Metallic polyphenol nanoparticles, a preparation method thereof and application thereof in preparing a drug for treating acute lung injury

Abstract

The application provides a metal polyphenol nanoparticle, a preparation method thereof and application thereof in preparing a medicine for treating acute lung injury. The metal polyphenol nanoparticle is self-assembled from metal ions and polyphenols. The metal ions are selected from Mg 2+ , Zn 2+ , Mn 2+ or Cu 2+ . The polyphenols are selected from epigallocatechin gallate EGCG, caffeic acid CA, chlorogenic acid CGA, gallic acid GA and luteolin LUT. The metal polyphenol nanoparticle is used for preparing a medicine for treating acute lung injury induced by sepsis, pneumonia, severe trauma or inhalation injury. The medicine further comprises a pharmaceutically acceptable carrier. The metal polyphenol nanoparticle provided by the application has the advantages of simple preparation process, high biological safety, synergistically enhanced anti-inflammatory and antioxidant activities of polyphenols and metal ions, and is superior to polyphenols or metal ions alone.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to a metal polyphenol nanoparticle, a preparation method thereof and application thereof in preparing a drug for treating acute lung injury. BACKGROUND

[0002] Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) formed by its progression are common severe respiratory system emergencies in clinic, have high morbidity and mortality, and pose a major threat to global public health safety. The core pathological features of such diseases are uncontrolled inflammation in the lungs, damage to alveolar epithelial cells and lung vascular endothelial cells, protein-rich fluid exudation in the alveoli, and significant reduction in lung compliance, ultimately leading to refractory hypoxemia and respiratory failure, which seriously endangers the lives of patients.

[0003] The causes of ALI / ARDS are complex and diverse, and can be induced by sepsis, severe trauma, pneumonia, inhalation injury, acute pancreatitis and other factors, among which sepsis is one of the most important inducers. In sepsis-induced ALI, lung capillary endothelial damage leads to abnormal increase in vascular permeability, a large amount of protein-rich exudate destroys the lung epithelial cell barrier and accumulates in the alveoli, further reducing the effective ventilation area, exacerbating hypoxemia, and forming a vicious cycle of "injury-inflammation-hypoxia".

[0004] At present, the treatment of ALI / ARDS in clinic is still mainly supportive intervention, including lung-protective mechanical ventilation, prone position ventilation and neuromuscular blocker application, which has improved the prognosis of patients to some extent, but the overall mortality rate is still high, and there is a lack of specific drug treatment regimen. This situation is mainly due to the complexity of the pathogenesis of ALI / ARDS, which involves the cross-regulation of multiple signaling pathways such as uncontrolled inflammation, excessive oxidative stress, apoptosis and necroptosis. Single target intervention is difficult to effectively block disease progression, therefore, developing new treatment methods that can intervene in multiple targets of the above pathological processes has become a top priority.

[0005] Natural products have become an important direction for drug research and development due to their multi-target action characteristics, relatively low toxicity and side effects, and rich sources. Among them, epigallocatechin gallate (EGCG) as the most abundant catechin component in green tea shows significant pharmacological activities, including strong antioxidant, anti-inflammatory, anti-apoptotic and other effects, and shows great potential in ALI treatment.

[0006] However, EGCG faces significant challenges in clinical transformation, its oral bioavailability is extremely low, and the conventional administration mode is difficult to achieve effective therapeutic concentration in the lung, which greatly limits its therapeutic effect. At the same time, the existing drug delivery system for pneumonia and ALI generally has the problems of insufficient targeting, low local drug concentration, obvious systemic side effects, etc., which cannot meet the clinical needs.

[0007] At present, the aerosol inhalation treatment of acute lung injury depends on mechanical ventilation and glucocorticoids, which has obvious side effects and poor prognosis. In the research of epigallocatechin gallate (EGCG), it is found that it has low solubility and high liver toxicity when administered intravenously, and lacks lung-targeted therapeutic effect. Therefore, developing a new drug delivery system that can improve the lung-targeted delivery efficiency of active ingredients such as EGCG, enhance the local drug concentration, and reduce systemic exposure is of great significance to improve the treatment effect of pneumonia and acute lung injury (ALI). SUMMARY

[0008] Based on the above-mentioned shortcomings and deficiencies in the prior art, the purpose of the present application is to provide a metal polyphenol nanoparticle and its preparation method and application in preparing a drug for treating acute lung injury, to solve the problems of low drug delivery efficiency and insufficient targeting of existing EGCG, and to achieve efficient treatment of acute lung injury. The polyphenol component extracted from Chinese herbal medicine has obvious antioxidant effect, and the present application further improves the stability and antioxidant property of polyphenol by self-assembling polyphenol component and metal ions with anti-inflammatory effect into metal polyphenol nanoparticles. Based on the pathogenesis of acute lung injury, the present application further screens out the metal polyphenol nanoparticle formula that shows the best therapeutic effect after driving the assembly of metal ions and polyphenol under alkaline conditions, and uses the aerosol inhalation nanoparticle drug to relieve the lung microvascular barrier damage caused by acute lung injury and explore its treatment mechanism.

[0009] The technical problems to be solved by the present application are:

[0010] 1) Exploration of the best metal polyphenol nanoparticle formula for treating acute lung injury after polyphenol and metal ions are complexed to form nanoparticles under alkaline conditions.

[0011] 2) Advantages of drug delivery in the form of nanoparticles compared to administration of polyphenol and metal ions alone.

[0012] 3) Mechanism research of nanoparticle treatment of acute lung injury.

[0013] Based on the above-mentioned objectives and technical problems to be solved, the present application provides the following technical solutions:

[0014] Technical solution 1 of the present application, a metal polyphenol nanoparticle, which is self-assembled from metal ions and polyphenol, generally formed by complexation under alkaline conditions.

[0015] the metal ion is selected from Mg 2+ ion, Zn 2+ ion, Mn 2+ ion or Cu 2+ ion.

[0016] the polyphenol is selected from any one of epigallocatechin gallate EGCG, caffeic acid CA, chlorogenic acid CGA, gallic acid GA, luteolin LUT.

[0017] Further, in some embodiments of the present application, the metal polyphenol nanoparticle is most preferably a combination of epigallocatechin gallate EGCG and Mg 2+ ion, i.e. an EGCG-Mg nanoparticle is provided; the EGCG-Mg nanoparticle is spheroid or spherical, with a particle size concentrated at 45-100 nm, a PDI of 0-0.25, and a potential distribution of -2-1.5 mV.

[0018] The technical solution 2 of the present application provides a preparation method of the above metal polyphenol nanoparticle, comprising the following steps:

[0019] 1) dissolving a polyphenol powder with an alkaline solution to obtain a polyphenol solution, and dissolving a metal salt with double-distilled water to obtain a metal ion solution; the final concentration of the polyphenol solution and the metal ion solution is 20-24 mM;

[0020] the alkaline solution is preferably a sodium hydroxide solution; the final concentration is preferably 24 mM; when the metal ion is Mg 2+ ion, the metal salt is preferably magnesium chloride hexahydrate;

[0021] 2) mixing equal volumes of the polyphenol solution and the metal ion solution, and oscillating the reaction;

[0022] 3) centrifuging, discarding the supernatant, adding double-distilled water to redissolve the precipitate, and dispersing by probe ultrasonic;

[0023] 4) repeating the operation of step 3), adding double-distilled water, physiological saline or culture medium after discarding the supernatant, and dispersing by water bath ultrasonic to obtain the metal polyphenol nanoparticle.

