CN112353815B - Micro-nano fiber membrane with extracellular vesicle slow-release function and preparation method and application thereof - Google Patents
Micro-nano fiber membrane with extracellular vesicle slow-release function and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a micro-nano fiber membrane with an extracellular vesicle slow-release function, and a preparation method and application thereof. The fiber membrane comprises a micro-nano fiber membrane, PEGylated phospholipid and extracellular vesicles; the micro-nano fiber membrane is connected with one end of the PEGylated phospholipid, and the extracellular vesicles are connected with the other end of the PEGylated phospholipid. The method comprises the following steps: and (3) carrying out surface modification on the micro-nano fiber membrane, soaking the micro-nano fiber membrane in a buffer solution containing PEGylated phospholipid, carrying out grafting reaction, and soaking the micro-nano fiber membrane in the buffer solution containing extracellular vesicles to obtain the micro-nano fiber membrane with the extracellular vesicles slow release function. The preparation method is simple, the reaction conditions are mild, the prepared micro-nano fiber membrane not only has good biocompatibility, but also can release extracellular vesicles rich in various bioactive factors to the wound surface, is beneficial to comprehensively improving pathological microenvironment of the wound surface, activates cells to participate in repair, and promotes the repair of the wound surface which is difficult to heal chronically. Is expected to be applied to the field of wound repair.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a micro-nano fiber membrane with an extracellular vesicle slow-release function, and a preparation method and application thereof.
Background
Wound healing is a complex process that can be roughly divided into 4 distinct but overlapping phases: clotting phase, inflammatory phase, proliferative phase, remodelling phase. However, many pathological factors including bacterial infection and foreign body reaction in clinic lead to the destruction of the normal physiological repair process of the wound surface, thus forming a wound surface which is difficult to heal. The root cause of the chronic difficult-to-heal wound repair difficulty is that the local physiological environment of the wound is seriously unbalanced, so that the clinical requirement cannot be met by delivering (loading) a single bioactive factor, thereby comprehensively improving the pathological microenvironment of the wound, activating cells to participate in repair and recovering the wound repair process, being the key for realizing the difficult-to-heal wound repair and also being the research and development trend of a new generation of wound repair material.
Extracellular vesicles include exosomes (exosomes), microvesicles (microvesicles) and apoptotic bodies (apoptoticbodies), containing large amounts of biologically active substances such as proteins, (growth factors, cytokines etc.), DNA, RNA and lipids etc., affecting target cell behaviour mainly by delivering self-carried contents (cargo), and in addition may also function by membrane surface molecules interacting with target cell surface receptors. Compared with stem cell therapy, the method has the advantages of smaller immunogenicity, more stable physicochemical properties and the like, and has great potential in the field of chronic refractory wound repair.
Due to the increasing demand for nanotechnology, electrospinning technology has received great attention. The electrostatic spinning nanofiber has a high specific surface area and high porosity because of the bionic natural extracellular matrix nanofiber structure, and has a huge prospect in the field of wound repair.
The extracellular vesicles are loaded on the micro-nano fiber repair material, so that bioactive factors can be delivered to the defect of the tissue through the extracellular vesicles, the purpose of comprehensively improving the pathological microenvironment of the defect and activating body cells to participate in repair of the defect is achieved (MSC-derived sEVs enhance patency and inhibit calcification of syntheticvascular grafts by immunomodulation in a rat model of hyperlipidemia). However, most of the existing materials load extracellular vesicles in a direct physical adsorption mode, and the materials and the extracellular vesicles lack effective interaction, so that the problems of low load, difficult enrichment and stopping exist.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a micro-nano fiber membrane with an extracellular vesicle slow-release function, and a preparation method and application thereof.
The invention provides a preparation method of a micro-nanofiber-based wound repair material with an extracellular vesicle slow-release function. According to the invention, the PEG phospholipid is grafted on the surface of the micro-nano fiber membrane, so that the extracellular vesicles are effectively loaded, and the slow release of the extracellular vesicles on the wound surface is realized.
The object of the invention is achieved by at least one of the following technical solutions.
