Bionic nano vesicle as well as preparation method and application thereof

Abstract

The invention discloses a bionic nano-vesicle preparation method, which comprises: dissolving epigallotein gallate and selenomethionine in deionized water, adding a formaldehyde solution, stirring in a dark environment, and carrying out centrifugal purification to obtain spherical nano-particles; extracting exosomes from the stem cells; and adding the spherical nano-particles and the exosome into deionized water, and stirring to obtain the bionic nano-vesicle. The bionic nano vesicles prepared by the invention can regulate and control macrophages and T cells, have anti-inflammatory and immune network regulation and control effects, and are applied to the protection effect on acute kidney injury.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a biomimetic nanovesicle and a preparation method and application thereof. Background Art

[0002] Acute nephritis is a common kidney disease caused by viruses, bacteria, or other pathogens, which can lead to kidney damage and even chronic kidney disease. Treatment for acute nephritis generally involves anti-infective therapy combined with immunomodulatory and anti-inflammatory therapies. Immunosuppressants are commonly used in immunomodulatory and anti-inflammatory treatments. These drugs block the proliferation of immune cells (such as T cells and B cells) or modulate immune signaling pathways, reducing abnormal immune activity and the release of inflammatory factors (such as TNF-α and IL-6), thereby alleviating immune damage to target organs such as the kidneys. Commonly used immunosuppressants include cyclosporine soft capsules, tacrolimus capsules, and glucocorticoids. However, immunosuppressants have numerous side effects that can affect patients' health. For example, cyclosporine soft capsules may cause liver dysfunction and hypertension; tacrolimus capsules may cause adverse reactions such as headaches and tremors; and glucocorticoids may cause adverse reactions such as infections. Furthermore, long-term use of immunosuppressants may increase patients' risk of developing other diseases.

[0003] Therefore, there is an urgent need for an anti-inflammatory drug with high biocompatibility, high drug loading efficiency and few side effects to achieve immune regulation. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide a method for preparing biomimetic nanovesicles. This method comprises spherical nanoparticles composed of epigallocatechin gallate and selenomethionine, which are then combined with stem cell exosomes to produce biomimetic nanovesicles. This method is simple to prepare and has high production efficiency. The biomimetic nanovesicles, composed of exosomes combined with nanoparticles, have high biocompatibility and can modulate the immune system upon entry into the human body, exhibiting superior anti-inflammatory effects.

[0005] To achieve the above object, the present invention provides the following technical solution: A method for preparing biomimetic nanovesicles comprises the following steps:

[0006] Epigallocatechin gallate and selenomethionine were dissolved in deionized water, added with formaldehyde solution, stirred in a dark environment, and purified by centrifugation to obtain spherical nanoparticles.

[0007] Extraction of exosomes from stem cells;

[0008] Spherical nanoparticles and exosomes were added into deionized water and stirred to obtain biomimetic nanovesicles.

[0009] Preferably, the mass ratio of epigallocatechin gallate to selenomethionine is 7-8:1.

[0010] Preferably, the concentration of the formaldehyde solution is 4%.

[0011] Preferably, the stem cells are human umbilical cord mesenchymal stem cells.

[0012] Preferably, exosomes are extracted from the human umbilical cord mesenchymal stem cells by gradient ultracentrifugation.

[0013] Preferably, the concentration of the spherical nanoparticles is 9-1 mg / ml, the concentration of the exosomes is 0.9-1.1 mg / ml, and the volume ratio of the spherical nanoparticles to the exosomes is 1:1-1.2.

[0014] Beneficial effects of the method for preparing biomimetic nanovesicles of the present invention:

[0015] The preparation method of the present invention has a simple preparation process, can be prepared by mixing and stirring, is easy to operate, and has high production efficiency.

[0016] The bionic nanocapsules prepared by the present invention have small particle size and strong biocompatibility and can smoothly enter organs such as the liver, spleen, and kidney of the human body.

