Wound dressing, preparation method and application thereof, and preparation method of exosome-like nanoparticle solution

Abstract

The invention relates to the technical field of wound dressings, in particular to a wound dressing, a preparation method and application thereof and a preparation method of an exosome-like nanoparticle solution. The invention provides a wound dressing. The wound dressing comprises exosome-like nanoparticles and recombinant mussel mucoprotein hydrogel wrapping the exosome-like nanoparticles, the recombinant mussel mucoprotein hydrogel has a three-dimensional network structure. According to the wound dressing, the local residence time of the exosome can be remarkably prolonged, rapid removal of the exosome in the body is effectively slowed down, and therefore the stability and the local using effect of the exosome are enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of wound dressings, and in particular to a wound dressing, a preparation method and application thereof, and a preparation method of an exosome-like nanoparticle solution. Background Art

[0002] Exosomes are extracellular vesicles secreted by cells with a diameter between 30 and 150 nm, and have various potential biomedical effects. Compared with animal exosomes, plant-derived exosomes are slightly larger in volume and have the advantages of low toxicity, low immunogenicity, high cell uptake rate, good biocompatibility and high stability. In recent years, researchers have extracted corresponding exosomes from plants such as fruits (such as apples, grapefruits), vegetables (such as carrots, broccoli), and Chinese herbal medicines (such as ginseng, turmeric). Exosomes from different sources have biological functions basically consistent with their source plants and can be used as natural therapeutic agents with biological activities such as anti-inflammatory, anti-tumor and anti-aging. Portulaca oleracea (PO) has been used to treat skin wounds very early in human history. Crushing it and applying it to the wound surface can quickly heal the wound. Its main components include polysaccharides, organic acids, esters, alkaloids, flavonoids, etc. These components endow Portulaca oleracea with multiple pharmacological functions, including anti-inflammatory, analgesic, antibacterial, hypoglycemic and lipid-lowering, anti-tumor, antioxidant and immune enhancement. It can be seen that Portulaca oleracea exosomes have broad application prospects in the field of wound healing. However, exosomes have problems such as low stability and difficulty in retention during clinical application. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a wound dressing, a preparation method and application thereof, and a preparation method of an exosome-like nanoparticle solution. The wound dressing of the present invention well solves the problems of low stability and difficulty in retention of exosomes during clinical application.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a wound dressing (PO-EVs@Mefp3), comprising exosome-like nanoparticles (PO-EVs) and a recombinant mussel adhesive protein (Mefp3) hydrogel encapsulating the exosome-like nanoparticles; the recombinant mussel adhesive protein hydrogel has a three-dimensional network structure.

[0006] Preferably, the raw material for preparing the recombinant mussel adhesive protein hydrogel is a recombinant mussel adhesive protein solution;

[0007] The raw material for preparing the exosome-like nanoparticles is an exosome-like nanoparticle solution;

[0008] The concentration of the recombinant mussel adhesive protein solution is 100-200 mg / mL;

[0009] The concentration of the exosome-like nanoparticle solution is 1-4 mg / mL.

[0010] Preferably, the volume ratio of the recombinant mussel adhesive protein solution to the exosome-like nanoparticle solution is (1-5):1.

[0011] Preferably, the preparation method of the exosome-like nanoparticle solution comprises the following steps:

[0012] After juicing purslane, the obtained first supernatant is subjected to stepwise centrifugation to obtain a second supernatant;

[0013] After centrifuging the second supernatant, the obtained precipitate is subjected to sucrose density gradient centrifugation to obtain a preliminary exosome-like filtrate;

[0014] After mixing the preliminary exosome-like filtrate and phosphate buffer solution, solid-liquid separation is carried out to obtain the exosome-like nanoparticle solution.

[0015] Preferably, the stepwise centrifugation includes first centrifugation, second centrifugation, and third centrifugation carried out in sequence;

[0016] The rotation speed of the first centrifugation is 200×g, the time is 10 min, and the temperature is 4°C;

[0017] The rotation speed of the second centrifugation is 2000×g, the time is 20 min, and the temperature is 4°C;

[0018] The rotation speed of the third centrifugation is 10,000×g, the time is 30 min, and the temperature is 4°C.

[0019] Preferably, the rotation speed of the centrifugation is 100,000×g, the time is 60 min, and the temperature is 4°C;

[0020] The mass concentration gradient of the sucrose solution used for the sucrose density gradient centrifugation is 15-45%;

[0021] The rotation speed of the sucrose density gradient centrifugation is 150,000×g, and the time is 60 min.

[0022] Preferably, the preliminary exosome-like filtrate is the supernatant obtained by centrifuging with a sucrose solution having a mass concentration of 30-45%;

[0023] The solid-liquid separation includes centrifugation and filtration carried out in sequence; the rotation speed of the centrifugation is 100,000×g, the time is 60-120 min, and the temperature is 4-8°C; the filtration uses a microporous membrane with a pore size of 0.22 μm.

