An engineered platelet membrane nanocarrier for regulating ischemic microenvironment and a preparation method thereof

Abstract

The application provides an engineered platelet membrane nanocarrier for regulating ischemic microenvironment and a preparation method thereof. The engineered platelet membrane nanocarrier is composed of a nanoparticle (DY) and a platelet membrane formed on the surface of the nanoparticle (DY), wherein the nanoparticle (DY) is self-assembled by long-chain molecules DSPE-PEG 2000 -Arg-TK-CY-09, TK represents a ROS-responsive material, a sulfur ketal bond, CY-09 represents an anti-inflammatory drug CY-09 for inhibiting the activation and assembly of NLRP3 inflammasome, Arg represents L-Arg for producing NO, and DSPE-PEG 2000 represents an amphiphilic polymer. The engineered platelet membrane nanocarrier (DY@PM) provided by the application has high stability and biological safety, can target a lesion site, and can synergistically regulate ischemic microenvironment through multiple targets, and has a wide application prospect in the biomedical field.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of medicine, and particularly relates to an engineered platelet membrane nanocarrier for regulating ischemic microenvironment and a preparation method thereof. BACKGROUND

[0002] Cardiovascular disease (CVD) is a global health problem, with a rising prevalence in developing countries. Among CVDs, ischemic heart disease is the leading cause of disability and death. Ischemic heart disease is caused by myocardial infarction (MI) after coronary artery obstruction. Clinically, percutaneous coronary intervention is often used to unblock the blood vessels or drug thrombolytic therapy, but the existing treatment methods are difficult to prevent heart failure, ultimately affecting the long-term survival rate of patients. Therefore, it is urgent to improve the treatment strategy for MI and seek more innovative and effective ways to preserve myocardial function, prevent adverse cardiac remodeling and ultimately heart failure.

[0003] Inflammation plays an important role in CVD. In the initial inflammatory stage, myocardial cells produce a large amount of reactive oxygen species (ROS), which destroy the cell microenvironment, causing myocardial cells to produce oxidative stress, further promoting myocardial inflammation and fibrosis. Excessive inflammatory response can cause a large number of myocardial cells to die, and various damage molecular patterns (DAMPs) and related inflammatory factors released further activate the nuclear factor kappa B (NF-κB) signaling pathway, prompting NACHT, LRR and PYD domain protein 3 (NLRP3) inflammasome to be assembled and activated, further activating cysteine-containing aspartate protease 1 (Caspase-1), promoting the secretion of inflammatory factors such as recombinant human interleukin-1β (IL-1β) and interleukin-18 (IL-18), thereby inducing myocardial cell pyroptosis by gasdermin D (GSDMD). This damage mechanism causes serious damage to the heart. As can be seen, NLRP3 inflammasome plays a core role in MI inflammation-induced myocardial cell death. Therefore, targeting the triggering mechanism of NLRP3 inflammasome is an important treatment strategy for myocardial infarction.

[0004] Nitric oxide (NO) as a multifunctional gaseous signaling molecule plays an important role in the homeostatic regulation of the cardiovascular system. Studies have shown that low-dose NO can not only eliminate oxidative stress and reduce myocardial cell apoptosis, but also increase cardiac contractility and heart rate, and improve myocardial activity. Studies have found that L-type arginine (L-Arg) is catalyzed by nitric oxide synthase to generate citrulline and NO, and NO further activates guanylate cyclase in cells to increase the concentration of cyclic guanosine monophosphate (cGMP) to achieve the purpose of vasodilation. At present, L-Arg has become an ideal drug for exogenous NO supply; CY-09 is a specific NLRP3 inhibitor that can directly bind to the ATP binding motif of the NLRP3 NACHT domain, inhibit the activity of NLRP3 ATPase, and thus inhibit the assembly and activation of NLRP3 inflammasome. However, L-Arg and CY-09 themselves do not have targetability to the lesion site, and the amount of L-Arg and CY-09 reaching the lesion site after tail vein injection is very small. Therefore, how to precisely enrich L-Arg and CY-09 in the myocardial ischemic site, and L-Arg can produce NO to enhance myocardial cell activity while synergizing with CY-09 to play an anti-inflammatory role to effectively treat MI is a difficult problem to be solved at present.

