Metal polyphenol nanoparticle, preparation method thereof and application of metal polyphenol nanoparticle in preparation of medicine for treating acute lung injury

Abstract

The invention provides a metal polyphenol nanoparticle, a preparation method thereof and an application of the metal polyphenol nanoparticle in preparation of a medicine for treating acute lung injury, the metal polyphenol nanoparticle is formed by self-assembly of metal ions and polyphenol, the metal ions are selected from Mg < 2 + >, Zn < 2 + >, Mn < 2 + > or Cu < 2 + >, and the polyphenol is selected from Mg < 2 + >, Zn < 2 + >, Mn < 2 + > or Cu < 2 + >. The polyphenol is selected from epigallocatechin gallate (EGCG), caffeic acid (CA), chlorogenic acid (CGA), gallic acid (GA) and luteolin (LUT). The metal polyphenol nanoparticles are used for preparing a medicine for treating acute lung injury induced by sepsis, pneumonia, serious trauma or inhalation injury, and the medicine further comprises a pharmaceutically acceptable carrier. The metal polyphenol nanoparticles provided by the invention are simple in preparation process and high in biological safety, polyphenol and metal ions synergistically enhance the anti-inflammatory and antioxidant activity, and the metal polyphenol nanoparticles are superior to the single use of polyphenol or metal ions.

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: 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.

[0010] 2) Advantages of drug delivery in the form of nanoparticles compared to administration of polyphenol and metal ions alone.

[0011] 3) Mechanism research of nanoparticle treatment of acute lung injury.

[0012] Based on the above-mentioned objectives and technical problems to be solved, the present application provides the following technical solutions: 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.

[0013] The metal ion is selected from Mg 2+ ion, Zn 2+ ion, Mn 2+ ion, or Cu 2+ ion.

[0014] The polyphenol is selected from any one of epigallocatechin gallate EGCG, caffeic acid CA, chlorogenic acid CGA, gallic acid GA, and luteolin LUT.

[0015] 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.

[0016] The technical solution 2 of the present application provides a preparation method of the metal polyphenol nanoparticle, comprising the following steps: 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; 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; 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.

[0017] The technical solution 3 of the present application provides an application of the metal polyphenol nanoparticle or the EGCG-Mg nanoparticle in the preparation of a drug for treating acute lung injury (ALI).

[0018] The acute lung injury is induced by sepsis, pneumonia, severe trauma, or inhalation injury.

[0019] The technical solution 4 of the present application provides a drug for treating acute lung injury, which comprises the metal polyphenol nanoparticle or the EGCG-Mg nanoparticle, and a pharmaceutically acceptable carrier.

[0020] The pharmaceutically acceptable carrier comprises at least any one of a dispersant, a stabilizer, a diluent, or a preservative.

[0021] The drug is an inhalant.

[0022] In some embodiments of the present application, the drug for treating acute lung injury, particularly 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 the inflammatory cell infiltration in lung tissue.

[0023] In some embodiments of the present application, the drug for treating acute lung injury, particularly for inhibiting the expression and secretion of inflammatory factors in an acute lung injury model animal, the inflammatory factors include TNF-α, IL-1β, IL-6 and sICAM-1.

[0024] In some embodiments of the present application, the drug for treating acute lung injury, particularly for improving the activity of antioxidant enzymes and the content of antioxidant substances in lung tissue of an acute lung injury model animal, the antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx), and the antioxidant substance is glutathione (GSH).

[0025] In some embodiments of the present application, the drug for treating acute lung injury, particularly 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 the oxidative stress damage of lung tissue.

[0026] In some embodiments of the present application, the drug for treating acute lung injury, particularly for reducing the cell concentration and protein concentration in bronchoalveolar lavage fluid of an acute lung injury model animal, and reducing the lung tissue dry / wet ratio, so as to reduce the lung microvascular barrier damage and pulmonary edema.

[0027] In summary, the present application adopts four kinds of 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 optimizing 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 the synergistic effect. Finally, the inventors used the atomization inhalation method in the acute lung injury (ALI) model of mice, so that the EM nanoparticles can be efficiently delivered to the lungs of mice, which not only can prolong the action time, reduce the frequency of administration, but also avoid the problems of liver toxicity, poor lung targeting and low bioavailability of EGCG when administered intravenously.

[0028] Compared with the prior art, the present application has at least the following improvements and beneficial effects: (1) The nanoparticles are directly targeted to the lungs by atomization inhalation, which can improve the local drug concentration and reduce the systemic side effects; (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; (3) The preparation process is simple, does not require carrier materials, and has high biological safety.