[0024] The present invention also provides a preparation method of the wound dressing according to the above technical solution, comprising the following steps:

[0025] After mixing the recombinant mussel adhesive protein solution and the exosome-like nanoparticle solution, loading is carried out to obtain the wound dressing.

[0026] The present invention also provides the use of the wound dressing described in the above technical solution or the wound dressing prepared by the preparation method described in the above technical solution in the preparation of a medical material for promoting wound healing.

[0027] The present invention provides a preparation method of an exosome-like nanoparticle solution, comprising the following steps:

[0028] After juicing purslane, the obtained first supernatant is subjected to stepwise centrifugation to obtain a second supernatant;

[0029] After centrifuging the second supernatant, the obtained precipitate is subjected to sucrose density gradient centrifugation to obtain a preliminary exosome-like filtrate;

[0030] After mixing the preliminary exosome-like filtrate and phosphate buffer solution, solid-liquid separation is carried out to obtain the exosome-like nanoparticle solution.

[0031] The present invention also provides a wound dressing, comprising exosome-like nanoparticles and a recombinant mussel adhesive protein hydrogel encapsulating the exosome-like nanoparticles; the recombinant mussel adhesive protein hydrogel has a three-dimensional network structure. The present invention utilizes the self-assembly function of recombinant mussel adhesive protein to encapsulate exosome-like nanoparticles and form a hydrogel material without the need for an additional cross-linking agent, and has good biocompatibility. At the same time, the recombinant mussel adhesive protein also has both interfacial adhesiveness and inherent adhesiveness of a coiled-coil structure, which can ensure that the wound dressing can be stably attached to the skin surface; in addition, after the wound dressing of the present invention contacts the skin wound surface, the wound dressing gradually absorbs water and swells, increasing in volume, and its three-dimensional network structure is beneficial to the outward movement of drugs, enabling the exosome-like nanoparticles loaded in the pore structure to be slowly released, thereby promoting angiogenesis, regulating the inflammatory response, promoting cell regeneration, reducing the burden of oxidative stress response on wound healing, accelerating the wound healing speed, reducing the formation of scar tissue, and after reaching the swelling equilibrium, the recombinant mussel adhesive protein hydrogel gradually degrades;

[0032] The present invention also provides a preparation method of an exosome-like nanoparticle solution, comprising the following steps: Juicing purslane, subjecting the obtained first supernatant to stepwise centrifugation to obtain a second supernatant; after centrifuging the second supernatant, subjecting the obtained precipitate to sucrose density gradient centrifugation to obtain a preliminary exosome-like filtrate; after mixing the preliminary exosome-like filtrate and phosphate buffer solution, solid-liquid separation is carried out to obtain the exosome-like nanoparticle solution. The preparation method of the present invention can enable the prepared exosome-like nanoparticle solution to have a high separation efficiency and purity. Brief Description of the Drawings

[0033] Figure 1 It is a schematic diagram of the preparation process of the exosome-like nanoparticle solution described in the present invention, as well as the TEM image and size distribution diagram of the exosome-like nanoparticles in the exosome-like nanoparticle solution described in Example 1;

[0034] Figure 2 It is the antibacterial test results of PBS buffer, fresh purslane juice, and the lysate of exosome-like nanoparticles in Example 1;

[0035] Figure 3 It is the cell activity and anti-inflammatory experimental results of the exosome-like nanoparticles described in Example 1;

[0036] Figure 4 It is a schematic diagram of the preparation process of the wound dressing described in the present invention, as well as the SEM images of the recombinant mussel adhesive protein and PO-EVs@Mefp3 described in Example 2;

[0037] Figure 5 It is a graph showing the change of the swelling rate of PO-EVs@Mefp3 described in Example 2 over time (a), the in vitro degradation curve (b), and the in vitro release curve of the loaded exosome-like nanoparticles (c);

[0038] Figure 6 It is the experimental results of PO-EVs@Mefp3 described in Example 2 promoting wound healing;

[0039] Figure 7 It is the morphological analysis results of the skin tissues near the wounds of each group of mice in the mouse wound model after sampling on the 14th day. Detailed Description of the Invention

[0040] The present invention provides a wound dressing, which includes exosome-like nanoparticles and a recombinant mussel adhesive protein hydrogel encapsulating the exosome-like nanoparticles; the recombinant mussel adhesive protein hydrogel has a three-dimensional network structure.

[0041] In the present invention, the preparation raw material of the recombinant mussel adhesive protein hydrogel is preferably a recombinant mussel adhesive protein solution; the preparation raw material of the exosome-like nanoparticles is preferably an exosome-like nanoparticle solution.

[0042] In the present invention, the volume ratio of the recombinant mussel adhesive protein solution to the exosome-like nanoparticle solution is preferably (1-5):1, more preferably (2-4):1. In the examples of the present invention, the volume ratio of the recombinant mussel adhesive protein solution to the exosome-like nanoparticle solution can be 3:1.

[0043] In the present invention, the concentration of the recombinant mussel adhesive protein solution is preferably 100-200 mg / mL, more preferably 120-160 mg / mL. In the examples of the present invention, the concentration of the recombinant mussel adhesive protein solution can be 150 mg / mL.

