4-octyl itaconic acid liposome loaded nano-particles wrapped by metal-phenolic aldehyde network coating as well as preparation method and application of 4-octyl itaconic acid liposome loaded nano-particles

Abstract

The invention discloses 4-octyl itaconic acid liposome loaded nano-particles wrapped by a metal-phenolic aldehyde network coating. The nano-particles are used for adjusting a myocarditis microenvironment after acute myocardial infarction. The drug-loaded nanoparticle provided by the invention can be divided into a drug-loaded liposome inner core and a shell layer; the inner core is functionalized liposome nanoparticles loaded with 4-octyl itaconic acid, and the shell layer is a metal-phenolic network coating formed by self-assembly of tannic acid and Mn < 2 + >. The invention further provides application of the drug-loaded nanoparticles in preparation of drugs for treating inflammation causing myocardial injury. The tannic acid of the shell layer of the drug-loaded nanoparticle has high affinity to an extracellular matrix of cardiac muscle tissue, and the drug-loaded nanoparticle injected from caudal vein can be efficiently enriched to a myocardial injury area to play roles in inhibiting pyroptosis of cardiac muscle cells, regulating macrophage functions and remodeling a damaged myocarditis microenvironment; accurate, effective and safe acute myocardial infarction treatment is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of composite nanomaterials, and in particular to a drug-loaded nanoparticle with myocardial inflammation microenvironment regulating function, a preparation method thereof, and an application of the drug in preparing an anti-acute myocardial infarction drug. Background Art

[0002] Acute myocardial infarction (AMI) is one of the cardiovascular diseases with sudden onset and high mortality rate, which seriously threatens the life and health of people around the world. The occlusion of coronary arteries can cause myocardial ischemia and hypoxia, leading to myocardial cell damage, and triggering immune inflammatory response, aggravating myocardial damage. Although drug thrombolytic therapy, percutaneous coronary intervention and coronary artery bypass grafting can restore blood supply to the infarcted myocardium in time, about 40% of AMI patients will still experience cardiac dysfunction after reperfusion therapy. Therefore, actively exploring new strategies for the treatment of AMI is a key issue that needs to be urgently solved in clinical practice.

[0003] Recent studies have found that after acute myocardial infarction, the most important form of programmed cell death in the injured myocardium is pyroptosis. Pyroptosis is a regulated inflammatory cell death characterized by the activation of inflammasomes, the formation of oligomeric pores in the cell membrane, and the release of inflammatory factors such as IL-1β. Pyroptosis of cardiomyocytes not only leads to the loss of cardiomyocytes, but also the release of inflammatory factors IL-1β triggers immune cell-mediated inflammatory responses. Among the immune cell populations that mediate AMI, monocytes / macrophages are the main populations involved in myocardial injury and repair. The inflammatory factor IL-1β is a key factor that stimulates the differentiation of monocytes / macrophages into inflammatory types. Proinflammatory monocytes / macrophages produce a large amount of reactive oxygen species (ROS), TNF-α, etc., which aggravate the occurrence of damage. However, repairing monocytes / macrophages can promote the dissolution of inflammation, the formation of new blood vessels, etc., and play a role in myocardial repair. Therefore, while inhibiting myocardial cell pyroptosis, regulating the function of monocytes / macrophages, reducing inflammatory responses, and promoting myocardial repair are expected to achieve better treatment of AMI.

[0004] Preclinical studies have confirmed that small molecule inhibitors of anti-pyroptosis can reduce the area of ​​myocardial infarction. However, no studies have yet revealed the regulatory effects of these small molecule inhibitors of anti-pyroptosis (such as VX-765 and MCC950) on macrophage function while inhibiting myocardial cell pyroptosis. This may be related to factors such as the low bioavailability, lack of targeting, and single target of small molecule inhibitors, making it difficult to effectively respond to the complex inflammatory microenvironment after myocardial infarction. In recent years, nano-drug delivery systems have developed rapidly in the diagnosis and treatment of cardiovascular diseases, providing unlimited potential for targeted intervention of cardiovascular diseases. Therefore, a targeted nano-drug delivery platform is constructed for the inflammatory microenvironment after myocardial infarction in order to achieve better AMI treatment. Summary of the invention

[0005] Purpose of the invention: In view of the shortcomings of current anti-inflammatory treatments for acute myocardial infarction, the purpose of the present invention is to provide a novel drug-loaded nanoparticle for regulating the myocardial inflammatory microenvironment and a preparation method and application thereof, so as to achieve accurate, safe and effective treatment of acute myocardial infarction.

