invention

[0006] In response to the problems of traditional nanozymes having single activity, limited therapeutic effect, and lack of active tumor targeting specificity, the present invention provides a multifunctional nanozyme and a preparation method and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: A multifunctional nanozyme uses N, P doped hollow carbon nanospheres HCNPNs as a carrier, simultaneously loads L-Arg (L-arginine) and GOx in the cavity and mesopores of the carrier, and couples the nucleic acid aptamer Apt that can recognize cell surface specific proteins on its surface.

[0008] Furthermore, the N, P dual-doped hollow carbon nanosphere HCNPNs material is a spherical particle with a particle size of 200 nm.

[0009] Furthermore, the GOx loading amount is 9.3 wt% of the N, P dual-doped hollow carbon nanosphere HCNPNs material, and the L-Arg loading amount is 13.6 wt% of the N, P dual-doped hollow carbon nanosphere HCNPNs material.

[0010] Furthermore, the nucleic acid aptamer Apt is coupled to the surface of the N, P doped hollow carbon nanosphere HCNPNs material by a covalent bonding method.

[0011] A method for preparing a multifunctional nanozyme comprises the following steps: Step 1, preparation of N, P doped hollow carbon nanosphere HCNPNs material: using SiO2 nanoparticles as a hard template, dopamine as a carbon source and a nitrogen source, and adenosine triphosphate as a phosphorus source, polycondensing on the hard template to obtain a precursor; under the protection of an inert gas, carbonizing the precursor at a high temperature, and completely removing SiO2 by etching with hydrofluoric acid to obtain N, P doped hollow carbon nanosphere HCNPNs material; Step 2, loading: using electrostatic interaction to load L-Arg (L-arginine) and GOx into the HCNPNs material; Step 3, surface modification with dicarboxyl polyethylene glycol (PEG): PEG is mixed and stirred with the N, P doped hollow carbon nanospheres HCNPNs material loaded with L-Arg and GOx to complete the surface modification; Step 4, using EDC / NHS reagents to covalently couple Apt that can recognize cell surface specific proteins to the surface of the material modified by PEG to prepare a multifunctional nanozyme.

[0012] Furthermore, the preparation method using SiO2 nanoparticles as a hard template is: ethanol, ammonia water and deionized water are mixed, tetrapropoxysilane (TEOS) is used as a silicon source, and polymerized to form SiO2 nanoparticles.

[0013] Furthermore, the mass ratio of dopamine to adenosine triphosphate is 8:1; the loading amount of L-Arg (L-arginine) is 10-15 wt%, and the loading amount of GOx is 5-15 wt%.

[0014] Furthermore, the high temperature carbonization temperature is 800° C. and the time is 3 h.

[0015] A use of the multifunctional nanozyme prepared by the preparation method as described above in the preparation of tumor targeting drugs.

[0016] A use of the multifunctional nanozyme prepared by the preparation method as described above in the preparation of a photothermal / catalytic combined therapeutic drug for breast cancer tumors.

[0017] A multifunctional nanozyme is used in tumor targeted therapy. The multifunctional nanozyme can actively identify specific tumor cells and perform photothermal / catalytic combined therapy on breast cancer tumors, effectively inhibiting tumor growth, thereby achieving precision medicine.

[0018] Compared with existing hollow carbon nanospheres (HCNs), the presence of heteroatoms in HCNPNs leads to a lower overpotential, so HCNPNs themselves have excellent POD enzyme properties. In addition, compared with HCNs, HCNPNs have a thinner mesoporous shell, resulting in larger hollow voids and lower density, so the photothermal conversion efficiency of the multifunctional nanozyme HCNPNs / AGPM is much higher than that of HCNs. And compared with traditional therapies, the catalytic therapy used is more effective and safer.

[0019] Compared with the prior art, the present invention has the following advantages: (1) The HCNPNs material selected in the present invention has the advantages of large specific surface area, rich pore structure, easy surface functionalization, good biocompatibility, good cell permeability, low density and low toxicity.

[0020] (2) The present invention performs PEG surface modification on HCNPNs materials, which can improve their dispersibility in physiological environments and biocompatibility.

[0021] (3) The present invention uses Apt as a targeting group and couples Apt to the surface of the multifunctional nanozyme through covalent binding. It has good stability and active targeting performance, and can enable the multifunctional nanozyme to accurately target specific tumor cells, efficiently enrich in the lesions, and effectively inhibit tumor growth, thereby achieving the purpose of targeted therapy.