[0029] (4) Compared with the preparation process of 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 deprotonation and metal ion complexation 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 atomization inhalation as a nano-inhaler. By reasonably controlling the particle size of the nanoparticles, the inventors can 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 more fully and directly play a role in the lesion. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Preparation, characterization and screening of 20 kinds of metal polyphenol nanoparticles; reference signs: Figure 1Figure 1A is a preparation process of metal polyphenol nanoparticles with mPEG-PLA nanoparticles as templates, Figure 1 Figure 1B is a particle size test of 20 kinds of metal polyphenol nanoparticles, Figure 1 Figure 1C is a potential test, Figure 1 Figure 1D is a PDI test, Figure 1 Figure 1E is a DPPH antioxidant test, Figure 1 Figure 1F is an ABTS antioxidant test, Figure 1 Figure 1G is a cytotoxicity test of metal polyphenol nanoparticles; Figure 2 Figure 2 is a characterization of EM nanoparticles; reference signs: Figure 2 Figure 2A is a preparation process of EM nanoparticles, Figure 2 Figure 2B is a TEM test of EM nanoparticles, Figure 2 Figure 2C is a particle size analysis result of EM nanoparticles, Figure 2 Figure 2D is an XPS test of EM nanoparticles and EGCG, Figure 2 Figure 2E-F is an EPR test of EM nanoparticles, EGCG, and MgCl2, Figure 2 Figure 2G-H is a DPPH and ABTS test of EM nanoparticles, Figure 2 Figure 2I is a catalase (CAT) mimetic activity of EM nanoparticles; Figure 2 Figure 2J is a superoxide dismutase (SOD) mimetic activity of EM nanoparticles; Figure 3 Figure 3 is a cellular uptake result of EM nanoparticles; Figure 4 Figure 4 is a result of in vitro anti-inflammatory and antioxidant concentration screening of nanoparticles; reference signs: Figure 4 Figure 4A is a column chart of TNF-α concentration of HUVEC inflammation model after nanoparticle intervention; Figure 4 Figure 4B is a column chart of G-CSF concentration of HUVEC inflammation model after nanoparticle intervention; Figure 4 Figure 4C is a ROS generation level of HUVEC oxidative stress model after nanoparticle intervention; Figure 4 Figure 4D is a fluorescence staining chart of total antioxidant capacity of cells after nanoparticle intervention; Figure 5 Figure 5 is a result of in vivo anti-inflammatory and antioxidant treatment concentration screening of nanoparticles; reference signs: Figure 5 Figure 5A is a column chart of IL-1β concentration in mouse BALF; Figure 5 Figure 5B is a column chart of IL-6 concentration in mouse BALF; Figure 5 Figure 5C is a column chart of IL-6 concentration in mouse serum; Figure 5 Figure 5D is a column chart of MDA content in mouse lung tissue; Figure 5 Figure 5E is a column chart of SOD activity in mouse lung tissue; Figure 5 Figure 5F is a column chart of GSH content in mouse lung tissue; Figure 6 Biological safety of nanoparticles and their components to HUVEC; reference signs: Figure 6 Figure 2A is a bar chart of HUVEC cell proliferation rate under a concentration gradient of nanoparticles; Figure 6 Figure 2B is a bar chart of HUVEC cell proliferation rate under a concentration gradient of EM nanoparticles and their components (EGCG, Mg 2+ ); Figure 7 Biological safety in vivo; reference signs: Figure 7 Figure 3A is a bar chart of mouse red blood cell (RBC) level; Figure 7 Figure 3B is a bar chart of mouse hemoglobin (HGB) level; Figure 7 Figure 3C is a bar chart of mouse neutrophil (Neu) level; Figure 7 Figure 3D is a bar chart of mouse urea (urea, a kidney function index) level; Figure 7 Figure 3E is a bar chart of mouse alanine aminotransferase (ALT, a liver function index) level; Figure 7 Figure 3F is a bar chart of mouse aspartate aminotransferase (AST, a liver function index) level; Figure 8 Anti-inflammatory effect verification of nanoparticles and components on human umbilical vein endothelial cell inflammation model (RT-qPCR and ELISA detection); reference signs: Figure 8 Figure 4A is a bar chart of relative expression amount of TNF-α mRNA in HUVEC; Figure 8 Figure 4B is a bar chart of relative expression amount of IL-6 mRNA in HUVEC; Figure 8 Figure 4C is a bar chart of relative expression amount of sICAM-1 mRNA in HUVEC; Figure 8 Figure 4D is a bar chart of ELISA detection of TNF-α concentration in cell supernatant; Figure 8 Figure 4E is a bar chart of ELISA detection of IL-6 concentration in cell supernatant; Figure 8 Figure 4F is a bar chart of ELISA detection of sICAM-1 concentration in cell supernatant; Figure 9 Nanoparticle antioxidant cell experiment; reference signs: Figure 9 Figure 5A is a fluorescence microscope observation chart of ROS in HUVEC oxidative stress model; Figure 9 Figure 5B is a bar chart of catalase (CAT) activity in HUVEC; Figure 9 Figure 5C is a bar chart of superoxide dismutase (SOD) activity in HUVEC; Figure 9 Figure 5D is a bar chart of glutathione peroxidase (Gpx) activity in HUVEC; Figure 9 Figure 5E is a bar chart of glutathione (GSH) content in HUVEC; Figure 9Figure 1 is a schematic diagram of the synergistic mechanism of intracellular antioxidant enzymes; Figure 10 Figure 2 is an in vivo uptake and anti-inflammatory experiment of the nanoparticles; the reference signs are as follows: Figure 10 Figure 3 is a schematic diagram of the inhalation uptake operation of the mice, Figure 10 Figure 4 is the distribution of the nanoparticles in the small animal live imaging and the statistical fluorescence intensity in the lung, Figure 10 Figure 5 is a schematic diagram of the treatment operation of the acute lung injury mice, Figure 10 Figure 6 is the concentration of the inflammatory factors in the mouse alveolar lavage fluid, Figure 10 Figure 7 is the concentration of the inflammatory factors in the mouse serum; Figure 11 Figure 8 is an in vivo antioxidant experiment of the nanoparticles; the reference signs are as follows: Figure 11 Figure 9 is a fluorescence microscope observation diagram of the DHE staining of the lung tissue of the mice; Figure 11 Figure 10 is a column chart of the content of the malondialdehyde (MDA) in the lung tissue of the mice; Figure 11 Figure 11 is a column chart of the activity of the superoxide dismutase (SOD) in the lung tissue of the mice; Figure 11 Figure 12 is a column chart of the activity of the catalase (CAT) in the lung tissue of the mice; Figure 11 Figure 13 is a column chart of the content of the glutathione (GSH) in the lung tissue of the mice; Figure 11 Figure 14 is a column chart of the activity of the glutathione peroxidase (Gpx) in the lung tissue of the mice; Figure 12 Figure 15 is the treatment effect of the nanoparticles on the acute lung injury; the reference signs are as follows: Figure 12 Figure 16 is the determination of the MPO in the lung tissue, the alveolar lavage fluid and the serum, Figure 12 Figure 17 is the concentration of the cells in the alveolar lavage fluid, Figure 12 Figure 18 is the concentration of the protein in the alveolar lavage fluid, Figure 12 Figure 19 is the dry / wet ratio of the lung. DETAILED DESCRIPTION

[0031] 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 combination with specific embodiments. It should be pointed out 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 pointed out 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.