[0006] As a metabolic byproduct of the tricarboxylic acid cycle, itaconate has shown great potential in regulating metabolism and inflammatory responses. Its derivative 4-octylitaconate (abbreviated as 4-OI) can play an anti-pyroptotic role by inhibiting the activation of inflammasomes (NLRP3). However, the efficacy of 4-OI is greatly limited due to its low bioavailability and lack of targeting. As the most widely used nano-drug carrier, liposomes can not only be used as drug / gene delivery carriers, but also facilitate functional modification to adapt to specific disease environments. To improve the bioavailability of 4-OI, we first synthesized liposomes loaded with 4-octylitaconate (4-OI@liposomes, 4oRL) using DSPC, DSPE-TK-PEG and cholesterol. In addition, high levels of ROS in the inflammatory microenvironment after myocardial infarction are one of the key factors promoting myocardial damage. Tannic acid (TA), a water-soluble polyphenol compound containing multiple phenolic hydroxyl structures, has been shown to have a variety of biological activities, such as scavenging oxygen free radicals, anti-inflammatory and antibacterial functions. Studies have shown that TA has a high affinity for the extracellular matrix of myocardial tissue. Inspired by metal phenolic network coating (MPN for short), we then used TA and Mn 2+ The self-assembled coating encapsulates 4oRL nanoparticles (MPN@4-OI@liposomes, M4oRL) to achieve myocardial targeting and effectively inhibit cardiomyocyte pyroptosis, while regulating macrophage function and stabilizing the inflammatory microenvironment after myocardial infarction, providing a promising strategy for the treatment of AMI.

[0007] In order to solve the above technical problems, the present invention discloses a 4-octylitaconic acid liposome nanoparticle wrapped with a metal-phenolic network coating, wherein the core of the nanoparticle is a liposome containing 4-octylitaconic acid, and the shell is a tannic acid-Mn 2+ Self-assembled metal phenolic network coating.

[0008] The present invention further discloses a method for preparing the 4-octylitaconic acid-loaded liposome nanoparticles coated with the metal-phenolic network coating, comprising the following steps:

[0009] (1) 4-Octyl itaconate (4-OI), 1,2-Distearoyl-sn-glycero-3-phosphorylcholine (DSPC), distearoyl phosphatidylethanolamine-thioketal-polyethylene glycol (DSPE-TK-PEG) and cholesterol are dissolved in a solvent respectively and mixed in a certain proportion, and then quickly injected into a PBS solution, and the obtained mixed solution is ultrasonicated and placed in 100-200W ultrasonication for 5-10 minutes, and the milky white suspension after ultrasonication is placed in a 35-50°C water bath and stirred for 3-6 hours to obtain a 4-octyl itaconate liposome solution, and the solution is centrifuged at a low speed and then transferred to a dialysis bag for dialysis to obtain 4-octyl itaconate liposome (4oRL);

[0010] (2) The 4-octylitaconic acid liposomes prepared in step (1) are mixed with tannic acid, MnCl2·4H2O and a buffer solution, and the coupling reaction is carried out for 1 to 6 hours. After the reaction is completed, the liposomes are washed and freeze-dried to obtain the drug-loaded nanoparticles having the function of regulating the myocardial inflammatory microenvironment.

[0011] Wherein, in step (1), the solvent is anhydrous ethanol.

[0012] In step (1), the molar mass ratio of 4-octylitaconic acid, DSPC, DSPE-TK-PEG and cholesterol is 10.32-24.77 mM: 0.4-0.6 mM: 0.04-0.1 mM: 0.3-0.6 mM.