[0022] (4) The multifunctional nanozyme HCNPNs / AGPM obtained in the present invention has GOx enzyme, POD enzyme, and nitric oxide synthase activities and can be used for tumor catalytic therapy.

[0023] (5) The multifunctional nanozyme HCNPNs / AGPM obtained in the present invention has excellent photothermal conversion ability. It can generate heat under 808 nm laser irradiation to achieve tumor photothermal therapy and enhance enzyme activity, thereby enhancing nanozyme-mediated tumor catalytic therapy.

[0024] (6) The Apt used in the present invention can be modified according to different target cells and specific antigens on the surface of different cells, and thus can be applied to the treatment of various cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the preparation and application of the multifunctional nanozyme of the present invention; Figure 2 TEM image of the HCNPNs material in Example 1 of the present invention; Figure 3 The EDS and mapping analysis results of the HCNPNs material in Example 1 of the present invention; Figure 4The Zata potential analysis results of HCNPNs, HCNPNs / A, HCNPNs / AG, HCNPNs / AGP and HCNPNs / AGPM materials in Example 1 of the present invention; Figure 5 The dynamic light scattering analysis results of HCNPNs, HCNPNs / A, HCNPNs / AG, HCNPNs / AGP and HCNPNs / AGPM materials in Example 1 of the present invention; Figure 6 The temperature variation results of the dispersion of HCNPNs / AGPM material in Example 2 of the present invention under irradiation with different 808 nm laser power densities; Figure 7 The temperature variation results of the dispersion of HCNPNs / AGPM materials with different concentrations under 808 nm laser irradiation in Example 2 of the present invention; Figure 8 The photothermal stability test results of the HCNPNs / AGPM material in Example 2 of the present invention after five times of 808 nm laser irradiation; Fig. 9 is the light-to-heat conversion efficiency of the HCNPNs / AGPM material in Example 2 of the present invention; Fig.10 The results of GOx enzyme activity analysis of HCNPNs / AGPM materials in Example 3 of the present invention; Fig.11 The ESR test result of POD enzyme activity of HCNPNs / G material in Example 4 of the present invention; Fig.12 The results of POD enzyme activity analysis of HCNPNs / AGPM materials in Example 5 of the present invention; Fig.13 The results of NOS enzyme activity analysis of HCNPNs / AGPM materials in Example 6 of the present invention; Fig.14 This is a comparison diagram of the targeting performance of MCF-7 cells after incubation with different materials for 4 hours in Example 7 of the present invention; Fig.15 This is a graph showing the viability of MCF-7 cells after being treated with various nanomaterials in Example 8 of the present invention; Fig.16 This is a graph showing the apoptosis results of MCF-7 cells after being treated with various nanomaterials in Example 8 of the present invention; Fig.17 This is a graph showing the change of material enrichment in the tumor site of mice over time after tail vein injection of each nanomaterial in Example 9 of the present invention; Fig.18 This is a graph showing the enrichment of the main organs and tumor tissue materials of mice after tail vein injection of each nanomaterial in Example 9 of the present invention; Fig.19 This is a graph showing the changes in material enrichment in the tumor sites of bilateral tumor-bearing mice over time after the HCNPNs / AGPM material was injected into the tail vein in Example 9 of the present invention; Fig. 20 This is a graph showing the weight changes of mice after treatment with various nanomaterials in Example 10 of the present invention; Fig.21 This is a graph showing the changes in tumor volume of mice after treatment with various nanomaterials in Example 10 of the present invention; Fig. 22 This is a diagram showing the dissection results of mouse tumor tissue after treatment with various nanomaterials in Example 10 of the present invention. DETAILED DESCRIPTION

[0026] In order to gain a deeper understanding of the present invention, we will provide a comprehensive and detailed description of the present invention. However, the present invention has multiple implementations and is not limited to the specific examples listed herein. The presentation of these examples is intended to deepen the comprehensive understanding of the disclosure of the present invention.

[0027] Example 1

[0028] Depend on Figure 1 As shown, a multifunctional nanozyme is carried by HCNPNs material, and L-Arg and GOx are loaded on its surface. After PEG surface modification, Apt that can recognize specific proteins on the cell surface is coupled on its surface to make it have targeting ability.