[0024] The technical solution 3 of the present application provides the use of the metal polyphenol nanoparticle or the EGCG-Mg nanoparticle in the preparation of a drug for treating acute lung injury (ALI).

[0025] The acute lung injury is induced by sepsis, pneumonia, severe trauma or inhalation injury.

[0026] The fourth technical solution of the present application provides a medicine for treating acute lung injury, wherein the medicine comprises metal polyphenol nanoparticles or EGCG-Mg nanoparticles and a pharmaceutically acceptable carrier.

[0027] The pharmaceutically acceptable carrier comprises at least one of a dispersing agent, a stabilizer, a diluent or a preservative.

[0028] The medicine is an inhalant.

[0029] In some embodiments of the present application, the medicine for treating acute lung injury, in particular, is used for reducing the concentration of myeloperoxidase (MPO) in lung tissue, bronchoalveolar lavage fluid (BALF) and serum of an acute lung injury model animal, so as to reduce inflammatory cell infiltration in lung tissue.

[0030] In some embodiments of the present application, the medicine for treating acute lung injury, in particular, is used for inhibiting the expression and secretion of inflammatory factors in an acute lung injury model animal, wherein the inflammatory factors include TNF-α, IL-1β, IL-6 and sICAM-1.

[0031] In some embodiments of the present application, the medicine for treating acute lung injury, in particular, is used for improving the activity of antioxidant enzymes and the content of antioxidant substances in lung tissue of an acute lung injury model animal, wherein the antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx), and the antioxidant substance is glutathione (GSH).

[0032] In some embodiments of the present application, the medicine for treating acute lung injury, in particular, is used for reducing the content of malondialdehyde (MDA) and the generation of reactive oxygen species (ROS) in lung tissue of an acute lung injury model animal, so as to reduce oxidative stress damage of lung tissue.

[0033] In some embodiments of the present application, the medicine for treating acute lung injury, in particular, is used for reducing the cell concentration and protein concentration in bronchoalveolar lavage fluid of an acute lung injury model animal, and reducing the dry / wet ratio of lung tissue, so as to reduce the destruction of lung microvascular barrier and pulmonary edema.

[0034] In summary, the present application uses four divalent metal ions (Mn 2+ , Zn 2+ , Mg 2+ , Cu 2+) 20 kinds of metal polyphenol nanoparticles were constructed with 5 kinds of polyphenols (such as epigallocatechin gallate EGCG, chlorogenic acid CGA, caffeic acid CA, gallic acid GA and luteolin LUT), and through layer-by-layer screening, it was finally found that the nanoparticles composed of EGCG and magnesium (EM) could reflect the highest antioxidant properties and the lowest cytotoxicity. Through optimization of the preparation scheme, while maintaining the dispersion stability (PDI) of the nanoparticles in the solvent, a simpler and higher-yield preparation scheme was adopted, which was more conducive to clinical transformation and quantitative production. In in vitro cell experiments, the inventors fully proved that the EM nanoparticles had better anti-inflammatory, antioxidant capacity and higher uptake efficiency than free EGCG due to synergistic effect. Finally, the inventors adopted the way of aerosol inhalation in the acute lung injury (ALI) model of mice, so that the EM nanoparticles could be delivered to the lungs of mice efficiently, which not only could prolong the action time and reduce the frequency of administration, but also could avoid the problems of liver toxicity, poor lung targeting and low bioavailability of EGCG when administered intravenously.

[0035] Compared with the prior art, the present application has at least the following improvements and beneficial effects:

[0036] (1) The nanoparticles are directly targeted to the lungs by aerosol inhalation, which improves the local drug concentration and reduces the systemic side effects;

[0037] (2) The polyphenol (preferably EGCG) and the metal ion (preferably Mg 2+ ) synergistically enhance the anti-inflammatory and antioxidant activities, which are superior to the use of EGCG or Mg 2+ alone;

[0038] (3) The preparation process is simple, does not require carrier materials, and has high biological safety.

[0039] (4) Compared with the preparation process of the metal polyphenol nanoparticles reported in the prior art, the present application designs a simpler preparation method for metal polyphenol nanoparticles, and through small-scale metal polyphenol nanoparticle screening, the metal polyphenol nanoparticles with the best anti-inflammatory and antioxidant status after the structure change of the phenolic hydroxyl group by deprotonation and complexation with metal ions are found. In addition, the nanoparticles prepared by the inventors have better stability and dispersibility than free polyphenol molecules, which are more conducive to the treatment of acute lung injury by aerosol inhalation in the form of nano-inhaler. By reasonably controlling the particle size of the nanoparticles, the inventors prolong the residence time of the nanoparticles in the lung tissue, avoid the problems of low drug bioavailability and drug restrictions for patients with liver dysfunction caused by intravenous administration, and enable the nanoparticles to fully and directly play a role in the lesion. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1Preparation, characterization, and screening of 20 metal polyphenol nanoparticles; Figure labels: Figure 1 In section A, the preparation process of metal polyphenol nanoparticles using mPEG-PLA nanoparticles as templates is described. Figure 1 B represents the particle size analysis of 20 types of metal polyphenol nanoparticles. Figure 1 C represents the potential test. Figure 1 D in the middle represents the PDI test. Figure 1 E represents the DPPH antioxidant activity assay. Figure 1 F represents the antioxidant activity assay using ABTS. Figure 1 G represents the cytotoxicity assay of metal polyphenol nanoparticles;

[0041] Figure 2 Characterization of EM nanoparticles; Figure labels: Figure 2 In section A, the preparation process of EM nanoparticles is described. Figure 2 B in the figure represents EM nanoparticles measured by TEM. Figure 2 C represents the particle size analysis results of EM nanoparticles. Figure 2 The image shows the XPS test results for EM nanoparticles and EGCG. Figure 2 The EPR test of EF in the middle part represents the EPR of EM nanoparticles, EGCG, and MgCl2. Figure 2 GH was determined by DPPH and ABTS of EM nanoparticles. Figure 2 In the middle, I represents the catalase (CAT) mimicry activity of EM nanoparticles; Figure 2 J represents the superoxide dismutase (SOD) mimicry activity of EM nanoparticles;

[0042] Figure 3 Results of cellular uptake of EM nanoparticles;

[0043] Figure 4 Screening results of in vitro anti-inflammatory and antioxidant concentrations of nanoparticles; Figure labels: Figure 4 In the middle A section, a bar chart shows the TNF-α concentration in the HUVEC inflammation model after nanoparticle intervention. Figure 4 In the middle B section, there is a bar chart showing the G-CSF concentration in the HUVEC inflammation model after nanoparticle intervention. Figure 4 In the figure, C represents the ROS generation level in the HUVEC oxidative stress model after nanoparticle intervention; Figure 4 In the middle, D is a fluorescence staining image of the total antioxidant capacity of cells after nanoparticle intervention;

