Optical treatment nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses an optical treatment nano-enzyme as well as a preparation method and application thereof. The preparation of a mesoporous silicon-loaded Cu9S5 nano composite material is taken as a main body, and CD47 antibody modification is carried out through electrostatic adsorption to obtain the optical treatment nano enzyme. On one hand, a CD47 receptor on the surface of a tumor cell can be specifically targeted through the anti-CD47 antibody, a CD47-SIRP alpha signal channel is blocked, and the phagocytic ability of macrophages to the tumor cell is recovered; and on the other hand, the nano-enzyme has photo-thermal conversion capacity and multiple enzyme activity, can realize fixed-point inhibition on in-situ tumors through near-infrared light irradiation, induces tumor cells to generate immunogenic cell death and increases exposure of endogenous CALR, so that phagocytosis of macrophages on the tumor cells is further enhanced, the immunotherapy effect is enhanced, and the immunotherapy effect is enhanced. The tumor metastasis is inhibited. The prepared nano enzyme for optical therapy can be applied to biomedical research.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a phototherapy nanozyme and a preparation method and application thereof. Background Art

[0002] Macrophages are a crucial component of the human innate immune system and serve as the body's first line of defense against invading foreign pathogens. When pathogens invade the body, macrophages recognize them through surface receptors, phagocytize and digest them, and then present them to T lymphocytes through antigen presentation. Macrophages, in turn, produce large quantities of inflammatory cytokines to regulate adaptive immunity. Thus, macrophage phagocytosis initiates the innate immune response, which in turn activates the adaptive immune response, playing a crucial role in mobilizing the body's immune response. Therefore, targeting macrophages and modulating their phenotype and function to enhance tumor immunotherapy efficacy has attracted increasing attention in tumor immunotherapy. However, within tumor tissue, tumor cells can evade macrophage recognition and phagocytosis through various pathways. One such mechanism is the upregulation of surface CD47 expression, which allows it to bind to the macrophage surface ligand signal regulatory protein α (SIRPα), transmitting an inhibitory "don't eat me" signal to the macrophage, thereby inhibiting macrophage phagocytosis and enabling immune escape. Therefore, using anti-CD47 or anti-SIRPα antibodies to block the CD47-SIRPα signaling pathway and inhibit the "don't eat me" signal is believed to enhance the ability of macrophages to recognize and phagocytose tumor cells, thereby improving the effectiveness of tumor immunotherapy.

[0003] However, macrophage phagocytosis is influenced not only by the "Don't eat me" signal provided by CD47 but also by the "Eat me" signal provided by calreticulin (CALR) expressed on the surface of tumor cells. CALR expressed on tumor cells binds to low-density lipoprotein receptor-related protein 1 (LRP1) on the surface of macrophages, providing the "Eat me" pro-phagocytic signal, thereby promoting macrophage phagocytosis of tumor cells. To maximize macrophage phagocytosis of tumor cells, in addition to blocking the CD47-SIRPa pathway, it is also necessary to enhance the "Eat me" signal provided by CALR. The synergistic effect of these two approaches can maximize the regulation of macrophage phagocytosis and improve anti-tumor efficacy. Studies have shown that phototherapy (such as photothermal therapy and photodynamic therapy) and reactive oxygen species (ROS) can increase endogenous CALR exposure by inducing immunogenic cell death in tumor cells. Therefore, the development of materials with both phototherapy and ROS generation capabilities is crucial for improving macrophage phagocytosis and exploring their applications in tumor immunotherapy. Summary of the Invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing a phototherapeutic nanozyme. This method utilizes nanotechnology to prepare a Cu9S5 nanocomposite material loaded with ultra-large mesoporous silica to obtain a phototherapeutic carrier loaded with anti-CD47 antibodies to construct a phototherapeutic nanozyme.

[0005] Another object of the present invention is to provide a phototherapeutic nanozyme obtained by the above preparation method.

[0006] Another object of the present invention is to provide applications of the above-mentioned phototherapeutic nanozyme.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] A method for preparing a phototherapeutic nanozyme comprises the following steps:

[0009] (1) Preparation of mesoporous silica-loaded Cu9S5 nanocomposite materials: synthesizing mesoporous silica nanoparticles, and then loading Cu9S5 into the pores or surface of mesoporous silica to obtain mesoporous silica-loaded Cu9S5 nanocomposite materials;

[0010] (2) Preparation of phototherapeutic nanozymes: The mesoporous silica-loaded Cu9S5 nanocomposite material obtained in step (1) is functionalized with polyethylene glycol and then modified with anti-CD47 antibodies through electrostatic adsorption to obtain phototherapeutic nanozymes.

[0011] The average particle size of the mesoporous silicon in step (1) is 300-400 nm, and the specific surface area is 450-550 m 2 / g, pore volume of 1.5~1.7cm 3 / g; preferably prepared by the following steps:

[0012] 1) uniformly mixing cetyltrimethylammonium bromide, urea and water to obtain solution A;

[0013] 2) mixing cyclohexane and ethyl orthosilicate to obtain solution B;

[0014] 3) adding solution B to solution A to react; after the reaction is completed, performing solid-liquid separation, washing the solid, extracting and refluxing to obtain mesoporous silicon.

[0015] In the reaction system described in step 3), hexadecyltrimethylammonium bromide, urea, water, cyclohexane and ethyl orthosilicate are preferably mixed in a ratio of 0.8-1.2 g: 0.2-0.4 g: 13-17 mL: 13-17 mL: 1.5-2.5 mL; more preferably in a ratio of 1 g: 0.3 g: 15 mL: 15 mL: 2 mL.

[0016] The reaction conditions in step 3) are preferably reacting in an oil bath at 60-80° C. for 15-18 h; more preferably reacting in an oil bath at 70° C. for 16 h.

[0017] The method for solid-liquid separation in step 3) is preferably centrifugation.

[0018] The centrifugal conditions are preferably 8000-12000 rpm for 5-15 min; more preferably 10000 rpm for 10 min.

[0019] The washing operation in step 3) is preferably performed by alternately washing with anhydrous ethanol and deionized water for 1 to 3 times.

[0020] The extraction reflux operation in step 3) is preferably performed by extraction reflux with a methanol solution containing NaCl.

[0021] The concentration of NaCl in the methanol solution containing NaCl is preferably 0.05 to 1.5 g / mL, and more preferably 0.08 g / mL.

[0022] The number of extraction reflux in step 3) is preferably 1 to 3 times.