[0017] The biomimetic nanovesicles prepared by the present invention can effectively regulate macrophages, causing them to transition from M1 to M2 when inflammation occurs, demonstrating inflammation-modulating capabilities. They can also inhibit the maturation of dendritic cells and alleviate overactivation of T cells, demonstrating superior immune network regulation. While simultaneously combating inflammation, they can also regulate the immune network, leading to applications in anti-inflammatory, immune network regulation, and acute kidney injury protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Example 1 Schematic diagram of the synthesis of biomimetic nanovesicles;

[0019] Figure 2 TEM (transmission electron microscopy) image of spherical nanoparticles in Example 1;

[0020] Figure 3 TEM (transmission electron microscopy) image of biomimetic nanovesicles in Example 1;

[0021] Figure 4 NTA (Nanoparticle Tracking Analysis) results of biomimetic nanovesicles in Example 1;

[0022] Figure 5 Example 1 Characterization of exosome surface proteins in biomimetic nanovesicles;

[0023] Figure 6 Example 2 Effects of different concentrations of biomimetic nanovesicles on polarization of M1 macrophages;

[0024] Figure 7 Example 2 Effects of different concentrations of biomimetic nanovesicles on polarization of M2 macrophages;

[0025] Figure 8 Example 2 Effects of different treatment groups on M1 macrophage polarization and quantitative analysis;

[0026] Figure 9 Example 2 Effects of different treatment groups on M2 macrophage polarization and quantitative analysis;

[0027] Figure 10 Example 2 Effects of different treatment groups on CD86 and CD206 gene expression;

[0028] Figure 11 Example 3: Mouse weight change trend diagram;

[0029] Figure 12 Example 3 Results of serum creatinine (CRE) and urea nitrogen (BUN) in mice;

[0030] Figure 13 Example 3 Typical images of hematoxylin-eosin (H&E) staining of mouse kidney tissue;

[0031] Figure 14 Example 4 Typical images of TUNEL staining of mouse kidney tissue;

[0032] Figure 15 Example 4 Results of immunofluorescence staining of mouse kidney tissue;

[0033] Figure 16 Example 4 Results of Detection of Inflammatory Factors in Mouse Kidney Tissue;

[0034] Figure 17 Example 4: Schematic diagram of biomimetic nanovesicles regulating the innate immune network in vivo;

[0035] Figure 18 Example 4: Diagram of biomimetic nanovesicles regulating the adaptive immune network in vivo. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although preferred embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to make the present invention more thorough and complete and to fully convey the scope of the present invention to those skilled in the art.

[0037] The present application provides a method for preparing biomimetic nanovesicles for regulating immune networks, comprising the following steps:

[0038] Epigallocatechin gallate and selenomethionine were dissolved in deionized water, added with formaldehyde solution, stirred in a dark environment, and purified by centrifugation to obtain spherical nanoparticles.

[0039] Extraction of exosomes from stem cells;

[0040] Spherical nanoparticles and exosomes were added into deionized water and stirred to obtain biomimetic nanovesicles.

[0041] In the present application, epigallocatechin gallate and selenomethionine form uniformly dispersed spherical nanoparticles under the action of formaldehyde, which combine with stem cell exosomes to form biomimetic nanovesicles. The biomimetic nanovesicles have anti-inflammatory and immune regulatory effects. Specifically, in an inflammatory environment, biomimetic nanovesicles can regulate the transformation of macrophages from M1 to M2. M2 macrophages secrete anti-inflammatory factors such as IL-10 and TGF-β to inhibit excessive inflammatory reactions and promote the disappearance of inflammation, thereby having an anti-inflammatory effect; they can also inhibit the maturation of dendritic cells, keeping them in an immature state that is conducive to immune tolerance, reducing antigen presentation, alleviating excessive activation of T cells, and inhibiting excessive immune activation, thereby having the effect of regulating the immune network.