[0044] Figure 5 Results of in vivo concentration screening for anti-inflammatory and antioxidant therapy using nanoparticles; Figure labels: Figure 5 In the middle A section, there is a bar chart showing the concentration of IL-1β in mouse BALF solution; Figure 5 B: Bar chart of IL-6 concentration in mouse BALF solution; Figure 5 C: Bar chart of IL-6 concentration in mouse serum; Figure 5D: Bar chart of MDA content in mouse lung tissue; Figure 5 Middle E: Bar chart of SOD activity in mouse lung tissue; Figure 5 F: Bar chart of GSH content in mouse lung tissue;

[0045] Figure 6 Biosafety of nanoparticles and their components for HUVECs; Figure labels: Figure 6 In the middle A section, there is a bar chart showing the proliferation rate of HUVEC cells under a nanoparticle concentration gradient. Figure 6 B represents EM nanoparticles and their components (EGCG, Mg). 2+ HUVEC cell proliferation rate bar chart under concentration gradient;

[0046] Figure 7 In vivo experimental biosafety; Figure labels: Figure 7 In the middle, A represents a bar chart showing the level of mouse red blood cells (RBCs); Figure 7 The bar chart in section B shows the mouse hemoglobin (HGB) level. Figure 7 The bar chart in the middle (C) shows the level of mouse neutrophils (Neu). Figure 7 The bar chart in section D shows the mouse urea (a renal function indicator) level. Figure 7 The bar chart in section E shows the levels of alanine aminotransferase (ALT, a liver function indicator) in mice. Figure 7 The bar chart in the middle (F) shows the levels of aspartate aminotransferase (AST, a liver function indicator) in mice.

[0047] Figure 8 Validation of the anti-inflammatory effect of nanoparticles and their components on a human umbilical vein endothelial cell inflammation model (RT-qPCR and ELISA detection); Figure labels: Figure 8 In the middle A bar chart, the relative expression level of TNF-α mRNA in HUVECs is shown. Figure 8 The bar chart in section B shows the relative expression levels of IL-6 mRNA in HUVECs. Figure 8 The bar chart in C represents the relative expression level of sICAM-1 mRNA in HUVECs; Figure 8 The bar chart in section D shows the ELISA detection of TNF-α concentration in cell supernatant. Figure 8 E in the middle is a bar chart of ELISA detection of IL-6 concentration in cell supernatant; Figure 8 The bar chart in Figure F shows the concentration of sICAM-1 in cell supernatant as detected by ELISA.

[0048] Figure 9 Nanoparticle antioxidant cell experiments; Figure labels: Figure 9 Image A in the image is a fluorescence microscopy observation of ROS in the HUVEC oxidative stress model. Figure 9 B is a bar chart of catalase (CAT) activity in HUVECs; Figure 9Figure 7C is a bar graph of glutathione peroxidase (Gpx) activity in HUVECs. Figure 9 Figure 7D is a bar graph of glutathione (GSH) content in HUVECs. Figure 9 Figure 7E is a bar graph of glutathione (GSH) content in HUVECs. Figure 9 Figure 7F is a schematic diagram of the synergistic mechanism of intracellular antioxidant enzymes.

[0049] Figure 10 Figure 8A is a schematic diagram of the inhalation procedure for mice. Figure 10 Figure 8B is a schematic diagram of the inhalation procedure for mice. Figure 10 Figure 8C is a schematic diagram of the inhalation procedure for mice. Figure 10 Figure 8D is a schematic diagram of the inhalation procedure for mice. Figure 10 Figure 8E is a schematic diagram of the inhalation procedure for mice. Figure 10 Figure 8F is a schematic diagram of the inhalation procedure for mice.

[0050] Figure 11 Figure 9A is a fluorescence microscope observation of DHE staining of lung tissue in mice. Figure 11 Figure 9B is a bar graph of the content of malondialdehyde (MDA) in lung tissue in mice. Figure 11 Figure 9C is a bar graph of the activity of superoxide dismutase (SOD) in lung tissue in mice. Figure 11 Figure 9D is a bar graph of the activity of catalase (CAT) in lung tissue in mice. Figure 11 Figure 9E is a bar graph of the content of glutathione (GSH) in lung tissue in mice. Figure 11 Figure 9F is a bar graph of the activity of glutathione peroxidase (Gpx) in lung tissue in mice. Figure 11

[0051] Figure 10A is a bar graph of the concentration of MPO in lung tissue, bronchoalveolar lavage fluid, and serum. Figure 12 Figure 10B is a bar graph of the concentration of cells in bronchoalveolar lavage fluid. Figure 12 Figure 10C is a bar graph of the concentration of protein in bronchoalveolar lavage fluid. Figure 12 Figure 10D is a bar graph of the dry / wet ratio of the lung. Figure 12 DETAILED DESCRIPTION Figure 12

[0052] ​​In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present application. These all belong to the protection scope of the present application. All raw materials of the present application, if not specially specified, represent that there is no special limitation on their sources, and they can be purchased on the market or prepared according to the conventional methods well known to those skilled in the art.

[0053] The embodiments of the present application are divided into the following parts:

[0054] First part: synthesis, characterization and screening of metal polyphenol nanoparticles in alkaline environment.

[0055] The synthesis conditions capable of preparing nanoparticles with target particle size (50-150 nm) are explored, and the nanoparticles are characterized by particle size determination and nanoparticle potential determination. In order to exclude the interference caused by different synthesis processes, under the unified synthesis conditions, the inventors synthesized 20 kinds of nanoparticles, and further screened the metal polyphenol nanoparticle formula with the best anti-inflammatory and antioxidant function in vitro experiments by DPPH and ABTS methods not relying on cells, and Human TNF-α ELISA, Human G-CSF ELISA, superoxide dismutase (SOD) and ROS fluorescence intensity determination relying on in vitro inflammatory cell model and oxidative stress cell model.

[0056] Second part: in vitro and in vivo anti-inflammatory and antioxidant experimental verification of metal polyphenol nanoparticles.

[0057] In the screening of the first part, the inventors have a preliminary understanding of the anti-inflammatory and antioxidant function of epigallocatechin gallate EGCG-Mg nanoparticles in vitro experiments. In the cell experiment of the second part, the inventors further explore whether the relieving effect of nanoparticles on cell inflammation and oxidative stress is stronger than that of epigallocatechin gallate (EGCG) and MgCl2 alone by establishing cell inflammation model and oxidative stress model. Since the in vivo environment of drugs is quite different from that in vitro, the inventors use LPS tracheal instillation method to establish a mouse acute lung injury model, and determine whether the anti-inflammatory and antioxidant effect of nanoparticles is better than that of epigallocatechin gallate (EGCG) and MgSO4 alone in the in vivo environment by measuring inflammatory factors in mouse serum and lung lavage fluid, and the expression of antioxidant-related enzymes in lung tissue.

[0058] Third part: verification of the therapeutic effect of metal polyphenol nanoparticles.

[0059] Inflammation of lung microvascular endothelial cells and oxidative stress stimulation are important factors for the increase of endothelial cell permeability, and further development of lung tissue inflammation factor infiltration, and eventually lead to the occurrence of pulmonary edema. In this part, the inventors further studied whether the expected therapeutic effect could be produced after the nano particles were inhaled by the acute lung injury model mice, that is, whether the anti-inflammatory and antioxidant properties of the nano particles had the ability to reverse the further development of acute lung injury. Therefore, the inventors determined the therapeutic effect of the nano particles by determining the concentration of myeloperoxidase (MPO) in the lung tissue and alveolar lavage fluid, HE staining and other methods.

