MMP-9 responsive hydrogel loaded with M2 macrophage exosome as well as preparation method and application of MMP-9 responsive hydrogel

Abstract

The invention discloses MMP-9 responsive hydrogel loaded with M2 macrophage exosomes as well as a preparation method and application of the MMP-9 responsive hydrogel loaded with the M2 macrophage exosomes, the M2 macrophage-derived exosomes (M2-Exos) are obtained by constructing a macrophage polarization model, meanwhile, the MMP-9 responsive hydrogel is used for loading and realizing controllable release of the M2-Exos, and the MMP-9 responsive hydrogel is used for wound inflammation regulation. The preparation method comprises the following steps: mixing oxidized dextran, oligopeptide Pro-Val-Gly-Leu-Iso-Gly and carboxymethyl chitosan according to a certain ratio, and carrying out cross-linking, so as to obtain the Schiff base gel responsive to the MMP-9. And loading the M2-Exos into the hydrogel, so as to obtain the MMP-9 responsive hydrogel loaded with the M2 macrophage derived exosome. The hydrogel can be kept stable at low MMP-9 concentration in the early stage of inflammation, and the content M2-Exos in the hydrogel can be quickly degraded and released when MMP-9 is greatly increased in the later stage of inflammation, so that the inflammatory reaction process of the diabetic wound surface is accurately regulated and controlled. The MMP-9 responsive hydrogel can effectively regulate inflammation, promote healing of diabetic wounds and relieve physiological and psychological burdens of patients caused by the diabetic wounds.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to an MMP-9 responsive hydrogel loaded with M2 macrophage exosomes, and a preparation method and application thereof. Background Art

[0002] With the rapid increase in the number of diabetic patients, chronic non-healing wounds are increasing, which has become a thorny problem that needs to be solved urgently in clinical practice. Chronic diabetic wounds usually stop at the inflammatory stage, which is characterized by microenvironmental disorder, long-term inflammatory state, and impaired repair cell function. Therefore, it is crucial to terminate inflammation in time to ensure a normal and orderly repair process. The dysregulation of macrophage M1-M2 phenotype conversion leads to excessive accumulation of M1 macrophages, which is the main pathological feature of chronic inflammation in diabetic wounds. However, in recent years, strategies targeting macrophages have generally focused on enhancing the activity of M2 macrophages or adding exogenous M2 macrophages, while ignoring the existence of M1 macrophages that have already accumulated in large quantities.

[0003] Exosomes are small extracellular vesicles that play an important role in intercellular communication. Studies have shown that exosomes from fibroblasts, endothelial cells, and adipose-derived mesenchymal stem cells can induce macrophages to switch from M1 to M2 phenotypes, thereby reducing inflammation and promoting diabetic wound healing. In addition, exosomes can obtain biomolecules from their donor cells and be endowed with the same biological functions as their parent cells. For example, M2-derived exosomes exhibit anti-inflammatory properties similar to M2 macrophages in inflammatory diseases. Based on this, we propose a strategy to use M2-Exos to regulate the inflammatory response in chronic diabetic wounds. However, the application of exosomes still faces challenges such as short half-life and instability.

[0004] To address these obstacles, some biomaterials including hydrogels are used for loading and continuous release of exosomes. However, these materials often cannot precisely control the release of exosomes, and their effects do not match the changes in the wound microenvironment. Therefore, in order to achieve the best therapeutic effect, it is necessary to develop a microenvironment-responsive exosome delivery system. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of the above-mentioned technology and provide an MMP-9 responsive hydrogel loaded with M2 macrophage exosomes and a preparation method and application thereof.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is a method for preparing an MMP-9 responsive hydrogel loaded with M2 macrophage exosomes, comprising the following steps:

[0007] S1. Extraction of exosomes from M2 macrophages, specifically: selecting exosome cells, inducing them into M2 macrophages, and extracting exosome solution;

[0008] S2. Intermediate solution configuration, specifically:

[0009] S201. Synthesis of oxidized dextran (OD);

[0010] S202. Oxidized dextran (OD), carboxymethyl chitosan (CMC) and Pro-Val-Gly-Leu-Iso-Gly (PG-6) were selected, and OD, CMC and PG-6 solutions were prepared for standby use;

[0011] S3. Preparation of MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes, specifically:

[0012] S301. preparing a mixture of response peptides and M2 macrophage exosomes;

[0013] S302. The OD solution is mixed with a mixture of the response peptide and exosomes, and then mixed with the CMC solution to obtain an MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes.