[0032] The embodiments of the present application are divided into the following parts: The first part: synthesis, characterization and screening of the metal polyphenol nanoparticles in the alkaline environment.

[0033] The synthesis conditions capable of preparing the target particle size nanoparticles (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 experiment 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.

[0034] Part II: Anti-inflammatory and antioxidant experiments of metal polyphenol nanoparticles in vitro and in vivo

[0035] 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 experiment. In the cell experiment of the second part, the inventors further explore whether the nanoparticles can alleviate the inflammation and oxidative stress of cells in cells stronger than epigallocatechin gallate (EGCG) and MgCl2 alone. Since the in vivo environment of drugs is quite different from 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 effects of nanoparticles are better than 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.

[0036] Part III: Verification of the therapeutic effect of metal polyphenol nanoparticles

[0037] The inflammatory response and oxidative stress stimulation of lung microvascular endothelial cells are important factors for the increase of endothelial cell permeability and the occurrence of lung tissue inflammatory factor infiltration, and ultimately lead to the occurrence of pulmonary edema. In this part, the inventors further study whether the expected therapeutic effect can be produced after the nanoparticles are inhaled by the acute lung injury model mice, that is, whether the anti-inflammatory and antioxidant properties of the nanoparticles have the ability to reverse the development of acute lung injury. Therefore, the inventors determine the therapeutic effect of the nanoparticles by measuring the concentration of myeloperoxidase (MPO) in lung tissue and lung lavage fluid, HE staining and other methods.

[0038] Specifically, the present application provides the following examples: Example 1: Preparation and characterization of metal polyphenol nanoparticles (1) Nanoparticle synthesis and anti-inflammatory and antioxidant experiment screening: 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 used to coat mPEG-PLA nanoparticles with metal polyphenol networks to prepare 20 different formulations of metal polyphenol nanoparticles. The specific method is as follows: 1) Preparation of mPEG-PLA vector: 20 mg of mPEG-PLA was dissolved in 1 mL of dichloromethane and 3 mL of 1% sodium cholate solution. The solution was emulsified into mPEG-PLA nanoparticles using 500 W ultrasonic probe. The solution was then placed in a 50 mL beaker, to which 40 mL of 0.5% sodium cholate solution was added. The mixture was then incubated under a fume hood for 1 hour. The solution was then removed and placed in a centrifuge tube for centrifugation. The precipitate after centrifugation was the mPEG-PLA nanoparticle vector.

[0039] 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 minutes, and centrifuge. The precipitate after centrifugation is the metal-polyphenol mPEG-PLA nanoparticle.

[0040] The combination of epigallocatechin gallate and magnesium ion is used as Example 1, and other combinations are used as Comparative Examples 1-19. See Table 1 below for details. The preparation process is referenced to Figure 1 Middle A.

[0041] Table 1 shows the preparation of 20 metal polyphenol nanoparticles (2) Characterization of nanoparticles: The 20 nanoparticles were characterized by particle size, potential, and PDI. Their antioxidant properties were preliminarily assessed using a cell-free assay using ABTS and DPPH. The biosafety of the different nanoparticles on human umbilical vein endothelial cells (HUVEC) at a consistent polyphenol concentration was determined using CCK-8 assays to measure cell proliferation.

[0042] The results of the test are as follows Figure 1 As shown: Figure 1 As shown in Figure B, the particle sizes of 20 kinds of nanoparticles were measured. Figure 1 As shown in C, the potential of the nanoparticles is measured, as shown in Figure 1 As shown in Figure D, the PDI of the nanoparticles was measured to gain a preliminary understanding of the dispersion stability of the nanoparticles. The results show that the particle size distribution of the 20 nanoparticles is 60-140nm, the potential distribution is -2~1.5mV, and the PDI distribution is around 0-0.25. Figure 1As shown in E-F, the polyphenols in the nanoparticles were quantified by DPPH and ABTS methods, respectively, and the polyphenol concentration in each nanoparticle was 12 μg / mL (it can be understood that the polyphenol concentration is quantified, that is, the concentration of the nanoparticles in the solvent is 12 μg / mL, and the same below), the antioxidant activity of the nanoparticles is preliminarily identified in a cell-free system, and a preliminary screening is carried out. As can be seen, the antioxidant activity of the 20 kinds of nanoparticles has a certain antioxidant activity, and relatively speaking, EGCG and Mg 2+ The antioxidant effect is the best when wrapped on the surface of mPEG-PLA. As shown in Figure 1 As shown in G, the polyphenols in the nanoparticles were quantified by CCK-8 method, and the polyphenol concentration in each nanoparticle was 60 μg / mL, and the cytotoxicity of the nanoparticles was preliminarily identified by HUVEC. According to the above results, from the preliminary screening results, EGCG and Mg 2+ The antioxidant activity is the highest and the cytotoxicity is the lowest when wrapped on the surface of mPEG-PLA. Figure 2 The yellow color in E-G indicates that the effect is better.

[0043] Optimization of the preparation process of Example 2 This embodiment removes the mPEG-PLA nanoparticles in the previously prepared metal polyphenol nanoparticles to prepare Mg 2+ Metal polyphenol nanoparticles formed with epigallocatechin gallate (EGCG) under alkaline conditions, this step refines the preparation scheme, makes the preparation process simpler, and the nanoparticle composition is simpler.

