A lipopolysaccharide-loaded exosome, its preparation method and application

Abstract

The present invention discloses a lipopolysaccharide-loaded exosome and its preparation method and application, which belongs to the field of biotechnology and comprises the following steps: when human umbilical vein endothelial cells grow to 80%-90% confluence, they are treated with ECM medium containing lipopolysaccharide to stimulate 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, and then centrifuged again, and then separated and purified to obtain a solution containing exosomes; a lipopolysaccharide solution is added to the solution containing exosomes and mixed well to obtain a mixture; the above mixture is subjected to ultrafiltration treatment to obtain lipopolysaccharide-loaded exosomes. The lipopolysaccharide-loaded exosomes have a more significant therapeutic effect in accelerating the skin wound healing cycle compared with the treatment with lipopolysaccharide alone or exosomes alone.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to an exosome loaded with lipopolysaccharide, a preparation method thereof, and an application thereof. Background Art

[0002] The skin has multiple functions such as protection, perception, and body temperature regulation. When skin injury occurs, a series of "injury" signals will be triggered at the wound site, that is, 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 various cell behaviors at the wound site, including fibroblasts, endothelial cells, adipocytes, and melanocytes; at the same time, macrophages affect the behavior of fibroblasts through signal transduction, resulting in collagen deposition and fibrosis.

[0003] During the process of skin wound healing, the migration of macrophages is crucial for regulating the physiological activities of endothelial cells and fibroblasts, promoting angiogenesis and fibrosis. However, the improvement of the wound healing speed is still an urgent problem to be solved. In recent years, exosomes, as a type of extracellular vesicles, have received extensive attention because they are rich in various information molecules such as genetic information fragments, proteins, and lipids, and have high specificity. Cells can quickly respond to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) during the occurrence of skin wounds to produce specific exosomes different from healthy cells, thereby regulating important functions in tissue repair. In this study, endothelial cells were stimulated with lipopolysaccharide, specific apoptotic exosomes were extracted, and the exosomes were co-loaded with lipopolysaccharide into macrophages, triggering the response of the non-canonical inflammasome of macrophages, thereby effectively promoting the healing of skin wounds.

[0004] Currently, most studies focus on the regulatory effects of the contents (information fragments, proteins, lipids, etc.) in exosomes on cells after phagocytosis, ignoring the specificity of the exosome vesicle membrane structure components and the specific binding function. However, exosomes themselves have good compatibility and targeting properties. Therefore, exploring the drug-loading function of exosome membranes will be of great significance for future clinical treatment. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of an exosome loaded with lipopolysaccharide to solve the problems proposed in the above background art.

[0006] To achieve the above purpose, the embodiments of the present invention provide the following technical solutions:

[0007] A preparation method of an exosome loaded with lipopolysaccharide, which includes the following steps:

[0008] Inoculate human venous endothelial cells in ECM complete medium for culture;

[0009] When human umbilical vein endothelial cells grow to a confluence of 80%-90%, they are treated with ECM medium containing lipopolysaccharide to stimulate apoptosis, and then replaced with RPMI 1640 medium for continued incubation, and then the cell supernatant is collected;

[0010] The cell supernatant is subjected to gradient centrifugation, and the supernatant is transferred to a new centrifuge tube, incubated with an exosome concentration reagent, and then centrifuged again, and then separated and purified to obtain a solution containing exosomes;

[0011] Lipopolysaccharide solution is added to the solution containing exosomes, mixed well, and placed at a temperature not higher than 4°C for low-temperature static settlement to obtain a mixture;

[0012] The above mixture is subjected to ultrafiltration treatment, the bottom waste liquid is discarded, and the upper concentrated liquid is collected to obtain lipopolysaccharide-loaded exosomes.

[0013] Preferably, in the ECM medium containing lipopolysaccharide, the concentration of lipopolysaccharide is 0.5-1.5 μg / mL.

[0014] Preferably, in the ECM medium containing lipopolysaccharide, the concentration of lipopolysaccharide is 1 μg / mL.