[0044] The present invention has no special limitation on the source of the recombinant mussel adhesive protein solution, and any source well-known to those skilled in the art can be used. In the examples of the present invention, the recombinant mussel adhesive protein solution is preferably prepared by conventional induction culture from the recombinant mussel adhesive protein plasmid in the prior art. The specific process is as follows: Transfer the recombinant mussel adhesive protein plasmid (Mefp3 plasmid) into the BL21 expression strain, pick a single colony on the LB plate, inoculate it into the LB liquid medium containing 100 μg / mL ampicillin, and place it in a shaker at 37 °C and 220 rpm for overnight culture to obtain an overnight culture; Take 10 mL of the overnight culture and inoculate it into 1 L of the LB liquid medium containing 100 μg / mL ampicillin, and place it in a shaker at 37 °C and 220 rpm for overnight culture. After the OD 600 value reaches 0.8-1.0, add 1 mL of a 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) solution, place it in a shaker for induction culture for 3 h, centrifuge at 4000×g for 15 min, collect the cells, and weigh them; Then, at a ratio of 1 g:10 mL, add an 8 M guanidine hydrochloride solution to the cells and react at 4 °C for 12 h. Centrifuge the resulting mixture, filter the obtained supernatant through a 0.22 μm filter, add 2 mL of Ni-NTA purification resin (purchased from Cytiva, product number 17531801) to the obtained filtrate, and shake it in a rotary shaker for 1 h. Then transfer the resulting mixture to a gravity chromatography column. After eluting the impurity proteins from the flowing protein lysate with a 20 mM imidazole solution with a volume 100-150 times that of the protein lysate, elute the target protein (recombinant mussel adhesive protein) with a 1 M imidazole solution with a volume 10-15 times that of the protein lysate. Put the collected protein solution into a dialysis bag and dialyze it with PBS buffer for 2-3 days, then desalt it using an ultrafiltration tube, concentrate it, measure the concentration of the recombinant mussel adhesive protein by Nanodrop 2000, and adjust the concentration of the recombinant mussel adhesive protein to 100-200 mg / mL.

[0045] In the present invention, the recombinant mussel adhesive protein can promote the adhesion and migration of skin cells, inhibit the release of inflammatory mediators, reduce the trauma of oxidative stress reaction to the skin, and accelerate wound healing; at the same time, the recombinant mussel adhesive protein also has good adhesiveness and can adhere effectively in a humid environment.

[0046] In the present invention, the concentration of the exosome-like nanoparticle solution is preferably 1 to 4 mg / mL, more preferably 2 to 3 mg / mL. In the examples of the present invention, the concentration of the exosome-like nanoparticle solution can be 2 mg / mL.

[0047] In the present invention, the diameter of the exosome-like nanoparticles in the exosome-like nanoparticle solution is preferably 30 to 200 nm, more preferably 50 to 200 nm.

[0048] In the present invention, the preparation method of the exosome-like nanoparticle solution preferably includes the following steps:

[0049] Juice purslane, and subject the obtained first supernatant to stepwise centrifugation to obtain a second supernatant;

[0050] After centrifuging the second supernatant, subject the obtained precipitate to sucrose density gradient centrifugation to obtain a preliminary exosome-like filtrate;

[0051] Mix the preliminary exosome-like filtrate and phosphate buffer, and perform solid-liquid separation to obtain the exosome-like nanoparticle solution.

[0052] In the present invention, purslane is juiced, and the obtained first supernatant is subjected to stepwise centrifugation to obtain a second supernatant.

[0053] In the present invention, the purslane is preferably fresh purslane.

[0054] Before performing the juicing, the present invention preferably washes the purslane, and the present invention has no special limitation on the washing process, and any process well-known to those skilled in the art can be used.

[0055] In the present invention, the juicing process is preferably to mix the crushed purslane and phosphate buffer (PBS buffer) and then perform juicing. The PBS buffer is preferably a PBS buffer with a concentration of 0.1 M and a pH of 7.0 to 7.4. The present invention has no special limitation on the dosage of the PBS buffer, and any dosage well-known to those skilled in the art can be used. The present invention has no special limitation on the juicing process, and any process well-known to those skilled in the art can be used.

[0056] After the juicing is completed, the present invention also preferably includes filtration, and the filtration is preferably performed using gauze; the present invention has no special limitation on the specification of the gauze, and any gauze specification well-known to those skilled in the art can be used.

[0057] In the present invention, the stepwise centrifugation preferably includes first centrifugation, second centrifugation, and third centrifugation performed in sequence; the rotation speed of the first centrifugation is preferably 200×g, the time is preferably 10 min, and the temperature is preferably 4°C; the rotation speed of the second centrifugation is preferably 2000×g, the time is preferably 20 min, and the temperature is preferably 4°C; the rotation speed of the third centrifugation is preferably 10,000×g, the time is preferably 30 min, and the temperature is preferably 4°C.

[0058] In the present invention, the function of the first centrifugation is to remove cells and larger cell debris, the function of the second centrifugation is to further remove cell debris and larger particles, and the function of the third centrifugation is to remove microvesicles and larger vesicles.