[0013] In step (1), the rapid injection method is to use a 200uL pipette to draw the drug mixture, avoid the appearance of bubbles at the tip of the gun, and then quickly inject it into the PBS solution. In some specific embodiments, the rapid injection method for the drug mixture containing 20.64mM is to use a 200uL pipette to draw the drug mixture, avoid the appearance of bubbles at the tip of the gun, and then quickly inject it into 1.8mL PBS solution, and the ratio of the volume of the mixed drug to the volume of PBS is preferably 1:10.

[0014] Preferably, in step (1), the ultrasonic condition is 100-200W for 5-10 min; the low-speed centrifugation condition is 1000-3000 rpm for 5-20 min.

[0015] In step (1), the specification of the dialysis bag is 10-20KD, the dialysis external solution is PBS solution, the dialysis external solution is changed every 1-3 hours, and the dialysis is performed for 10-24 hours.

[0016] In step (2), the mass ratio of the 4-octylitaconic acid liposomes, tannic acid and MnCl2·4H2O is 1-3 mg: 1 mg: 0.25 mg, the volume of the buffer solution is 3-5 mL, and the pH is 8-8.5.

[0017] The present invention further discloses the use of the above-mentioned metal-phenolic network coating-encapsulated 4-octylitaconic acid liposome nanoparticles (M4oRL) in the preparation of a drug for treating myocardial damage caused by aseptic inflammation after myocardial infarction, wherein the inflammation mainly includes non-infectious inflammation caused by factors such as ischemia-reperfusion injury, oxidative stress and immune response.

[0018] The present invention further discloses the use of the 4-octylitaconic acid-loaded liposome nanoparticles wrapped by the metal-phenolic network coating in the preparation of a drug for alleviating or treating myocardial damage caused by acute myocardial infarction.

[0019] Beneficial effects: Compared with the prior art, this application has the following advantages:

[0020] (1) The present invention provides a drug-loaded nanoparticle having the function of regulating the myocardial inflammatory microenvironment, comprising a core and an outer shell coating. The core is a liposome loaded with 4-octylitaconic acid, which can effectively solve the defect of low bioavailability of 4-octylitaconic acid. The outer shell coating is tannic acid and Mn 2+ The self-assembled metal phenolic network coating, on the one hand, has a high affinity of tannic acid to the extracellular matrix of myocardial tissue, which can enhance the targeting of M4oRL nanoparticles to the myocardium; on the other hand, the metal phenolic network coating has the function of scavenging ROS and can synergize with 4-OI to effectively achieve multiple adjustments to the myocardial inflammatory microenvironment, thereby exerting a better myocardial protective effect.

[0021] (2) The nanodrug M4oRL of the present invention can significantly inhibit hypoxia-induced pyroptosis of H9c2 cardiomyocytes in vitro and improve cell viability, which is mainly manifested in: effectively reducing the level of mitochondrial ROS in H9c2 cells caused by hypoxia; reducing the cell apoptosis rate; and reducing the levels of intracellular NLRP3, Cleaved-Caspase-1, Cleaved-GSDMD and Cleaved-IL-1β proteins.

[0022] (3) The nanodrug M4oRL of the present invention can regulate the function of LPS-induced RAW 264.7 macrophages in vitro, mainly manifested in: effectively reducing the level of ROS in macrophages under LPS stimulation, inhibiting the secretion of inflammatory factors IL-1β and TNF-α; and effectively increasing the level of anti-inflammatory factor IL-10.

[0023] (4) The nanomedicine M4oRL described in the present invention can be efficiently enriched in the myocardial infarction area after being injected via the tail vein in the acute myocardial infarction mouse model.

[0024] (5) In the acute myocardial infarction mouse model, the nanodrug M4oRL of the present invention can effectively inhibit ischemia-induced cardiomyocyte pyroptosis, reduce the proportion of pro-inflammatory monocytes / macrophages, increase the proportion of reparative monocytes / macrophages, regulate the inflammatory microenvironment, thereby improving cardiac function and showing a more excellent myocardial protective effect, compared with the normal saline control group, the 4-octylitaconate treatment group and the 4oRL treatment group.