[0014] Furthermore, the extraction of exosomes from M2 macrophages in step S1 comprises the following steps:

[0015] S101. RAW264.7 cells are selected as the source of exosome cells, and after subculture, the RAW264.7 cells are screened for cells in good condition, and interleukin (IL-4) is added to induce M2 macrophages;

[0016] S102. Identify the phenotype of M2 macrophages. After confirming that M2 macrophages are successfully obtained, culture them continuously for 24 hours using a culture medium containing exosome-free serum, remove impurities such as cell debris, and collect the cell culture supernatant;

[0017] S103. Take the cell culture supernatant obtained in S102, control the centrifugal acceleration to 10,000×g, the temperature to 4°C, centrifuge for 10 min, and transfer to a new centrifuge tube;

[0018] S104. Vortex the bottled magnetic beads for 30 seconds, take out 350 μL and add it to a centrifuge tube. Centrifuge at 3,000 × g and 4°C for 2 minutes, and discard the supernatant.

[0019] S105. Add 10 mL of phosphate buffered saline (PBS) precooled at 4°C and filtered through 0.22 μm, shake and mix for 30 seconds, control the centrifugal acceleration to 3,000 × g, centrifuge at 4°C for 5 minutes, and discard the supernatant;

[0020] S106. Take and add 4 mL Buffer EXA, 1 mL Buffer EXB and 14.65 mL supernatant, place in a rotary mixer, and rotate and mix at 4°C for 40 min;

[0021] S107. Remove the centrifuge tube, place it on a magnetic rack, and let it stand at 4°C for 10 min until the magnetic beads aggregate, then discard the supernatant;

[0022] S108. For the objects retained in S107, centrifuge at 3,000 × g and 4°C for 1 min, and discard the residual liquid;

[0023] S109. Add 0.5 mL of Buffer EXE and mix well. Centrifuge at 7,000 × g and 4 °C for 2 min. Transfer the supernatant to an EP tube.

[0024] S110. Rinse the syringe filters A and B with EXE and filter the exosome solutions respectively to obtain purified exosome solutions, which are frozen and stored for future use.

[0025] Furthermore, the step S2 is the preparation of the intermediate solution, comprising the following steps:

[0026] S201. Synthesizing oxidized dextran (OD), specifically comprising:

[0027] ① Dissolve 5g of dextran in 250mL of distilled water to obtain Dex solution;

[0028] ② NaIO4 was dissolved in 5 mL of distilled water and then added dropwise to the Dex solution, and the mixture was stirred at room temperature in the dark for 3.5 h;

[0029] ③ 3 mL of ethylene glycol was added to the mixture to react the unreacted NaIO4, and the mixture was stirred for another 1 h. The resulting product was dialyzed against distilled water (MWCO 1400 Da) for 3 days and lyophilized to obtain synthetic oxidized dextran (OD);

[0030] S202. Oxidized dextran (OD), carboxymethyl chitosan (CMC) and Pro-Val-Gly-Leu-Iso-Gly (PG-6) are selected respectively, and a 5% OD solution, a 3% CMC solution and a 20 mg / mL PG-6 solution are prepared respectively using a phosphate buffer solution (PBS) at pH = 7.4. The specific steps are as follows:

[0031] (1) Prepare 10 mL of 5% OD by adding 10 mL of PBS solution into a brown bottle containing 500 mg of OD and dissolve by ultrasonication / oscillation;

[0032] (2) Prepare 10 mL of 3% CMC by adding 10 mL of PBS solution into a brown bottle containing 300 mg of CMC and dissolve it by ultrasonication / oscillation;

[0033] (3) Prepare 5 mL of the response peptide with a concentration of 20 mg / mL by adding 5 mL of PBS solution to a brown bottle containing 100 mg of the response peptide and shaking to dissolve.

[0034] Furthermore, in step S202, the OD solution is prepared at a temperature of 20-25°C, and the CMC solution is prepared at a temperature of 45-60°C.

[0035] Furthermore, the step S3 is to prepare an MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes, and the specific contents are as follows:

[0036] S301. Prepare a mixture of response peptides and M2 macrophage exosomes

[0037] Add 0.7 mL of PBS solution and 0.5 mL of 3 × 10 10 particles / mL of M2 macrophage exosome solution, oscillate and dissolve to obtain 1.2mL of a mixed solution of the response peptide and M2 macrophage exosomes;

[0038] S302. Preparation of MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes

[0039] Take 300 μL of 5% OD solution, mix it with the mixture obtained from S201, and then mix it with 1.5 mL of 3% CMC solution. At room temperature, 3 mL of MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes can be cross-linked to obtain 5×10 exosomes per ml of hydrogel. 9 particles.

[0040] Furthermore, the mixed solution of the response peptide and M2 macrophage exosomes in step S301 contains 1.5×10 10 particles, and the response peptide concentration was 20 mg / mL.

[0041] The second object of the present invention is to provide an MMP-9 responsive hydrogel loaded with M2 macrophage exosomes prepared by the above-mentioned preparation method.

[0042] The third object of the present invention is to provide the use of the MMP-9 responsive hydrogel loaded with M2 macrophage exosomes in regulating the inflammatory response process of diabetic wounds and promoting the healing of diabetic wounds.