[0059] After obtaining the second supernatant, in the present invention, the second supernatant is centrifuged, and the obtained precipitate is subjected to sucrose density gradient centrifugation to obtain a preliminary exosome-like filtrate.

[0060] In the present invention, the rotation speed of the centrifugation is preferably 100,000×g, the time is preferably 60 min, and the temperature is preferably 4°C.

[0061] In the present invention, the function of the centrifugation is to precipitate exosomes.

[0062] In the present invention, the mass concentration gradient of the sucrose solution used in the sucrose density gradient centrifugation is preferably 15-45%, and the specific mass concentration gradient settings are preferably 15%, 20%, 30%, and 45% in ascending order from low to high.

[0063] In the present invention, the rotation speed of the sucrose density gradient centrifugation is preferably 150,000×g, and the time is preferably 60 min.

[0064] In the present invention, the preliminary exosome-like filtrate is preferably the supernatant obtained by centrifugation using a sucrose solution with a mass concentration of 30-45%.

[0065] In the present invention, the function of the sucrose density gradient centrifugation is to enable exosomes to migrate to the gradient layer with the same density as theirs during centrifugation, providing the concentration and purity of exosomes.

[0066] After obtaining the preliminary exosome-like filtrate, in the present invention, the preliminary exosome-like filtrate and phosphate buffer solution are mixed, and solid-liquid separation is performed to obtain the exosome-like nanoparticle solution.

[0067] In the present invention, the phosphate buffer is preferably a PBS buffer with a concentration of 0.1 M and a pH of 7.0 - 7.4. There is no special limitation on the dosage of the PBS buffer in the present invention. The dosage well-known to those skilled in the art is adopted for mixing, and it is only necessary to ensure that the mass concentration of the finally prepared exosome-like nanoparticle solution is within the range of 1 - 4 mg / mL.

[0068] In the present invention, the solid-liquid separation preferably includes centrifugation and filtration carried out in sequence. In the present invention, the rotation speed of the centrifugation is preferably 100,000×g; the time of the centrifugation is preferably 60 - 120 min; the temperature of the centrifugation is preferably 4 - 8°C. In the present invention, the filtration preferably uses a microporous membrane with a pore size of 0.22 μm.

[0069] After the solid-liquid separation is completed, if the obtained exosome-like nanoparticle solution is not used immediately, it is preferably stored in a refrigerator at -80°C.

[0070] In the present invention, since skin wound healing is a dynamic process involving many bioactive molecules, incomplete healing or necrosis will both damage the skin barrier function. The recombinant mussel adhesive protein hydrogel, as a hydrophilic three-dimensional network structure gel, has great advantages in biocompatibility, degradability and swelling property. It can be used both as a cell support material in the process of tissue repair and regeneration and as an efficient carrier for drug delivery. Forming a complex with exosome-like nanoparticles, it can protect the exosome-like nanoparticles from being cleared by the in vivo immune effect and can also slow down the release of the exosome-like nanoparticles. The synergistic effect of the two can improve the tissue retention time and curative effect of the exosome-like nanoparticles.

[0071] The present invention also provides a preparation method of the wound dressing described in the above technical solution, including the following steps:

[0072] After mixing the recombinant mussel adhesive protein solution and the exosome-like nanoparticle solution, loading is carried out to obtain the wound dressing.

[0073] The present invention has no special limitation on the mixing process, and the process well-known to those skilled in the art can be adopted.

[0074] In the present invention, the loading is preferably carried out in an incubator at 37°C for 3 - 5 h.

[0075] The present invention also provides the application of the wound dressing described in the above technical solution or the wound dressing prepared by the preparation method described in the above technical solution in the preparation of a medical material for promoting wound healing. The present invention has no special limitation on the application method, and the method well-known to those skilled in the art can be adopted.

[0076] The present invention provides a method for preparing an exosome-like nanoparticle solution, comprising the following steps:

[0077] Juice purslane, and subject the obtained first supernatant to stepwise centrifugation to obtain a second supernatant;

[0078] After centrifuging the second supernatant, subject the obtained precipitate to sucrose density gradient centrifugation to obtain a preliminary exosome-like filtrate;

[0079] Mix the preliminary exosome-like filtrate and phosphate buffer solution, and perform solid-liquid separation to obtain the exosome-like nanoparticle solution.

[0080] In the present invention, the method for preparing the exosome-like nanoparticle solution preferably refers to the method for preparing the exosome-like nanoparticle solution described in the above technical solution, and will not be elaborated herein.