[0029] The preparation method of the multifunctional nanozyme comprises the following steps: Step 1, preparation of HCNPNs material: water (17.2 mL), ethanol (92 mL) and NH3·H2O (1.45 mL) were mixed. After stirring for 3 min, 3.5 mL TEOS was added, and then stirred for 3.5 h. The precipitate was collected by centrifugation and dried at 60 °C to obtain a SiO2 hard template. Then 100 mg SiO2 and 100 mg dopamine were dissolved in 30 mL Tris-HCl buffer. After 8 h, 12.5 mg ATP was added and stirred for 12 h. The precipitate was collected by centrifugation, washed with ethanol 3 times, and dried at 60 °C to obtain a carbon precursor. The carbon precursor was then carbonized at 800 °C in a N2 atmosphere for 3 h. Finally, SiO2 was removed by HF etching to obtain HCNPNs.

[0030] Depend on Figure 2 The TEM image shows that the obtained HCNPNs material has a hollow structure, spherical particles, good dispersion, and a particle size of 200 nm. Figure 3 The EDS analysis results show that the HCNPNs material has been completely carbonized and SiO2 has been removed. Its specific surface area is 244 m 2 g −1.

[0031] Step 2, loading of L-Arg and GOx: L-Arg and GOx were loaded into HCNPNs material by using electrostatic interaction. Specifically, 10 mg of synthesized HCNPNs was immersed in a 5 mg mL −1 20 mL L-Arg solution was added. After continuous stirring for 12 h, the mixture was centrifuged and washed to remove unbound L-Arg to obtain L-Arg-loaded HCNPNs (HCNPNs / A). 5 mg HCNPNs / A and 1.5 mg GOx were dispersed in water. After stirring for 8 h, the mixture was centrifuged to obtain HCNPNs / AG.

[0032] Depend on Figure 4 and Figure 5 It can be seen that due to the loading of L-Arg and GOx, the hydrated particle size of HCNPNs / AG increased from 200 nm to 260 nm, and the Zeta potential increased from − 32.4 mV to − 1.3 mV. The above results show that L-Arg and GOx were successfully loaded into HCNPNs. The GOx loading amount was 9.3 wt% of the HCNPNs material, and the L-Arg loading amount was 13.6 wt% of the HCNPNs material.

[0033] Step 3, PEG surface modification: HCNPNs / AG (2.0 mg) and PEG (6.0 mg) were dispersed in 10.0 mL of distilled water under ultrasound. After continuous stirring for 12 h, unbound PEG was removed by centrifugation to obtain HCNPNs / AGP.

[0034] Depend on Figure 4 and Figure 5 It can be seen that due to the loading of PEG on the surface of HCNPNs / AG, the hydrated particle size of HCNPNs / AGP increased to 280 nm, while the Zeta potential decreased to − 21 mV. The above results indicate that the surface of HCNPNs / AG was successfully modified with PEG.

[0035] Step 4, Apt coupling: Activate HCNPNs / AGP (2.0 mL, 1.0 mg mL −1), the reaction was carried out under magnetic stirring for 30 min, and the activated HCNPNs / AGP material was obtained by centrifugation, followed by washing 3 times. After that, the MUC1 protein nucleic acid aptamer (Apt-M) (4 OD) was denatured at 85°C for 10 min, and then refolded in an ice bath for 10 min. The activated HCNPNs / AGP material was reacted with Apt-M under shaking conditions for 5 h, and then centrifuged and washed with water 3 times to obtain Apt-M modified HCNPNs / AGPM material. At the same time, for the subsequent control experiments, the random sequence (Apt-C) without targeting performance was reacted under the same conditions to prepare HCNPNs / AGPC material; HCNPNs / AGPM and HCNPNs / AGPC were labeled with the fluorescent molecule rhodamine B to prepare HCNPNs / RGPM and HCNPNs / RGPC materials for flow cytometry experiments and in vivo imaging experiments.

[0036] Depend on Figure 4 and Figure 5 It can be seen that the hydrated particle size of HCNPNs / AGPM increased to 300 nm and the Zeta potential decreased to − 44.2 mV due to the successful connection of Apt-M to the surface of HCNPNs / AGP.

[0037] Example 2

[0038] In order to study the photothermal performance of the multifunctional nanozyme, HCNPNs / AGPM dispersions with different concentrations were irradiated with 808 nm laser at 1.5 W cm −2 The power density was irradiated for 10 min, and the temperature change was recorded simultaneously.

