Adipose-derived mesenchymal stem cell exosome-icariin drug delivery system, preparation method and application

Abstract

The present invention provides an adipose-derived mesenchymal stem cell exosome-icariin drug delivery system, a preparation method and an application, and relates to the field of biomedical technology. The adipose-derived mesenchymal stem cell exosome-icariin drug delivery system comprises adipose-derived mesenchymal stem cell exosomes and icariin, and the adipose-derived mesenchymal stem cell exosomes and icariin are mixed to form a suspension; the suspension is incubated, centrifuged, and the supernatant is collected to obtain the product. The preparation method of the adipose-derived mesenchymal stem cell exosome-icariin drug delivery system of the present invention is simple, and the obtained adipose-derived mesenchymal stem cell exosome-icariin drug delivery system realizes the synergy and superposition of the effectiveness of the trinity of "natural targeting + exosomes + effective compounds", and realizes the synergistic regulatory effect on macrophage polarization.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to an adipose-derived mesenchymal stem cell exosome-icariin drug delivery system, a preparation method and an application thereof. Background Art

[0002] Adipose-derived stem cells (ADSC) are stem cells with multidirectional differentiation potential isolated from adipose tissue. It is a mesenchymal stem cell derived from adipose tissue that can differentiate into mesenchymal cells, such as osteocytes, chondrocytes or adipocytes. ADSC cells can stably proliferate in vitro with a low apoptosis rate. They are easy to obtain, a large number of stem cells can be obtained from a small amount of tissue, suitable for large-scale culture, and have little damage to the body. Moreover, they have a wide range of sources, a large amount of reserves in the body, and are suitable for autologous transplantation. They have gradually become one of the new research hotspots in recent years. Compared with ADSCs, exosomes derived from adipose stem cells (ADSCs-EXO) have many advantages such as low immunogenicity, high yield, low invasiveness and easy acquisition, which can overcome the disadvantages of clinical application of ADSCs.

[0003] Exosomes carry natural bioactive molecules, including proteins, small molecule nucleic acids and active lipids, and are responsible for the transmission and exchange of proximal or remote intercellular substances and information. Therefore, exosomes can be used as a natural biological nanocarrier to load active proteins, small molecule drugs or therapeutic nucleic acids for vaccine or nanomedicine development. Currently, many animal experiments and clinical studies have confirmed that ADSCs-EXO can regulate macrophage polarization and treat inflammatory diseases.

[0004] Icariin (ICA) is a natural active ingredient extracted from the traditional Chinese medicine Herba Epimedii. As a flavonoid compound, it has the effects of increasing cardiovascular and cerebrovascular blood flow, promoting hematopoiesis, immune function and bone metabolism, and also has the effects of tonifying the kidney and strengthening yang, and anti-aging. However, due to the low solubility and poor permeability of icariin, its bioavailability is low, and its use alone is not conducive to being absorbed by cells and fully exerting its efficacy.

[0005] There are currently no reports on exosomes derived from adipose stem cells loaded with icariin, so there is an urgent need to provide an adipose mesenchymal stem cell exosome-icariin drug delivery system. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides an adipose-derived mesenchymal stem cell exosome-icariin drug delivery system (ADSCs-EXO-ICA), a preparation method and an application thereof. The drug delivery system has a high encapsulation rate for icariin, is simple to operate and has a simple preparation method. The ADSCs-EXO-ICA drug delivery system realizes the synergy and superposition of the effectiveness of the trinity of "natural targeting + exosomes + effective compounds".

[0007] The first object of the present invention is to provide an adipose-derived mesenchymal stem cell exosome-icariin drug delivery system, comprising adipose-derived mesenchymal stem cell exosomes and icariin, wherein the icariin is encapsulated in the adipose-derived mesenchymal stem cell exosomes.

[0008] Preferably, the mass ratio of the adipose-derived mesenchymal stem cell exosomes to the icariin is 1-20:1-10.

[0009] Preferably, the mass ratio of the adipose-derived mesenchymal stem cell exosomes to the icariin is 1:1.

[0010] Preferably, the method for preparing adipose-derived mesenchymal stem cell exosomes comprises the following steps:

[0011] (1) Inoculating human adipose-derived mesenchymal stem cells into culture medium and collecting the culture supernatant;

[0012] (2) subjecting the supernatant to gradient centrifugation and collecting the centrifugal supernatant;

[0013] (3) filtering the centrifugal supernatant and performing refrigerated centrifugation to obtain refrigerated centrifugal supernatant;

[0014] (4) Centrifuge the frozen supernatant, discard the supernatant, add PBS to resuspend the precipitate.