[0015] Preferably, the volume ratio of the solution containing exosomes to the lipopolysaccharide solution is 1000:(0.5-1.5).

[0016] Preferably, the concentration of lipopolysaccharide in the lipopolysaccharide solution is 0.5-1.5 μg / mL.

[0017] Preferably, the concentration of lipopolysaccharide in the lipopolysaccharide solution is 1 μg / mL.

[0018] Another object of the present invention is to provide lipopolysaccharide-loaded exosomes prepared by the above preparation method.

[0019] Another object of the present invention is to provide an application of the above lipopolysaccharide-loaded exosomes in the preparation of a drug for promoting skin wound healing.

[0020] The lipopolysaccharide-loaded exosomes prepared by the present invention can moderately increase the proportion of M1 macrophages, contribute to wound healing, the exosome membrane structure has a drug-loading function, can load lipopolysaccharide into macrophages, and the genetic material fragments, proteins, lipids, etc. contained in apoptotic exosomes have clinical treatment prospects. The lipopolysaccharide-loaded exosomes have a more significant therapeutic effect in accelerating the skin wound healing cycle compared with the treatment with lipopolysaccharide alone or exosomes alone. Description of the Drawings

[0021] Figure 1The flowchart of the preparation method of lipopolysaccharide-loaded exosomes provided by the embodiments of the present invention;

[0022] Figure 2 The TEM detection result diagram of lipopolysaccharide-loaded exosomes; in the figure, A is the TEM image with a scale of 1μm; B is the TEM image with a scale of 100nm;

[0023] Figure 3 The NTA detection result diagram of lipopolysaccharide-loaded exosomes; in the figure, A is the particle concentration curve graph; B is the particle volume distribution curve graph;

[0024] Figure 4 The treatment schematic diagram of lipopolysaccharide-loaded exosomes;

[0025] Figure 5 The flow cytometry detection diagram of endothelial cell apoptosis; in the figure, A is the apoptosis flow cytometry diagram at 0h of lipopolysaccharide stimulation; B is the apoptosis flow cytometry diagram at 6h of lipopolysaccharide stimulation; C is the apoptosis flow cytometry diagram at 24h after removing lipopolysaccharide stimulation;

[0026] Figure 6 The diagram of macrophages taking up exosomes;

[0027] Figure 7 The uptake diagram of lipopolysaccharide and lipopolysaccharide-loaded exosomes; in the figure, A is the membrane protein binding diagram of lipopolysaccharide alone; B is the uptake diagram of lipopolysaccharide-loaded exosomes;

[0028] Figure 8 The tube formation effect diagram of the conditioned medium for macrophages polarized to 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;

[0029] Figure 9 The diagram of the healing trend of animal skin wounds. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0031] Exosomes, a type of extracellular vesicles, have important physiological functions in intercellular communication and regulation of cell metabolism. However, current research mainly focuses on the physiological effects of exosomes produced by mesenchymal stem cells, and little is known about the mechanism of action of exosomes produced by terminally differentiated cells in the body. Stem cells are pluripotent undifferentiated cells, and their therapeutic potential has been widely recognized. However, the extraction and culture of stem cells are technical challenges. In contrast, terminally differentiated cells are the basic workers of physiological activities in the human body and participants in pathological development. Studying the metabolic secretion, intercellular communication, and functional regulation of terminally differentiated cells under stress conditions will provide new perspectives, new approaches, and new methods for understanding the mechanisms of disease development, tissue repair, and disease treatment.

[0032] Lipopolysaccharide (LPS) is a major component of Gram-negative bacteria. The traditional view holds that LPS, as a non-specific immunogen, interacts with host effector cells (such as monocytes and macrophages) after entering the microcirculation. Under normal circumstances, LPS cannot directly enter cells but is transported to the membrane surface of immune cells and binds to the membrane protein CD14. Subsequently, CD14 transports LPS to form a protein complex with Toll-like receptor 4 and myeloid differentiation protein 2, activating the expression of downstream cytokines and inducing an immune inflammatory response, which further leads to toxic pathophysiological activities in the body. However, this view ignores the beneficial effects of the cascade reaction of immune stimulation caused by appropriate amounts of LPS, namely, activating macrophages, increasing the secretion of cytokines, promoting phagocytic activity, and exerting an immune regulatory effect beneficial to the body. The embodiments of the present invention establish a dialectical view: the toxic mechanism of bacterial products may provide new ideas for clinical disease treatment. For example, botulinum toxin, a neurotoxin produced by Clostridium botulinum, is widely used in the medical and cosmetic fields to treat muscle spasms, hyperhidrosis, trigeminal neuralgia, etc.