The invention provides a micro-nanofiber membrane with an extracellular vesicle slow-release function (micro-nanofiber-based chronic refractory wound repair material with a slow-release function), which comprises a micro-nanofiber membrane (a micro-nanofiber membrane obtained by an electrostatic spinning technology), PEGylated phospholipid and extracellular vesicles; the micro-nanofiber membrane is attached to a pegylated phospholipid, which is attached (loaded) to an extracellular vesicle. And grafting PEG phospholipid with a long-chain structure on the surface of the micro-nano fiber membrane, wherein the PEG phospholipid can realize the loading of extracellular vesicles.
Further, the micro-nano fiber membrane is an electrospun polymer; the micro-nano fiber membrane is made of more than one of L-polylactic acid (PLLA), polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL) and the like; the diameter of the micro-nano fiber membrane is 400-2000nm.
Further, the PEGylated phospholipid is a hydrophobic phospholipid connected by a PEG chain; the chain length of the PEG chain is 500-5000; the PEGylated phospholipid is connected with a group on the surface of the micro-nanofiber membrane; the groups on the surface of the micro-nano fiber membrane are more than one of carboxyl, amino, hydroxyl and the like. The PEGylated phospholipid is connected with groups (such as carboxyl, amino, hydroxyl and the like) capable of reacting with the groups on the surface of the micro-nanofiber membrane through a PEG chain.
Further, the pegylated phospholipids are capable of intercalating into lipid bilayers of extracellular vesicle membranes via hydrophobic interactions.
Further, the extracellular vesicles are one or more of exosomes (exosomes), microvesicles (microvesicles) and apoptotic bodies (apoptotics); the extracellular vesicles have a diameter of 50-1000nm.
The invention provides a method for preparing the micro-nano fiber membrane with the extracellular vesicle slow-release function, which comprises the following steps:
(1) Preparing micro-nano fibers by an electrostatic spinning method, and weaving the micro-nano fibers into a micro-nano fiber film;
(2) Carrying out surface modification treatment on the micro-nanofiber membrane in the step (1) by using plasma treatment, wet chemical method and other technologies, so that groups (such as amino, carboxyl, hydroxyl and the like) capable of reacting with PEGylated phospholipid are generated on the surface of the micro-nanofiber membrane, and the groups are activated to obtain a modified micro-nanofiber membrane;
(3) Soaking the modified micro-nano fiber membrane in the step (2) in a buffer solution containing PEGylated phospholipid, performing grafting reaction in a shaking table, and taking out to obtain the micro-nano fiber membrane with the surface grafted with the PEGylated phospholipid;
(4) And (3) immersing the micro-nano fiber membrane grafted with the PEG phospholipid on the surface in the step (3) in a buffer solution containing extracellular vesicles to obtain the micro-nano fiber membrane with the extracellular vesicles sustained release function (the micro-nano fiber membrane loaded with the extracellular vesicles and having the extracellular vesicles sustained release function).
Further, the surface modification treatment method in the step (2) is one or more of plasma treatment and wet chemical method.
Further, in the buffer solution containing the PEGylated phospholipid in the step (3), the concentration of the PEGylated phospholipid is 2-10mg/mL, the pH value of the buffer solution containing the PEGylated phospholipid is 5.0-6.0, the time of the grafting reaction is more than or equal to 12 hours, and the rotating speed of a shaking table is 10-60rpm.
Preferably, the grafting reaction in step (3) takes from 12 to 24 hours.
Further, in the buffer solution containing the extracellular vesicles in the step (4), the concentration of the extracellular vesicles is 5-15 mug/ml, the pH value of the buffer solution containing the extracellular vesicles is 6.8-7.6, the temperature of the buffer solution containing the extracellular vesicles is 4-37 ℃, and the time of soaking the micro-nano fiber membrane grafted with the PEGylated phospholipid on the surface in the buffer solution containing the extracellular vesicles is more than or equal to 4 hours.
Preferably, the temperature of the buffer containing extracellular vesicles in step (4) is 4 ℃.
Preferably, the micro-nanofiber membrane grafted with PEGylated phospholipid on the surface in the step (4) is soaked in the buffer solution containing extracellular vesicles for 4-16 hours.
The invention provides an application of a micro-nano fiber membrane with an extracellular vesicle slow-release function in preparing a medicine or a carrier material of a bioactive substance for repairing a chronic wound surface.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The micro-nanofiber-based wound repair material prepared by the invention can effectively load extracellular vesicles, realize slow release of the extracellular vesicles, improve pathological microenvironment of wound and promote chronic difficult-to-heal wound repair;
(2) The preparation method of the micro-nanofiber-based wound repair material provided by the invention is simple to operate and mild in condition.