[0015] Further preferably, the culturing time in step (1) is 40-60 h.

[0016] Further preferably, the specific process of the gradient centrifugation in step (2) is as follows: the culture supernatant of step (1) is centrifuged at 200-400 g for 5-15 min to obtain supernatant No. I; supernatant No. I is centrifuged at 1500-2500 g for 15-45 min to obtain supernatant No. II; supernatant No. II is centrifuged at 8000-15000 g for 15-45 min to obtain centrifuged supernatant.

[0017] Further preferably, the temperature of the gradient centrifugation in step (2) is 2-6°C.

[0018] Further preferably, the filtration in step (3) is filtration through a 0.22 μm filter.

[0019] Further preferably, the refrigerated centrifugation in step (3) is performed in an ultraspeed refrigerated centrifuge.

[0020] Further preferably, the centrifugation temperature in step (4) is 2-6°C.

[0021] Further preferably, the centrifugation in step (4) is performed at 100,000-160,000 g for 100-180 min.

[0022] The second object of the present invention is to provide a method for preparing an adipose-derived mesenchymal stem cell exosome-icariin drug delivery system, the method for preparing the drug delivery system comprising the following steps:

[0023] (A) Adipose-derived mesenchymal stem cell exosomes and icariin were mixed to form a suspension;

[0024] (B) Incubate the suspension, centrifuge, and collect the supernatant.

[0025] Preferably, in step (A), the mass ratio of the adipose-derived mesenchymal stem cell exosomes to the icariin is 1-20:1-10.

[0026] Preferably, in step (A), the mass ratio of the adipose-derived mesenchymal stem cell exosomes to the icariin is 1:1.

[0027] Preferably, the incubation in step (B) is performed at room temperature for 2-4 hours, and the centrifugation is performed at 12000-15000 g for 30-45 minutes.

[0028] The present invention also relates to the use of the adipose-derived mesenchymal stem cell exosome-icariin drug delivery system or the adipose-derived mesenchymal stem cell exosome-icariin drug delivery system prepared by the preparation method of the adipose-derived mesenchymal stem cell exosome-icariin drug delivery system in the preparation of drugs for treating arthritis.

[0029] Compared with the prior art, the present invention has the following beneficial effects: utilizing the natural macrophage targeting of adipose-derived mesenchymal stem cell exosomes, the bioavailability of icariin can be improved, and the polarization of macrophages can be regulated. The prepared adipose-derived mesenchymal stem cell exosome-icariin drug delivery system (ADSCs-EXO-ICA) realizes the synergy and superposition of the effectiveness of the trinity of "natural targeting + exosomes + effective compounds". BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Morphological images of ADSCs-EXO and ADSCs-EXO-ICA under electron microscope;

[0031] Figure 2Western blot images of ADSCs-EXO and ADSCs-EXO-ICA;

[0032] Figure 3 Uptake of ADSCs-EXO by M1 macrophages for 8 h;

[0033] Figure 4 Fluorescence imaging results of macrophage M1 surface markers MHCII and iNOS;

[0034] Figure 5 Fluorescence imaging results of macrophage M2 surface markers CD163 and CD206;

[0035] Figure 6 Effects of different concentrations of ADSCs-EXO-ICA on the proliferation activity of RAW264.7;

[0036] Figure 7 Effect of ADSCs-EXO-ICA on the relative expression of INOS;

[0037] Figure 8 Effect of ADSCs-EXO-ICA on the relative expression of Arg1;

[0038] Fig. 9 Effect of ADSCs-EXO-ICA on the content of inflammatory factor TNF-α in the serum of CIA rats;

[0039] Fig.10 Effect of ADSCs-EXO-ICA on the content of inflammatory factor IL-1β in the serum of CIA rats;

[0040] Fig.11 Effects of ADSCs-EXO-ICA on the inflammatory score results of CIA rats;

[0041] Fig.12 Effect of ADSCs-EXO-ICA on the bone surface area / bone volume (BS / BV) value in CIA rats. DETAILED DESCRIPTION