[0043] The advantages of the present invention compared with the prior art are:

[0044] 1) The present invention uses exosomes as the background, and aims to transform the excessively accumulated pro-inflammatory M1 macrophages in the wound into anti-inflammatory M2 macrophages through exosome-guided cell reprogramming technology, thereby improving the inflammatory microenvironment of diabetic wounds and effectively promoting wound healing;

[0045] 2) The present invention combines M2 macrophage exosomes with MMP-9 responsive hydrogel to prepare an intelligent hydrogel dressing that can intelligently respond to the progress of inflammatory response according to changes in MMP-9 content in the wound. The dressing can remain stable in the low MMP-9 environment of the wound in the early stage of inflammation, and quickly degrade and release its content M2-Exos when MMP-9 increases significantly in the late stage of inflammation, thereby achieving precise regulation of the inflammatory response process of diabetic wounds. At the same time, it also solves the problems of short half-life and instability faced by exosomes in vitro, and improves the bioavailability of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a preparation process and application flow chart of the present invention;

[0047] Figure 2 : These are the SEM (a), NTA (b) and WB (c) identification results of M2 macrophage exosomes extracted in Example 1 of the present invention;

[0048] Figure 3 is a SEM image of the MMP-9 responsive hydrogel in Example 4 of the present invention;

[0049] Figure 4 1 is a responsive degradation diagram (a) and a statistical result (b) of the MMP-9 responsive hydrogel in Example 4 of the present invention;

[0050] Figure 5 : is a diagram of the in vitro degradation and release of exosomes of the MMP-9 responsive hydrogel in Example 6 of the present invention (a), and the internalization of the released exosomes by M1 macrophages (b);

[0051] Figure 6 Graph (a) and statistical results (b) of the exosome clearance of the MMP-9 responsive hydrogel on one side of the wound of diabetic mice in Example 6 of the present invention;

[0052] Figure 7 The wound healing promoting effect (a) and the wound healing rate statistical result (b) of the MMP-9 responsive hydrogel in Example 7 of the present invention when used in diabetic mice;

[0053] Figure 8This is the epithelial regeneration and anti-inflammatory effect of the MMP-9 responsive hydrogel in Example 7 of the present invention on the wound surface of diabetic mice. Figure (a) is the HE and Masson staining results of the wound surface tissue, Figure (b) is the immunofluorescence staining result of the wound surface, and Figure (c) is the statistical result. DETAILED DESCRIPTION

[0054] The MMP-9 responsive hydrogel loaded with M2 macrophage exosomes of the present invention and its preparation method and application are further described in detail below in conjunction with the accompanying drawings and examples.

[0055] Example 1

[0056] The preparation and extraction of M2 macrophage-derived exosomes (M2-Exos) includes the following steps:

[0057] Step 1) RAW264.7 cells were cultured in a 37°C, 5% CO2 incubator with high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. IL-4 (20 ng / mL, Peprotech) was added to the culture medium for 24 hours, and the supernatant was collected;

[0058] Step 2) Sample processing: The supernatant was centrifuged at 10,000 × g at 4°C for 10 min and transferred to a new centrifuge tube;

[0059] Step 3) Preparation of magnetic beads: Vortex the bottled magnetic beads for 30 seconds, take out 350 μL and add it to a centrifuge tube. Control the centrifugal acceleration to 3,000×g, centrifuge at 4°C for 2 minutes, and discard the supernatant.

[0060] Step 4) Washing the magnetic beads: Add 10 mL of PBS precooled at 4°C and filtered through 0.22 μm, vortex and mix for 30 seconds, control the centrifugal acceleration to 3,000×g, centrifuge at 4°C for 5 minutes, and discard the supernatant.

[0061] Step 5) Exosome adsorption: Add 4 mL Buffer EXA, 1 mL Buffer EXB and 14.65 mL supernatant, place in a rotating mixer, and rotate and mix at 4°C for 40 min.

[0062] Step 6) Magnetic bead collection: Remove the centrifuge tube, place it on a magnetic rack, and let it stand at 4°C for 10 minutes until the magnetic beads aggregate, and discard the supernatant.

[0063] Step 7) Control the centrifugal acceleration to 3,000×g, centrifuge at 4°C for 1 min, and discard the residual liquid.

[0064] Step 8) Elute exosomes: Add 0.5 mL of Buffer EXE and mix well. Centrifuge at 7,000 × g and 4 °C for 2 min. Transfer the supernatant to an EP tube.

[0065] Step 9) Filtration and impurity removal: After washing the needle filters A and B with EXE, filter the exosome solution separately to obtain a purified exosome solution, which can be used immediately or frozen for later use.