Drawings
FIG. 1a is a scanning electron microscope image of extracellular vesicles in example 1.
FIG. 1b is a graph showing the particle size distribution of extracellular vesicles in example 1.
FIG. 1c is a graph showing the results of the electrophoresis identification of extracellular vesicles in example 1.
Fig. 2 is a schematic diagram of a micro-nanofiber-based chronic refractory wound repair material with an extracellular vesicle slow-release function prepared in example 2.
FIG. 3 is a scanning electron microscope image of the micro-nanofiber membrane before and after the treatment in example 2.
FIG. 4 is an infrared plot of the micro-nanofiber membrane before and after treatment in example 2.
FIG. 5 is a graph showing the release rate of extracellular vesicles on a micro-nanofiber membrane in example 2.
FIG. 6 is a graph showing migration effect of fibroblasts in example 2.
FIGS. 7a and 7b are graphs showing proliferation results of fibroblasts and keratinocytes, respectively, in example 2.
FIGS. 8a, 8b and 8c are graphs showing the results of expression of the genes involved in wound repair of fibroblasts in example 2.
Fig. 9a, 9b, 9c and 9d are graphs showing polarization results of macrophages in example 2.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but are not intended to limit the practice and protection of the invention. It should be noted that the following processes, if not specifically described in detail, can be realized or understood by those skilled in the art with reference to the prior art. The reagents or apparatus used were not manufacturer-specific and were considered conventional products commercially available.
The following examples take rat-derived adipose mesenchymal stem cell-derived extracellular vesicles as an example, and extracellular vesicles were obtained and identified, but are not limited to adipose mesenchymal stem cells.
Example 1: preparation and identification of rat-derived adipose mesenchymal stem cell-derived extracellular vesicles
(1) The supernatant of rat adipose-derived mesenchymal stem cells was collected.
The proliferation medium of rat adipose-derived mesenchymal stem cells consisted of 10wt% fetal bovine serum, 1wt% diabody and alpha-MEM. The density of the plate is 20,000cells/cm 2 Algebraic p3-p5 are used. For the extraction of extracellular vesicles, the present invention was carried out by culturing with a medium containing no extracellular vesicles when the cell confluency reached 80%, the composition was 10wt% of fetal bovine serum containing no extracellular vesicles, 1wt% of diabody and 1wt% of alpha-MEM,cell culture medium was collected after two days of culture.
(2) Extraction of extracellular vesicles of rat adipose-derived mesenchymal stem cells.
Extracellular vesicles of rat adipose-derived mesenchymal stem cells are extracted by a series of gradient centrifugation at 4 ℃. (1) 300 Xg, 10 minutes, cells and debris were removed; (2) 2000 Xg, 10 minutes, dead cells and debris were removed; (3) 10000 Xg, 30 minutes, cell debris is removed; (4) 100000 Xg, 70 min, precipitation (extracellular vesicles and protein components) was seen; (5) discarding the supernatant, retaining the precipitate, and adding PBS buffer (the concentration is 0.01mol/L, and the concentration of the PBS buffer is 0.01mol/L below); (6) 100000 Xg, 70 minutes, the sediment was seen as a purer concentration of extracellular vesicles.
(3) Characterization of extracellular vesicles of rat adipose mesenchymal stem cells.
Three characterization methods were used in combination for extracellular vesicle characterization. Wherein Transmission Electron Microscopy (TEM) is used for extracellular vesicle morphology characterization; western blot (Western blot) was used for extracellular vesicle marker identification; nanoparticle Tracking (NTA) was used for particle size analysis.
(1) Particle size analysis: as shown in FIG. 1b, the particle size of the extracted material is in the range of 50-200 nm.
(2) Morphological characterization: as shown in fig. 1a, the extracted material has a saucer-like structure.
(3) Identification of the marker: as shown in FIG. 1c, the extracted material was positive for the vesicle membrane markers CD 9, CD 63, TSG 101 expression.
The extracted substance is determined to have an extracellular vesicle structure according to three experimental results of particle size distribution, morphological characterization and marker identification, and can be modified by hydrophobic insertion.
Example 2: preparation and characterization of PLLA micro-nanofiber membranes.