[0033] In addition, the traditional mainstream view that the pro-inflammatory effect of M1 macrophages is not conducive to wound healing ignores the combined effect of M1 and M2, that is, an increase in the M1 / M2 ratio is associated with more angiogenesis. In a large number of academic studies, excessive emphasis has been placed on increasing the conversion rate of M2 to promote anti-inflammatory and tissue repair, while ignoring the bridging role of M1 macrophages in the tissue repair process. A large number of cytokines secreted by M1 macrophages can promote the formation of new blood vessels, preparing for the subsequent repair by M2 macrophages. Artificially regulating physiological processes within the body's tolerance threshold will have important clinical therapeutic significance.

[0034] Therefore, the embodiments of the present invention focus on extracellular vesicles produced by the stress apoptosis of endothelial cells under mild LPS stimulation. These extracellular vesicles can not only enhance the activation of M1 macrophages but also synergistically deliver LPS into macrophages, increasing the M1 / M2 ratio and promoting angiogenesis and tissue healing.

[0035] For the specific technical solution, please refer to the following embodiments; the following embodiments are some specific implementation cases of the present invention in actual applications, but are not limited thereto.

[0036] Example 1: As Figure 1 shown, this example provides a method for preparing lipopolysaccharide-loaded exosomes, which includes the following steps:

[0037] S1. Inoculate human umbilical vein endothelial cells (HUVEC) in a culture dish containing ECM complete medium, and place it in an incubator at 37°C and 5% CO2 for culture;

[0038] S2. When the human umbilical vein endothelial cells grow to 80%-90% confluence, treat them with ECM medium containing lipopolysaccharide (the concentration of lipopolysaccharide is 1 μg / mL) for 6 h to stimulate apoptosis, and then replace it with RPMI 1640 medium and continue to incubate for a certain period of time, a total of 24 hours, and then collect the cell supernatant;

[0039] S3. Perform gradient centrifugation on the above cell supernatant: Specifically, first centrifuge at 300 g for 5 min (4°C) to remove cells; then centrifuge at 2000 g for 10 min (4°C) to remove cell debris; then centrifuge at 14000 g for 30 min (4°C) to remove large-volume particles; after gradient centrifugation, transfer the supernatant to a new centrifuge tube, add an exosome concentration reagent (use the WSR0024 reagent of Viskers Biotechnology (Wuhan) Co., Ltd.) for incubation, and then perform centrifugation at 12000 g for 30 min (4°C) again, and then separate and purify with an SEC separation column to obtain a PBS solution containing exosomes;

[0040] 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 well, and place it in a refrigerator at 4°C for low-temperature standing for 6 h to obtain a mixture;

[0041] S5. Take out the above mixture, place it in an ultrafiltration tube, and centrifuge at 3000 g for 15 min for ultrafiltration treatment, discard the waste liquid at the bottom, and collect the upper concentrated solution to obtain lipopolysaccharide-loaded exosomes. The transmission electron microscope (TEM) detection results of the lipopolysaccharide-loaded exosomes are as Figure 2 shown, and the results show that the exosomes are round or oval vesicles; in addition, the nanoparticle tracking analysis (NTA) detection results are as Figure 3 shown, and the results show that the average diameter of the peak of exosome particles is 141.7 nm and the concentration is 4.1E+6. The schematic diagram of the treatment with lipopolysaccharide-loaded exosomes is as Figure 4 shown.