[0042] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0043] Example 1

[0044] Extraction of adipose-derived mesenchymal stem cell exosomes (ADSCs-EXO): Human adipose-derived mesenchymal stem cells were inoculated into a special culture medium for mesenchymal stem cells without exosomes. When the cells grew to 80% of the T75 culture flask, the culture was continued for 48 hours, and the culture supernatant was collected; the culture supernatant was centrifuged at 300g and 4°C for 10 minutes to collect supernatant No. Ⅰ; centrifuged at 2000g for 20 minutes at 4°C to collect supernatant No. Ⅱ; centrifuged at 12000g for 40 minutes at 4°C to obtain the centrifuged supernatant; the centrifuged supernatant was filtered with a 0.22μm filter and placed in an ultraspeed refrigerated centrifuge for centrifugation; the refrigerated centrifuged supernatant was centrifuged at 100000g and 4°C for 70 minutes; centrifuged at 150000g for 80 minutes at 4°C, the supernatant was discarded, PBS was added to resuspend the precipitate, and a solution containing hADSCs exosomes was obtained; the supernatant was completely discarded, and 100 microliters of sterile PBS was added. The precipitate was resuspended by pipetting for 3 times, and the resuspended liquid was collected to obtain adipose-derived mesenchymal stem cell exosomes. The adipose-derived mesenchymal stem cell exosomes were placed in a 1.5 ml EP tube and stored in a -80°C refrigerator for subsequent experiments.

[0045] Preparation of ADSCs-EXO-ICA: The collected adipose-derived mesenchymal stem cell exosomes and icariin (ICA) were mixed in a 1:1 ratio to prepare a suspension of the two. The suspension was incubated at room temperature for 2 hours, centrifuged at 13500g for 40 minutes to remove free icariin, and the supernatant was collected to obtain ADSCs-EXO-ICA.

[0046] Experimental Example 1: Verification of Icariin-loaded Exosomes from Adipose-derived Mesenchymal Stem Cells

[0047] (1) Transmission electron microscopy morphology observation of ADSCs-EXO and ADSCs-EXO-ICA samples

[0048] Experimental process: 10 μL of the ADSCs-EXO sample and the ADSCs-EXO-ICA sample in Example 1 were taken respectively, and the following steps were performed for each sample: the sample was dripped on the copper grid to precipitate for 1 min, and the floating liquid was absorbed by filter paper; 10 μL of uranyl acetate was dripped on the sample to precipitate for 1 min, and the floating liquid was absorbed by filter paper; it was dried at room temperature for several minutes, and the electron microscope was used for imaging at 100 kV to obtain the transmission electron microscope imaging results, as shown in FIG. Figure 1 shown.

[0049] (2) Particle size analysis of ADSCs-EXO samples and ADSCs-EXO-ICA samples

[0050] Experimental process: (1) Take out 10 μL of exosomes and dilute to 30 μL; (2) Use standard samples to perform instrument performance test and pass the test before loading samples. Note that gradient dilution is required to avoid clogging of the injection needle by the sample; (3) After the sample is tested, the particle size and concentration information of the ADSCs-EXO sample and ADSCs-EXO-ICA sample in Example 1 can be obtained. The average particle size of the ADSCs-EXO-ICA sample is 180 nm. The results are shown in Table 1.

[0051] Table 1 Analysis results of particle size and concentration of ADSCs-EXO-ICA samples

[0052]

[0053] (3) Exosome surface molecular markers

[0054] Western blot was used to detect the expression of exosome-specific proteins CD81, TSG101 and Calnexin. The specific steps were as follows: an appropriate amount of exosomes were taken for SDS-PAGE gel electrophoresis, and after transfer and blocking, CD81, TSG101 and Calnexin primary antibodies were added for overnight incubation, followed by secondary antibody incubation, washing, and exposure to images. The results were as follows: Figure 2 shown.

[0055] Experimental Example 2 Statistics of the encapsulation efficiency of icariin in adipose-derived mesenchymal stem cell exosomes

[0056] Methods: According to the preparation method of ADSCs-EXO-ICA sample in Example 1, adipose-derived mesenchymal stem cell exosomes-icariin drug delivery system with a mass ratio of ADSCs-EXO to ICA of 1:3, 1:10, 10:1, and 20:1 was prepared. The encapsulation efficiency of adipose-derived mesenchymal stem cell exosomes for icariin was determined by HPLC. The chromatographic column was C18 (4.6 mm × 250 mm, 5 μm); the column temperature was 30 ° C; the mobile phase was acetonitrile-water = 30﹕70; the flow rate was 1.5 mL / min; the detection wavelength was 270 nm; and the injection volume was 20 μL. Using the normalization method and regression equation, the encapsulation efficiency of adipose-derived mesenchymal stem cell exosomes for icariin was obtained to be 91.7%. The results are shown in Table 2.