[0044] The specific preparation steps are as follows: 1) Dissolve epigallocatechin gallate (EGCG) powder with 66 mM NaOH to obtain an EGCG solution, dissolve magnesium chloride hexahydrate (MgCl2·6H2O) powder with double distilled water (ddH2O) to obtain a MgCl2 solution, and the final concentration of the two solutions after dissolution is 24 mM; 2) Add 500 μL of EGCG solution to a 1.5 mL centrifuge tube, then add an equal volume of MgCl2 solution, and place the centrifuge tube on a shaker for 30 min to promote the reaction of the two; 3) Place the centrifuge tube in a centrifuge and centrifuge at room temperature, 9400g, 10 min; 4) After centrifugation, discard the supernatant, take the lower sediment, and add 1 mL of double distilled water to the centrifuge tube; 5) Use a probe ultrasonic machine to promote the dispersion of the bottom sediment into water; 6) After dispersion, centrifuge again under the same conditions as above; 7) After centrifugation, discard the supernatant and add double-distilled water (or physiological saline, culture medium) to the precipitate below. Seal the centrifuge tube with sealing film and perform water bath sonication to promote the dispersion of the precipitate below in the solvent to finally obtain EGCG-Mg nanoparticles.

[0045] The results of the test are as follows Figure 2 As shown: 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 and removing the mPEG-PLA carrier to form EGCG-Mg nanoparticles (abbreviated as EM). Figure 2 As shown in Figure B, the morphology of EM was observed using a transmission electron microscope (TEM). It can be seen that EM has a spherical or spherical structure, and the particle size is relatively uniform with no obvious agglomeration phenomenon, indicating that EM has a relatively regular morphology and good dispersion. Figure 2 As shown in Figure C, the particle size of EM was statistically analyzed and the particle size of nanoparticles was mainly distributed in the range of 30-120 nm, mainly concentrated in the range of 45-100 nm, showing a single peak distribution feature. Figure 2 As shown in D, by comparing EM with EGCG through X-ray photoelectron spectroscopy (XPS), it was found 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 2 As shown in Figure EF, the scavenging effect of EM nanoparticles on hydroxyl radicals and singlet oxygen was compared with that of EGCG and MgCl2 using electron paramagnetic resonance (EPR). From the analysis of the amplitude of the spectrum, it was found that the amplitude of EM nanoparticles was smaller than that of EGCG and MgCl2, which indicates that the scavenging efficiency of EM nanoparticles on hydroxyl radicals and singlet oxygen per unit time is higher than that of EGCG and MgCl2. Figure 2 As shown in Figure GH, the antioxidant activity of EM nanoparticles was found to be related to the concentration of polyphenols in the nanoparticles through DPPH and ABTS assays in a cell-free system. The antioxidant activity increased with the increase of the polyphenol concentration in the nanoparticles. Figure 3 Figures IJ show the antioxidant activity of EGCG-Mg nanoparticles, which demonstrates the antioxidant biological function of EM.

[0046] In summary, EGCG-Mg nanoparticles (EM) were successfully prepared by adjusting the alkaline environment during the preparation process and removing the mPEG-PLA carrier.

[0047] Example 3 is a human umbilical vein endothelial cell (HUVEC) uptake experiment of Cy5-labeled EM nanoparticles Firstly, EGCG-Mg nanoparticles were labeled with Cy5, and then the Cy5-labeled nanoparticles were put into human umbilical vein endothelial cells for 1 hour, 3 hours, 6 hours, and 9 hours. The uptake of the nanoparticles by the human umbilical vein endothelial cells was observed under a fluorescence microscope.

[0048] The experimental results are shown in Table 1. Figure 3 As can be seen from Table 1, the uptake of the Cy5-labeled nanoparticles by the human umbilical vein endothelial cells was good, and the fluorescence intensity analysis showed that the uptake of the nanoparticles by the endothelial cells increased with time, indicating that the uptake of the nanoparticles by the cells was time-dependent. Figure 4

[0049] Example 4: Selection of therapeutic concentration of nanoparticles in in vitro and in vivo experiments and verification of biological safety (cell and mouse levels) (1) Selection of therapeutic concentration of nanoparticles in in vitro experiments: 1) Human umbilical vein endothelial cell inflammation model: human umbilical vein endothelial cells (HUVECs) 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, nanoparticles at low, medium, and high therapeutic doses (EM1: 30 μg / mL, EM2: 60 μg / mL, EM3: 90 μg / mL) were selected according to the therapeutic doses in previous studies and were put into the human umbilical vein endothelial cell inflammation model.

[0050] Measurement of TNF-α and G-CSF concentrations: after 4 hours of LPS addition, the cell supernatant was taken and the concentrations of TNF-α and G-CSF in the cell supernatant of the control group, LPS group, EM1 group, EM2 group, and EM3 group were measured using human Human TNF-α ELISA and Human G-CSF ELISA kits, respectively.

[0051] 2) Human umbilical vein endothelial cell oxidative stress model: human umbilical vein endothelial cells (HUVECs) 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, nanoparticles at low, medium, and high therapeutic doses (EM1: 30 μg / mL, EM2: 60 μg / mL, EM3: 90 μg / mL) were selected according to the therapeutic doses in previous studies and were put into the human umbilical vein endothelial cell oxidative stress model.

[0052] ​Extraction of protein and determination of SOD activity: 24 hours after the addition of LPS, the protein in the cells was extracted with a cell lysis solution (containing protease inhibitors and phosphatase inhibitors) on ice. In order to calculate the content of superoxide dismutase (SOD) in the unit protein next, the protein was quantified by BCA method, and then the content of SOD in the control group, LPS group, EM1 group, EM2 group, EM3 group was 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.

