Preparation method and application of psoralen-loaded exosome

Abstract

The invention discloses a preparation method of a bakuchiol-loaded exosome and application of the bakuchiol-loaded exosome. The method comprises the following steps: S1, mixing supernate of a collected umbilical cord mesenchymal stem cell hUMSC culture supernate sample with Exosome Concentration Solution, oscillating and uniformly mixing through a vortex oscillator, and then standing at 4 DEG C to capture exosome particles; s2, centrifuging, discarding the supernatant, re-suspending the precipitate, centrifuging, reserving the supernatant, namely an exosome particle crude product, transferring to a purification column, and purifying to obtain purified exosome particles; and S3, mixing the exosome particles obtained in S2 with bakuchiol BAK, and carrying out drug loading treatment by using an ultrasonic cell crusher. The drug-loaded exosome can inhibit inflammation, promote collagen and angiogenesis and accelerate wound healing through subcutaneous injection near a wound, the treatment effect of the exosome is maximized, and the drug-loaded exosome has practical value in the field of skin wound healing.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, specifically to the technical field of exosome drug delivery, and more specifically to a method for preparing exosomes loaded with bakuchiol and an application thereof. Background Art

[0002] Skin wound healing is a complex biological process involving the interaction of different tissues, cells and molecules. During the wound healing process, it may be difficult to repair the wound due to individual or environmental reasons, and it may be difficult to heal for a long time, leading to serious consequences such as impaired skin barrier function, tissue infection and necrosis, and severe local or systemic dysfunction. Improper repair will generate pathological scars, affecting the beauty of the skin and the psychological trauma of the patient. Currently, common traditional wound treatments include wound dressings, debridement of damaged tissues, and autologous skin transplantation, depending on the severity and size of the wound.

[0003] Bakuchiol is the main component of the volatile oil of the traditional Chinese medicine Psoralea corylifolia, and is an easily oxidized isoprenoid phenol terpenoid compound. It is soluble in organic solvents such as methanol, ethanol, and DMSO, and has strong fat solubility. Literature reports show that bakuchiol has the effects of lowering blood sugar and blood lipids, anti-inflammatory, promoting collagen upregulation, antibacterial, antioxidant, liver protection, anti-cancer, and playing an important role in the treatment of mental disorders. Bakuchiol has been used in traditional Chinese and Indian medicine for centuries and has a variety of pharmacological effects, including the effect of promoting wound healing. However, since bakuchiol is easily oxidized, has poor stability, and is an oil-soluble substance, it is not suitable for direct application to wounds.

[0004] Mesenchymal stem cells and their exosomes have become important tools in the context of tissue engineering and regenerative medicine. Exosomes derived from human umbilical cord mesenchymal stem cells contain α-2-macroglobulin, which can promote angiogenesis and cell proliferation and differentiation, inhibit inflammatory response, regulate cell adhesion and migration, stimulate tissue cell regeneration, and thus accelerate skin wound healing. At the same time, they can also be used as a delivery carrier for multiple drug ingredients to achieve a superimposed effect.

[0005] Therefore, it is necessary to find a delivery carrier that can enhance the effectiveness of bakuchiol at the wound surface or inside the body, which can help bakuchiol and the related growth factors and peptides contained in the exosomes of umbilical cord mesenchymal stem cells to more effectively reduce the inflammatory response at the wound surface, while promoting the growth and healing of wound tissue in a short period of time and reducing the risk of infection.

[0006] Chinese patent CN116869960A discloses a method for preparing a mesenchymal stem cell-derived exosome-encapsulated reovirus complex, which uses mesenchymal stem cell-derived exosomes as a vehicle to encapsulate reovirus (an oncolytic virus) and is prepared by extrusion. Although this method can protect the product from the hindrance of neutralizing antibodies to the greatest extent, and deliver it to tumor cells, enhancing the tumor targeting of reovirus in vivo, the amount of mesenchymal stem cells used is large, and the efficiency of obtaining exosomes is not high. It is necessary to manually squeeze 30 to 50 times in the extrusion device until the encapsulation is completed, and the single output is <1ml, and it is easy to cause loss of raw materials. The pharmaceutical preparation involved in this patent is an anti-tumor drug preparation, and the actual method of use is not clear.