[0025] (6) The present invention provides a method for preparing the above-mentioned drug-loaded nanoparticles having the function of regulating myocardial inflammatory microenvironment. The method is simple to operate, does not require the support of large-scale equipment, is low in cost, and is easy to realize the industrial production of the nanomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A preparation process for drug-loaded nanoparticles having the function of regulating myocardial inflammatory microenvironment;

[0027] Figure 2 The particle size (A), Zeta potential (B), transmission electron microscopy (C) and FT-IR (D) images of 4oRL and M4oRL nanomedicines;

[0028] Figure 3 The antioxidant capacity test (TEAC) (A), ·O2- and ·OH scavenging ability test (B and C) of 4oRL and M4oRL nanomedicines, and the drug release of M4oRL nanomedicine in different buffers (D);

[0029] Figure 4 The results showed that 4-OI, 4oRL and M4oRL nanomedicines could scavenge hypoxia-induced mitochondrial reactive oxygen species and inhibit apoptosis in H9c2 cardiomyocytes;

[0030] Figure 5 The results show that 4-OI, 4oRL and M4oRL nanomedicines inhibited the hypoxia-induced NLRP3 and IL-1β protein expressions in H9c2 cardiomyocytes;

[0031] Figure 6 The results are as follows: 4-OI, 4oRL and M4oRL nanomedicines modulate the function of pro-inflammatory RAW 264.7 macrophages;

[0032] Figure 7 The results of myocardial targeting of IR780-labeled M4oRL nanodrug (abbreviated as MIrRL) in an acute myocardial infarction mouse model;

[0033] Figure 8 The results of 4-OI, 4oRL and M4oRL nanomedicines regulating the myocardial inflammatory microenvironment after acute myocardial infarction include reducing ROS levels, inhibiting cardiomyocyte pyroptosis, reducing pro-inflammatory macrophages, and increasing reparative macrophages;

[0034] Fig. 9 The results are for the cardioprotective effects of 4-OI, 4oRL and M4oRL nanomedicines in an acute myocardial infarction mouse model. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent. Unless otherwise specified, the reagents used in the following examples are all commercially available conventional reagents.

[0036] The present invention is based on the drug-loaded liposome nano-platform, and further uses tannic acid and Mn 2+ The metal phenolic network coating formed by self-assembly is modified to construct a drug-loaded nanoparticle with cardiac targeting, which can regulate the inflammatory microenvironment after acute myocardial infarction. The purpose of the present invention is to deliver M4oRL nanomedicine to the myocardial injury area through tail vein injection. In the microenvironment of high reactive oxygen and low pH in the infarction area, M4oRL nanoparticles dissociate and release 4-octylitaconic acid to inhibit the activation of myocardial cell inflammasome NLRP3. At the same time, the dissociated tannic acid can act synergistically to regulate the function of pro-inflammatory macrophages, further comprehensively regulate the inflammatory microenvironment, and thus more effectively treat acute myocardial infarction.

[0037] Example 1 Synthesis of 4-octylitaconic acid loaded liposome nanodrug M4oRL wrapped in metal-phenolic network coating.