[0023] The Cu9S5 in step (1) is preferably Cu9S5 nanoparticles with an average particle size of 5 to 50 nm, which are preferably prepared by the following steps:

[0024] A. Stir cuprous chloride, oleylamine, and oleic acid in an oil bath at 132-140° C. for 10-30 minutes and mix well; then cool to room temperature under nitrogen to obtain a copper precursor;

[0025] B. Stir sulfur powder and octadecene in an oil bath at 202-210° C. for 10-30 minutes until uniform; when cooled to 180-185° C. with nitrogen, quickly inject the copper precursor obtained in step A to react;

[0026] C. After the reaction is completed, nitrogen is passed through and cooled to room temperature, and then acetone is added to precipitate it. The solid-liquid separation is performed, and the obtained solid is washed with anhydrous ethanol to obtain Cu9S5 nanoparticles.

[0027] The cuprous chloride, oleylamine and oleic acid described in step A are preferably mixed in a mass ratio of 1:3-5:4-6; more preferably in a mass ratio of 1:4:5.

[0028] The stirring reaction time in step A is preferably 20 minutes.

[0029] The room temperature in step A is preferably 10-35°C; more preferably 20-30°C; and most preferably 24-26°C.

[0030] The sulfur powder and octadecene described in step B are preferably mixed in a ratio of 0.32 g: 30-50 mL; more preferably in a ratio of 0.32 g: 40 mL.

[0031] The amount of the copper precursor in step B is preferably such that the mass ratio of the reaction raw materials, cuprous chloride and sulfur powder, is 1:0.3-0.4; preferably, the mass ratio of the reaction raw materials, cuprous chloride and sulfur powder, is 1:0.32.

[0032] The amount of acetone used in step C is preferably the same as the volume of octadecene in step B.

[0033] The solid-liquid separation method in step C is preferably centrifugation.

[0034] The centrifugal condition is preferably centrifuged at 10000 rpm for 10 min.

[0035] The number of washing steps in step C is preferably 1 to 3 times.

[0036] The steps of loading described in step (1) are preferably as follows:

[0037] 1) dispersing mesoporous silica in an organic solvent to obtain a dispersion A;

[0038] 2) dispersing Cu9S5 nanoparticles in an organic solvent to obtain dispersion B;

[0039] 3) Mixing dispersion A and dispersion B, stirring and reacting;

[0040] 4) After the reaction is completed, the solid and liquid are separated, and the obtained solid is washed to obtain a mesoporous silicon-loaded Cu9S5 nanocomposite material.

[0041] The organic solvent in step 1) is preferably anhydrous ethanol.

[0042] The amount of the organic solvent used in step 1) is preferably calculated based on the concentration of mesoporous silica being 0.08 to 0.3 g / mL.

[0043] The organic solvent in step 2) is preferably cyclohexane.

[0044] The amount of the organic solvent used in step 2) is preferably calculated based on the concentration of Cu9S5 nanoparticles being 0.08 to 0.15 g / mL.

[0045] The amounts of dispersion A and dispersion B described in step 3) are preferably mixed according to a mass ratio of mesoporous silicon to Cu9S5 nanoparticles of 1:1 to 1.5.

[0046] The stirring reaction conditions in step 3) are preferably 400-500 rpm for 24-48 h; more preferably 450 rpm for 36 h.

[0047] The specific steps of step (2) are preferably as follows:

[0048] ① Dispersing the mesoporous silica-loaded Cu9S5 nanocomposite obtained in step (1) in an organic solvent, adding polyethylene glycol modified by a silane coupling agent, stirring the reaction, and performing solid-liquid separation to obtain a solid which is a polyethylene glycol-modified mesoporous silica-loaded Cu9S5 nanocomposite;

[0049] ② Disperse the polyethylene glycol-modified mesoporous silica-loaded Cu9S5 nanocomposite material obtained in step ① in water, add anti-CD47 antibody, mix evenly, incubate at 2-8°C, separate the solid and liquid, and obtain a solid as the optical therapeutic nanozyme.

[0050] The organic solvent described in step ① is preferably anhydrous alcohol.

[0051] The amount of the organic solvent in step ① is preferably calculated based on the concentration of the mesoporous silica-loaded Cu9S5 nanocomposite material of 0.005 to 0.015 g / mL; more preferably, the concentration of the mesoporous silica-loaded Cu9S5 nanocomposite material is 0.01 g / mL.

[0052] The amount of the silane coupling agent-modified polyethylene glycol described in step ① is preferably calculated based on 0.08 to 0.15 times the mass of the mesoporous silica-loaded Cu9S5 nanocomposite material; more preferably calculated based on 0.1 times the mass of the mesoporous silica-loaded Cu9S5 nanocomposite material.

[0053] The stirring reaction time in step ① is preferably 2 to 4 hours; more preferably 3 hours.

[0054] The method of solid-liquid separation described in step ① is preferably centrifugation.

[0055] The centrifugal condition is preferably centrifugal at 10000 rpm for 10 minutes.

[0056] The polyethylene glycol-modified mesoporous silica-loaded Cu9S5 nanocomposite material and the anti-CD47 antibody described in step ② are preferably mixed in a mass ratio of 5 to 40:1.

[0057] The incubation time in step ② is preferably 10 to 15 hours, more preferably 12 hours.

[0058] The solid-liquid separation method in step ② is preferably centrifugation.

[0059] The centrifugal condition is preferably centrifugal at 10000 rpm for 10 minutes.

[0060] A phototherapy nanozyme is obtained by the above preparation method. The phototherapy nanozyme has photothermal conversion performance. Under the irradiation of a laser with a wavelength of 650nm to 1350nm, the illumination condition is preferably 0.3W / cm 2 Up to 1W / cm 2 The temperature can be raised to no less than 45°C within 10 minutes; the phototherapeutic nanozyme has peroxidase activity and can catalyze the production of various reactive oxygen species, such as hydroxyl radicals, singlet oxygen, and superoxide anions; the phototherapeutic nanozyme has peroxidase activity and can consume glutathione.

[0061] The application of the above-mentioned phototherapeutic nanozymes in the preparation of immunotherapeutic drugs that regulate macrophage phagocytosis and sensitize the immune system.

[0062] The present invention has the following advantages and effects compared to the prior art:

[0063] 1. The phototherapeutic nanozyme prepared by the present invention uses mesoporous silica-loaded Cu9S5 nanocomposite as a carrier to load anti-CD47 antibody (aCD47). The loading amount of aCD47 can be controlled by adjusting the pore size of the mesoporous silica. Moreover, this antibody loading mediated by electrostatic interaction can better maintain its biological function unaffected compared with chemical modification.

[0064] 2. The phototherapeutic nanozyme prepared by the present invention has both photothermal conversion ability and dual enzyme-mimicking activity, which can mediate photothermal therapy and the production of reactive oxygen species, thereby increasing the exposure of endogenous CALR.