[0007] Chinese patent CN114085817A discloses a method for preparing exosomes encapsulating microRNA, wherein the exosomes encapsulating microRNA are directly secreted by mesenchymal stem cells, and the exosomes are separated from the cell culture medium by differential centrifugation at different speeds. The exosomes prepared by this patented method have a high content and fewer steps, and can reduce inflammatory cells in lung tissue and reduce the level of inflammatory factors. However, during the preparation process, the pre-transfection solution needs to be electroporated, followed by multiple centrifugation for a total of about 95-130 minutes, and the mixed culture time is about 20-24 hours, which takes a long preparation time. At the same time, since the viscosity of the biological fluid is strongly correlated with the purity of the isolated exosomes, this method is not suitable for biological samples with high viscosity. The patent describes lung diseases, and the drug dosage form is an aerosol dosage form. Summary of the invention

[0008] The technical problem to be solved by the present invention is to optimize the exosome preparation method, improve the efficiency of obtaining exosomes, save the consumption of umbilical cord mesenchymal stem cell culture and the amount of cells used, and reduce the loss of raw materials. At the same time, it solves the low efficiency of original wound healing, the limited way of drug application (commonly used dressing (fibroblast epidermal growth factor), skin penetration, promotes skin surface wound healing, and the low efficiency of drug use below the dermis.

[0009] The present invention first provides a method for preparing exosomes loaded with bakuchiol, which comprises the following steps:

[0010] S1: Mix the collected supernatant of the hUMSC culture supernatant sample with the Exosome Concentration Solution, vortex and mix evenly, and then place at 4°C to capture exosome particles;

[0011] S2 is then centrifuged to discard the supernatant, and the precipitate is resuspended and centrifuged again to retain the supernatant, which is the crude exosome particles, and transferred to a purification column for purification to obtain purified exosome particles;

[0012] S3 mixes the exosome particles obtained in S2 with bakuchiol BAK and performs drug loading treatment using an ultrasonic cell disruptor.

[0013] The hUMSC culture supernatant sample collected in S1 was specifically processed as follows: after being transferred from -80°C to 4°C for thawing, it was centrifuged at 4°C, 3000g, for 10 min, the precipitate was discarded to remove the cell debris in the supernatant, and the supernatant was used for subsequent steps.

[0014] Specifically, the supernatant and Exosome Concentration Solution (ECS reagent) were mixed in a ratio of 4-6:1, vortexed and mixed for 1 min, and then placed at 4°C for 16 h to capture exosome particles; then centrifuged at 4°C, 10,000g, 60 min, discarded the supernatant, and resuspended in PBS. Centrifuged again at 4°C, 12,000g, 2 min, and the supernatant was retained to obtain the crude exosome particles.

[0015] Preferably, the purification in S2 is to transfer the crude exosome particles to the upper chamber of the purification column Exosome PurificationFilter, centrifuge at 4°C, 3000g, 10min; collect the liquid filtered to the bottom of the EPF column after centrifugation, which is the purified exosome particles; optionally, pack it into 1.5ml centrifuge tubes and store it at -80°C for subsequent experiments.

[0016] In an embodiment, the exosome particles obtained in S2 can express specific proteins CD63, CD81 and TSG101, and the particle size is concentrated in the range of 80-100 nm.

[0017] Preferably, the ultrasonic parameters in step S3 are set as follows: 20% amplitude, 30s on / off, 6 cycles in total, and a 2min cooling time between each cycle.

[0018] Optionally, the method further comprises the following steps:

[0019] After S4 ultrasonic treatment, the mixed solution was incubated at 37°C for 1 h to restore the exosome membrane structure; the free drug was separated using an ultrafiltration tube and centrifuged at 5000×g for 10 min.

