Application of environmental response type anti-oxidation system to regulation and control of mitochondrial homeostasis and heart regeneration

Abstract

The invention provides application of an environmental response type anti-oxidation system to regulation and control of mitochondrial homeostasis and heart regeneration. The present invention relates to a reduced glutathione-modified polydopamine melanin nanoparticle, and particularly provides a reduced glutathione-modified polydopamine melanin nanoparticle, a method for promoting transformation of M1 macrophages to M2 macrophages by using the reduced glutathione-modified polydopamine melanin nanoparticle, a method for promoting damage repair of target cells, and a pharmaceutical composition.

Description

Technical Field

[0001] The present application belongs to the field of biomaterial technology, and specifically relates to reduced glutathione-modified polydopamine particles, a preparation method thereof, and their application in regulating macrophage transformation and mitochondrial transfer. Background Art

[0002] Ischemic heart disease is one of the leading causes of death in humans, with high clinical morbidity and mortality. Myocardial regeneration and the treatment of myocardial infarction still face significant challenges, primarily due to the limited regenerative and repair capacity of damaged myocardium. A key cause of cell death (apoptosis, ferroptosis, etc.) in damaged myocardial cells is mitochondrial dysfunction, and timely replenishment of mitochondria in damaged myocardial cells is crucial for maintaining their activity.

[0003] In the early stages of myocardial infarction, immune cells, especially macrophages, interact most with cardiomyocytes. M2 macrophages can trigger endogenous myocardial repair through the release of regenerative growth factors. Mitochondria secreted by M2 macrophages can also transfer to cardiomyocytes and aid their survival. Therefore, regulating macrophage polarization toward M2 macrophages and promoting the transfer of their mitochondria to cardiomyocytes are important strategies for protecting cardiomyocytes. Summary of the Invention

[0004] According to a first aspect of the present application, reduced glutathione-modified polydopamine melanin nanoparticles are provided, comprising polydopamine melanin nanoparticles and reduced glutathione bound to the polydopamine melanin nanoparticles.

[0005] In some embodiments, the particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 40-200 nm.

[0006] In some embodiments, the average particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 90-120 nm.

[0007] In some embodiments, the D90 particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 60 nm-140 nm.

[0008] According to the second aspect of the present application, there is provided a method for preparing reduced glutathione-modified polydopamine melanin nanoparticles, comprising the following steps:

[0009] 1) mixing ammonia water, ethanol, and deionized water, adding dopamine hydrochloride, and stirring to react to obtain a solution containing polydopamine melanin nanoparticles;

[0010] 2) adding reduced glutathione (GSH) to the solution containing polydopamine melanin nanoparticles in step 1), stirring for reaction, and then dialyzing to finally obtain a suspension of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles; and

[0011] 3) Optionally, the suspension obtained in step 2) is freeze-dried.

[0012] In some embodiments, in step 1) and step 2), the ratio of ammonia water, ethanol, deionized water, dopamine hydrochloride and GSH is 2-3 mL: 32-48 mL: 72-108 mL: 40-60 mg: 32-48 mg.

[0013] In some embodiments, the stirring reaction temperature in step 1) is room temperature, and the stirring reaction time is 16-32 hours.

[0014] In some embodiments, in step 2), the stirring reaction temperature is 45-55° C., and the stirring reaction time is 6-10 h.

[0015] In some embodiments, in step 2), the size of the dialysis bag is 1000-2000 Da, and the dialysis time is 2-4 days.

[0016] According to the third aspect of the present application, a method for promoting the transformation of M1 macrophages to M2 macrophages is provided, comprising: culturing M1 macrophages in the presence of reduced glutathione-modified polydopamine melanin nanoparticles according to the first aspect or reduced glutathione-modified polydopamine melanin nanoparticles prepared according to the method of the second aspect to obtain M2 macrophages.