[0065] 3. The phototherapeutic nanozyme prepared in this invention has the advantage of dual regulation, restoring the phagocytic function of macrophages through the synergistic action of aCD47 and CALR. Compared with the traditional single CD47 blocking method, this strategy more effectively activates the immune system and improves the therapeutic effect.

[0066] 4. The optical therapeutic nanozyme prepared by the present invention has precise targeting ability and can specifically target the CD47 receptor on the surface of tumor cells, which improves the accuracy of treatment and reduces the impact on normal cells.

[0067] 5. The phototherapeutic nanozyme prepared by the present invention has the effect of phototherapy sensitizing immunotherapy. On the one hand, it has phototherapy function. Through near-infrared light irradiation, it can achieve targeted inhibition of in situ tumors, while inducing immunogenic cell death in tumor cells, increasing the exposure of endogenous CALR, and enhancing the immunotherapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is a transmission electron microscope image of the mesoporous silicon prepared in Example 1.

[0069] Figure 2This is the nitrogen adsorption-desorption isotherm diagram of the mesoporous silicon prepared in Example 1.

[0070] Figure 3 This is a transmission electron microscope image of Cu9S5 prepared in Example 1.

[0071] Figure 4 Transmission electron microscope image (a) and element distribution map (b) of the phototherapeutic nanozyme CMC prepared in Example 1.

[0072] Figure 5 The graph shows the adsorption rate of CM on aCD47 at different CM and aCD47 (w:w) feed ratios.

[0073] Figure 6 This is the transmission electron microscope image of MSN@Cu9S5-2 prepared in Comparative Example 1.

[0074] Figure 7 (a) The heat transfer time constant curve and (b) thermal stability test diagram of the phototherapy nanozyme CMC prepared in Example 1; wherein, the oblique straight line in a is the cooling fitting curve.

[0075] Figure 8 Figure 1 shows the test results of the phototherapy nanozyme CMC's catalytic reactive oxygen species production ability (a) and catalytic glutathione ability (b). The curves in a are 0 min, 2 min, 4 min, 6 min, 8 min, 10 min, 12 min, 14 min and 16 min from bottom to top; the curves in b are 0 μg / mL, 50 μg / mL, 100 μg / mL and 150 μg / mL from top to bottom.

[0076] Figure 9 This is a statistical graph of the average fluorescence intensity of the PBS, CM, CMC, and pre-blocking groups in testing their targeting ability to 4T1 tumor cells.

[0077] Figure 10 Statistical graph of the average fluorescence intensity of exogenous calreticulin expressed on the surface of 4T1 cells after 4T1 tumor cells were treated with PBS, NIR, CMC, CM+NIR, and CMC+NIR groups.

[0078] Figure 11 The bar graph shows the test results of the ability of macrophages to phagocytose tumor cells induced by different particles.

[0079] Figure 12 For CMC and MSN aCD47&CALR Histogram of the ability of macrophages to mediate phagocytosis of tumor cells.

[0080] Figure 13 (a) Line graph of mouse tumor volume and (b) line graph of weight change.

[0081] Figure 14 It is a line graph of mouse tumor volume.

[0082] Figure 15 CD8 + T cells account for CD45 + Histogram of cell percentages.

[0083] Figure 16 Photos and HE staining of the lungs in different experimental groups. DETAILED DESCRIPTION

[0084] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0085] Example 1:

[0086] A method for preparing a phototherapeutic nanozyme comprises the following steps:

[0087] (1) Preparation of ultra-large mesoporous silica-loaded Cu9S5 nanocomposites (MSN@Cu9S5)

[0088] 1) Preparation of ultra-large mesoporous silica nanoparticles: 1 g of hexadecyltrimethylammonium bromide, 0.3 g of urea, and 15 mL of water were mixed and stirred at room temperature for 30 minutes until uniform. A mixed solution of 15 mL of cyclohexane and 2 mL of ethyl orthosilicate (liquid, analytical grade) was then added and reacted in a 70°C oil bath for 16 hours. The supernatant was discarded after centrifugation at 10,000 rpm for 10 minutes. The mixture was washed alternately with anhydrous ethanol and deionized water three times, and then extracted and refluxed three times with a methanol solution containing NaCl (0.16 g of NaCl dispersed in 20 mL of methanol) to obtain mesoporous silica nanoparticles. The resulting mixture was dispersed in 10 mL of ethanol (liquid, analytical grade) to obtain an anhydrous ethanol solution of mesoporous silica nanoparticles for later use.

[0089] Transmission electron microscopy images of prepared mesoporous silica nanoparticles are shown in Figure 2. Figure 1 As shown: The average particle size of the prepared mesoporous silica nanoparticles is about 320nm, and they are uniformly spherical. Using a specific surface area microporous tester, it can be measured that the specific surface area of ​​the prepared mesoporous silica nanoparticles is 506.33m 2 / g, pore volume 1.61cm 3 / g (as shown in Table 1 and Figure 2 Its large specific surface area and mesoporous structure are conducive to the subsequent loading of photothermal materials and antibodies.

[0090] Table 1

[0091] <![CDATA[Surface area (m 2 / g)]]> <![CDATA[Pore volume (cm 3 / g)]]> MSN 506.33 1.61 <![CDATA[MSN@Cu9S5]]> 244.10 0.69

[0092] 2) Preparation of Cu9S5 Nanocrystals: 1g of cuprous chloride (solid, analytical grade), 4g of oleylamine (liquid, analytical grade), and 5g of oleic acid (liquid, analytical grade) were stirred in an oil bath at 132-140°C for 20 minutes until uniform. The mixture was cooled to room temperature under nitrogen to obtain a copper precursor. Subsequently, 0.32g of sulfur powder (solid, analytical grade) and 40mL of octadecene (liquid, analytical grade) were stirred in an oil bath at 202-210°C for 20 minutes until uniform. After cooling to 182°C under nitrogen, the mixture was rapidly injected into the copper precursor prepared above and allowed to react for 10 minutes. After the reaction was completed, the mixture was cooled to room temperature under nitrogen. 40mL of acetone was then added to precipitate the mixture. The mixture was centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, and the mixture was washed three times with anhydrous ethanol to obtain Cu9S5 nanoparticles. The mixture was dispersed in 10mL of cyclohexane (liquid, analytical grade) to obtain a cyclohexane solution of Cu9S5 nanoparticles for later use.

[0093] Transmission electron microscopy images of the prepared Cu9S5 nanoparticles are shown in Figure 2. Figure 3 As shown: the average particle size of the prepared Cu9S5 nanoparticles is about 10 nm.

