MMP-9 Responsive Hydrogel Loaded with M2 Macrophage Exosomes, Preparation Method and Application Thereof

Abstract

The present invention discloses an MMP-9 responsive hydrogel loaded with M2 macrophage exosomes, its preparation method and application. By constructing a macrophage polarization model, exosomes derived from M2 macrophages (M2-Exos) are obtained. At the same time, the MMP-9 responsive hydrogel is used to load and achieve the controlled release of M2-Exos for wound inflammation regulation. By mixing oxidized dextran with the responsive peptide Pro-Val-Gly-Leu-Iso-Gly and carboxymethyl chitosan in a certain proportion and crosslinking, a Schiff base gel responsive to MMP-9 is obtained. M2-Exos are loaded into the hydrogel to obtain an MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes. This hydrogel can remain stable at low MMP-9 concentrations in the early stage of inflammation, while rapidly degrading and releasing its content M2-Exos when MMP-9 is significantly elevated in the late stage of inflammation, precisely regulating the inflammatory response process of diabetic wounds. The MMP-9 responsive hydrogel described in the present invention can effectively regulate inflammation, promote the healing of diabetic wounds, and reduce the physiological and psychological burdens caused by diabetic wounds to patients.

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 preparation, specifically:

[0009] S201. Synthesize oxidized dextran (OD);

[0010] S202. Select oxidized dextran (OD), carboxymethyl chitosan (CMC), and the response peptide Pro-Val-Gly-Leu-Iso-Gly (PG-6) respectively, and prepare OD, CMC, and PG-6 solutions respectively for standby;

[0011] S3. Fabricate an MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes, the specific content is:

[0012] S301. Prepare a mixture of the response peptide and M2 macrophage exosomes

[0013] Add 0.7 mL of PBS solution and 0.5 mL of M2 macrophage exosome solution with a concentration of 3×10 10 particles / mL to a brown bottle containing 24 mg of the response peptide, shake and dissolve to obtain 1.2 mL of the mixture of the response peptide and M2 macrophage exosomes;

[0014] S302. Preparation of an MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes

[0015] Take 300 μL of 5% OD solution, mix it evenly with the mixture obtained in S301, and then mix it evenly with 1.5 mL of 3% CMC solution. At room temperature, crosslinking can obtain 3 mL of an MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes, and each milliliter of the hydrogel contains 5×10 9 particles.

[0016] Furthermore, the extraction of M2 macrophage-derived exosomes in the step S1 includes the following steps:

[0017] S101. Select RAW264.7 cells as the exosome cell source. After the RAW264.7 cells are passaged and cultured, cells in good condition are screened out, and interleukin (IL-4) is added to induce M2 macrophages;

[0018] S102. Identify the M2 macrophage phenotype. After confirming that M2 macrophages are successfully obtained, continuously culture with a medium containing exosome-free serum for 24 hours, remove cell debris impurities, and collect the cell culture supernatant;

[0019] 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 it to a new centrifuge tube;

[0020] S104. Shake the bottled magnetic beads evenly for 30 s, take 350 μL and add it to the centrifuge tube, control the centrifugal acceleration at 3,000×g, centrifuge at 4°C for 2 min, and discard the supernatant;

[0021] S105. Add 10 mL of phosphate buffered saline (PBS) pre-cooled at 4°C and filtered through 0.22 μm, shake and mix evenly for 30 s, control the centrifugal acceleration at 3,000×g, centrifuge at 4°C for 5 min, and discard the supernatant;

[0022] S106. Take and add 4 mL of BufferEXA, 1 mL of Buffer EXB and 14.65 mL of supernatant, place it in a rotary mixer, and mix at 4°C for 40 min;

[0023] S107. Remove the centrifuge tube, place it on a magnetic stand, let it stand at 4°C for 10 min, wait for the magnetic beads to aggregate, and discard the supernatant;

[0024] S108. For the object retained in S107, control the centrifugal acceleration at 3,000×g, centrifuge at 4°C for 1 min, and discard the residual liquid;

[0025] S109. Add 0.5 mL of BufferEXE and mix evenly, control the centrifugal acceleration at 7,000×g, centrifuge at 4°C for 2 min, and transfer the supernatant to an EP tube;

[0026] S110. After rinsing the needle filters A and B with EXE, filter the exosome solution respectively to obtain a purified exosome solution, freeze and store for later use.

[0027] Further, the step S2 is the preparation of the intermediate solution, including the following steps:

[0028] S201. Synthesize oxidized dextran (OD), specifically including:

[0029] ① Dissolve 5 g of dextran in 250 mL of distilled water to obtain a Dex solution;

[0030] ② Dissolve NaIO 4 in 5 mL of distilled water, then add it dropwise to the Dex solution, and then stir the mixture in the dark at room temperature for 3.5 h;

[0031] ③ Add 3 mL of ethylene glycol to the mixture to make the unreacted NaIO 4React, stir the mixture for another 1 h, dialyze the obtained product against distilled water for 3 days and lyophilize it, with a MWCO of 1400 Da, to obtain synthetic oxidized dextran (OD).

