Aggregation-induced luminescence nanoparticle-modified montmorillonite composite, preparation method, and application thereof

Abstract

The present invention discloses a montmorillonite composite modified with aggregation-induced luminescent nanoparticles, and a preparation method and application thereof. The present invention uses pharmaceutical-grade calcium-montmorillonite as a carrier to load aggregation-induced luminescent nanoparticles. Preparation process: the aggregation-induced luminescent nanoparticles are dissolved with dimethyl sulfoxide, the calcium-montmorillonite is dispersed in deionized water, and fully ultrasonicated to obtain a montmorillonite suspension. Deionized water is added to the montmorillonite suspension for dilution, and then the aggregation-induced luminescent nanoparticles are added, mixed at a constant speed, centrifuged, and the precipitate is freeze-dried to obtain a composite. The present invention utilizes calcium-montmorillonite to load a very small amount of aggregation-induced luminescent nanoparticles to enhance the hemostatic effect of the montmorillonite composite hemostatic material, and the prepared composite has no obvious cytotoxicity, no hemolysis, good biocompatibility, high safety performance, and good antibacterial effect; and the preparation method of the present invention has simple steps, is easy to operate, and is conducive to large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of hemostatic materials, and in particular to an aggregation-induced luminescence nanoparticle-modified montmorillonite composite, a preparation method thereof, and an application thereof. Background Art

[0002] Chronic wounds, such as burns and trauma, are highly susceptible to bacterial infection. Treatment of wound infections caused by Gram-positive bacteria, such as Staphylococcus aureus, is often challenged by limited hemostasis and bacterial resistance. According to statistics from the World Health Organization, more than 25 million people worldwide suffer from wound infections each year, and the cost of treating wound infections exceeds tens of billions of dollars. Wound infections have become a major and thorny issue threatening future human health.

[0003] Montmorillonite, as a biomaterial approved by the U.S. Food and Drug Administration (FDA), is the most effective hemostatic agent among natural silicate minerals. However, montmorillonite has been reported to have a weak antibacterial effect, and its biocompatibility and hemostatic effect still need to be improved. Aggregation-induced luminescence nanoparticles are used as a photosensitizer to activate photosensitizers to produce reactive oxygen species and kill pathogenic microorganisms. This treatment method has the unique advantages of being non-invasive, highly effective, and precisely controllable. The prior art has disclosed the use of montmorillonite loaded with a certain photosensitizer as a hemostatic material to improve the efficacy of montmorillonite. The antibacterial properties of montmorillonite are excellent, but in the hemostatic material, the mass of montmorillonite and photosensitizer is relatively small, which leads to excessive dosage of photosensitizer. As a synthetic organic compound, the photosensitizer has a relatively high synthesis cost, and secondly, excessive dosage will cause certain toxicity. Therefore, it is necessary to develop a method that combines montmorillonite with a photosensitizer of a specific structure to reduce the loading amount of the photosensitizer while having good hemostatic properties, antibacterial effects and biocompatibility. It can help solve the bacterial infection risks faced by traditional wound materials and has good application prospects in the field of wound healing. Summary of the Invention

[0004] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide an aggregation-induced luminescence nanoparticle-modified montmorillonite composite, a preparation method thereof, and an application thereof.

[0005] The aggregation-induced luminescence nanoparticle-modified montmorillonite composite of the present invention uses pharmaceutical-grade calcium-montmorillonite as a carrier to load aggregation-induced luminescence nanoparticles. The structural formula of the aggregation-induced luminescence nanoparticles is:

[0006]

[0007] Furthermore, the mass ratio of the aggregation-induced luminescence nanoparticles to the calcium-montmorillonite is (0.5-2.0):100.

[0008] Furthermore, pharmaceutical grade calcium montmorillonite is used as a carrier.

[0009] A method for preparing the above-mentioned aggregation-induced luminescence nanoparticle-modified montmorillonite composite comprises the following steps:

[0010] S1: Dispersing montmorillonite in deionized water by ultrasonication to prepare a uniform montmorillonite suspension;

[0011] S2: dissolving the aggregation-induced luminescence nanoparticles in DMSO to prepare an aggregation-induced luminescence nanoparticle solution;

[0012] S3: The montmorillonite suspension is mixed with the aggregation-induced luminescence nanoparticle solution under certain temperature and oscillation speed conditions, the obtained material is centrifuged, and the precipitate is freeze-dried to obtain the aggregation-induced luminescence nanoparticle modified montmorillonite complex MMT-TTPy.