[0060] Specifically, the present application provides the following examples:

[0061] Example 1: Preparation and characterization of metal polyphenol nanoparticles

[0062] (1) Nanoparticle synthesis and anti-inflammatory and antioxidant experiment screening:

[0063] In this example, five polyphenols (epigallocatechin gallate EGCG, caffeic acid CA, chlorogenic acid CGA, gallic acid GA, and luteolin LUT) and four ions (magnesium ion, copper ion, manganese ion, and zinc ion) were selected to coat the metal polyphenol network on the mPEG-PLA nanoparticles to prepare 20 different formulations of metal polyphenol nanoparticles. The specific method is as follows:

[0064] 1) Preparation of mPEG-PLA carrier: 20 mg of mPEG-PLA was dissolved in 1 mL of dichloromethane and 3 mL of 1% sodium cholate solution, and was emulsified into mPEG-PLA nanoparticles by 500w probe ultrasonic, then was placed in a 50 mL beaker, 40 mL of 0.5% sodium cholate solution was added to the beaker, and was ventilated under the fume hood for 1 hour. Then it was taken out and centrifuged in a centrifuge tube. The precipitate after centrifugation was the mPEG-PLA nanoparticle carrier.

[0065] 2) Take 1 mL of mPEG-PLA nanoparticle carrier, add 100 μL of metal ion solution and 100 μL of polyphenol solution, shake for 30 min, then centrifuge, and take the precipitate after centrifugation as the metal-polyphenol mPEG-PLA nanoparticle.

[0066] The combination of epigallocatechin gallate and magnesium ion was used as Example 1, and the other combinations were used as Comparative Examples 1-19. The specific preparation process is shown in Table 1 below. Figure 1

[0067] Table 1 is the preparation of 20 kinds of metal polyphenol nanoparticles

[0068]

[0069] ​(2) Characterization of nanoparticles:

[0070] The particle size, potential, and PDI of the 20 nanoparticles were characterized. Antioxidant activity of the 20 nanoparticles was preliminarily assessed using non-cellular experiments with ABTS and DPPH assays. The biosafety of different nanoparticles on human umbilical vein endothelial cells (HUVECs) at a uniform polyphenol concentration was determined using a CCK-8 assay to detect cell proliferation rate.

[0071] The measurement results are as follows Figure 1 As shown: Figure 1 As shown in Figure B, the particle size of 20 types of nanoparticles was measured, such as... Figure 1 As shown in Figure C, the potential of the nanoparticles was measured, as follows. Figure 1 As shown in Figure D, the PDI of the nanoparticles was measured to preliminarily understand their dispersion stability. The results show that the particle size distribution of the 20 nanoparticles ranged from 60 to 140 nm, the potential distribution ranged from -2 to 1.5 mV, and the PDI distribution ranged from approximately 0 to 0.25. Figure 1 As shown in Figure EF, the polyphenols in the nanoparticles were quantified using the DPPH and ABTS methods, respectively. The polyphenol concentration of each nanoparticle was 12 μg / mL (which can be understood as quantification based on polyphenol concentration, i.e., the concentration of the nanoparticle in the solvent is 12 μg / mL, the same below). The antioxidant activity of the nanoparticles was preliminarily identified and screened in a cell-free system. It can be seen that all 20 nanoparticles exhibited certain antioxidant activity, with EGCG and Mg showing relatively higher activity. 2+ The antioxidant effect is best when coated on the surface of mPEG-PLA. For example... Figure 1 As shown in Figure G, the polyphenols in the nanoparticles were quantified using the CCK-8 assay, with a polyphenol concentration of 60 μg / mL for each nanoparticle. HUVEC was used for preliminary identification of the nanoparticle cytotoxicity. Based on the above results, the preliminary screening results indicate that EGCG and Mg... 2+ When coated on the surface of mPEG-PLA, it exhibits the highest antioxidant activity and the lowest cytotoxicity. Figure 2 In EG (a traditional Chinese medicine), the more yellow the color, the better the effect.

[0072] Example 2: Optimization of the preparation process

[0073] In this embodiment, the mPEG-PLA nanoparticles inside the previously prepared metal polyphenol nanoparticles were removed, and a nanoparticle composed solely of Mg was prepared. 2+ Metal polyphenol nanoparticles can be formed by combining epigallocatechin gallate (EGCG) under alkaline conditions. This step improves the preparation scheme, making the preparation process simpler and the nanoparticle composition purer.

[0074] The specific preparation steps are as follows:

[0075] 1) EGCG solution was obtained by dissolving epigallocatechin gallic acid (EGCG) powder in 66 mM NaOH. MgCl2 solution was obtained by dissolving magnesium chloride hexahydrate (MgCl2·6H2O) powder in double-distilled water (ddH2O). The final concentration of both solutions after dissolution was 24 mM.

[0076] 2) Add 500 μL of EGCG solution to a 1.5 mL centrifuge tube, then add an equal volume of MgCl2 solution. Place the centrifuge tube on a shaker and shake for 30 min to promote the reaction.

[0077] 3) Place the centrifuge tubes in a centrifuge and centrifuge under the following conditions: room temperature, 9400g, 10min;

[0078] 4) After centrifugation, discard the supernatant, take the precipitate at the bottom, and add 1 mL of double-distilled water to the centrifuge tube;

[0079] 5) Use a probe ultrasonic machine to promote the dispersion of the bottom sediment into the water;

[0080] 6) After dispersing, centrifuge again under the same conditions as above;

[0081] 7) After centrifugation, discard the supernatant and add double-distilled water (or physiological saline, culture medium) to the precipitate at the bottom. After sealing the centrifuge tube with sealing film, perform water bath sonication to promote the dispersion of the precipitate at the bottom in the solvent, and finally obtain EGCG-Mg nanoparticles.

[0082] The measurement results are as follows Figure 2 As shown:

[0083] like Figure 2 As shown in Figure A, the preparation process of the nanoparticles was optimized by adjusting the alkaline environment of the preparation process to remove the mPEG-PLA support, thus forming EGCG-Mg nanoparticles (EM for short). Figure 2 As shown in Figure B, the morphology of EMs was observed using transmission electron microscopy (TEM). The EMs exhibit a near-spherical or spherical structure with relatively uniform particle size and no obvious aggregation, indicating that the EMs have a relatively regular morphology and good dispersibility. Figure 2 As shown in Figure C, statistical analysis of the particle size of EM revealed that the nanoparticles are mainly distributed in the range of 30-120 nm, with a concentration in the 45-100 nm range, exhibiting a unimodal distribution characteristic. Figure 2 As shown in Figure D, X-ray photoelectron spectroscopy (XPS) was used to compare EM with EGCG, revealing that the surface of EM nanoparticles also contained Mg. 2+ Characteristic peaks indicate the presence of Mg on the surface of EM nanoparticles. 2+ ;like Figure 2The hydroxyl radical and singlet oxygen scavenging effects of the EM nanoparticles were compared with EGCG and MgCl2 by electron paramagnetic resonance (EPR) as shown in E-F. The amplitude of the spectrum was analyzed to show that the amplitude of the EM nanoparticles was smaller than that of EGCG and MgCl2, which indicates that the scavenging efficiency of the hydroxyl radical and singlet oxygen of the EM in unit time is higher than that of EGCG and MgCl2. As shown in Figure 2 As shown in G-H, the antioxidant activity of the EM nanoparticles was found to be related to the polyphenol concentration in the nanoparticles by DPPH and ABTS assays in a cell-free system, and the antioxidant activity of the nanoparticles increased with the increase of the polyphenol concentration in the nanoparticles. As shown in Figure 3 As shown in I-J, the antioxidant activity of the EGCG-Mg nanoparticles was determined, which shows the antioxidant biological function of the EM.