[0020] Specifically, the mass ratio of bakuchiol to exosomes is from 1:1 to 20:1, preferably from 1:1 to 5:1; most preferably, the mass ratio of bakuchiol to exosomes is 5:1.

[0021] The present invention also provides bakuchiol-loaded exosomes obtained by the preparation method.

[0022] The present invention further provides the use of the exosomes in the preparation of a drug for regulating cell proliferation and migration and / or promoting wound healing. Specifically, the exosomes are prepared into a reagent suitable for subcutaneous injection near the wound.

[0023] The present invention provides a method for preparing bakuchiol-loaded exosomes and an effect thereof on promoting skin wound healing, wherein the drug-loaded exosomes include exosomes and bakuchiol contained in the exosomes, wherein the concentration of the exosomes is 1 mg / ml based on the drug-loaded exosomes. The exosomes of the present invention can regulate cell proliferation and migration ability and promote wound healing. BAK-EXO at a concentration of 20 μg / mL was used to act on HSF cells and HUVEC cells, and it was observed that 20 μg / mL of BAK-EXO promoted the proliferation and migration of HSF cells and HUVEC cells, and could promote wound healing with significant effects. The drug-loaded exosomes of the present invention can be injected subcutaneously near the wound, can inhibit inflammation, promote collagen and angiogenesis, accelerate wound healing, maximize the therapeutic effect of exosomes, and have practical value in the field of skin wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The culture morphology of hUMSCs (scale bar = 200 μm), where (A) is the growth state of primary cells after recovery; (B) is the growth state of primary cells with 80% confluence; (C) is the growth state of cells 1-2 days after passage; (D) is the growth state of cells 3 days after passage.

[0025] Figure 2 Characterization of hUMSCs. (A) EXO morphology under transmission electron microscopy; (B) EXO surface specific protein marker expression; (C) EXO nanoparticle average diameter distribution and particle concentration.

[0026] Figure 3 The following are representative HPLC chromatograms and BAK standard curves: (A) 100 μg / mL BAK standard solution chromatogram; (B) EXO:BAK=1:5 solution chromatogram; (C) EXO:BAK=1:0.5 solution chromatogram; (D) BAK standard curve.

[0027] Figure 4 Characterization of BAK-EXO. (A) BAK-EXO particle morphology (B) BAK-EXO nanoparticle diameter distribution (C) BAK-EXO protein blot analysis.

[0028] Figure 5The effect of BAK-EXO on the migration ability of HSF cells. Among them, the Transwell experiment is a cell migration or invasion experiment used to study the movement of cells through porous membranes. This experiment further proved through the Transwell experiment that BAK-EXO promoted the proliferation of vascular endothelial cells in 12 hours and enhanced the migration ability of cells.

[0029] Figure 6 This is the effect of BAK-EXO on the migration ability of HUVEC cells.

[0030] Figure 7 Effect of BAK-EXO on the angiogenic ability of HUVECs.

[0031] Figure 8 Expression of cytokines in HSF and HUVEC culture supernatants. Among them, (A) PEDF; (B) IL-8; (C) IL-1β; (D) MMP-9; (E) PG-E2; (F) TNF-α; (G) VEGF; (H) COL-Ⅰ; (I) COL-Ⅲ. DETAILED DESCRIPTION

[0032] The present invention is described below through specific implementation modes in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0033] 1. Collection of hUMSC supernatant

[0034] Take out hUMSC cells with a fusion rate of 80% from the incubator and place them in a clean bench for experiments. Discard the culture supernatant, wash with PBS three times, discard the liquid, and add trypsin for digestion. Observe the cell morphology under a microscope. When the cells are round and movable, add trypsin and an equal volume of complete culture medium to terminate the digestion. Transfer the cell suspension to a centrifuge tube, balance it and centrifuge it at 1000rpm / min for 4min. Discard the supernatant, add an appropriate amount of complete culture medium, blow it evenly with a pipette, transfer it to a cell culture bottle, and place it in an incubator at 37℃ and 5% CO2 for culture. Subculture every 2-3 days. Collect the MSC supernatant before subculture, divide it into centrifuge tubes, and store it at -80℃.