[0013] Furthermore, the concentration of the aggregation-induced luminescence nanoparticles is 0.01-10 mg / mL.

[0014] Furthermore, in step S3, the mixture is mixed at a constant speed of 800-1200 rpm at 30-40° C. for 2-4 hours.

[0015] Furthermore, in step S3, centrifugation is performed at 1300 rpm-1500 rpm for 8-12 minutes.

[0016] Furthermore, in step S3, the precipitate is freeze-dried for 20-28 hours.

[0017] The aggregation-induced luminescence nanoparticle-modified montmorillonite composite can be used as a hemostatic agent.

[0018] The present invention utilizes calcium-based montmorillonite loaded with a very small amount of aggregation-induced luminescent nanoparticles to enhance the hemostatic effect of the montmorillonite composite hemostatic material. The prepared MMT-TTPy composite hemostatic material has no obvious cytotoxicity, no hemolysis, good biocompatibility, high safety performance, and good antibacterial effect. Moreover, the preparation method of the present invention is simple in steps, easy to operate, and conducive to large-scale production.

[0019] The present invention utilizes calcium-based montmorillonite with a lamellar structure to load aggregation-induced luminescence nanoparticles. The positively charged aggregation-induced luminescence nanoparticles are loaded on the surface of the montmorillonite. The flaky montmorillonite surface is negatively charged and has abundant surface hydroxyl groups, which is conducive to the efficient loading of functional nanoparticles. The encapsulation rate of the aggregation-induced luminescence nanoparticles can reach 100%. Moreover, the aggregation-induced luminescence nanoparticles have high biocompatibility and are irradiated with white light (100mW / cm 2, 30min) can achieve photodynamic therapy sterilization, and under the premise of ensuring the hemostatic performance of montmorillonite, it also improves the cell safety and biocompatibility of mineral materials, and enhances the antibacterial effect of montmorillonite, so that it can be used in the field of hemostasis and wound healing, and develop hemostatic products with social and economic significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For phase analysis of montmorillonite;

[0021] Figure 2a 、 2b 2c are the structures of the aggregation-induced luminescence nanoparticles of Comparative Examples 1 and 2 and the present invention;

[0022] Figure 3 These are optical images of the suspensions and centrifuged samples of Examples 1-5;

[0023] Figure 4a 、 4b , 4c, 4d are the antibacterial results of aggregation-induced luminescence nanoparticles compounded with montmorillonite;

[0024] Figure 5 The linear relationship between the relative fluorescence intensity of Examples 1-5 and time is shown;

[0025] Figure 6 is the cytotoxicity graph of Examples 1-5;

[0026] Figure 7 Graph showing the hemolytic test results of Examples 1-5;

[0027] Figure 8 is a graph of in vitro coagulation time of Examples 1-5;

[0028] Figure 9 Flow chart for the preparation of aggregation-induced emission nanoparticle-modified montmorillonite composites. DETAILED DESCRIPTION

[0029] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0030] The pharmaceutical grade montmorillonite in the examples of this specification is a pharmaceutical grade montmorillonite product of Shanghai Aladdin Biochemical Technology Co., Ltd. Figure 1 As shown, it is consistent with the standard card PDF#13-0135, and the molecular formula is Ca 0.2 (Al,Mg)2Si4O 10 (OH)2·4H2O is calcium-based montmorillonite.

[0031] The aggregation-induced luminescence nanoparticles in the comparative examples and embodiments were prepared by Guangzhou Carbon Water Biotechnology Co., Ltd. according to the structural formula designed by the applicant. Figure 2a 、 2b , as shown in 2c.

[0032] Preparation of montmorillonite suspension

[0033] Example 1:

[0034] 1 g of montmorillonite was dispersed in 10 mL of deionized water and ultrasonicated for 30 min to obtain a montmorillonite suspension with a concentration of 100 mg / mL.

[0035] Examples 2-5

[0036] The preparation process of aggregation-induced emission nanoparticles (TTPy structure) modified montmorillonite composites, such as Figure 9 shown.