[0032] S202. Select oxidized dextran (OD), carboxymethyl chitosan (CMC), and Pro-Val-Gly-Leu-Iso-Gly (PG-6) respectively, and prepare a 5% OD solution, a 3% CMC solution, and a 20 mg / mL PG-6 solution respectively with phosphate buffer solution (PBS) at pH = 7.4. The specific steps are as follows:

[0033] (1) Prepare 10 mL of a 5% OD solution, that is, add 10 mL of PBS solution to a brown bottle containing 500 mg of OD, and dissolve it by ultrasonic oscillation to obtain it;

[0034] (2) Prepare 10 mL of a 3% CMC solution, that is, add 10 mL of PBS solution to a brown bottle containing 300 mg of CMC, and dissolve it by ultrasonic oscillation to obtain it;

[0035] (3) Prepare 5 mL of a 20 mg / mL response peptide solution, that is, add 5 mL of PBS solution to a brown bottle containing 100 mg of the response peptide, and dissolve it by oscillation to obtain it.

[0036] Furthermore, in step S202, the preparation temperature of the OD solution is 20 - 25 °C, and the preparation temperature of the CMC solution is 45 - 60 °C.

[0037] Furthermore, in step S301, the mixture of the response peptide and M2 macrophage exosomes contains 1.5×10 10 particles of exosomes, and the concentration of the response peptide is 20 mg / mL.

[0038] The second object of the present invention is to provide an MMP-9 responsive hydrogel loaded with M2 macrophage exosomes prepared by the above preparation method.

[0039] The third object of the present invention is to provide the application of the MMP-9 responsive hydrogel loaded with M2 macrophage exosomes in the preparation of drugs for regulating the inflammatory reaction process of diabetic wounds and promoting the healing of diabetic wounds.

[0040] The advantages of the present invention compared with the prior art are as follows:

[0041] 1) Based on exosomes, the present invention aims to transform the pro-inflammatory M1 macrophages that are excessively aggregated in the wound into anti-inflammatory M2 macrophages through exosome-guided cell reprogramming technology, improve the inflammatory microenvironment of diabetic wounds, and effectively promote wound healing;

[0042] 2) The present invention combines M2 macrophage exosomes with an MMP-9 responsive hydrogel to prepare an intelligent hydrogel dressing that can intelligently respond to the inflammatory reaction process according to the change in the content of MMP-9 in the wound surface. This dressing can remain stable in the low-MMP-9 environment of the wound surface in the early stage of inflammation, while rapidly degrading and releasing its content M2-Exos when MMP-9 increases significantly in the late stage of inflammation, achieving precise regulation of the inflammatory reaction process of diabetic wounds. At the same time, it also solves the problems faced by exosomes in vitro, such as short half-life and instability, and improves the bioavailability of exosomes. Brief Description of the Drawings

[0043] Figure 1 is the process flow chart of the preparation process and application of the present invention;

[0044] Figure 2 is the SEM (a), NTA (b) and WB (c) identification result diagrams of the M2 macrophage exosomes extracted in Example 1 of the present invention;

[0045] Figure 3 is the SEM diagram of the MMP-9 responsive hydrogel in Example 4 of the present invention;

[0046] Figure 4 is the responsive degradation diagram (a) and statistical result (b) of the MMP-9 responsive hydrogel in Example 4 of the present invention;

[0047] Figure 5 is the diagram (a) of the MMP-9 responsive hydrogel releasing exosomes in vitro in Example 6 of the present invention, and the internalization of the released exosomes by M1 macrophages (b);

[0048] Figure 6 is the diagram (a) of the clearance of exosomes on one side of the wound surface of diabetic mice by the MMP-9 responsive hydrogel in Example 6 of the present invention and the statistical result (b);

[0049] Figure 7 is the promotion of wound healing effect (a) and the statistical result of wound healing rate (b) when the MMP-9 responsive hydrogel is applied to diabetic mice in Example 7 of the present invention;

[0050] Figure 8 is the promotion of epithelial regeneration and anti-inflammatory effect of the MMP-9 responsive hydrogel on the wound surface of diabetic mice in Example 7 of the present invention. Figure (a) is the HE and Masson staining results of the wound tissue, Figure (b) is the immunofluorescence staining result of the wound surface, and Figure (c) is the statistical result. Detailed Embodiments

[0051] The following further elaborates on the MMP-9 responsive hydrogel loaded with M2 macrophage exosomes of the present invention, its preparation method and application in conjunction with the accompanying drawings and embodiments.

[0052] Example 1

[0053] Preparation and extraction of M2 macrophage-derived exosomes (M2-Exos), including the following steps:

[0054] Step 1) Culture RAW264.7 cells in a high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in an incubator at 37°C and 5% CO2. After adding IL-4 (20 ng / mL, Peprotech) to the medium for 24 h, collect the supernatant;

[0055] Step 2) Sample treatment: Control the centrifugal acceleration of the supernatant at 10,000×g and centrifuge at 4°C for 10 min, then transfer it to a new centrifuge tube;

[0056] Step 3) Magnetic bead preparation: Shake the bottled magnetic beads evenly for 30 s, take 350 μL and add it to the centrifuge tube, control the centrifugal acceleration at 3,000×g and centrifuge at 4°C for 2 min, then discard the supernatant.