[0017] According to the fourth aspect of the present application, a method for promoting damage repair of target cells is provided, comprising: co-culturing a macrophage population comprising M1 macrophages with damaged target cells in the presence of reduced glutathione-modified polydopamine melanin nanoparticles of the first aspect or reduced glutathione-modified polydopamine melanin nanoparticles prepared according to the method of the second aspect.

[0018] In some embodiments, the target cell is a cardiomyocyte, an endothelial cell, or a neuron.

[0019] According to the fifth aspect of the present application, a pharmaceutical composition for repairing damaged target cells in an individual is provided, which comprises the reduced glutathione-modified polydopamine melanin nanoparticles of the first aspect or the reduced glutathione-modified polydopamine melanin nanoparticles prepared according to the method of the second aspect, and a delivery vehicle for delivering the nanoparticles to the site where the damaged target cells are located.

[0020] In some embodiments, the delivery vehicle is a hydrogel, eg, an injectable hydrogel.

[0021] In some embodiments, the target cell is a cardiomyocyte, an endothelial cell, or a neuron.

[0022] In some embodiments, the individual has experienced myocardial infarction, ischemic heart disease, or ischemia-reperfusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following further describes the embodiments illustrated in this document with reference to the accompanying drawings. It should be understood that the drawings are only intended to enable those skilled in the art to better understand the technical solutions of the present application and are not intended to limit the scope of the technical solutions of the present application.

[0024] Figure 1 Characterization diagrams of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles prepared according to one embodiment of the present application are shown, wherein the left figure is a scanning electron microscope (SEM) image of the GSH-PDA nanoparticles; the right figure is an energy dispersive spectroscopy (EDS) elemental analysis diagram of the GSH-PDA nanoparticles.

[0025] Figure 2 The diagram shows Fourier transform infrared spectroscopy (FT-IR) of dopamine (DA), reduced glutathione (GSH) and glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles prepared according to one embodiment of the present application.

[0026] Figure 3 The antioxidant performance test results of glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles with different loading concentrations prepared according to the examples of the present application are shown compared with pure polydopamine melanin (PDA) nanoparticles, wherein a shows the scavenging rate of H2O2, b shows the scavenging rate of O2·-, and c shows the scavenging rate of ·OH.

[0027] Figure 4 The immunofluorescence staining observation results of the GSH-PDA nanoparticles prepared in the examples of the present application promoting the transformation of M1 macrophages into M2 macrophages are shown, wherein green represents the staining of CD86 (M1) and CD206 (M2) expressed by macrophages, and blue represents DAPI staining, which is used to mark the cell nucleus.

[0028] Figure 5The results of immunofluorescence staining observations of GSH-PDA nanoparticles prepared in an example of the present application promoting the transfer of M2 macrophage mitochondria to cardiomyocytes are shown, wherein a represents an immunofluorescence staining image of cardiomyocytes, wherein green represents CD206 staining expressed by M2 macrophages, red represents mitochondria of M2 macrophages labeled with Mitochondria Tracker Green (Mito tracker), and blue represents DAPI staining; b is the ratio of transferred red-labeled macrophage mitochondria contained in cardiomyocytes detected by flow cytometry, wherein F4 / 80 is a marker for macrophages and MitochondriaTracker Green (Mito tracker) is a marker for mitochondria.

[0029] Figure 6 The results of JC-1 staining for detecting the mitochondrial membrane potential of myocardial cells after co-culturing M2 macrophages with damaged myocardial cells in the presence of GSH-PDA nanoparticles prepared in an example of the present application are shown. JC-1 monomers aggregate to form polymers, and the myocardial mitochondria exhibit red fluorescence. JC-1 exists in monomeric form in apoptotic or necrotic cells, and the myocardial mitochondria exhibit green fluorescence.