[0084] In summary, by adjusting the alkaline environment of the preparation process and removing the mPEG-PLA carrier, the EGCG-Mg nanoparticles (EM) were successfully prepared.

[0085] Example 3 is a Cy5-labeled EM nanoparticle human umbilical vein endothelial cell (HUVEC) uptake experiment

[0086] First, Cy5-labeled epigallocatechin gallate (EGCG)-Mg nanoparticles were prepared, and then the Cy5-labeled nanoparticles were introduced into human umbilical vein endothelial cells for 1 hour, 3 hours, 6 hours, and 9 hours, and the uptake of the nanoparticles by the human umbilical vein endothelial cells was observed under a fluorescence microscope.

[0087] As shown in Figure 3 The Cy5-labeled nanoparticles were introduced into human umbilical vein endothelial cells for 1 hour, 3 hours, 6 hours, and 9 hours, and the uptake of the nanoparticles by the human umbilical vein endothelial cells was observed. It can be seen that the human umbilical vein endothelial cells have good uptake of the Cy5-labeled nanoparticles, and Figure 4 From the fluorescence intensity analysis, it can be seen that the endothelial cells take up more and more nanoparticles over time, indicating that the uptake of the nanoparticles by the cells is time-dependent.

[0088] Example 4: Selection of treatment concentration of nanoparticles in in vitro and in vivo experiments and verification of biological safety (cell + mouse level)

[0089] (1) Selection of treatment concentration of nanoparticles in in vitro experiments:

[0090] 1) Human umbilical vein endothelial cell inflammation model: human umbilical vein endothelial cells (HUVEC) were stimulated with 1 μg / mL LPS (0111: B4) for 4 hours to establish a human umbilical vein endothelial cell inflammation model. One hour before modeling, according to the therapeutic dose in previous studies, three treatment doses (EM1: 30 μg / mL, EM2: 60 μg / mL, EM3: 90 μg / mL) of nanoparticles were selected and added to the human umbilical vein endothelial cell inflammation model.

[0091] Determination of TNF-α, G-CSF concentration: after adding LPS for 4 hours, the cell supernatant was taken, and the concentration of TNF-α and G-CSF in the cell supernatant of the control group, LPS group, EM1 group, EM2 group, and EM3 group was determined by human Human TNF-α ELISA and Human G-CSF ELISA kit, respectively.

[0092] 2) Human umbilical vein endothelial cell oxidative stress model: human umbilical vein endothelial cells (HUVEC) were stimulated with 1 μg / mL LPS for 24 hours to establish a human umbilical vein endothelial cell oxidative stress model. One hour before modeling, according to the therapeutic dose in previous studies, three treatment doses (EM1: 30 μg / mL, EM2: 60 μg / mL, EM3: 90 μg / mL) of nanoparticles were selected and added to the human umbilical vein endothelial cell oxidative stress model.

[0093] Extraction of protein and determination of SOD activity: after adding LPS for 24 hours, the protein in the cells was extracted on ice with cell lysis solution (containing protease inhibitor and phosphatase inhibitor). In order to calculate the content of superoxide dismutase (SOD) in unit protein next, the protein needs to be quantified by BCA method first, and then the content of SOD in the control group, LPS group, EM1 group, EM2 group, and EM3 group is determined by superoxide dismutase (SOD) detection kit, and then divided by the protein mass, so as to obtain the expression of SOD in unit mass of protein.

[0094] Determination of active oxygen level: after adding LPS for 24 hours, the supernatant of each group was removed, washed with PBS for 2 times, and then DCFH-DA fluorescent dye was added. After incubation in the incubator for 30 minutes, the DCFH-DA fluorescent dye was removed, washed with PBS for 2 times, and then the well plate was placed under the fluorescence microscope to observe the fluorescence intensity of each group of cells.

[0095] Experimental results: the optimal anti-inflammatory concentration in vitro is as follows Figure 4As shown in the middle A-B, by quantifying the polyphenol concentration in the nanoparticles, anti-inflammatory experiments were performed on HUVEC inflammatory models using nanoparticles with polyphenol concentrations of 30, 60, and 90 μg / mL. The contents of TNF-α and G-CSF in the cell supernatant were determined using the HUMAN TNF-α ELISA kit and the HUMAN G-CSF ELISA kit, and it was found that the anti-inflammatory effect of the nanoparticles was dose-dependent on the polyphenol concentration in the nanoparticles. The optimal concentration for in vitro antioxidant experiments is as follows Figure 4 As shown in the middle C, in order to determine the appropriate antioxidant nanoparticle concentration and the relationship between the antioxidant effect of the nanoparticles and the polyphenol concentration thereof, a HUVEC oxidative stress model was established, the polyphenol in the nanoparticles was quantified, and the anti-cell ROS generation level was determined by grouping. The fluorescence intensity of ROS was observed by fluorescence microscopy to evaluate the antioxidant degree of the nanoparticles at different concentrations. It was observed by fluorescence intensity that the antioxidant property of the nanoparticles in the cells increased with the increase of the polyphenol concentration in the nanoparticles. Figure 5 As shown in the middle D, the total antioxidant capacity of the cells was determined after the cells were treated with nanoparticles at different concentrations.

[0096] (2) Selection of nanoparticle treatment concentration in in vivo experiments:

[0097] 6-8 weeks old C57BL / 6 mice were purchased and LPS (0111: B4) 5 mg / kg was dropped into the trachea to establish a mouse acute lung injury model. When modeling, the water column method was used to verify that the LPS was dropped into the trachea. Specifically, a retention needle was used to cannulate the trachea of the mouse, and then the metal needle core was removed and a 1 mL syringe with a 50 μL water column was installed on the retention needle. If the retention needle and the syringe are installed well, the water column in the syringe moves up and down with the breathing of the mouse, indicating that the retention needle is in the trachea of the mouse. Grouping: control group, LPS group, EM1 group, EM2 group, and EM3 group (EM1: 10 mg / kg, EM2: 30 mg / kg, EM3: 60 mg / kg).

[0098] After LPS was dropped into the trachea for 1 hour, different concentrations of nanoparticles were inhaled by atomization for the first time to treat acute lung injury mice; after LPS was dropped into the trachea for 12 hours, different concentrations of nanoparticles were inhaled by atomization for the second time to treat acute lung injury mice; after LPS was dropped into the trachea for 24 hours, the serum, alveolar lavage fluid, and lung tissue of the mice in each group were taken. The concentration of IL-6 in the serum was determined using the Mouse IL-6 ELISA kit, and the concentrations of TNF-α, IL-1β, and IL-6 in the alveolar lavage fluid were determined using the Mouse TNF-α ELISA kit, the Mouse IL-1β ELISA kit, and the Mouse IL-6 ELISA kit to determine the anti-inflammatory effect of different nanoparticle concentrations.