Referring to FIG. 2, the following example was performed by electrospinning L-polylactic acid (PLLA) and modifying DSPE-PEG-NH on PLLA micro-nanofiber membrane 2 The PEGylated phospholipids are used for example to modify extracellular vesicles, but not only for PLLA and DSPE-PEG-NH 2 。
DSPE-PEG-NH 2 The structural formula of (2) is as follows:
(1) And (3) preparing the micro-nano fiber membrane.
(1) Preparing a micro-nano fiber film: dissolving the L-polylactic acid (PLLA) in a solvent, spinning by electrostatic spinning equipment to obtain a PLLA micro-nano fiber membrane, and drying in vacuum for 48 hours to remove the residual organic solvent.
(2) Exposing carboxyl groups on the surface of the micro-nanofiber membrane: and placing the PLLA micro-nanofiber membrane prepared by the method on a shaking table, soaking the PLLA micro-nanofiber membrane in a NaOH solution to expose carboxyl groups on the surface of the micro-nanofiber membrane, and washing the PLLA micro-nanofiber membrane with ultrapure water for three times to obtain the micro-nanofiber membrane with exposed carboxyl groups.
(3) Activating carboxyl on the surface of the micro-nano fiber film: and placing the prepared micro-nanofiber membrane with exposed carboxyl on a shaking table, soaking in MES buffer solution (pH is 6.0) of EDC and NHS, activating the carboxyl on the surface of the micro-nanofiber membrane, and washing with ultrapure water for three times to obtain the micro-nanofiber membrane with activated carboxyl.
④DSPE-PEG-NH 2 Grafting: placing the prepared carboxyl activated micro-nanofiber membrane on a shaking table, and soaking in DSPE-PEG-NH 2 In MES buffer solution of (B), washing with ultrapure water for three times, sterilizing with alcohol and ultraviolet to obtain grafted DSPE-PEG-NH 2 Is a micro-nanofiber membrane.
(5) Extracellular vesicle-loaded: the obtained DSPE-PEG-NH is grafted 2 The micro-nano fiber membrane is soaked in PBS buffer solution containing sterile extracellular vesicles, and is rinsed twice by the PBS buffer solution to obtain the micro-nano fiber membrane with a slow release function.
The solvent used in the step (1) is hexafluoroisopropanol, and the mass volume ratio of the solvent to PLLA is 10:1g/mL.
The process parameters used in the step (1) are as follows: the temperature is 25 ℃, the relative humidity is 60%, the flow rate is 0.6mL/h, the needle head is vertical to the ground, the distance is 15cm, the spinning voltage is 12kV, the stainless steel roller is wrapped by tin paper to receive the nanofiber, the rotating speed of the roller is 1.5 revolutions per minute, and the spinning time is 15-20 minutes.
The concentration of NaOH solution in the step (2) is 0.1M, the soaking time is 15min, the temperature is room temperature, and the rotating speed of the shaking table is 50rpm.
The EDC concentration in the step (3) is 0.5mg/ml, the NHS concentration is 0.25mg/ml, the soaking time is 20min, the temperature is room temperature, and the rotation speed of the shaking table is 50rpm.
DSPE-PEG-NH in step (4) 2 The concentration was 10mg/ml, the soaking time was 16h, the temperature was room temperature, and the shaking table rotation speed was 50rpm.
The concentration of extracellular vesicles in step (5) was 10. Mu.g/ml, the soaking time was 6h, and the temperature was 4 ℃.
(2) Characterization of micro-nanofiber membranes.
The scanning electron microscope observes the DSPE-PEG-NH 2 Morphology features of modified PLLA micro-nanofiber membranes and unmodified PLLA micro-nanofiber membranes, infrared appearance was subjected to DSPE-PEG-NH 2 Chemical bond of modified PLLA micro-nanofiber membrane with unmodified PLLA micro-nanofiber membrane.
(1) Morphology observation: will go through DSPE-PEG-NH 2 Modified PLLA fiber membranes (PLLA-DSPE in FIG. 3) and unmodified PLLA fiber membranes (PLLA in FIG. 3) were attached to a sample stage with a conductive adhesive, and taken out after metal spraying for 60s, and photographed by an on-machine observation. The results are shown in FIG. 3. The fiber membrane diameter before modification is 867.7 +/-60 nm, the fiber diameter after modification is 734.8 +/-80 nm, and no obvious difference exists; and the globular grafts can be seen on the fiber membrane after modification.