[0005] With the in-depth study of nanodrug delivery systems, its precise targeting, controllable drug release and other advantages have become a hot spot in the field of drug delivery. SUMMARY

[0006] The purpose of the present application is to provide an engineered platelet membrane nanocarrier for multi-target synergistic treatment of MI.

[0007] The engineered platelet membrane nanocarrier provided by the present application is composed of a nanoparticle (DY) and a platelet membrane formed on the surface of the nanoparticle (DY), wherein the nanoparticle (DY) is self-assembled by a long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09, TK represents a ROS-responsive material, sulfolactone bond, CY-09 represents an anti-inflammatory drug CY-09 that inhibits the activation and assembly of NLRP3 inflammasome, and Arg represents L-Arg that produces NO, DSPE-PEG 2000 represents an amphiphilic polymer; the nanoparticle (DY) is regular spherical; and the particle size of the engineered platelet membrane nanocarrier is 130-160 nm.

[0008] Specifically, the long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 is prepared by a method comprising the following steps:

[0009] 1) Anti-inflammatory drug CY-09 inhibiting NLRP3 inflammasome activation and assembly reacts with TK-2OH to obtain HO-TK-CY-09 (CY-TK);

[0010] 2) L-type arginine L-Arg reacts with amphiphilic polymer DSPE-PEG 2000 -COOH to obtain DSPE-PEG 2000 -Arg(DA);

[0011] 3) The carboxyl end of activated DSPE-PEG 2000 -Arg reacts with the hydroxyl group of HO-TK-CY-09 to obtain DSPE-PEG 2000 -Arg-TK-CY-09.

[0012] In the above method step 1), the structure of TK-2OH is as follows:

[0013]

[0014] Wherein, m and n are the same or different, and can be an integer of 1-6.

[0015] In step 1), the carboxyl end of CY-09 is activated first, and then the esterification reaction occurs between the carboxyl-activated CY-09 and the hydroxyl group at one end of TK-2OH to obtain HO-TK-CY-09 (CY-TK);

[0016] The molar ratio of CY-09 to TK-2OH can be 1:0.5-2.5;

[0017] The temperature of the esterification reaction can be room temperature, and the time can be 12-30h.

[0018] In step 2), the carboxyl end of DSPE-PEG 2000 -COOH is activated first, and then the reaction occurs between the carboxyl-activated DSPE-PEG 2000 -COOH and L-Arg to obtain DSPE-PEG 2000 -Arg(DA);

[0019] The molar ratio of DSPE-PEG 2000 -COOH to L-Arg can be 1:1-2.5;

[0020] The temperature of the reaction can be room temperature, and the time can be 12-30h.

[0021] In step 3), DMAP and EDC are used to activate the carboxyl end of DSPE-PEG 2000 -Arg.

[0022] DSPE-PEG2000 The molar ratio of Arg to HO-TK-CY-09 can be 1:2-5.

[0023] The temperature of the esterification reaction can be room temperature, and the time can be 12-30 h.