[0038] Schematic diagram of the synthesis process of M4oRL nanomedicine Figure 1As shown. First, the liposomes loaded with 4-octylitaconic acid were synthesized by the improved ethanol injection method. 4-OI, DSPC, DSPE-TK-PEG and cholesterol were dissolved in ethanol solvent respectively, and 20.64mM 4-OI and DSPC, DSPE-TK-PEG and cholesterol of different molar masses were mixed evenly. Then, the above four drug mixtures were taken with a 200uL pipette and quickly injected into 1.8mL PBS. The suspension was placed under 200W ultrasound, with ultrasound time: interval time = 5s: 3s, instrument protection temperature: 30℃, total working time: 10min, 4-OI nanoparticles (abbreviated as 4oRL) were spontaneously formed to obtain a milky white suspension. The obtained milky white suspension was centrifuged at 2000rpm for 10min, transferred to a 10KD dialysis bag, dialyzed for 12h, and the dialysis external fluid was replaced every 2h to obtain purified 4oRL nanoparticles. The particle size of 4oRL was measured by dynamic light scattering to be approximately 145 nm ( Figure 2 A), the surface Zeta potential is about -27mV ( Figure 2 B) If Figure 2 As shown in Figure C, 4oRL nanoparticles are uniformly spherical under electron transmission electron microscopy. High performance liquid chromatography showed that when 20.64mM 4-OI was mixed with 0.57mM DSPC, 0.38mM cholesterol, and 0.05mM DSPE-TK-PEG, the drug loading rate of 4-OI in 4oRL nanoparticles could reach about 11%. In the second step, a metal-phenolic network coating-coated 4-octylitaconic acid liposome nanodrug (abbreviated as M4oRL) was synthesized by self-assembly. Dissolve tannic acid (abbreviated as TA) and MnCl2·4H2O in ultrapure water, add 2 mL of the synthesized 4oRL nanoparticles to 2.38 mL of PBS stirred at 700 rpm, stir for 2 min, add the fully dissolved TA and MnCl2·4H2O to the PBS suspension at a mass ratio of 1 mg:0.25 mg, stir for 2 min, adjust the pH of the reaction system with 0.5 mL of MOPS buffer (0.1 M, pH 8.0), and react for 1 h to obtain M4oRL nanoparticles. Centrifuge with PBS at 18000 rpm for 25 min, wash 3 times, remove the remaining reactants, and freeze-dry to obtain purified M4oRL nanoparticles. Dynamic light scattering measured the particle size of M4oRL to be approximately 153 nm ( Figure 2 A), the surface Zeta potential is about -22mV ( Figure 2 B) If Figure 2 As shown in Figure C, M4oRL nanoparticles are uniformly spherical under transmission electron microscopy, and the presence of surface coating is visible. As shown in Figure D, Fourier transform infrared spectroscopy detection indicates that TA and Mn on 4oRL nanoparticles 2+A metal phenolic network coating was formed, further suggesting the successful preparation of M4oRL nanoparticles.

[0039] Example 2 Antioxidant function of nanodrug M4oRL.

[0040] To further demonstrate the improvement of the performance of single functional liposomes by modification of metal phenolic network coating, empty liposomes (abbreviated as MRL) coated with metal phenolic network coating were introduced as a control. According to the instructions of the total antioxidant capacity detection kit, the total antioxidant capacity (abbreviated as TEAC) of 4-OI, 4oRL, M4oRL and MRL was determined. The results showed that 4-OI and 4oRL did not have ABTS scavenging function, while MPN-modified M4oRL and MRL showed significant ABTS scavenging ability ( Figure 3 A) In addition, the above drugs were further tested for their effects on ·O2 - and ·OH scavenging capacity, the results are as follows Figure 3 BC, EPR signal intensity decreased after adding M4oRL, MRL or TA compared with the control group. These results indicate that the modification of metal phenolic network coating endows single functional liposomes with the ability to scavenge reactive oxygen species.

[0041] Example 3 Responsiveness of nanodrug M4oRL to active oxygen and pH.

[0042] The M4oRL nanodrug was dispersed in PBS at pH 7.4 and placed in a 10KD dialysis bag. The basic dialysis external fluid was PBS containing 0.5% Tween-80 and pH 7.4. The pH and reactive oxygen level of the dialysis external fluid were adjusted with dilute hydrochloric acid and hydrogen peroxide. The dialysis bag containing the M4oRL nanodrug was completely immersed in the dialysis external fluid and placed in a constant temperature water bath at 37°C and stirred. The dialysis external fluid was taken out at different time points, the 4-OI content therein was detected, and fresh dialysis external fluid of equal volume and conditions was supplemented to measure the responsiveness of the nanodrug M4oRL to reactive oxygen and pH. The results are shown in Figure 2. Figure 3 As shown in D, under dual stimulation (low pH and high ROS), the 4-OI release rate of M4oRL was significantly higher than that under single low pH or single ROS stimulation conditions.