[0030] Figure 7 The results of detecting the concentration of mitochondrial ATP in cardiomyocytes are shown after M2 macrophages are co-cultured with damaged cardiomyocytes in the presence of GSH-PDA nanoparticles prepared in an example of the present application. DETAILED DESCRIPTION

[0031] Hereinafter, the inventive concept of the present application will be further elaborated according to specific embodiments. However, the specific embodiments listed are only for illustrative purposes and are not intended to limit the scope of the present application. Those skilled in the art will recognize that the specific features in any of the following embodiments can be used in any other embodiment as long as it does not deviate from the inventive concept described herein.

[0032] In one aspect, the present application provides reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles, which include polydopamine melanin nanoparticles (PDA) and reduced glutathione bound to the polydopamine melanin nanoparticles.

[0033] In some embodiments, the particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 40-200 nm, for example, 40 nm, 50 nm, 60 nm, 80 nm, 100 nm, 150 nm, 200 nm, or any value within a range consisting thereof.

[0034] In some embodiments, the average particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 90-120 nm, for example, the average particle size is 90 nm, 100 nm, 110 nm or 120 nm or any value within a range composed thereof.

[0035] In some embodiments, the D90 particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 60 nm to 140 nm. The term "particle size D90" as used herein refers to the particle size range of 90% of the nanoparticles extending from the center of the particle size distribution graph to both sides, and can also be understood as the particle size range of the remaining nanoparticles after excluding the 5% of nanoparticles with the smallest particle size and the 5% of nanoparticles with the largest particle size.

[0036] On the other hand, the present application provides a method for preparing the above-mentioned GSH-modified polydopamine melanin nanoparticles, comprising the following steps:

[0037] 1) Mixing appropriate amounts of ammonia water, ethanol, and deionized water, adding dopamine hydrochloride, and stirring to react to obtain a solution containing polydopamine melanin nanoparticles;

[0038] 2) adding reduced glutathione (GSH) to the solution containing polydopamine melanin nanoparticles in step 1), stirring for reaction, and then dialyzing to finally obtain a suspension of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles; and

[0039] 3) Optionally, the suspension obtained in step 2) is freeze-dried.

[0040] According to the preparation method of the above-mentioned hydrogel of the present application, polydopamine melanin nanoparticles are prepared by oxidative self-polymerization of dopamine hydrochloride under alkaline conditions, and then reduced glutathione with the function of inhibiting the generation of reactive oxygen species is modified onto the polydopamine melanin nanoparticles to obtain glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles.

[0041] In some embodiments, in step 1) and step 2), the ratio of ammonia water, ethanol, deionized water, dopamine hydrochloride and GSH is 2-3 mL: 32-48 mL: 72-108 mL: 40-60 mg: 32-48 mg.

[0042] In some embodiments, in step 1) and step 2), the ratio of ammonia water, ethanol, deionized water, dopamine hydrochloride and GSH is 2.5 mL:40 mL:90 mL:50 mg:40 mg.

[0043] In some embodiments, the stirring reaction time in step 1) is 16-32 hours, for example, 24 hours.

[0044] In some embodiments, the temperature of the stirring reaction in step 1) is room temperature.

[0045] In some embodiments, in step 2), the stirring reaction temperature is 45-55°C, for example 50°C.

[0046] In some embodiments, in step 2), the stirring reaction time is 6-10 h, for example, 7 h.

[0047] In some embodiments, in step 2), the size of the dialysis bag is 1000-2000 Da, for example, 1000 Da.

[0048] In some embodiments, in step 2), the dialysis time is 2-4 days, for example, 3 days.

[0049] On the other hand, the present application provides a method for promoting the transformation of M1 macrophages to M2 macrophages, comprising: culturing M1 macrophages in the presence of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles provided herein to obtain M2 macrophages.

[0050] On the other hand, the present application provides a method for promoting damage repair of target cells, comprising: co-culturing a macrophage population comprising M1 macrophages with damaged target cells in the presence of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles provided by the present application.

[0051] In some embodiments, the method of promoting damage repair of target cells of the present application comprises: co-culturing M2 macrophages with target cells in the presence of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles provided herein.