[0094] 3) 10 mL of the anhydrous ethanol solution of the mesoporous silica nanoparticles obtained in step 1) was placed in an ultrasonic cleaner (Jiemeng brand ultrasonic cleaner, 180w) for ultrasonic dispersion for 30 minutes, and then 10 mL of the cyclohexane solution of the Cu9S5 nanoparticles prepared in step 2) was added. The mixture was stirred at 450 rpm for 36 hours. After the reaction was completed, the mixture was washed with anhydrous ethanol by centrifugation (9000 rpm, 10 minutes) to obtain MSN@Cu9S5 nanoparticles.

[0095] Transmission electron microscopy images of the prepared MSN@Cu9S5 are shown in Figure 2. Figure 4 As shown: the cavity of the prepared MSN@Cu9S5 is evenly distributed with Cu9S5 nanoparticles. The element distribution results show that MSN@Cu9S5 is mainly composed of three elements: Cu, S, and Si.

[0096] The specific surface area of ​​the mesoporous silica nanoparticles was measured by a surface area microporous tester. The specific surface area of ​​the mesoporous silica nanoparticles was reduced to 244.10 m 2 / g, the pore volume is reduced to 0.69 cm 3 / g (as shown in Table 1 and Figure 2 As shown), it shows that a large number of Cu9S5 nanoparticles are loaded on the surface or in the pores of mesoporous silicon, which lays the foundation for the subsequent preparation of phototherapeutic nanozymes with high photothermal conversion performance and enzyme activity (see effect verification examples 1 and 2 for details).

[0097] (2) Preparation of phototherapeutic nanozymes:

[0098] Contains PEG 2000 - Preparation of ethanol solution of Saline: Prepared with reference to Example 1 "Preparation of silane coupling agent modified polyethylene glycol (PEG2000-Saline)" in the Chinese patent "CN201911042030.8-A kind of azide group modified Janus nanoparticles and its preparation method and application".

[0099] Disperse 0.1 g of MSN@Cu9S5 in 10 mL of anhydrous ethanol and add 10 mL of PEG 2000 -Saline ethanol solution (1 mg / mL) was mixed and stirred at room temperature for 3 hours, and the upper reaction liquid was discarded after centrifugation at 10000 rpm for 10 minutes to obtain PEG-MSN@Cu9S5 nanoparticles (abbreviated as CM).

[0100] The CM nanoparticles were dissolved in 9 mL of ultrapure water and then evenly mixed with Anti-CD47 antibody (purchased from Bioxcell, product number: BE0270). The mass ratio of Anti-CD47 antibody to PEG-MSN@Cu9S5 was 1:5 to 1:40. The mixture was incubated at 4°C for 12 hours. After centrifugation at 10,000 rpm for 10 minutes, the upper reaction liquid was discarded to obtain phototherapeutic nanozyme (abbreviated as CMC).

[0101] Determination of Anti-CD47 Antibody Loading on CMC Surface: CM nanoparticles were dissolved in 9 mL of ultrapure water and then uniformly mixed with Anti-CD47 antibody (purchased from Bioxcell, Catalog No. BE0270). The mass ratios of Anti-CD47 to PEG-MSN@Cu9S5 were 1:5, 1:10, 1:20, and 1:40, respectively. The mixture was incubated at 4°C for 12 hours. The supernatant was centrifuged at 10,000 rpm for 10 minutes, and the free aCD47 concentration in the supernatant was determined by the BCA assay. The steps for testing protein content by the BCA assay are as follows:

[0102] a. Use the protein standard included in the kit to create a standard curve according to the procedure. The BCA kit includes a protein standard whose absorbance at 562 nm shows a linear relationship with protein concentration within a certain concentration range. Dilute the protein standard into a gradient concentration range, then measure the absorbance using a microplate reader and the BCA kit, and fit a standard curve to the protein mass.

[0103] b. Treat the supernatant after the reaction according to the above steps, measure the absorbance at 562 nm using a microplate reader, and then apply it to the standard curve for verification.

[0104] The adsorption efficiency of the antibody was then calculated using the subtraction method, i.e., the amount of aCD47 loaded on the CM surface = the total amount of aCD47 input - the amount of free aCD47 in the supernatant. Figure 5 As shown in the figure, the adsorption efficiency of aCD47 is high. At various feed ratios, almost 100% of aCD47 can be adsorbed onto the mesoporous silica surface.

[0105] Example 2

[0106] The preparation of a FITC-labeled phototherapeutic nanozyme (CMC-FITC) comprises the following steps:

[0107] (1) The CM nanoparticles obtained in Example 1 were dissolved in 1 mL of carbonate buffer (0.1 M, pH = 9) and ultrasonically dispersed in an ultrasonic cleaner (Jiemeng brand ultrasonic cleaner, 180W) for 10 minutes. 100 μL of 0.07 mg / mL FITC solution was added and stirred at room temperature for 6 hours. After the reaction was completed, the unreacted FITC was removed by centrifugation to obtain FITC-labeled CM nanoparticles (CM-FITC).

[0108] (2) The CM-FITC nanoparticles obtained in step 1) were dissolved in 9 mL of ultrapure water and then evenly mixed with Anti-CD47 (purchased from Bioxcell, product number: BE0270) at a mass ratio of 1:5. The mixture was incubated at 4°C for 12 hours, centrifuged at 10,000 rpm for 10 minutes, and the upper reaction liquid was discarded to obtain phototherapy nanozyme (abbreviated as CMC-FITC).

[0109] The preparation of FITC-labeled phototherapeutic nanozymes is mainly to verify that the prepared phototherapeutic nanozymes can specifically target the surface of tumor cells.

[0110] Comparative Example 1

[0111] The preparation of mesoporous silica-supported Cu9S5 nanocomposite material 2 (MSN@Cu9S5-2) includes the following steps:

[0112] 1) Preparation of ultra-large mesoporous silica nanoparticles: 1 g of hexadecyltrimethylammonium bromide, 0.3 g of urea, and 15 mL of water were mixed and stirred at room temperature for 30 minutes until uniform. A mixed solution of 10 mL of cyclohexane and 2 mL of ethyl orthosilicate (liquid, analytical grade) was then added and reacted in a 70°C oil bath for 16 hours. The supernatant was discarded after centrifugation at 10,000 rpm for 10 minutes. The mixture was washed three times with anhydrous ethanol and deionized water, and then refluxed with ammonium nitrate three times to obtain mesoporous silica nanoparticles. The particles were then dispersed in 10 mL of ethanol (liquid, analytical grade) for later use.

[0113] 2) The preparation process of Cu9S5 nanocrystals is the same as that of Example 1.