[0024] The above multi-target synergistic treatment of myocardial infarction engineered platelet membrane nanocarrier is prepared by a method comprising the following steps:

[0025] a) Preparation of long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09

[0026] 1) Anti-inflammatory drug CY-09 and TK-2OH were reacted to inhibit NLRP3 inflammasome activation and assembly, to obtain HO-TK-CY-09 (CY-TK);

[0027]

[0028] Wherein, m, n are the same or different, which can be an integer of 1-6, and specifically can be 3;

[0029] 2) L-arginine L-Arg of L type was reacted with amphiphilic polymer DSPE-PEG 2000 -COOH to obtain DSPE-PEG 2000 -Arg(DA);

[0030] 3) The carboxyl end of DSPE-PEG 2000 -Arg was esterified with the hydroxyl group of HO-TK-CY-09 to obtain DSPE-PEG 2000 -Arg-TK-CY-09;

[0031] b) The long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 was prepared into regular spherical nanoparticles (DY) by thin film hydration method;

[0032] c) The nanoparticles formed by the long-chain molecule and the platelet membrane were prepared into engineered platelet membrane nanocarriers (DY@PM) by co-extrusion through polycarbonate porous membrane.

[0033] In the above method step b), the specific operation of the thin film hydration method is as follows: the long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 (1.5-4.5 mg) was dissolved in 10 mL chloroform, placed in a 100 mL single-neck flask, and the organic solvent was removed by reduced pressure distillation at this time to form a thin film; then 3 mL of PBS solution was added to the single-neck flask, and the nanoparticles (DY) were prepared by ultrasonic treatment (room temperature, power 100) for 30 min.

[0034] The long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 or the long-chain molecule DSPE-PEG 2000 The regular spherical nanoparticles (DY) made of -Arg-TK-CY-09 also belong to the protection scope of the present application.

[0035] The long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 or the long-chain molecule DSPE-PEG 2000 The application of the regular spherical nanoparticles (DY) made of -Arg-TK-CY-09 or the engineered platelet membrane nanocarrier in the preparation of a drug for preventing / treating MI also belongs to the protection scope of the present application.

[0036] The application, the drug utilizes the specific factors on the platelet membrane surface, targets the ischemic site of the organism, in the microenvironment of the infarct myocardium with high levels of ROS and biological esterase, cleaves the TK ketal bond, hydrolyzes the ester bond, and releases CY-09 which specifically inhibits the activity of NLRP3 inflammasome and L-Arg which generates NO.

[0037] The present application also provides a drug for preventing and / or treating MI, which contains the engineered platelet membrane nanocarrier or the long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 or the long-chain molecule DSPE-PEG 2000 The regular spherical nanoparticles (DY) made of -Arg-TK-CY-09.

[0038] The present application connects the anti-inflammatory drug CY-09 which inhibits the activation and assembly of NLRP3 inflammasome and L-Arg which generates NO through the ROS-responsive material ketal bond, and combines with the amphiphilic polymer DSPE-PEG 2000 -COOH to prepare a long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09. We use the thin film hydration method to prepare the long-chain molecule into regular spherical nanoparticles (DY), and then use the polycarbonate porous membrane to co-extrude with the platelet membrane to obtain the engineered platelet membrane nanocarrier (DY@PM) for multi-target synergistic treatment of MI.

[0039] The engineered platelet membrane nanocarrier (DY@PM) provided by the present application has high stability and biological safety, can target the lesion site, and multi-target synergistically regulates the ischemic microenvironment, and has a broad application prospect in the biomedical field. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Mass spectrum of CY-TK prepared for the embodiment 1 of the present application.

[0041] Figure 2 DSPE-PEG prepared for the embodiment 1 of the present application 2000 Mass spectrum of -Arg(DA).

[0042] Figure 3 Long chain molecule DSPE-PEG prepared for the embodiment 1 of the present application 2000 NMR hydrogen spectrum of -Arg-TK-CY-09 (deuterated DMSO).

[0043] Figure 4 Long chain molecule DSPE-PEG prepared for the embodiment 1 of the present application 2000 XPS diagram of -Arg-TK-CY-09, wherein a): F element; b): N element; c): S element.

[0044] Figure 5 Particle size stability curve of DY in PBS solution.

[0045] Figure 6 DLS particle size distribution of DY@PM, with a particle size of about 154 nm.

[0046] Figure 7 SEM diagram of DY@PM.