[0057] Step 4) Wash the magnetic beads: Add 10 mL of PBS pre-cooled at 4°C and filtered through 0.22 μm, shake evenly for 30 s, control the centrifugal acceleration at 3,000×g and centrifuge at 4°C for 5 min, then discard the supernatant.

[0058] Step 5) Exosome adsorption: Add 4 mL of Buffer EXA, 1 mL of Buffer EXB and 14.65 mL of supernatant, place it in a rotary mixer, and mix at 4°C for 40 min.

[0059] Step 6) Magnetic bead collection: 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.

[0060] Step 7) Control the centrifugal acceleration at 3,000×g and centrifuge at 4°C for 1 min, then discard the residual liquid.

[0061] Step 8) Elute the exosomes: Add 0.5 mL of Buffer EXE and mix evenly, control the centrifugal acceleration at 7,000×g and centrifuge at 4°C for 2 min, then transfer the supernatant to an EP tube.

[0062] Step 9) Filter out impurities: Rinse the syringe filters A and B with EXE and then filter the exosome solution respectively to obtain a purified exosome solution, which can be used immediately or stored frozen for later use.

[0063] Example 2

[0064] For the exosomes obtained in Example 1, the following related identifications were carried out:

[0065] 1. Electron microscopy identification (SEM)

[0066] (1) Take 10 μL of the exosome solution and drop it on a copper grid. Incubate at room temperature for 10 min, wash with sterile distilled water, and blot dry the excess liquid with absorbent paper;

[0067] (2) Pipette 10 μL of 2% uranyl acetate and drop it on the copper grid for negative staining for 1 min. Blot off the floating liquid with filter paper and dry it under an incandescent lamp for 2 min;

[0068] (3) Place the copper grid under a transmission electron microscope and observe it at 80 kV for imaging.

[0069] 2. NTA (nanoparticle tracking analysis)

[0070] Take the nanoparticle suspension irradiated with a laser light source and detect the scattered light of the nanoparticles. Calculate the nanoparticle concentration by counting the number of scattered particles. That is, use the particle matrix ZetaView PMX 110 to measure the concentration of the isolated exosomes under the emission light of 405 nm, dilute the exosomes with PBS to 1x10 7 particles / mL~1x10 9 particles / mL, and measure its size and mass. At the same time, analyze the particle movement trajectory of the exosomes.

[0071] 3. Exosome protein extraction

[0072] (1) Take 50 μL of exosome lysis solution and 150 μL of exosome solution in a ratio of 1:3 and mix well. Take 1 mL of cell suspension and centrifuge it. Add 1 mL of lysis solution to the cell pellet and mix well, then boil at 100 °C for 5 min.

[0073] (2) Transfer it to an ice-water mixture and cool for 5 min. Control the centrifugal acceleration at 12,000×g and centrifuge at 4 °C for 5 min. Transfer the supernatant to a new EP tube.

[0074] 4. Western Blot detection

[0075] (1) Protein concentration determination.

[0076] Determine the exosome protein concentration according to the instructions of the BCA method protein concentration determination kit. The brief process is as follows: Mix solution A and solution B in the kit in a ratio of 50:1 to prepare the working solution. Dilute the protein standard product to 0.5 mg / mL with sterile ddH 2 O, and then use ddH 2The standard product was diluted by a factor of O. The diluted standard product and the sample were added to a 96-well plate, and then 200 μL of the prepared working solution was added to each well. Incubate at 37 °C for 30 min. The absorbance value was detected at 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader. A standard curve was plotted, and the protein concentration in the sample was calculated using the regression equation.

[0077] (2) Electrophoresis and transfer.

[0078] Using a polyacrylamide gel preparation kit, prepare an SDS-PAGE gel with an appropriate concentration according to the molecular weight of the target antibody. Calculate the required sample volume based on the protein concentration in the sample, add it to the polyacrylamide gel wells, transfer it to an electrophoresis tank, and perform electrophoresis. Electrophoresis was carried out at a constant voltage of 80 V until the indicator (bromophenol blue) in the sample entered the separating gel, and then at a constant voltage of 120 V until the indicator reached the bottom of the gel. Stop electrophoresis, peel off the polyacrylamide gel, cut off the stacking gel, retain the separating gel, place it on a PVDF membrane, and clamp the filter paper and sponge to prepare a sandwich structure. Insert it into an electrotransfer tank, add electrotransfer solution, and perform electrotransfer at a constant current of 200 mA for 2 h. Take out the PVDF membrane, wash the membrane 3 times with TBST for 5 min each time, and then place it in 5% skim milk powder (prepared with TBST) at room temperature for 1 h of blocking.

[0079] (3) Antibody incubation

[0080] