Lipopolysaccharide-loaded exosome as well as preparation method and application thereof

Abstract

The invention discloses a lipopolysaccharide-loaded exosome and a preparation method and application thereof, and belongs to the technical field of biology, and the preparation method comprises the following steps: when human vein endothelial cells grow to a convergence degree of 80%-90%, treating and stimulating apoptosis by using a lipopolysaccharide-containing ECM culture medium, then replacing with an RPMI 1640 culture medium to continue incubation, and then collecting a cell supernatant; carrying out gradient centrifugation treatment on the cell supernatant, transferring the supernatant to a new centrifuge tube, adding an exosome concentration reagent for incubation, then carrying out centrifugation treatment again, and then carrying out separation and purification to obtain a solution containing exosomes; adding the lipopolysaccharide solution into the solution containing the exosome, and fully and uniformly mixing to obtain a mixed solution; and carrying out ultrafiltration treatment on the mixed solution to obtain the lipopolysaccharide-loaded exosome. Compared with single lipopolysaccharide treatment or single exosome treatment, the lipopolysaccharide-loaded exosome has a more remarkable treatment effect in the aspect of accelerating the healing cycle of a skin wound.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to lipopolysaccharide-loaded exosomes and a preparation method and application thereof. Background Art

[0002] The skin has multiple functions such as protection, perception and regulation of body temperature. When skin damage occurs, a series of "damage" signals are triggered at the wound, namely the exposure of pathogen-associated molecular patterns (PAMPs) and the rapid release of damage-associated molecular patterns (DAMPs) by damaged cells, thereby recruiting inflammatory cells. Among them, macrophages regulate the behavior of multiple cells at the wound site, including fibroblasts, endothelial cells, adipocytes and melanocytes; at the same time, macrophages affect the behavior of fibroblasts through signal transduction, leading to collagen deposition and fibrosis.

[0003] In the process of skin wound healing, the migration of macrophages is crucial for regulating the physiological activities of endothelial cells and fibroblasts and promoting angiogenesis and fibrosis. However, the improvement of wound healing speed is still an urgent problem to be solved. In recent years, exosomes, as an extracellular vesicle, have attracted widespread attention because they are rich in genetic information fragments, proteins, lipids and other information molecules, and are highly specific. When responding to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) in the process of skin wounds, cells can quickly respond to produce specific exosomes that are different from healthy cells, thereby regulating and important functions in tissue repair. In this study, endothelial cells were stimulated by lipopolysaccharide, specific apoptotic exosomes were extracted, and exosomes were synergistically loaded with lipopolysaccharide into macrophages, triggering the response of non-classical inflammasomes of macrophage cells, thereby effectively promoting the healing of skin wounds.

[0004] Currently, most studies have explored the regulatory effects of the contents of exosomes (information fragments, proteins, lipids, etc.) on cells after engulfment of exosomes, ignoring the specificity of the structural components of the exosome vesicle membrane and its specific binding function. Exosomes themselves have good compatibility and targeting, so exploring the drug-loading function of exosome membranes will be of great significance for future clinical treatments. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing exosomes loaded with lipopolysaccharide to solve the problems raised in the above background technology.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions: A method for preparing exosomes loaded with lipopolysaccharide, comprising the following steps: Human venous endothelial cells were seeded and cultured in ECM complete medium; When human venous endothelial cells grow to 80%-90% confluence, they are treated with ECM medium containing lipopolysaccharide to stimulate their apoptosis, and then replaced with RPMI 1640 medium for continued incubation, and then the cell supernatant is collected; The cell supernatant is subjected to gradient centrifugation, and the supernatant is transferred to a new centrifuge tube, and an exosome concentration reagent is added for incubation, followed by another centrifugation, and then separated and purified to obtain a solution containing exosomes; Adding the lipopolysaccharide solution to the solution containing exosomes, mixing thoroughly, and placing the solution at a temperature not higher than 4° C. to obtain a mixed solution; The mixed solution is subjected to ultrafiltration treatment, the waste liquid at the bottom is discarded, and the upper concentrated liquid is collected to obtain exosomes loaded with lipopolysaccharide.

[0007] Preferably, in the ECM culture medium containing lipopolysaccharide, the concentration of lipopolysaccharide is 0.5-1.5 μg / mL.

[0008] Preferably, in the ECM culture medium containing lipopolysaccharide, the concentration of lipopolysaccharide is 1 μg / mL.