[0081] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in 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 in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0082] Example 1

[0083] As Figure 1 shown in a of, mix fresh and clean purslane with a PBS buffer solution having a concentration of 0.1 M and a pH value of 7.0 - 7.4 until the purslane is submerged by the PBS buffer solution, then juice it, and filter it with a gauze to obtain a first supernatant;

[0084] Centrifuge the first supernatant at 200×g and 4 °C for 10 min, then centrifuge the obtained supernatant at 2000×g and 4 °C for 20 min, and further centrifuge the obtained supernatant at 10000×g and 4 °C for 30 min to obtain a second supernatant;

[0085] Centrifuge the second supernatant at 100000×g and 4 °C for 60 min, then put the obtained precipitate into a sucrose density gradient solution (with mass concentrations of 15%, 20%, 30% and 45% in sequence, sucrose is purchased from Macklin Reagent Company, product number S818046), and then centrifuge it at 150000×g for 60 min, and collect the supernatant obtained by centrifuging with a sucrose solution having a mass concentration of 30 - 45% to obtain a preliminary exosome-like filtrate;

[0086] The preliminary filtrate of exosome-like particles was diluted with PBS buffer at a concentration of 0.1 M and a pH value of 7.0 - 7.4, and then centrifuged at 100,000×g for 60 min and filtered through a microporous membrane with a pore size of 0.22 μm to obtain a pure exosome-like nanoparticle solution (the concentration of the exosome-like nanoparticle solution was measured to be 2.15 mg / mL by a BCA protein concentration assay kit (purchased from Thermo Fisher Scientific, catalog number 23227)), and it was stored in a refrigerator at -80 °C;

[0087] 10 μL of the exosome-like nanoparticle solution was dropped onto a copper grid, incubated at room temperature for 10 min, washed with sterile distilled water, the excess liquid was blotted with absorbent paper, 10 μL of 2 vol% uranyl acetate was pipetted onto the copper grid for counterstaining for 1 min, the floating liquid was blotted with filter paper, and after drying for 2 min, the copper grid was observed under a transmission electron microscope (Hitachi transmission electron microscope (HT7700, Hitachi));

[0088] The exosome-like nanoparticle solution was irradiated with a laser light source, and the scattered light of the exosome-like nanoparticles was detected. The concentration of the exosome-like nanoparticle solution was calculated by counting the number of scattered particles; the exosome-like nanoparticle solution was diluted with PBS buffer at a concentration of 0.1 M and a pH value of 7.0 - 7.4 to a concentration of exosome-like nanoparticles of 1×10 7 particles / mL - 1×10 9 particles / mL, and its size and mass were measured, and the movement trajectory of the exosome-like nanoparticles was analyzed simultaneously;

[0089] Among them, Figure 1 b in is the TEM image of exosome-like nanoparticles in the exosome-like nanoparticle solution. As can be seen from Figure 1 b in, the exosome-like nanoparticles in the exosome-like nanoparticle solution are visible in the fields of view of 100 nm, 200 nm, 500 nm, and 1 μm, showing a morphology similar to exosomes (tea saucer-shaped or hemispherical with a concave side);

[0090] Figure 1 c in is that the average particle size of exosome-like nanoparticles in the exosome-like nanoparticle solution is 157.0 nm, the main particle size peak is 112.1 nm, the percentage of the main peak is 72.6%, and the final concentration is 2.3×10 9 particles / mL.

[0091] Antibacterial ability test:

[0092] Staphylococcus aureus (ATCC6538) and Escherichia coli (ATCC25922) were used as indicator bacteria. Single colonies of the above strains were picked and inoculated into LB liquid medium (purchased from Jiangsu KeyGen Biotech Co., Ltd., product number KGH2105) respectively, and cultured in a shaker at 37°C for 12 h. The bacterial solution was taken out and the OD was measured. 600 The bacterial solution was diluted to 10 6 CFU / mL with PBS buffer at a concentration of 0.1 M and a pH value of 7.0 - 7.4. 100 μL of the diluted bacterial solution was taken and spread on an LB agar plate. Macropores were made on the agar plate by the Oxford cup method. The PBS buffer at a concentration of 0.1 M and a pH value of 7.0 - 7.4 was used as a negative control, and fresh purslane juice was used as a positive control. After the exosome-like nanoparticle solution was lysed with RIPA lysis buffer, the supernatant was collected by centrifugation at 4°C and 12,000 rpm (recorded as exosome-like nanoparticle lysate). 200 μL of each of the above three solutions was added to the wells. After diffusion at 4°C for 2 h, it was placed in an incubator at 37°C for 16 - 18 h, and the size of the inhibition zone was measured using a vernier caliper.

[0093] Figure 2 were the antibacterial test results of PBS buffer, fresh purslane juice, and exosome-like nanoparticle lysate. Among them, a was the antibacterial effect of fresh purslane juice and its exosomes on Staphylococcus aureus, b was the antibacterial effect of fresh purslane juice and its exosomes on Escherichia coli, and c was the statistical result of the inhibition zone; Figure 2 It can be seen that fresh purslane juice has a significant inhibitory effect on Staphylococcus aureus and Escherichia coli. The inhibitory effect of exosome-like nanoparticle lysate on the two indicator bacteria is less than that of fresh purslane juice, but it is significantly different from the PBS buffer group. The inhibition zones reached 10 mm and 7.5 mm respectively, indicating that the exosome-like nanoparticles have antibacterial effects.