[0042] Example 2: This example provides a method for preparing lipopolysaccharide-loaded exosomes, which includes the following steps:

[0043] S1. Inoculate human umbilical vein endothelial cells (HUVEC) in a culture dish containing ECM complete medium, and place it in an incubator at 37°C and 5% CO2 for culture;

[0044] S2. When the human umbilical vein endothelial cells grow to 80%-90% confluence, treat them with ECM medium containing lipopolysaccharide (the concentration of lipopolysaccharide is 0.5 μg / mL) for 6 h to stimulate apoptosis, and then replace it with RPMI 1640 medium to continue incubation for a certain period of time, a total of 24 hours, and then collect the cell supernatant;

[0045] S3. Perform gradient centrifugation on the above cell supernatant: Specifically, first centrifuge at 300 g for 5 min (4°C) to remove cells; then centrifuge at 2000 g for 10 min (4°C) to remove cell debris; then centrifuge at 14000 g for 30 min (4°C) to remove large-volume particles; after gradient centrifugation, transfer the supernatant to a new centrifuge tube, add an exosome concentration reagent (use WSR0024 reagent from Viskers Biotechnology (Wuhan) Co., Ltd.) for incubation, and then centrifuge again at 12000 g for 30 min (4°C), and then separate and purify with an SEC separation column to obtain a PBS solution containing exosomes;

[0046] 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 well, and place it in a refrigerator at 4°C for low-temperature static standing for 6 h to obtain a mixed solution;

[0047] S5. Take out the above mixed solution, place it in an ultrafiltration tube, centrifuge at 3000 g for 15 min for ultrafiltration treatment, discard the bottom waste liquid, and collect the upper concentrated solution to obtain lipopolysaccharide-loaded exosomes.

[0048] Example 3: This example provides a method for preparing lipopolysaccharide-loaded exosomes, which includes the following steps:

[0049] S1. Inoculate human umbilical vein endothelial cells (HUVEC) in a culture dish containing ECM complete medium, and place it in an incubator at 37°C and 5% CO2 for culture;

[0050] S2. When the human umbilical vein endothelial cells grow to 80%-90% confluence, treat them with ECM medium containing lipopolysaccharide (the concentration of lipopolysaccharide is 1.5 μg / mL) for 6 h to stimulate apoptosis, and then replace it with RPMI 1640 medium to continue incubation for a certain period of time, a total of 24 hours, and then collect the cell supernatant;

[0051] S3. Perform gradient centrifugation on the above cell supernatant: Specifically, first centrifuge at 300 g for 5 min (4 °C) to remove cells; then centrifuge at 2000 g for 10 min (4 °C) to remove cell debris; then centrifuge at 14000 g for 30 min (4 °C) to remove large-volume particles. After gradient centrifugation, transfer the supernatant to a new centrifuge tube, add an exosome concentration reagent (use the WSR0024 reagent of Vesix Bioscience (Wuhan) Co., Ltd.) for incubation, and then centrifuge again at 12000 g for 30 min (4 °C), and then separate and purify with an SEC separation column to obtain a PBS solution containing exosomes;

[0052] 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 well, and place it in a refrigerator at 4 °C for low-temperature static standing for 6 h to obtain a mixture;

[0053] S5. Take out the above mixture, place it in an ultrafiltration tube, centrifuge at 3000 g for 15 min for ultrafiltration treatment, discard the waste liquid at the bottom, and collect the upper concentrated solution to obtain exosomes loaded with lipopolysaccharide.

[0054] Example 4: This example provides an application of the lipopolysaccharide-loaded exosomes prepared in Example 1 above. The lipopolysaccharide-loaded exosomes are applied to promote skin injury healing, and in vitro experiments are carried out to verify the effectiveness of the lipopolysaccharide-loaded exosomes in related applications.