[0042] Example 1 Preparation of biomimetic nanovesicles

[0043] like Figure 1 As shown, the preparation steps of multifunctional biomimetic nanovesicles are:

[0044] S1: Dissolve 22.875 mg of epigallocatechin gallate (EGCG) and 3.26 mg of selenomethionine (SeMet) in 10 mL of deionized water. Add 75 μL of 4% formaldehyde solution and stir in the dark for 5 hours. Purify by centrifugation to obtain spherical nanoparticles (ES). In this example, the mass ratio of EGCG to SeMet is 7:1.

[0045] S2: Exosomes (EVs) derived from mesenchymal stem cells were extracted using gradient ultracentrifugation.

[0046] S3: Take 100 μL of 10 mg / ml spherical nanoparticle solution and 100 μL of 1 mg / ml exosome solution, add them to 800 μL of deionized water, and stir at low temperature overnight to obtain biomimetic nanovesicles (ES-EVs).

[0047] The spherical nanoparticles and biomimetic nanovesicles prepared in Example 1 were characterized as follows:

[0048] like Figure 2As shown, the spherical nanoparticles prepared in step S1 can be observed under a transmission electron microscope (TEM) image to have clear boundaries and uniform dispersion of the particles.

[0049] like Figure 3 As shown, the TEM image of the spherical nanoparticles prepared in step S3 shows a combination of ES and EV, in which ES is wrapped inside by EV to form an ES-EV coating structure, indicating that exosomes coat the spherical nanoparticles to form biomimetic nanovesicles.

[0050] like Figure 4 As shown in the figure, the biomimetic nanovesicles were analyzed by nanoparticle tracking analysis (NTA). The spectrum showed that there was a single peak at about 170 nm, which was a symmetrical Gaussian distribution with a narrow peak width and a high peak value, indicating that the particle size of the biomimetic nanovesicles was about 170 nm and the particle size uniformity was high.

[0051] like Figure 5 As shown in the figure, the surface protein characterization analysis of human umbilical cord mesenchymal stem cells, exosomes and biomimetic nanovesicles showed that the multidimensional protein characterization data of exosomes and biomimetic nanovesicles were consistent, indicating that the biomimetic nanovesicles were successfully synthesized. Figure 3 The results information is consistent.

[0052] It should be noted that, by varying the volume of deionized water in the final step, different concentrations of biomimetic nanovesicles can be prepared. Alternatively, by dissolving the prepared biomimetic nanovesicles in different volumes of deionized water, different concentrations of biomimetic nanovesicle solutions can be prepared.

[0053] Example 2 Anti-inflammatory effect of biomimetic nanovesicles

[0054] The first set of experiments:

[0055] Macrophages are an important component of the innate immune system and can exhibit different functional states depending on the different stimuli within the immune microenvironment. They can be functionally divided into M1 and M2 macrophages. M1 macrophages promote inflammation, while M2 macrophages play a role in regulating the immune system and tissue remodeling. M1 macrophages are marked by positive expression of CD86, while M2 macrophages are marked by positive expression of CD206.

[0056] Biomimetic nanovesicle solutions at concentrations of 5 μg / mL, 10 μg / mL, and 20 μg / mL were prepared as in Example 1. The effects of these different concentrations of biomimetic nanovesicle solutions on the expression of CD86 and CD206 were calculated. RAW264.7 cells were activated with lipopolysaccharide (LPS) to simulate an inflammatory state, and the following experimental groups were set up:

[0057] Blank control group: RAW264.7 cells were not treated with LPS and no biomimetic nanovesicles were administered;

[0058] Inflammation control group: RAW264.7 cells were treated with LPS and not administered with biomimetic nanovesicles;

[0059] Experimental group 1 (5 μg / mL): RAW264.7 cells were treated with LPS and administered with biomimetic nanovesicles at a concentration of 5 μg / mL;

[0060] Experimental group 2 (10 μg / mL): RAW264.7 cells were treated with LPS and administered with biomimetic nanovesicles at a concentration of 10 μg / mL;

[0061] Experimental group 3 (20 μg / mL): RAW264.7 cells were treated with LPS and administered with biomimetic nanovesicles at a concentration of 20 μg / mL;

[0062] The results are as follows Figure 6 As shown in the data, the CD86 expression rate of the blank control group (6.83%) was the lowest, and the expression rate of the inflammation control group (27.5%) was the highest. As the concentration of the biomimetic nanovesicle solution increased, the CD86 expression rates of experimental groups 1, 2, and 3 gradually decreased. When the concentration of the biomimetic nanovesicles was 20 μg / mL, the CD86 expression rate (19.6%) was the lowest.