[0052] In some embodiments, the target cells described herein are selected from cardiomyocytes, endothelial cells, or neurons.

[0053] On the other hand, the present application provides a pharmaceutical composition for repairing damaged target cells in an individual, which comprises the reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles provided by the present application, and a delivery vehicle for delivering the nanoparticles to the site where the damaged target cells are located.

[0054] As a specific application example, in the early stages of myocardial infarction, macrophages are the primary cell type interacting with damaged myocardial cells. Delivering the nanoparticles of this application to the damaged myocardium via a suitable delivery vehicle can promote the transformation of M1 macrophages to M2 macrophages, thereby protecting and repairing the damaged myocardium through mitochondrial translocation. Because macrophages are widely present in the body and participate in immune defense and injury repair in multiple tissues, it is expected that delivery via different delivery vehicles can achieve injury repair effects in different parts of the body.

[0055] In some embodiments, the delivery vehicle is a hydrogel, for example, an injectable hydrogel. For example, the GSH-PDA nanoparticles of the present application can be included in a hydrogel to prepare an injectable hydrogel that can be delivered to a damaged area.

[0056] In some embodiments, the damaged target cell is selected from a damaged cardiomyocyte, endothelial cell, or neuron.

[0057] In some embodiments, the individual having the damaged cells has experienced myocardial infarction, ischemic heart disease, or ischemia-reperfusion.

[0058] This application uses the regulation of macrophages and the interaction between macrophages and cardiomyocytes as key regulatory methods, and provides a GSH-PDA nanoparticle that can promote this regulation. By combining the amino group of polydopamine with the sulfhydryl group of reduced glutathione (GSH), glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles were prepared. These nanoparticles can effectively promote the transformation of M1 macrophages to M2 macrophages, further promoting the transfer of mitochondria from transformed macrophages to cardiomyocytes, thereby promoting mitochondrial homeostasis and myocardial regeneration in damaged cardiomyocytes.

[0059] Example

[0060] The following embodiments are provided to facilitate a better understanding of the embodiments of the present application, but are not intended to limit them in any way. The experimental methods used in the following embodiments are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0061] Material Description

[0062] Dopamine hydrochloride: Aladdin (Product No.: D103111)

[0063] Reduced glutathione (GSH): Macklin (Cat. No. G6268)

[0064] Dialysis bag (1000Da): Beijing Solebeau Technology Co., Ltd. (Cat. No.: YA1049)

[0065] Example 1: Preparation of polydopamine melanin nanoparticles

[0066] Take 2.5 ml of ammonia water, 40 ml of ethanol and 90 ml of deionized water, stir for 30 minutes to mix evenly; then add 50 mg of dopamine hydrochloride to the above mixed solution and stir for 24 hours to obtain polydopamine melanin (PDA) nanoparticles.

[0067] Example 2: Preparation of glutathione-modified polydopamine melanin nanoparticles

[0068] Take 2.5ml of ammonia water, 40ml of ethanol, and 90ml of deionized water and stir for 30 minutes to mix thoroughly. Then, add 50mg of dopamine hydrochloride to the mixed solution and stir at room temperature for 24 hours. Then add 40mg of reduced glutathione (GSH) to the solution and stir at 50°C for 7 hours. Finally, use a 1000Da dialysis bag to dialyze the resulting mixed solution for 3 days to obtain the final suspension of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles. The solid content of GSH-PDA nanoparticles in this suspension is 100μg / ml. It is then freeze-dried for subsequent experimental testing.