[0047] Figure 8 Fluorescence image of myocardial cells taking DY@PM. (Blue (DAPI): cell nucleus; green (Dio): platelet membrane; red (Cy5): nanoparticle DY; Merge (yellow)): proving that the nanoparticle successfully wraps the platelet membrane.

[0048] Figure 9 Effect of engineered platelet membrane nanocarrier at different concentrations on the viability of myocardial cells.

[0049] Figure 10 ROS scavenging ability of engineered platelet membrane nanocarrier.

[0050] Figure 11 In a), fluorescence imaging of NO in myocardial cells; in b), quantitative statistical analysis of green fluorescence intensity.

[0051] Figure 12 In a), confocal image of the ability of DY@PM to reduce NLRP3 inflammasome; in b), quantitative statistical analysis of fluorescence intensity.

[0052] Figure 13Distribution images of engineered platelet membrane nanocarriers tail vein injected into myocardial ischemia model in major organs. DETAILED DESCRIPTION

[0053] The application will be further described in conjunction with the preferred embodiments thereof, given solely by way of illustration of the present application and not by way of limitation. The following examples provided serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation of the present application.

[0054] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0055] Example 1, DSPE-PEG 2000 Preparation of long chain molecule DSPE-PEG

[0056] First, CY-TK is synthesized from CY-09 and TK-2OH, second, L-Arg and DSPE-PEG 2000 -COOH are synthesized to form DSPE-PEG 2000 -Arg(DA), and finally, CY-TK and DSPE-PEG 2000 -Arg(DA) ester are synthesized to form long chain molecule DSPE-PEG 2000 -Arg-TK-CY-09.

[0057] The specific operation is as follows:

[0058] 1. Synthesis of CY-TK

[0059] (1) First, compound CY-09 (0.1 mmol, 2.34 mg), DMAP (0.1 mmol, 12.22 mg), EDC·HCL (0.1 mmol, 19.17 mg) are sequentially added to DCM and stirred for 30 min to activate the carboxyl end of CY-09.

[0060] (2) Then, the above liquid is slowly added to a solution of TK-2OH (m, n = 3) (0.15 mmol, 33.6 mg) dissolved in DCM, and stirring is continued for 24 h.

[0061] (3) Finally, the solution is extracted to remove water, placed in anhydrous sodium sulfate overnight, and the purified sticky yellow-green product is obtained by silica gel chromatography (silica gel, dichloromethane: anhydrous methanol = 30:1).

[0062] Figure 1Mass spectrum of CY-TK.

[0063] 2. DSPE-PEG 2000 Synthesis of Arg

[0064] (1) Activation of the carboxyl end of DSPE-PEG 2000 -COOH, EDC (0.015 mmol, 2.9 mg) and DSPE-PEG 2000 -COOH (0.015 mmol, 42.38 mg) were added to the DMSO solution, after stirring for 30 min, NHS (0.076 mmol, 8.75 mg) was added and stirring was continued for 3 h;

[0065] (2) L-Arg (0.015 mmol, 2.613 mg) dissolved in DMSO was slowly added to the above solution, and stirring was continued for 24 h;

[0066] (3) The solution obtained after stirring was placed in a dialysis bag and dialyzed for 48 h, and freeze-dried for 72 h to obtain a white solid powder (DA).

[0067] Figure 2 Mass spectrum of DA.

[0068] 3. DSPE-PEG 2000 Synthesis of Arg-TK-CY-09

[0069] (1) Activation of the carboxyl end of DSPE-PEG 2000 -Arg, DSPE-PEG 2000 -Arg (0.0069 mmol, 20 mg), DMAP (0.0069 mmol, 0.843 mg), EDC-HCl (0.0345 mmol, 6.614 mg) were added to the DMF solution in sequence and stirred for 30 min;

[0070] (2) CY-TK (0.0276 mmol, 17.372 mg) dissolved in DMF solution was added to the above solution, and stirring was continued for 24 h;

[0071] (3) The solution was slowly dropped into cold ether, and stirred vigorously, and after standing, the precipitate was collected, and the precipitate was placed in a vacuum drying oven to remove the ether, to obtain light green solid DSPE-PEG 2000 -Arg-TK-CY-09.

