Engineered platelet membrane nano-carrier for regulating and controlling ischemic microenvironment and preparation method of engineered platelet membrane nano-carrier

Abstract

The invention provides an engineered platelet membrane nano-carrier for regulating and controlling an ischemic microenvironment and a preparation method of the engineered platelet membrane nano-carrier. The engineered platelet membrane nano-carrier is composed of nano-particles (DY) and a platelet membrane formed on the surface of the nano-particles (DY), the nano-particles (DY) are formed by self-assembly of long-chain molecules DSPE-PEG2000-Arg-TK-CY-09, TK represents a thioketal bond of an ROS response material, CY-09 represents an anti-inflammatory drug CY-09 for inhibiting activation and assembly of NLRP3 inflammasomes, Arg represents L-Arg for generating NO, and the platelet membrane is formed by self-assembly of long-chain molecules DSPE-PEG2000-Arg-TK-CY-09. And the DSPE-PEG2000 represents an amphiphilic polymer. The engineered platelet membrane nano-carrier (DY at PM) provided by the invention has relatively high stability and biological safety, can target a focus part, can cooperatively regulate and control an ischemic microenvironment in a multi-target manner, and has a wide application prospect in the field of biomedicine.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to an engineered platelet membrane nanocarrier for regulating ischemic microenvironment and a preparation method thereof. Background Art

[0002] Cardiovascular disease (CVD) is a global health problem, and its prevalence continues to rise in developing countries. Among CVD, ischemic heart disease is the leading cause of disability and death. Ischemic heart disease is caused by myocardial infarction (MI) after coronary artery obstruction. Percutaneous coronary intervention or drug thrombolysis is often used clinically, but existing treatments are difficult to prevent heart failure, which ultimately affects the long-term survival rate of patients. Therefore, there is an urgent need to improve the treatment strategy of 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, cardiomyocytes produce a large amount of reactive oxygen species (ROS). These ROS damage the cell microenvironment, causing oxidative stress in cardiomyocytes, and further promoting myocardial inflammation and fibrosis. Excessive inflammatory response can lead to the death of a large number of cardiomyocytes. Various damage molecular patterns (DAMPs) and the released related inflammatory factors further activate the nuclear factor κB (NF-κB) signaling pathway, prompting the assembly and activation of NACHT, LRR and PYD domain protein 3 (NLRP3) inflammasomes, 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 glycosylated sildenafil D (GSDMD)-induced cardiomyocyte pyroptosis. This damage mechanism causes serious damage to the heart. It can be seen that NLRP3 inflammasomes play a core role in cardiomyocyte death caused by MI inflammation. Therefore, targeting the triggering mechanism of NLRP3 inflammasome is an important therapeutic strategy for myocardial infarction.

[0004] As a multifunctional gas signaling molecule, nitric oxide (NO) plays an important role in the homeostatic regulation of the cardiovascular system. Studies have shown that low doses of NO can not only eliminate oxidative stress and reduce cardiomyocyte apoptosis, but also increase cardiac contractility and heart rate, and improve myocardial vitality. Studies have found that L-arginine (L-Arg) generates citrulline and NO under the catalysis of nitric oxide synthase, and NO further activates guanylate cyclase in cells, increases the concentration of cyclic guanine monophosphate (cGMP), and achieves 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 inflammasomes. However, L-Arg and CY-09 themselves do not have targeting to the lesion site, and the amount reaching the lesion site after tail vein injection is very small. Therefore, how to accurately enrich L-Arg and CY-09 in the area of ​​myocardial ischemia, and how to enable L-Arg to produce NO in the lesion site to enhance myocardial cell vitality and cooperate with CY-09 to exert anti-inflammatory effects to effectively treat MI is a difficult problem that needs to be solved urgently.

[0005] With the in-depth research on nanodrug delivery systems, they have become a hot topic in the field of drug delivery due to their advantages such as precise targeting and controllable drug release. Summary of the invention

[0006] The purpose of the present invention is to provide an engineered platelet membrane nanocarrier for the synergistic treatment of MI with multiple targets.