[0099] Lung tissue was divided into left and right lungs. The left lung was immersed in 4% paraformaldehyde for HE staining; the right lung was placed in a tissue homogenizer tube, and 1 mL of cell lysis buffer (containing protease inhibitors and phosphatase inhibitors) was added. The tube was then placed in a tissue homogenizer and homogenized at 4°C. After homogenization, the tissue homogenate was centrifuged at 12,000 rpm at 4°C for 15 minutes, and the supernatant was transferred to a new centrifuge tube. The homogenate was quantified using the BCA method, and the expression levels of SOD, MDA, and GSH per unit mass of protein in each group were determined using a superoxide dismutase (SOD) assay kit, a malondialdehyde (MDA) assay kit, and a glutathione (GSH) assay kit. Based on these findings, an appropriate therapeutic concentration of anti-inflammatory and antioxidant nanoparticles was determined.

[0100] Experimental results: such as Figure 5 The figure shows the optimal concentration for anti-inflammatory and antioxidant effects in in vivo experiments. Figure 5 Figures A and B show the determination of IL-1β and IL-6 in mouse BALF solution. Figure 5 The value of IL-6 in mouse serum was measured in the middle C. Analysis showed that nanoparticle nebulization at a concentration of 60 mg / kg could effectively and stably exert anti-inflammatory effects in an ALI mouse model. Figure 6 In Figures D, E, and F, the levels of MDA (malondialdehyde), superoxide dismutase (SOD), and glutathione (GSH) in the lung tissue of an ALI mouse model were measured. Analysis revealed that nanoparticles at concentrations of 10, 30, and 60 mg / kg all exhibited good antioxidant effects in mouse lung tissue, but the nanoparticles at a concentration of 60 mg / kg showed the best antioxidant effect.

[0101] (3) Biosafety verification of nanoparticles in in vitro experiments:

[0102] At appropriate therapeutic concentrations, it is necessary to understand the biosafety of drugs in cell experiments to avoid cytotoxic effects caused by drug administration. This experiment used the CCK-8 assay to determine the effect of nanoparticles at different concentrations on the proliferation of human umbilical vein endothelial cells (HUVECs). Different concentrations (10, 30, 60, 90, 120, 150 μg / mL) of nanoparticles and their components (EGCG, MgCl2) were added to HUVECs and cultured in an incubator for 24 hours. The cell proliferation rate was measured to determine the cytotoxic effects of different nanoparticles and their components on HUVECs.

[0103] Experimental results: such as Figure 6 The image shows the biosafety determination of nanoparticles and their components in HUVEC. Figure 6 The cell proliferation rate of HUVEC was determined by the CCK-8 assay, and it was found that nanoparticles at concentrations of 90 μg / mL and below had good safety for HUVECs.Figure 7 In the middle B is the CCK-8 method to determine the biological safety of different nanoparticle concentrations and their components, found that the concentration of polyphenols in nanoparticles is 90 μg / mL and below have good safety on HUVEC.

[0104] (4) Nanoparticle in vivo experiment biological safety verification:

[0105] At the appropriate therapeutic concentration, it is necessary to understand the biological safety of drugs in animal experiments to avoid the occurrence of systemic toxicity due to drug use. 6-8 weeks of male C57BL / 6 mice were grouped: control group, EM group, EGCG group, MgSO4 group. The therapeutic concentration of EM, EGCG, MgSO4 was used for aerosol inhalation administration in mice, and aerosolized twice within 24 hours, i.e. every 12 hours. After 24 hours, the heart, spleen, lung, liver, kidney, whole blood, serum of each group of mice were taken out for liver function, kidney function, blood routine test and HE staining observation of important organs.

[0106] The experimental results are shown in Table 1 as follows: Figure 7 The biological safety of nanoparticles and their components in mice. Figure 7 In the middle A-C is the blood biochemical test, found that after aerosol inhalation of nanoparticles, compared with the control group, the number of red blood cells, hemoglobin and neutrophils did not change significantly. Figure 7 In the middle D is the kidney function test, after analysis found that after aerosol inhalation of nanoparticles, it will not cause damage to the kidney function of mice. Figure 8 In the middle E-F is the liver function test, after the determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) analysis found that nanoparticles did not cause damage to the liver function of mice.

[0107] Example 5 is the anti-inflammatory and antioxidant effect verification of EM nanoparticles in HUVEC inflammation and oxidative stress model (compared with single component)

[0108] (1) In the human umbilical vein endothelial cell inflammation model, the anti-inflammatory experiment verification of nanoparticles:

[0109] After adding the nanoparticles for 1 hour, the human umbilical vein endothelial cells were stimulated with 1 μg / mL LPS for 4 hours to establish the inflammation model. The cells were divided into control group, LPS group, EM group, EGCG group, and MgCl2 group. After adding 1 μg / mL LPS to each group for 4 hours, the cell supernatant was taken, and the concentration of inflammatory factors (TNF-α, IL-6, and sICAM-1) in the cell supernatant was determined by ELISA kit. At the same time, the mRNA in each group of cells was extracted to determine the expression of inflammatory factors (TNF-α, IL-6, and sICAM-1) in human umbilical vein endothelial cells by RT-qPCR.

[0110] Experimental results: Figure 8 Fig. 4A-C: mRNA in HUVEC cells was extracted, and the expression of inflammatory-related factors (TNF-α, IL-6, and sICAM-1) in the cells was detected by RT-qPCR. Figure 9 Fig. 4D-F: The cell culture supernatant was taken, and the secretion of TNF-α, IL-6, and sICAM-1 in the supernatant was determined by ELISA kit to analyze the anti-inflammatory effect of different treatment methods. The statistical results show that the anti-inflammatory effect of EM nanoparticles is better than that of its components.

[0111] (2) In the oxidative stress model of human umbilical vein endothelial cells, the antioxidant experiment of nanoparticles verifies:

[0112] After adding the nanoparticles for 1 hour, the human umbilical vein endothelial cells were stimulated with 1 μg / mL LPS for 24 hours to establish the oxidative stress model. The cells were divided into control group, LPS group, EM group, EGCG group, and MgCl2 group. After adding 1 μg / mL LPS to each group for 24 hours, the supernatant was discarded, and the cells were washed twice with PBS, and the cells were lysed on ice with cell lysis solution to extract the protein in the cells. The protein concentration of each group was quantified by BCA method. Then the superoxide dismutase (SOD) detection kit, catalase (CAT) detection kit, glutathione peroxidase (Gpx), and glutathione (GSH) detection kit were used to detect the concentrations of superoxide dismutase, catalase, glutathione, and glutathione peroxidase. The expression content of SOD, CAT, GSH, and Gpx in each group was calculated, and it was determined whether the antioxidant performance of the nanoparticles was better than that of epigallocatechin gallate (EGCG) and MgCl2 alone. In addition to detecting the expression of antioxidant-related enzymes in each group of cells, the generation of ROS in the cells is also an important standard to reflect the oxidative stress state of the cells, therefore, the fluorescence intensity of ROS in each group of cells was observed by fluorescence microscope, and the generation of ROS in each group of cells was quantified by flow cytometry.

[0113] Experimental results: as shown in Figure 9 In the middle A, the intracellular ROS was labeled with DCFH-DA dye, and the intracellular fluorescence intensity was observed by fluorescence microscope. It was found that the antioxidant intensity of the nanoparticles was significantly stronger than that of its components. Figure 9 In the middle B-E, it can be seen that the antioxidant property of EM nanoparticles in cells is higher than that of its components and has statistical difference. Figure 10 In the middle G, it is a schematic diagram of the antioxidant effect of each antioxidant enzyme in cells.