[0114] 3) 10 mL of the anhydrous ethanol solution (aqueous phase) of the mesoporous silica nanoparticles obtained in step 1) was ultrasonically dispersed in an ultrasonic cleaner (Jiemeng brand ultrasonic cleaner, 180W) for 30 minutes, and then 10 mL of the cyclohexane solution (oil phase) of the Cu9S5 nanoparticles prepared in step 2) was added. The mixture was stirred at 450 rpm for 36 hours. After the reaction, the mixture was washed with anhydrous ethanol by centrifugation (9000 rpm, 10 minutes) to obtain MSN@Cu9S5 nanoparticles.

[0115] The transmission electron microscopy images of the prepared CMC are shown in Figure 2. Figure 6 As shown in the figure: the prepared Cu9S5 nanoparticles cannot be evenly dispersed in the cavity structure of CMC, indicating that the distribution of Cu9S5 in the pores can be effectively improved by changing the template cleaning agent in the mesoporous silica and adjusting the ratio of the water and oil phases.

[0116] Comparative Example 2

[0117] Double antibody modified mesoporous silica (MSN aCD47&CALR )

[0118] The mesoporous silica particles (i.e., CM, PEG-MSN@Cu9S5 nanoparticles) prepared in Example 1 were dispersed in 1.5 mL of water, and then anti-CD47 antibody (purchased from Bioxcell, catalog number: BE0270) and calreticulin CALR (purchased from Abcam) were added and evenly mixed. The mass ratio of anti-CD47, CALR and PEG-MSN@Cu9S5 was 1:1:5, respectively. The mixture was incubated at 4°C for 12 hours, and the precipitate was collected after centrifugation at 10,000 rpm for 10 minutes to obtain MSN. aCD47&CALR .

[0119] MSN aCD47&CALR The preparation is mainly to verify that the prepared optical therapeutic nanozyme can induce macrophages to phagocytose tumor cells more effectively than simply using chemically modified two antibodies.

[0120] Effect verification

[0121] Effect Verification Example 1: CMC Photothermal Performance Test

[0122] 1 mL of CMC aqueous solution (concentration of 200 μg / mL) was placed in a transparent container and irradiated with a 1064 nm laser (Changchun New Industries) (0.50 W / cm 2), record the temperature rise with an infrared thermal imager (FLIR), and after reaching the highest temperature and stabilizing, turn off the laser until the CMC aqueous solution returns to room temperature, and record its cooling process. Repeat three times. The CMC photothermal conversion efficiency η is calculated using formula (1-1):

[0123]

[0124] Among them, T max Indicates the maximum temperature; T surr Indicates ambient temperature; Q dis It represents the heat loss caused by the container absorbing light, and its value is about 0mW; I represents the laser power density; A 808 represents the absorbance of the sample at 1064 nm; h represents the heat transfer coefficient; s represents the surface area of ​​the container. The hs value can be obtained by fitting the cooling curve (see Figure 7 The inclined straight line of a in (1-2) is obtained by formula (1-2):

[0125]

[0126] in, τ s is the time constant, T is the temperature of CMC aqueous solution, m D and c D is the mass of the solvent (1 g) and the heat capacity (4.2 J g -1 ℃ -1 ).

[0127] Figure 7 The curve of temperature change of CMC aqueous solution under 1064nm laser irradiation is shown as follows. Figure 7 As can be seen from a, after irradiation of CMC aqueous solution with 1064nm laser for 600s, the temperature reaches the maximum value and tends to be stable, and its photothermal conversion efficiency η is 50.27%. Figure 7 As can be seen from b, the CMC aqueous solution can be repeatedly heated and cooled, and has good photothermal stability.

[0128] Effect verification example 2: CMC simulated enzyme activity test

[0129] (1) Peroxidase (POD) activity test of CMC: O-phenylenediamine (OPD) was used as an indicator to detect the nanozyme activity of CMC at the solution level to determine its ability to produce reactive oxygen species (ROS). OPD is easily oxidized by various oxidants (such as reactive oxygen species) to produce the yellow fluorescent substance 2,3-diaminophenazine (OPDox). CMC (final concentration in the reaction system is 50 μg / mL), OPD (final concentration in the reaction system is 1 mM) and H2O2 (final concentration in the reaction system is 10 mM) were added to a 0.01 M PBS solution with a pH value of 6.5. After mixing evenly, the absorbance at 420 nm was measured using a UV-visible spectrometer at 0, 2, 4, 6, 8, 10, 12, 14 and 16 minutes.

[0130] The ability of CMC to catalyze the generation of reactive oxygen species, such as Figure 8 As shown in a: As the reaction time increases, the absorption value at 420 nm gradually increases, indicating that CMC has the ability to catalyze the production of ROS.

[0131] (2) Glutathione mimetic activity test of CMC: CMC was added to a 1.2 mM aqueous solution of glutathione GSH. The concentrations of CMC were 0, 50, 100, and 150 μg / mL, respectively. After 30 seconds of reaction, the mixture was centrifuged at 10,000 rpm and 4°C for 10 minutes. The supernatant was taken and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 50 μg mL) dissolved in PBS (pH = 6.5) was added. -1 ) solution for color development reaction, and the absorption value at 410 nm was detected using an ultraviolet-visible spectrometer, wherein the maximum absorption wavelength was 410 nm.

[0132] The ability of CMC to catalyze glutathione Figure 8 As shown in b: CMC has excellent glutathione consumption ability, which can destroy the cell's antioxidant defense system, thereby further enhancing the effect of reactive oxygen species.

[0133] Effect Verification Example 3: CMC targeting ability test on mouse breast cancer cells (4T1 tumor cells, purchased from the American Type Culture Collection ATCC)

[0134] First, 10 × 10 4 Cells were cultured in 500 μL DMEM (containing 10% primary bovine serum) in a CO2 incubator (37°C, 5% CO2 concentration) for 12 hours. The DMEM medium was then removed and the 24 wells were divided into 4 groups, with 3 wells in each group. The 3 wells were used as parallel experiments. The 4 groups were named PBS group, CM group, CMC group, and pre-blocking group.

[0135] Among them, 0.5 mL of 10 mM PBS buffer (pH 7.2) was added to each of the three wells in the PBS group;

[0136] 0.5 mL of 200 μg / mL CM-FITC solution (solvent: DMEM medium) was added to each of the three wells in the CM group. CM-FITC was prepared in step (1) of Example 2.

[0137] 0.5 mL of 200 μg / mL CMC-FITC solution (solvent: DMEM medium) was added to each of the three wells in the CMC group. CMC-FITC was prepared in step (2) of Example 2.

