A nano-traditional Chinese medicine that effectively blocks macrophage pyroptosis and its application in anti-pulmonary fibrosis

Abstract

The present invention provides a nano-traditional Chinese medicine that can block macrophage pyroptosis and its application in anti-pulmonary fibrosis. The albumin nanomedicine loaded with formononetin targets the lesion site and is captured by macrophages based on the affinity of albumin to SPARC protein in the pulmonary fibrosis microenvironment. The natural product formononetin inhibits GSDMD-mediated macrophage pyroptosis, downregulates the related inflammatory fibrosis network, significantly prolongs survival, and reduces the toxic side effects of the drug, providing a valuable mechanism and promising therapeutic strategy for the treatment of pulmonary fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to the construction of a nano Chinese medicine capable of effectively blocking macrophage pyroptosis and its application in anti-pulmonary fibrosis. Technical Background

[0002] Pulmonary fibrosis (PF) is a progressive interstitial lung disease. The median survival of patients is 2 to 4 years, and it is called a "tumor-like disease." With the in-depth study of the new coronavirus, data show that most critically ill patients will have varying degrees of pulmonary fibrosis. As one of the important sequelae, PF can seriously affect lung function, eventually causing respiratory failure or even death. Although lung transplantation is the only way to cure pulmonary fibrosis, it is difficult to promote due to its high price and the risks of immune rejection and infection. Most patients can only use two FDA-approved drugs, pirfenidone (PFD) and nintedanib, for palliative treatment, with little effect. Therefore, it is necessary to develop new drug products to improve the clinical benefits of PF patients.

[0003] Cell pyroptosis, also known as cell inflammatory necrosis, is a new type of programmed cell death discovered and confirmed in recent years. It can be mediated by inflammasomes, manifested by continuous cell swelling until the cell membrane ruptures, leading to the release of cell contents and activating a strong inflammatory response. More and more evidence shows that macrophage pyroptosis can upregulate the level of inflammatory response, trigger an uncontrolled immune response, and accelerate the development of fibrotic diseases. Studies have shown that the levels of Caspase-1 and IL-1β are significantly increased in the bronchoalveolar lavage fluid (BALF) of PF patients. In the process of defending against microorganisms and airborne particles, alveolar macrophages initiate innate immune responses and host defense mechanisms. NLRP3 inflammasomes are formed, activating Caspase-1 to cleave the perforated protein gasdermin D (GSDMD) to form large holes on the plasma membrane, thereby promoting alveolar macrophage pyroptosis. The leakage of related inflammatory factors causes the persistence of fibrogenic stress signals. Therefore, inhibiting macrophage pyroptosis and related signaling pathways and regulating the inflammatory microenvironment may provide a new immunotherapy strategy for PF.

[0004] Formononetin (FMN) is a type of phytoestrogen rich in the traditional Chinese medicine Astragalus, which has anti-inflammatory, anti-tumor, lipid metabolism regulation and other pharmacological effects. Studies have found that FMN can improve myocardial ischemia / reperfusion injury in rats by inhibiting the ROS-TXNIP-NLRP3 pathway, and its application in colitis can significantly reduce the levels of NLRP3 and IL-1β in macrophages. Therefore, FMN is expected to be a potential macrophage pyroptosis inhibitor to achieve the purpose of treating pulmonary fibrosis, which is a reasonable drug choice.

[0005] However, the strong hydrophobicity and poor oral bioavailability of FMN lead to rapid metabolism and poor stability of the drug in the body. These shortcomings have caused many problems in the treatment of FMN in PF. Therefore, it is necessary to use appropriate drug delivery strategies to accurately and efficiently make the drug work at the lesion site.

[0006] SPARC protein (acidic and cysteine-rich secretory protein) is a matrix cell molecule that regulates the interaction between cells and their surrounding extracellular matrix (ECM), and controls basic functions such as cell adhesion, proliferation and differentiation. Studies have shown that SPARC protein is overexpressed in the microenvironment of atherosclerosis, fibrotic lesions and various invasive cancers. In lung diseases, SPARC drives the pathological response of non-small cell lung cancer and idiopathic pulmonary fibrosis by promoting microvascular remodeling and excessive deposition of ECM proteins. This also makes it possible to actively target nanomedicines at the site of pulmonary fibrosis lesions. Studies have found that the highly expressed SPARC protein in the tumor microenvironment has a high natural affinity with BSA, which has led to the birth of albumin-bound paclitaxel as the first nanotechnology-based anti-tumor drug on the market. However, there have been no reports on effective anti-PF treatment strategies that utilize SPARC overexpression in the PF lung interstitial microenvironment.