[0035] 1-2 days after primary cell recovery, hUMSCs (human umbilical cord mesenchymal stem cells) have a spindle-shaped or fusiform morphology and can grow into colonies locally ( Figure 1 Middle A). Around 3 days, the cells formed sheets and 80% of them were fused, growing in a spiral shape ( Figure 1 After subculturing, the cells showed a uniform long spindle shape, similar to fibroblasts, and grew in a typical spiral arrangement ( Figure 1C, D). There was no obvious change in cell morphology during proliferation and passaging.

[0036] 2. Extraction, purification and identification of hUMSC-Exo

[0037] The collected hUMSC culture supernatant samples were transferred from -80℃ to 4℃ for thawing, centrifuged at 4℃, 3000g, 10min, and the precipitate was discarded to remove the cell debris in the supernatant. The supernatant and Exosome Concentration Solution (ECS reagent) were mixed in a ratio of 5:1, vortexed and mixed for 1min, and then placed at 4℃ for 16h to capture exosome particles. Then centrifuged at 4℃, 10000g, 60min, discarded the supernatant, and resuspended in PBS. Centrifuged again at 4℃, 12000g, 2min, and the supernatant was retained. The supernatant (exosome crude particles) was transferred to the upper chamber of the purification column Exosome Purification Filter (EPF column) and centrifuged at 4℃, 3000g, 10min. After centrifugation, the liquid filtered to the bottom of the EPF column was collected, which was the purified exosome particles. After being divided into 1.5ml centrifuge tubes, it was stored at -80℃ for subsequent experiments.

[0038] TEM (transmission electron microscopy to observe the morphology and size of exosomes); NTA (nanoparticle tracking analyzer (NanoSight NS500, Zetaview, UK) to detect the size and concentration of exosomes); and Western blot (to detect exosome marker proteins CD9, CD63, CD81, TSG101) were performed.

[0039] The results of identification after separation and purification of exosomes showed that transmission electron microscopy observed the typical round-shaped morphology of exosomes with a concave center ( Figure 2(A). After exosomes are formed and released, they will form their own unique marker proteins on the outer membrane. The exosome membrane is rich in the tetraspanin family (CD63, CD81 and CD9) involved in exosome transport, the heat shock protein family (HSP60, HSP70, HSPA5, CCT2 and HSP90) and some cell-specific proteins. Among them, CD63, CD9, CD81, TSG101, HSP70, ALIX, etc. are the most commonly used extracellular vesicle markers. These marker proteins are actually mainly involved in the formation and secretion of vesicles. They are produced in cells and located near the intracellular membrane structure. For example, tetraspanins (CD9, CD63 and CD81) are directly involved in the sorting of extracellular vesicle contents; TSG101 is a protein related to the ESCRT complex, and the ESCRT complex is a driver of membrane formation and rupture. ALIX is directly involved in the process of cutting and detaching the plasma membrane to form an independent membrane structure during the formation of membrane vesicles. WB experiments showed that exosomes can express specific proteins CD63, CD81 and TSG101 ( Figure 2 (B). Nanoparticle tracking analysis revealed that the exosome particle size was concentrated between 80-100 nm ( Figure 2 Middle C). The characterization results were consistent with the expected exosome characteristics.

[0040] 3. Preparation of drug-loaded exosomes and determination of drug loading efficiency

[0041] The study used ultrasonic treatment for drug loading, exosomes and BAK were mixed in different proportions, and the drug loading rate was detected by HPLC. The exosome and BAK mixture was treated with drug loading using an ultrasonic cell disruptor. The ultrasonic parameters were set as follows: 20% amplitude, 30s on / off, 6 cycles in total, and 2min cooling time between each cycle. After ultrasonic treatment, the mixed solution was incubated at 37°C for 1h to restore the exosome membrane structure. The free drug was separated using an ultrafiltration tube at 5000×g for 10min.