[0053] Determination of active oxygen level: 24 hours after the addition of LPS, the supernatant of each group was removed, washed with PBS for 2 times, and then DCFH-DA fluorescent dye was added, and after incubation in the incubator for 30 minutes, the DCFH-DA fluorescent dye was removed, washed with PBS for 2 times, and then the hole plate was placed under the fluorescence microscope to observe the fluorescence intensity of each group of cells.

[0054] Experimental results: the best anti-inflammatory concentration in vitro experiment is shown in A-B of Figure 4 , by quantifying the polyphenol concentration in the nanoparticles, anti-inflammatory experiments were carried out on HUVEC inflammatory model using nanoparticles with polyphenol concentration of 30, 60 and 90 μg / mL, and the contents of TNF-α and G-CSF in the cell supernatant were determined by HUMAN TNF-α ELISA kit and HUMAN G-CSF ELISA kit, and it was found that the anti-inflammatory effect of nanoparticles was dose-dependent with the polyphenol concentration in nanoparticles. The best antioxidant concentration in vitro experiment is shown in C of Figure 4 : In order to determine the appropriate antioxidant nanoparticle concentration and the relationship between the antioxidant effect of nanoparticles and the polyphenol concentration in nanoparticles, HUVEC oxidative stress model was established, the polyphenol in nanoparticles was quantified and the anti-cell ROS generation level was determined by grouping. The fluorescence intensity of ROS was observed by fluorescence microscope to evaluate the antioxidant degree of nanoparticles with different concentrations. It was observed by fluorescence intensity that the antioxidant property of nanoparticles in cells increased with the increase of polyphenol concentration in nanoparticles. Figure 5 D of the middle, the protein in the cells was taken, and the change of total antioxidant capacity of the cells after treatment with nanoparticles with different concentrations was determined.

[0055] (2) Selection of treatment concentration of nanoparticles in in vivo experiment: 6~8 weeks C57BL / 6 mice were purchased and acute lung injury model was established by intratracheal instillation of LPS (0111: B4) 5mg / kg. When modeling, water column method was used to verify the LPS drop into the trachea, specifically, first intubate the mouse trachea with a retention needle, then remove the metal needle core, install a 50 μL water column on the 1 milliliter syringe needle of the retention needle, if the retention needle and the syringe are installed, the water column in the syringe moves up and down with the mouse's breathing, indicating that the retention needle is in the mouse trachea. Grouping: control group, LPS group, EM1 group, EM2 group, EM3 group (EM1: 10mg / kg, EM2: 30mg / kg, EM3: 60mg / kg).

[0056] After 1 hour of LPS intratracheal instillation, different concentrations of nanoparticles were used for the first treatment of acute lung injury mice by inhalation; after 12 hours of LPS intratracheal instillation, different concentrations of nanoparticles were used for the second treatment of acute lung injury mice by inhalation; after 24 hours of LPS intratracheal instillation, the serum, alveolar lavage fluid and lung tissue of each group of mice were taken. The concentration of IL-6 in the serum was determined by Mouse IL-6 ELISA kit, and the concentrations of TNF-α, IL-1β and IL-6 in the alveolar lavage fluid were determined by Mouse TNF-α ELISA kit, Mouse IL-1β ELISA kit and Mouse IL-6 ELISA kit to determine the anti-inflammatory effect of different concentrations of nanoparticles.

[0057] The lung tissue was divided into left lung and right lung, the left lung was placed in 4% paraformaldehyde for HE staining; the right lung was placed in a tissue grinder tube, 1 mL of cell lysis solution (containing protease inhibitor and phosphatase inhibitor) was added to the tube, then the tissue grinder tube was placed in a tissue grinder, and the grinding was carried out at 4°C, after grinding, the tissue homogenate was placed in a centrifuge, centrifuged at 12000 rpm, 4°C, 15 minutes, and the supernatant was placed in a new centrifuge tube. The homogenate was quantified by BCA method, and then the expression contents of SOD, MDA and GSH in unit mass protein of each group were determined by superoxide dismutase (SOD) detection kit, malondialdehyde (MDA) detection kit and glutathione (GSH) detection kit. In summary, the appropriate anti-inflammatory and antioxidant nanoparticle treatment concentration was determined.

[0058] The experimental results are shown in Table 1. Figure 5 Table 1 shows the in vivo experiment anti-inflammatory and antioxidant optimal concentration selection. Figure 5 Table 1 shows the determination of IL-1β and IL-6 in the BALF of mice, Figure 5 Table 1 shows the determination of IL-6 in the serum of mice, and it is found that the concentration of 60mg / kg of nanoparticles can effectively and stably play an anti-inflammatory role in the ALI mouse model.Figure 6 MDA (Malondialdehyde), SOD (Superoxide dismutase), GSH (Glutathione) in lung tissue of ALI mouse model, it was found that the nano-particles with the concentration of 10, 30, 60 mg / kg could play a good antioxidant effect in the lung tissue of mice, but the antioxidant effect of the nano-particles with the concentration of 60 mg / kg was better.

[0059] (3) Biological safety verification of nano-particles in vitro experiment: Under the appropriate therapeutic concentration, it is necessary to understand the biological safety of the drug in the cell experiment to avoid the cytotoxicity effect caused by the drug. In this experiment, CCK-8 method was used to determine the effect of nano-particles at different concentrations on the proliferation of human umbilical vein endothelial cells (HUVEC), and different concentrations (10, 30, 60, 90, 120, 150 μg / mL) of nano-particles and its components (EGCG, MgCl2) were put into HUVEC, and cultured in the incubator for 24 hours. The cell proliferation rate was determined to judge the cytotoxicity of different nano-particles and its components to HUVEC.

