Metal-doped hollow mesoporous carbon nano-enzyme anti-tumor delivery system and application thereof

Abstract

The invention discloses a metal-doped hollow mesoporous carbon nano-enzyme anti-tumor delivery system and application thereof, and belongs to the technical field of medicines. The anti-tumor delivery system is composed of a metal ion doped nano enzyme, a photosensitizer and a biocompatible group, according to the delivery system, POD-like activity and GSH expression of depleting the tumor microenvironment are exerted through the metal ion doped nano-enzyme, meanwhile, mild heating of the tumor microenvironment is achieved by utilizing the excellent photothermal conversion performance of the metal ion doped nano-enzyme and combining with NIR light, the photosensitizer can generate ROS under stimulation of the NIR light to oxidize and kill tumor cells, and the tumor microenvironment can be effectively protected. And similar POD enhanced synergistic photothermal therapy and photodynamic therapy are realized. According to the nano enzyme capable of adjusting the oxidation-reduction level, a photothermal therapy and a photodynamic therapy are combined to realize efficient tumor growth inhibition, and an important reference is provided for multi-mode tumor treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and relates to a metal-doped hollow mesoporous carbon nanoszyme antitumor delivery system and application thereof, in particular to preparation of a hollow mesoporous carbon nanoszyme antitumor delivery system for regulating redox levels and application thereof in the synergistic aspects of photothermal therapy and photodynamic therapy for tumors. BACKGROUND

[0002] The International Agency for Research on Cancer of the World Health Organization test results show that malignant tumors are still one of the diseases that damage human life and health, and are characterized by abnormal cell metabolism, rapid growth and metastasis. With the in-depth and rapid development of tumor phototherapy research, aiming at the photochemical reaction and photothermal heating effect triggered by laser, the generated reactive oxygen species (ROS) and heat energy are used to kill tumor cells, and at the same time, significant progress has been made in clinical treatment. Among them, ROS-mediated tumor cell apoptosis has become a hot spot in tumor treatment because it can greatly affect the stromal cells to regulate the metabolic level, blood supply pathway and immune response of tumors. Although ROS-mediated antitumor therapy has become a very promising cancer treatment, related adverse events such as the antioxidant system of the tumor microenvironment and the complex ROS generation pathway significantly limit its clinical application. Therefore, it is urgent to develop new ROS generators to resist the antioxidant system of the tumor microenvironment and promote more efficient antitumor therapy.

[0003] At present, photothermal therapy, photodynamic therapy, biological catalytic therapy and other treatment methods have been confirmed to cause different degrees of tumor cell apoptosis. Among them, ROS-based antitumor therapy shows great potential in tumor invasion and metastasis. Nanoszyme is a kind of artificial enzyme that combines the unique characteristics of nanomaterials with the catalytic function of natural enzymes. Nanoszyme-mediated tumor biological catalytic therapy is a very promising treatment method, which utilizes the tumor microenvironment to trigger enzyme activity and drive in situ generation of reactive oxygen species. Among them, H2O2 and GSH are two important biomolecules present in the tumor microenvironment, which have important influence on maintaining the redox homeostasis and the efficacy of nanoszyme catalytic therapy. H2O2, as the main catalytic substrate of nanoszyme, can induce tumor cells to produce reactive oxygen species to kill cells. On the contrary, GSH is the main antioxidant in the body and plays an important role in resisting oxidative stress. Maintaining the balance of H2O2 and GSH concentrations in the tumor microenvironment is a key factor to achieve the ideal effect of nanoszyme therapy.

[0004] In addition, the biocompatibility of nanomaterials is crucial for their safe and efficient anti-tumor effect. Since the size, morphology, surface properties, pore geometry, and surface modification of nanomaterials are the main parameters affecting the biocompatibility of nanomaterials, it is necessary to design and synthesize nanoskeleton materials with appropriate morphology and size and to functionalize the surface to achieve safe anti-tumor efficacy in vivo. Therefore, it is of great significance for clinical tumor treatment to safely achieve efficient killing of solid tumors by destroying the redox balance of tumor microenvironment through the biocatalytic function of nanozymes, inducing tumor cells to produce ROS. SUMMARY

[0005] The purpose of the present application is to construct a metal-doped hollow mesoporous carbon nanozyme anti-tumor delivery system to stimulate tumor oxidative stress by adjusting the redox level of tumor microenvironment and achieve synergistic anti-tumor effect of photothermal therapy / photodynamic therapy by loading photosensitizer.