[0043] Example 4 Effect of nanodrug M4oRL on pyroptosis of H9c2 cardiomyocytes.

[0044] According to previous studies, a hypoxic environment was constructed using Anning bag, and a serum-free conditioned medium was used to establish a hypoxia-induced H9c2 cell injury model. With the help of this cell model, the pyroptosis and cell protection effects of the nanodrug M4oRL on H9c2 cardiomyocytes were explored.

[0045] H9c2 cells were cultured at 5×105 1000 cells / well were inoculated in 6-well plates. The control group (Control) was treated with normoxia and normal temperature and conventional culture medium; the hypoxia group (Hyp) was treated with hypoxia and conditioned medium with an equal amount of PBS; the 4-OI group (4-OI) was treated with hypoxia and conditioned medium containing 75 μM 4-OI; the 4oRL nanodrug group (4oRL) was treated with hypoxia and conditioned medium containing 75 μM 4-OI and 4oRL nanodrug; the M4oRL nanodrug group (M4oRL) was treated with hypoxia and conditioned medium containing an equal amount of M4oRL nanodrug. The 6-well plate with replaced conditioned medium was placed in a hypoxic chamber and hypoxia was continued for 12 hours. After the end of hypoxia, the protein levels of NLRP3 (Abcam, ab263899) and IL-1β (Abcam, ab254360) were detected by Western blot using MitoSOX mitochondrial superoxide red fluorescent probe (Yisheng Biotechnology, Catalog No. 40778ES50), Annexin V-FITC / PI double staining cell apoptosis detection kit (Keygen Biotechnology, Catalog No. KGA1102-100), and cells in each group.

[0046] Figure 4 As shown in A-4D, compared with the Control group, the mitochondrial ROS level of H9c2 cardiomyocytes in the Hyp group was significantly increased, and the cell apoptosis rate was significantly increased, indicating that hypoxia caused significant damage to H9c2 cells. After treatment with 4-OI, 4oRL or M4oRL, the mitochondrial ROS level and cell apoptosis rate were significantly reduced, and the reduction in the M4oRL group was more significant. These results show that compared with the treatment of 4-OI and 4oRL alone, the M4oRL nanomedicine has a stronger inhibitory effect on the increase of mitochondrial reactive oxygen species in H9c2 cells induced by hypoxia, and can more effectively reduce cell apoptosis, exerting a superior cardiomyocyte protection effect.

[0047] like Figure 5 As shown in A and 5B, compared with the hypoxia group (Hyp), the expression levels of pyroptosis-related markers NLRP3 and activated IL-1β in H9c2 cells were significantly reduced after treatment with 4-OI, 4oRL, and M4oRL, and the reduction in the M4oRL group was the most significant. These results indicate that M4oRL treatment can more effectively inhibit hypoxia-induced pyroptosis in H9c2 cells, thereby further exerting a superior cell protection effect.

[0048] Example 5 Effect of nanodrug M4oRL on the function of pro-inflammatory RAW264.7 macrophages.

[0049] RAW 264.7 macrophages were cultured at 1×10 6100 cells / well were inoculated in a 6-well plate, RAW264.7 cells were stimulated with 200 ng / mL LPS, and then treated with PBS, 4-OI, 4oRL and M4oRL. The control group was treated with regular culture medium, and the stimulation time was 24 h. After the cell treatment, the intracellular reactive oxygen species were detected according to the instructions of the 2,7-dichlorofluorescein diacetate (DCFH-DA) (MedChemExpress, HY-D0940) reagent; the cell supernatant was collected and the levels of IL-1β (Novus Biologicals, VAL601), TNF-α (Absin, abs520010) and IL-10 (Novus Biologicals, VAL605) were detected according to the instructions of the ELISA kit. The results are shown in Figure 6 As shown in A-6E, M4oRL nanomedicine can significantly reduce the production of reactive oxygen species in macrophages induced by LPS, inhibit the secretion of pro-inflammatory factors IL-1β and TNF-α, and increase the level of anti-inflammatory factor IL-10.