[0037] Preparation of aggregation-induced emission nanoparticle suspension

[0038] 10 mg of aggregation-induced emission nanoparticles (TTPy) were dissolved in 1 mL of DMSO to obtain a 10 mg / mL aggregation-induced emission nanoparticle solution.

[0039] Example 2:

[0040] A method for preparing an aggregation-induced luminescence nanoparticle-modified montmorillonite composite comprises the following steps: 160 μL of deionized water is added to 800 μL of a 100 mg / mL montmorillonite suspension for dispersion, followed by the addition of 40 μL of a 10 mg / mL aggregation-induced luminescence nanoparticle solution. The resulting 1 mL mixture is placed in a thermomixer and mixed at 37°C and 1000 rpm for 3 hours. The resulting material is centrifuged at 14,000 rpm for 10 minutes, and the precipitate is freeze-dried for 24 hours to obtain the aggregation-induced luminescence nanoparticle-modified montmorillonite composite labeled with 0.5% TTPy.

[0041] Example 3:

[0042] A method for preparing an aggregation-induced luminescence nanoparticle-modified montmorillonite composite comprises the following steps: 560 μL of deionized water is added to 400 μL of a 100 mg / mL montmorillonite suspension for dispersion, followed by the addition of 40 μL of a 10 mg / mL aggregation-induced luminescence nanoparticle solution. The resulting 1 mL mixture is placed in a thermomixer and mixed at 37°C and 1000 rpm for 3 hours. The resulting material is centrifuged at 14,000 rpm for 10 minutes, and the precipitate is freeze-dried for 24 hours to obtain the aggregation-induced luminescence nanoparticle-modified montmorillonite composite labeled with 1.0% TTPy.

[0043] Example 4:

[0044] A method for preparing an aggregation-induced luminescence nanoparticle-modified montmorillonite composite comprises the following steps: 693 μL of deionized water is added to 267 μL of a 100 mg / mL montmorillonite suspension for dispersion, followed by the addition of 40 μL of a 10 mg / mL aggregation-induced luminescence nanoparticle solution. The resulting 1 mL mixture is placed in a thermomixer and mixed at 37°C and 1000 rpm for 3 hours. The resulting material is centrifuged at 14,000 rpm for 10 minutes, and the precipitate is freeze-dried for 24 hours to obtain the aggregation-induced luminescence nanoparticle-modified montmorillonite composite labeled with 1.5% TTPy.

[0045] Example 5:

[0046] A method for preparing an aggregation-induced luminescence nanoparticle-modified montmorillonite composite comprises the following steps: 760 μL of deionized water is added to 200 μL of a 100 mg / mL montmorillonite suspension for dispersion, followed by the addition of 40 μL of a 10 mg / mL aggregation-induced luminescence nanoparticle solution. The resulting 1 mL mixture is placed in a thermomixer and mixed at 37°C and 1000 rpm for 3 hours. The resulting material is centrifuged at 14,000 rpm for 10 minutes, and the precipitate is freeze-dried for 24 hours to obtain the aggregation-induced luminescence nanoparticle-modified montmorillonite composite labeled with 2.0% TTPy.

[0047] The optical images of the suspensions prepared in Examples 1-5 and after centrifugation are as follows: Figure 3 As shown, the preparation methods of Examples 1-5 are simple and easy to implement, and the encapsulation efficiency of the aggregation-induced luminescence nanoparticles can reach 100%, which greatly improves the utilization rate of the aggregation-induced luminescence nanoparticles. Figure 2c The structure of the aggregation-induced luminescence nanoparticles shows that the aggregation-induced luminescence nanoparticles, as a cationic dye, can be bound to the montmorillonite surface through electrostatic interaction. + groups and Si-O groups on the surface of montmorillonite.

[0048] Aggregation-induced emission nanoparticles ( Figure 2a Preparation of modified montmorillonite composites

[0049] Comparative Example 1:

[0050] A composite of aggregation-induced emission nanoparticles (AIE1) modified with montmorillonite was prepared by adding 900 μL of deionized water to 50 μL of a 0.5 mg / mL montmorillonite suspension for dispersion, followed by the addition of 50 μL of a 0.5 mg / mL aggregation-induced emission nanoparticle solution. The resulting 1 mL mixture was placed in a thermomixer and mixed at 37°C and 1000 rpm for 30 minutes. The resulting material was centrifuged at 14000 rpm for 10 minutes, and the precipitate was freeze-dried for 24 hours to obtain the aggregation-induced emission nanoparticle-modified montmorillonite composite, labeled MMT-AIE1.