[0006] The technical scheme adopted by the present application is as follows: The metal-doped hollow mesoporous carbon nanozyme anti-tumor delivery system comprises metal ion-doped nanozyme, photosensitizer and biocompatible group.

[0007] Preferably, the metal ion-doped nanozyme is formed by oxidation and polymerization of metal ions and dopamine, wherein the metal ion is one of Cu 2+ , Mn 2+ , Mg 2+ , Fe 3+ , Fe 2+ , Zn 2+ , Ag + .

[0008] Preferably, the photosensitizer is one of IR780, chlorin e6 (Ce6), hexaethyl pyropheophorbide a (HPPH) and chlorophyll-a (Chl-a).

[0009] Preferably, the biocompatible group is one of amino polyethylene glycol 2000 (NH2-PEG 2000 ), hyaluronic acid and folic acid.

[0010] The preparation method of the metal-doped hollow mesoporous carbon nanozyme anti-tumor delivery system comprises the following steps:

[0011] (1) Preparation of metal-doped hollow mesoporous carbon nanoszyme: tetraethyl orthosilicate (TEOS) is added to a mixed solution of ethanol and water, and ammonia water is added to adjust the pH of the solution to be alkaline. After stirring for a period of time, centrifugation is performed to obtain solid silica nanoparticles (SiO2). Hydrochloric acid dopamine, metal ions and ammonia water are added to the dispersion of SiO2, and after uniform mixing under alkaline conditions for a period of time, centrifugation is performed to obtain metal-doped PDA@SiO2. The dried product is subjected to carbonization treatment under the protection of nitrogen. Hydrofluoric acid is added to remove residual SiO2, and after centrifugation and drying, a black solid is obtained, which is the metal-doped hollow mesoporous carbon nanoszyme.

[0012] (2) Preparation of biocompatible group modified nanoszyme: the metal-doped hollow mesoporous carbon nanoszyme is subjected to surface carboxylation using a sulfuric acid solution and ammonium persulfate, and the carboxylated metal-doped hollow mesoporous carbon nanoszyme is activated using EDC and NHS. A biocompatible group solution is added and reacted for a period of time, and centrifugation is performed to obtain the biocompatible group modified hollow mesoporous carbon nanoszyme.

[0013] (3) Preparation of drug-loaded nanoszyme: a photosensitizer is dissolved in a methanol solution, and the biocompatible group modified hollow mesoporous carbon nanoszyme is added dropwise. After reacting for a period of time, centrifugation is performed to obtain the drug-loaded nanoszyme.

[0014] Preferably, in step (1), the volume ratio of TEOS to the mixed solution of ethanol and water is 1:25-1:75, the molar ratio of metal ions to hydrochloric acid dopamine is 0.5:1-1.5:1, the pH value of the alkaline environment is 8-10, the volume ratio of water to ethanol is 1:1-1:5, and the reaction time is 1-5 h. The metal ion is preferably Cu 2+ , the heating rate of carbonization is 1-10℃ / min, the carbonization temperature is 500-900℃, and the concentration of hydrofluoric acid is 1%-25%.

[0015] Preferably, in step (2), the concentration of sulfuric acid is 10%-50%, the mass ratio of added ammonium persulfate to metal-doped hollow mesoporous carbon nanoszyme is 2:1-10:1, the mass ratio of carboxylated metal-doped hollow mesoporous carbon nanoszyme to EDC and NHS is 1:4:2-1:16:2, the mass ratio of biocompatible group to carboxylated metal-doped hollow mesoporous carbon nanoszyme is 1:1-1:10, and the reaction time is 8-24 h. The biocompatible group is preferably NH2-PEG 2000 .

[0016] Preferably, in step (3), the mass ratio of the photosensitizer to the biocompatible group modified hollow mesoporous carbon nanoszyme is 0.5:1-5:1, and the reaction time is 8-24 h. The photosensitizer is preferably IR780.