[0138] 0.5 mL of 10 μg / mL Anti-CD47 solution (solvent: DMEM medium, pre-blocking treatment for 1 h) was first added to each of the three wells in the pre-blocking group. The Anti-CD47 solution was then aspirated, and then 0.5 mL of 200 μg / mL CMC-FITC solution (solvent: DMEM medium) was added to each of the three wells in the pre-blocking group.

[0139] One hour later, the mean fluorescence intensity of each group was measured using flow cytometry.

[0140] The test results are as follows Figure 9 As shown in the results, CMC can specifically target the CD47 receptor on the surface of tumor cells, and this targeting effect is mainly mediated by binding to the CD47 receptor on the surface of 4T1 tumor cells.

[0141] Effect Verification Example 4: Verification of Endogenous Calreticulin Expression Triggered by CMC-Mediated Phototherapy

[0142] First, 4T1 tumor cells were added to a 24-well plate, and then 10 × 10 4 cells / 500 μL DMEM (containing 10% primary bovine serum) in a CO2 incubator (37°C, CO2 concentration of 5%). After 12 hours of culture, the DMEM medium was removed and the 24 wells were divided into 4 groups, with 3 wells in each group, and named as PBS group, CM group, CMC group, and CMC+NIR group respectively.

[0143] Among them, 0.5 mL of 10 mM PBS buffer (pH 7.2) was added to each of the three wells in the PBS group;

[0144] 0.5 mL of 200 μg / mL CM solution (solvent: DMEM medium) was added to each of the three wells in the CM group. The CM was prepared in step (2) of Example 1.

[0145] 0.5 mL of 200 μg / mL CMC solution (solvent: DMEM medium) was added to each of the three wells in the CMC group. The CMC was prepared in step (2) of Example 1.

[0146] In the three wells of the CMC+NIR group, 0.5 mL of 200 μg / mL CMC solution (solvent: DMEM medium) was added. After culturing for 1 hour, the cells were illuminated using a 1064 nm laser (intensity: 0.50 W / cm 2 ) irradiation for 5-10 minutes.

[0147] After 6 hours of culture, the DMEM medium was removed, the cells were washed three times with PBS, and then 1 μL of anti-APC-CALR antibody was added and stained for 20 minutes at 4°C. The mean fluorescence intensity of each group was measured using flow cytometry.

[0148] The test results are as follows Figure 10 As shown, CMC has nanozyme activity that can induce endogenous CALR exposure. After 1064nm laser irradiation, the exposure of endogenous CALR induced by CMC is further increased.

[0149] Effect verification example 5: CMC-induced macrophage phagocytosis of tumor cells

[0150] (1) Macrophage extraction and staining:

[0151] Six-week-old BALB / c mice (purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd.) were exsanguinated from their eyeballs and their carotid arteries cut to allow for thorough exsanguination. Leg bones were then removed and bone marrow was removed from the leg bones using a 2.5 mL syringe. Bone marrow was then blown out of the leg bones into DMEM medium and centrifuged at 450 × g for 5 min to collect the cells. Cells were then resuspended in 1 mL of erythrocyte lysis buffer and incubated at 4°C for 10 min. Lysis was terminated by adding 11 mL of 1× PBS and collected by centrifugation at 450 × g for 5 min. Finally, DMEM medium containing M-CSF (10 ng / mL) was added and cultured for 7 days to obtain mature BMDMs. Mature macrophages were stained with eFluor 670 dye (purchased from eBioscience, Cat. No. 65-0840) to obtain eFluor 670-labeled macrophages (eFluor 670-BMDM). Specific staining procedures were performed according to the product instructions.

[0152] (2) 4T1 tumor cell staining:

[0153] 4T1 tumor cells were stained using the CFDA SE Cell Proliferation and Tracing Detection Kit (purchased from beyotime, catalog number: C0051) to obtain CFSE dye-labeled 4T1 tumor cells (CFSE-4T1). The specific staining steps were performed according to the product instructions.

[0154] (3) Test of macrophage phagocytic ability of tumor cells

[0155] 2 × 10 were seeded per well in a 24-well plate. 5 CFSE-4T1 (mouse 4T1 tumor cells) were then divided into 4 groups, with 3 wells in each group. The 4 groups were named PBS group, NIR group, CMC group, CM+NIR group, and CMC+NIR group respectively.

[0156] Among them, 0.5 mL of 10 mM PBS buffer (pH 7.2) was added to each of the three wells in the PBS group;

[0157] 0.5 mL of 200 μg / mL CM solution (solvent: DMEM medium) was added to each of the three wells in the CM group. The CM was prepared in step (2) of Example 1.

[0158] 0.5 mL of 200 μg / mL CM solution (solvent: DMEM medium) was added to each of the three wells in the CM+NIR group. After culturing for 1 hour, the cells were visualized using a 1064 nm laser (intensity: 0.50 W / cm 2 ) irradiation for 5-10 minutes.

[0159] 0.5 mL of 200 μg / mL CMC solution (solvent: DMEM medium) was added to each of the three wells in the CMC group. The CMC was prepared in step (2) of Example 1.

[0160] In the three wells of the CMC+NIR group, 0.5 mL of 200 μg / mL CMC solution (solvent: DMEM medium) was added. After culturing for 1 hour, the cells were illuminated using a 1064 nm laser (intensity: 0.50 W / cm 2 ) irradiation for 5-10 minutes.

[0161] After being treated under the above conditions for 4 hours, the cells in each group were digested with 0.25% trypsin and then resuspended in 0.5 mL DMEM medium.

[0162] Take a 24-well plate and seed each well with 5×10 4eFluor670-BMDM were added with 0.5 mL of DMEM medium and cultured in a CO2 incubator (37°C, CO2 concentration of 5%) for 24 hours. The DMEM medium was then removed and the above-mentioned tumor cell resuspension was added. After 6 hours of culture in each group, the percentage of macrophages phagocytosing tumor cells was tested by flow cytometry.

[0163] The test results are as follows Figure 11 As shown in the results, CMC has a better ability to induce macrophages to phagocytose tumor cells under laser irradiation than other groups.

[0164] Effect Verification Example 6: CMC and MSN aCD47&CALR Comparison of the ability of macrophages to phagocytose tumor cells

[0165] The extraction and staining of macrophages and the staining of 4T1 tumor cells were the same as those in effect verification example 5;

[0166] 2 × 10 were seeded per well in a 24-well plate. 5 CFSE-4T1 (mouse 4T1 tumor cells) were then divided into 3 groups, with 3 wells in each group. The 3 groups were named PBS group, MSN group, aCD47&CALR group and CMC+NIR group;

[0167] Among them, 0.5 mL of 10 mM PBS buffer (pH 7.2) was added to each of the three wells in the PBS group;

[0168] MSN aCD47&CALR 0.5 mL of 200 μg / mL MSN was added to each of the three wells in the group. aCD47&CALR Solution (solvent is DMEM culture medium), the MSN aCD47&CALR Prepared in Comparative Example 2;

[0169] In the three wells of the CMC+NIR group, 0.5 mL of 200 μg / mL CMC solution (solvent: DMEM medium) was added. After culturing for 1 hour, the cells were illuminated using a 1064 nm laser (intensity: 0.50 W / cm 2 ) irradiation for 5-10 minutes.