[0009] Preferably, the volume ratio of the solution containing exosomes to the lipopolysaccharide solution is 1000:(0.5-1.5).

[0010] Preferably, the concentration of lipopolysaccharide in the lipopolysaccharide solution is 0.5-1.5 μg / mL.

[0011] Preferably, the concentration of lipopolysaccharide in the lipopolysaccharide solution is 1 μg / mL.

[0012] Another object of the present invention is to provide an exosome loaded with lipopolysaccharide prepared by the above preparation method.

[0013] Another object of the present invention is to provide a use of the above-mentioned lipopolysaccharide-loaded exosomes in the preparation of a drug for promoting the healing of skin injuries.

[0014] The lipopolysaccharide-loaded exosomes prepared by the present invention can moderately increase the proportion of M1 macrophages, which is helpful for wound healing. The exosome membrane structure has a drug-carrying function and can load lipopolysaccharide into macrophages. The genetic material fragments, proteins, lipids, etc. contained in the apoptotic exosomes have clinical treatment prospects. The lipopolysaccharide-loaded exosomes have a more significant therapeutic effect in accelerating the healing cycle of skin wounds compared with lipopolysaccharide treatment alone or exosome treatment alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 An operation flow chart of a method for preparing lipopolysaccharide-loaded exosomes provided in an embodiment of the present invention; Figure 2The TEM test results of exosomes loaded with lipopolysaccharide are shown in Figure 1. In the figure, A is a TEM image with a scale of 1 μm; B is a TEM image with a scale of 100 nm; Figure 3 The NTA test results of exosomes loaded with lipopolysaccharide are shown in the figure; in the figure, A is the particle concentration curve; B is the particle volume distribution curve; Figure 4 Schematic diagram of treatment with exosomes loaded with lipopolysaccharide; Figure 5 Figure 1 is a flow cytometry image of endothelial cell apoptosis. In the figure, A is a flow cytometry image of apoptosis at 0 h after LPS stimulation; B is a flow cytometry image of apoptosis at 6 h after LPS stimulation; C is a flow cytometry image of apoptosis at 24 h after LPS stimulation was removed. Figure 6 Diagram for exosome uptake by macrophages; Figure 7 Figure 1 is an uptake diagram of lipopolysaccharide and exosomes loaded with lipopolysaccharide; in the figure, A is a membrane protein binding diagram of lipopolysaccharide alone; B is an uptake diagram of exosomes loaded with lipopolysaccharide; Figure 8 The effect diagram of the tube formation of the conditioned medium of macrophage polarization M1; in the figure, A is the blank group; B is the EV group; C is the LPS group; D is the LPS-EV group; Fig. 9 This is a chart showing the healing trend of animal skin wounds. DETAILED DESCRIPTION

[0016] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0017] Exosomes, a type of extracellular vesicle, have important physiological functions of intercellular communication and regulation of cell metabolism. However, most current research focuses on the physiological effects of exosomes produced by mesenchymal stem cells, while little is known about the mechanism of action of exosomes produced by terminal cells of the body. Stem cells are a type of multipotent undifferentiated cells, and their therapeutic prospects have been widely recognized. However, stem cell extraction and culture are technical difficulties. Compared with stem cells, terminal cells are the basic workers of human physiological activities and participants in the development of pathology. Research on the metabolic secretion, intercellular communication, and functional regulation of terminal cells under stress conditions will provide new perspectives, new approaches, and new methods for the development mechanism of diseases, tissue repair mechanisms, and disease treatment mechanisms.

[0018] Lipopolysaccharide (LPS) is the main component of Gram-negative bacteria. The traditional view is that LPS, as a non-specific immunogen, interacts with host effector cells (monocytes, macrophages, etc.) after entering the microcirculation, that is, LPS cannot reach the cell directly under normal circumstances, but is transported to the membrane surface of the immune cell and combines with the protein CD14 on the membrane surface. CD14 then transports LPS to Toll-like receptor 4 and myeloid differentiation protein 2 to form a protein complex, activates the expression of downstream cytokines, induces immune inflammatory response, and further leads to toxic pathophysiological activities of the body. However, this ignores the beneficial effects of the cascade reaction of immune stimulation caused by an appropriate amount of LPS, that is, activating macrophages, increasing the secretion of cytokines, promoting cell phagocytic activity, and playing an immunomodulatory role that is beneficial to the body. The embodiment of the present invention establishes a dialectical viewpoint: the toxic mechanism of bacterial products may provide new ideas for the treatment of clinical diseases. For example, botulinum toxin is a neurotoxin produced by Clostridium botulinum, which is widely used in the fields of medicine and cosmetology to treat muscle spasms, hyperhidrosis, trigeminal neuralgia, etc.

[0019] In addition, the traditional mainstream view is that the pro-inflammatory effect of M1 macrophages is not conducive to wound healing, ignoring the combined effect of M1 and M2, that is, the increase in the M1 / M2 ratio is associated with more vascularization. A large number of academic studies have emphasized that increasing the M2 conversion rate is beneficial to anti-inflammatory and promoting tissue repair, ignoring the role of M1 macrophages in the process of tissue repair. The large amount of cytokines secreted by M1 macrophages can promote the formation of new blood vessels, prepare for the later repair of M2 macrophages, and artificially regulate physiological processes within the body's tolerance threshold, which will have important clinical therapeutic significance.

[0020] Therefore, the embodiments of the present invention focus on the extracellular vesicles produced by stress apoptosis of endothelial cells under micro-stimulation of lipopolysaccharide, which can not only amplify the activation of M1 macrophages, but also synergistically load lipopolysaccharide into macrophages, and increase the M1 / M2 ratio to promote the healing of angiogenic tissue.

[0021] The specific technical solutions refer to the following embodiments; the following embodiments are some specific implementation cases of the present invention in practical applications, but are not limited to them.

[0022] Example 1: Figure 1 As shown, this embodiment provides a method for preparing exosomes loaded with lipopolysaccharide, which comprises the following steps: S1. Human venous endothelial cells (HUVECs) were seeded in a culture dish containing complete ECM medium and cultured in an incubator at 37°C and 5% CO2. S2. When human venous endothelial cells grow to 80%-90% confluence, they are treated with ECM medium containing lipopolysaccharide (the concentration of lipopolysaccharide is 1 μg / mL) for 6 h to stimulate their apoptosis, and then replaced with RPMI 1640 medium for a certain period of time, a total of 24 hours, and then the cell supernatant is collected; S3. The cell supernatant was subjected to gradient centrifugation: specifically, first centrifuged at 300g for 5min (4°C) to remove cells; then centrifuged at 2000g for 10min (4°C) to remove cell debris; then centrifuged at 14000g for 30min (4°C) to remove large particles; after gradient centrifugation, the supernatant was transferred to a new centrifuge tube, and an exosome concentration reagent (WSR0024 reagent from Wisc Biotech (Wuhan) Co., Ltd.) was added for incubation, and then centrifuged at 12000g for 30min (4°C) again, and then separated and purified using a SEC separation column to obtain a PBS solution containing exosomes; S4. Add 1 μL of lipopolysaccharide solution (the concentration of lipopolysaccharide is 1 μg / mL) to the above 1 mL of PBS solution containing exosomes, mix thoroughly, and place in a refrigerator at 4°C for 6 hours to obtain a mixed solution; S5. Take out the above mixed solution, place it in an ultrafiltration tube and centrifuge it at 3000g for 15 minutes to perform ultrafiltration treatment, discard the waste liquid at the bottom, collect the upper concentrated liquid, and obtain the exosomes loaded with lipopolysaccharide. The transmission electron microscopy (TEM) test results of the exosomes loaded with lipopolysaccharide are as follows: Figure 2 As shown in the figure, the results showed that exosomes were round or oval vesicles; in addition, the results of nanoparticle tracking analysis (NTA) were as follows Figure 3 As shown in the figure, the results showed that the peak average diameter of exosome particles was 141.7nm and the concentration was 4.1E+6. Figure 4 shown.

[0023] Example 2: This example provides a method for preparing exosomes loaded with lipopolysaccharide, which comprises the following steps: S1. Human venous endothelial cells (HUVECs) were seeded in a culture dish containing complete ECM medium and cultured in an incubator at 37°C and 5% CO2. S2. When human venous endothelial cells grow to 80%-90% confluence, they are treated with ECM medium containing lipopolysaccharide (the concentration of lipopolysaccharide is 0.5 μg / mL) for 6 hours to stimulate their apoptosis, and then replaced with RPMI 1640 medium for a certain period of time, a total of 24 hours, and then the cell supernatant is collected; S3. The cell supernatant was subjected to gradient centrifugation: specifically, first centrifuged at 300g for 5min (4°C) to remove cells; then centrifuged at 2000g for 10min (4°C) to remove cell debris; then centrifuged at 14000g for 30min (4°C) to remove large particles; after gradient centrifugation, the supernatant was transferred to a new centrifuge tube, and an exosome concentration reagent (WSR0024 reagent from Wisc Biotech (Wuhan) Co., Ltd.) was added for incubation, and then centrifuged at 12000g for 30min (4°C) again, and then separated and purified using a SEC separation column to obtain a PBS solution containing exosomes; S4. Add 0.5 μL of lipopolysaccharide solution (the concentration of lipopolysaccharide is 0.5 μg / mL) to the above 1 mL of PBS solution containing exosomes, mix thoroughly, and place in a refrigerator at 4°C for 6 hours to obtain a mixed solution; S5. The mixed solution was taken out and placed in an ultrafiltration tube for centrifugation at 3000 g for 15 min to perform ultrafiltration treatment, the waste liquid at the bottom was discarded, and the concentrated liquid at the top was collected to obtain exosomes loaded with lipopolysaccharide.

[0024] Example 3: This example provides a method for preparing exosomes loaded with lipopolysaccharide, which comprises the following steps: S1. Human venous endothelial cells (HUVECs) were seeded in a culture dish containing complete ECM medium and cultured in an incubator at 37°C and 5% CO2. S2. When human venous endothelial cells grow to 80%-90% confluence, they are treated with ECM medium containing lipopolysaccharide (the concentration of lipopolysaccharide is 1.5 μg / mL) for 6 hours to stimulate their apoptosis, and then replaced with RPMI 1640 medium for a certain period of time, a total of 24 hours, and then the cell supernatant is collected; S3. The cell supernatant was subjected to gradient centrifugation: specifically, first centrifuged at 300g for 5min (4°C) to remove cells; then centrifuged at 2000g for 10min (4°C) to remove cell debris; then centrifuged at 14000g for 30min (4°C) to remove large particles; after gradient centrifugation, the supernatant was transferred to a new centrifuge tube, and an exosome concentration reagent (WSR0024 reagent from Wisc Biotech (Wuhan) Co., Ltd.) was added for incubation, and then centrifuged at 12000g for 30min (4°C) again, and then separated and purified using a SEC separation column to obtain a PBS solution containing exosomes; S4. Add 1.5 μL of lipopolysaccharide solution (the concentration of lipopolysaccharide is 1.5 μg / mL) to the above 1 mL of PBS solution containing exosomes, mix thoroughly, and place in a refrigerator at 4°C for 6 hours to obtain a mixed solution; S5. The mixed solution was taken out and placed in an ultrafiltration tube for centrifugation at 3000 g for 15 min to perform ultrafiltration treatment, the waste liquid at the bottom was discarded, and the concentrated liquid at the top was collected to obtain exosomes loaded with lipopolysaccharide.

[0025] Example 4: This example provides an application of the lipopolysaccharide-loaded exosomes prepared in the above-mentioned Example 1. The lipopolysaccharide-loaded exosomes are used to promote the healing of skin injuries, and in vitro experiments are performed to verify the effectiveness of the lipopolysaccharide-loaded exosomes in related applications.

[0026] This embodiment is a basic biological experiment, with exosomes as the research focus. Exosomes (EV) are a type of nano-scale extracellular vesicles that play an important biological role in intercellular communication. In recent years, they have become a new research hotspot, especially exosomes secreted by various mesenchymal stem cells have great prospects for the treatment of clinical diseases. However, different cell types and different environments lead to huge differences in the information contents of the secreted extracellular vesicles, so the embodiment of the present invention raises a question: whether all exosomes have the function of treating diseases, and whether there are signals that promote the development of diseases. In addition, exosomes are produced by cell exocytosis, and compared with liposomes, membranes with more biological characteristics may also become an effective carrier. The purpose of this embodiment is to prove that apoptotic exosomes produced by human venous endothelial cells under LPS stimulation conditions have special contents and can be loaded with LPS and engulfed by cells, resulting in damage to local tissue cells. This provides a new direction for in-depth research on the occurrence and development mechanism of diseases, and is of great significance for exploring new treatment methods for various diseases.

[0027] (I) In vitro cell experiment: divided into blank group, LPS group, EV group, and LPS-EV group. The blank group is an M0 macrophage group treated with PBS solution; the LPS group, EV group, and LPS-EV group are M1 macrophage groups, and an increase in the M1 / M2 ratio is conducive to angiogenesis; among them, the LPS group was treated with lipopolysaccharide alone, the EV group was treated with exosomes alone, and the LPS-EV group was treated with exosomes loaded with lipopolysaccharide prepared in Example 1.

[0028] The embodiment of the present invention emphasizes a concept that different cells produce different exosomes, and the same cell produces different exosomes under different environments. Therefore, the exosomes produced by apoptotic cells can be called apoptotic exosomes (ApoEVs). Through in vitro experiments, the stimulation conditions are controlled, the state changes of endothelial cells are observed, and the apoptotic exosomes secreted by them are collected to further explore how exosomes, as an important part of intercellular communication, regulate inflammation in macrophages, and accidentally discover that exosomes can load lipopolysaccharide.

[0029] biological functions and promotes angiogenesis and wound healing by increasing the M1 / M2 ratio.

[0030] like Figure 5 As shown, endothelial cell apoptosis was induced. The apoptotic state of endothelial cells was continuously monitored in the experiment. The flow cytometry results at 0h, 6h, and 24h showed that the apoptotic proportion of endothelial cells was gradually increasing. It also proved that after 6h of high-dose lipopolysaccharide (1μg / mL) stimulation, even if lipopolysaccharide was removed, irreversible cell damage still existed and eventually led to cell apoptosis. Figure 5 It can be seen that at 0h of LPS stimulation, the late apoptosis rate of endothelial cells was 1.06%; at 6h of LPS stimulation, the late apoptosis rate of endothelial cells was 10.28%; and at 24h of LPS stimulation removal, the late apoptosis rate of endothelial cells was 20.72%.

[0031] Apoptotic exosomes were labeled with exosome red tracer (WSR0001-2 reagent from Wisc Biotech (Wuhan) Co., Ltd.) and incubated with M0 macrophages for 6 hours. The cells were then fixed with 4% paraformaldehyde, the nuclei were stained with DAPI, and the cytoskeleton was stained with phalloidin; the results were taken by immunofluorescence microscopy. Figure 6 As shown. Figure 6 It can be seen that exosomes are engulfed by macrophages. The high-density round particles gathered around the cell nucleus in the figure are exosomes; the results show that exosomes have the ability to be taken up by macrophages, further indicating that exosomes enter the cells.

[0032] FITC-labeled lipopolysaccharide (FITC-LPS) was co-incubated with macrophages M0 in an incubator for 6 hours, and membrane proteins were labeled with CD14. Confocal imaging was performed, and the results were as follows: Figure 7 As shown in A, FITC-LPS can be seen to be uniformly adhered to the cell membrane surface, which is significantly different from the distribution of intracellular LPS. Intracellular LPS is distributed in the cytoplasmic region except for the nucleus. There is no FITC-LPS distribution on the membrane protein marked by CD14.

[0033] In addition, exosomes loaded with lipopolysaccharide (FITC labeled) were incubated with M0 macrophages for 6 hours. The cells were then washed with PBS and fixed and blocked with 100% methanol and QuickBlock blocking solution (PBS), respectively. Macrophage membrane proteins were stained with CD14Rabbit mAb, labeled with ABflo-594 secondary antibody, and photographed with a confocal microscope. The results are shown in Figure 2. Figure 7 As shown in B, in the figure, FITC-LPS green tiny particles are distributed in the cytoplasm. The conventional mechanism of lipopolysaccharide acting on macrophages is to recognize macrophage membrane proteins, and the signal is transferred to cause M0 macrophages to polarize into M1. FITC-labeled lipopolysaccharide has a green fluorescence effect, which can locate the distribution area of ​​lipopolysaccharide in the cell, whether it is on the outer surface of the cell membrane or distributed in the cytoplasm. Figure 7 The results showed that under the action of exosomes, lipopolysaccharide can enter the interior of macrophages.

[0034] The cytokines secreted by M1 macrophages have angiogenic effects, and angiogenesis is the first step in any tissue repair. Therefore, after culturing the four groups of macrophages, the conditioned medium was extracted for tube formation experiments. The bottom of the 24-well plate was evenly covered with 60 μL of matrix gel, incubated at 37°C for 30 minutes, 20,000 HUVECs were added to each well, and M1 conditioned medium and ECM complete medium were mixed in a 1:3 ratio and added to the wells, and cultured at 37°C for 3 hours. The experimental results are shown in Figure 2. Figure 8 As shown. Figure 8 It can be seen that the endothelial cells in the blank group were extremely loose, with very few connections and extremely incomplete tubules; the endothelial cells in the EV group were arranged more regularly, with fewer connections and less complete tubules; the endothelial cells in the LPS group were arranged more regularly, with fewer connections and more complete tubules; the endothelial cells in the LPS-EV group were arranged extremely regularly and orderly, with many connections and very complete tubules. LPS-EV formed the best tube, with the largest number of endothelial cells proliferating and migrating into tubes, and uniform tube wall thickness and pore size.

[0035] (II) In vivo animal experiments: divided into blank group, LPS group, EV group, and LPS-EV group.

[0036] Skin wounds were prepared on day 0; on days 3, 5, and 7, the wounds were photographed, and the blank group, LPS group, EV group, and LPS-EV group were injected with 0.2 mL of PBS solution, lipopolysaccharide solution alone, exosome solution alone, and lipopolysaccharide-loaded exosomes prepared in Example 1 into the local area around the wound for treatment, respectively, to establish a non-infected skin injury model in mice, and local wound photographs were taken on days 0, 3, 5, 7, and 10 to observe the wound healing process. The results are shown in Figure 2 Fig. 9 Among them, the wound healing in the blank group was the worst, and that in the LPS-EV group was the best.

[0037] The skin window healing speed gradient effect of the embodiment of the present invention is very obvious in animal experiments, especially at 5 days, 7 days, and 10 days, the reduction of wound area and the coverage of new epithelium can be observed by naked eyes. The embodiment of the present invention uses a simple local infiltration treatment method, and this type of small molecule therapeutic drug can also be loaded with degradable slow-release materials, which has the development prospect of achieving the dual effects of wound coverage protection and promoting tissue repair.

[0038] Based on the above-mentioned ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above-mentioned description. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. A method for preparing exosomes loaded with lipopolysaccharide, characterized in that: The following steps are involved: Human venous endothelial cells were seeded and cultured in ECM complete medium; When human venous endothelial cells grow to 80%-90% confluence, they are treated with ECM medium containing lipopolysaccharide to stimulate their apoptosis, and then replaced with RPMI 1640 medium for continued incubation, and then the cell supernatant is collected; The cell supernatant is subjected to gradient centrifugation, and the supernatant is transferred to a new centrifuge tube, and an exosome concentration reagent is added for incubation, followed by another centrifugation, and then separated and purified to obtain a solution containing exosomes; Adding the lipopolysaccharide solution to the solution containing exosomes, mixing thoroughly, and placing the solution at a temperature not higher than 4° C. to obtain a mixed solution; The mixed solution is subjected to ultrafiltration treatment, the waste liquid at the bottom is discarded, and the upper concentrated liquid is collected to obtain exosomes loaded with lipopolysaccharide.

2. The method for preparing lipopolysaccharide-loaded exosomes according to claim 1, characterized in that: The concentration of lipopolysaccharide in the ECM culture medium containing lipopolysaccharide is 0.5-1.5 μg / mL.

3. The method for preparing lipopolysaccharide-loaded exosomes according to claim 2, characterized in that: The concentration of lipopolysaccharide in the ECM medium containing lipopolysaccharide was 1 μg / mL.

4. The method for preparing lipopolysaccharide-loaded exosomes according to claim 1, characterized in that: The volume ratio of the solution containing exosomes to the lipopolysaccharide solution was 1000:(0.5-1.5).

5. The method for preparing lipopolysaccharide-loaded exosomes according to claim 4, characterized in that: The concentration of lipopolysaccharide in the lipopolysaccharide solution is 0.5-1.5 μg / mL.

6. The method for preparing lipopolysaccharide-loaded exosomes according to claim 5, characterized in that: The concentration of lipopolysaccharide in the lipopolysaccharide solution was 1 μg / mL.

7. An exosome loaded with lipopolysaccharide prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the lipopolysaccharide-loaded exosomes as claimed in claim 7 in the preparation of a medicament for promoting the healing of skin injuries.

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