[0094] Anti-inflammatory ability test:

[0095] RAW264.7 cells were grown in DMEM medium containing 10% FBS and 1% penicillin-streptomycin (purchased from Thermo Fisher Scientific Inc., product number 11965092) (temperature 37°C, carbon dioxide concentration 5%). Before the anti-inflammatory experiment, a CCK-8 kit (purchased from MCE, product number HY-K0301) was used to detect the effect of exosome-like nanoparticles on cell viability, and the operation steps were carried out according to the kit instructions. RAW264.7 cells were transferred to a 12-well plate with an inoculation density of 5×10 6Cells were seeded at a density of [[ID=]], and 1 mL of culture medium was not added to each well until the volume reached 2 mL, and the cells were cultured for 24 h. The cells were washed 3 times with sterile PBS. In the blank control group, the medium was replaced with 2 mL of culture medium. In the experimental group, 500 ng / well of LPS was added. In the exosome-like nanoparticle group, 500 ng / well of LPS and 200 μg / well of exosome-like nanoparticles were added. In the negative control group, 500 ng / well of LPS and an equal volume of PBS were added. According to the above different treatments, the cells were co-incubated for 24 h, then the cells were washed 3 times with sterile PBS. The RNAiso Easy kit (purchased from Takara, catalog number TCH020) was used to extract cell RNA. After removing gDNA, fluorescence quantitative PCR was performed. Using β-actin (purchased from Yijin Biotechnology, catalog number MQP026493) as an internal reference, the relative expression levels of inflammatory factors IL-6 (purchased from Yijin Biotechnology, catalog number MQP088375), IL-1β (purchased from Yijin Biotechnology, catalog number MQP092978), and TNF-α (purchased from Yijin Biotechnology, catalog number MQP096302) were detected respectively;

[0096] Figure 3 The cell viability and anti-inflammatory experimental results of the exosome-like nanoparticles are shown in the figure. Among them, a shows the effect of exosome-like nanoparticles at different concentrations on cell viability, b shows the effect of Portulaca oleracea exosome-like nanoparticles on the relative expression level of IL-1β, c shows the effect of Portulaca oleracea exosome-like nanoparticles on the relative expression level of IL-6, and d shows the effect of Portulaca oleracea exosome-like nanoparticles on the relative expression level of TNF-α; Figure 3 It can be seen that in the range of 10 - 100 μg / mL, the exosome-like nanoparticles can promote cell proliferation and have no adverse effect on cell viability, indicating good biocompatibility, providing a basis and potential possibility for their application in the biomedical field. Compared with the LPS and PBS groups, the relative expression levels of IL-6, IL-1β, and TNF-α in the experimental groups containing exosome-like nanoparticles were significantly reduced, indicating that the exosome-like nanoparticles have anti-inflammatory effects, providing theoretical support for their use as wound dressings to promote wound healing in clinical practice.

[0097] Example 2

[0098] Preparation of the PO-EVs@Mefp3 hydrogel:

[0099] Transfer the recombinant mussel adhesive protein plasmid (Mefp-3 plasmid) into the BL21 expression strain. Pick a single colony on the LB plate and inoculate it into an LB liquid medium containing 100 μg / mL ampicillin. Place it in a shaker and culture overnight at 37°C and 220 rpm to obtain an overnight culture. Take 10 mL of the overnight culture and inoculate it into 1 L of LB liquid medium containing 100 μg / mL ampicillin. Place it in a shaker and culture overnight at 37°C and 220 rpm. Wait until the OD 600 value reaches 0.8 - 1.0, then add 1 mL of IPTG solution with a concentration of 0.5 mM, place it in a shaker for induced culture for 3 h, centrifuge at 4000×g for 15 min, collect the bacterial cells and weigh them. Then, in a ratio of 1 g:10 mL, add an 8 M guanidine hydrochloride solution to the bacterial cells and react at 4°C for 12 h. Centrifuge the obtained mixture, filter the obtained supernatant through a 0.22 μm filter, add 2 mL of Ni-NTA resin to the obtained filtrate, place it on a rotary shaker and shake for 1 h, then transfer the obtained mixture to a gravity chromatography column. Elute the miscellaneous proteins from the flowing protein lysate with an imidazole solution with a concentration of 20 mM at 150 times the volume, and elute the target protein (recombinant mussel adhesive protein) with an imidazole solution with a concentration of 1 M at 15 times the volume. Load the collected protein solution into a dialysis bag and dialyze it with PBS buffer for 2 - 3 days, then desalt it using an ultrafiltration tube, concentrate it to obtain a recombinant mussel adhesive protein solution. Measure the concentration of the recombinant mussel adhesive protein solution by Nanodrop 2000 and adjust the concentration of the recombinant mussel adhesive protein to 150 mg / mL.