[0063] Figure 7 As shown in the data, compared with the CD206 expression rate of the blank control group (2.33%), the CD206 expression rate of the inflammation control group (2.3%) was slightly lower. As the concentration of biomimetic nanovesicles increased, the CD206 expression rates of experimental groups 1, 2, and 3 also gradually increased. When the concentration of biomimetic nanovesicles was 20 μg / mL, the CD206 expression rate (5.73%) was the highest.

[0064] It can be seen from this that when inflammation occurs, the expression rate of CD86 increases, and the expression rate of CD206 decreases. Through the addition of bionic nano vesicles, the expression rate of CD86 decreases, and the expression rate of CD206 increases. Explanation: adding bionic nano vesicles to an inflammatory environment can significantly reduce M1 type macrophage group (CD86 expression positive cell group), and significantly increase M2 type macrophage group (CD206 expression positive cell group). And then show that bionic nano vesicles ES-EVs can promote M1 type macrophages to change to M2 type macrophages, and along with the increase of concentration its controllable ability also strengthens, and in the present embodiment, the bionic nano vesicles with a concentration of 20 μg / mL have optimal anti-inflammatory effect.

[0065] The second set of experiments:

[0066] Exosomes, spherical nanoparticles, and biomimetic nanovesicles were prepared at the same concentration of 20 μg / mL as in Example 1. RAW264.7 cells were activated by lipopolysaccharide (LPS) to simulate an inflammatory state, and the following experimental groups were set up:

[0067] Blank control group: RAW264.7 cells were not treated with LPS and no drug was administered;

[0068] Inflammation control group: RAW264.7 cells were treated with LPS and no drug was administered;

[0069] EV control group: RAW264.7 cells were treated with LPS and exosomes were administered;

[0070] ES control group: RAW264.7 cells were treated with LPS and administered with spherical nanoparticles;

[0071] ES-EV experimental group: RAW264.7 cells were treated with LPS and administered with biomimetic nanovesicles;

[0072] The results are as follows Figure 8 As shown in the figure, the expression rate of CD86 in the inflammation control group (69.0%) was higher than that in the blank control group (8.2%). Compared with the inflammation control group (69.0%), the expression rates of CD86 in the EV control group (57.9%), ES control group (56.7%), and ES-EV experimental group (49.7%) were all lower, among which the ES-EV experimental group had the most significant decrease.

[0073] Figure 9As shown in the data, compared with the CD206 expression rate of the inflammation control group (2.73%), the CD206 expression rate of the EV control group (2.67%) decreased slightly, while the CD206 expression rate of the ES control group (3.02%) and the CD206 expression rate of the ES-EV experimental group (4.48%) increased, among which the increase in the ES-EV experimental group was the most significant.

[0074] Figure 10 As shown in the data, compared with the blank control group, the inflammation control group significantly increased the expression levels of M1 and CD86 mRNA; compared with the inflammation control group, the expression of M1 and CD86 mRNA in the RAW264.7 cells of the EV control group, ES control group and ES-EV experimental group were significantly decreased, among which the decrease in the ES-EV experimental group was the most obvious; compared with the inflammation control group, the expression of M2 and CD206 mRNA in the RAW264.7 cells of the EV control group, ES control group and ES-EV experimental group were increased, among which the increase in the ES-EV experimental group was the most obvious.