[0057] Table 2 Results of encapsulation efficiency of icariin by adipose-derived mesenchymal stem cell exosomes

[0058]

[0059] The experimental results show that the encapsulation rate of icariin in adipose-derived mesenchymal stem cell exosomes at different mass ratios is above 90%.

[0060] Experimental Example 3 Results of M1 macrophage uptake of ADSCs-EXO and ADSCs-EXO-ICA

[0061] ADSCs-EXO and ADSCs-EXO-ICA were stained and labeled according to the instructions of the PKH67 dye kit. PKH67-labeled exosomes were collected after ultracentrifugation at 100,000 g for 70 min, and then co-cultured with macrophages for 8 h. Subsequently, 4% paraformaldehyde was added to fix the macrophages. After permeabilization, washing, and nuclear staining, laser confocal microscopy was used for observation and photography. Figure 3 shown.

[0062] Experimental Example 4 Fluorescence imaging results of macrophage M1 surface markers (MHCII, iNOS) and macrophage M2 surface markers (CD163, CD206)

[0063] An appropriate amount of macrophages were plated in a confocal culture dish, induced with LPS+IFN-γ and IL-4+IL-13 for 24 hours, and then 4% paraformaldehyde was added to fix the macrophages. After permeabilization and overnight incubation with MHCII, iNOS, CD206, and CD163 antibodies, the corresponding fluorescent secondary antibodies were incubated, and nuclear staining was performed before observation under a confocal microscope and photographed. The results are shown in Figure 2. Figure 4 , Figure 5 shown.

[0064] Experimental Example 5 Effects of different concentrations of ADSCs-EXO-ICA on the proliferation activity of RAW264.7

[0065] The fifth generation RAW264.7 cells were seeded in a 96-well plate at a density of 3000 per well. After the cells adhered to the wall, the original culture medium was discarded. After LPS+IFN-γ induction for 24 hours, 12.5, 25, 50, 100, and 200 μg / mL ADSCS-EXO-ICA were added to the RAW264.7 cells. After incubation for 24 hours, the reaction solution was prepared according to the CCK-8 manual, and 100 μl of solution was added to each well. Note that the solution should be added slowly and evenly to avoid the generation of bubbles during the whole process, thereby preventing errors in the experimental results. 37 ℃, 5% CO 2 Incubate in the incubator for 4 h and measure the absorbance OD value at 450 nm. The results are shown in Table 3. Figure 6 shown.

[0066] Table 3 Effects of different concentrations of ADSCs-EXO-ICA on the proliferation activity of RAW264.7

[0067]

[0068] Note: Compared with the BLANK group,#### P<0.0001; compared with the LPS+IFN-γ group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0069] The experimental results showed that ADSCs-EXO-ICA had a concentration-dependent promoting effect on RAW264.7.

[0070] Experimental Example 6 Regulatory effect of ADSCs-EXO-ICA on macrophage polarization

[0071] (1) RT-qPCR detection of Arg1 and iNOS mRNA expression

[0072] Experimental process: Take the above 6 groups of macrophages and add Trizol to lyse for 5 minutes, add 100ul chloroform to the above lysate, let stand at room temperature for 5 minutes, centrifuge at 4℃, 12000 rpm, 15min, take the upper solution, add an equal volume of isopropanol, let stand at room temperature for 10min, centrifuge at 4℃, 12000 rpm, 10min, discard the supernatant, add 1mL of 75% ethanol to each tube, let stand for 2min, centrifuge at 4℃, 7500 rpm, 5min, resuspend the RNA precipitate in 20 μL RNA-free water, which is the total RNA, and use a nucleic acid quantifier to detect the total RNA content. According to the reverse transcription kit, the extracted RNA was reverse transcribed into cDNA, and then the reaction system was configured using the QPCR SYBRGreen Master Mi kit instructions, and the qPCR reaction was performed, GAPDH was used as the internal reference, and the expression level was calculated by the 2^-ΔΔCt method after the reaction. The drug concentrations were: ICA 40ug / mL, ADSCs-EXO 40ug / mL, and ADSCs-EXO-ICA 40ug / mL.