[0007] CN108853083A provides the use of formononetin in the preparation of drugs for treating pulmonary fibrosis. However, due to hydrophobicity and body metabolism, the concentration of oral formononetin in blood and tissue is low, and the therapeutic effect is limited. In order to improve the therapeutic effect, high concentration and frequent administration are required, which usually leads to serious adverse reactions.

[0008] Based on the above analysis, we intend to prepare albumin nanomedicine loaded with FMN and explore its efficacy and potential mechanism as an anti-pulmonary fibrosis treatment. Summary of the invention

[0009] The purpose of the present invention is to construct a nano-Chinese medicine that can effectively block macrophage pyroptosis. According to the physicochemical properties of the natural product FMN, BSA is used as a carrier to construct a nano-Chinese medicine FMN@BSA NPs with a reasonable size that carries FMN, so as to achieve the purpose of targeted treatment of pulmonary fibrosis by blocking macrophage pyroptosis.

[0010] The present invention adopts an anti-solvent precipitation method to induce FMN and BSA to self-assemble into nanoparticles through intermolecular hydrogen bonds and hydrophobic forces, thereby increasing the drug loading content and avoiding the rapid clearance of small molecules in the body and systemic side effects.

[0011] The present invention explores the pathological microenvironment of high expression of SPARC protein in pulmonary fibrosis lesions, and based on the principle of high affinity between SPARC and BSA, evaluates the feasibility of lung-targeted drug delivery of FMN@BSA NPs via tail vein injection in vivo.

[0012] The cells used in the present invention are primary macrophages BMDMs extracted from the thigh bone marrow of mice and induced to differentiate. The bleomycin (BLM)-induced mouse pulmonary fibrosis model selected is a classic modeling method.

[0013] The present invention describes a method for preparing nano traditional Chinese medicine, as well as related preparation characterization, in vivo targeting evaluation and pharmacodynamic evaluation.

[0014] The present invention demonstrates through confocal microscopy, western blotting and other experiments that the constructed FMN@BSA NPs serve as potential pyroptosis inhibitors, blocking lipopolysaccharide (LPS) and nigericin-induced macrophage pyroptosis by intervening in the Caspase-1 / GSDMD pathway.

[0015] The present invention proves through comprehensive efficacy experiments that the nano Chinese medicine has significant anti-inflammatory and anti-fibrosis effects on pulmonary fibrosis model mice, has good in vivo drug safety, and significantly prolongs the survival period of animals.

[0016] Specifically, the present invention provides the following technical solutions:

[0017] A Chinese medicine nanoparticle that can effectively block macrophage pyroptosis is used in the preparation of a drug for the targeted treatment of pulmonary fibrosis. Bovine serum albumin (BSA) is used as a carrier to carry the natural product formononetin (FMN) to form FMN@BSA NPs.

[0018] The traditional Chinese medicine nanoparticles, FMN and BSA are self-assembled into nanoparticles through intermolecular hydrogen bonding and hydrophobic forces:

[0019]

[0020] The traditional Chinese medicine nanoparticles are administered by intravenous injection, and BSA gives the nano traditional Chinese medicine a long circulation characteristic and has good biocompatibility.

[0021] The traditional Chinese medicine nanoparticles achieve targeted lung delivery of drugs based on the principle that SPARC protein is highly expressed in pulmonary fibrosis lesions and has high affinity with BSA.

[0022] The traditional Chinese medicine nanoparticles, FMN@BSA NPs, act as pyroptosis inhibitors, blocking GSDMD-mediated macrophage pyroptosis and inhibiting related inflammatory fibrosis signaling networks.

[0023] The Chinese medicine nanoparticles exert excellent anti-inflammatory and anti-fibrosis effects in an animal model of pulmonary fibrosis, and significantly prolong the survival period of the animals.

[0024] In the traditional Chinese medicine nanoparticles, the mass ratio of FMN to BSA is 1-20:10-60, and the preferred mass ratio is 1:20.

[0025] The traditional Chinese medicine nanoparticles are prepared by an anti-solvent precipitation method, thereby avoiding the harm of drug excipients to organisms and improving the hydrophobicity of FMN.