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

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

[0009] 1) The anti-inflammatory drug CY-09, which inhibits the activation and assembly of NLRP3 inflammasome, reacts with TK-2OH to obtain HO-TK-CY-09 (CY-TK);

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

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

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

[0013]

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

[0015] In step 1), the carboxyl end of CY-09 is first activated, and then the activated carboxyl end of CY-09 is subjected to an esterification reaction with the hydroxyl 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 12-30 hours.

[0018] In step 2), DSPE-PEG is first activated 2000 -COOH at the carboxyl end, and then the carboxyl group is activated and DSPE-PEG 2000 -COOH reacts with L-Arg to obtain DSPE-PEG 2000 -Arg(DA);

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

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

[0021] In step 3), DMAP and EDC are used to activate DSPE-PEG 2000 -Arg carboxyl terminus;

[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 hours.

[0024] The engineered platelet membrane nanocarrier for multi-target synergistic treatment of myocardial infarction 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) The anti-inflammatory drug CY-09, which inhibits the activation and assembly of NLRP3 inflammasome, reacts with TK-2OH to obtain HO-TK-CY-09 (CY-TK);

[0027]

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

[0029] 2) L-arginine L-Arg and amphiphilic polymer DSPE-PEG 2000 -COOH reaction to obtain DSPE-PEG 2000 -Arg(DA);

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

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

[0032] c) The nanoparticles formed by long-chain molecules and platelet membranes were co-extruded through a polycarbonate porous membrane to prepare engineered platelet membrane nanocarriers (DY@PM).

[0033] In step b) of the above method, the specific operation of the thin film hydration method is: 2000 -Arg-TK-CY-09 (1.5-4.5 mg) was dissolved in 10 mL of chloroform and placed in a 100 mL single-mouth bottle. The organic solvent was removed by vacuum distillation to form a thin film. Then 3 mL of PBS solution was added to the single-mouth bottle, and nanoparticles (DY) were prepared by ultrasound (room temperature, power 100) for 30 minutes.

[0034] The above long chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 or the long-chain molecule DSPE-PEG 2000 - Regular spherical nanoparticles (DY) made of Arg-TK-CY-09 also fall within the protection scope of the present invention.

[0035] The above long chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 or the long-chain molecule DSPE-PEG 2000 The use of regular spherical nanoparticles (DY) made of -Arg-TK-CY-09 or the engineered platelet membrane nanocarrier in the preparation of drugs for preventing / treating MI also falls within the protection scope of the present invention.

[0036] The application of the drug is to utilize specific factors on the surface of the platelet membrane to target the ischemic site of the organism, cleave the TK thioketal bond and hydrolyze the ester bond in the infarcted myocardial microenvironment with high levels of ROS and biological esterase, and release CY-09 that specifically inhibits the activity of the NLRP3 inflammasome and L-Arg that generates NO.

[0037] The present invention also provides a drug for preventing and / or treating MI, wherein the drug 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 -Regular spherical nanoparticles made of Arg-TK-CY-09 (DY).

[0038] The present invention connects the anti-inflammatory drug CY-09 that inhibits the activation and assembly of NLRP3 inflammasomes and L-Arg that produces NO through the ROS-responsive material thioketal bond, and connects it with the amphiphilic polymer DSPE-PEG 2000 -COOH combined to prepare a long-chain molecule DSPE-PEG that can self-assemble into nanoparticles 2000 -Arg-TK-CY-09. We used thin film hydration to prepare long-chain molecules into regular spherical nanoparticles (DY), and then used polycarbonate porous membranes and platelet membranes to co-extrude and push to obtain engineered platelet membrane nanocarriers (DY@PM) for multi-target synergistic treatment of MI.

[0039] The engineered platelet membrane nanocarrier (DY@PM) provided by the present invention has high stability and biosafety, can reach the lesion site in a targeted manner, and can coordinately regulate the ischemic microenvironment through multiple targets, thus having broad application prospects in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the mass spectrum of CY-TK prepared in embodiment 1 of the present invention.

[0041] Figure 2 DSPE-PEG prepared for implementation 1 of the present invention 2000 -Mass spectrometry of -Arg(DA).

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

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

[0044] Figure 5 This is the particle size stability curve of DY in PBS solution.