[0039] Depend on Figure 6 It can be seen that under 808 nm laser irradiation, the temperature of HCNPNs / AGPM dispersions with different concentrations will rise, and the temperature rise of the dispersion is related to the concentration. After 808 nm laser irradiation, the HCNPNs / AGPM dispersion (200 µg mL −1 ) rapidly increased from 25°C to 80.6°C, indicating that the multifunctional nanozyme has excellent photothermal conversion ability.

[0040] In order to study the relationship between the photothermal capacity of the multifunctional nanozyme and the laser power, HCNPNs / AGPM dispersion (100 µg mL −1 ) were irradiated with 808 nm laser at different power densities for 10 min, and the temperature changes were recorded simultaneously.

[0041] Depend on Figure 7It can be seen that under 808 nm laser irradiation, the temperature of HCNPNs / AGPM dispersion will rise, and the temperature rise of the dispersion is related to the power density. −2 After laser irradiation, the temperature of HCNPNs / AGPM dispersion increased rapidly from 28°C to 80°C.

[0042] In order to study the photothermal stability of the multifunctional nanozyme, HCNPNs / AGPM dispersion (100 µg mL −1 ) Irradiate with 808nm laser for 10 min, turn off the laser, wait for it to cool to room temperature, and then turn on the laser again. Repeat five times while recording the temperature change.

[0043] Depend on Figure 8 It can be seen that after 5 cycles of laser switching process, the photothermal conversion capacity did not decrease significantly, indicating that the multifunctional nanozyme has good photothermal stability.

[0044] According to HCNPNs / AGPM dispersion (100 µg mL −1 ) at 808 nm laser (1.5 W cm −2 ) The photothermal conversion efficiency of the multifunctional nanozyme was calculated based on the heating and cooling curves under irradiation.

[0045] Depend on Fig. 9 It can be seen that the calculated photothermal conversion efficiency (η) of HCNPNs / AGPM is 35.6%, which is much higher than the composite material based on hollow carbon nanospheres (HCNs). This shows that N and P doping can greatly improve the photothermal performance of HCNPNs, ultimately resulting in HCNPNs / AGPM having strong photothermal conversion ability and excellent photostability, indicating that the multifunctional nanozyme has the potential for application in tumor photothermal therapy.

[0046] Example 3

[0047] To evaluate the GOx enzymatic activity of HCNPNs / AGPM, glucose solution (1 mg mL −1 ), and measure the pH value of the solution at different times.

[0048] Depend on Fig.10 It can be seen that, similar to GOx, the pH value of the glucose solution treated with HCNPNs / AGPM was significantly reduced, indicating that HCNPNs / AGPM can catalyze glucose to produce gluconic acid and H2O2. These results show that HCNPNs / AGPM has GOx activity, and the catalytic process can increase the H2O2 content and enhance the subsequent catalytic treatment.

[0049] Example 4

[0050] To evaluate the POD enzyme catalytic ability of HCNPNs / AGPM, PBS buffer ([H2O2] = 1.4 mM, [Glu] = 125 µg mL −1 ), the ability of different dispersions to produce •OH was determined by ESR.

[0051] Depend on Fig.11 It can be seen that the typical •OH (1:2:2:1) signal was not detected in the HCNs dispersion. However, the •OH signal can be clearly detected in the HCNPNs dispersion. This result shows that the doping of heteroatoms gives the HCNPNs material POD enzyme catalytic activity, which makes HCNPNs a nanomaterial superior to HCNs. In addition, the •OH signal of HCNPNs / G is significantly enhanced, which indicates that the POD enzyme catalysis is further amplified after loading GOx.

[0052] Example 5

[0053] The TA method was used to detect the ability of HCNPNs / AGPM to produce •OH under different substrates, different pH values, and different laser irradiation conditions.

[0054] Depend on Fig.12 It can be seen that HCNPNs / AGPM has excellent GOx and POD enzyme activities. In the acidic environment of laser irradiation, HCNPNs / AGPM can catalyze H2O2 and glucose to produce a large amount of •OH. This shows that HCNPNs / AGPM has both GOx and POD enzyme activities in a slightly acidic environment and can be used for catalytic treatment of tumors in a slightly acidic environment.