[0055] This example is a basic biological experiment with exosomes as the research focus. Exosomes (EVs) are nanoscale extracellular vesicles that play an important biological role in cell-to-cell communication and have become a new research hotspot in recent years. In particular, exosomes secreted by various mesenchymal stem cells have great prospects for the treatment of clinical diseases. However, due to the differences in cell types and the environment, there are huge differences in the information inclusions of the extracellular vesicles secreted. Therefore, this example of the present invention raises a question: Do all exosomes have the function of treating diseases, and are there also signals that promote the development of diseases? In addition, exosomes are produced by cell exocytosis, and compared with liposomes, the more biologically characteristic membrane may also become an effective carrier. The research purpose of this example is to prove that apoptotic exosomes produced by human umbilical vein endothelial cells under LPS stimulation have special inclusions and can load LPS and be phagocytosed by cells together, resulting in damage to local tissue cells. This provides a new direction for in-depth study of the occurrence and development mechanisms of diseases and is of great significance for exploring new treatment methods for various diseases.

[0056] (1) In vitro cell experiments: divided into a blank group, an LPS group, an EV group, and an LPS-EV group. The blank group was the M0 macrophage group, treated with PBS solution; the LPS group, the EV group, and the LPS-EV group were the M1 macrophage groups, and an increased M1 / M2 ratio was 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 the lipopolysaccharide-loaded exosomes prepared in Example 1.

[0057] An embodiment of the present invention emphasizes a concept that different cells produce different exosomes, and the same cell produces different exosomes under different environments. Then, the exosomes produced by apoptotic cells can be called apoptotic exosomes (ApoEVs). Through in vitro experiments, by controlling the stimulation conditions, observing the state changes of endothelial cells, collecting the secreted apoptotic exosomes, and further exploring how exosomes, as an important part of intercellular communication, regulate macrophage inflammation, and accidentally finding that exosomes can load lipopolysaccharide.

[0058] Biological functions, and promote angiogenesis and wound healing by increasing the M1 / M2 ratio.

[0059] As Figure 5 shown, endothelial cell apoptosis was induced, and the apoptotic state of endothelial cells was continuously monitored in the experiment. The flow cytometry detection results at 0 h, 6 h, and 24 h showed that the apoptotic ratio of endothelial cells was gradually increasing. At the same time, it was also proved that after stimulation with high-dose lipopolysaccharide (1 μg / mL) for 6 h, even if the lipopolysaccharide was removed, irreversible cell damage still existed and ultimately led to cell apoptosis. As Figure 5 can be seen, the late apoptosis rate of endothelial cells was 1.06% at 0 h of lipopolysaccharide stimulation; the late apoptosis rate of endothelial cells was 10.28% at 6 h of lipopolysaccharide stimulation; the late apoptosis rate of endothelial cells was 20.72% at 24 h after removing lipopolysaccharide stimulation.

[0060] Apoptotic exosomes were labeled with an exosome red tracer (using the WSR0001-2 reagent of Wisks Biotechnology (Wuhan) Co., Ltd.) and co-incubated with M0 macrophages for 6 h. Subsequently, the cells were fixed with 4% paraformaldehyde, the nucleus was stained with DAPI, and the cytoskeleton was stained with phalloidin; the results were photographed by an immunofluorescence microscope as Figure 6 shown. As Figure 6 can be seen, the exosomes were phagocytosed by macrophages, and the high-density round particles aggregated around the nucleus in the figure were exosomes; the results showed that exosomes had the ability to be taken up by macrophages, further indicating that exosomes entered the cells.

[0061] FITC-labeled lipopolysaccharide (FITC-LPS) was co-incubated with macrophage M0 in an incubator for 6 h. CD14 was used to label the membrane protein, and confocal microscopy was performed. The results are shown in Figure 7 Figure A of Figure 7 ; it can be seen that FITC-LPS adhered evenly to the cell membrane surface, showing obvious differences from the intracellular LPS distribution. Intracellular LPS was distributed in the cytoplasmic region except the nucleus. There was no FITC-LPS distribution on the CD14-labeled membrane protein.