[0170] After being treated under the above conditions for 4 hours, the cells in each group were digested with 0.25% trypsin and then resuspended in 0.5 mL DMEM medium.

[0171] Take a 24-well plate and seed each well with 5×10 4eFluor670-BMDM were added with 0.5 mL of DMEM medium and cultured in a CO2 incubator (37°C, CO2 concentration of 5%) for 24 hours. The DMEM medium was then removed and the above-mentioned tumor cell resuspension was added. After 6 hours of culture in each group, the percentage of macrophages phagocytosing tumor cells was tested by flow cytometry.

[0172] The test results are as follows Figure 12 As shown, CMC under laser irradiation is compared with MSN aCD47&CALR , which has a better ability to induce macrophages to phagocytose tumor cells.

[0173] Effect Verification Example 7: 4T1 Mouse Breast Cancer Orthotopic Model and Treatment Experiment

[0174] Female BALB / c mice aged about 6 weeks were divided into 5 groups, with 6 mice in each group. They were anesthetized with sodium pentobarbital and then injected with 5×10 5 4T1 cells.

[0175] Tumor-bearing mice with a 4T1 mouse breast cancer orthotopic model were divided into the following groups: sterile PBS buffer group, light irradiation group (abbreviated as NIR group), CM group, CMC group, and CMC+NIR group, with 6 mice in each group. CM and CMC were prepared as in Example 1.

[0176] When the tumor grows to 60-100 mm 3 When (tumor volume formula: tumor volume = length × width 2 / 2),

[0177] The corresponding drug injection treatment was performed, and each group was administered with intratumoral injection. The drug dosage was sterile PBS buffer group (50 μL / mouse), NIR group (1064 nm laser, 0.50 W / cm 2 The mice were irradiated for 10 minutes at a temperature of 50°C at the tumor site), CMC group (50 μL / mouse), CM+NIR group (50 μL / mouse, irradiated as in the NIR group 4 hours after injection), and CMC+NIR group (50 μL / mouse, irradiated as in the NIR group 4 hours after injection). The drugs were administered every 2 days for a total of 2 doses.

[0178] Starting from the 6th day, the tumor volume and body weight changes of each group of mice were recorded and counted.

[0179] The tumor size and weight changes of mice Figure 13 As shown, Figure 13 a in the figure is a line graph showing the changes in tumor volume. Figure 13Figure b is a line graph of weight changes. It can be seen that compared with other groups, the use of laser-induced CMC can significantly inhibit tumor growth, and there is basically no obvious change in the weight of mice.

[0180] Effect Verification Example 8: Comparison of the therapeutic effects of CMC on 4T1 mouse breast cancer under different light conditions

[0181] The tumor-bearing mice with 4T1 mouse breast cancer orthotopic model were divided into the following groups: sterile PBS buffer group, CMC+NIR low temperature group, and CMC+NIR high temperature group, with 6 mice in each group.

[0182] When the tumor grows to 60-100 mm 3 When (tumor volume formula: tumor volume = length × width 2 / 2).

[0183] The corresponding drug injection treatment was performed, and each group was administered with intratumoral injection. The drug dosage was sterile PBS buffer group (50 μL / mouse), CMC+NIR high temperature group (CMC 50 μL / mouse, 1064 nm laser, 0.50 W / cm 2 , irradiation for 10 minutes, tumor site temperature 50°C), CMC+NIR low temperature group (CMC 50 μL / mouse, 1064 nm laser, 0.50 W / cm 2 , irradiation for 10 minutes, tumor site temperature 43°C) was administered every 2 days, for a total of 2 times.

[0184] Starting from the 6th day, the tumor volume and body weight changes of each group of mice were recorded and counted.

[0185] The tumor size and weight changes of mice Figure 14 As shown in the figure, compared with the PBS group, the CMC+NIR high-temperature and low-temperature groups showed significant inhibition of tumor growth, but there was no significant difference in the inhibition of tumor growth between the CMC+NIR high-temperature and low-temperature groups. This shows that CMC can effectively inhibit tumor growth under low-temperature conditions, avoiding damage to surrounding normal tissues and causing inflammation, and is expected to achieve safe and effective treatment effects.

[0186] Effect Verification Example 9: Analysis of Related Immune Cells in Tumors

[0187] (1) The construction and treatment process of the 4T1 mouse breast cancer orthotopic model were the same as those in effect verification example 6.

[0188] On the third day after the end of the treatment experiment, the tumor-bearing mice were killed by bleeding from the eyeballs and dislocating the neck. The tumors were removed and ground into 5 mL of PBS solution containing 0.2% bovine serum albumin (PBA). The tumor suspension was filtered through a 200-mesh filter and centrifuged at 450 g for 5 min at 4°C. 2 mL of red blood cell lysis buffer was then added to the lower precipitate, lysed at 4°C for 10 min, and then 10 mL of percoll was added and centrifuged at 450 g for 5 min at 4°C. The supernatant was discarded. 6 mL of 40% Percoll (purchased from Sigma, catalog number: P1644, 40% was diluted by volume with 0.1 mM PBS, pH = 7.2) was added to the tumor suspension and centrifuged at 450 g for 20 min at room temperature. The supernatant was discarded. 30 μL of the suspension was added with CD16 / 32 antibody and blocked at 4°C for 15 min. The corresponding antibodies (CD11c: 0.2 μL, CD3: 0.4 μL, CD4: 0.1 μL, CD8a: 0.2 μL, CD45: 0.3 μL) were then added and incubated at 4°C in the dark for 20 min. The cells were washed twice with PBA and detected using a flow cytometer.

[0189] The test results are as follows Figure 15 As shown in the figure, the percentage of CD8+T cells in CD45-positive cells was significantly increased in the CMC+NIR group compared with the other groups, indicating that CMC-mediated photothermal therapy can activate the body's immune system and kill tumors.

[0190] Effect Verification Example 10: Verification of the Effect of CMC-mediated Phototherapy on Inhibiting Lung Metastasis

[0191] The construction and treatment process of the 4T1 mouse orthotopic breast cancer model were similar to those in Example 5. Lung metastasis of the tumor was observed after the initiation of tumor implantation. Twenty-four days after the initiation of tumor implantation, the mice were euthanized, and the lungs were removed. After fixation with Bouins' fixative, the lung nodules were photographed and counted. Lung metastasis was also observed after HE staining.