[0069] The prepared glutathione-modified polydopamine melanin nanoparticles were freeze-dried, and the sample surface was sprayed with gold. The sample was placed under an electron gun, and the morphology of the nanoparticles was observed using a scanning electron microscope (Zeiss Supra55). Figure 1 As shown in the middle left figure, the formed particles are uniformly dispersed GSH-PDA nanospheres with a diameter of about 40-200nm, an average particle size of 100nm, and a D90 particle size of 60-140nm. The elements on the surface of GSH-PDA nanoparticles were further analyzed using energy dispersive spectroscopy (EDS). The results are as follows: Figure 1 As shown in the middle right picture, GSH-PDA nanoparticles are not only rich in N, C, and O, which are common elements in the formation process of polydopamine melanin, but also S element is detected, proving that GSH is successfully repaired to polydopamine melanin nanoparticles.

[0070] In addition, in order to investigate the changes in the chemical bonds on the surface of GSH-PDA nanoparticles, Fourier transform infrared spectroscopy (FT-IR) measurements were performed on dopamine (DA), reduced glutathione (GSH) and prepared GSH-PDA nanoparticles. Figure 2 As shown, the 1616cm -1 The absorption characteristic peak at 3225 cm represents the stretching vibration of the C=C double bond in the aromatic ring; -1The strong absorption characteristic peak at represents the stretching vibration of OH and NH in GSH-PDA, proving that GSH is modified on PDA.

[0071] Detection of antioxidant properties of GSH-PDA nanoparticles

[0072] PDA nanoparticles were prepared according to the method of Example 1, freeze-dried, and then water was added to reconstitute a suspension containing PDA nanoparticles, wherein the solid contents of the PDA nanoparticles were 100 μg / ml, 150 μg / ml, and 200 μg / ml, respectively. Freeze-dried samples of GSH-PDA nanoparticles were prepared according to the method of Example 2, and then the freeze-dried samples were reconstituted with water until the solid contents of the GSH-PDA nanoparticles were 50 μg / ml, 100 μg / ml, and 200 μg / ml, respectively.

[0073] Then the antioxidant performance was tested. O2·-, ·OH and H2O2 are the main components of reactive oxygen species (ROS). O2·- detection kit, hydroxyl radical (·OH) detection kit and H2O2 detection kit were used to detect O2·-, hydroxyl radical and H2O2 respectively. The antioxidant performance of pure PDA and GSH-PDA nanoparticles with different concentrations (50, 100 and 200 μg / mL) was verified. Figure 4 b), ·OH( Figure 4 c) and H2O2( Figure 4 The scavenging effect of a).

[0074] The results showed that PDA melanin nanoparticles alone exhibited significant antioxidant activity, with the scavenging efficiency for O2·-, ·OH, and H2O2 increasing significantly with increasing concentration. At 100 μg / mL, PDA achieved a scavenging rate of 48% for O2·-, 60% for hydroxyl radicals, and 47.3% for H2O2. Glutathione (GSH) modification significantly enhanced the antioxidant properties of PDA. At the same concentration, GSH-PDA achieved a scavenging rate of 64% for O2·-, 78% for hydroxyl radicals, and 73% for H2O2.

[0075] Example 3: GSH-PDA nanoparticles promote the transformation of macrophages

[0076] 1. Induction of M0 macrophages isolated from 8-week-old C57BL / 6 mice into M1 macrophages

[0077] Mice were anesthetized with sodium pentobarbital, and the tibia and femur were aseptically isolated. The bone marrow was flushed into a 50 ml centrifuge tube using RPMI-1640 medium. The cell suspension was centrifuged at 300 g for 5 min. The cells were then resuspended in RPMI-1640 medium containing 20 ng / ml M-CSF, 2 mM L-glutamine, 1% penicillin-streptomycin, and 10% FBS, plated, and cultured for 8 days. Subsequently, 10 ng / mL of lipopolysaccharide (LPS) was added to induce M1 macrophages.