[0017] Preferably, step (1) is specifically: TEOS is added to a mixed solution of ethanol and water, then ammonia water is added to adjust the pH, stirred at room temperature for 2.5h, centrifuged at 11000rpm for 15min to obtain a reaction precipitate. The precipitate is dispersed in a mixed solution of water and ethanol, ammonia water is added to adjust the pH, dopamine hydrochloride is added, and after stirring at 25℃ for 1h, CuCl2·2H2O solution is added, the reaction system is stirred at room temperature, then centrifuged at 11000rpm for 15min to obtain a reaction precipitate, the reaction precipitate is washed with distilled water and ethanol solution for 3 times, the reaction precipitate is dried, sieved and calcined in a vacuum tube furnace at a rate of 5℃ / min to 800℃ for 3h, the sample is collected, dispersed in a dilute hydrofluoric acid solution, stirred at room temperature overnight, then centrifuged at 11000rpm for 15min to obtain a black solid, which is a copper-doped hollow mesoporous carbon nanoscale enzyme (CHC).

[0018] Preferably, step (2) is specifically: ammonium persulfate and sulfuric acid solution are added to the CHC water dispersion, and refluxed at 60℃ in a water bath for 5h to obtain a carboxylated copper-doped hollow carbon carrier (CHC-COOH), EDC and NHS are dissolved in a PBS solution at pH 7.4 to activate the carboxyl groups in CHC-COOH, after activation, NH2-PEG-containing PBS aqueous solution at pH 7.4 is added dropwise, and after the reaction is completed, centrifugation is performed at 11000rpm for 10-15min to obtain a solid, which is a NH2-PEG-modified CHC nanoscale enzyme (CHC-PEG).

[0019] Preferably, step (3) is specifically: the CHC-PEG nanoscale enzyme is dispersed in a methanol solution, IR780 methanol solution is added dropwise, after the reaction is completed, centrifugation is performed at 10000rpm for 10min to obtain a solid, which is an IR780-loaded CHC-PEG nanoscale enzyme (IR780 / CHCP)

[0020] The metal-doped hollow mesoporous carbon nanoscale enzyme delivery system described in the application is applied to tumor multi-mode synergistic therapy.

[0021] The application adopts a metal ion-doped hollow mesoporous carbon nanoscale enzyme as a carrier, loads a photosensitizer IR780, and further modifies a biocompatible group on the surface of the carrier to constitute a nanoscale enzyme anti-tumor delivery system that can adjust the redox level of the tumor microenvironment.

[0022] Among them, Cu 2+ As a doping ion, a CHC nanoscale enzyme with a particle size of about 180nm is constructed. Cu 2+The doping of CHC nanozymes endows them with excellent POD-like enzyme activity and the ability to consume GSH, while also improving their photothermal conversion efficiency. Furthermore, CHC nanozymes, through their POD-like enzyme activity, catalyze the generation of large amounts of the strong oxidizing agent ·OH, causing oxidative damage to tumor cells. Simultaneously, the GSH-consuming ability of CHC nanozymes can alleviate the antioxidant effect of the tumor microenvironment, thus more efficiently exerting the oxidative damage effect of ·OH. IR780, as a photosensitizer, can generate singlet oxygen (…) under NIR light irradiation. 1 O2) enables photodynamic killing of tumors; NH2-PEG, as a biocompatible group, can be bound to the surface of CHC nanozyme through amide bonds, enabling CHC nanozyme to exhibit good biocompatibility in vivo.

[0023] Once inside the body, this delivery system exhibits POD-like enzyme activity, catalyzing the highly expressed H2O2 in the tumor microenvironment to generate the highly oxidizing ·OH. Simultaneously, the nanozyme effectively depletes GSH in the tumor microenvironment, reducing the reducing effect of GSH on highly oxidizing substances and enhancing the oxidative activity of free radicals. Under 808nm laser irradiation, the nanozyme generates localized heat, and the loaded photosensitizer IR780 can be excited by the laser to produce… 1 O2 co-induces tumor cell apoptosis. This nanodelivery system leverages nanozymes to regulate the tumor microenvironment, exert photodynamic and photothermal effects, achieving synergistic photothermal and photodynamic therapy on tumors, resulting in excellent anti-tumor therapeutic effects.

[0024] The beneficial effects of this invention are:

[0025] (1) The CHC nanozyme constructed in this invention generates a large amount of ·OH through the regulation of the tumor microenvironment and the catalytic reaction of POD-like enzymes, which greatly enhances the effect of nanozyme-mediated biocatalytic anti-tumor therapy.

[0026] (2) This invention introduces a photosensitizer, which, under NIR light irradiation, excites the generation of… 1 O2 oxidizes the DNA and proteins of tumor cells, while the excellent photothermal properties of CHC nanozymes can generate heat energy, achieving mild thermotherapy while enhancing the POD-like enzyme activity of CHC, catalyzing the production of more ROS, and significantly improving the killing effect of nanozymes on tumor cells. At the same time, CHC nanozymes exhibit GSH-consuming capacity, which can further enhance the oxidative killing effect of ROS on tumor cells. In vivo pharmacodynamic studies revealed that under the synergistic effect of nanozyme-mediated photothermal therapy and photodynamic therapy, tumor growth was severely inhibited, achieving a synergistic effect of dual therapy in tumor treatment. Attached Figure Description

[0027] Figure 1 This is a transmission electron microscope image of the CHC nanozyme prepared in Example 1.

[0028] Figure 2 (A) Particle size and (B) Zeta potential of CHC, CHC-COOH and CHC-PEG prepared in Example 1.

[0029] Figure 3 Fourier infrared spectra of CHC, CHC-COOH and CHC-PEG prepared in Example 1.

[0030] Figure 4 XPS electron spectrogram of CHC-PEG nanoszyme prepared in Example 1.

[0031] Figure 5 Hemolysis data of CHC and CHC-PEG nanoszyme prepared in Example 1.

[0032] Figure 6 Photothermal heating diagram of CHC nanoszyme prepared in Example 1 (A) heating diagram under different laser power irradiation and (B) heating diagram of CHC nanoszyme with different concentrations. (C) Photothermal stability diagram of CHC nanoszyme.

[0033] Figure 7 POD-like enzyme activity diagram of CHC-PEG nanoszyme prepared in Example 1 (A) UV-visible spectrogram of CHC-PEG nanoszyme with different concentrations reacting with TMB (B) Michaelis-Menten equation fitting curve of CHC-PEG nanoszyme with TMB as reaction substrate (C) Michaelis-Menten equation fitting curve of CHC-PEG nanoszyme with H2O2 as reaction substrate (D) Michaelis-Menten equation fitting parameters of POD-like enzyme activity of CHC-PEG nanoszyme.

[0034] Figure 8 (A) UV spectrogram and (B) fluorescence spectrogram of CHC-PEG and IR780 / CHCP nanoszyme and IR780 prepared in Example 1 and Example 2.

[0035] Figure 9 Cell toxicity of CHC-PEG and IR780 / CHCP nanoszyme prepared in Example 1 and Example 2 (A) without NIR laser light irradiation (B) with NIR laser light irradiation.

[0036] Figure 10 Live / dead cell staining diagram of CHC-PEG nanoszyme prepared in Example 1, the scale is 50 μm.

[0037] Figure 11 Intracellular reactive oxygen species generation diagram of CHC-PEG and IR780 / CHCP nanoszyme prepared in Example 1 and Example 2, the scale is 50 μm.

[0038] Figure 12 Intracellular GSH depletion plot of CHC-PEG nanoszyme and IR780 prepared in Example 1, scale bar is 50 pm.

[0039] Figure 13 (A) Mouse tumor growth curve and (B) Mouse body weight change curve of CHC-PEG and IR780 / CHCP nanoszyme and IR780 prepared in Example 1 and Example 2. DETAILED DESCRIPTION

[0040] The application will be further described with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of this application. Furthermore, it should be understood that various modifications can be made to the application by those skilled in the art upon reading the teachings of this application, and such modifications are intended to come within the scope of the appended claims.

[0041] Example 1

[0042] Preparation and characterization of CHC nanoszyme

[0043] (1) Preparation of CHC-PEG nanoszyme

[0044] 2.00 mL of TEOS was accurately added to a mixed solution of 100 mL of ethanol and water (V 水 :V 乙醇 = 1:4), followed by 2 mL of ammonia water, so that the reaction system was in an alkaline environment (pH at 8-10), stirred at room temperature for 2.5 h, centrifuged at 11000 rpm for 15 min, and the reaction precipitate was SiO2. 100 mg of SiO2 was redispersed in a mixed solution of 6 mL of water and 10 mL of ethanol, and 0.75 mL of NH3·H2O was added to the system. At the same time, 100 mg of dopamine hydrochloride was dissolved in 2 mL of water, and the dopamine hydrochloride solution was added dropwise into the SiO2 dispersion under stirring, and after stirring at room temperature for 1 h, CuCl2·2H2O aqueous solution (100 mg) was added and stirred at room temperature for 11 h. Centrifuged at 11000 rpm for 15 min, the precipitate was placed in a vacuum dryer and dried for 12 h, ground through a 200 mesh sieve and placed in a vacuum tube furnace, and the furnace temperature was raised to 800℃ at a rate of 5℃ / min and calcined for 3 h, the sample was collected, dispersed in dilute HF aqueous solution, stirred magnetically (500 rpm, 24 h) at 25℃, the internal silica was removed, the precipitate was collected by centrifugation (11000 rpm, 15 min), and washed with distilled water three times and ethanol three times, and dried to obtain copper ion-doped hollow mesoporous carbon nanoszyme (CHC).

[0045] 500 mg of CHC-supported probe was ultrasonically dispersed in 20 mL of distilled water. 3.42 g of ammonium persulfate was weighed, dissolved in 5 mL of distilled water, and added to the CHC dispersion. Then, 1.67 mL of concentrated sulfuric acid was added dropwise, and the mixture was refluxed at 60 °C with stirring for 5 h. After cooling to room temperature, the reaction solution was centrifuged at 11000 rpm for 15 min and washed twice with distilled water and anhydrous ethanol to obtain a carboxylated copper-doped hollow carbon support (CHC-COOH). 30 mg of CHC-COOH was weighed and dispersed in 30 mL of pH 7.4 PBS. 68 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and approximately 38 mg of N-hydroxysuccinimide were added with stirring. 5 mg of NH2-PEG was added... 2000 Dissolve in 5 mL of pH 7.4 PBS and slowly add to the above reaction solution. Continue to stir the mixture magnetically overnight (400 rpm), centrifuge (11000 rpm, 15 min) to collect the precipitate, and wash the precipitate twice with distilled water and anhydrous ethanol, respectively, to obtain CHC-PEG.

[0046] (2) Characterization of CHC-PEG nanozymes

[0047] The morphology and particle size of CHC nanozymes were observed using transmission electron microscopy, and the results are shown in the attached figure. Figure 1 As shown, the nanozyme exhibits a distinct cavity structure, uniform particle size of approximately 160 nm, and an outer wall thickness of approximately 8-10 nm.

[0048] The particle size and potential of CHC, CHC-COOH, and CHC-PEG nanozymes were measured using dynamic light scattering method. The results are shown in the attached figure. Figure 2 As shown, the CHC-PEG nanozyme has a particle size of approximately 188 nm, a uniform particle size distribution, and a potential of approximately -11.8 mV. This demonstrates the successful preparation of the CHC-PEG nanozyme.

[0049] The surface modification of CHC-PEG nanozymes was analyzed using Fourier transform infrared spectroscopy, and the results are shown in the attached figure. Figure 3 As shown, the C=O absorption peak in CHC-PEG (1710 cm⁻¹) -1 The reduced strength indicates that NH2-PEG has been modified on the surface of CHC nanozyme via amide bonds (-CO-NH2).

[0050] The elemental composition and chemical valence state of the CHC-PEG nanozyme surface were determined by X-ray photoelectron spectroscopy (XPS), and the results are shown in the attached figure. Figure 4 As shown, copper elements are incorporated into CHC-PEG nanozymes in the form of Cu. + and Cu 2+ Two valence states exist, and Cu element accounts for 1.95% of the elemental composition of CHC-PEG nanozyme.

[0051] The blood compatibility of CHC-PEG nanosidase was investigated by in vitro hemolysis experiment, and the results are shown in the following table 1. Figure 5 As shown in the following table 1, the hemolysis percentage of CHC-PEG was only 3.94% at a concentration of 1000 μg / mL, while the hemolysis percentage of CHC was 34.15%, indicating that the modification of NH2-PEG significantly improved the biological safety of nanosidase.

[0052] The photothermal performance of CHC nanosidase was evaluated by irradiating CHC nanosidase with a NIR laser, and the results are shown in the following figure 2. Figure 6 As shown in the following figure 2, the photothermal effect of CHC nanosidase had a power and concentration dependence of NIR laser, and CHC nanosidase still had the same warming effect after repeated irradiation with NIR laser, indicating that CHC nanosidase had good warming stability.

[0053] The POD-like activity of CHC-PEG nanosidase was investigated by TMB, and the results are shown in the following figure 3. Figure 7 As shown in the following figure 3, the POD-like activity of CHC-PEG nanosidase had a concentration dependence, and the Michaelis-Menten equation fitting of the POD-like activity of CP nanosidase with TMB and H2O2 as reaction substrates respectively indicated that CHC-PEG nanosidase had excellent POD-like activity.

[0054] (3) Preparation of HC-PEG nanosidase

[0055] HC-PEG is a PEG-modified hollow mesoporous carbon carrier without doping copper ions, and the preparation method of HC-PEG is the same as that of CHC-PEG, except that no CuCl2·2H2O aqueous solution is added during the preparation process.

[0056] Example 2

[0057] Preparation of IR780 / CHCP nanosidase

[0058] About 10 mg of CHC-PEG and 10 mg of IR780 were weighed and ultrasonically dispersed in 5 mL of methanol, respectively. The methanol solution of IR780 was slowly added to the CHC-PEG dispersion, and stirred at 25°C in the dark overnight, then centrifuged (10000 rpm, 10 min), and the precipitate was collected and washed with methanol until the supernatant had no UV absorption of IR780, to obtain IR780 / CHCP.

[0059] The UV-Vis-NIR absorption spectra of CHC-PEG, IR780 and IR780 / CHCP were scanned by UV spectrophotometer, and the fluorescence spectra of CHC-PEG, IR780 and IR780 / CHCP were scanned by fluorescence spectrometer, and the results are shown in the following figure 4. Figure 8As shown, IR780 has been successfully loaded into the mesoporous channels and cavities of CHC-PEG by adsorption equilibrium method.

[0060] Example 3

[0061] In vitro cytotoxicity of nanozyme on 4T1 tumor cells

[0062] Take 4T1 tumor cells in the logarithmic growth phase, 5 x 10 3 cells / well were seeded in 96-well plates, 5 replicates were set for each group and placed in a 37°C, 5% CO2 cell incubator. After the cells adhered, incubate with DMEM medium containing CHC-PEG, IR780, IR780 / CHCP for 24h. For the NIR laser irradiation group, irradiate the cells with a laser power of 1.0 W / cm 2 for 3min after 6h of incubation. The concentration of IR780 was 0.01, 0.1, 1, 2.5μg / mL and 5μg / mL (the concentrations of the remaining groups were converted from the concentration of the IR780 group). After incubation, add 20μL of 5mg / mL MTT solution to each well and incubate at 37°C, 5% CO2 for 3h. Discard the solution in the 96-well plate, add 150μL of dimethyl sulfoxide solution to each well, shake for 15min on a shaker, and measure the absorbance of each well at 570nm on a microplate reader and calculate the cell survival rate. The results are shown in Figure 2. Figure 9 As shown, CHC-PEG alone does not produce significant cytotoxicity to 4T1 cells, and the IR780 / CHCP nanozyme group significantly reduces the survival rate of 4T1 cells. At the same time, the use of NIR laser irradiation further reduces the survival rate of 4T1 cells.

[0063] Example 4

[0064] In vitro survival study of nanozyme on 4T1 tumor cells

[0065] Take 4T1 tumor cells in the logarithmic growth phase and seed them in 24-well plates with cell slides. After 24h of incubation, add CHC-PEG (50μg / mL) prepared with serum-free medium, and after 2h of incubation, irradiate with NIR light at 1.0 W / cm 2 for 2min, 4min and 6min, respectively, and then incubate for 30min. Add 100μL of Calcein-AM (2μM) fluorescent dye working solution to each well and stain in the dark for 50min. Then add 100μL of PI fluorescent dye (4μM) to each well and stain in the dark for 20min. Use CLSM to observe cell survival. The results are shown in Figure 3. Figure 10As shown, after irradiation with NIR laser, CHC-PEG exhibited good photothermal heating effect, which could generate some heat in 4T1 cells and cause a certain degree of thermal damage to the cells. In addition, the elevated temperature could enhance the POD-like activity of the carrier, thereby producing more ROS to kill tumor cells.

[0066] Example 5

[0067] Intracellular reactive oxygen species production of nanoscale enzyme

[0068] 4T1 tumor cells in the logarithmic growth phase were seeded in 24-well plates with cell slides. After 24 h of incubation, the culture medium was replaced with DMEM medium containing CHC-PEG and IR780 / CHCP and incubated for 2 h. The NIR laser irradiation group (CHC-PEG(+) and IR780 / CHCP(+)) was irradiated with a laser power of 1.0 W / cm 2 for 3 min at the end of incubation. The culture medium was discarded, and the cells were washed with PBS for 3 times. 500 μL of 10 μM DCFH-DA staining solution was added and incubated for 30 min to stain the intracellular reactive oxygen species. After incubation, the cells were washed with PBS for 3 times. 500 μL of 10 μg / mL Hoechst 33258 staining solution was added and incubated for 20 min to stain the cell nucleus. After staining, the cells were washed with PBS for 3 times, mounted with an anti-fluorescence quenching mounting medium, and placed under a confocal laser microscope for fluorescence imaging. The results are shown in FIG. 6. Figure 11 As shown in FIG. 6, the green fluorescence of the CHC-PEG treatment group alone was slightly enhanced compared with the control group. The green fluorescence of the CHC-PEG(+) group was higher than that of the non-irradiation group, indicating that NIR laser irradiation could further enhance the production of intracellular reactive oxygen species. The green fluorescence of the IR780 / CHCP(+) group was higher than that of the other groups, indicating that the IR780 / CHCP(+) nanoscale enzyme had excellent intracellular reactive oxygen species production capacity.

[0069] Example 6

[0070] Intracellular glutathione (GSH) depletion of nanoscale enzyme

[0071] The 4T1 tumor cells in the logarithmic growth phase were inoculated in a 24-well plate with cell climbing sheets, incubated for 24 h, and then incubated with DMEM medium containing IR780, HC-PEG and CHC-PEG for 4 h. After incubation, the medium was discarded, washed with PBS for 3 times, 500 μL of 75 μM naphthalene-2,3-dimethyl formaldehyde (NDA) was added and incubated in the dark for 30 min, then washed with PBS for 3 times, 500 μL of 10 μg / mL Hoechst 33258 dye solution was added and incubated for 25 min to stain the nucleus. After staining, the cells were washed with PBS for 3 times, sealed with an anti-fluorescence quenching sealing solution, and placed under a confocal laser microscope for fluorescence photography. The results are shown in FIG. 6. Figure 12 As shown in FIG. 6, the IR780 alone group had little effect on the intracellular GSH content, and the GSH content of the nanoenzyme treatment group showed different degrees of reduction, and the green fluorescence intensity in the CHC-PEG group was the lowest, indicating that the metal-doped nanoenzyme could effectively deplete the intracellular GSH.

[0072] Example 7

[0073] Nanoenzyme for mouse tumor treatment

[0074] The 4T1 tumor cells in the logarithmic growth phase were inoculated in a 24-well plate with cell climbing sheets, incubated for 24 h, and then incubated with DMEM medium containing IR780, HC-PEG and CHC-PEG for 4 h. After incubation, the medium was discarded, washed with PBS for 3 times, 500 μL of 75 μM naphthalene-2,3-dimethyl formaldehyde (NDA) was added and incubated in the dark for 30 min, then washed with PBS for 3 times, 500 μL of 10 μg / mL Hoechst 33258 dye solution was added and incubated for 25 min to stain the nucleus. After staining, the cells were washed with PBS for 3 times, sealed with an anti-fluorescence quenching sealing solution, and placed under a confocal laser microscope for fluorescence photography. The results are shown in FIG. 6. 6 The 4T1 tumor cells in the logarithmic growth phase were inoculated in a 24-well plate with cell climbing sheets, incubated for 24 h, and then incubated with DMEM medium containing IR780, HC-PEG and CHC-PEG for 4 h. After incubation, the medium was discarded, washed with PBS for 3 times, 500 μL of 75 μM naphthalene-2,3-dimethyl formaldehyde (NDA) was added and incubated in the dark for 30 min, then washed with PBS for 3 times, 500 μL of 10 μg / mL Hoechst 33258 dye solution was added and incubated for 25 min to stain the nucleus. After staining, the cells were washed with PBS for 3 times, sealed with an anti-fluorescence quenching sealing solution, and placed under a confocal laser microscope for fluorescence photography. The results are shown in FIG. 6. 3 The mice were randomly divided into Control, CHC-PEG, IR780(+), CHC-PEG(+), IR780 / CHCP(+) groups after the tumor volume of the mice was about 100 mm Figure 13 As shown in FIG. 6, the IR780 alone group had little effect on the intracellular GSH content, and the GSH content of the nanoenzyme treatment group showed different degrees of reduction, and the green fluorescence intensity in the CHC-PEG group was the lowest, indicating that the metal-doped nanoenzyme could effectively deplete the intracellular GSH.