[0192] The test results are as follows Figure 16 As shown, compared with the other groups, fewer metastatic nodules were observed in the CMC+NIR group after treatment, indicating the powerful effect of CMC-mediated phototherapy in inhibiting the metastasis of tumor cells to the lungs.

[0193] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for preparing a phototherapeutic nanozyme, characterized in that The steps include: (1) Preparation of mesoporous silica-loaded Cu9S5 nanocomposite materials: synthesizing mesoporous silica nanoparticles, and then loading Cu9S5 into the pores or surface of mesoporous silica to obtain mesoporous silica-loaded Cu9S5 nanocomposite materials; (2) Preparation of phototherapeutic nanozymes: The mesoporous silica-loaded Cu9S5 nanocomposite material obtained in step (1) is functionalized with polyethylene glycol and then modified with anti-CD47 antibodies through electrostatic adsorption to obtain phototherapeutic nanozymes.

2. The method for preparing the phototherapeutic nanozyme according to claim 1, wherein: The average particle size of the mesoporous silicon in step (1) is 300-400 nm, and the specific surface area is 450-550 m 2 / g, pore volume of 1.5~1.7cm 3 / g; The Cu9S5 described in step (1) is Cu9S5 nanoparticles with an average particle size of 5 to 50 nm; The steps for loading the load described in step (1) are as follows: 1) dispersing mesoporous silica in an organic solvent to obtain a dispersion A; 2) dispersing Cu9S5 nanoparticles in an organic solvent to obtain dispersion B; 3) Mixing dispersion A and dispersion B, stirring and reacting; 4) After the reaction is completed, the solid and liquid are separated, and the obtained solid is washed to obtain a mesoporous silicon-loaded Cu9S5 nanocomposite material.

3. The method for preparing the phototherapeutic nanozyme according to claim 2, wherein: The mesoporous silicon described in step (1) is prepared by the following steps: 1) uniformly mixing cetyltrimethylammonium bromide, urea and water to obtain solution A; 2) mixing cyclohexane and ethyl orthosilicate to obtain solution B; 3) adding solution B to solution A to react; after the reaction is completed, performing solid-liquid separation, washing the solid, extracting and refluxing to obtain mesoporous silicon; The Cu9S5 described in step (1) is prepared by the following steps: A. Stir cuprous chloride, oleylamine, and oleic acid in an oil bath at 132-140° C. for 10-30 minutes and mix well; then cool to room temperature under nitrogen to obtain a copper precursor; B. Stir sulfur powder and octadecene in an oil bath at 202-210° C. for 10-30 minutes until uniform; when cooled to 180-185° C. with nitrogen, quickly inject the copper precursor obtained in step A to react; C. After the reaction is completed, nitrogen is passed through and cooled to room temperature, and then acetone is added to precipitate it. The solid-liquid separation is performed, and the obtained solid is washed with anhydrous ethanol to obtain Cu9S5 nanoparticles.

4. The method for preparing the phototherapeutic nanozyme according to claim 3, wherein: In the reaction system described in step 3), cetyltrimethylammonium bromide, urea, water, cyclohexane and ethyl orthosilicate are mixed in a ratio of 0.8-1.2 g: 0.2-0.4 g: 13-17 mL: 13-17 mL: 1.5-2.5 mL; The cuprous chloride, oleylamine and oleic acid described in step A are mixed in a mass ratio of 1:3-5:4-6; The sulfur powder and octadecene described in step B are mixed in a ratio of 0.32 g: 30-50 mL; The amount of the copper precursor in step B is based on the mass ratio of the reaction raw materials cuprous chloride and sulfur powder of 1:0.3-0.

4.

5. The method for preparing the phototherapeutic nanozyme according to claim 3, wherein: The reaction conditions in step 3) are as follows: reacting in an oil bath at 60-80° C. for 15-18 h; The solid-liquid separation method in step 3) is centrifugation; The washing operation in step 3) is to wash alternately with anhydrous ethanol and deionized water 1 to 3 times; The extraction reflux operation in step 3) is extraction reflux with a methanol solution containing NaCl; The stirring reaction time described in step A is 20 minutes; The solid-liquid separation method in step C is centrifugation.

6. The method for preparing the phototherapeutic nanozyme according to claim 2, wherein: The organic solvent in step 1) is anhydrous ethanol; The amount of the organic solvent in step 1) is calculated based on the concentration of mesoporous silica being 0.08 to 0.3 g / mL; The organic solvent in step 2) is cyclohexane; The amount of the organic solvent used in step 2) is calculated based on the concentration of Cu9S5 nanoparticles being 0.08 to 0.15 g / mL; The stirring reaction conditions in step 3) are stirring at 400-500 rpm for 24-48 hours.

7. The method for preparing the phototherapeutic nanozyme according to claim 1, wherein: The specific steps of step (2) are as follows: ① Dispersing the mesoporous silica-loaded Cu9S5 nanocomposite obtained in step (1) in an organic solvent, adding polyethylene glycol modified by a silane coupling agent, stirring the reaction, and performing solid-liquid separation to obtain a solid which is a polyethylene glycol-modified mesoporous silica-loaded Cu9S5 nanocomposite; ② Disperse the polyethylene glycol-modified mesoporous silica-loaded Cu9S5 nanocomposite material obtained in step ① in water, add anti-CD47 antibody, mix evenly, incubate at 2-8°C, separate the solid and liquid, and obtain a solid as the optical therapeutic nanozyme.

8. The method for preparing the phototherapeutic nanozyme according to claim 7, wherein: The organic solvent described in step ① is anhydrous alcohol; The amount of the organic solvent in step ① is calculated based on the concentration of the mesoporous silica-supported Cu9S5 nanocomposite material being 0.005 to 0.015 g / mL; The amount of the silane coupling agent-modified polyethylene glycol described in step ① is calculated as 0.08 to 0.15 times the mass of the mesoporous silica-supported Cu9S5 nanocomposite material; The stirring reaction time in step ① is 2 to 4 hours; The solid-liquid separation method in step ① is centrifugation; The polyethylene glycol-modified mesoporous silica-loaded Cu9S5 nanocomposite material described in step ② and the anti-CD47 antibody are mixed in a mass ratio of 5 to 40:1; The incubation time in step ② is 10 to 15 hours; The solid-liquid separation method in step ② is centrifugation.

9. A phototherapeutic nanozyme, characterized by: The method is obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the phototherapeutic nanozyme according to claim 9 in the preparation of immunotherapeutic drugs for regulating macrophage phagocytosis and sensitization.

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