[0066] Example 2

[0067] The exosomes obtained in Example 1 were subjected to relevant identification, including the following contents:

[0068] 1. Scanning electron microscope (SEM)

[0069] (1) 10 μL of exosome solution was dropped onto a copper grid, incubated at room temperature for 10 min, washed with sterile distilled water, and the excess liquid was absorbed with absorbent paper;

[0070] (2) Pipette 10 μL of 2% uranyl acetate onto the copper grid for negative staining for 1 min, remove the floating liquid with filter paper, and dry under an incandescent lamp for 2 min;

[0071] (3) Place the copper mesh under a transmission electron microscope and observe it at 80 kV.

[0072] 2.NTA (Nanoparticle Tracking Analysis)

[0073] The nanoparticle suspension was irradiated with a laser light source, and the scattered light of the nanoparticles was detected. The concentration of the nanoparticles was calculated by counting the number of scattered particles. The concentration of the separated exosomes was measured under 405nm emission light using the particle matrix ZetaViewPMX 110, and the exosomes were diluted to 1x10 with PBS. 7 particles / mL~1x10 9 particles / mL, and measured their size and mass. At the same time, the particle motion trajectory of exosomes was analyzed.

[0074] 3. Exosome Protein Extraction

[0075] (1) Take 50 μL of exosome lysis buffer and 150 μL of exosome solution at a ratio of 1:3 and mix well. Centrifuge 1 mL of cell suspension, add 1 mL of lysis buffer to the cell pellet, mix well, and boil at 100°C for 5 min.

[0076] (2) Transfer to an ice-water mixture and cool for 5 min. Centrifuge at 12,000 × g and 4°C for 5 min. Transfer the supernatant to a new EP tube.

[0077] 4. Western Blot Detection

[0078] (1) Protein concentration determination.

[0079] The exosome protein concentration was determined according to the instructions of the BCA protein concentration determination kit. The brief process is as follows: Mix solution A and solution B in the kit at a ratio of 50:1 to prepare a working solution. Use sterile ddH2O to dilute the protein standard to 0.5 mg / mL, then use ddH2O to dilute the standard in multiples, add the diluted standard and sample to a 96-well plate, then add 200 μL of the prepared working solution to each well, incubate at 37°C for 30 minutes, use a microplate reader to detect the absorbance at 562 nm, draw a standard curve, and use a regression equation to calculate the protein concentration in the sample.

[0080] (2) Electrophoresis and membrane transfer.

[0081] Use the polyacrylamide gel preparation kit to prepare SDS-PAGEGel of the corresponding concentration according to the molecular weight of the target antibody. According to the protein concentration in the sample, calculate the required sample volume, add it to the polyacrylamide gel well, transfer it to the electrophoresis tank, and perform electrophoresis. Perform 80V constant voltage electrophoresis until the indicator (bromophenol blue) in the sample enters the separation gel, and perform 120V constant voltage electrophoresis until the indicator reaches the bottom of the gel. Stop electrophoresis, peel off the polyacrylamide gel, cut off the concentrated gel, retain the separation gel, place it on the PVDF membrane, and clamp the filter paper and sponge to prepare a sandwich structure, insert the electrotransfer tank, add the electrotransfer solution, and electrotransfer at 200mA constant current for 2h. Take out the PVDF membrane, wash the membrane 3 times with TBST for 5min each time, and place it in 5% skim milk powder (prepared with TBST), room temperature, and block for 1h.

[0082] (3) Antibody incubation

[0083] Primary antibody incubation: Remove the PVDF membrane from the blocking solution, wash it with TBST three times for 5 minutes each time, transfer it to the prepared primary antibody solution, rotate and shake on a horizontal shaker at 4℃, and incubate overnight.

[0084] Secondary antibody incubation: Wash the membrane three times with TBST for 5 minutes each time, and transfer to the prepared secondary antibody solution. The secondary antibody is diluted with 5% skim milk powder (prepared with TBST). Incubate at room temperature for 1.5 hours.

[0085]

[0086] (4) Develop and take photos

[0087] (a) Washing: After the secondary antibody incubation, wash the PVDF membrane three times with TBST for 5 min each time.

[0088] (b) Lift the PVDF membrane and drain the excess washing solution from the membrane.

[0089] (c) Spread the membrane flat on a developing plate, add the prepared ECL reaction solution, and place it in a chemiluminescence imager.

[0090] (d) Photographing: Prepare the luminescent liquid, place the strips into an exposure machine (GE), drip the luminescent liquid on them for exposure, and collect pictures. If necessary, use Image J software to analyze the grayscale value of the protein strips.

[0091] The results are as follows Figure 1 As shown in Figure 2, the purified exosomes were in the shape of a saucer or a hemispherical shape with one side concave, with an average particle size of 121.1 nm and a concentration of 3.0*10 10 WB results showed that the purified exosomes expressed TSG101 and CD9, but did not express the endoplasmic reticulum-specific marker Calnexin and the classic activation phenotype markers iNOS and Arginase of macrophages, confirming that the extracted extracellular vesicles were exosomes.