[0075] These results demonstrate that the EV control group, ES control group, and ES-EV experimental group all induced the transition of macrophages from M1 to M2 in inflammatory environments, with the ES-EV experimental group demonstrating a stronger induction ability than both the EV and ES control groups. This highlights that the biomimetic nanovesicles derived from the combination of spherical nanoparticles and stem cell exosomes have a stronger ability to induce macrophages in inflammatory environments than either stem cell exosomes or spherical nanoparticles alone, significantly enhancing their anti-inflammatory effects and demonstrating superior potential for inflammation regulation.

[0076] Example 3 Protective effect of biomimetic nanovesicles on acute kidney injury

[0077] Blank control group: mice were not treated with cisplatin and were not administered with biomimetic nanovesicles;

[0078] Induction control group: mice were treated with cisplatin and not administered with biomimetic nanovesicles;

[0079] Drug-administered experimental group: mice were treated with cisplatin and administered with biomimetic nanovesicles;

[0080] The experimental group was treated with cisplatin to establish an acute kidney injury (AKI) model in mice. The mice were then administered biomimetic nanovesicles via the tail vein for three consecutive days after cisplatin injection. The concentration of the biomimetic nanovesicles was 20 μg / mL. After cisplatin treatment, substances such as myoglobin accumulate in the kidneys through systemic circulation and metabolism, leading to oxidative damage and impaired renal function. Renal function status was determined using key renal function indicators (body weight, blood urea nitrogen (BUN), and serum creatinine (CRE)).

[0081] The results are as follows Figure 11 As shown in the figure, the weight of mice in the blank control group changed little, while the weight of mice in the induced control group dropped sharply. Compared with the induced control group, the weight of mice in the drug-treated experimental group showed a significant rebound. Figure 12 As shown, the blank control group had lower blood urea nitrogen (BUN) and serum creatinine (CRE) levels. Compared with the blank control group, the induction control group showed a sharp increase in BUN and CRE levels, while the treatment group showed a significant decrease in BUN and CRE levels. This indicates that renal function indicators in mice in the blank control group remained stable. After cisplatin treatment, body weight decreased, while BUN and CRE levels increased. After administration of biomimetic nanovesicles, body weight recovered, BUN and CRE levels decreased, and renal function indicators recovered. This suggests that biomimetic nanovesicles have great potential in treating acute kidney injury (AKI).

[0082] The mouse kidney tissues were subjected to histopathological evaluation and hematoxylin-eosin (H&E) staining. Figure 13 As shown, the kidneys of mice in the induced control group showed renal tubular casts accompanied by obstructions within the tubules (indicated by black asterisks). Tubulointerstitial damage is caused by excessive death of renal tubular epithelial cells and impaired tubular repair. These tubular casts, caused by the deposition of denatured proteins, are often a hallmark of kidney disease, indicating pathological damage to the renal tissue of the induced control mice. In contrast, mice in the drug-treated experimental group showed significantly reduced damage, highlighting the potential therapeutic effects of biomimetic nanovesicles.

[0083] like Figure 14 As shown, the number of apoptotic cells in the kidneys was analyzed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Green markers represent TUNEL-positive cells. The blank control group showed minimal green markers (apoptotic cell counts) in the kidneys, while the induced control group showed a significant increase in green markers (apoptotic cell counts). The treatment group showed a significant decrease in green markers (apoptotic cell counts) compared to the induced control group. This indicates that cisplatin-treated mice in the induced control group experienced kidney damage, with a significant increase in the number of apoptotic cells. However, treatment with biomimetic nanovesicles significantly alleviated kidney damage and decreased apoptotic cell counts.

[0084] In summary, biomimetic nanovesicles have a significant protective effect in acute kidney injury and play a therapeutic role in kidney damage.

[0085] Example 4 Bionic nanovesicles regulate the immune network in vivo

[0086] Using the blank control group, induction control group, and treatment experimental group established in Example 3, the macrophage phenotype was analyzed by immunofluorescence staining to evaluate the polarization state of macrophages infiltrating the kidneys of mice under different treatments. CD86 was used as an M1 marker, and CD206 as an M2 marker.

[0087] The results are as follows Figure 15 As shown in the upper part of the picture, the blue fluorescence (DAPI) of the blank control group is evenly distributed with a small amount of green fluorescence (F4 / 80), and no red fluorescence (CD86) is observed; the blue fluorescence (DAPI) of the induced control group is reduced while a large area of ​​red fluorescence (CD86) is increased; compared with the induced control group, the red fluorescence (CD86) of the drug-treated experimental group almost disappears, and the green fluorescence (F4 / 80) is significantly increased. In the lower part of the picture, the blue fluorescence (DAPI) and green fluorescence (F4 / 80) of the blank control group are evenly distributed, and no red fluorescence (CD206) is observed; the blue fluorescence (DAPI) and green fluorescence (F4 / 80) of the induced control group are reduced and the distribution is relatively uneven, and no red fluorescence (CD206) is observed; compared with the induced control group, a small amount of red fluorescence (CD206) is increased in the drug-treated experimental group. These results indicate that the immune system of the blank control group was in a balanced state. However, cisplatin-treated mice induced renal damage, with renal macrophages exhibiting M1 polarization (increased CD86). However, after administration of biomimetic nanovesicles, renal macrophages in the experimental group exhibited M2 polarization (increased CD206). This suggests that biomimetic nanovesicles can induce the transition of macrophages from M1 to M2 phenotypes in response to renal injury, demonstrating an anti-inflammatory effect.

[0088] The increased levels of cytokines such as TNF-α, IL-6, IFN-γ, and IL-1β secreted by M1 macrophages promote the deterioration of renal injury and non-bacterial inflammation, while M2 macrophages secrete anti-inflammatory factors such as IL-10 and TGF-β to inhibit excessive inflammatory response and promote inflammation resolution. Therefore, the renal function status of mice can be determined by detecting the expression of the above-mentioned pro-inflammatory cytokines in mouse serum. Figure 16 As shown, the expression of proinflammatory cytokines (TNF-α, IL-6, IFN-γ, and IL-1β) in the blank control group was at a low level; compared with the blank control group, the expression of proinflammatory cytokines in the induced control group mice showed a significant increase; compared with the induced control group, the expression of proinflammatory cytokines in the treatment group significantly decreased. This indicates that when renal injury occurs, the expression of proinflammatory cytokines such as TNF-α, IL-6, IFN-γ, and IL-1β in mice increases sharply. After administration of biomimetic nanovesicles, the expression of proinflammatory cytokines is significantly reduced. This suggests that when renal injury occurs, biomimetic nanovesicles can inhibit the expression of inflammatory mediators, promote the secretion of anti-inflammatory factors, effectively alleviate the inflammatory pathology, and improve renal function.

[0089] Flow cytometry was used to evaluate the immune tolerance response induced by biomimetic nanovesicles, specifically, by assessing the innate immunity, antigen presentation, and adaptive immune systems in the spleen of mice.

[0090] The results are as follows Figure 17 As shown, the expression rate of CD86 in the spleen of mice in the blank control group (11.0%) was the lowest, the expression rate of CD86 in the induced control group (27.4%) increased, and the expression rate of CD86 in the drug-treated experimental group (14.2%) decreased; at the same time, the expression rate of C206 in the spleen of mice in the drug-treated experimental group (38.6%) was significantly increased compared with the expression rate of CD206 in the blank control group (28.1%) and the expression rate of CD206 in the induced control group (27.8%). This shows that the M1 and M2 macrophages in the blank control group mice were at a low level. After treatment with cisplatin, the kidneys were damaged and the macrophages in their spleens were M1 polarized. After administration of biomimetic nanovesicles, the macrophages changed from M1 to M2 polarization. This shows that biomimetic nanovesicles can dynamically regulate polarization according to the immune status in the body and have a regulatory effect on the innate immune system in mice.

[0091] CD80 is expressed in large quantities when dendritic cells are stimulated by antigens. After recognizing the CD28 molecule on the surface of T cells, it can complete antigen presentation, thereby activating T cells and exerting immune effects. In addition, T cells are divided into CD4 + (helper T cells) and CD8 + (Cytotoxic T cells) have two core subtypes, of which CD8 + T cells express CD8 co-receptors on their surface, and initial CD8 + T cells need to be activated by antigen presentation by dendritic cells (DCs) to differentiate into effector cytotoxic T cells (CTLs) or memory T cells. CTLs maintain the body's defense balance through direct killing and immune regulation. In this example, the mature dendritic cell population is marked by positive expression of CD80, and the T cell population is marked by positive expression of CD8.

[0092] like Figure 18As shown, the CD80 expression rate in the spleen of mice in the blank control group was low (8.27%), the CD80 expression rate in the induced control group (15.5%) increased, and the drug-treated experimental group (10.2%) decreased compared with the induced control group. The CD8 expression rate in the blank control group (1.68%) was low, the CD8 expression rate in the induced control group (4.37%) increased, and the CD8 in the drug-treated experimental group (2.1%) was lower than that in the induced control group. This shows that the CD80 expression rate and CD8 expression rate in the spleen of healthy mice in the blank control group are in a balanced state. After treatment with cisplatin, the control group mice induced kidney damage and were in an inflammatory state. The immune network in the body was stimulated by antigens, which promoted the maturation of dendritic cells and increased the expression of CD80, promoted the presentation of antigens, and activated CD8 + The biomimetic nanovesicles inhibited the maturation of dendritic cells, maintaining them in an immature state conducive to immune tolerance (tDCs), reducing CD80 expression and inhibiting antigen presentation, thereby alleviating T cell overactivation. This suggests that the biomimetic nanovesicles can dynamically regulate polarization based on the immune status in vivo and have a regulatory effect on the adaptive immune system in mice.

[0093] In summary, when kidney is injured, biomimetic nanovesicles can effectively regulate the body's immune network and restore damaged kidney function.

[0094] It is understandable that the above embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, the above technical features can be freely combined, and several deformations and improvements can be made, all of which fall within the scope of protection of the present invention. Therefore, all equivalent changes and modifications made to the scope of the claims of the present invention should fall within the scope of coverage of the claims of the present invention.

Claims

1. A method for preparing biomimetic nanovesicles, characterized in that: The following steps are involved: Epigallocatechin gallate and selenomethionine were dissolved in deionized water, added with formaldehyde solution, stirred in a dark environment, and purified by centrifugation to obtain spherical nanoparticles. Extraction of exosomes from stem cells; Spherical nanoparticles and exosomes were added into deionized water and stirred to obtain biomimetic nanovesicles.

2. The method for preparing biomimetic nanovesicles according to claim 1, characterized in that: The mass ratio of epigallocatechin gallate to selenomethionine is 7-8:

1.

3. The method for preparing biomimetic nanovesicles according to claim 1, characterized in that: The concentration of the formaldehyde solution is 4%.

4. The method for preparing biomimetic nanovesicles according to claim 1, wherein: The stem cells are human umbilical cord mesenchymal stem cells.

5. The method for preparing biomimetic nanovesicles according to claim 4, characterized in that: Exosomes were extracted from the human umbilical cord mesenchymal stem cells by gradient ultracentrifugation.

6. The method for preparing biomimetic nanovesicles according to claim 1, characterized in that: The concentration of the spherical nanoparticles is 9-11 mg / ml, the concentration of the exosomes is 0.9-1.1 mg / ml, and the volume ratio of the spherical nanoparticles to the exosomes is 1:1-1.

2.

7. A biomimetic nanovesicle, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the biomimetic nanovesicles according to claim 7 in anti-inflammatory treatment.

9. Use of the biomimetic nanovesicles according to claim 7 in regulating immune networks.

10. Use of the biomimetic nanovesicles according to claim 7 in protecting against acute kidney injury.

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