[0055] Example 6

[0056] The NOS enzyme activity of HCNPNs / AGPM was detected by Griess method. First, different nanomaterials were added to a 96-well plate, H2O2 and glucose were added, and laser irradiation was performed after mixing. Then, the mixture was mechanically shaken at 37°C. Finally, the absorbance at 540 nm was detected by a microplate reader at predetermined time intervals using the Griess detection kit to calculate the release of NO.

[0057] Depend on Fig.13It can be seen that in PBS without H2O2 and glucose, HCNPNs / AGPM released almost no NO, indicating that HCNPNs / AGPM can only release NO under ROS triggering. In addition, the GOx activity of HCNPNs / AGPM can catalyze the decomposition of glucose into additional H2O2, ultimately catalyzing the production of more NO. Most importantly, under laser irradiation, the NO generation efficiency of HCNPNs / AGPM was further improved, indicating that the photothermal properties of HCNPNs / AGPM are conducive to improving NOS enzyme activity and ultimately catalyzing the production of more NO.

[0058] These results indicate that HCNPNs / AGPM is a powerful and multifunctional nanozyme that can directly generate heat and highly cytotoxic •OH and NO upon laser irradiation, thus promising excellent photothermal / catalytic combined therapeutic effects.

[0059] Example 7

[0060] MCF-7 cells were seeded in 6-well plates and cultured for 24 h. −1 ), HCNPNs / RGPM (100 µg mL −1 ), HCNPNs / RGPM (100 µg mL −1 ) + 2 OD Apt-M, HCNPNs / RGPM (100µg mL −1 ) + 2 OD Apt-C medium was used to replace the medium in the 6-well plate. After culturing for 4 h, the fluorescence intensity of cells in each group was analyzed by flow cytometry.

[0061] Depend on Fig.14 It can be seen that due to the targeting effect of Apt-M on the MUC1 protein on the surface of MCF-7 cells, HCNPNs / AGPM can specifically bind to MCF-7 cells, so the intake of HCNPNs / AGPM by MCF-7 cells increases. MCF-7 cells were first co-incubated with Apt-M and then co-incubated with HCNPNs / AGPM, and there was no obvious change in the fluorescence signal, indicating that Apt-M can bind to and occupy the MUC1 protein site on the surface of MCF-7 cells, resulting in a sharp decrease in the number of HCNPNs / AGPM entering MCF-7 cells, and HCNPNs / AGPM cannot effectively play its targeting role.

[0062] Example 8

[0063] MCF-7 cells were seeded in 96-well plates and cultured for 24 h. The original culture medium was discarded, and fresh culture medium containing each group of nanomaterials was added and cultured for another 4 h. Subsequently, the cells were slowly washed with PBS, and all laser treatment groups were treated with a power intensity of 1.5 W cm−2 The cells were irradiated with 808 nm laser for 10 min. After culturing for another 12 h, the cell viability was detected by MTT assay.

[0064] Depend on Fig.15 It can be seen that the cell viability of the catalytic treatment group (HCNPNs / AP) dropped to about 75%, indicating that catalytic treatment can kill tumor cells to a certain extent. After the introduction of GOx, the cell viability of the enhanced catalytic treatment group (HCNPNs / AGP) further dropped to about 60%. In addition, the cell viability of the combined treatment group (HCNPNs / AGP + Laser or HCNPNs / AGPC + Laser) was significantly reduced, indicating that the combined treatment group can produce a large amount of highly toxic •OH and local hyperthermia to induce MCF-7 cell death, and has a high photothermal / catalytic combined treatment effect. The most exciting result is that the cell activity of the targeted photothermal / catalytic combined treatment group (HCNPNs / AGPM + Laser) decreased significantly, indicating that the specific targeting effect of Apt-M leads to an increase in the number of multifunctional nanozymes in cells, which ultimately greatly improves the treatment effect.

[0065] The lethal mechanism of HCNPNs / AGPM was studied by flow cytometry. MCF-7 cells were inoculated in 12-well plates and cultured for 24 h. The original culture medium was discarded and fresh culture medium containing each group of nanomaterials was added. After another 4 h of culture, the culture medium was replaced with fresh culture medium. The power intensity of all laser treatment groups was 1.5 W cm −2 After 8 h of culture, the cells were collected, stained with Annexin V-FITC / PI kit, and analyzed by flow cytometry.