[0051] Aggregation-induced emission nanoparticles ( Figure 2b Preparation of modified montmorillonite composites

[0052] Comparative Example 2:

[0053] A composite of aggregation-induced emission nanoparticles (AIE2) modified with montmorillonite was prepared by adding 700 μL of deionized water to 250 μL of a 1.0 mg / mL montmorillonite suspension for dispersion, followed by the addition of 50 μL of a 1.0 mg / mL aggregation-induced emission nanoparticle solution. The resulting 1 mL mixture was placed in a thermomixer and mixed at 37°C and 1000 rpm for 30 minutes. The resulting material was centrifuged at 14,000 rpm for 10 minutes, and the precipitate was freeze-dried for 24 hours to obtain the aggregation-induced emission nanoparticle-modified montmorillonite composite, labeled MMT-AIE2.

[0054] In vitro antibacterial test:

[0055] Staphylococcus aureus (ATCC 25923) was used as a Gram-positive bacterial model to evaluate the antibacterial activity of the samples. A single strain of Staphylococcus aureus was dispersed in 5 mL of Luria–Bertani liquid medium and shaken at 37°C for 24 h to obtain an initial bacterial concentration of 2×10 9 CFU / mL. Subsequently, the bacterial suspension was serially diluted with PBS to 10 6 Then, 100 μL of the diluted bacterial suspension was mixed with 100 μL of the material solution to obtain a 500 μg / mL (based on the concentration of montmorillonite) material solution. The mixed bacterial sample was incubated in the dark for 5 minutes and then heated at 100 mw / cm 2 The concentrations of Comparative Example 1 and Comparative Example 2 were 2.5 μg / mL and 10 μg / mL (based on the concentrations of AIE1 and AIE2, respectively). The mixed bacterial samples were incubated in the dark for 5 min and then irradiated under white light at 100 mw / cm 2Irradiate under white light for 15 minutes. Then, serially dilute the bacterial suspension 10-fold with PBS. Spread 100 μL of the diluted bacteria onto the corresponding solid agar plate and incubate at 37°C for 14-16 hours. Bacterial survival rate was used as an indicator to evaluate the antibacterial activity of the material against bacteria.

[0056] Bacterial survival rate = average number of colonies in the sample / average number of colonies in the control group × 100%

[0057] The antibacterial effects of Examples 1-5 and Comparative Examples 1 and 2 on Staphylococcus aureus are shown in Table 1. Figure 4a 、 4b , 4c and 4d. Figure 4a The results of bacterial plating showed that the antibacterial effects of AIE1 and MMT-AIE1 were not significant. Figure 4b The results of bacterial plating showed that AIE2 had a significant antibacterial effect, but the effect of MMT-AIE2 was still not obvious. Figure 4c ,d The results of the bacterial plating show that MMT-TTPy has a significant antibacterial effect. The results of the bacterial survival rate can be intuitively seen that MMT has a certain antibacterial effect under light, and the bacterial survival rate is 79.46%. After compounding with TTPy, the antibacterial effect of MMT-TTPy is significantly improved. Among them, the bacterial survival rates of the materials corresponding to Example 4 and Example 5, 1.5% TTPy and 2.0% TTPy are 10.64% and 3.73%, respectively. This result shows that finding aggregation-induced luminescent nanoparticles (TTPy structure) suitable for compounding with montmorillonite is very critical to improving the antibacterial properties of montmorillonite. In addition, excellent antibacterial properties can be achieved at a low TTPy compound ratio (1.5%) and a low concentration dose.