[0100] As Figure 4 shown in a of the figure, take 50 μL of the exosome-like nanoparticle solution described in Example 1 and mix it evenly with 150 μL of the recombinant mussel adhesive protein solution, and place it at 37°C for 3 h to obtain a wound dressing (denoted as PO-EVs@Mefp3);

[0101] First, place the wound dressing at -80°C overnight, and then perform freeze-drying treatment to obtain a powdery sample. Fix the powdery sample on the stage with conductive glue, use an ear syringe to blow away the surface debris, perform vacuum sputtering gold treatment and place it in the scanning chamber, and observe the microscopic morphology of the powdery sample with a field emission scanning electron microscope (SEM) and select different magnifications for scanning;

[0102] Figure 4 b in the figure is the SEM image of the recombinant mussel adhesive protein (Mefp3) at a magnification of 500 times and the SEM images of the PO-EVs@Mefp3 from top to bottom and from left to right at magnifications of 2k times, 5k times, and 2OK times respectively. From Figure 4It can be seen that after the Mefp3 forms a hydrogel, it presents a loose and porous three-dimensional network structure, indicating the potential for drug loading and delivery; for the PO-EVs@Mefp3, the exosome-like nanoparticles are successfully attached to the porous structure of the recombinant mussel adhesive protein hydrogel, which is beneficial to maintaining its biological activity, enhancing stability and therapeutic effect.

[0103] Testing of the swelling properties, in vitro degradation behavior and exosome release rate of the hydrogel:

[0104] The swelling behavior of the hydrogel can absorb wound exudate, nutrients, etc., which is beneficial to the transmission of metabolites and the diffusion and release of drugs. Prepare an appropriate amount of the PO-EVs@Mefp3 hydrogel, and accurately weigh the initial mass of each sample and record it as Wd. Immerse the samples in 10 mL of PBS (pH = 7.4) and place them in an incubator at 37°C. At fixed time points (such as 0, 1, 2, 4, 6, 8, 10, 12 hours, etc.), take out the samples, first gently blot the surface moisture with absorbent paper, and then accurately weigh the mass of each sample after swelling using an electronic balance and record it as Ws. Repeat each group of samples three times. Record the mass after swelling at each time point and calculate the swelling rate or expansion rate. The formula is: equilibrium swelling rate SR = (Ws - Wd) / Wd × 100%. Plot a curve of the swelling rate or expansion rate versus time to visually display the swelling process of the hydrogel. Figure 5 In a, the swelling curve of the PO-EVs@Mefp3 hydrogel is shown, as Figure 5 0As shown in a, with the extension of the soaking time, the swelling rate of the PO-EVs@Mefp3 hydrogel first increases and then tends to balance, reaching the swelling equilibrium at about 4 h, and its equilibrium swelling rate is 174%.

[0105] The degradation rate reflects the degradation rate and stability of the hydrogel sample in the in vitro environment. Take an appropriate amount of the PO-EVs@Mefp3 hydrogel, weigh the initial weight and record it as Wt. Immerse it in 10 mL of PBS (pH = 7.4) and place it in an incubator at 37°C. Take out the PO-EVs@Mefp3 hydrogel at the same time every day, remove the excess moisture on the surface with filter paper, then place it in an oven and dry it to a constant weight, and weigh the corresponding mass and record it as W0. Repeat each group of samples three times, and record from the first day to the 14th day. The degradation rate calculation formula for the PO-EVs@Mefp3 hydrogel is (Wt / W0) × 100%. Figure 5 In b, the in vitro degradation curve of the PO-EVs@Mefp3 hydrogel is shown, as Figure 5 As shown in b, the PO-EVs@Mefp3 hydrogel can degrade about 80% within 7 days, and with the extension of time, the degradation rate tends to slow down.

[0106] The PO-EVs@Mefp3 hydrogels were immersed in a 24-well plate containing sterile PBS. PBS supernatant was collected daily and an equal volume of fresh sterile PBS was added to continue soaking. PBS supernatant was collected for a total of 14 days as the test sample. The samples were assayed using a BCA protein concentration assay kit; refer to the instructions for the procedure. Figure 5 c in the figure is the in vitro release curve of exosome-like nanoparticles in the PO-EVs@Mefp3 hydrogel. Figure 5 As shown in c, the hydrogel wound dressing loaded with exosome-like nanoparticles has a sustained release performance during the 14-day test period and can continuously release within the service life of the hydrogel dressing; at the same time, the release amount in the first 48 hours reaches about 40%. This feature helps to eliminate and kill pathogens in and around the wound site in the early stage of use of the dressing, creating a sterile microenvironment for wound healing.

[0107] The above results indicate that the PO-EVs@Mefp3 can achieve the slow release of exosome-like nanoparticles through the three-dimensional network structure, effectively preventing the rapid clearance of exosomes in the body, thereby improving the effect of wound repair.

[0108] Application of the PO-EVs@Mefp3 in a mouse wound model:

[0109] After 7 days of adaptive feeding, BALB / c mice (male, 8 weeks old, weighing 18-22 g) were randomly divided into three groups: control group, Mefp3 group, and PO-EVs@Mefp3 group. Each mouse was marked, and the grouping date was D0. On D0, a wound model was created in each mouse. A full-thickness circular skin wound with a diameter of 6 mm was created after shaving the hair in the middle of the back, which was convenient for operation. The wound in the control group was covered with gauze and secured with adhesive tape. The wound in the Mefp3 group was covered with Mefp3 hydrogel without purslane exosomes, and the wound in the PO-EVs@Mefp3 group was fixed with adhesive tape. The gauze / dressing material of the mice was changed every two days (i.e., D0 / D3 / D6 / D9 / D12), and the wound recovery was observed and photographed. All mice were euthanized on D14, and skin tissue samples from the wound (near the wound and a portion of the surrounding normal tissue) were collected and fixed with 4% paraformaldehyde for 24 h for subsequent HE staining and Masson staining analysis.