[0060] The experimental results are shown in Figure 6 , which are the biological safety determination of nano-particles and its components in HUVEC. Figure 6 A is the cell proliferation rate determined by CCK-8 method, and it was found that the nano-particles with the concentration of 90 μg / mL and below had good safety to HUVEC. Figure 7 B is the biological safety of different nano-particle concentrations and its components determined by CCK-8 method, and it was found that the polyphenol concentration in the nano-particles was 90 μg / mL and below, which had good safety to HUVEC.

[0061] (4) Biological safety verification of nano-particles in vivo experiment: Under the appropriate therapeutic concentration, it is necessary to understand the biological safety of the drug in the animal experiment to avoid the systemic toxicity effect caused by the drug. 6-8 weeks old male C57BL / 6 mice were grouped: control group, EM group, EGCG group, MgSO4 group. The mice were given aerosol inhalation of therapeutic concentration of EM, EGCG, MgSO4, and aerosolized twice within 24 hours, i.e. aerosolized once 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.

[0062] The experimental results are shown in Figure 7 , which are the biological safety determination of nano-particles and its components in mice. Figure 7Biochemical tests A-C showed that the number of red blood cells, hemoglobin, and neutrophils did not change significantly after nano-particle nebulization compared with the control group. Figure 7 Biochemical tests D showed that nano-particle nebulization did not cause kidney damage in mice. Figure 8 Biochemical tests E-F showed that nano-particles did not cause liver damage in mice.

[0063] Example 5: Anti-inflammatory and antioxidant effects of EM nanoparticles in HUVEC inflammation and oxidative stress models (compared with single components) (1) Anti-inflammatory experiment of nanoparticles in a human umbilical vein endothelial cell inflammation model: After adding nano-particle drugs to human umbilical vein endothelial cells for 1 hour, the cells were stimulated with 1 μg / mL LPS for 4 hours to establish an endothelial cell inflammation model. The groups were control, LPS, EM, EGCG, and MgCl2. After adding 1 μg / mL LPS to each group for 4 hours, the cell supernatant was collected, and the concentration of inflammatory factors (TNF-α, IL-6, sICAM-1) in the cell supernatant was determined using an ELISA kit. At the same time, the mRNA in each group of cells was extracted, and the expression of inflammatory factors (TNF-α, IL-6, sICAM-1) in human umbilical vein endothelial cells was determined by RT-qPCR.

[0064] Experimental results: Figure 8 A-C: mRNA was extracted from HUVEC cells, and the expression of inflammatory-related factors (TNF-α, IL-6, sICAM-1) in the cells was detected by RT-qPCR. Figure 9 D-F: The cell culture supernatant was collected, and the secretion of TNF-α, IL-6, and sICAM-1 in the supernatant was determined using an ELISA kit to analyze the anti-inflammatory effects of different treatments. The statistical results showed that the anti-inflammatory effect of EM nanoparticles was better than that of its components.

[0065] (2) Anti-oxidant experiment of nanoparticles in a human umbilical vein endothelial cell oxidative stress model: After adding the nanoparticle drug for 1 hour in human umbilical vein endothelial cells, the human umbilical vein endothelial cells were stimulated by 1 μg / mL LPS for 24 hours to establish an oxidative stress model of endothelial cells. Grouping: control group, LPS group, EM group, EGCG group, MgCl2 group. After adding 1 μg / mL LPS to each group for 24 hours, the supernatant was discarded, 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 concentration of superoxide dismutase, catalase, glutathione, glutathione peroxidase was detected by superoxide dismutase (SOD) detection kit, catalase (CAT) detection kit, glutathione peroxidase (Gpx), glutathione (GSH) detection kit. The expression content of SOD, CAT, GSH, Gpx in each group per unit protein was calculated, and then it could be concluded whether the antioxidant performance of the nanoparticles was better than that of 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, it is necessary to observe the fluorescence intensity of ROS in each group of cells by fluorescence microscope, and at the same time use flow cytometry to quantify the generation of ROS in each group of cells.

[0066] Experimental results: as shown in Figure 9 In Fig. A, the intracellular ROS was labeled with DCFH-DA dye, and the intracellular fluorescence intensity was observed by fluorescence microscope. It was found by analysis that the antioxidant intensity of the nanoparticles was significantly higher than that of its components. Figure 9 In Figs. B-E, it can be seen that the antioxidant property of EM nanoparticles in the cells is higher than that of its components and has statistical difference. Figure 10 Fig. G is a schematic diagram of the antioxidant effect of each antioxidant enzyme in the cells.

[0067] 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 (1) In vivo uptake of nanoparticles: In this embodiment, Cy5-labeled EGCG-Mg nanoparticles (Cy5-EM) were used, and C57BL / 6 mice were made to ingest Cy5-EM by atomization inhalation. One hour, 3 hours, 6 hours and 12 hours after atomization, 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 the uptake and distribution of Cy5-EM in the lung tissue were observed by fluorescence microscope.

[0068] Figure 10 Fig. A is a schematic diagram of the photographing time of mouse atomization uptake. Figure 10Fig. 2 shows the distribution and metabolism of the nanoparticles in the mouse body after the mouse was nebulized and inhaled. Figure 10 Fig. 3 shows the fluorescence intensity of the nanoparticles in the mouse lung.

[0069] Fig. 4 shows the anti-inflammatory and anti-oxidation experiments of the nanoparticles in the mouse acute lung injury model. Figure 10 Fig. 5 shows the schematic diagram of the mouse lung injury model and the treatment administration time.

[0070] 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. After tracheal instillation of LPS for 1 hour, start the first nebulization and inhalation administration, and after 12 hours of model establishment, the second nebulization and inhalation administration. After 24 hours of model establishment, take the mouse serum, lung lavage fluid and lung tissue, and leave for anti-inflammatory and anti-oxidation related determination experiments.