[0045] Figure 6 This is the DLS particle size distribution of DY@PM, and the particle size is about 154nm.

[0046] Figure 7 This is the SEM image of DY@PM.

[0047] Figure 8 Fluorescence image of DY@PM in myocardial cells (blue (DAPI): cell nucleus; green (Dio): platelet membrane; red (Cy5): nanoparticle DY; Merge (yellow)): proves that the nanoparticles successfully encapsulate the platelet membrane.

[0048] Fig. 9 Effects of different concentrations of engineered platelet membrane nanocarriers on cardiomyocyte viability.

[0049] Fig.10 The ability of engineered platelet membrane nanocarriers to scavenge ROS.

[0050] Fig.11 a) is the fluorescence imaging of NO in cardiomyocytes; b) is the quantitative statistical analysis of green fluorescence intensity.

[0051] Fig.12 (a) is a confocal image of DY@PM's ability to reduce NLRP3 inflammasomes; (b) is a quantitative statistical analysis of fluorescence intensity.

[0052] Fig.13This is an image of the distribution of engineered platelet membrane nanocarriers in major organs after tail vein injection into a myocardial ischemia model. DETAILED DESCRIPTION

[0053] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0054] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0055] Example 1, DSPE-PEG 2000 Preparation of long-chain molecules of -Arg-TK-CY-09

[0056] First, CY-TK was synthesized from CY-09 and TK-2OH, and then L-Arg and DSPE-PEG 2000 Synthesis of DSPE-PEG with -COOH 2000 -Arg(DA), and finally CY-TK and DSPE-PEG 2000 -Arg(DA) esterification to synthesize long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09.

[0057] The specific operations are 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), and EDC·HCL (0.1 mmol, 19.17 mg) were added to DCM in sequence and stirred for 30 min to activate the carboxyl end of CY-09.

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

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

[0062] Figure 1This is the mass spectrum of CY-TK.

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

[0064] (1) Activate DSPE-PEG under nitrogen protection 2000 -COOH carboxyl end, EDC (0.015mmol, 2.9mg) and DSPE-PEG 2000 -COOH (0.015 mmol, 42.38 mg) was added to the DMSO solution, stirred for 30 min, then NHS (0.076 mmol, 8.75 mg) was added and stirring continued for 3 h;

[0065] (2) Slowly add L-Arg (0.015 mmol, 2.613 mg) dissolved in DMSO to the above solution and stir for 24 h;

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

[0067] Figure 2 This is the mass spectrum of DA.

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

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

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

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

[0072] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of the long-chain molecule (deuterated DMSO).

[0073] Figure 4This is the XPS graph of the long-chain molecule, where a): F element; b): N element; c): S element (DA does not contain F and S elements, CY-TK does. After the two are successfully esterified and CY-TK is removed by cold ether, DY will also contain F and S elements).

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

[0075] (1) Long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 (3.5 mg) was dissolved in 10 mL of chloroform, and the organic solvent was removed by vacuum distillation to form a thin film;

[0076] (2) adding the film obtained above into PBS (3 mL), and obtaining a PBS solution of nanoparticles (DY) by ultrasonication (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. The mixture is co-extruded through a polycarbonate porous membrane for 20 times to prepare a stable engineered platelet membrane nanocarrier (DY@PM).

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

[0079] We used DLS to measure 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 wrapped in platelet membranes. In addition, we used the self-assembly ability of long-chain molecules to load Cy5 dye (red), and stained 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 Merge, indicating that the platelet membrane was stably present on the surface of nanoparticles formed by long-chain molecules.

[0080] Figure 5 This is the particle size stability curve of DY in PBS solution.

[0081] Figure 6 This is the DLS particle size distribution of DY@PM, and the particle size is about 154nm.

[0082] Figure 7 This is the SEM image of DY@PM.

[0083] Figure 8 Fluorescence image of DY@PM in myocardial cells (blue (DAPI): cell nucleus; green (Dio): platelet membrane; red (Cy5): nanoparticle DY; Merge (yellow)): proves that the nanoparticles successfully encapsulate the platelet membrane.