[0026] The preparation method of the traditional Chinese medicine nanoparticles comprises the following steps: 1) dissolving FMN and BSA in appropriate solvents respectively; 2) adding the FMN solution dropwise into the BSA solution and stirring; 3) dropping an organic solvent into the mixed solution and stirring; 4) dialyzing and freeze-drying to obtain the nanoparticles.

[0027] The preparation method of the traditional Chinese medicine nanoparticles comprises the following steps: 1) dissolving FMN and BSA in suitable solvents DMSO and ddH2O respectively; 2) adding the FMN solution dropwise into the BSA solution and stirring; 3) dropping EtOH into the mixed solution and stirring; 4) dialyzing and freeze-drying to obtain the nanoparticles.

[0028] The preparation method of the traditional Chinese medicine nanoparticles comprises the following steps: 1) dissolving FMN and BSA in DMSO and ddH2O respectively and vortexing; 2) adding dropwise the DMSO solution (5 mg / mL) containing FMN into the BSA aqueous solution (30 mg / mL) under strong stirring, and stirring for 3 hours at room temperature in the dark; 3) dropping 2 mL EtOH into the mixed solution at a speed of 1 mL / min using an automatic injection pump, and stirring for 3 hours; 4) transferring to a 20 kDa dialysis bag and dialyzing for 48 hours to remove free drugs and organic solvents, followed by freeze-drying and weighing, and re-dispersing with sterile PBS and storing at 4°C for later use. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a single factor screening experiment of FMN:BSA weight (w:w)

[0030] Considering the particle size, PdI and drug loading, the optimal feed ratio was finally determined to be FMN:BSA=1:20 (w:w).

[0031] Figure 2 It is the characterization of nano Chinese medicine

[0032] Figure A is the particle size distribution diagram and photo of FMN@BSA NPs

[0033] Figure B is the UV absorption spectra of FMN@BSA NPs at different concentrations

[0034] Figure C (i) is the scanning electron microscopy image of FMN@BSA NPs

[0035] (ii) Transmission electron microscopy image of FMN@BSA NPs

[0036] Note: Scale bar: 100 nm.

[0037] Figure 3 This is the result of nano-TCM inhibiting pyroptosis of macrophages in vitro

[0038] Figure A shows the exposure of PI-labeled cell contents after treatment with Control, LPS, LPS+Nigericin, LPS+Nigericin+FMN@BSA, and LPS+Nigericin+Z-VAD-FMK, and the pyroptosis ballooning of cells was observed in the bright field.

[0039] Figure B is the expression and quantitative analysis of cell pyroptosis-related proteins GSDMD-N and Cleaved-Caspase-1 after treatment with Control, LPS, LPS+Nigericin, LPS+Nigericin+FMN@BSA, and LPS+Nigericin+Z-VAD-FMK.

[0040] Note: *P<0.05, **P<0.01, ***P<0.001. Scale bar: 20 μm.

[0041] Figure 4 It is the in vivo targeting result of nano-TCM

[0042] Figure A shows the expression and quantitative analysis results of SPARC protein in lung tissues of healthy mice and PF mice. Figure B shows the fluorescence change images of DID in the main organs of PF mice after treatment with DID-FMN@BSA and DID-FMN@BSA+block.

[0043] Figure C is the fluorescence quantitative analysis of DID in major organs of living PF mice after treatment with DID-FMN@BSA and DID-FMN@BSA+block.

[0044] Note: *P<0.05, **P<0.01, ***P<0.001.

[0045] Figure 5 In vivo pharmacodynamics evaluation of nano-TCM

[0046] Figure A shows the hematoxylin-eosin (H&E) staining of mouse lung tissue after treatment with PBS, BLM, BLM-FMN@BSA, and BLM-PFD.

[0047] Figure B is the weight change curve of mice during PBS, BLM, BLM-FMN@BSA and BLM-PFD treatment. Figure C is the hydroxyproline content of mouse lung tissue after PBS, BLM, BLM-FMN@BSA and BLM-PFD treatment. Figure D is the survival curve analysis of mice within 40 days after PBS, BLM, BLM-FMN@BSA and BLM-PFD treatment. Note: *P<0.05, **P<0.01, ***P<0.001. Scale bar: 100μm. DETAILED DESCRIPTION

[0048] Example 1: Preparation and characterization of nano-traditional Chinese medicine