[0078] 2. Immunofluorescence Staining to Detect Macrophage Transformation

[0079] The freeze-dried sample of GSH-PDA nanoparticles prepared according to the method of Example 2 was re-dissolved to obtain a suspension containing GSH-PDA nanoparticles, which was added to the M1 macrophage culture medium so that the concentration of M1 macrophages in the culture medium after addition was 2×10 6 / mL, and the concentration of GSH-PDA nanoparticles was 100 μg / mL; a blank control group (Control, in which an equal volume of PBS was added) and a PDA-added control group (PDA, in which a suspension of PDA nanoparticles prepared as above was added to make the concentration of PDA nanoparticles in the culture medium 100 μg / mL) were also provided.

[0080] After culturing for 24 hours, the cells were fixed with 4% paraformaldehyde and the expression of CD86 on M1 macrophages and CD206 on M2 macrophages was detected. Figure 4 As shown in the results, PDA in the culture medium can effectively promote the transformation of M1 macrophages to M2 macrophages; compared with the addition of PDA, the addition of GSH-PDA can more effectively promote the polarization of M1 macrophages to M2 macrophages, and the number of M2 macrophages expressing CD206 positive in the GSH-PDA group is greater.

[0081] Example 4: GSH-PDA nanoparticles promote the transfer of M2 macrophage mitochondria to cardiomyocytes

[0082] (1) Cell culture

[0083] Primary isolated cardiomyocytes from newborn SD rats (age: within 24 hours) were treated with 200 μM H2O2 for 12 hours. Mitochondria from M2 macrophages obtained according to the method of Example 3 were then labeled with Mitochondria Tracker Green (Mito tracker) and co-cultured with H2O2-treated cardiomyocytes (Injured CMs) at a ratio of 1:1 for 24 hours. A suspension containing GSH-PDA nanoparticles obtained by reconstitution of a freeze-dried sample of GSH-PDA nanoparticles prepared according to the method of Example 2 was simultaneously added to the culture system, such that the concentration of M2 macrophages in the culture system after addition was 2×10 6 / mL, and the concentration of GSH-PDA nanoparticles was 100 μg / mL; a blank control group (Control, in which an equal volume of PBS was added and damaged cardiomyocytes were co-cultured) and a PDA-added control group (PDA, in which a suspension of PDA nanoparticles prepared as above was added and damaged cardiomyocytes were cultured under conditions where the concentration of PDA nanoparticles in the culture medium was 100 μg / mL) were also provided.

[0084] (2) Detection of the effect of GSH-PDA nanoparticles on the transfer of M2 macrophage mitochondria to cardiomyocytes

[0085] After culturing for 24 hours, cell samples were collected and fixed with 4% paraformaldehyde. Mitochondrial transfer was observed by immunofluorescence staining to detect whether there were labeled macrophage mitochondria in the cardiomyocytes.

[0086] The results are as follows Figure 5 As shown, (a) represents an immunofluorescence staining image of cardiomyocytes, and (b) represents the proportion of red-labeled macrophage mitochondria transferred into non-macrophages. In the co-culture of macrophages and cardiomyocytes alone, the proportion of red-labeled macrophage mitochondria transferred into cardiomyocytes was 37.7%. After the addition of PDA, the proportion of red-labeled macrophage mitochondria transferred into cardiomyocytes was 45.2%. After the addition of GSH-PDA, the proportion of red-labeled macrophage mitochondria transferred into cardiomyocytes was 68.7%. Therefore, it can be seen that after the co-culture of macrophages and cardiomyocytes alone, macrophage mitochondria transferred into cardiomyocytes. When GSH-PDA was added to the cell culture system, the transfer of macrophage mitochondria into cardiomyocytes was significantly promoted. Flow cytometry was used to observe the content of labeled mitochondria in cardiomyocytes. The results showed that under H2O2 injury conditions, the addition of PDA and GSH-PDA both promoted the transfer of mitochondria from M2 macrophages into cardiomyocytes, and the GSH-PDA group was more pronounced than the PDA group.