[0050] Example 6 Cardiac targeting of nanodrug M4oRL.

[0051] The myocardial infarction model of mice was established by ligating the left anterior descending coronary artery through thoracotomy; after thoracotomy in the sham group, the ligature passed through the anterior wall of the left ventricle but did not ligate the left anterior descending coronary artery. The nanodrug M4oRL (abbreviated as MIrRL) labeled with the fluorescent dye IR780 demonstrated myocardial targeting. MIrRL nanodrug was injected into the tail vein 30 minutes after the coronary artery of mice was ligated, and the cardiac fluorescence distribution was detected using a small animal in vivo optical imaging system. Figure 7 As shown in A, 4 to 12 hours after the tail vein injection of MIrRL, the fluorescence intensity of the precardiac area of ​​the acute myocardial infarction mouse gradually increased. After 12 hours, the heart of the acute myocardial infarction mouse was removed for in vitro fluorescence photography, as shown in Figure 7 As shown in B, a high-intensity fluorescence signal was clearly observed below the cardiac ligation area. These results suggest that M4oRL nanomedicine has targeting to the injured myocardium, providing strong support for its application in targeted therapy of myocardial injury.

[0052] Example 7 Regulation of the inflammatory microenvironment by the nanodrug M4oRL after myocardial infarction.

[0053] In the mouse myocardial infarction model, 4oRL and M4oRL nanomedicines were injected into the tail vein, and an equal amount of PBS was used as a control. 4-OI was injected intraperitoneally. After treatment, myocardial tissue was obtained for staining of reactive oxygen species, myocardial cell pyroptosis markers, and pro-inflammatory and anti-inflammatory macrophage markers. Figure 8 As shown, the MI group showed a significantly increased ROS level compared with the Sham group ( Figure 8A, E), NLRP3-positive cardiomyocytes ( Figure 8 B, F), iNOS positive macrophages ( Figure 8 C, H), and CD206-positive macrophages (D, G) were significantly reduced, indicating the formation of an inflammatory microenvironment in the myocardial tissue in the injured area. Compared with the MI group and the 4-OI and 4oRL treatment groups, the M4oRL treatment group showed lower ROS levels, NLRP3-positive cardiomyocytes and iNOS-positive macrophages, and the proportion of CD206-positive macrophages was further increased. These results suggest that M4oRL treatment can more effectively inhibit cardiomyocyte pyroptosis and regulate the myocardial inflammatory microenvironment.

[0054] Example 8 Effects of nanodrug M4oRL on cardiac function in mice with myocardial infarction.

[0055] After the myocardial infarction mice were treated with PBS, 4-OI, 4oRL and M4oRL nanomedicines, the cardiac function status was evaluated by small animal ultrasound on the 28th day. Fig. 9 As shown, compared with the Sham group, the MI group showed a significant decrease in left ventricular ejection fraction (EF) and fractional shortening (FS), as well as a significant increase in left ventricular internal diameter, which reflected the decline in cardiac function caused by myocardial damage after myocardial infarction. However, in mice treated with M4oRL nanomedicine, EF and FS values ​​were significantly increased, and the left ventricular internal diameter was significantly reduced, showing a significant improvement compared with the PBS treatment group. The results suggest that M4oRL has a significant myocardial protective effect in the recovery of cardiac function and can effectively inhibit the decline of cardiac function after myocardial infarction.