[0049] Nano-traditional Chinese medicine FMN@BSA NPs were prepared by inducing self-assembly of FMN and BSA by anti-solvent precipitation. First, FMN and BSA were dissolved in DMSO and ddH2O, respectively, and vortexed. Under strong stirring, the DMSO solution containing FMN (5 mg / mL) was added dropwise to the BSA aqueous solution (30 mg / mL), stirred at room temperature in the dark for 3 hours, and then 2 mL of EtOH was added to the mixture at a rate of 1 mL / min using an automatic syringe pump. After stirring for 3 hours, it was transferred to a 20 kDa dialysis bag and dialyzed for 48 hours to remove free drugs and organic solvents. Then, it was freeze-dried and weighed, and redispersed with sterile PBS and stored at 4°C for later use. FMN@BSA labeled with fluorescent dye DID was obtained by a similar procedure as above. An ethanol solution containing DID was added during the self-assembly process of FMN and BSA. The morphology of FMN@BSA NPs was then observed by transmission electron microscopy and scanning electron microscopy, respectively. The hydrodynamic diameter and PdI of FMN@BSA NPs were measured using a DLS dynamic light scattering detector (Malvern, UK). The characteristic peaks of BSA and FMN in NPs were observed at 270 and 300-330 nm, respectively, by a UV spectrophotometer (PerkinElmer, USA). In order to quantify the ability to load FMN, FMN@BSA was dissolved in a mixed solution of 2 mL DMSO / ethanol (9:1, v / v) and sonicated for 10 minutes to completely extract FMN. The concentration of FMN was detected by high performance liquid chromatography (Aglient, USA), and the loading and encapsulation efficiency were calculated.

[0050] EE (%) = (drug content in nanoparticles) / (total amount of drug input) × 100%.

[0051] LC (%) = (drug content in nanoparticles) / (total weight of nanoparticles) x 100%.

[0052] The results showed that the nano-TCM FMN@BSA NPs had uniform particle size and stable distribution, with a PdI of 0.122 and a hydrated particle size of 148.8 nm. The appearance of FMN@BSA NPs was a white emulsion, and there was an obvious Tyndall effect after laser irradiation, which was consistent with the characteristics of nano-scale particles ( Figure 2 A). UV spectrum analysis shows ( Figure 2 B), the UV absorption peak of FMN@BSA NPs at 270 nm is consistent with that of BSA, indicating that the nanoparticles have the structural characteristics of albumin, and the UV absorption band of FMN can be seen around 300-330 nm in FMN@BSA, indicating that albumin is successfully loaded with FMN. Figure 2 C shows that FMN@BSA NPs are spherical and evenly distributed under scanning electron microscopy and transmission electron microscopy, with a particle size between 120-150nm. The encapsulation efficiency and drug loading rate of FMN detected by HPLC were 52.5% and 4.75%, respectively.

[0053] Example 2: Nano-Chinese medicine inhibits pyroptosis of macrophages in vitro

[0054] We used LPS+Nigericin to stimulate BMDMs to construct a macrophage pyroptosis model and explore the inhibitory effect of FMN@BSA NPs on macrophage pyroptosis. BMDMs were placed in a confocal dish (5×10 5 Cells / dish) were added and cultured overnight at 37°C in complete medium. BMDMs were first stimulated with ultrapure LPS (100 ng / mL) for 3 hours, and the specified dose of FMN@BSA (FMN 50 μM) or positive control drug Z-VAD-FMK was added for pre-incubation for 1 hour, and finally stimulated with nigericin (10 μM) for 2 hours. Subsequently, propidium iodide PI and Hoechst 33342 dyes were used to label pyroptotic cells and cell nuclei, respectively, at room temperature. Fluorescence and bright field images were observed and photographed under a confocal microscope (Leica, Germany).

[0055] At the same time, protein immunoblotting analysis of pyroptosis-related proteins (GSDMD-N, Cleaved-Caspase-1) of BMDMs was performed in vitro. The cells treated as described above were collected and total protein was extracted with lysis buffer. Equal amounts of protein from different samples were loaded into SDS-polyacrylamide gel to separate the target proteins. The proteins were then transferred to PVDF membranes, blocked, incubated with primary antibodies overnight at 4°C, and incubated in secondary antibodies at room temperature for 2 hours. Finally, the bands were imaged using ECL immunoblotting substrates and quantified using Image J software.