[0066] Depend on Fig.16 It can be seen that the cells in each treatment group migrated from the Q4 zone (living cells) to the Q3 zone (early apoptosis) and Q2 zone (late apoptosis), indicating that apoptosis triggered the cell death pathway. The apoptosis rate in the combined treatment group was significantly higher than that in the catalytic treatment group, indicating that the combined treatment group significantly promoted apoptosis by increasing the production of ROS and NO. Under the targeted effect, the apoptosis rate of photothermal / catalytic combined therapy was better than that of single treatment. The results show that the cytotoxicity of HCNPNs / AGPM multifunctional nanozymes is mainly related to apoptosis, and HCNPNs / AGPM multifunctional nanozymes have very good therapeutic effects and are expected to be used in in vivo tumor treatment.

[0067] Example 9

[0068] To evaluate the accumulation efficiency of HCNPNs / AGPM in mouse tumor tissue, MCF-7 cells were injected subcutaneously in the right groin of mice to establish a unilateral tumor-bearing mouse model. 3 Afterwards, HCNPNs / RGPC or HCNPNs / RGPM (200 µL, 3 mg mL −1 ), in vivo fluorescence imaging was performed at different times after injection, and the in vivo biological distribution of HCNPNs / AGPM was analyzed based on the in vivo fluorescence intensity.

[0069] Depend on Fig.17 It can be seen that the fluorescence signal of HCNPNs / RGPM in the tumor site of unilateral tumor-bearing mice gradually increased with the blood circulation of the nanomaterials and reached a peak 6 hours after tail vein injection, indicating the effective accumulation of HCNPNs / RGPM in tumors. Therefore, the optimal treatment time point is 6 hours after injection.

[0070] In addition, the mice were dissected 6 h after injection, and the main organs (heart, liver, spleen, lung, and kidney) of the mice were taken together with the tumor for fluorescence imaging.

[0071] Depend on Fig.18 It can be seen that the fluorescence signals of the main organs and tumor tissues of mice showed that the accumulation ability of HCNPNs / RGPM material in mouse tumor tissues far exceeded that of HCNPNs / RGPC material. This is because the targeting effect of Apt-M enhanced the accumulation ability of the multifunctional nanozyme.

[0072] To further evaluate the specific targeting ability of HCNPNs / AGPM in mouse tumor tissues, MCF-7 cells were injected subcutaneously in the right groin of mice, and HepG2 cells were injected subcutaneously in the left groin to establish a bilateral tumor-bearing nude mouse model. The tumor volume reached about 200 mm 3 After that, HCNPNs / RGPM (200 μL, 3 mg mL −1 ), in vivo fluorescence imaging was performed at different times after injection, and the specific targeting ability of HCNPNs / AGPM was analyzed based on in vivo fluorescence imaging.

[0073] Depend on Fig.19 It can be seen that during the observation period, the fluorescence intensity of the HepG2 tumor site in bilateral tumor-bearing mice was always lower than that of the MCF-7 tumor site. This is because HCNPNs / RGPM can recognize the MUC1 protein overexpressed on the surface of MCF-7 cells for transport, and more materials are accumulated in the tumor site. This shows that the multifunctional nanozyme has excellent active targeting ability, which ensures the large accumulation of materials in mouse tumors and is expected to achieve good therapeutic effects in vivo.

[0074] Example 10

[0075] MCF-7 cells were injected subcutaneously in the right groin of mice to establish a unilateral tumor-bearing mouse model. 3 The mice were randomly divided into six groups (5 mice in each group) and the mice in each group were injected with the same dose (200 μL, 3 mg mL −1 ) of different nanomaterials. Six hours after injection, mice in the laser irradiation group were exposed to 808 nm (1.5 W cm −2 ) laser irradiation for 5 min. The weight and tumor volume of mice were recorded every two days during the treatment. After 17 days, all mice were euthanized, and the tumor tissues were removed and photographed.

[0076] Depend on Fig. 20 It can be seen that the average body weight of mice in each group did not change significantly during the treatment, indicating that the multifunctional nanozyme has good biosafety and the adverse reactions during the treatment can be ignored.

[0077] Depend on Fig.21 It can be seen that during the treatment period, the tumor in the control group grew rapidly. However, only slight tumor growth inhibition was observed in the enhanced catalytic treatment group and the combined treatment group. Due to the targeting effect of Apt-M, the targeted photothermal / catalytic combined treatment showed a significant anti-tumor effect, tumor growth was significantly inhibited, and the tumor volume gradually decreased. This satisfactory anti-tumor effect is due to the effective accumulation of multifunctional nanozymes in tumors, which successfully achieved targeted photothermal / catalytic combined tumor treatment.