[0073] The relative expression results of INOS and Arg1 are shown in Table 4. Figure 7 , Figure 8 .

[0074] Table 4 Relative expression of INOS and Arg1 ( ±s, n=10)

[0075]

[0076] Note: In INOS, compared with the Blank group, ****P<0.0001; compared with the LPS+IFN-γ group, #### P<0.0001; compared with the ICA group, ^^ P<0.01, ++++P<0.0001; compared with the ADSCs-EXO group, & P<0.05, &&&& P<0.0001. In Arg1, compared with the Blank group, ****P<0.0001; compared with the LPS+IFN-γ group, #### P<0.0001; compared with the ICA group, &&&& P<0.0001; compared with the ADSCs-EXO group, ++++ P<0.0001.

[0077] According to the results, ADSCs-EXO-ICA has the effect of reducing the relative expression of INOS and increasing the relative expression of Arg1. Whether compared with the ICA group or the ADSCs-EXO group, the effect of ADSCs-EXO-ICA is significantly different. Therefore, the ADSCs-EXO-ICA drug delivery system achieves the synergy and superposition of the three-in-one effectiveness of "natural targeting + exosomes + effective compounds".

[0078] (2) Determination of serum inflammatory factors TNF-α and IL-1β

[0079] Experimental procedure: ELISA kits were used according to the manufacturer's instructions to detect the levels of TNF-α and IL-1β in the cell supernatant.

[0080] The results are shown in Table 5. Fig. 9 , Fig.10 .

[0081] Table 5 The levels of inflammatory factors TNF-α and IL-1β in serum ( ±s, n=10)

[0082]

[0083] Note: Compared with Control (normal group), *P<0.05; Compared with Model (model group), # P<0.05; compared with the ICA group, + P<0.05; compared with ADSCs-EXO group, & P<0.05.

[0084] According to the results, compared with the model group, ADSCs-EXO-ICA can significantly inhibit the levels of inflammatory cytokines TNF-α and IL-1β in serum, and the anti-inflammatory effect is better than that of ADSCs-EXO and ICA alone, and the effect is equivalent to MTX. The above experimental results show that ADSCs-EXO-ICA plays a synergistic role in anti-inflammatory.

[0085] Experimental Example 7 In vivo efficacy experiment of ADSCs-EXO-ICA

[0086] Objective: To study the effects of ADSCs-EXO-ICA on rats with arthritis model.

[0087] method:

[0088] (1) Animals: 70 SPF male SD rats, 2 months old, were selected.

[0089] (2) Modeling process: An equal volume of bovine type II collagen solution (2 mg / mL) and an equal volume of complete Freund's adjuvant (CFA) were mixed in an ice bath to form an emulsion with a final concentration of 1 mg / mL. On day 0, 100uL of the above emulsion was injected intradermally at the base of the rat's tail and back for primary immunization. An equal volume of bovine type II collagen solution (2 mg / mL) and an equal volume of incomplete Freund's adjuvant (IFA) were mixed in an ice bath to form an emulsion with a final concentration of 1 mg / mL. On day 14, booster immunization was performed once at different parts of the base of the tail to establish a collagen-induced experimental rheumatoid arthritis animal model in rats.

[0090] (3) Animal grouping and dosing: The animals were divided into normal group (Blank group), CIA group (model group), methotrexate group, ICA group, ADSCs-EXO group, and ADSCs-EXO-ICA group, with 10 rats in each group. Except for the normal group, the rats in the other groups were modeled according to the method in (2). On the 14th day, the blank control group and the model group were injected with normal saline through the tail vein, the methotrexate group was injected with methotrexate (MTX) (2 mg / kg) through the tail vein, the ICA group was injected with ICA (100 ug / rat) through the tail vein, the ADSCs-EXO group was injected with ADSCs-EXO (100 ug / rat), and the ADSCs-EXO-ICA group was injected with ADSCs-EXO-ICA (100 ug / rat). The above drugs were administered once every 2 days for 2 consecutive weeks.

[0091] The effects of ADSCs-EXO-ICA on the inflammation score results are shown in Table 6, Fig.11 .