[0056] The results show that: Figure 3As shown in A, we used PI staining to show pyroptotic cells, and used confocal microscopy to observe fluorescence and cell morphology under bright field. The loss of cell membrane integrity caused by LPS+Nigericin increased the number of PI-positive cells, and cell ballooning occurred. However, the number of PI-positive cells in the LPS+Nigericin+FMN@BSA group was significantly reduced, and no cell ballooning occurred. Western Blot results are shown in Figure 3 As shown in B, after LPS+Nigericin treatment, the expression levels of GSDMD-N and Cleaved-Caspase-1 increased significantly, and the expression levels decreased accordingly after pre-incubation with FMN@BSA NPs. The above results show that nano-TCM FMN@BSA NPs have the ability to inhibit macrophage pyroptosis and protect BMDMs by blocking the Caspase-1 / GSDMD-dependent pyroptosis pathway.

[0057] Example 3: In vivo targeting results of nano-TCM

[0058] Male C57 mice aged 6 to 8 weeks were used to establish a bleomycin (BLM)-induced pulmonary fibrosis model. In brief, mice were anesthetized and tracheotomized, and 50 μL of PBS solution containing BLM was dripped into the trachea using a micro-liquid atomizer, with a single dose of 2.5 U / kg. The healthy control group was given 50 μL of PBS solution in the same way (n=3). The mouse lung tissue samples were taken out on the ninth day after modeling, homogenized and protein was extracted, and the SPARC protein content was detected. The above-mentioned Western Blot method was used for detection and quantitative analysis. Another batch of PF model mice were taken and injected with 100 μL of fluorescently labeled DID-FMN@BSA NPs (DID 1 mg / mL) through the tail vein on the ninth day of modeling. At the same time, the SPARC-Block group was established as a control, and 50 μg / mouse Anti-SPARC antibody was injected intraperitoneally 1 hour in advance. The biodistribution behavior of the nanoparticles in vivo was non-invasively monitored using the IVIS small animal in vivo imaging system (PerkinElmer, USA) at 1, 2, 4, 8, 12, and 24 hours. The main organs (heart, liver, spleen, lung, and kidney) of the two groups of mice were taken in vitro 24 hours after injection to photograph the fluorescence distribution of each tissue, and the LivingImage software was used for semi-quantitative analysis.

[0059] The results showed that: Considering the pathophysiological characteristics of PF, we detected the expression of SPARC in lung tissues of healthy mice and PF model mice by Western Blot. Figure 4A shows that the content of SPARC in the lung tissue of PF model mice is significantly higher than that of healthy mice. After quantitative analysis by Image J software, the expression level is about twice that of healthy mice. The above results show that SPARC is overexpressed in PF lung tissue, which also makes the active targeting strategy of nanomedicine in PF lesions possible. IVIS small animal in vivo imaging results are shown in Figure 4 As shown in B, the fluorescence signal in the lungs of mice injected with DID-FMN@BSA gradually increased over time, and the fluorescence signal intensity reached a peak at 8h, while the accumulation and retention of particles in the lungs of mice in the DID-FMN@BSA+Block group, in which the binding between SPARC and BSA was blocked, was significantly impaired. The continuous accumulation of DID-FMN@BSA in the lung tissues of PF model mice is attributed to the high affinity between SPARC and BSA. It is worth noting that the accumulation of NPs in PF lungs was not completely eliminated by blocking the interaction between SPARC and BSA, and it may still be affected by the increased demand for albumin caused by the upregulation of metabolism in the inflammatory environment. The fluorescence distribution of each tissue after 24h is shown in Figure 2. Figure 4 As shown in C, the fluorescence intensity of the lung tissue of mice in the DID-FMN@BSA group was the highest, about 2.2 times that of the DID-FMN@BSA+Block group.

[0060] Example 4: In vivo pharmacodynamic evaluation of nano-TCM

[0061] To evaluate the effectiveness of nano-TCM as an anti-pulmonary fibrosis drug, we conducted a detailed study on the therapeutic effect of FMN@BSA NPs in a BLM-induced PF mouse model. The mice were randomly divided into four groups. The PF model mice were modeled as before, and the control group was given 50 μL PBS solution in the same way. Five days after modeling, the mice began to receive tail vein injections of FMN@BSA (FMN 10 mg / kg) and positive control drug pirfenidone (PFD 10 mg / kg) once every 2 days (n=13). On the 21st day, 5 mice in each group were sacrificed and samples were collected for further analysis. The remaining 8 mice were observed for survival until 40 days after modeling. The weight changes of mice in each group were recorded during modeling and treatment, and they were weighed every three days. The collected mouse lung tissues were fixed with 4% paraformaldehyde for more than 48 hours, embedded in paraffin, and sectioned. H&E staining was performed, and the degree of lung tissue inflammation and collagen deposition was observed using an inverted microscope. The method for detecting hydroxyproline content in the lung followed the instructions of the HYP detection kit (Nanjing Jiancheng Bioengineering Institute).