[0056] In summary, this application first prepared liposomes loaded with 4-octylitaconic acid and successfully improved the bioavailability of 4-octylitaconic acid. In order to further achieve targeted delivery to the heart and better cope with the high reactive oxygen state in the inflammatory microenvironment, this study prepared 4-OI nanoparticles modified with a metal phenolic network coating formed by self-assembly of tannic acid and Mn2+. The M4oRL nanodrug modified with the metal phenolic network coating exhibits excellent antioxidant properties and cardiac targeting properties. In vitro experimental verification showed that the M4oRL nanodrug can effectively inhibit the pyroptosis of H9c2 cells and regulate the function of RAW 264.7 proinflammatory cells. In the myocardial infarction mouse model, thanks to the targeting of the M4oRL nanodrug and its enhanced antioxidant properties, the inflammatory microenvironment in the myocardial injury area was effectively regulated, the cardiac function of the mice was significantly improved, and an excellent myocardial protective effect was shown.

[0057] Finally, it should be noted that the above-described embodiments are only preferred embodiments of the present invention. It should be noted that any person skilled in the art can make several improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. A 4-octylitaconic acid-loaded liposome nanoparticle coated with a metal-phenolic network coating, characterized in that: The core of the nanoparticle is a liposome loaded with 4-octylitaconic acid, and the shell is tannic acid-Mn 2+ Self-assembled metal phenolic network coating.

2. The method for preparing the 4-octylitaconic acid loaded liposome nanoparticles wrapped with the metal-phenolic network coating according to claim 1, characterized in that: The steps include: (1) Dissolving 4-octyl itaconic acid, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-thioketal-polyethylene glycol and cholesterol in a solvent respectively, mixing them in a certain proportion, and then rapidly injecting them into a PBS solution. The obtained mixed solution is ultrasonicated, and the milky white suspension after ultrasonication is placed in a 35-50° C. water bath and stirred for 3-6 h to obtain a 4-octyl itaconic acid-loaded liposome solution. The solution is centrifuged at a low speed to obtain a precipitate, and the precipitate is transferred to a dialysis bag for dialysis to obtain a 4-octyl itaconic acid-loaded liposome. (2) The 4-octylitaconic acid liposomes prepared in step (1) are mixed with tannic acid, MnCl2•4H2O and a buffer solution, and the coupling reaction is carried out for 1 to 6 hours. After the reaction is completed, the mixture is washed and freeze-dried to obtain the drug-loaded nanoparticles having the function of regulating the myocardial inflammatory microenvironment.

3. The preparation method according to claim 2, characterized in that: In step (1), the solvent is anhydrous ethanol.

4. The preparation method according to claim 2, characterized in that: In step (1), the molar mass ratio of 4-octylitaconic acid, DSPC, DSPE-TK-PEG and cholesterol is 10.32-24.77 mM: 0.4-0.6 mM: 0.04-0.1 mM: 0.3-0.6 mM.

5. The preparation method according to claim 2, characterized in that: In step (1), the rapid injection method is to use a 200 uL pipette to aspirate the drug mixture, avoiding the formation of bubbles at the tip of the pipette, and then rapidly inject it into the PBS solution.

6. The preparation method according to claim 2, characterized in that: In step (1), the ultrasonic condition is placed at 100-200 W for 5-10 min; the low-speed centrifugation condition is 1000-3000 rpm for 5-20 min.

7. The preparation method according to claim 2, characterized in that: In step (1), the specification of the dialysis bag is 10-20 KD, the dialysis external solution is PBS solution, the dialysis external solution is changed every 1-3 hours, and the dialysis is performed for 10-24 hours.

8. The preparation method according to claim 2, characterized in that: In step (2), the mass ratio of the 4-octylitaconic acid liposomes, tannic acid and MnCl2·4H2O is 1-3 mg: 1 mg: 0.25 mg, the volume of the buffer solution is 3-5 mL, and the pH is 8-8.

5.

9. Use of the metal-phenolic network coating-coated 4-octylitaconic acid loaded liposome nanoparticles according to claim 1 in the preparation of a drug for alleviating or treating myocardial injury mediated by inflammatory response; the myocardial injury mediated by inflammatory response includes allergic inflammation, infectious inflammation and nonspecific inflammation.

10. The use according to claim 9, characterized in that: The myocardial injury mediated by the inflammatory response is acute myocardial infarction.

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