[0071] (2) Anti-inflammatory experiment of the nanoparticles in vivo: Take the mouse serum, dilute 5 times with PBS, and 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 concentrations of sICAM-1, IL-6 and TNF-α in the serum and lung lavage fluid to judge the anti-inflammatory effect of different treatment drugs. Put the mouse lung tissue into a grinding tube, add 340 μL of Buffer RL in the tissue RNA extraction kit to the tube, then put the grinding tube into a tissue grinder, grind at 4°C, after grinding, centrifuge the grinding tube containing the tissue homogenate at 4°C, 12000 rpm for 15 minutes, and take the supernatant into a centrifuge tube. The obtained mRNA is reverse transcribed to obtain cDNA, and then determined by RT-qpCR to know the expression content of inflammatory factors in the lung tissue.

[0072] Experimental grouping: control, LPS, EM, EGCG, MgSO4.

[0073] Experimental results: Figure 10 E-G, by evaluating the inflammatory factors (sICAM-1, IL-6, TNF-α) 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 11H-J: To evaluate the systemic anti-inflammatory effect of different treatments on mice after acute lung injury by measuring inflammatory factors (sICAM-1, IL-6, TNF-α) in mouse serum. Through analysis, it was found that the anti-inflammatory effect of the EM nanoparticle group was significantly stronger than that of the EGCG and MgSO4 single treatment groups.

[0074] (3) In vivo antioxidant experiment of nanoparticles: The mouse lung tissue was divided into left and right lungs. The left lung was placed in a -80°C freezer, ready for staining with tissue ROS dye DHE and making into frozen sections for observation of ROS production in the tissue under a fluorescence fiber microscope. The right lung tissue of each group of mice was placed in a tissue grinding tube, 1 mL of cell lysis solution (containing protease inhibitors and phosphatase inhibitors) was added to the grinding tube, and then the tissue grinding tube was placed in a tissue grinder, and the tissue was ground at 4°C. After grinding, the tissue homogenate was placed in a centrifuge, 4°C, 12000 rpm, 15 min for centrifugation, and the supernatant was taken after centrifugation and placed in a centrifuge tube for testing. The supernatant of each group was quantified by BCA method. Prepare superoxide dismutase (SOD) assay kit, catalase (CAT) assay kit, glutathione (GSH) assay kit, glutathione peroxidase (Gpx) assay kit, malondialdehyde (MDA) assay kit to determine the content of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (Gpx), malondialdehyde (MDA) in lung tissue, to determine the level of oxidative stress in lung tissue of different treatment groups.

[0075] The experimental results are shown in Figure 11 , wherein Figure 11 A: Antioxidant treatment of mouse lung tissue, DHE dye was used to stain the frozen sections of mouse lung tissue, and a fluorescence microscope was used to scan and observe the changes in fluorescence intensity in the lung tissue of each group. Figure 11 B: The oxidative stress level of lung tissue was evaluated by measuring malondialdehyde (MDA). Figure 12 C-F: The content of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (Gpx) in lung tissue was used to evaluate the level of antioxidant enzyme secretion in lung tissue after treatment. The more antioxidant enzymes are secreted, the better the lung tissue can be protected in the oxidative stress environment. Through statistical analysis, it was found that the lung tissue of the EM nanoparticle treatment group was better protected.

[0076] Example 7 is the verification of the treatment effect of EGCG-Mg nanoparticles on acute lung injury (ALI) in mice (determination of lung injury and pulmonary edema indicators) An acute lung injury model was established in 6-8 week old male C57BL / 6 mice using intratracheal instillation of 5 mg / kg LPS. Groups were divided into control, LPS, EM, EGCG, and MgSO4 groups. The first nebulized inhalation dose was administered 1 hour after intratracheal instillation of LPS, followed by the second nebulized inhalation dose 12 hours after model establishment. Twenty-four hours after model establishment, serum, bronchoalveolar lavage fluid, and lung tissue were collected for experiments to determine the extent of lung tissue damage.

[0077] (1) Determination of myeloperoxidase (MPO): The Mouse MPO ELISA kit was used to detect the MPO content in the serum and alveolar lavage fluid of each group; the MPO content in the lung tissue was determined using an MPO content detection kit.

[0078] The experimental results are as follows Figure 12 Figures AC show: The level of lung tissue damage was assessed by measuring MPO in lung tissue, alveolar lavage fluid, and serum. Figure 12 In middle A), the LPS group was significantly higher than the control group, indicating that the acute lung injury model was successfully established by LPS induction. A large number of inflammatory cells infiltrated the lung tissue due to the inflammatory response, and the damage was severe. The MPO level in the EM group was significantly reduced compared with the LPS group, indicating that EGCG-Mg nanoparticles (EM) can effectively reduce the infiltration of inflammatory cells in the lung tissue, alleviate lung tissue damage, and have a significant anti-inflammatory effect. Although the MPO levels of the EGCG group and the MgSO4 group were lower than those of the LPS group, they were higher than those of the EM group, indicating that the MPO levels of simple EGCG or Mg 2+ In terms of reducing lung tissue inflammation and damage, the effect is weaker than that of EM nanoparticles, which reflects the synergistic effect of nanoparticles. Figure 12 Middle B), the MPO content in the control group was extremely low, there were few inflammatory cells in the alveolar lavage fluid, and the lung tissue was normal. The MPO content in the LPS group increased sharply, indicating that a large number of inflammatory cells infiltrated into the alveolar cavity due to inflammation, and the alveoli were severely damaged. Compared with the LPS group, the MPO content in the EM group decreased significantly (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 inflammatory effect of the alveolar lavage fluid microenvironment. EGCG group and MgSO4 group: The MPO content was higher than that in the EM group, indicating that its inhibitory effect on the infiltration of inflammatory cells in the alveolar cavity was not as good as EM nanoparticles, further proving the anti-inflammatory advantage of EM in the local alveoli. In serum MPO ( Figure 12MPO level, less inflammation-related substances in serum, and stable systemic inflammatory state. LPS group: significantly higher than the control group, indicating that the inflammatory response triggered by lung tissue damage has affected the whole body, and inflammatory factors have entered the blood circulation, increasing the level of systemic inflammation. EM group: compared with the LPS group, the MPO level is significantly reduced, indicating that EM not only reduces local lung inflammation but also alleviates systemic inflammatory response caused by lung damage, effectively improving the overall inflammatory state. EGCG group and MgSO4 group: MPO level is higher than that of the EM group, indicating that its improvement effect on systemic inflammation is not as good as that of EM nanoparticles, which reflects the advantage of EM in regulating systemic inflammation.