[0084] Example 4: Biosafety Evaluation of Engineered Platelet Membrane Nanocarriers

[0085] In order to evaluate the biosafety of engineered platelet membrane nanocarriers, we co-incubated the engineered platelet membrane nanocarriers with cardiomyocytes (H9C2) and determined the effect of engineered platelet membrane nanocarriers on the vitality of H9C2 by CCK-8 method. The results showed that the engineered platelet membrane nanocarriers had almost no effect on the vitality of cardiomyocytes and had high biosafety, so they can be used in subsequent experiments.

[0086] Fig. 9 Effects of different concentrations of engineered platelet membrane nanocarriers on cardiomyocyte viability.

[0087] Example 5: Evaluation of the ROS-scavenging ability of engineered platelet membrane nanocarriers

[0088] We used the potassium iodide (KI) method (KI reacts with H2O2 to produce a yellow solution with a maximum absorption value at 352nm) to evaluate the ability of engineered platelet membrane nanocarriers to remove H2O2. The specific operation method is as follows: the control group is a simple H2O2 solution (500μM), and the experimental group is a H2O2 solution (500μM) and an engineered platelet membrane nanocarrier (10μM) reacted at 37℃ for 12h. 100μL of liquid was taken from the control group and the experimental group and placed in a 96-well plate. At the same time, 100μL of KI (1M) solution was added to each of the two groups to react in the dark for 5min, and the absorbance of the two groups was measured at a wavelength of 352nm using an enzyme marker.

[0089] Fig.10 To demonstrate the ability of engineered platelet membrane nanocarriers to scavenge ROS, it can be found that the absorbance value of the experimental group was significantly reduced, indicating a decrease in H2O2 content.

[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 cardiomyocytes (H9C2), we used the NO fluorescent probe DAF-FM DA to detect the production of NO in cardiomyocytes. The specific operation is as follows: first, 3.5×10 4Cardiomyocytes (H9C2) of 100 μg / mL were seeded in 24-well plates and cultured overnight. The control group was normal cultured cells, and the experimental groups were divided into H2O2 group: 500 μL H2O2 (500 μM) stimulated cardiomyocytes for 4 h to establish an oxidative stress model; H2O2+DY@PM group: After the oxidative stress model was established, the model group was treated with 500 μL DMEM medium rich in engineered platelet membrane nanocarriers (10 μM). DAF-FM DA probe was added to the control group and the experimental group, incubated in the dark for 20 min, washed 3 times with PBS, added DAPI and incubated in the dark for 5 min, washed 3 times with PBS again, and observed under a laser confocal fluorescence microscope. The results showed that weak green fluorescence was visible in the control group, the green fluorescence of the H2O2 group was enhanced, and the green fluorescence of the H2O2+DY@PM group was the strongest. Image J quantitative analysis confirmed that the engineered platelet membrane nanocarrier had a good ability to produce NO in vitro.

[0092] Fig.11 a) Fluorescence imaging of NO in cardiomyocytes (DAF-FM shows fluorescence due to the interaction of DA and NO; the more NO there is, the stronger the fluorescence is); b) Quantitative statistical analysis of green fluorescence intensity.

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

[0094] After adding engineered platelet membrane nanocarriers to cardiomyocytes (H9C2), we used cell immunofluorescence to detect the expression of NLRP3 inflammasome in cardiomyocytes (H9C2). 4Cardiomyocytes (H9C2) of 100 μg / mL were seeded in 24-well plates and cultured overnight. The control group was normal cultured cells, and the experimental groups were as follows: LPS+ATP group: 500 μL LPS solution (10 μg / mL) stimulated cardiomyocytes for 4 hours, and then 500 μL ATP solution (8 mM) stimulated for 2 hours to establish a myocardial inflammation injury model; LPS+ATP+DY@PM group: After LPS solution stimulation, 500 μL DMEM medium rich in engineered platelet membrane nanocarriers (10 μM) was used for 4 hours, and then ATP solution (8 mM) was added to stimulate for 2 hours. The control group and the experimental group were added with 1:500 (NLRP3 inflammasome primary antibody: 1% goat serum) diluted NLRP3 inflammasome primary antibody, incubated at 4℃ overnight, washed 3 times with PBS, 5min each time; then added 1:500 (secondary antibody fluorescent probe: 1% goat serum) diluted goat anti-rabbit IgG (H+L) Fluor594 (secondary antibody fluorescent probe), incubated at 37℃ in the dark for 1h, washed 3 times with PBS, 5min each time; added DAPI-containing sealing agent to seal the slices, and observed under laser confocal fluorescence microscope. Under normal culture conditions, H9C2 cells can be seen with weak red fluorescence. The increase in NLRP3 inflammasomes after inflammatory damage leads to enhanced red fluorescence, but the addition of engineered platelet membrane nanocarriers after inflammatory damage significantly reduces the red fluorescence intensity. The red fluorescence intensity was quantified by Image J, which confirmed that the engineered platelet membrane nanocarrier has a certain anti-inflammatory ability in vitro.