[0058] In vitro reactive oxygen species production experiment:

[0059] The commonly used reactive oxygen species (ROS) indicator 2,7-dichlorofluorescein diacetate (DCFH-DA) was used to detect the sample at 100 mW / cm 2 ROS generation under white light irradiation. In this experiment, 0.5 mL of ethanol-dissolved DCFH-DA (1×10 -3 mol·L -1 ) added 2 mL 1×10 -2 mol·L -1 The solution was stirred at room temperature for 30 minutes in 10 mL of NaOH. The hydrolyzate was then neutralized with 10 mL of 1× PBS and stored in the dark. At this point, DCFH-DA was hydrolyzed to 2,7-dichlorofluorescein (DCFH). The ROS indicator (4×10-5 mol·L) in PBS was then added. -1) was further diluted to 5×10 - 6 mol·L -1 Immediately after irradiation with white light, the fluorescence spectrum of the solution was measured at an excitation wavelength of 488 nm and an emission wavelength of 490-700 nm.

[0060] The linear relationship between the relative fluorescence intensity of Examples 1-5 and time is shown in Figure 5 As shown, from Figure 5 It can be seen that MMT can produce very small amounts of reactive oxygen species under visible light, and the ability of the MMT-TTPy complex to produce reactive oxygen species increases with increasing TTPy content. This suggests that the antibacterial mechanism of TTPy-MMT is mainly through the generation of reactive oxygen species under light, thereby killing bacteria.

[0061] Cytotoxicity assay

[0062] This experiment uses the Cell Counting Kit-8 (CCK-8) method to analyze the cytotoxicity of samples using human immortalized fibroblasts (BJ cells) as the research object. RPMI-1640 basic culture medium, 1% double antibody, and 10% fetal bovine serum are used to prepare a complete culture medium for culturing BJ cells. Take the normally frozen BJ cells and shake them continuously in a 37°C water bath until they are thawed. Pipette 1mL of BJ cell freezing solution and add it to a 15mL centrifuge tube with 10mL of complete culture medium. Shake to mix, centrifuge at 1000rpm for 5min, remove the supernatant and resuspend the cells with complete culture medium, and transfer the cell suspension to a culture dish. After the cells are cultured until they are in a good state of adherent growth, they are digested with trypsin to detach the cells from the adherent state and resuspend them. Adjust the density of the cell suspension to 1×10 per well. 4 The cells were seeded in 96-well plates at a cell density of 100 and incubated at 37°C with 5% CO 2 Incubate in a cell culture incubator until cells are well adhered and in production. Discard the original culture medium, add 100 μL of fresh complete culture medium to the control wells, and add 100 μL of culture medium containing different concentrations of samples to the test wells. After 24 hours of incubation, cytotoxicity is measured by adding complete culture medium containing 10% CCK-8 reagent to each well. Incubate for another 1 hour, and measure the absorbance (OD) of the culture medium at 450 nm using a microplate reader. At the same time, wells without added sample materials serve as blank controls, and cell viability is calculated by measuring the absorbance. Cell viability is calculated as follows:

[0063] Cell viability = (OD experimental well - OD blank well) / (OD control well - OD blank well) × 100%

[0064] The toxicity of Examples 1-5 was evaluated by setting a concentration gradient of 2.5, 5, 10, 20, and 40 μg / mL. The test results are shown in the following table. Figure 6 As shown. In the cytotoxicity rating, a cell survival rate greater than 75% is designated as reaching Biosafety Level 1. The cell survival rates of the five samples in Examples 1-5 were all above 75% within the experimental concentration range, indicating that both MMT and the MMT-TTPy complex possess good biocompatibility. Furthermore, the cell survival rate of the MMT-TTPy complex was significantly improved compared to that of MMT, indicating that TTPy binding to the MMT surface can enhance the biocompatibility of MMT.

[0065] Hemolytic test:

[0066] Preparation of 2% red blood cell suspension: Take 1 mL of fresh anticoagulated rabbit blood, centrifuge at 2500 rpm for 5 min, remove the supernatant, wash three times with phosphate buffered saline (PBS), take 500 μL of the washed blood into a 50 mL centrifuge tube, and add PBS to 50 mL.

[0067] Hemolytic test: Examples 1-5 were prepared with PBS to prepare material solutions with concentrations of 2.5, 5, 10, 20, and 40 μg / mL, respectively. Take 500 μL of each concentration solution and mix it evenly with 500 μL of the prepared 2% red blood cell suspension. Set up a positive control group: 500 μL of deionized water mixed with 500 μL of 2% red blood cell suspension; a negative control group: 500 μL of PBS solution mixed with 500 μL of 2% red blood cell suspension, with 3 parallel tubes in each group. The samples were incubated in a 37°C water bath for 1 hour, centrifuged at 2500 rpm, and the supernatant was taken and its OD value was measured using a microplate reader (540 nm). The lower the hemolytic activity, the higher the biocompatibility. If the hemolysis rate is less than 5%, it is considered that no hemolysis occurs.