[0062] The results showed that: by scanning the H&E stained sections of the right middle lobe of the mouse lung ( Figure 5A), we found that BLM stimulation led to severe alveolar hemorrhage, edema, alveolar septal thickening and inflammatory cell infiltration, while these changes were significantly reduced in the BLM-FMN@BSA group. Figure 5 As shown in B, compared with the mice in the sham operation group, the average body weight of the BLM group decreased significantly after 21 days, from 23.6g to 17.3g. The average body weight of the model mice treated with PFD changed from 23.5g to 21.9g. Although the weight loss caused by BLM was slightly improved, the systemic condition of the mice was still poor due to the inherent gastrointestinal and hepato-renal toxicity of PFD. The average body weight of mice treated with FMN@BSA NPs did not change significantly and showed an upward trend, which was statistically different from the PFD group, proving the effectiveness and in vivo safety of FMN@BSA treatment. Hydroxyproline (HYP) has the highest content in collagen. By measuring HYP, we can understand the collagen deposition in the tissue. As an important indicator for judging the degree of fibrosis, the HYP content results are shown in Figure 2. Figure 5 As shown in C, compared with the PBS group, the HYP content in the lung tissue of mice in the BLM group increased by about 2 times to 0.72 μg / mg. After FMN@BSA treatment, the HYP content decreased to 0.48 μg / mg. The Kaplan-Meier survival curve is shown in Figure 5 As shown in D, all untreated BLM group mice suffered from acute respiratory failure and died within 32 days due to pulmonary fibrosis. Both FMN@BSA and PFD treatments significantly prolonged the survival time of PF model mice, with the survival rate of BLM-FMN@BSA mice reaching 50%, which was higher than the 25% of PFD. We speculate that the reason is that FMN@BSA has better biocompatibility. The above results all indicate that FMN@BSA NPs can reduce collagen deposition in PF lung tissue and effectively delay the progression of pulmonary fibrosis. These results support the application of FMN@BSA NPs as a potential new anti-pulmonary fibrosis drug.

Claims

1. Application of a traditional Chinese medicine nanoparticle that can effectively block macrophage pyroptosis in the preparation of a drug for the targeted treatment of pulmonary fibrosis. The traditional Chinese medicine nanoparticle uses bovine serum albumin (BSA) as a carrier and carries the natural product formononetin (FMN) to form FMN@BSANPs.

2. The use according to claim 1, wherein the FMN and BSA of the traditional Chinese medicine nanoparticles are self-assembled through intermolecular hydrogen bonding and hydrophobic forces.

3. The use according to claim 1, wherein the Chinese medicine nanoparticles are administered by intravenous injection.

4. The use according to claim 1, wherein the mass ratio of FMN to BSA in the traditional Chinese medicine nanoparticles is 1-20:10-60.

5. The use according to claim 1, wherein the traditional Chinese medicine nanoparticles FMN@BSANPs are prepared by an anti-solvent precipitation method.

6. The use according to claim 1, wherein the method for preparing the traditional Chinese medicine nanoparticles comprises the following steps: 1) Dissolve FMN and BSA in appropriate solvents respectively; 2) Add the FMN solution dropwise into the BSA solution and stir; 3) dropping an organic solvent into the mixed solution and stirring; 4) dialyzing and freeze-drying to obtain the product.

7. The use according to claim 1, wherein the preparation method of the traditional Chinese medicine nanoparticles comprises the following steps: 1) Dissolve FMN and BSA in appropriate solvents DMSO and ddH2O, respectively; 2) Add the FMN solution dropwise into the BSA solution and stir; 3) Add EtOH dropwise into the mixed solution and stir; 4) dialyze and freeze-dry to obtain the product.

8. The method for preparing the Chinese medicine nanoparticles according to any one of claims 1 to 7, comprising the following steps: 1) Dissolve FMN and BSA in appropriate solvents respectively; 2) Add the FMN solution dropwise into the BSA solution and stir; 3) dropping an organic solvent into the mixed solution and stirring; 4) dialyzing and freeze-drying to obtain the product.

9. The Chinese medicine nanoparticles obtained by the preparation method according to claim 8.

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