[0078] Depend on Fig. 22 It can be seen that after 17 days of treatment, the tumors in the targeted photothermal / catalytic combined treatment group almost disappeared, and the tumors in 2 mice were completely eliminated, which intuitively proved that HCNPNs / AGPM multifunctional nanozymes can significantly inhibit tumor growth and have potential for clinical application.

[0079] The sequences of Apt-M and Apt-C in the above examples are as follows: The Apt-M sequence is: NH2-(CH2)6-GCAGTTGATCCTTTGGATACCCTGGGTTTTTTTTTT; The Apt-C sequence is: NH2-(CH2)6-ATTGCACTTACTATATTGCACTTACTATATTGCAC.

[0080] The contents not described in detail in the specification of the present invention belong to the prior art known to the professional and technical personnel in the field. Although the illustrative specific embodiments of the present invention are described above to facilitate the understanding of the present invention by the technical personnel in the field, it should be clear that the present invention is not limited to the scope of the specific embodiments. For the ordinary technical personnel in the field, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

Claims

1. A multifunctional nanozyme, characterized in that: Using N, P doped hollow carbon nanospheres HCNPNs as carriers, L-Arg and GOx were simultaneously loaded in the cavities and mesopores of the carriers, and the nucleic acid aptamer Apt that can recognize specific cell surface proteins was coupled on its surface.

2. A multifunctional nanozyme according to claim 1, characterized in that: The N, P double-doped hollow carbon nanosphere HCNPNs material is a spherical particle with a particle size of 200 nm.

3. A multifunctional nanozyme according to claim 1, characterized in that: The GOx loading amount is 9.3 wt% of the N, P dual-doped hollow carbon nanosphere HCNPNs material, and the L-Arg loading amount is 13.6 wt% of the N, P dual-doped hollow carbon nanosphere HCNPNs material.

4. A multifunctional nanozyme according to claim 1, characterized in that: The nucleic acid aptamer Apt is coupled to the surface of the N, P double-doped hollow carbon nanosphere HCNPNs material by a covalent bonding method.

5. A multifunctional nanozyme according to claim 1, characterized in that: The multifunctional nanozyme has GOx enzyme, POD enzyme, and nitric oxide synthase activities and has multiple catalytic capabilities.

6. A method for preparing a multifunctional nanozyme, characterized in that: The following steps are involved: Step 1, preparation of N, P doped hollow carbon nanosphere HCNPNs material: using SiO2 nanoparticles as a hard template, dopamine as a carbon source and a nitrogen source, and adenosine triphosphate as a phosphorus source, polycondensing on the hard template to obtain a precursor; Under the protection of inert gas, the precursor is carbonized at high temperature, and SiO2 is completely removed by hydrofluoric acid etching to obtain N, P double-doped hollow carbon nanospheres HCNPNs material; Step 2, loading: using electrostatic interaction to load L-Arg and GOx into HCNPNs material; Step 3, surface modification with dicarboxyl polyethylene glycol (PEG): PEG is mixed and stirred with the N, P doped hollow carbon nanosphere HCNPNs material loaded with L-Arg and GOx to complete the surface modification; Step 4, using EDC / NHS reagents to covalently couple Apt that can recognize cell surface specific proteins to the surface of the material modified by PEG to prepare a multifunctional nanozyme.

7. The method for preparing a multifunctional nanozyme according to claim 5, characterized in that: The method for preparing SiO2 nanoparticles as a hard template is as follows: ethanol, ammonia water and deionized water are mixed, tetrapropoxysilane is used as a silicon source, and polymerized to form SiO2 nanoparticles.

8. The method for preparing a multifunctional nanozyme according to claim 5, characterized in that: The mass ratio of dopamine to adenosine triphosphate is 8:1; the loading amount of L-Arg is 10-15 wt%, and the loading amount of GOx is 5-15 wt%; the temperature of the high-temperature carbonization is 800°C, and the time is 3 h.

9. Use of a multifunctional nanozyme prepared by the preparation method according to any one of claims 5 to 8 in the preparation of tumor targeting drugs.

10. Use of a multifunctional nanozyme prepared by the preparation method according to any one of claims 5 to 8 in the preparation of a photothermal / catalytic combined therapeutic drug for breast cancer tumors.

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