(2) Chemical bond change: to further confirm grafting, the mixture was subjected to a 0.1M NaOH shaker at 50rpm for 15min, 0.5mol/LEDC and 0.25mol/LNHS shaker at 50rpm for 20min, 10g/ml DSPE-PEG-NH 2 The shaking table 50rpm was used to treat the PLLA nanofiber membrane for 16 hours and the untreated PLLA nanofiber membrane was placed on an infrared spectrometer for detection. As a result, as shown in FIG. 4, it can be seen that the DSPE-PEG-NH was passed only 2 The treated fiber film has characteristic peaks of amide bonds. PLLA in fig. 4 represents an untreated PLLA nanofiber membrane, naOH represents a NaOH-treated PLLA nanofiber membrane, edc\nhs represents an edc\nhs-treated PLLA nanofiber membrane,PLLA-DSPE means passing DSPE-PEG-NH 2 Treated PLLA nanofiber membranes.
Labelling the extracted extracellular vesicles with PKH 26 dye, loading the stained extracellular vesicles on DSPE-PEG-NH 2 The modified PLLA micro-nanofiber membrane and the surface of the PLLA micro-nanofiber membrane which is not modified are rinsed twice with PBS buffer after 6 hours, 200 microliter of PBS buffer is added, 200 microliter of PBS buffer is taken out at the detection time point at 37 ℃ to detect fluorescence value, and new 200 microliter of PBS buffer is added. The detection result is subjected to image J treatment as shown in FIG. 5, and it can be seen that DSPE-PEG-NH is subjected to 2 The release rate of the modified PLLA micro-nanofiber membrane extracellular vesicles is significantly slowed. PLLA-EXO in FIG. 5 represents PLLA micro-nanofiber membranes carrying stained extracellular vesicles; PLLA-DSPE-EXO represents stained extracellular vesicles loaded and passed through DSPE-PEG-NH 2 Modified PLLA micro-nanofiber membranes.
Example 3: application of PLLA micro-nano fiber membrane
The prepared micro-nano fiber membrane is cultured into fibroblasts, and the fibroblasts are taken out and observed at 24h and 48h, and the result is shown in FIG. 6, and the migration condition of the fibroblasts can be seen through DSPE-PEG-NH 2 The modified micro-nanofiber membrane can promote migration of fibroblasts. PLLA-DSPE-EXO in FIG. 6 represents extracellular vesicles loaded and passed through DSPE-PEG-NH 2 A modified PLLA micro-nanofiber membrane; PLLA means neither extracellular vesicles were loaded nor passed through DSPE-PEG-NH 2 A modified PLLA micro-nanofiber membrane; PLLA-DSPE indicated no extracellular vesicles loaded but with DSPE-PEG-NH passed 2 Modified PLLA micro-nanofiber membranes.
Culturing fibroblast and keratinocyte with the prepared micro-nanofiber membrane, taking out and observing proliferation of cells on day 1, day 3 and day 7, and observing the results as shown in FIG. 7a and FIG. 7b 2 The modified micro-nanofiber membrane can promote proliferation of fibroblasts and keratinocytes. FIG. 7a and FIG. 7b show PLLA with neither extracellular vesicles loaded nor DSPE-PEG-NH passed 2 A modified PLLA micro-nanofiber membrane; PLLA-DSPE-EXO represents extracellular vesicles loaded and passed through DSPE-PEG-NH 2 A modified PLLA micro-nanofiber membrane; PLLA-DSPE indicated no extracellular vesicles loaded but with DSPE-PEG-NH passed 2 Modified PLLA micro-nanofiber membranes.
Culturing fibroblasts and macrophages with the prepared micro-nano fiber membrane, taking out the fibroblasts on the 1 st day and the 7 th day, taking out the macrophages on the 2 nd day, and observing the gene expression condition of the cells, wherein the results are shown in FIG. 8a, FIG. 8b, FIG. 8c, FIG. 9a, FIG. 9b, FIG. 9c and FIG. 9d, and can be seen that DSPE-PEG-NH is passed 2 The modified micro-nano fiber membrane can promote the parallel expression of genes related to the wound repair of the fibroblast; promote the expression of macrophage anti-inflammatory gene and inhibit the expression of cell pro-inflammatory gene. The PLLA in FIGS. 8a, 8b, 8c, 9a, 9b, 9c and 9d represent PLLA micro-nanofiber membranes that were neither loaded with extracellular vesicles nor modified with DSPE-PEG-NH2, and PLLA-DSPE represent membranes that were not loaded with extracellular vesicles but were modified with DSPE-PEG-NH 2 Modified PLLA micro-nanofiber membrane, PLLA-DSPE-EXO represents extracellular vesicle loaded and passes through DSPE-PEG-NH 2 Modified PLLA micro-nanofiber membranes.