[0092] Example 3 Preparation of intermediate solution and MMP-9 responsive hydrogel

[0093] The configuration of the intermediate solution includes the following contents:

[0094] 1. Synthesis of Oxidized Dextran (OD)

[0095] Dextran (Dex) was oxidized with NaIO4 to obtain oxidized dextran OD. 5 g of dextran was dissolved in 250 mL of distilled water. NaIO4 was dissolved in 5 mL of distilled water and then added dropwise to the Dex solution. The mixture was then stirred for 3.5 h at room temperature in the dark. Finally, 3 mL of ethylene glycol was added to the mixture to react unreacted NaIO4. The mixture was stirred for another 1 h. The product was dialyzed for 3 days against distilled water (MWCO 1400 Da) and lyophilized to obtain OD.

[0096] 2. Use PBS solution with pH = 7.4 to prepare OD solution with a mass fraction of 5%, CMC with a mass fraction of 1.5%, and PG-6 solution with a concentration of 20 mg / mL.

[0097] (1) Prepare 10 mL of 5% OD solution by adding 10 mL of PBS solution into a brown bottle containing 500 mg of OD and dissolve by ultrasonication / oscillation. The temperature during the process should not exceed 25°C.

[0098] (2) Prepare 10 mL of 3% CMC solution by adding 10 mL of PBS solution into a brown bottle containing 300 mg of CMC, at a temperature not exceeding 60°C, and dissolve by ultrasonication / oscillation;

[0099] (3) Prepare 5 mL of 20 mg / mL response peptide solution by adding 5 mL of PBS solution to a brown bottle containing 100 mg of response peptide and shaking to dissolve.

[0100] The preparation method of MMP-9 responsive hydrogel is as follows: at room temperature, 100 μL 5% OD and 400 μL 20 mg / mL responsive peptide are mixed evenly to form liquid A, and then mixed evenly with 500 μL 3% CMC (liquid B) to prepare MMP-9 responsive hydrogel; wherein the preferred hydrogel raw material ratio of the present invention is: 100 mL 5% OD + 400 mL 2% PG-6 + 500 mL 1.5% CMC.

[0101] Example 4

[0102] The MMP-9 responsive hydrogel obtained in Example 3 was characterized and analyzed, including the following contents:

[0103] 1. Scanning / Transmission Microscopy (SEM)

[0104] (1) Sample preparation:

[0105] Gel sample preparation: Cut the freshly prepared gel sample into 1*1*1cm 3 A certain amount of distilled water was added to the square gel block and allowed to stand in a 4°C refrigerator overnight. After swelling equilibrium, the sample was quickly frozen in liquid nitrogen for 30 minutes and then immediately placed in a freeze dryer for 24 hours. The block gel sample was directly glued to the conductive glue.

[0106] (2) Gold spraying test:

[0107] An Oxford Quorum SC7620 sputtering coater was used to spray gold for 45 seconds at a current of 10 mA. A TESCAN MIRALMS scanning electron microscope was then used to photograph the sample morphology for testing. The accelerating voltage during morphology photography was 3 kV.

[0108] The results are as follows Figure 3 As shown, the hydrogel presents a cross-linked three-dimensional network structure.

[0109] 2. Gel Degradation Analysis

[0110] Phosphate buffer solution (PBS) and 10, 20, and 50 ng / mL MMP-9 response peptide solutions were added to the constant weight hydrogel samples, respectively, and the samples were placed in a 37°C incubator for immersion. The weight changes of the hydrogel samples after immersion for different times were tested, and the percentage of weight loss relative to the original hydrogel sample was calculated, which was defined as the degradation rate of the hydrogel. The specific calculation formula is:

[0111] Degradation rate (%) = (W0-W t ) / W0×100%

[0112] Where W0 is the initial weight of the hydrogel, W t is the weight of the hydrogel measured at different immersion times.

[0113] The preferred raw material ratio of the hydrogel of the present invention is: 100 mL 5% OD + 400 mL 2% PG-6 + 500 mL 1.5% CMC.

[0114] The responsive degradation of MMP-9 responsive hydrogel is shown in Figure 2. Figure 4 As shown in the figure, in PBS solution, the hydrogel was basically stable within 7 days. After adding MMP-9 on the 7th day, the degradation rate of the hydrogel was significantly accelerated. Especially on the 7th to 10th day, the degradation rate of the 50ng / mL MMP-9 concentration group was significantly faster than that of the other groups. At 21 days, the degradation rates of the hydrogels in the PBS, 10ng / mL, 20ng / mL and 50ng / mL MMP-9 groups were 40%, 47%, 49% and 60%, respectively.