[0114] Example 6 is the observation of EM nanoparticle uptake in mice and the verification of anti-inflammatory and antioxidant effects in acute lung injury (ALI) model

[0115] (1) Nanoparticle in vivo uptake:

[0116] In this embodiment, Cy5-labeled EGCG-Mg nanoparticles (Cy5-EM) were used, and C57BL / 6 mice were given Cy5-EM by nebulization inhalation. One hour, 3 hours, 6 hours, and 12 hours after nebulization, the distribution of Cy5-EM in the mouse body was observed by small animal live imaging instrument. After each observation, the lung tissue of the mouse was taken out and left for frozen section, and the uptake and distribution of Cy5-EM in the lung tissue were observed by fluorescence microscope.

[0117] Figure 10 In the middle A, the photographing time of mouse nebulization is shown. Figure 10 In the middle B, after the mouse was nebulized, the distribution and metabolism of nanoparticles in the mouse body were observed by national imaging at different times. Figure 10 In the middle C, the fluorescence intensity of nanoparticles in the mouse lung was counted.

[0118] Subsequently, in the mouse acute lung injury model, the anti-inflammatory and antioxidant experiments of nanoparticles were verified, Figure 10 In the middle D, the schematic diagram of establishing a mouse lung injury model and treatment administration time is shown.

[0119] Prepare 6-8 weeks old male C57BL / 6 mice, and establish an acute lung injury model by tracheal instillation of LPS (dose of 5 mg / kg). Grouping: control group, LPS group, EM group, EGCG group, MgSO4 group. One hour after tracheal instillation of LPS, the first nebulization inhalation administration was started, and the second nebulization inhalation administration was given 12 hours after the model was established. 24 hours after the model was established, the mouse serum, alveolar lavage fluid and lung tissue were taken out for anti-inflammatory and antioxidant related determination experiments.

[0120] (2) Nanoparticle in vivo anti-inflammatory experiment:

[0121] Take the mouse serum, dilute 5 times with PBS, then measure, take the mouse lung lavage fluid for measurement. Prepare Mouse sICAM-1 ELISA kit, Mouse IL-6 ELISA kit and Mouse TNF-α ELISA kit to determine the concentration of sICAM-1, IL-6, TNF-α in serum and lung lavage fluid to judge the anti-inflammatory effect of different treatment drugs. Take the mouse lung tissue into the grinding tube, add 340 μL of Buffer RL in the tissue RNA extraction kit to the tube, then put the grinding tube into the tissue grinder, grind at 4°C, after grinding, put the grinding tube containing the tissue homogenate into the centrifuge, centrifuge at 4°C, 12000 rpm for 15 minutes, take the supernatant into the centrifuge tube. The mRNA obtained by reverse transcription is cDNA, and the expression content of inflammatory factors in the lung tissue can be determined by RT-qpCR.

[0122] Experimental grouping: control, LPS, EM, EGCG, MgSO4.

[0123] Experimental results: Figure 10 E-G in the mouse lung lavage fluid to evaluate the anti-inflammatory effect of different treatment methods on the lung tissue of the mouse acute lung injury model. Figure 11 H-J: Through the analysis of inflammatory factors (sICAM-1, IL-6, TNF-α) in mouse serum, the anti-inflammatory effect of different treatment methods on the whole body of mice after acute lung injury was evaluated. It was found that the anti-inflammatory effect of EM nanoparticle group was significantly stronger than that of EGCG and MgSO4 single treatment group.

[0124] (3) Nanoparticle in vivo antioxidant experiment:

[0125] Mice lung tissue was divided into left and right lungs. The left lung was placed in a -80°C freezer for staining with the tissue ROS dye DHE and prepared as frozen sections for observation of ROS production under a fluorescence fiber microscope. Right lung tissue from each group of mice was placed in tissue homogenizer tubes, and 1 mL of cell lysis buffer (containing protease inhibitors and phosphatase inhibitors) was added. The tubes were then placed in a tissue homogenizer and homogenized at 4°C. After homogenization, the tissue homogenate was centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected and placed in centrifuge tubes for analysis. Protein quantification of the supernatant was performed using the BCA method. We prepared superoxide dismutase (SOD) assay kits, catalase (CAT) assay kits, glutathione (GSH) assay kits, glutathione peroxidase (Gpx) assay kits, and malondialdehyde (MDA) assay kits to determine the levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (Gpx), and malondialdehyde (MDA) in lung tissue, thereby determining the level of oxidative stress in lung tissue of different treatment groups.

[0126] Experimental results are as follows Figure 11 As shown, where Figure 11 In the study A: Antioxidant treatment of mouse lung tissue was performed by staining frozen sections of mouse lung tissue with DHE dye and observing the changes in fluorescence intensity in each group of lung tissue using a fluorescence microscope. Figure 11 B: Assessing oxidative stress levels in lung tissue by measuring malondialdehyde (MDA). Figure 12 In the study of CF (Chemical Metabolic Evolution), the levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (Gpx) in lung tissue were used to assess the level of antioxidant enzyme secretion in lung tissue after treatment. Higher levels of antioxidant enzyme secretion resulted in better protection of lung tissue under oxidative stress. Statistical analysis revealed that the lung tissue of the EM nanoparticle treatment group showed better protection.

[0127] Example 7 is a validation of the therapeutic effect of EGCG-Mg nanoparticles on acute lung injury (ALI) in mice (determination of lung injury and pulmonary edema indices).

[0128] An acute lung injury model was established in 6-8 week old male C57BL / 6 mice using LPS at a dose of 5 mg / kg via intratracheal instillation. The mice were divided into four groups: control group, LPS group, EM group, EGCG group, and MgSO4 group. One hour after intratracheal instillation of LPS, the first nebulized inhalation administration was initiated, followed by a second nebulized inhalation administration 12 hours after model establishment. Twenty-four hours after model establishment, serum, bronchoalveolar lavage fluid, and lung tissue were collected from the mice for experiments to determine the degree of lung tissue damage.

[0129] (1) Determination of myeloperoxidase (MPO): The MPO content in serum and bronchoalveolar lavage fluid of each group was detected by Mouse MPO ELISA kit; the MPO content in lung tissue was determined by MPO content detection kit.