The above examples are only preferred embodiments of the present invention, and are merely for illustrating the present invention, not for limiting the present invention, and those skilled in the art should not be able to make any changes, substitutions, modifications and the like without departing from the spirit of the present invention.
Claims (5)
1. The application of the micro-nano fiber membrane with the extracellular vesicle slow-release function in preparing a medicine for repairing a chronic wound surface is characterized in that the micro-nano fiber membrane with the extracellular vesicle slow-release function comprises a micro-nano fiber membrane, PEGylated phospholipid and extracellular vesicles; the micro-nano fiber membrane is connected with one end of the PEGylated phospholipid, and the extracellular vesicles are connected with the other end of the PEGylated phospholipid; the micro-nano fiber membrane is an electrospun polymer; the micro-nano fiber membrane is made of L-polylactic acid; the PEGylated phospholipid is a hydrophobic phospholipid connected with a PEG chain; the PEGylated phospholipid is connected with a group on the surface of the micro-nanofiber membrane; the PEGylated phospholipids are capable of intercalating into fines by hydrophobic interactionsIn the lipid bilayer of the extracellular vesicle membrane; the diameter of the micro-nano fiber membrane is 400-2000nm; the chain length of the PEG chain is 500-5000; the group on the surface of the micro-nano fiber membrane is carboxyl; the PEGylated phospholipid is DSPE-PEG-NH 2 。
2. The application of the micro-nanofiber membrane with the extracellular vesicle slow-release function in preparing a medicine for chronic wound repair according to claim 1, wherein the preparation method of the micro-nanofiber membrane with the extracellular vesicle slow-release function comprises the following steps:
(1) Preparing micro-nano fibers by an electrostatic spinning method, and weaving the micro-nano fibers into a micro-nano fiber film;
(2) Carrying out surface modification treatment on the micro-nanofiber membrane in the step (1) to enable groups capable of reacting with PEG phospholipid to be generated on the surface of the micro-nanofiber membrane, and activating the groups to obtain the modified micro-nanofiber membrane;
(3) Soaking the modified micro-nano fiber membrane in the step (2) in a buffer solution containing PEGylated phospholipid, performing grafting reaction in a shaking table, and taking out to obtain the micro-nano fiber membrane with the surface grafted with the PEGylated phospholipid;
(4) And (3) immersing the micro-nano fiber membrane grafted with the PEGylated phospholipid on the surface in the step (3) in a buffer solution containing extracellular vesicles to obtain the micro-nano fiber membrane with the extracellular vesicles slow release function.
3. The application of the micro-nano fiber membrane with the extracellular vesicle slow-release function in preparing a medicine for repairing a chronic wound surface according to claim 2, wherein in the preparation method step (2), the surface modification treatment method is one of a plasma treatment method and a wet chemical method.
4. The application of the micro-nano fiber membrane with the extracellular vesicle slow-release function in preparing a medicine for repairing chronic wounds, according to claim 2, wherein in the preparation method step (3), the concentration of PEGylated phospholipid in the buffer solution containing PEGylated phospholipid is 2-10mg/mL, the pH value of the buffer solution containing PEGylated phospholipid is 5-6, the grafting reaction time is more than or equal to 12 hours, and the rotating speed of a shaking table is 10-60rpm.
5. The use of a micro-nanofiber membrane with an extracellular vesicle slow release function in preparation of a medicine for chronic wound repair according to claim 2, wherein in the preparation method step (4), the concentration of extracellular vesicles in the buffer solution containing extracellular vesicles is 5-15 mug/ml, the pH value of the buffer solution containing extracellular vesicles is 6.8-7.6, the temperature of the buffer solution containing extracellular vesicles is 4-37 ℃, and the time of soaking the micro-nanofiber membrane grafted with PEGylated phospholipids on the surface of the buffer solution containing extracellular vesicles is more than or equal to 4 hours.
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