[0110] Figure 6 The experimental results of PO-EVs@Mefp3 promoting wound healing are shown in Figure 1, where a is a representative picture of the skin wounds of each group of mice at 0 days, 3 days, 9 days, and 14 days, and b is the statistical result of the wound healing area. Figure 6It can be seen that the wound healing rate after treatment with the PO-EVs@Mefp3 is the highest, significantly higher than that of the control group and the Mefp3 group.

[0111] Figure 7 The morphological analysis results of the skin tissues near the wounds of each group of mice were sampled on the 14th day. Among them, a is the HE staining result, and b is the Masson staining result. Compared with the control group and the Mefp3 group, after the skin wounds of the mice treated with the PO-EVs@Mefp3 dressing healed, the epidermal layer was thicker, there were more new blood vessels, and skin appendages such as hair follicles appeared, and the content of collagen fibers was higher. Thus, it can be seen that the PO-EVs@Mefp3 can effectively promote wound healing.

[0112] The above is only the preferred embodiment of the present invention, and it does not impose any form of limitation on the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention.

Claims

1. A wound dressing, characterized in that, Comprising exosome-like nanoparticles and a recombinant mussel adhesive protein hydrogel encapsulating the exosome-like nanoparticles; the recombinant mussel adhesive protein hydrogel has a three-dimensional network structure.

2. The wound dressing according to claim 1, characterized in that, The raw material for preparing the recombinant mussel adhesive protein hydrogel is a recombinant mussel adhesive protein solution; The raw material for preparing the exosome-like nanoparticles is an exosome-like nanoparticle solution; The concentration of the recombinant mussel adhesive protein solution is 100 - 200 mg / mL; The concentration of the exosome-like nanoparticle solution is 1 - 4 mg / mL.

3. The wound dressing according to claim 2, wherein, The volume ratio of the recombinant mussel adhesive protein solution to the exosome-like nanoparticle solution is (1 - 5):

1.

4. The wound dressing according to claim 2 or 3, wherein The preparation method of the exosome-like nanoparticle solution includes the following steps: After juicing purslane, subject the obtained first supernatant to stepwise centrifugation to obtain a second supernatant; After centrifuging the second supernatant, subject the obtained precipitate to sucrose density gradient centrifugation to obtain a preliminary exosome-like filtrate; After mixing the preliminary exosome-like filtrate and phosphate buffer solution, perform solid-liquid separation to obtain the exosome-like nanoparticle solution.

5. The wound dressing according to claim 4, wherein The stepwise centrifugation includes first centrifugation, second centrifugation, and third centrifugation performed in sequence; The rotation speed of the first centrifugation is 200×g, the time is 10 min, and the temperature is 4°C; The rotation speed of the second centrifugation is 2000×g, the time is 20 min, and the temperature is 4°C; The rotation speed of the third centrifugation is 10,000×g, the time is 30 min, and the temperature is 4°C.

6. The wound dressing according to claim 4, wherein The rotation speed of the centrifugation is 100,000×g, the time is 60 min, and the temperature is 4°C; The mass concentration gradient of the sucrose solution used in the sucrose density gradient centrifugation is 15 - 45%; The rotation speed of the sucrose density gradient centrifugation is 150,000×g, and the time is 60 min.

7. The wound dressing according to claim 6, wherein The preliminary exosome-like filtrate is the supernatant obtained by centrifuging with a sucrose solution having a mass concentration of 30 - 45%; The solid-liquid separation includes centrifugation and filtration performed in sequence; the rotation speed of the centrifugation is 100,000×g, the time is 60 - 120 min, and the temperature is 4 - 8°C; the filtration uses a microporous membrane with a pore size of 0.22 μm.

8. The preparation method of the wound dressing according to any one of claims 1 to 7, characterized in that, Including the following steps: After mixing the recombinant mussel adhesive protein solution and the exosome-like nanoparticle solution, perform loading to obtain the wound dressing.

9. Use of the wound dressing according to any one of claims 1 - 7 or the wound dressing prepared by the preparation method according to claim 8 in the preparation of a medical material for promoting wound healing.

10. A method for preparing an exosome-like nanoparticle solution, characterized in that, Including the following steps: After juicing purslane, subject the obtained first supernatant to stepwise centrifugation to obtain a second supernatant; After centrifuging the second supernatant, subject the obtained precipitate to sucrose density gradient centrifugation to obtain a preliminary exosome-like filtrate; After mixing the preliminary exosome-like filtrate and phosphate buffer solution, perform solid-liquid separation to obtain the exosome-like nanoparticle solution.

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