[0079] In summary, EGCG-Mg nanoparticles (EM) can effectively reduce MPO levels from lung tissue, alveolar lavage fluid to serum, reduce lung tissue inflammation 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.

[0080] (2) Lung dry / wet ratio determination: After the serum of the mouse is taken, the chest is opened, and the blood vessels are perfused to remove the residual blood in the lung tissue. After perfusion, the mouse is dissected, the lung tissue is taken out, the surface blood is washed off with normal saline, the right middle lobe of the lung tissue of each group of mice is separated and placed in a centrifuge tube, and then the centrifuge tube is temporarily stored in liquid nitrogen. After the right middle lobe of the lung tissue of all mice is taken out, the lung tissue is placed on filter paper and the surface liquid is absorbed, and then the lung tissue is placed on a balance for weighing, which is the wet weight of the lung tissue. After weighing the wet weight, the lung tissue is placed back into the centrifuge tube, which is then temporarily stored in liquid nitrogen. The oven is turned on and the temperature is raised to 65°C. The centrifuge tube containing the lung tissue is taken out of the liquid nitrogen and placed in the oven. After 24 hours, the dried lung tissue is weighed.

[0081] Figure 12 F, the lung edema level of the mouse lung injury model was evaluated by lung dry / wet ratio determination. Among them, 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 alleviate pulmonary edema. The effect of EGCG and MgSO4 groups was not as good, which again verified the protective advantage of EM for lung injury.

[0082] (3) Cell quantity and protein concentration in the bronchoalveolar lavage fluid: The bronchoalveolar lavage fluid of each group of mice was taken and placed in a centrifuge, centrifuged at 1000 rpm for 3 minutes at 4°C. The supernatant obtained after centrifugation was placed in a new centrifuge tube, and the cell precipitate was re-dissolved with 200 μL PBS. After re-dissolution, the cell number in the bronchoalveolar lavage fluid of each group was determined by a cell counter. The supernatant obtained after centrifugation was used to determine the protein concentration in the supernatant by the BCA method.

[0083] Figure 12 The lung microvascular endothelial cell junction damage level was evaluated by the cell content and protein content in the bronchoalveolar lavage fluid. Figure 12 The cell quantity in the bronchoalveolar lavage fluid of the control group was at a low level, reflecting the normal lung tissue state. The cell quantity in the LPS group was significantly increased, indicating that LPS successfully induced lung injury, causing a large number of inflammatory cells to exude. The cell quantity in 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 simple EGCG or MgSO4, reflecting the synergistic anti-inflammatory advantage of nanoparticles. ​ The protein concentration in the bronchoalveolar lavage fluid of the control group was low due to the normal lung microvascular endothelium. The protein concentration in 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 in the EM group was significantly lower than that in the LPS group, proving that EM can strengthen the endothelial barrier and prevent protein exudation, and the repair effect is outstanding. The repair capacity of the EGCG group and the MgSO4 group is weaker than that of EM, highlighting the synergistic protection value of nanoparticles.

[0084] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the application. 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 application is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. A metal polyphenol nanoparticle, characterized in that: The metal polyphenol nanoparticles are EGCG-Mg nanoparticles, which are composed of epigallocatechin gallate EGCG and Mg 2+ The EGCG-Mg nanoparticles are spherical 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.

2. A method for preparing metal polyphenol nanoparticles as claimed in claim 1, characterized in that: The following steps are involved: 1) dissolving polyphenol powder in an alkaline solution to obtain a polyphenol solution, and dissolving a metal salt in double distilled water to obtain a metal ion solution; the final concentrations of the polyphenol solution and the metal ion solution are both 20-24 mM; 2) Mix equal volumes of polyphenol solution and metal ion solution and shake for reaction; 3) Centrifuge, discard the supernatant, take the precipitate, add double-distilled water to reconstitute, and then disperse with ultrasonic probe; 4) Repeat the operation in step 3), discard the supernatant, add double-distilled water, physiological saline or culture medium, and disperse in a water bath using ultrasonic waves to obtain metal polyphenol nanoparticles.

3. Use of the metal polyphenol nanoparticles according to claim 1 in preparing a drug for treating acute lung injury, characterized in that: The acute lung injury is induced by sepsis, pneumonia, severe trauma or inhalation injury; the drug comprises the metal polyphenol nanoparticles according to claim 1, and a pharmaceutically acceptable carrier.

4. The use of the metal polyphenol nanoparticles according to claim 3 in preparing a drug for treating acute lung injury, characterized in that: The pharmaceutically acceptable carrier includes at least any one of a dispersant, a stabilizer, a diluent or a preservative.

5. Use of the metal polyphenol nanoparticles according to claim 3 in preparing a drug for treating acute lung injury, characterized in that: The medicine is an inhaler.

Images