[0068] Hemolysis rate (%) = (OD experimental group - OD negative group) / (OD positive group - OD negative group) × 100%

[0069] The hemolytic results of Examples 1-5 are shown in Figure 7 As shown, from Figure 7 It can be seen that the hemolysis rate of the MMT-TTPy complex is lower than that of MMT, indicating that TTPy binds to the surface of MMT and can improve the biocompatibility of MMT.

[0070] In vitro bleeding time determination:

[0071] 8 mg each of MMT and MMT-TTPy composite hemostatic material were weighed into a 2 mL centrifuge tube and preheated in a 37°C water bath for 3 minutes. 200 μL of anticoagulated whole blood from New Zealand rabbits was added to the sample powder at the bottom of the tube. Then, 2.5 μL of 0.2 mol / L CaCl₂ solution was quickly added to the mixture to calcify the blood and trigger coagulation. The mixture was quickly incubated in a 37°C water bath. The centrifuge tube was shaken every 10 seconds, and the blood flow was observed until coagulation occurred. The hemostasis time was recorded. Three replicates were performed for each material.

[0072] Table 1 Bleeding time of MMT and MMT-TTPy composite hemostatic materials

[0073]

[0074]

[0075] Depend on Figure 8 As shown in Table 1, MMT itself has a good hemostatic effect. After compounding with TTPy, it can effectively improve the hemostatic speed. This may be because TTPy is positively charged. After compounding with MMT, it can react with the Ca2+ adsorbed on the surface of montmorillonite. 2+ There is a weak interaction between them, which favors Ca 2+ Stability, Ca 2+ It can activate coagulation factors and promote coagulation cascade reaction, thereby effectively enhancing the hemostatic effect of montmorillonite.

[0076] Any matters not mentioned above shall be subject to the existing technology.

[0077] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art of the present invention may make various modifications or additions to the described specific embodiments or replace them in similar ways, but they will not deviate from the direction of the present invention or exceed the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent replacements, improvements, etc. made to the above embodiments based on the technical essence of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of an aggregation-induced luminescence nanoparticle-modified montmorillonite composite in the preparation of a hemostatic agent, characterized in that: The aggregation-induced luminescence nanoparticle-modified montmorillonite composite uses calcium-based montmorillonite as a carrier and loads aggregation-induced luminescence nanoparticles. The aggregation-induced luminescence nanoparticles are TTPy, and the structural formula is: 。 2. The use according to claim 1, characterized in that: The mass ratio of aggregation-induced emission nanoparticles to calcium-montmorillonite is (0.5-2.0):

100.

3. The use according to claim 1, characterized in that: It uses pharmaceutical grade calcium montmorillonite as the carrier.

4. The use according to claim 1 or 2, characterized in that The preparation of the aggregation-induced luminescence nanoparticle-modified montmorillonite composite comprises the following steps: S1: Dispersing montmorillonite in deionized water by ultrasonication to prepare a uniformly dispersed montmorillonite suspension; S2: dissolving the aggregation-induced luminescence nanoparticles in DMSO to prepare an aggregation-induced luminescence nanoparticle solution; S3: The montmorillonite suspension is mixed with the aggregation-induced luminescence nanoparticle solution under certain temperature and oscillation speed conditions, the obtained material is centrifuged, and the precipitate is freeze-dried to obtain the aggregation-induced luminescence nanoparticle modified montmorillonite complex MMT-TTPy.

5. The use according to claim 4, characterized in that: The concentration of the aggregation-induced luminescence nanoparticles is 0.01-10 mg / mL.

6. The use according to claim 4, wherein: In step S3, the mixture was mixed at a constant speed of 800-1200 rpm at 30-40° C. for 2-4 h.

7. The use according to claim 4, wherein: In step S3, centrifuge at 1300 rpm-1500 rpm for 8-12 minutes.

8. The use according to claim 4, characterized in that: In step S3, the precipitate is freeze-dried for 20-28 hours.

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