[0042] High performance liquid chromatography (HPLC) was used with methanol-water (90:10) as the mobile phase, Agilent HC-C18 (250×4.6mm, 5μm) as the chromatographic column, flow rate of 1.0ml / min, column temperature of 30℃, detection wavelength of 254nm, and injection volume of 10μl.

[0043] In the process of preparing drug-loaded exosomes, the concentration of bakuchiol loaded by exosomes was determined by HPLC. According to the chromatographic conditions of 3.2.2.1, linear regression was performed with the concentration of bakuchiol as the horizontal axis and the peak area as the vertical axis. Figure 3A, B, and C are representative chromatograms, with retention times of 8.31 min, 8.11 min, and 8.12 min, respectively. The results showed that bakuchiol had a good linear relationship in the range of 12.5-1000 μg / mL, and the standard curve was y=32943x+253914, R2=0.9997 ( Figure 3 In D), the minimum detection limit of bakuchiol was 12.5 μg / mL.

[0044] High performance liquid chromatography showed that ( Figure 3 ), as the drug concentration increases, the drug loading of exosomes gradually increases, and the encapsulation rate generally shows a downward trend. As the drug loading capacity of exosomes gradually saturates, its encapsulation efficiency is limited. When the mass ratio of bakuchiol to exosomes increases from 0.5:1 to 20:1, the encapsulation rate generally shows a downward trend, and the drug loading concentration shows an increasing trend. After the mass ratio of bakuchiol to exosomes increases from 1:1 to 5:1, the concentration of bakuchiol increases significantly; when the mass ratio increases from 5:1 to 20:1, the drug loading concentration increases, but the encapsulation rate gradually decreases; when the mass ratio of bakuchiol to exosomes is 1:1, the encapsulation rate is the highest, about 24.20%, but its drug loading concentration is the lowest; when the mass ratio of bakuchiol to exosomes is 20:1, the drug loading concentration is optimal, which can reach 1.27mg / ml. Finally, considering the drug loading capacity and encapsulation efficiency, the sample with an encapsulation efficiency of 16.02% when the mass ratio of bakuchiol to exosomes was 5:1 was selected for subsequent experiments.

[0045] 4. Identification of drug-loaded exosomes

[0046] TEM observation, NTA identification, and Western blot detection were performed in the same manner as hUMSC-Exo identification.

[0047] BAK-EXO has a typical concave round shape of exosomes under TEM. NTA results show that the particle size of the analyzed particles is mostly concentrated in the range of 30-200nm, which is consistent with the expected characteristics of exosomes. WB experiments detected exosome markers CD9 and CD81 in the samples, which are consistent with the characteristics of exosomes (see Figure 4 ).

[0048] The cell scratch test is a simple and effective method to measure cell migration and repair ability, and can effectively simulate the wound healing process in vitro. BAK-EXO, BAK, and EXO at a concentration of 20 μg / mL were used to act on HSF cells to observe their effects on the proliferation and migration of HSF in the transverse plane to evaluate the ability of BAK-EXO to promote wound healing. The results showed that ( Figure 5), the cell migration ability of BAK-EXO group, BAK group and EXO group at 12h and 24h were significantly different from those of NC group. Among them, BAK-EXO group had the most significant effect in enhancing the migration ability of HUVEC cells, and BAK and EXO had similar effects in promoting migration.

[0049] In the angiogenesis experiment, human umbilical vein endothelial cells formed a vascular network on the matrix gel. The lumen formation reflects the early process of capillary formation and is also an indicator for in vitro examination of the integrity of endothelial cell function. Figure 6 The results showed that after BAK-EXO treatment, the total number of lumens and vascular coverage area of ​​HUVEC cells increased significantly compared with the control group, with significant differences (p<0.05), indicating that drug-loaded exosomes effectively promoted HUVEC angiogenesis. Under a fluorescent inverted microscope, the formation of small tubes in each group ( Figure 7 ). Compared with the EXO group and the BAK group, more tubule structures were observed in HUVEC treated with BAK-EXO solution, and the length of the tubules was significantly higher than that in the NC group (p<0.01), indicating that BAK-EXO treatment enhanced the tube formation ability of endothelial cells. Exosomes and drug-loaded exosomes had a better effect on improving angiogenesis.