[0115] Example 5 Preparation of MMP-9 responsive hydrogel loaded with M2 macrophage exosomes

[0116] This example is based on the method described in Example 3, and the preferred hydrogel raw material ratio is: 100mL 5% OD + 400mL 2% PG-6 + 500mL 1.5% CMC. In this example, when preparing the response peptide solution, M2 macrophage exosomes are used for dissolution, and the specific steps are as follows:

[0117] (1) Prepare 1.2 mL of a mixture of response peptide and M2 macrophage exosomes by adding 0.7 mL of PBS solution and 0.5 mL of 3×10 10 particles / mL of M2 macrophage exosome solution, oscillate and dissolve. 10 particles, and the response peptide concentration was still 20 mg / mL.

[0118] (2) Take 300 mL of 5% OD and 1.2 mL of the mixture of response peptide and exosomes and mix them evenly, then mix them evenly with 1.5 mL of 3% CMC. Cross-linking can be done at room temperature to obtain 3 mL of MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes, where each milliliter of hydrogel is loaded with 5×10 exosomes. 9 particles.

[0119] Example 6 Evaluation of the responsive degradation function of MMP-9 responsive hydrogel loaded with M2 macrophage exosomes

[0120] 1. In vitro evaluation:

[0121] Exo containing fluorescent markers were prepared by Dil staining, and the M2 macrophage exosomes containing Dil markers were spread on the bottom of a 24-well plate. The culture medium without exosome serum and different concentrations of MMP-9 were added to it. After incubation for 24 hours, the supernatant was extracted, filtered through a 0.22mm filter, and added to M1 macrophages for further 24 hours. The internalization of exosomes and the polarization of M1 were observed under different MMP-9 concentrations. The collected supernatant was subjected to NTA, and the results showed that the exosome content in the 50ng / mL group was the highest ( Figure 5 a). At the same time, cell immunofluorescence showed that the exosomes in the group with a MMP-9 concentration of 50 ng / mL were most significantly internalized by macrophages, which was significantly different from the control group and the 10 ng / mL group ( Figure 5 b).

[0122] 2. In vivo evaluation:

[0123] Establishment of diabetic wound model in mice:

[0124] (1) Anesthesia: Ten 8-week-old male diabetic mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (administered at a dose of 0.1 mL / 20 g);

[0125] (2) Surgery: After anesthesia, the hair on the back of the mouse was removed, and a 10 mm diameter trephine punch was used to mark the position 1 cm away from the midline of the spine on both sides of the back of the mouse, and the marked skin was fully excised;

[0126] Postoperative drug administration of diabetic wounds in mice: On the day of modeling, the wounds on both sides of the back of each mouse were administered separately. On one side, 25 μL of Dil-labeled M2-Exos were subcutaneously injected at 3, 6, 9, and 12 o'clock around the wound edge with a microsyringe, totaling 100 μL; on the other side, 100 μL of synthesized hydrogel was used to cover the wound. The hydrogel was loaded with an equal amount of Dil-labeled M2 macrophage exosomes.

[0127] Evaluation of the sustained-release effect of M2 macrophage exosomes on the wounds of diabetic mice: 1 day, 3 days, 7 days, and 10 days after surgery were selected as time nodes. The mice were anesthetized again and placed in a small animal in vivo imager to detect the red fluorescence distribution and intensity of the wounds on both sides of the back.

[0128] The results showed that after four-point injection of free M2-Exos, the fluorescence signal was gradually eliminated within 3 days, but the M2-Exos released by the MMP-9 responsive hydrogel loaded with M2 macrophage exosomes were still retained in large quantities on the 7th day ( Figure 6 a). This result indicates that MMP-9 responsive hydrogel loaded with M2 macrophage exosomes can reduce the rapid clearance and degradation of M2-Exos in the early stage of mouse wounds ( Figure 6b), thereby increasing the bioavailability of M2-Exos.

[0129] Example 7 In vivo application and therapeutic effect evaluation of MMP-9 responsive hydrogel loaded with M2 macrophage exosomes

[0130] The mice used in this example are spontaneous type II diabetic db / db mice for research purposes, purchased from Weishanglide (Beijing) and approved by the Animal Ethics Committee.

[0131] 1. Modeling: 72 7-week-old male diabetic mice were divided into 4 groups, namely PBS group, simple exosome injection group (Exo group), simple MRH group (MRH group) and hydrogel-loaded exosome group (Exo@MRH group), with 18 mice in each group.

[0132] Anesthesia was performed by intraperitoneal injection of 1% sodium pentobarbital solution (administered at a dose of 0.1 mL / 20 g). After anesthesia, the hair on the back of the mouse was shaved, and a 10-mm diameter trephine punch was used to mark the position 1 cm away from the midline of the spine on both sides of the mouse's back, and the marked skin was fully excised.