[0095] Fig.12 Middle a) is a confocal image of DY@PM's ability to reduce NLRP3 inflammasomes (red: NLRP3 inflammasome secondary antibody fluorescent probe: goat anti-rabbit IgG (H+L) Fluor 594. The more NLRP3 inflammasomes there are, the stronger the red fluorescence.)

[0096] b) Quantitative statistical analysis of fluorescence intensity.

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

[0098] In order to evaluate the ability of engineered platelet membrane nanocarriers to target the ischemic site of myocardial infarction, we used small animal in vivo imaging to observe the distribution of nanocarriers in different tissues. This part of the experiment was conducted in the MI mouse model. First, the mouse MI model was constructed as follows: 10-week-old C57 male mice were taken, and the mice were fasted for one day before surgery. After the mice were anesthetized by inhalation of isoflurane gas, the limbs of the mice were fixed on the operating table in a supine position. After the chest was depilated and disinfected with iodine, the skin was incised in the middle and lower third of the mouse's neck, the trachea was separated and cut, and the anesthesia ventilator was intubated to assist breathing. Use forceps to bluntly separate the fascia at the junction of the pectoralis major and the serratus anterior muscles, and probe into the chest cavity at the fourth rib. Use a chest opener to open the chest cavity, use a sterilized cotton ball to push the lungs to fully expose the heart, use forceps to carefully tear open the cardiac capsule to expose the anterior wall of the left ventricle, and use 7-0 sutures to ligate the left anterior descending branch of the coronary artery 1mm below the starting end of the left coronary artery. The ligation depth is about 0.5mm and the width is 1mm. After ligation, if the myocardium below the ligation site changes from red to light white or cyanosis occurs, and the activity of the ventricular wall decreases to disappear, it indicates that the mouse MI model has been successfully established. After the mouse circulation is stable, use 4-0 sutures to ligate and close the chest cavity, suture the skin and disinfect it with iodine. Place the mouse on a heating pad and continue to intubate and connect the ventilator to assist breathing, and observe the mouse status.

[0099] In order to detect the targeted enrichment effect of engineered platelet membrane nanocarriers in the myocardial ischemic site in MI mice, sham operation group and MI group mice were randomly selected, and engineered platelet membrane nanocarriers loaded with cy5 (0.2 mg / mL) were injected through the tail vein within 15 minutes after modeling. The fluorescence distribution of engineered platelet membrane nanocarriers in the heart, liver, spleen, lung, and kidney were observed using small animal in vivo imaging technology 6 hours and 12 hours after injection.

[0100] Fig.13 This is an image of the distribution of engineered platelet membrane nanocarriers in major organs after tail vein injection into a myocardial ischemia model. The results show that engineered platelet membrane nanocarriers have the ability to target myocardial ischemia sites.

[0101] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An engineered platelet membrane nanocarrier, comprising a nanoparticle (DY) and a platelet membrane formed on the surface of the nanoparticle (DY), wherein: The nanoparticles (DY) are connected by long-chain molecules DSPE-PEG 2000 -Arg-TK-CY-09 self-assembly, TK represents the thioketal bond of the ROS-responsive material, CY-09 represents the anti-inflammatory drug CY-09 that inhibits the activation and assembly of NLRP3 inflammasomes, Arg represents L-Arg that produces NO, DSPE-PEG 2000 Represents an amphiphilic polymer.