[0087] (3) Detection of the stability of mitochondria in myocardial cells after GSH-PDA promoted mitochondrial transfer into myocardial cells:

[0088] Collect the cardiomyocytes after co-culture of M2 macrophages and H2O2-treated cardiomyocytes under the condition of adding GSH-PDA in (1) for 24 hours (M2+GSH-PDA, and provide the control (Control) of cardiomyocytes treated with H2O2 for 24 hours under the condition of adding equal volume of PBS and the control (GSH-PDA) of cardiomyocytes treated with H2O2 for 24 hours) by centrifugation, add 1 mL of JC-1 staining working solution, and mix thoroughly. Incubate in a cell culture incubator at 37°C for 20 minutes. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1×) according to the ratio of adding 4 mL of distilled water to every 1 mL of JC-1 staining buffer (5×), and place it in an ice bath. After the incubation at 37°C, remove the supernatant and wash twice with JC-1 staining buffer (1×). Add 2 mL of cell culture medium, which may contain serum and phenol red. Observe by flow cytometry.

[0089] JC-1 staining was used to detect mitochondrial membrane potential (MMP) in cardiomyocytes. Figure 6 As shown, the proportion of mitochondrial membrane potential multimers in injured cardiomyocytes alone was 66.1%, the proportion of mitochondrial membrane potential multimers in injured cardiomyocytes cultured with GSH-PDA alone was 77.7%, and the proportion of mitochondrial membrane potential multimers in injured cardiomyocytes co-cultured with M2 macrophages under the condition of GSH-PDA addition was 81.4%. This indicates that GSH-PDA can significantly enhance the mitochondrial membrane potential of injured cardiomyocytes, and the repair effect of mitochondrial membrane potential in injured cardiomyocytes co-cultured with M2 macrophages is even stronger when GSH-PDA is added. It is speculated that GSH-PDA can further protect mitochondria in cardiomyocytes from ROS damage by promoting the transfer of M2 mitochondria.

[0090] (4) Detection of ATP energy metabolism in myocardial mitochondria after GSH-PDA promoted mitochondrial transfer to myocardial cells:

[0091] Collect the cardiomyocytes after co-culture of M2 macrophages and H2O2-treated cardiomyocytes under the condition of adding GSH-PDA in (1) for 24 hours (M2+GSH-PDA, and provide a control (Control) of cardiomyocytes treated with H2O2 and cultured for 24 hours under the condition of adding equal volume of PBS and a control (GSH-PDA) of cardiomyocytes treated with H2O2 and cultured for 24 hours with GSH-PDA alone). Add lysis solution at a ratio of 200 μl of lysis solution per well of a 6-well plate (i.e., equivalent to 1 / 10 of 2 ml of cell culture solution) to lyse the cells. In order to fully lyse the cells, a pipette can be used to repeatedly blow or shake the culture plate to allow the lysis solution to fully contact and lyse the cells. Usually, the cells will lyse immediately after contacting the lysis solution. After lysis, centrifuge at 4°C and 12000g for 5 minutes, and take the supernatant for subsequent determination. Melt the reagents to be used in an ice bath, and dilute the ATP standard solution with ATP detection lysis solution to an appropriate concentration gradient. The specific concentration depends on the ATP concentration in the sample. The initial test can detect concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μM. In subsequent experiments, the concentration range of the standard can be appropriately adjusted according to the concentration of ATP in the sample.

[0092] Prepare ATP Assay Working Solution: Prepare an appropriate amount of ATP Assay Working Solution, using 100 μL of ATP Assay Working Solution for each sample or standard. Thaw the reagents in an ice bath. Take an appropriate amount of ATP Assay Reagent and dilute it with ATP Assay Reagent Diluent at a ratio of 1:9. For example, add 100 μL of ATP Assay Reagent to 900 μL of ATP Assay Reagent Diluent to make 1 mL of ATP Assay Working Solution. This diluted ATP Assay Reagent is used as the ATP Assay Working Solution for subsequent experiments.