[0133] 2. Postoperative medication: The PBS group was injected with 100 μL PBS around the wound surface using the subcutaneous four-point injection method;

[0134] The Exo group was injected with 100 μL of M2-exo in the same way; the MRH group and Exo@MRH group used 100 μL of synthesized hydrogel to cover the wound surface, and each 100 μL of Exo@MRH group was loaded with the same amount of Exo as that of the Exo group.

[0135] The wound surfaces of the four groups were covered with sterile transparent films to keep the wound surfaces moist.

[0136] 3. Photo record: On the 1st, 3rd, 7th, 10th, 14th and 21st day after surgery, the wound healing of all mice in each group was observed and photographed; the initial area of ​​the surgical wound was recorded as A0, and the actual wound area on the nth day was recorded as A t , calculated according to the formula:

[0137] Wound healing rate = (A0-A t ) / A0×100%

[0138] Image J software was used to calculate the wound area and draw the wound healing curves of each group of mice ( Figure 7 b);

[0139] The wounds of the four groups of diabetic mice healed gradually over time, and the wound area of ​​the Exo@MRH group was the smallest at each time point, and the wound healing speed of the diabetic mice in the PBS group was the slowest. After quantification according to the formula described in the method, it was found that Exo@MRH treatment in each group could promote faster healing of diabetic wounds ( Figure 7 a), the difference was statistically significant (P<0.05).

[0140] 4. Histological Analysis:

[0141] On the 1st, 3rd, 7th, 10th, 14th and 21st day after surgery, 3 mice from each group were killed by cervical dislocation. A circular incision was made 1 cm away from the wound edge with the center of the wound as the center, and the full-thickness skin of the mice was cut off. The tissue was fixed with 4% paraformaldehyde, embedded and sliced. HE staining and Masson staining were performed.

[0142] HE results showed that the narrowest scar width was observed in the Exo@MRH group on days 7, 10, and 14 after injury. On day 14, the wound surface of the Exo@MRH group was completely closed. Compared with the control group, the new epithelium in the Exo@MRH group was neatly arranged, and keratinocytes were evenly distributed throughout the epithelium, achieving epithelial re-formation. Masson staining results showed that the dermis in the Exo@MRH group was thinner and flatter, with lower collagen fiber density and more regular arrangement compared with the control group ( Figure 8 a).

[0143] 5. Wound tissue immunofluorescence: Tissue sections at each time point were obtained as described in step 4 above, and tissue CD86 / CD206 immunofluorescence co-staining was performed. The results showed that compared with the PBS group, the Exo@MRH group showed a large amount of CD206 expression on the 10th day, and the CD86 expression was significantly reduced on the 14th day, indicating that Exo@MRH can effectively intervene M1 macrophages into M2 cells, reverse the excessive inflammatory response of diabetic wounds, and promote the smooth progress of wound repair process ( Figure 8 b and Figure 8 c).

[0144] The working principle of the present invention is to mix oxidized dextran (OD) with short peptide Pro-Val-Gly-Leu-Iso-Gly (PG-6) and carboxymethyl chitosan (CMC) in a certain proportion, and cross-link to obtain a Schiff base gel responsive to MMP-9. M2-Exos are loaded into the hydrogel to obtain an MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes, and an intelligent hydrogel dressing that can intelligently respond to the inflammatory response process according to the change of MMP-9 content in the wound is prepared. The dressing can remain stable in the low MMP-9 environment of the wound in the early stage of inflammation, and quickly degrade and release its content M2-Exos when MMP-9 increases in large quantities in the late stage of inflammation, thereby achieving precise regulation of the inflammatory response process of the diabetic wound, and also solving the problems of short half-life and instability faced by exosomes in vitro, and improving the bioavailability of exosomes.

[0145] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. A method for preparing an MMP-9 responsive hydrogel loaded with M2 macrophage exosomes, characterized in that: The following steps are involved: S1. Extraction of exosomes from M2 macrophages, specifically: selecting exosome cells, inducing them into M2 macrophages, and extracting exosome solution; S2. Intermediate solution configuration, specifically: S201. Synthesis of oxidized dextran (OD); S202. Oxidized dextran (OD), carboxymethyl chitosan (CMC) and Pro-Val-Gly-Leu-Iso-Gly (PG-6) were selected, and OD, CMC and PG-6 solutions were prepared for standby use; S3. Preparation of MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes, specifically: S301. preparing a mixture of response peptides and M2 macrophage exosomes; S302. The OD solution is mixed with a mixture of the response peptide and exosomes, and then mixed with the CMC solution to obtain an MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes.