[0050] ELISA was used to detect the secretion of related factors by BAK-EXO in HUVEC and HSF cultured in vitro in promoting wound healing experiments. Figure 8 The results showed that compared with the NC group, the expressions of PEDF, IL-1β, IL-8, TNF-α, MMP-9, and PG-E2 in the BAK-EXO group, EXO group, and BAK group were significantly reduced, and the expressions of VEGF, COL-Ⅰ, and COL-Ⅲ were significantly increased, and the effect in the BAK-EXO group was more significant. This shows that BAK-EXO has the effect of inhibiting proinflammatory factors and promoting angiogenic factors, promoting the proliferation and migration of HSF and HUVEC, improving the angiogenesis ability of HUVEC, the degree of wound healing, the inhibitory effect of inflammation, and the amount of angiogenesis, and has a certain promoting effect on wound healing.

Claims

1. A method for preparing exosomes loaded with bakuchiol, characterized in that: The steps include: S1: Mix the collected supernatant of the hUMSC culture supernatant sample with the Exosome Concentration Solution, vortex and mix evenly, and then place at 4°C to capture exosome particles; S2 is then centrifuged to discard the supernatant, and the precipitate is resuspended and centrifuged again to retain the supernatant, which is the crude exosome particles, and transferred to a purification column for purification to obtain purified exosome particles; S3 mixes the exosome particles obtained in S2 with bakuchiol BAK and performs drug loading treatment using an ultrasonic cell disruptor.

2. The preparation method according to claim 1, characterized in that The specific operation of the hUMSC culture supernatant sample collected in S1 is as follows: transfer from -80 °C to 4 °C for thawing, centrifuge at 4 °C, 3000 g, 10 min, discard the precipitate to remove the cell debris in the supernatant, and take the supernatant for subsequent steps.

3. The preparation method according to claim 1, characterized in that: The supernatant and Exosome Concentration Solution (ECS reagent) were mixed in a ratio of 4-6:1, vortexed and mixed for 1 min, and then placed at 4 °C for 16 h to capture exosome particles; then centrifuged at 4 °C, 10,000 g, 60 min, discarded the supernatant, and resuspended in PBS. Centrifuged again at 4 °C, 12,000 g, 2 min, and the supernatant was retained to obtain the crude exosome particles.

4. The preparation method according to claim 1, characterized in that: The purification in S2 is to transfer the crude exosome particles to the upper chamber of the purification column Exosome Purification Filter, centrifuge at 4 °C, 3000 g, 10 min; after centrifugation, collect the liquid filtered to the bottom of the EPF column, which is the purified exosome particles; optionally, divide it into 1.5 ml centrifuge tubes and store it at -80 °C for subsequent experiments.

5. The preparation method according to claim 4, characterized in that: The exosome particles obtained by S2 can express specific proteins CD63, CD81 and TSG101, and the particle size is concentrated in the range of 80-100nm.

6. The preparation method according to claim 1, characterized in that: The ultrasonic parameters in step S3 were set as follows: 20% amplitude, 30 s on / off, 6 cycles in total, and a 2 min cooling time between each cycle.

7. The preparation method according to claim 1, characterized in that: The following steps are also included: After S4 ultrasonic treatment, the mixed solution was incubated at 37 °C for 1 h to restore the exosome membrane structure; the free drug was separated using an ultrafiltration tube and centrifuged at 5000×g for 10 min.

8. The preparation method according to claim 7, characterized in that: The mass ratio of bakuchiol to exosomes is 1:1 to 20:1, preferably 1:1 to 5:1; most preferably, the mass ratio of bakuchiol to exosomes is 5:

1.

9. Exosomes loaded with bakuchiol obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the exosomes according to claim 9 in the preparation of a drug for regulating cell proliferation and migration ability and / or promoting wound healing; specifically, the exosomes are prepared into an agent suitable for subcutaneous injection near a wound.

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