[0072] Figure 3 NMR hydrogen spectrum of long-chain molecule (deuterated DMSO).

[0073] Figure 4XPS spectra of long-chain molecules, wherein a) F element; b) N element; c) S element (DA does not have F and S elements, CY-TK has F and S elements, and after successful esterification synthesis, CY-TK is removed by cold ether, and DY also has F and S elements).

[0074] Example 2, Preparation of engineered platelet membrane nanocarriers

[0075] (1) Dissolve long-chain molecule DSPE-PEG 2000 - Arg-TK-CY-09 (3.5 mg) in 10 mL chloroform, remove the organic solvent by reduced pressure distillation to form a thin film;

[0076] (2) Add the thin film obtained above to PBS (3 mL) to obtain a PBS solution of nanoparticles (DY) by ultrasonic method (room temperature, power 100, time 30 min);

[0077] (3) Take 1 mL of nanoparticles (DY) formed by long-chain molecules and add 7 μg of platelet membranes to prepare stable engineered platelet membrane nanocarriers (DY@PM) by co-extrusion through a polycarbonate porous membrane for 20 times.

[0078] Example 3, Characterization of engineered platelet membrane nanocarriers

[0079] We used DLS to determine the particle size stability of nanoparticles in PBS for 7 days and the particle size of engineered platelet membrane nanocarriers (DY@PM), and used scanning electron microscopy to observe the morphology of nanoparticles after wrapping platelet membranes. In addition, we used the self-assembly ability of long-chain molecules to load Cy5 dye (red), and dyed platelet membranes with Dio (green), and used laser confocal fluorescence microscopy to determine the co-localization of platelet membranes and nanoparticles. The results showed that a large amount of yellow appeared after merging, indicating that platelet membranes were stably present on the surface of nanoparticles formed by long-chain molecules.

[0080] Figure 5 The particle size stability curve of DY in PBS solution.

[0081] Figure 6 The DLS particle size distribution of DY@PM, with a particle size of about 154 nm.

[0082] Figure 7 The SEM image of DY@PM.

[0083] Figure 8 The fluorescence image of myocardial cells taking up DY@PM. (Blue (DAPI): cell nucleus; green (Dio): platelet membrane; red (Cy5): nanoparticle DY; Merge (yellow)): proving that nanoparticles successfully wrapped platelet membranes.

[0084] Example 4, Biological safety identification of engineered platelet membrane nanocarriers

[0085] To evaluate the biological safety of engineered platelet membrane nanocarriers, we co-incubated engineered platelet membrane nanocarriers with myocardial cells (H9C2) and determined the effect of engineered platelet membrane nanocarriers on the viability of H9C2 by CCK-8 method. The results showed that engineered platelet membrane nanocarriers had little effect on the viability of myocardial cells, had high biological safety, and therefore could be used for subsequent experiments.

[0086] Figure 9 The effect of different concentrations of engineered platelet membrane nanocarriers on the viability of myocardial cells.

[0087] Example 5, Evaluation of the ability of engineered platelet membrane nanocarriers to remove ROS

[0088] We evaluated the ability of engineered platelet membrane nanocarriers to remove H2O2 by potassium iodide (KI) method (KI reacts with H2O2 to produce a yellow solution, and has a maximum absorption value at 352 nm). The specific operation method is as follows: the control group is a simple H2O2 solution (500 μM), and the experimental group is a H2O2 solution (500 μM) and engineered platelet membrane nanocarriers (10 μM) reacted at 37°C for 12 h. 100 μL of liquid was taken from the control group and the experimental group and placed in a 96-well plate, and 100 μL of KI (1 M) solution was added to both groups to react in the dark for 5 min. The absorbance of the two groups was determined by a microplate reader at a wavelength of 352 nm.