[0075] The above is only the best specific embodiment of the present application, but the scope of protection of the present application is not limited to this. Any modification, equivalent replacement and improvement made by any person skilled in the art within the technical range disclosed by the present application shall be included in the protection scope of the present application.

Claims

1. A metal-doped hollow mesoporous carbon nanotube enzyme antitumor delivery system, characterized in that, The antitumor delivery system consists of metal ion-doped nanozymes, photosensitizers, and biocompatible groups.

2. The metal-doped hollow mesoporous carbon nanotube enzyme antitumor delivery system according to claim 1, characterized in that, The metal ion-doped nanozyme is formed by the oxidative polymerization of metal ions and dopamine, wherein the metal ion is Cu. 2+ Mn 2+ Mg 2+ Fe 3+ Fe 2+ Zn 2+ Ag + One of them.

3. The metal-doped hollow mesoporous carbon nanotube enzyme antitumor delivery system according to claim 1, characterized in that, The photosensitizer is one of IR780, dihydroporphyrin E6, pyromethoxyphylla α-hexyl ether, and chlorophyll-a.

4. The metal-doped hollow mesoporous carbon nanotube enzyme antitumor delivery system according to claim 1, characterized in that, The biocompatible group is amino-modified polyethylene glycol. 2000 One of hyaluronic acid and folic acid.

5. A method for preparing the metal-doped hollow mesoporous carbon nanotube enzyme antitumor delivery system according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Tetraethyl orthosilicate was added to a mixed solution of ethanol and water, and ammonia was added to adjust the pH of the solution to be alkaline. After stirring for a period of time, the solid silica nanoparticles were obtained by centrifugation. (2) The silica nanoparticles obtained in step (1) are uniformly mixed with ammonia, dopamine hydrochloride and metal ions, centrifuged and dried. The dried product is carbonized under nitrogen protection, hydrofluoric acid is added to remove residual silica, and after centrifugation and drying, metal-doped hollow mesoporous carbon nanozymes are obtained. (3) The metal-doped hollow mesoporous carbon nanozymes obtained in step (2) are subjected to surface carboxylation treatment, and then co-incubated with a biocompatible group solution to obtain biocompatible group-modified hollow mesoporous carbon nanozymes. (4) The hollow mesoporous carbon nanozyme modified with the biocompatible group obtained in step (3) is co-incubated with a photosensitizer to obtain a drug-loaded hollow mesoporous carbon nanozyme.

6. The preparation method according to claim 5, characterized in that, In step (1), the volume ratio of tetraethyl orthosilicate to the mixed solution of ethanol and water is 1:25 to 1:75, the pH value of the alkaline environment is 8 to 10, the volume ratio of water to ethanol is 1:1 to 1:5, and the reaction time is 1 to 5 hours.

7. The preparation method according to claim 5, characterized in that, In step (2), the molar ratio of the metal ion to dopamine hydrochloride is 0.5:1 to 1.5:1, and the metal ion is Cu. 2+ The heating rate for carbonization is 1-10℃ / min, the carbonization temperature is 500-900℃, and the concentration of hydrofluoric acid is 1%-25%.

8. The preparation method according to claim 5, characterized in that, In step (3), the mass ratio of the biocompatible group to the carboxylated metal-doped hollow mesoporous carbon nanozyme is 1:1 to 1:10, and the reaction time is 8 to 24 hours.

9. The preparation method according to claim 5, characterized in that, In step (4), the mass ratio of the photosensitizer to the biocompatible group-modified hollow mesoporous carbon nanozyme is 0.5:1 to 5:1, the reaction time is 8 to 24 hours, and the photosensitizer is IR780.

10. The metal-doped hollow mesoporous carbon nanozyme antitumor delivery system according to any one of claims 1-4 is used in the preparation of a system that improves the redox microenvironment of tumor tissue and achieves synergistic application of enzyme-like catalysis / photothermal therapy / photodynamic therapy.

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