2. The engineered platelet membrane nanocarrier according to claim 1, characterized in that: The nanoparticles (DY) are regular spherical; The particle size of the engineered platelet membrane nanocarrier is 130-160 nm.

3. The engineered platelet membrane nanocarrier according to claim 1, characterized in that: The long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 is prepared by a method comprising the following steps: 1) The anti-inflammatory drug CY-09, which inhibits the activation and assembly of NLRP3 inflammasome, reacts with TK-2OH to obtain HO-TK-CY-09 (CY-TK); 2) L-arginine L-Arg and amphiphilic polymer DSPE-PEG 2000 -COOH reaction to obtain DSPE-PEG 2000 -Arg(DA); 3) Activated DSPE-PEG 2000 The carboxyl end of -Arg undergoes esterification reaction with the hydroxyl group of HO-TK-CY-09 to obtain DSPE-PEG 2000 -Arg-TK-CY-09, Wherein, the structural formula of the TK-2OH is as follows: Wherein, m and n are the same or different and are integers of 1-6.

4. A method for preparing the engineered platelet membrane nanocarrier according to any one of claims 1 to 3, comprising the following steps: a) Preparation of long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 1) The anti-inflammatory drug CY-09, which inhibits the activation and assembly of NLRP3 inflammasome, reacts with TK-2OH to obtain HO-TK-CY-09 (CY-TK); in, m and n are the same or different and are integers from 1 to 6; 2) L-arginine L-Arg and amphiphilic polymer DSPE-PEG 2000 -COOH reaction to obtain DSPE-PEG 2000 -Arg(DA); 3) Activated DSPE-PEG 2000 The carboxyl end of -Arg undergoes esterification reaction with the hydroxyl group of HO-TK-CY-09 to obtain DSPE-PEG 2000 -Arg-TK-CY-09; b) Using thin film hydration method, the long-chain molecule DSPE-PEG 2000 -Arg-TK-CY-09 was prepared into regular spherical nanoparticles (DY); c) The nanoparticles formed by the long-chain molecules are co-extruded with the platelet membrane through a polycarbonate porous membrane to prepare an engineered platelet membrane nanocarrier.

5. The method according to claim 4, characterized in that In step a) 1), the carboxyl end of CY-09 is first activated, and then the activated carboxyl end of CY-09 is subjected to an esterification reaction with the hydroxyl end of TK-2OH to obtain HO-TK-CY-09 (CY-TK); 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 12-30h; In step a) 2), DSPE-PEG is first activated 2000 -COOH at the carboxyl end, and then the carboxyl group is activated and DSPE-PEG 2000 -COOH reacts with L-Arg to obtain DSPE-PEG 2000 -Arg(DA); DSPE-PEG 2000 The molar ratio of -COOH to L-Arg is 1:1-2.5; The reaction temperature is room temperature and the reaction time can be 12-30h; In step 3), DMAP and EDC are used to activate DSPE-PEG 2000 -Arg carboxyl terminus; 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 hours.

6. The long-chain molecule DSPE-PEG of claim 4 2000 -Arg-TK-CY-09.

7. The long-chain molecule DSPE-PEG according to claim 6 2000 -Regular spherical nanoparticles made of Arg-TK-CY-09 (DY).

8. The long-chain molecule DSPE-PEG of claim 6 2000 -Use of Arg-TK-CY-09, the nanoparticles according to claim 7 or the engineered platelet membrane nanocarriers according to claim 1 in the preparation of drugs for preventing and / or treating myocardial infarction.

9. The use according to claim 8, characterized in that: The drug utilizes specific factors on the surface of the platelet membrane to target the ischemic site of the organism, and in the infarcted myocardial microenvironment with high levels of ROS and biological esterase, it cleaves the TK thioketal bond and hydrolyzes the ester bond, releasing CY-09 that specifically inhibits the activity of the NLRP3 inflammasome and L-Arg that generates NO.

10. A drug for preventing and / or treating myocardial infarction, the drug comprising the engineered platelet membrane nanocarrier according to claim 1 or the long-chain molecule DSPE-PEG according to claim 6 2000 -Arg-TK-CY-09 or the long-chain molecule DSPE-PEG 2000 -Regular spherical nanoparticles made of Arg-TK-CY-09 (DY).

Images