[0062] In addition, exosomes loaded with lipopolysaccharide (already labeled with FITC) were co-incubated with M0 macrophages for 6 h. Subsequently, the cells were washed with PBS and fixed and blocked with 100% methanol and QuickBlock blocking solution (PBS) respectively. The macrophage membrane protein was stained with CD14 Rabbit mAb and labeled with ABflo-594 secondary antibody, and confocal microscopy was performed. The results are shown in Figure 7 Figure B of Figure 7 . In the figure, green tiny particles of FITC-LPS were distributed in the cytoplasm. The conventional mechanism of lipopolysaccharide acting on macrophages is to recognize macrophage membrane proteins, transfer signals, and cause M0 macrophages to polarize into M1. FITC-labeled lipopolysaccharide has a green fluorescence effect and can locate the distribution area of lipopolysaccharide in cells, whether it is on the outer surface of the cell membrane or distributed in the cytoplasm. Figure 7 The results show that under the action of exosomes, lipopolysaccharide can enter the interior of macrophages.

[0063] Cytokines secreted by M1 macrophages have an angiogenic effect, and angiogenesis is the first step in the start of any tissue repair. Therefore, after culturing 4 groups of macrophages, the conditioned medium was extracted for tube formation experiments. 60 μL of Matrigel was evenly spread on the bottom of a 24-well plate and incubated at 37 °C for 30 minutes. 20,000 HUVECs were added to each well. The M1 conditioned medium and ECM complete medium were mixed and added to the wells at a ratio of 1:3, and cultured at 37 °C for 3 hours. The experimental results are shown in Figure 8 Figure shown in 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 relatively regularly, with few connections and relatively incomplete tubules; the endothelial cells in the LPS group were arranged relatively regularly, with fewer connections and relatively complete tubules; the endothelial cells in the LPS-EV group were arranged extremely regularly and orderly, with a large number of connections and very complete tubules. The LPS-EV group had the best tube formation effect, with the largest number of proliferating endothelial cells, the largest number of migrating and tube-forming cells, and uniform wall thickness and pore size.

[0064] (2) In vivo animal experiments: divided into blank group, LPS group, EV group, and LPS-EV group.

[0065] Prepare a skin wound in 0 days; at 3 days, 5 days, and 7 days, take pictures of the wound. The blank group, LPS group, EV group, and LPS-EV group were respectively injected with 0.2 mL of PBS solution, a separate lipopolysaccharide solution, a separate exosome solution, and the lipopolysaccharide-loaded exosomes prepared in Example 1 in the local area around the wound for treatment. Establish a mouse non-infected skin injury model, and take local pictures of the wound on days 0, 3, 5, 7, and 10 to observe the wound healing process. The results are as Figure 9 shown. Among them, the wound healing of the blank group was the worst, and the LPS-EV group had the best healing.

[0066] In the animal experiment of the embodiment of the present invention, the gradient effect of the skin window healing speed is very obvious. Especially at 5 days, 7 days, and 10 days, the reduction of the wound area and the coverage of the newly formed epithelium can be observed with the naked eye. The embodiment of the present invention uses a simple local infiltration treatment method, and such small molecule therapeutic drugs can also be loaded with biodegradable and sustained-release materials, having the development prospect of realizing the dual effects of wound coverage protection and tissue repair promotion.

[0067] Taking the above-mentioned ideal embodiment of the present invention as an inspiration, through the above description, relevant staff can completely make various changes and modifications without departing from the technical idea of the present invention. The technical scope of the present invention is not limited to the content in 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 concentration of lipopolysaccharide in the ECM medium containing lipopolysaccharide is 1 μg / mL; 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 lipopolysaccharide solution to the solution containing exosomes, mixing thoroughly, and placing at a temperature not higher than 4° C. to obtain a mixed solution; the volume ratio of the solution containing exosomes to the lipopolysaccharide solution is 1000:(0.5-1.5); the concentration of lipopolysaccharide in the lipopolysaccharide solution is 0.5-1.5 μg / mL; 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 lipopolysaccharide solution was 1 μg / mL.

3. An exosome loaded with lipopolysaccharide prepared by the preparation method according to any one of claims 1 to 2.

4. Use of the lipopolysaccharide-loaded exosomes as claimed in claim 3 in the preparation of a medicament for promoting the healing of skin injuries.

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