2. The preparation method according to claim 1, characterized in that: The extraction of exosomes from M2 macrophages in step S1 comprises the following steps: S101. RAW264.7 cells are selected as the source of exosome cells, and after subculture, the RAW264.7 cells are screened for cells in good condition, and interleukin (IL-4) is added to induce M2 macrophages; S102. Identify the phenotype of M2 macrophages. After confirming that M2 macrophages are successfully obtained, culture them continuously for 24 hours using a culture medium containing exosome-free serum, remove impurities such as cell debris, and collect the cell culture supernatant; S103. Take the cell culture supernatant obtained in S102, control the centrifugal acceleration to 10,000×g, the temperature to 4°C, centrifuge for 10 min, and transfer to a new centrifuge tube; S104. Vortex the bottled magnetic beads for 30 seconds, take out 350 μL and add it to a centrifuge tube. Centrifuge at 3,000 × g and 4°C for 2 minutes, and discard the supernatant. S105. Add 10 mL of phosphate buffered saline (PBS) precooled at 4°C and filtered through 0.22 μm, shake and mix for 30 seconds, control the centrifugal acceleration to 3,000 × g, centrifuge at 4°C for 5 minutes, and discard the supernatant; S106. Take and add 4 mL Buffer EXA, 1 mL Buffer EXB and 14.65 mL supernatant, place in a rotary mixer, and rotate and mix at 4°C for 40 min; S107. Remove the centrifuge tube, place it on a magnetic rack, and let it stand at 4°C for 10 min until the magnetic beads aggregate, then discard the supernatant; S108. For the objects retained in S107, centrifuge at 3,000 × g and 4°C for 1 min, and discard the residual liquid; S109. Add 0.5 mL of Buffer EXE and mix well. Centrifuge at 7,000 × g and 4 °C for 2 min. Transfer the supernatant to an EP tube. S110. Rinse the syringe filters A and B with EXE and filter the exosome solutions respectively to obtain purified exosome solutions, which are frozen and stored for future use.

3. The preparation method according to claim 1, characterized in that: The step S2 is the preparation of the intermediate solution, comprising the following steps: S201. Synthesizing oxidized dextran (OD), specifically comprising: ① Dissolve 5g of dextran in 250mL of distilled water to obtain Dex solution; ② NaIO4 was dissolved in 5 mL of distilled water and then added dropwise to the Dex solution, and the mixture was stirred at room temperature in the dark for 3.5 h; ③ 3 mL of ethylene glycol was added to the mixture to react the unreacted NaIO4, and the mixture was stirred for another 1 h. The resulting product was dialyzed against distilled water (MWCO 1400 Da) for 3 days and lyophilized to obtain synthetic oxidized dextran (OD); S202. Oxidized dextran (OD), carboxymethyl chitosan (CMC) and Pro-Val-Gly-Leu-Iso-Gly (PG-6) are selected respectively, and a 5% OD solution, a 3% CMC solution and a 20 mg / mL PG-6 solution are prepared respectively using a phosphate buffer solution (PBS) at pH = 7.

4. The specific steps are as follows: (1) Prepare 10 mL of 5% OD by adding 10 mL of PBS solution into a brown bottle containing 500 mg of OD and dissolve by ultrasonication / oscillation; (2) Prepare 10 mL of 3% CMC by adding 10 mL of PBS solution into a brown bottle containing 300 mg of CMC and dissolve it by ultrasonication / oscillation; (3) Prepare 5 mL of the response peptide with a concentration of 20 mg / mL by adding 5 mL of PBS solution to a brown bottle containing 100 mg of the response peptide and shaking to dissolve.

4. The preparation method according to claim 3, characterized in that: In step S202, the OD solution is prepared at a temperature of 20-25°C, and the CMC solution is prepared at a temperature of 45-60°C.

5. The preparation method according to claim 1, characterized in that: The step S3 is to prepare an MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes, and the specific contents are as follows: S301. Prepare a mixture of response peptides and M2 macrophage exosomes Add 0.7 mL of PBS solution and 0.5 mL of 3 × 10 10 particles / mL of M2 macrophage exosome solution, oscillate and dissolve to obtain 1.2mL of a mixed solution of the response peptide and M2 macrophage exosomes; S302. Preparation of MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes Take 300 μL of 5% OD solution, mix it with the mixture obtained from S201, and then mix it with 1.5 mL of 3% CMC solution. At room temperature, 3 mL of MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes can be cross-linked to obtain 5×10 exosomes per ml of hydrogel. 9 particles.

6. The preparation method according to claim 5, characterized in that: The mixed solution of the response peptide and M2 macrophage exosomes in step S301 contains 1.5×10 10 particles, and the response peptide concentration was 20 mg / mL.

7. An MMP-9 responsive hydrogel loaded with M2 macrophage exosomes prepared according to the preparation method according to any one of claims 1 to 6.

8. Use of the MMP-9 responsive hydrogel loaded with M2 macrophage exosomes according to claim 7 in regulating the inflammatory response process of diabetic wounds and promoting the healing of diabetic wounds.

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