[0089] Figure 10 The ability of engineered platelet membrane nanocarriers to remove ROS can be found that the absorbance value of the experimental group is significantly reduced, indicating that the content of H2O2 is reduced.

[0090] Example 6, Evaluation of the ability of engineered platelet membrane nanocarriers to produce NO in vitro

[0091] After adding engineered platelet membrane nanocarriers to myocardial cells (H9C2), we used NO fluorescent probe DAF-FM DA to detect the production of NO in myocardial cells. The specific operation is as follows: first, 3.5 x 10 4Cardiomyocytes (H9C2) per mL were seeded in 24-well plates and cultured overnight. The control group consisted of normally cultured cells. The experimental groups were divided into two groups: the H2O2 group (500 μL H2O2 (500 μM) stimulated cardiomyocytes for 4 h to establish an oxidative stress model) and the H2O2+DY@PM group (after establishing the oxidative stress model, the model group was treated with 500 μL of DMEM medium rich in engineered platelet membrane nanocarriers (10 μM). Both the control and experimental groups were added to the DAF-FM DA probe, incubated in the dark for 20 min, washed three times with PBS, and then incubated with DAPI in the dark for 5 min, followed by three more washes with PBS. Observation was performed under a laser confocal fluorescence microscope. The results showed that the control group exhibited weak green fluorescence, the H2O2 group showed enhanced green fluorescence, and the H2O2+DY@PM group showed the strongest green fluorescence. Quantitative analysis using ImageJ confirmed that the engineered platelet membrane nanocarriers had a good ability to generate NO in vitro.

[0092] Figure 11 a) Fluorescence imaging of NO in cardiomyocytes (DAF-FM: fluorescence produced by the interaction of DA and NO; the more NO present, the stronger the fluorescence) b) Quantitative statistical analysis of green fluorescence intensity.

[0093] Example 7: Evaluation of the in vitro anti-inflammatory capacity of engineered platelet membrane nanocarriers

[0094] After adding engineered platelet membrane nanocarriers to cardiomyocytes (H9C2), we used immunofluorescence to detect the expression of NLRP3 inflammasomes in H9C2 cardiomyocytes. First, 3.5 × 10⁻⁶ platelet membrane nanocarriers were added to the cells. 4H9C2 myocardial cells were seeded in 24-well plates and cultured overnight. The control group was normal cultured cells, and the experimental group was divided into: LPS+ATP group: 500 μL of LPS solution (10 μg / mL) was used to stimulate myocardial cells for 4 h, and then 500 μL of ATP solution (8 mM) was used to stimulate for 2 h to establish a myocardial inflammation injury model; LPS+ATP+DY@PM group: after LPS solution stimulation, 500 μL of DMEM medium containing engineered platelet membrane nanocarriers (10 μM) was used to treat for 4 h, and then ATP solution (8 mM) was added to stimulate for 2 h. The control group and the experimental group were added with 1:500 (NLRP3 inflammasome primary antibody: 1% goat serum) diluted NLRP3 inflammasome primary antibody, and incubated at 4°C overnight, washed with PBS for 3 times, each time for 5 min; then 1:500 (secondary antibody fluorescent probe: 1% goat serum) diluted goat anti-rabbit IgG (H+L) Fluor 594 (secondary antibody fluorescent probe) was added, and incubated at 37°C for 1 h, and washed with PBS for 3 times, each time for 5 min; DAPI-containing mounting medium was added for mounting, and laser confocal fluorescence microscopy was used for observation. Under normal culture conditions, weak red fluorescence was observed in H9C2 cells, and after inflammation injury, the increase of NLRP3 inflammasome led to an increase in red fluorescence, but after inflammation injury, the addition of engineered platelet membrane nanocarriers led to a significant decrease in red fluorescence intensity. The red fluorescence intensity was quantified by Image J, thereby confirming that the engineered platelet membrane nanocarriers had certain anti-inflammatory ability in vitro.