[0092] Table 6 Effect of ADSCs-EXO-ICA on inflammation score results

[0093]

[0094] Note: Compared with Control (normal group), *P<0.01; Compared with Model (model group), # P<0.05; compared with the ICA group, ^ P<0.05; compared with ADSCs-EXO group, +P<0.05.

[0095] The results of arthritis scoring showed that the model group was significantly different from the normal group (P<0.01), indicating that the model was successfully established; whether compared with the ICA group or ADSCs-EXO, the effect of ADSCs-EXO-ICA in reducing the severity of arthritis was statistically significant (P<0.05); the anti-inflammatory effect of ADSCs-EXO-ICA was equivalent to that of MTX. The ADSCs-EXO-ICA drug delivery system can alleviate the severity of arthritis, and the anti-inflammatory synergistic effect of the trinity of ADSCs-EXO-ICA "natural targeting + exosomes + effective compounds" was verified by the appearance of the ankle joint in the in vivo experiment.

[0096] Experimental Example 8 Effect of ADSCs-EXO-ICA on Bone Surface Area / Bone Volume (BS / BV) Value

[0097] Experimental process: After sampling the model rats in Experimental Example 7, the imaging changes of the ankle joints of the rats in each group were observed: the left ankle joints of the rats in each group were scanned and measured using micro-CT, and the hind limbs of the rats were scanned at a voltage of 70 kV, a current of 200 μA, and a scanning resolution of 10.2 μm. The three-dimensional image reconstruction was performed using NRecon software, and the three-dimensional analysis and drawing were performed using DataViewer, CTan, and CTvox software.

[0098] The bone surface area (BS) and bone volume (BV) of each group were measured, and BS / BV was calculated. The results are shown in Table 7. Fig.12 .

[0099] Table 7 Effect of ADSCs-EXO-ICA on bone surface area / bone volume (BS / BV) values

[0100]

[0101] Note: Compared with the normal group, **P<0.0001; compared with the model group, # P<0.05; compared with ICA, + P<0.05; compared with ADSCs-EXO, & P<0.05.

[0102] From the above results, it can be seen that ADSCs-EXO-ICA can significantly reduce the BS / BV of the ankle joints of arthritic rats and inhibit bone destruction. Its effect in preventing bone destruction is better than that of ADSCs-EXO and ICA alone. The effect of ADSCs-EXO-ICA on the bone surface area / bone volume value is equivalent to that of MTX.

[0103] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Application of an adipose-derived mesenchymal stem cell exosome-icariin drug delivery system in the preparation of a drug for treating rheumatoid arthritis, characterized in that: The drug-carrying system comprises adipose-derived mesenchymal stem cell exosomes and icariin, wherein the icariin is encapsulated in the adipose-derived mesenchymal stem cell exosomes; the mass ratio of the adipose-derived mesenchymal stem cell exosomes to the icariin is 1:1; The steps for preparing the adipose-derived mesenchymal stem cell exosomes are as follows: (1) Inoculating human adipose-derived mesenchymal stem cells into culture medium and collecting the culture supernatant; (2) subjecting the supernatant to gradient centrifugation and collecting the centrifugal supernatant; (3) filtering the centrifugal supernatant and performing refrigerated centrifugation to obtain refrigerated centrifugal supernatant; (4) Centrifuging the frozen centrifugal supernatant, discarding the supernatant, and adding PBS to resuspend the precipitate to obtain adipose-derived mesenchymal stem cell exosomes; wherein the specific process of the gradient centrifugation in step (2) is as follows: taking the culture supernatant of step (1) and centrifuging it at 200-400 g for 5-15 min to obtain supernatant No. I; centrifuging supernatant No. I at 1500-2500 g for 15-45 min to obtain supernatant No. II; centrifuging supernatant No. II at 8000-15000 g for 15-45 min to obtain centrifuged supernatant; The preparation steps of the drug delivery system are as follows: (A) Adipose-derived mesenchymal stem cell exosomes and icariin were mixed to form a suspension; (B) Incubate the suspension, centrifuge, and collect the supernatant.

2. The use according to claim 1, characterized in that: The culture time in step (1) is 40-60 h.

3. The use according to claim 1, characterized in that: The temperature of the gradient centrifugation in step (2) is 2-6°C.

4. The use according to claim 1, characterized in that: The incubation in step (B) is performed at room temperature for 2-4 hours, and the centrifugation is performed at 12000-15000 g for 30-45 minutes.

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