[0130] Experimental results are as follows Figure 12 As shown in AC: The level of lung tissue damage was assessed by measuring MPO in lung tissue, bronchoalveolar lavage fluid, and serum. (The remaining text appears to be incomplete and requires further context.) Figure 12 In the EGCG group (A), the MPO level was significantly higher than that in the control group, indicating that LPS successfully induced an acute lung injury model. The lung tissue showed extensive inflammatory cell infiltration due to the inflammatory response, resulting in severe damage. Compared to the LPS group, the MPO level in the EM group was significantly lower, indicating that EGCG-Mg nanoparticles (EM) can effectively reduce inflammatory cell infiltration in lung tissue, alleviate lung tissue damage, and have a significant anti-inflammatory effect. Although the MPO levels in the EGCG and MgSO4 groups were lower than those in the LPS group, they were higher than those in the EM group, suggesting that EGCG or MgSO4 alone... 2+ In reducing lung tissue inflammation and damage, its effect was weaker than that of EM nanoparticles, demonstrating the synergistic effect of nanoparticles. In bronchoalveolar lavage fluid MPO (… Figure 12 In the control group (B), MPO content was extremely low, with few inflammatory cells in the bronchoalveolar lavage fluid and normal lung tissue. In the LPS group, MPO content increased sharply, indicating a large amount of inflammatory cell infiltration in the alveolar cavity due to inflammation, resulting in severe alveolar damage. Compared to the LPS group, the EM group showed a significant decrease in MPO content (P<0.0001), indicating that EM can effectively inhibit the infiltration of inflammatory cells in the alveolar cavity, reduce alveolar damage, and significantly improve the inflammation of the bronchoalveolar lavage fluid microenvironment. In the EGCG and MgSO4 groups, MPO content was higher than in the EM group, indicating that their inhibitory effect on alveolar inflammatory cell infiltration was not as strong as that of EM nanoparticles, further demonstrating the anti-inflammatory advantage of EM in the alveolar region. Serum MPO content ( Figure 12 In the control group (C), MPO levels were low, and serum levels of inflammatory substances were low, reflecting a stable systemic inflammatory state. In the LPS group, MPO levels were significantly higher than in the control group, indicating that the inflammatory response caused by lung tissue damage had affected the whole body, with inflammatory factors entering the bloodstream and systemic inflammation levels increasing. In the EM group, compared to the LPS group, MPO levels were significantly lower, indicating that EM not only reduces local lung inflammation but also alleviates the systemic inflammatory response caused by lung damage, effectively improving the overall inflammatory state. In the EGCG and MgSO4 groups, MPO levels were higher than in the EM group, indicating that their effect on improving systemic inflammation was not as good as that of EM nanoparticles, demonstrating the advantage of EM in regulating systemic inflammation.

[0131] In summary, EGCG-Mg nanoparticles (EM) can effectively reduce MPO levels from the lung tissue, alveolar lavage fluid to the serum level, reduce lung tissue inflammation and injury and systemic inflammatory response, and show good anti-inflammatory treatment potential in acute lung injury models, and the effect is better than that of simple EGCG or MgSO4.

[0132] (2) Lung dry / wet ratio determination: After the serum of the mice was taken, the mouse chest was opened, and the blood was removed from the lung tissue by vascular perfusion. After perfusion, the mouse was dissected, the lung tissue was taken out, the surface blood was washed off with normal saline, the right middle lobe of the lung tissue of each group of mice was separated and placed in a centrifuge tube, and then the centrifuge tube was temporarily stored in liquid nitrogen. After the right middle lobe of the lung tissue of all mice was taken out, all the lung tissues were placed on filter paper and the surface liquid was absorbed, and then the lung tissues were placed on a balance for weighing, which was the wet weight of the lung tissue. After weighing the wet weight, the lung tissue was placed back into the centrifuge tube, and then the centrifuge tube was temporarily stored in liquid nitrogen. The oven was opened, and when the temperature in the oven reached 65°C, the centrifuge tube containing the lung tissue was taken out of the liquid nitrogen and placed in the oven. After 24 hours, the dried lung tissue was weighed.

[0133] Figure 12 The lung edema level of the mouse lung injury model was evaluated by lung dry / wet ratio determination. The dry / wet ratio of the control group was stable, representing normal lung tissue water metabolism balance; the dry / wet ratio of the LPS group increased, which was a manifestation of increased lung microvascular permeability and pulmonary edema caused by inflammation; the dry / wet ratio of the EM group decreased, indicating that EM could reduce lung fluid retention, repair microvessels, and significantly reduce lung edema. The effects of EGCG and MgSO4 groups were not good, which again verified the protective advantage of EM on lung injury.

[0134] (3) Cell content and protein content determination in alveolar lavage fluid: After the alveolar lavage fluid of each group of mice was taken, the alveolar lavage fluid was placed in a centrifuge, centrifuged at 4°C, 1000 rpm for 3 minutes, and the supernatant obtained after centrifugation was placed in a new centrifuge tube. The cell pellet was resuspended with 200 μL PBS, and the cell number in the alveolar lavage fluid of each group was determined by a cell counter after resuspension. The protein concentration in the supernatant obtained after centrifugation was determined by BCA method.

[0135] Figure 12 The lung microvascular endothelial cell junction damage level was evaluated by the cell content and protein content in the alveolar lavage fluid. Figure 12The cell amount of the control group was at a low level, reflecting the normal lung tissue state. The cell amount of the LPS group was significantly increased, indicating that LPS successfully induced lung injury, causing a large number of inflammatory cells to exude. The cell amount of the EM group was significantly reduced compared with the LPS group, indicating that the EGCG-Mg nanoparticles (EM) can effectively repair the lung microvascular endothelial junction, reduce the infiltration of inflammatory cells into the alveolar cavity, and the effect is better than that of pure EGCG or MgSO4, reflecting the synergistic anti-inflammatory advantage of nanoparticles. ​ The protein concentration of the control group was low due to the normal lung microvascular endothelium. The protein concentration of the LPS group was suddenly increased, resulting from the collapse of the endothelial barrier and the leakage of plasma protein into the alveolar cavity. The protein concentration of the EM group was significantly lower than that of the LPS group, proving that EM can strengthen the endothelial barrier and prevent protein exudation. The repair effect of the EGCG group and the MgSO4 group is weaker than that of EM, highlighting the synergistic protective value of nanoparticles.

[0136] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. Those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without having to go through creative labor. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Use of metal polyphenol nanoparticles in the preparation of a medicament for treating acute lung injury, characterized in that, The metal polyphenol nanoparticle is an EGCG-Mg nanoparticle, the EGCG-Mg nanoparticle is formed by epigallocatechin gallate EGCG and Mg 2+ ionically self-assembled; The EGCG-Mg nanoparticle is spheroid or spherical, with a particle size of 45-100 nm, a PDI of 0-0.25, and a potential distribution of -2-1.5 mV; The preparation method of the metal polyphenol nanoparticle comprises the following steps: 1) dissolving polyphenol powder with an alkaline solution to obtain a polyphenol solution, and dissolving a metal salt with double-distilled water to obtain a metal ion solution; the final concentration of the polyphenol solution and the metal ion solution is 20-24 mM; 2) mixing equal volumes of the polyphenol solution and the metal ion solution, and oscillating the reaction; 3) centrifuging, discarding the supernatant, adding double-distilled water to redissolve the precipitate, and dispersing by probe ultrasonic; 4) repeating the operation of step 3), adding double-distilled water, physiological saline or a culture medium after discarding the supernatant, and dispersing by water bath ultrasonic to obtain the metal polyphenol nanoparticle; The drug is an inhalant.

2. Use of the metal polyphenol nanoparticle according to claim 1 in the preparation of a medicament for treating acute lung injury, characterized in that, The acute lung injury is induced by sepsis, pneumonia, severe trauma or inhalation injury.

3. A medicament for treating acute lung injury, characterized by, The drug comprises the metal polyphenol nanoparticle of claim 1 and a pharmaceutically acceptable carrier, and the drug is an inhalant.

4. The medicament for treating acute lung injury according to claim 3, characterized by, The pharmaceutically acceptable carrier is at least one of a dispersant, a stabilizer, a diluent or a preservative.

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