[0095] Figure 12 Figure 8a is a confocal image showing the ability of DY@PM to reduce NLRP3 inflammasome (red: NLRP3 inflammasome secondary antibody fluorescent probe: goat anti-rabbit IgG (H+L) Fluor 594, the more NLRP3 inflammasome, the stronger the red fluorescence).

[0096] Figure 8b is a quantitative statistical analysis of fluorescence intensity.

[0097] Example 8, Evaluation of the ability of engineered platelet membrane nanocarriers to target infarct myocardium

[0098] To evaluate the ability of the engineered platelet membrane nanocarrier to target the ischemic site of myocardial infarction, we observed the distribution of the nanocarrier in different tissues using small animal live imaging. In this part of the experiment, the mouse MI model was constructed as follows: 10-week-old C57 male mice were used. The mice were fasted for one day before the operation. After the mice were anesthetized by inhaling isoflurane gas, the four limbs of the mouse were fixed on the operating table in a supine position. The chest was disinfected with iodophor, and the skin was incised at the lower one-third of the neck. The trachea was separated and incised, and a tracheal tube was inserted for assisted respiration with an anesthetic respirator. The fascia at the junction of the pectoralis major muscle and the anterior serratus muscle was bluntly separated with forceps, and the fascia was inserted into the chest at the fourth rib. The chest was opened with a chest opener, and the lungs were pushed aside with a sterile cotton ball to expose the heart. The pericardium was carefully torn with forceps to expose the anterior wall of the left ventricle. At a distance of 1 mm from the left auricle below the starting end of the left coronary artery, the left anterior descending branch of the coronary artery was ligated with 7-0 suture. The ligation depth was about 0.5 mm, and the width was 1 mm. After ligation, if the myocardium below the ligation site changes from red to pale or appears purple, and the activity of the ventricular wall is reduced to disappearance, it indicates that the mouse MI model is successfully constructed. After the circulation of the mouse stabilized, the chest was closed with 4-0 suture, the skin was sutured, and the skin was disinfected with iodophor. The mouse was placed on a heating pad and continued to be connected to the respirator for assisted respiration. The state of the mouse was observed.

[0099] To detect the targeted enrichment effect of the engineered platelet membrane nanocarrier in the ischemic myocardium of the MI mouse, sham and MI mice were randomly selected. Within 15 minutes after modeling, the engineered platelet membrane nanocarrier loaded with cy5 (0.2 mg / mL) was injected through the tail vein. At 6 hours and 12 hours after injection, the fluorescence distribution of the engineered platelet membrane nanocarrier in the heart, liver, spleen, lung, and kidney was observed using small animal live imaging technology.

[0100] Figure 13 The distribution images of the engineered platelet membrane nanocarrier in the main organs after tail vein injection into the myocardial ischemia model. The results show that the engineered platelet membrane nanocarrier has the ability to target the myocardial ischemic site.

[0101] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range of equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application.

Claims

1. An engineered platelet membrane nanocarrier, consisting of a nanoparticle and a platelet membrane formed on the surface of the nanoparticle, wherein, The nanoparticles are formed by long-chain molecules DSPE-PEG 2000 - Arg-TK-CY-09 self-assembles, TK represents a ROS-responsive material, a thio-ketone bond, CY-09 represents an anti-inflammatory drug that inhibits the activation and assembly of NLRP3 inflammasome, Arg represents L-Arg that produces NO, DSPE-PEG 2000 represents an amphiphilic polymer; The long-chain molecule DSPE-PEG 2000 - Arg-TK-CY-09 is prepared by a method comprising the following steps: 1) Anti-inflammatory drug CY-09 inhibiting NLRP3 inflammasome activation and assembly reacts with TK-2OH to obtain HO-TK-CY-09; 2) L-arginine L-Arg with amphiphilic polymer DSPE-PEG 2000 -COOH reaction to obtain DSPE-PEG 2000 -Arg; 3) Activated DSPE-PEG 2000 - esterification reaction between the carboxyl end of Arg and the hydroxyl group of HO-TK-CY-09, to obtain DSPE-PEG 2000 - Arg-TK-CY-09, The structural formula of TK-2OH is as follows: Wherein, m, n are the same or different, integers from 1 to 6.

