Mesoporous hollow carbon drug loading system with cascade enzyme catalytic activity and preparation and application thereof

Abstract

The invention discloses a mesoporous hollow carbon drug loading system with cascade enzyme catalytic activity and preparation and application thereof, and belongs to the technical field of medicines. The drug loading system is composed of mesoporous hollow carbon, an anti-tumor drug, gold nano-enzyme and a hydrophilic functional polymer. The preparation method comprises the following steps: preparing gold nano-enzyme; the preparation method comprises the following steps: preparing MHC-COOH and MHC-SH; and preparing the drug-loaded nano enzyme. The drug loading system provided by the invention realizes efficient loading and stable dispersion of chemotherapeutic drugs. The gold nano-enzyme with the size consistent with that of the mesoporous channel of the carrier is used as a'gated 'switch and has multiple functions. The drug loading system has the effect of enhancing PTT; the premature release of chemotherapeutic drugs is prevented, the toxic and side effects on normal tissues are reduced, and the reduction / pH / NIR multi-responsive drug release behavior is realized; under the irradiation of near-infrared light, the drug release speed is further accelerated, the GSH consumption rate and the catalytic rates of the gold nano-enzyme and MHC are further improved, and a nano delivery system for enhancing chemotherapy through photo-thermal and cascade enzyme catalysis is achieved.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity and a preparation and application thereof. Background Art

[0002] At present, malignant tumors have become a major threat to human health. Accordingly, various powerful means of treating tumors have been developed, including surgical resection, chemotherapy, radiotherapy, etc. Among them, chemotherapy has become a new cancer treatment method. However, due to the lack of tumor-specific targeting ability and multidrug resistance, this conventional method still has disadvantages such as high toxicity of anticancer drugs and poor efficacy of individualized chemotherapy. Photothermal therapy (PTT) has attracted much attention in cancer treatment as a non-invasive, effective treatment method with few systemic side effects. Near-infrared (NIR) light (700-1200nm) has a large penetration depth and is less harmful to the human body. It can be absorbed by photothermal agents and converted into heat that is toxic to tumor cells in tumor tissues. The resulting high temperature can accelerate the release of antitumor drugs from the carrier, promote the uptake of the drug-carrying system by tumor cells, and make cancer cells more sensitive to chemotherapy drugs. Therefore, the combined treatment effect of chemotherapy-PTT will be significantly better than that of a single treatment method.

[0003] With the development of PTT technology, different types of PTT photothermal agents have been developed, such as gold nanoparticles, platinum nanoparticles, metal organic framework materials and carbon-based materials. Among these PTT photothermal agents, mesoporous carbon nanoparticles (MCN) have attracted much attention due to their advantages such as large specific surface area, adjustable pore size, high photothermal conversion efficiency, high porosity, easy surface functionalization and good biocompatibility, and supramolecular π-π stacking. Mesoporous hollow carbon (MHC), as a representative of carbon carriers, has many unique advantages. MHC has the characteristics of large specific surface area, high carrying capacity, and easy surface functionalization. At the same time, compared with other mesoporous carbon materials, MHC has a lighter density and higher thermal conversion efficiency due to its unique hollow structure.

[0004] Nanozymes are nanomaterials with enzyme-mimicking properties. They have the characteristics of high catalytic activity, strong stability, and easy mass production. Therefore, they have been widely used in drug delivery and combined tumor therapy in recent years. Carbon-based nanozymes have multiple enzymatic activities. Carbon-based nanozymes can consume the reduced GSH overexpressed in the tumor microenvironment and oxidize it into oxidized glutathione (GSSG), thereby breaking the redox homeostasis of the tumor microenvironment. In an acidic environment, carbon-based nanozymes exhibit peroxidase-like properties, catalyzing the overexpressed hydrogen peroxide in the tumor microenvironment to produce hydroxyl radicals (·OH), thereby increasing the oxidative stress level of the tumor microenvironment, achieving tumor cell killing, and enhancing the anti-tumor effects of other therapies.

[0005] However, the application of MHC-based chemotherapy-PTT drug delivery system still faces some unresolved problems. The release of drugs before the target site will have obvious toxic side effects on normal cells, leading to adverse consequences. Therefore, the development of stimulus-responsive delivery system using special sensitive stimuli in the tumor microenvironment, such as high intracellular GSH levels, lysosomal pH, enzymes, heat and near-infrared light, can achieve responsive drug release in the tumor microenvironment, thereby reducing toxic side effects on normal cells.

[0006] In the prior art, there is a lack of mesoporous hollow carbon "gated" drug delivery systems with selective GSH consumption and ROS generation, so as to achieve photothermal cascade enzyme catalysis to produce ·OH in the tumor microenvironment, so as to realize photothermal and cascade enzyme catalysis enhanced chemotherapy nano-delivery system for combined treatment of tumors. Summary of the invention

[0007] The purpose of the present invention is to provide a mesoporous hollow carbon drug delivery system with cascade enzyme catalytic activity and its preparation and application; this mesoporous hollow carbon drug delivery system with continuous GSH consumption and cascade enzyme catalytic production of ·OH is used for synergistic tumor treatment, by utilizing the system's dual enzyme activity to catalyze the production of ·OH under tumor microenvironment conditions, and realizing reduction / pH / NIR multi-responsive drug release, thereby ultimately achieving a nano-delivery system for photothermal and cascade enzyme catalytic enhanced chemotherapy.

[0008] A mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity, which consists of mesoporous hollow carbon, anti-tumor drugs, gold nanozymes and hydrophilic functionalized polymers; the anti-tumor drugs are one or more of doxorubicin hydrochloride, paclitaxel, vinorelbine, cisplatin and methotrexate; the hydrophilic functionalized polymer is thiol-modified hyaluronic acid, thiol-modified polyethylene glycol 2000 or thiol-modified folic acid.

[0009] The preparation of the above-mentioned mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity is achieved by the following steps: 1. Preparation of gold nanozymes: 20-40 mL HAuCl4 solution and 20-40 mL sodium citrate solution were mixed and reacted for 5-30 min, and then 0.5-2 mL NaHB4 solution was added to continue the reaction to obtain a wine red solution, which was dialyzed and freeze-dried to obtain gold nanozyme; 2. Preparation of carboxyl-modified mesoporous hollow carbon nanozymes (MHC-COOH) and thiol-modified mesoporous carbon nanozymes (MHC-SH); 3. Preparation of drug-loaded nanozymes: Disperse the above 30-50 mg of thiol-modified mesoporous carbon nanozyme into 5-15 mL of phosphate buffer (PBS) at pH 7.4, then add 10 mL of anti-tumor drug solution with a concentration of 1 mg / mL-5 mg / mL, sonicate for 0.5-2 h, and then continue stirring for 2-12 h, collect the precipitate by centrifugation, and obtain D@MHC-SH drug carrier; Take the gold nanozyme obtained in step 1, add distilled water to prepare a gold nanozyme suspension with a concentration of 1 mg / mL; 30-50 mg of D@MHC-SH drug carrier was dispersed in 10 mL of PBS with a pH of 7.4, 5-15 mL of 1 mg / mL gold nanozyme suspension was added and stirred for 2-12 h, and the precipitate was collected by centrifugation to obtain drug-loaded D@MHC-Au, and then 10-30 mg of hydrophilic functionalized polymer was added to obtain D@MHC-Au-P, which is a mesoporous hollow carbon drug-loading system with cascade enzyme catalytic activity, completing the preparation.

[0010] Furthermore, the concentrations of the HAuCl4 solution and the sodium citrate solution in step 1 are both (2-3)×10 -4 mol / L; the concentration of the NaHB4 solution is 0.75mol / L ~0.1mol / L.

[0011] Furthermore, the temperature for continuing the reaction in step 1 is 10-50° C., and the reaction time is 0.5-6 h; the dialysis uses a dialysis bag with Mw=1000Da, and the dialysis time is 4-24 h.

[0012] Furthermore, the carboxyl-modified mesoporous hollow carbon nanozyme is prepared as described in step 2, and the specific process is as follows: ① Preparation of mesoporous core-shell silica spheres: 200-250 mL of anhydrous ethanol and 20-50 mL of deionized water were mixed, and then 4-6 mL of NH3·H2O was added. After stirring for 30 min, 4-6 mL of TEOS (tetraethyl orthosilicate) was added dropwise. The mixture was stirred in a water bath at 30 °C for 2 h. The sample was collected after centrifugation. After washing with anhydrous ethanol and distilled water twice, solid silica spheres were obtained. 200-400 mg of solid silica spheres were dispersed in 70-100 mL of distilled water to obtain solution A. 400-500 mg of CTAB (hexadecyltrimethylammonium chloride) was dissolved in 50-80 mL of water. 80-100 mL of anhydrous ethanol, 1-2 mL of NH3·H2O and solution A were added in sequence. After stirring for 1 h, 0.5-1 mL of TEOS was added dropwise. The mixture was reacted for 6 h. The sample was collected after centrifugation. The sample was washed with anhydrous ethanol and distilled water twice, dried, and then heated at 600 °C. o C and vacuum calcined to remove the template to obtain mesoporous core-shell silicon spheres; ② Solidification, calcination and removal of template of carbon source: Add carbon source precursor solution to the above mesoporous core-shell silicon spheres, and then heat at 60 o C and 80 o C for 12 h, and then placed in a vacuum tube furnace for 2 o C / min and the temperature was raised to 800 o C, calcined for 3 h, and finally dispersed in 1% hydrofluoric acid solution, stirred for 6 h to remove silica in the pores, and then washed and dried to obtain a mesoporous hollow carbon carrier (MHC); ③. Carboxylation modification: Add 0.4~0.6g of mesoporous hollow carbon carrier into a round-bottom flask containing 20~40mL of 100mg / mL ammonium persulfate solution and 3~5mL of 98% sulfuric acid, react at 60℃ with stirring and reflux for 3h, obtain a black solid after centrifugation, wash with purified water 3~5 times, and obtain carboxylation-modified mesoporous hollow carbon nanozyme (MHC-COOH) after vacuum drying.

[0013] Furthermore, the carbon source precursor solution includes: 2 mL furfuryl alcohol, 20 mg oxalic acid and 50 mg dopamine hydrochloride.

[0014] Further, the thiol-modified mesoporous carbon nanozyme is prepared as described in step 2, and the specific process is as follows: a. Disperse 100-300 mg of carboxyl-modified mesoporous hollow carbon nanoenzyme into 30-70 mL of ethylenediamine, then add 10-30 mg of HATU and continue the reaction for 4 h to obtain amino-modified MHC (MHC-NH2). After centrifugal washing and anhydrous ethanol washing twice each, distilled water is added to prepare a MHC-NH2 dispersion with a concentration of 10-50 mg / mL; b. Preparation of MHC-SH: 150-300 mg of 3-mercaptopropionic acid was dispersed in distilled water, and EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added for activation for 1 h to obtain an activated 3-mercaptopropionic acid solution. Then 10-50 mL of MHC-NH2 dispersion was added and stirred for 12 h. After centrifugation and water washing, thiol-modified mesoporous carbon nanozyme (MHC-SH) was obtained.

[0015] Furthermore, the concentrations of EDC and NHS are both 0.5 mg / mL to 5 mg / mL.

[0016] Furthermore, the solute in the anti-tumor drug solution is one or more of doxorubicin hydrochloride, paclitaxel, vinorelbine, cisplatin, and methotrexate, and the solvent is an aqueous medium or a good solvent.

[0017] An application of a mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity, which can release drugs in a reduction / pH / NIR multi-responsive manner, is used in the combined treatment of tumors with photothermal and cascade enzyme catalytic enhanced chemotherapy.

[0018] Advantages of the present invention: The present invention discloses a mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity, which efficiently loads anti-tumor drugs inside the MHC carrier, and modifies the gold nanoenzyme on the surface of the MHC through a gold-sulfur bond, thereby preventing the premature release of the drug in the carrier. When the drug-loaded nanoplatform is delivered to the tumor site and internalized into the tumor cells, the gold-sulfur bond is cut under the action of high concentration of glutathione, resulting in rapid release of the drug at the tumor site. The separated gold nanoparticles can catalyze the oxidation of glucose to generate H2O2 and glucuronic acid, aggravating the acidic microenvironment. By utilizing the catalytic action of peroxidase and glutathione oxidase, the glutathione in the tumor microenvironment is significantly reduced, and a large number of hydroxyl free radicals are generated, amplifying the oxidative stress of the tumor tissue. In addition, under near-infrared light irradiation, the drug release rate is further accelerated, and the glutathione consumption rate and the catalytic rate of the gold nanoparticles and the mesoporous hollow carbon are further improved, thereby realizing the combined treatment of tumors with photothermal and cascade enzyme catalytic enhanced chemotherapy. MHC nanozyme is a nitrogen-doped mesoporous hollow carbon with excellent POD-like enzyme activity. The carrier has obvious hollow cavities and mesoporous channels, which can efficiently load drugs. The cavity size is adjustable from 50 nm to 150 nm, and the mesopore size is adjustable from 2 to 10 nm. It has the ability to continuously consume glutathione and excellent photothermal conversion efficiency. The particle size of the gold nanozyme modified on the carrier surface is consistent with the mesoporous channel of the MHC carrier, and is modified to the surface of MHC through gold-sulfur bonds, which can be used as a "gated" switch to prevent the premature release of drugs; the gold nanozyme has glucose oxidase-like activity and photothermal conversion efficiency.

[0019] The mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity prepared by the present invention can utilize the cavity structure inside the carrier to achieve efficient loading and stable dispersion of chemotherapy drugs. The use of gold nanozymes that are consistent with the size of the carrier mesoporous channels as a "gated" switch has multiple functions. First, gold nanozymes with excellent photothermal conversion efficiency can enhance the PTT effect; second, gold nanozymes can prevent the premature release of chemotherapy drugs, reduce toxic side effects on normal tissues, and achieve reduction / pH / NIR multi-responsive drug release behavior; third, gold nanozymes have GOx-like enzyme activity, which can effectively catalyze the conversion of glucose into hydrogen peroxide and glucuronic acid in the tumor microenvironment, further reduce the pH value of the tumor microenvironment, and continuously generate hydroxyl radicals.

[0020] When the drug-loaded D@MHC-Au-P drug delivery system is delivered to the tumor site and internalized into tumor cells, the gold-sulfur bond breaks under the action of high concentrations of GSH in the tumor microenvironment, resulting in rapid release of the drug at the tumor site. The separated gold nanozyme can catalyze the oxidation of glucose to produce hydrogen peroxide and glucuronic acid, aggravating the acidic microenvironment. Through the catalytic action of MHC-like POD enzymes and GSH oxidase, the GSH in the tumor microenvironment is significantly reduced, and a large amount of ·OH is produced, amplifying the oxidative stress of tumor tissues. In addition, under near-infrared light irradiation, the drug release rate is further accelerated, and the GSH consumption rate and the catalytic rate of gold nanozymes and MHC are further increased, thereby achieving the synergistic anti-tumor effect of PTT-enhanced catalytic therapy and chemotherapy.

[0021] The invention is suitable for preparing a mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron microscopy image of the gold nanozyme prepared in Example 1.

[0023] Figure 2 This is the hydration particle size result of the gold nanozyme prepared in Example 1 (determined by DLS method).

[0024] Figure 3 The gold nanozyme prepared in Example 1 catalyzes glucose to reduce the pH value of the solution and produce hydrogen peroxide content at different glucose concentrations.

[0025] Figure 4 Part A is the temperature rise curve of gold nanozyme at different power densities of 100 μg / mL; Part B is the temperature rise curve of gold nanozyme at different concentrations of 2 W / cm 2 The heating curve diagram below.

[0026] Figure 5 The photostability graph of 100 μg / mL gold nanozyme under NIR light irradiation.

[0027] Figure 6 This is the transmission electron microscopy image of the prepared MHC-COOH.

[0028] Figure 7 This is the transmission electron microscopy image of the prepared MHC-Au-P.

[0029] Figure 8 Nitrogen isotherms (see part A) and pore size distributions (see part B) of MHC, MHC-COOH and MHC-Au-P.

[0030] Fig. 9Part A shows the Zeta potential data of different samples; Part B shows the particle size distribution and PDI data of MHC-COOH, MHC-Au and MHC-Au-P.

[0031] Fig.10 For different concentrations of MHC-COOH (see Part A) and MHC-Au-P (see Part B) at 1 W / cm 2 The heating curve diagram below.

[0032] Fig.11 Cyclic photothermal stability graphs of 100 μg / mL MHC-COOH (see part A) and MHC-Au-P (see part B) under 5 NIR light irradiations.

[0033] Fig.12 The linear time data of the photothermal curves of MHC-COOH (see part A) and MHC-Au-P (see part B) versus -lnθ.

[0034] Fig.13 Part A shows the drug release curves of D@MHC-COOH and D@MHC-Au-P; Part B shows the drug release curves of D@MHC-COOH and D@MHC-Au-P in pH 5.5 PBS; Part C shows the drug release curves of D@MHC-Au-P under different conditions (n=3).

[0035] Fig.14 This is a graph showing the effects of different pH and near-infrared light on the activity of MHC-COOH POD enzymes.

[0036] Fig.15 This is a data graph showing the consumption capacity of GSH by MHC-COOH at different concentrations.

[0037] Fig.16 The absorbance change curves of TMB in glucose solution (5 mM) at pH 7.4 (see part A) and pH 5.5 (see part B) (n=3).

[0038] Fig.17 At 1.0 W / cm 2 Live / dead cell staining of MHC-Au-P prepared under laser irradiation.

[0039] Fig.18 At 1.0 W / cm 2 Photothermal heating curve of MHC-Au-P at 4T1 cell level under laser irradiation.

[0040] Fig.19 CLSM images of 4T1 cells incubated with different samples for 2 h.

[0041] Fig. 20 CLSM images of 2',7'-dichlorofluorescein (DCF) in 4T1 cells after treatment with different drug groups.

[0042] Fig.21 The graph shows the GSH content in 4T1 cells after incubation with MHC-Au-P for different time periods.

[0043] Fig. 22 CLSM images of 4T1 cells after treatment with different samples (equivalent to a DOX dose of 5 μg / mL).

[0044] Fig.23 Cell viability graphs of 4T1 cells incubated with different samples for 24 h without NIR irradiation (see part A) and with NIR irradiation (see part B).

[0045] Fig.24 Cell survival rate of 4T1 cells co-incubated with D@MHC-Au-P at different glucose concentrations.

[0046] Fig.25 Parts AE are tumor images and tumor growth curves of mice; Part F is a curve of weight changes of mice in each group; Part G is a bar graph of relative tumor mass; Part H is a graph of the relative mass of tumors at 1 W / cm 2 Thermal imaging of mice in the saline group and D@MHC-Au-P group under high-power near-infrared laser irradiation. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any combination of the specific implementation modes.

[0048] Specific implementation method one: This implementation method is a mesoporous hollow carbon drug delivery system with cascade enzyme catalytic activity, which is composed of mesoporous hollow carbon, anti-tumor drugs, gold nanozymes and hydrophilic functionalized polymers; the anti-tumor drugs are one or more of doxorubicin hydrochloride, paclitaxel, vinorelbine, cisplatin, and methotrexate; the hydrophilic functionalized polymer is thiol-modified hyaluronic acid, thiol-modified polyethylene glycol 2000 or thiol-modified folic acid.

[0049] In this embodiment, the mesoporous hollow carbon has peroxidase (POD)-like enzyme activity and glutathione consumption ability.

[0050] In this embodiment, the gold nanozyme has glucose oxidase (GOx)-like enzyme activity.

[0051] The thiol-modified polyethylene glycol 2000 in this embodiment: PEG-SH, Mw=2000.

[0052] When the anti-tumor drug in this embodiment is a composition, the ratio of each component is arbitrary.

[0053] Specific implementation method 2: In this implementation method, a mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity is prepared, which is achieved by the following steps: 1. Preparation of gold nanozymes: 20-40 mL HAuCl4 solution and 20-40 mL sodium citrate solution were mixed and reacted for 5-30 min, and then 0.5-2 mL NaHB4 solution was added to continue the reaction to obtain a wine red solution, which was dialyzed and freeze-dried to obtain gold nanozyme; 2. Preparation of carboxyl-modified mesoporous hollow carbon nanozymes (MHC-COOH) and thiol-modified mesoporous carbon nanozymes (MHC-SH); 3. Preparation of drug-loaded nanozymes: Disperse the above 30-50 mg of thiol-modified mesoporous carbon nanozyme into 5-15 mL of phosphate buffer (PBS) at pH 7.4, then add 10 mL of anti-tumor drug solution with a concentration of 1 mg / mL-5 mg / mL, sonicate for 0.5-2 h, and then continue stirring for 2-12 h, collect the precipitate by centrifugation, and obtain D@MHC-SH drug carrier; Take the gold nanozyme obtained in step 1, add distilled water to prepare a gold nanozyme suspension with a concentration of 1 mg / mL; 30-50 mg of D@MHC-SH drug carrier was dispersed in 10 mL of PBS with a pH of 7.4, 5-15 mL of 1 mg / mL gold nanozyme suspension was added and stirred for 2-12 h, and the precipitate was collected by centrifugation to obtain drug-loaded D@MHC-Au, and then 10-30 mg of hydrophilic functionalized polymer was added to obtain D@MHC-Au-P, which is a mesoporous hollow carbon drug-loading system with cascade enzyme catalytic activity, completing the preparation.

[0054] In this embodiment, HATU is used as a condensing agent.

[0055] The centrifugation in this embodiment is performed at 12000 rpm for 30 min.

[0056] The ultrasound in step 3 of this embodiment has a power of 90 W and lasts for 20 minutes.

[0057] Specific embodiment 3: The difference between this embodiment and specific embodiment 2 is that the concentrations of the HAuCl4 solution and the sodium citrate solution in step 1 are both (2-3)×10 -4 mol / L; the concentration of the NaHB4 solution is 0.75mol / L ~ 0.1mol / L. Other steps and parameters are the same as those in the second embodiment.

[0058] Specific embodiment 4: The difference between this embodiment and specific embodiment 2 is that the temperature of the continued reaction in step 1 is 10-50°C, the reaction time is 0.5-6h, the dialysis uses a dialysis bag with Mw=1000Da, and the dialysis time is 4-24h. The other steps and parameters are the same as those in specific embodiment 2.

[0059] Specific embodiment 5: The difference between this embodiment and specific embodiment 2 is that the carboxyl-modified mesoporous hollow carbon nanozyme is prepared in step 2, and the specific process is as follows: ① Preparation of mesoporous core-shell silica spheres: 200-250 mL of anhydrous ethanol and 20-50 mL of deionized water were mixed, and then 4-6 mL of NH3·H2O was added. After stirring for 30 min, 4-6 mL of TEOS (tetraethyl orthosilicate) was added dropwise. The mixture was stirred in a water bath at 30 °C for 2 h. The sample was collected after centrifugation. After washing with anhydrous ethanol and distilled water twice, solid silica spheres were obtained. 200-400 mg of solid silica spheres were dispersed in 70-100 mL of distilled water to obtain solution A. 400-500 mg of CTAB (hexadecyltrimethylammonium chloride) was dissolved in 50-80 mL of water. 80-100 mL of anhydrous ethanol, 1-2 mL of NH3·H2O and solution A were added in sequence. After stirring for 1 h, 0.5-1 mL of TEOS was added dropwise. The mixture was reacted for 6 h. The sample was collected after centrifugation. The sample was washed with anhydrous ethanol and distilled water twice, dried, and then heated at 600 °C. o C and vacuum calcined to remove the template to obtain mesoporous core-shell silicon spheres; ② Solidification, calcination and removal of template of carbon source: Add carbon source precursor solution to the above mesoporous core-shell silicon spheres, and then heat at 60 o C and 80 o C for 12 h, and then placed in a vacuum tube furnace for 2 o C / min and the temperature was raised to 800 o C, calcined for 3 h, and finally dispersed in 1% hydrofluoric acid solution, stirred for 6 h to remove silica in the pores, and then washed and dried to obtain a mesoporous hollow carbon carrier (MHC); ③, Carboxylation modification: add 0.4~0.6g of mesoporous hollow carbon carrier to a round-bottom flask containing 20~40mL of 100mg / mL ammonium persulfate solution and 3~5mL of 98% sulfuric acid by mass, react at 60°C, stirring and reflux for 3h, centrifuge to obtain a black solid, wash with purified water 3~5 times, and vacuum dry to obtain a carboxyl-modified mesoporous hollow carbon nanozyme (MHC-COOH). Other steps and parameters are the same as those in the first embodiment.

[0060] Specific embodiment 6: This embodiment differs from specific embodiment 5 in that the carbon source precursor solution includes: 2 mL furfuryl alcohol, 20 mg oxalic acid and 50 mg dopamine hydrochloride. Other steps and parameters are the same as those of specific embodiment 5.

[0061] Specific embodiment 7: The difference between this embodiment and specific embodiment 1 is that the thiol-modified mesoporous carbon nanozyme is prepared in step 2, and the specific process is as follows: a. Disperse 100-300 mg of carboxyl-modified mesoporous hollow carbon nanoenzyme into 30-70 mL of ethylenediamine, then add 10-30 mg of HATU and continue the reaction for 4 h to obtain amino-modified MHC (MHC-NH2). After centrifugal washing and anhydrous ethanol washing twice each, distilled water is added to prepare a MHC-NH2 dispersion with a concentration of 10-50 mg / mL; b. Preparation of MHC-SH: 150-300 mg of 3-mercaptopropionic acid was dispersed in distilled water, and EDC and NHS were added for activation for 1 h to obtain an activated 3-mercaptopropionic acid solution, and then 10-50 mL of MHC-NH2 dispersion was added, stirred for 12 h, and centrifuged and washed to obtain thiol mesoporous carbon nanozyme (MHC-SH). Other steps and parameters are the same as those in the first embodiment.

[0062] Specific embodiment eight: This embodiment differs from specific embodiment seven in that the concentrations of EDC and NHS are both 0.5 mg / mL to 5 mg / mL. Other steps and parameters are the same as those of specific embodiment seven.

[0063] Specific embodiment 9: This embodiment differs from specific embodiment 7 in that the solute in the anti-tumor drug solution is one or more of doxorubicin hydrochloride, paclitaxel, vinorelbine, cisplatin, and methotrexate, and the solvent is an aqueous medium or a good solvent. Other steps and parameters are the same as those of specific embodiment 7.

[0064] Specific embodiment ten: This embodiment is an application of a mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity, which can release drugs in multiple responsiveness to reduction / pH / NIR and is used in the combined treatment of tumors with photothermal and cascade enzyme catalytic enhanced chemotherapy.

[0065] Embodiment 1: A preparation of a mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity is achieved by the following steps: 1. Preparation of gold nanozymes: 25 mL of HAuCl4 solution and 25 mL of sodium citrate solution were mixed and reacted for 15 min, and then 0.75 mL of NaHB4 solution was added to continue the reaction to obtain a wine red solution, which was dialyzed and freeze-dried to obtain gold nanozyme; 2. Preparation of carboxyl-modified mesoporous hollow carbon nanozymes (MHC-COOH) and thiol-modified mesoporous carbon nanozymes (MHC-SH); 3. Preparation of drug-loaded nanozymes: The above 40 mg of thiol-modified mesoporous carbon nanozyme was dispersed in 10 mL of phosphate buffer (PBS) at pH 7.4, and then 10 mL of 3 mg / mL doxorubicin hydrochloride (DOX) aqueous solution was added. After ultrasonication for 1 h, stirring was continued for 12 h, and the precipitate was collected by centrifugation to obtain the D@MHC-SH drug carrier. Take the gold nanozyme obtained in step 1, add distilled water to prepare a gold nanozyme suspension with a concentration of 1 mg / mL; 40 mg of D@MHC-SH drug carrier was dispersed in 10 mL of PBS with a pH of 7.4, 10 mL of 1 mg / mL gold nanozyme suspension was added and stirred for 8 h, and the precipitate was collected by centrifugation to obtain drug-loaded D@MHC-Au, and then 20 mg of thiol-modified polyethylene glycol 2000 (PEG-SH, Mw=2000) was added to obtain D@MHC-Au-P, which is a mesoporous hollow carbon drug-loading system with cascade enzyme catalytic activity, completing the preparation.

[0066] At the same time, the supernatant of each step was collected, the drug content in the supernatant was determined, and the drug loading amount was calculated. The drug loading amount of DOX in D@MHC-Au-P was 35.3%.

[0067] The concentrations of the HAuCl4 solution and the sodium citrate solution in step 1 of the embodiment are both 2.5×10 -4 mol / L; the concentration of the NaHB4 solution is 0.1 mol / L.

[0068] The temperature for continuing the reaction in step 1 of the embodiment is 30° C., and the reaction time is 2 h. The dialysis uses a dialysis bag with Mw=1000 Da, and the dialysis time is 8 h.

[0069] The carboxyl-modified mesoporous hollow carbon nanozyme is prepared as described in step 2 of the embodiment, and the specific process is as follows: ①. Preparation of mesoporous core-shell silica spheres: 210 mL of anhydrous ethanol and 30 mL of deionized water were mixed, and then 5 mL of NH3·H2O was added. After stirring for 30 min, 5 mL of TEOS (tetraethyl orthosilicate) was added dropwise. The mixture was stirred in a water bath at 30 °C for 2 h. The sample was collected after centrifugation. After washing with anhydrous ethanol and distilled water twice, solid silica spheres were obtained. 300 mg of solid silica spheres were dispersed in 90 mL of distilled water to obtain solution A. 450 mg of CTAB (hexadecyltrimethylammonium chloride) was dissolved in 60 mL of water. 90 mL of anhydrous ethanol, 1.7 mL of NH3·H2O and solution A were added in sequence. After stirring for 1 h, 0.75 mL of TEOS was added dropwise. The mixture was reacted for 6 h. The sample was collected after centrifugation. The sample was washed with anhydrous ethanol and distilled water twice, dried, and then heated at 600 °C. o C and vacuum calcined to remove the template to obtain mesoporous core-shell silicon spheres; ② Solidification, calcination and removal of template of carbon source: Add carbon source precursor solution to the above mesoporous core-shell silicon spheres, and then heat at 60 o C and 80 o C for 12 h, and then placed in a vacuum tube furnace for 2 o C / min and the temperature was raised to 800 o C, calcined for 3 h, and finally dispersed in 1% hydrofluoric acid solution, stirred for 6 h to remove silica in the pores, and then washed and dried to obtain a mesoporous hollow carbon carrier (MHC); ③. Carboxylation modification: 0.5 g of the mesoporous hollow carbon carrier was added to a round-bottom flask containing 30 mL of 100 mg / mL ammonium persulfate solution and 3.2 mL of 98% sulfuric acid, and the mixture was reacted at 60 ° C under stirring and reflux conditions for 3 h. A black solid was obtained after centrifugation. The solid was washed with purified water 5 times and vacuum dried to obtain a carboxyl-modified mesoporous hollow carbon nanozyme (MHC-COOH); the carbon source precursor solution: 2 mL of furfuryl alcohol, 20 mg of oxalic acid and 50 mg of dopamine hydrochloride.

[0070] The thiol-modified mesoporous carbon nanozyme is prepared as described in step 2 of the embodiment, and the specific process is as follows: a. Disperse 200 mg of carboxyl-modified mesoporous hollow carbon nanoenzyme into 50 mL of ethylenediamine, then add 20 mg of HATU and continue the reaction for 4 h to obtain amino-modified MHC (MHC-NH2). After centrifugal washing and anhydrous ethanol washing twice each, distilled water was added to prepare a 25 mg / mL MHC-NH2 dispersion; b. Preparation of MHC-SH: 200 mg of 3-mercaptopropionic acid was dispersed in distilled water, and EDC and NHS were added for activation for 1 h to obtain an activated 3-mercaptopropionic acid solution, and then 20 mL of MHC-NH2 dispersion was added, stirred for 12 h, and centrifuged and washed to obtain thiol-modified mesoporous carbon nanozyme (MHC-SH); The concentrations of EDC and NHS were both 2.5 mg / mL.

[0071] Embodiment 2: The difference between this embodiment and embodiment 1 is that the concentrations of the HAuCl4 solution and the sodium citrate solution in step 1 are both 2.0×10 -4 mol / L; 1 mL of NaHB4 solution was added to continue the reaction; the dialysis time was 24 h; the rest was the same as in Example 1.

[0072] Embodiment 3: The difference between this embodiment and embodiment 1 is that the concentrations of the HAuCl4 solution and the sodium citrate solution in step 1 are both 3.0×10 -4 mol / L; react for 30 min, then add 1 mL NaHB4 solution to continue the reaction; dialysis time is 16 h; other aspects are the same as in Example 1.

[0073] Example 4 (comparative reference): The difference between this embodiment and embodiment 1 is that in step 3, 40 mg of thiol-modified mesoporous carbon nanozyme is dispersed in 10 mL of phosphate buffer (PBS) at pH 7.4, and then 10 mL of 1 mg / mL gold nanozyme suspension is added dropwise and stirred for 8 hours. The precipitate is collected by centrifugation to obtain MHC-Au, and then 20 mg of thiol-modified polyethylene glycol 2000 (PEG-SH, Mw=2000) is added to obtain MHC-Au-P; the rest is the same as in embodiment 1.

[0074] Example 5 (comparative reference): Preparation of D@MHC-COOH: 40 mg of MHC-COOH mesoporous carbon nanozyme was dispersed in 10 mL of phosphate buffer (PBS) at pH 7.4, and then 10 mL of a 3 mg / mL aqueous solution of doxorubicin hydrochloride (DOX) was added. After ultrasonication for 1 h, stirring was continued for 12 h, and the precipitate was collected by centrifugation to obtain the DOX-loaded MHC-COOH system, namely D@MHC-COOH.

[0075] result: Characterization of gold nanozymes: The particle size of the gold nanozymes obtained in Examples 1 to 3 was characterized by transmission electron microscopy. Figure 1As shown, the prepared gold nanozyme is spherical with uniform particle size of about 3~4nm.

[0076] The particle size of the gold nanozymes obtained in Examples 1 to 3 was measured using a Malvern laser particle size analyzer. Figure 2 As shown, the hydrated particle size of the prepared gold nanozyme is about 8 nm, and the particle size is uniform.

[0077] Evaluation of the enzyme-like properties of gold nanozymes: The catalytic effect of the glucose oxidase (GOx)-like gold nanozyme was evaluated by detecting the pH value and hydrogen peroxide concentration changes in glucose solutions of different concentrations. Glucose solutions with concentrations of 0 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 400 μg / mL, 750 μg / mL and 1000 μg / mL were prepared respectively, and the gold nanozymes prepared in Examples 1 to 3 were added to the above solutions respectively, reacted at 37°C for 12 h, sampled and centrifuged, and the pH value of the solution was detected by a pH meter, and the absorbance of the supernatant was measured; ammonium molybdate can react with hydrogen peroxide to form a stable yellow complex with characteristic absorption at 330 nm. The ammonium molybdate method was used to detect the hydrogen peroxide level in the solution, and a curve of the hydrogen peroxide level changing with the concentration of the glucose solution was drawn. The results are shown in Figure 2. Figure 3 As shown in the figure, as the glucose concentration in the solution increases, the hydrogen peroxide level gradually increases, and basically reaches a peak when the glucose concentration is 1000 μg / mL, producing hydrogen peroxide close to 100 μg / mL; the pH value of the solution is detected by a pH detector. As the glucose content increases, the gold nanozyme can effectively reduce the pH value of the solution and increase the acidity of the solution, providing a guarantee for the subsequent catalytic reaction of the POD-like enzyme, and laying the foundation for the enhanced catalysis of the nanozyme.

[0078] Evaluation of the photothermal heating properties of gold nanozymes: The gold nanozymes were irradiated with NIR laser to evaluate the photothermal performance of the gold nanozymes obtained in Examples 1 to 3. The results are shown in the attached figure. Figure 4 As shown in AB, gold nanozymes have a good temperature-raising effect. The gold nanozyme suspensions of different concentrations were heated by 808 nm near-infrared laser at 2 W / cm 2 After 5 min of irradiation, the results are as follows Figure 4 As shown in A, at a concentration of 200 μg / mL, the temperature of the gold nanozyme suspension increased by about 40°C, showing a concentration-dependent temperature rise characteristic. The power-dependent temperature rise curves of gold nanozymes at different power densities are shown in Figure 4 B. Subsequently, the stability of the gold nanozyme was further studied by repeated near-infrared irradiation; after 5 times of near-infrared irradiation, the temperature-raising ability of the gold nanozyme did not change significantly, as shown in Figure 5As shown, it is proved that gold nanozymes have excellent photostability.

[0079] Transmission electron microscopy was used to characterize the electron microscopic morphology of carboxyl-modified mesoporous hollow carbon nanozyme (MHC-COOH). Figure 6 As shown in the figure, MHC-COOH is a uniform spherical structure with an average diameter of 140-150nm, rich mesoporous pores and obvious hollow structure, and the layer thickness of the carrier is about 30nm. The transmission electron microscope was used to characterize the electron microscopic morphology of MHC-Au-P. The results are shown in Figure 7 As shown in the figure, many gold nanozymes can be clearly observed on the surface of MHC. The particle size of gold nanozymes is similar to the mesoporous particle size of MHC carrier, both of which are 3~5nm. Therefore, gold nanozymes can block the mesoporous channels of MHC to prevent the release of drugs before reaching the target site.

[0080] The carriers were characterized by surface area and pore size analyzers. The isotherms, distribution curves and related parameters of MHC, MHC-COOH and MHC-Au-P were as follows: Figure 8 As shown in Table 1; the results show that with each step of modification, the specific surface area and pore volume of MHC-Au-P gradually decrease, proving that the gold nanozyme has been grafted onto MHC-Au-P.

[0081] The particle size and Zeta potential of different nanoparticles were measured using a Malvern laser particle size and potential analyzer. Fig. 9 The Zeta potential of MHC-COOH is -29.4 mV, while the Zeta potential of MHC-NH2 after modification with ethylenediamine is 12.1 mV. When the surface of the carrier is modified with thiol and gold nanozyme, the Zeta potential of MHC-Au is significantly reduced to -32.4 mV. After MHC-Au-P is grafted with PEG-SH, the Zeta potential of MHC-Au-P increases to -13.7 mV. The hydrated particle sizes of MHC-COOH, MHC-Au and MHC-Au-P are shown in Fig. 9 As shown in Figure B, after each step of carrier modification, the particle size increased by approximately, but the PDI value of the carrier gradually decreased, indicating that MHC-Au-P had good dispersibility after PEG grafting.

[0082] MHC-COOH and MHC-Au-P were irradiated with NIR laser to evaluate the photothermal properties of the carrier. Fig.10 As shown in the figure, MHC-COOH has obvious concentration-dependent temperature rise characteristics. Under the same conditions, the temperature rise of MHC-Au-P is higher than that of MHC-COOH, which indicates that MHC-Au-P has a better photothermal temperature rise effect than MHC-COOH.

[0083] In addition, the photostability of near-infrared photothermal agents is a key factor affecting PTT. The stability results of MHC-COOH and MHC-Au-P under repeated near-infrared irradiation are shown in Fig.11 ; After 5 repeated NIR light irradiation, the temperature rising performance of MHC-COOH and MHC-Au-P did not change significantly, proving that MHC-COOH and MHC-Au-P have excellent photothermal stability under repeated near-infrared laser irradiation.

[0084] At 2.0W / cm 2 100 μg / mL carriers (MHC-COOH and MHC-Au-P) were irradiated to further calculate the η values ​​of MHC-COOH and MHC-Au-P, as Fig.12 As shown. The η values ​​of MHC-COOH and MHC-Au-P are 30.28% and 33.65%, respectively. The above results prove that the temperature-raising effect of MHC-Au-P is better than that of MHC-COOH and gold nanozymes, indicating that the gold nanozymes and MHC-COOH in MHC-Au-P can play a role in joint temperature-raising.

[0085] Weigh 2 mg of D@MHC-Au-P and disperse them in 5 mL of pH 7.4 PBS, pH 5.5 PBS, pH 7.4 PBS (containing 10 mM GSH), and pH 5.5 PBS (containing 10 mM GSH), respectively. Shake them in a constant temperature shaker (37°C, 100 rpm) in the dark. Centrifuge and take out 1 mL of supernatant at a specific time point, and measure the absorbance at 482 nm. Add the measured solution back into the dispersion and continue shaking. At the specified time, shake the supernatant at 2 W / cm 2 The light was irradiated for 5 minutes and the absorbance was measured after sampling.

[0086] Fig.13 A is the drug release curve of D@MHC-COOH and D@MHC-Au-P under different pH conditions in the release medium. Compared with PBS at pH 7.4, D@MHC-COOH and D@MHC-Au-P release DOX faster at pH 5.5 (simulating tumor microenvironment conditions). This is because the electrostatic interaction between MHC-COOH and DOX is weaker in an acidic environment, which accelerates the release of the drug from the mesoporous skeleton. However, under the same pH conditions, the release percentage of DOX in D@MHC-Au-P is slower than that in D@MHC-COOH, indicating that gold nanozymes can act as a "switch" to prevent premature release of drugs in D@MHC-Au-P. Fig.13As shown in Figure B, there is no significant difference in the release behavior of D@MHC-COOH when containing or not containing 10 mM GSH under pH 5.5 PBS. In the absence of GSH, the release rate of D@MHC-Au-P is 20.0%. In contrast, the cumulative release of D@MHC-Au-P under 10 mM GSH conditions is 38.1%. These results indicate that D@MHC-Au-P has obvious reduction-responsive release properties, which is due to the gold-sulfur bonds in D@MHC-Au-P. Under the action of high GSH, the gold-sulfur bonds between MHC and gold nanozymes are broken, causing the gold nanozymes to fall off the surface of the carrier, thereby promoting the release of DOX from D@MHC-Au-P. In addition, at 2 W / cm 2 The D@MHC-Au-P samples were irradiated with near-infrared light at 1, 4, and 9 h to study the drug release behavior, e.g. Fig.13 C. After 5 min of near-infrared irradiation in pH 5.5 PBS, the release curves of DOX all increased sharply. D@MHC-Au-P showed that the DOX release of 10 mM GSH under near-infrared irradiation was 58.2%, which was much higher than that under the conditions of no GSH (30.7%) and no NIR irradiation (38.1%). The above results indicate that near-infrared irradiation has a synergistic effect on reducing conditions. Therefore, D@MHC-Au-P has reduction / pH / near-infrared multi-responsive release characteristics.

[0087] Evaluation of GSH consumption effect, enzyme-like catalysis and cascade catalysis effect: The POD-like enzyme catalytic ability of MHC-COOH was tested by 3,3',5,5'-tetramethylbenzidine (TMB): 50 μg / mL MHC-COOH was mixed with 2 mM TMB and 100 mM hydrogen peroxide, incubated at 37°C for 30 min, and the absorbance of the supernatant was detected at 652 nm. The results of the POD-like enzyme catalytic ability of MHC-COOH under near-infrared irradiation at 808 nm in PBS at pH 7.4 and pH 5.5 are shown in Fig.14 .

[0088] The experimental results are as follows Fig.14 As shown in the figure, the absorbance in pH 5.5 PBS is significantly higher than that in pH 7.4 PBS, indicating that the weakly acidic environment improves the POD-like enzyme activity of the system. In addition, the POD-like enzyme activity of MHC-COOH is significantly enhanced under near-infrared irradiation, indicating that near-infrared irradiation can enhance the enzyme activity of MHC-COOH.

[0089] The 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) method was used to detect the thiol groups on GSH to determine the content of GSH. 200 μL of MHC-COOH (50, 100, and 200 μg / mL) was mixed with 10 mM GSH for 12 h, and then GSH was diluted and reacted with DTNB. The absorbance at 412 nm was measured to detect the GSH consumption capacity of MHC-COOH at different times. Fig.15 , at a concentration of 10 mM, 50 µg / mL MHC-COOH could consume approximately 4 mM of GSH, and 200 µg / mL MHC-COOH could almost completely consume 10 mM of GSH within 12 h, causing the NTB solution to change from yellow to almost colorless.

[0090] Gold nanozymes with GOx-like enzyme activity can catalyze glucose to produce hydrogen peroxide, while MHC-Au-P with POD-like activity can react with hydrogen peroxide to produce ·OH, thereby using the ·OH generated by the cascade catalytic reaction to kill tumor cells. MHC-Au-P samples were co-incubated with glucose solutions under different pH conditions to evaluate the cascade reaction generated by MHC-Au-P catalysis. The experimental results are shown in Fig.16 Gold nanozymes, MHC-COOH, and MHC-Au-P (corresponding to a concentration of 50 μg / mL) were added to PBS solutions of different pH 7.4 and pH 5.5 containing TMB (2 mM) and glucose solution (5 mM), respectively. The solution was heated in a 50°C water bath for 5 min to simulate 808 nm laser irradiation. The absorbance change of the supernatant was measured at 652 nm to determine the efficiency of the cascade reaction.

[0091] from Fig.16 It can be seen that neither MHC-COOH nor Au alone can convert TMB into blue ox-TMB, but MHC-Au-P can generate blue ox-TMB. At the same time, MHC-Au-P has stronger POD-like enzyme activity under acidic conditions, and the reaction rate at pH 5.5 is significantly stronger than that at pH 7.4. Near-infrared irradiation was simulated at 50°C. The increase in temperature leads to accelerated oxidation of TMB to blue ox-TMB, which is related to the increase in the GOx-like enzyme activity of gold nanozymes and the POD-like enzyme activity of MHC-COOH at high temperatures. The above results show that MHC-Au-P has the ability to catalyze glucose to produce hydrogen peroxide and glucuronic acid in the tumor microenvironment to reduce the intracellular pH, thereby catalyzing the formation of •OH, thereby killing tumor cells.

[0092] Evaluation of cell photothermal effect: 4T1 tumor cells in the logarithmic growth phase were inoculated in a 24-well plate. After incubation for 24 h, a series of concentrations of MHC-Au-P prepared in serum-free culture medium were added. After incubation for 2 h, the cells were illuminated at 1.0 W / cm 2 After irradiation with NIR light for 2 min, 4 min, 6 min and 8 min, 100 μL of Calcein-AM and PI fluorescent dye working solution were added to each well for staining, and the cell survival was observed using a laser confocal microscope (CLSM). The results are shown in the attached figure. Fig.17 As shown in the figure, obvious green fluorescence was observed in the control group. After irradiation with NIR laser, the number of dead cells with red fluorescence gradually increased with the extension of irradiation time. Within 6 min, all cells showed red fluorescence, and the green fluorescence basically disappeared. Therefore, the results of Calcein-AM / PI experiments showed that MHC-Au-P had a good photothermal effect and could kill tumor cells under PTT irradiation.

[0093] 4T1 tumor cells in the logarithmic growth phase were inoculated in 6-well plates for 24 h, and different concentrations of D@MHC-Au-P were added to the cells for 2 h. The cells were washed and resuspended in 150 μL PBS. 2 The cells were irradiated with near infrared light for 5 min, and the temperature of the cells was recorded with a thermal infrared imager. Fig.18 As shown in the figure, under 808 nm irradiation, the cell temperature increased significantly with the increase of D@MHC-Au-P concentration, while the temperature of blank 4T1 cells did not increase significantly. After incubating 4T1 cells with 50 μg / mL D@MHC-Au-P for 2 h and irradiating with near-infrared for 2 min, the cell temperature increased by more than 10°C. Tumor cells will be ablated at temperatures above 42°C.

[0094] 4T1 cells were seeded in 24-well plates and incubated for 24 h. The old culture medium was discarded and the cells were cultured with serum-free free DOX, MHC-Au-P and D@MHC-Au-P (DOX concentration was 2.5 μg / mL). After 2 h of culture, the culture medium was discarded and the cells were washed three times with cold PBS. For the NIR irradiation groups (DOX + L and D@MHC-Au-P + L), 2.0 W / cm 2 The samples were irradiated with NIR light for 3 min and cultured for 1 h. After that, they were fixed with 4% formaldehyde for 15 min, washed with PBS, stained with Hoechst 33258 for 15 min, washed with PBS three times, and observed under CLSM. The cell uptake results are shown in Fig.19As shown. MHC-Au-P only showed blue fluorescence stained with Hoechst, indicating that the gold nanozyme does not emit fluorescence under the DOX excitation wavelength. There was no significant difference in the red fluorescence intensity of the free DOX group before and after near-infrared irradiation. In contrast, the red fluorescence in the cytoplasm of the D@MHC-Au-P group was significantly enhanced after near-infrared irradiation, indicating that near-infrared irradiation can promote the separation of gold nanozymes from MHC carriers and promote the release of DOX.

[0095] 4T1 tumor cells were seeded in a 24-well plate with a cell slide. After incubation for 24 h, the cells were incubated with MHC-COOH, MHC-Au-P, and MHC-Au-P+Glu (containing 5 mM glucose) for 2 h. The concentration of the carrier was 50 μg / mL. For the NIR irradiation group (MHC-Au-P + Glu + L), 1.0 W / cm 2 After irradiating the cells with a laser power of 100 nm for 3 min, the culture medium was discarded, the cells were washed three times with PBS, incubated with 10 μM DCFH-DA dye solution for 30 min, then incubated with 10 μg / mL Hoechst33258 dye solution for 20 min, washed three times with PBS, and fluorescence images were taken using CLSM.

[0096] The study examined the ability of MHC-Au-P to catalyze hydrogen peroxide and generate ROS in cells by evaluating the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) in cells. Fig. 20 In the control group, almost no green fluorescence was observed, while in the MHC-COOH group, there was a weak green fluorescence, which indicated that MHC-COOH could catalyze hydrogen peroxide to generate a certain amount of ·OH in the tumor microenvironment, indicating that MHC-COOH has a certain catalytic effect similar to POD enzymes. The green fluorescence of the MHC-Au-P group was stronger, because the gold nanozyme could catalyze glucose to continuously supplement hydrogen peroxide for the POD enzyme-catalyzed reaction, resulting in more ·OH production. Under 5 mM glucose and 808 nm near-infrared light irradiation (MHC-Au-P + Glu + L), the MHC-Au-P group showed obvious green fluorescence due to the enhanced photothermal conversion and self-supply of hydrogen peroxide of MHC-Au-P, resulting in increased ·OH production, proving that MHC-Au-P has the ability of PTT-enhanced cascade catalysis to anti-tumor effect.

[0097] The 4T1 cells were seeded in 24-well plates and incubated for 24 h. The old culture medium was discarded, and the cells were incubated with 50 µg / mL of MHC-Au-P for 3 h, 6 h, and 9 h. After 6 h and 9 h, the cells were washed three times with PBS, stained with 50 µM naphthalene-2,3-dicarboxaldehyde (NDA) for 10 min, and then washed three times. The cells were stained with Hoechst 33258 for 20 min, washed three times with PBS, and the fluorescence was detected by CLSM.

[0098] The experimental results are as Fig.21 shown. After incubation with NDA, the green fluorescence of GSH was widely distributed in 4T1 cells. The green fluorescence gradually decreased after incubation of 4T1 cells with MHC-Au-P for 3 h and 6 h, and almost completely disappeared after 9 h, indicating that MHC-Au-P has excellent GSH consumption ability at the cellular level.

[0099] The 4T1 cells were seeded in 24-well plates and incubated for 24 h. The old culture medium was discarded, and the cells were incubated with PBS, DOX, MHC-Au-P, and D@MHC-Au-P (DOX dose was 5 µg / mL) for 6 h, respectively. The cells in the PTT group were irradiated with near-infrared laser (1.0 W / cm2) for 5 min. Finally, the cells were stained with DAPI and Rh123, and the damage of mitochondrial membrane was detected by CLSM. Rh123 is a fluorescent probe widely used for the detection of mitochondrial membrane potential (MMP). When MMP decreases, the green fluorescence intensity of Rh123 decreases. The CLSM detection results of Rh123 in different treatment groups are as Fig. 22 . Compared with the control group, the green fluorescence of Rh123 in each treatment group was significantly decreased, which may be related to chemotherapy and PTT-induced hyperthermia promoting apoptosis. The apoptosis of the chemotherapy combined with PTT group (D@MHC-Au-P + L) was more obvious, and the green fluorescence intensity was the weakest.

[0100] The 4T1 cells were seeded in 96-well plates at a density of 5×10 3 / well, with 5 replicates in each group. After the cells adhered, the cells were incubated with the culture medium of DOX, MHC-Au-P, and D@MHC-Au-P for 24 h. For the NIR light irradiation group, the cells were irradiated with a 1.0 W / cm 2 NIR laser for 5 min. The concentrations of DOX equivalent in each group were 0.01, 0.1, 1, 2.5 µg / mL, and 5 µg / mL, respectively. After incubation, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated at 37 °C for 4 h. The solution in the well plate was discarded, 150 μL of dimethyl sulfoxide was added, and the absorbance was measured at 570 nm to calculate the cell survival rate. The combination index (CI) was used to evaluate the synergistic effect of chemotherapy and PTT. CI < 0.9 indicates a synergistic effect, 0.9 < CI < 1.1 indicates an additive effect, and CI > 1.1 indicates an antagonistic effect.

[0101] like Fig.23 As shown in Figures A and 23B, in the MHC-Au-P group, the cell survival rate was close to 60% at a concentration of 18.3 μg / mL. The D@MHC-Au-P group showed a concentration-dependent cytotoxic effect with a cell survival rate of 40%, which was due to the incomplete release of DOX in the absence of NIR irradiation. Under NIR light irradiation, the cell survival rate of the free DOX group was basically the same as that of the D@MHC-Au-P group. However, for the other three groups, the cell survival rate was significantly reduced. In the MHC-COOH group, the cell survival rate under NIR light irradiation dropped to 58%. For the MHC-Au-P group, the cell survival rate under NIR light was further reduced. Encouragingly, the survival rate of D@MHC-Au-P cells under NIR irradiation was significantly lower than that of the DOX group, because NIR irradiation would increase the temperature of 4T1 cells and promote the release of DOX. The heat of NIR irradiation would damage tumor cells and increase the sensitivity of cells to chemotherapeutic drugs, thereby producing the greatest cytotoxicity.

[0102] In addition, the study investigated the survival rate of D@MHC-Au-P cells under different concentrations of glucose, such as Fig.24 As shown. The experimental results show that with the increase of glucose concentration, the viability of D@MHC-Au-P cells is significantly reduced. This is because the gold nanozyme with GOx-like enzyme activity can catalyze glucose in cancer cells to produce hydrogen peroxide and glucuronic acid, thereby reducing the energy supply of tumor cells, optimizing catalytic conditions, accelerating the catalytic action of POD-like enzymes, and promoting the production of ·OH. Therefore, the above data show that D@MHC-Au-P has good potential as a nano-delivery system for synergistic chemotherapy-cascade enzyme catalysis-photothermal anti-tumor.

[0103] BALB / c mice were subcutaneously injected with 4T1 cells (18-20 g). When the tumor volume increased to 100 cm 3 The D@MHC-Au-P group and the saline group were given respectively. The tumor site was irradiated with near-infrared light, and the temperature rise of the tumor site in each group was recorded with a thermal imager. The experimental results are shown in Fig.25 H. D@MHC-Au-P + L group was treated with 1 W / cm 2 After 3 minutes of continuous irradiation, the temperature of the tumor tissue increased to 51.2°C, which can promote DOX release and tumor ablation. In contrast, the temperature of the saline group increased to 39.5°C under the same power irradiation, indicating that D@MHC-Au-P has excellent temperature-raising ability.

[0104] In order to prove that D@MHC-Au-P has a good synergistic anti-tumor effect in vivo, 4T1 tumor-bearing mice were randomly divided into normal saline group, no DOX group, D@MHC-Au-P group, cascade catalysis combined with hyperthermia group (MHC-Au-P + L group) and chemotherapy-cascade catalysis combined with hyperthermia group (D@MHC-Au-P + L). The dosage of DOX in each group was 5 mg / kg (100 μL). In the hyperthermia group, the DOX was irradiated at 1 W / cm at 6 hours after administration on the 1st, 4th and 7th day after injection. 2 The tumor was irradiated with 808 nm laser for 3 min. After 15 days of experimental measurement, all mice were killed by dislocation of the neck, and the complete tumor tissue was dissected out, washed with physiological saline, and dried with filter paper. The tumor mass of each control group was weighed, and the tumor inhibition rate (TIR) ​​was calculated according to the following formula.

[0105] Where W saline : Average mass of tumor tissue in the saline group, Wt est : The average mass of tumor tissue in other experimental groups.

[0106] The results of the pharmacodynamic experiments are shown in 25A-E. The tumor growth curve and tumor images show that the tumor in the saline group grew rapidly, while the other treatment groups all had a certain degree of inhibitory effect on tumor tissue. For the D@MHC-Au-P group, due to the low release of DOX in D@MHC-Au-P and the cascade enzyme catalysis of MHC-Au-P, the tumor inhibition rate of D@MHC-Au-P was 53.0%. The therapeutic effect of the free DOX group was similar to that of the D@MHC-Au-P group. There was no significant difference between the two groups, but the mice lost weight significantly after being given free DOX ( Fig.25 F), indicating that free DOX has obvious systemic toxicity, and there is no significant weight loss in the other groups of mice, indicating that D@MHC-Au-P and MHC-Au-P have good biosafety. Under PTT combined with nanozyme catalysis therapy, the tumor inhibition effect of the MHC-Au-P + L group was significant, with a tumor inhibition rate of 85.1%. Excitingly, the tumor inhibition effect of D@MHC-Au-P + L was the best at 94.7%, showing the combined antitumor effect of chemotherapy-PTT-nanocatalysis. Similarly, the relative tumor volume histogram obtained after 15 days of treatment also confirmed that the synergistic anticancer effect of D@MHC-Au-P under near-infrared irradiation was much better than that of any treatment group (see Fig.25 G). In summary, D@MHC-Au-P can achieve synergistic antitumor effects of chemotherapy, PTT, and nanozyme catalysis under the irradiation of NIR light.

[0107] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity, characterized in that It consists of mesoporous hollow carbon, anti-tumor drugs, gold nanozymes and hydrophilic functionalized polymers; the anti-tumor drugs are one or more of doxorubicin hydrochloride, paclitaxel, vinorelbine, cisplatin and methotrexate; the hydrophilic functionalized polymer is thiol-modified hyaluronic acid, thiol-modified polyethylene glycol 2000 or thiol-modified folic acid.

2. A method for preparing a mesoporous hollow carbon drug-carrying system having cascade enzyme catalytic activity as claimed in claim 1, characterized in that It is implemented as follows:

1. Preparation of gold nanozymes: 20-40 mL HAuCl4 solution and 20-40 mL sodium citrate solution were mixed and reacted for 5-30 min, and then 0.5-2 mL NaHB4 solution was added to continue the reaction to obtain a wine red solution, which was dialyzed and freeze-dried to obtain gold nanozyme; 2. Preparation of carboxyl-modified mesoporous hollow carbon nanozymes and thiol-modified mesoporous carbon nanozymes; 3. Preparation of drug-loaded nanozymes: The above 30-50 mg of thiol-modified mesoporous carbon nanozyme was dispersed into 5-15 mL of pH 7.4 phosphate buffer, and then 10 mL of 1 mg / mL-5 mg / mL anti-tumor drug solution was added. After ultrasonication for 0.5-2 h, stirring was continued for 2-12 h, and the precipitate was collected by centrifugation to obtain the D@MHC-SH drug carrier. Take the gold nanozyme obtained in step 1, add distilled water to prepare a gold nanozyme suspension with a concentration of 1 mg / mL; 30-50 mg of D@MHC-SH drug carrier was dispersed in 10 mL of PBS with a pH of 7.4, 5-15 mL of 1 mg / mL gold nanozyme suspension was added and stirred for 2-12 h, and the precipitate was collected by centrifugation to obtain drug-loaded D@MHC-Au, and then 10-30 mg of hydrophilic functionalized polymer was added to obtain D@MHC-Au-P, which is a mesoporous hollow carbon drug-loading system with cascade enzyme catalytic activity, completing the preparation.

3. The method for preparing a mesoporous hollow carbon drug-carrying system having cascade enzyme catalytic activity according to claim 2, characterized in that The concentrations of the HAuCl4 solution and the sodium citrate solution in step 1 are both (2-3)×10 -4 mol / L; the concentration of the NaHB4 solution is 0.75mol / L ~0.1mol / L.

4. The method for preparing a mesoporous hollow carbon drug-carrying system having cascade enzyme catalytic activity according to claim 2, characterized in that The temperature for continuing the reaction in step 1 is 10-50° C., and the reaction time is 0.5-6 h. The dialysis uses a dialysis bag with Mw=1000Da, and the dialysis time is 4-24 h.

5. The method for preparing a mesoporous hollow carbon drug-carrying system having cascade enzyme catalytic activity according to claim 2, characterized in that The preparation of carboxyl-modified mesoporous hollow carbon nanozyme described in step 2 is as follows: ① Preparation of mesoporous core-shell silica spheres: 200-250 mL of anhydrous ethanol and 20-50 mL of deionized water were mixed, and then 4-6 mL of NH3·H2O was added. After stirring for 30 min, 4-6 mL of TEOS was added dropwise. The mixture was stirred in a water bath at 30°C for 2 h. The sample was collected after centrifugation. After washing with anhydrous ethanol and distilled water twice, solid silica spheres were obtained. 200-400 mg of solid silica spheres were dispersed in 70-100 mL of distilled water to obtain solution A. 400-500 mg of CTAB was dissolved in 50-80 mL of water. 80-100 mL of anhydrous ethanol, 1-2 mL of NH3·H2O and solution A were added in sequence. After stirring for 1 h, 0.5-1 mL of TEOS was added dropwise. The mixture was reacted for 6 h. The sample was collected after centrifugation. The sample was washed with anhydrous ethanol and distilled water twice, respectively, and then dried. Then, the sample was heated to 600 °C. o C and vacuum calcined to remove the template to obtain mesoporous core-shell silicon spheres; ② Solidification, calcination and removal of template of carbon source: Add carbon source precursor solution to the above mesoporous core-shell silicon spheres, and then heat at 60 o C and 80 o C for 12 h, and then placed in a vacuum tube furnace for 2 o C / min and the temperature was raised to 800 o C, calcined for 3 h, and finally dispersed in 1% hydrofluoric acid solution, stirred for 6 h to remove silica in the pores, and then washed and dried to obtain a mesoporous hollow carbon carrier; ③. Carboxylation modification: Add 0.4~0.6g of mesoporous hollow carbon carrier into a round-bottom flask containing 20~40mL of 100mg / mL ammonium persulfate solution and 3~5mL of 98% sulfuric acid, react at 60℃ with stirring and reflux for 3h, obtain a black solid after centrifugation, wash with purified water 3~5 times, and obtain carboxylation-modified mesoporous hollow carbon nanozyme after vacuum drying.

6. The method for preparing a mesoporous hollow carbon drug-carrying system having cascade enzyme catalytic activity according to claim 5, characterized in that The carbon source precursor solution includes: 2 mL furfuryl alcohol, 20 mg oxalic acid and 50 mg dopamine hydrochloride.

7. The method for preparing a mesoporous hollow carbon drug-carrying system having cascade enzyme catalytic activity according to claim 1, characterized in that The specific process of preparing the thiol-modified mesoporous carbon nanozyme in step 2 is as follows: a. Disperse 100-300 mg of carboxyl-modified mesoporous hollow carbon nanoenzyme into 30-70 mL of ethylenediamine, then add 10-30 mg of HATU and continue the reaction for 4 h to obtain amino-modified MHC, wash it with water by centrifugation and with anhydrous ethanol twice each, and then add distilled water to prepare a MHC-NH2 dispersion with a concentration of 10-50 mg / mL; b. Preparation of MHC-SH: 150-300 mg of 3-mercaptopropionic acid was dispersed in distilled water, and EDC and NHS were added for activation for 1 h to obtain an activated 3-mercaptopropionic acid solution. Then 10-50 mL of MHC-NH2 dispersion was added and stirred for 12 h. After centrifugation and water washing, thiol-modified mesoporous carbon nanozyme was obtained.

8. The method for preparing a mesoporous hollow carbon drug-carrying system having cascade enzyme catalytic activity according to claim 7, characterized in that The concentrations of the EDC and NHS are both 0.5 mg / mL to 5 mg / mL.

9. The method for preparing a mesoporous hollow carbon drug-carrying system having cascade enzyme catalytic activity according to claim 7, characterized in that The solute in the anti-tumor drug solution is one or more of doxorubicin hydrochloride, paclitaxel, vinorelbine, cisplatin and methotrexate, and the solvent is an aqueous medium or a good solvent.

10. The use of a mesoporous hollow carbon drug-carrying system having cascade enzyme catalytic activity as claimed in claim 1, characterized in that A mesoporous hollow carbon drug-carrying system with cascade enzyme catalytic activity, which can release drugs in a reduction / pH / NIR multi-responsive manner and is used in the combined treatment of tumors with photothermal and cascade enzyme catalytic enhanced chemotherapy.

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