[0093] The concentration of mitochondrial ATP in cardiomyocytes was measured using an ATP energy detection kit ((S0026)-Beyotime), expressed as μmol ATP per μg of cardiomyocyte protein. Figure 7 As shown in the results, GSH-PDA more significantly promoted the ATP energy metabolism of cardiomyocyte mitochondria after co-culture of M2 macrophages and cardiomyocytes, and the mitochondrial ATP metabolism of cardiomyocytes co-cultured with M2 macrophages under the condition of adding GSH-PDA was stronger, indicating that GSH-PDA can further protect the mitochondrial energy metabolism in cardiomyocytes by promoting the transfer of M2 macrophage mitochondria, thereby promoting myocardial remodeling and regeneration.

[0094] The above description is merely a specific embodiment of the invention covered by this application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of this application. Therefore, the scope of protection of this application should be based on the scope of protection of the claims.

Claims

1. Reduced glutathione-modified polydopamine melanin nanoparticles, comprising polydopamine melanin nanoparticles and reduced glutathione bound to the polydopamine melanin nanoparticles.

2. The reduced glutathione-modified polydopamine melanin nanoparticles according to claim 1, wherein the particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 40-200 nm, Optionally, the average particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 90-120 nm. Optionally, the D90 particle size of the reduced glutathione-modified polydopamine melanin nanoparticles is 60-140 nm.

3. A method for preparing reduced glutathione-modified polydopamine melanin nanoparticles, comprising the following steps: 1) mixing ammonia water, ethanol, and deionized water, adding dopamine hydrochloride, and stirring to react to obtain a solution containing polydopamine melanin nanoparticles; 2) adding reduced glutathione (GSH) to the solution containing polydopamine melanin nanoparticles in step 1), stirring for reaction, and then dialyzing to finally obtain a suspension of reduced glutathione-modified polydopamine melanin (GSH-PDA) nanoparticles; as well as 3) Optionally, the suspension obtained in step 2) is freeze-dried.

4. The method according to claim 3, wherein: In step 1) and step 2), the ratio of ammonia water, ethanol, deionized water, dopamine hydrochloride and GSH is 2-3 mL: 32-48 mL: 72-108 mL: 40-60 mg: 32-48 mg; Optionally, the stirring reaction temperature in step 1) is room temperature, and the stirring reaction time is 16-32h; Optionally, in step 2), the stirring reaction temperature is 45-55° C., and the stirring reaction time is 6-10 h; Optionally, in step 2), the size of the dialysis bag is 1000-2000 Da, and the dialysis time is 2-4 days.

5. Methods for promoting the transformation of M1 macrophages to M2 macrophages, including: M1 macrophages are cultured in the presence of reduced glutathione-modified polydopamine melanin nanoparticles according to claim 1 or 2 or reduced glutathione-modified polydopamine melanin nanoparticles prepared according to the method of claim 3 or 4 to obtain M2 macrophages.

6. A method for promoting damage repair in target cells, comprising: In the presence of reduced glutathione-modified polydopamine melanin nanoparticles according to claim 1 or 2 or reduced glutathione-modified polydopamine melanin nanoparticles prepared according to the method of claim 3 or 4, a macrophage population comprising M1 macrophages is co-cultured with damaged target cells.

7. The method according to claim 6, wherein: The target cells are myocardial cells, endothelial cells or neurons.

8. A pharmaceutical composition for repairing damaged target cells in an individual, comprising the reduced glutathione-modified polydopamine melanin nanoparticles according to claim 1 or 2 or the reduced glutathione-modified polydopamine melanin nanoparticles prepared according to the method of claim 3 or 4, and a delivery vehicle for delivering the nanoparticles to the site where the damaged target cells are located.

9. The method of claim 8, wherein the delivery vehicle is a hydrogel, eg, an injectable hydrogel. Optionally, the target cells are cardiomyocytes, endothelial cells or neurons.

10. The method of claim 8 or 9, wherein the subject has experienced myocardial infarction, ischemic heart disease, or ischemia-reperfusion.

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