2. The engineered platelet membrane nanocarriers of claim 1, wherein, The nanoparticles are regular spherical; The particle size of the engineered platelet membrane nanocarrier is 130-160 nm.

3. The method for preparing the engineered platelet membrane nanocarrier of claim 1 or 2, comprising the following steps: a) Preparation of long-chain molecule DSPE-PEG 2000 - Arg-TK-CY-09 1) Anti-inflammatory drug CY-09 inhibiting NLRP3 inflammasome activation and assembly reacts with TK-2OH to obtain HO-TK-CY-09; wherein, m, n are the same or different, integers from 1 to 6; 2) L-arginine L-Arg with amphiphilic polymer DSPE-PEG 2000 -COOH to obtain DSPE-PEG 2000 -Arg; 3) Activated DSPE-PEG 2000 - esterification of the carboxyl end of Arg with the hydroxyl group of HO-TK-CY-09 to obtain DSPE-PEG 2000 - Arg-TK-CY-09; b) long chain molecules DSPE-PEG 2000 - Arg-TK-CY-09 was prepared into regular spherical nanoparticles; c) The nanoparticles formed by long-chain molecules and the platelet membrane are co-extruded through a polycarbonate porous membrane to obtain the engineered platelet membrane nanocarrier.

4. The method of claim 3, wherein, In step a) 1), the carboxyl terminal of CY-09 is activated first, then the hydroxyl group at one end of CY-09 after carboxyl activation reacts with TK-2OH to obtain HO-TK-CY-09; The molar ratio of CY-09 to TK-2OH is 1:0.5-2.5; The temperature of the esterification reaction is room temperature, and the time is 12-30 h; In step a) 2) the DSPE-PEG is first activated 2000 at the carboxyl end of -COOH, and the carboxyl is activated after the DSPE-PEG 2000 -COOH is reacted with L-Arg to give DSPE-PEG 2000 -Arg; DSPE-PEG 2000 the molar ratio of -COOH to L-Arg is 1 : 1 - 2.5; The temperature of the reaction is room temperature, and the time is 12-30 h; In step 3), DMAP, EDC was used to activate DSPE-PEG 2000 - carboxy terminus of Arg DSPE-PEG 2000 - the molar ratio of Arg to HO-TK-CY-09 is 1 : 2-5; The temperature of the esterification reaction is room temperature, and the time is 12-30 h.

5. The long chain molecule DSPE-PEG prepared in step a) of the method of claim 3 2000 - Arg-TK-CY-09.

6. The long-chain molecule DSPE-PEG of claim 5 2000 - Regularly spherical nanoparticles made of Arg-TK-CY-09.

7. The long-chain molecule DSPE-PEG of claim 5 2000 - Arg-TK-CY-09, the nanoparticle of claim 6 or the engineered platelet membrane nanocarrier of claim 1 for use in the preparation of a medicament for the prevention and / or treatment of myocardial infarction.

8. A medicine for preventing and / or treating myocardial infarction, the medicine containing the engineered platelet membrane nanocarrier of claim 1 or the long-chain molecule DSPE-PEG of claim 5 2000 - Arg-TK-CY-09 or a regular spherical nanoparticle made of the long-chain molecule DSPE-PEG 2000 - Arg-TK-CY-09 or a regular spherical nanoparticle made of the long-chain molecule DSPE-PEG

Images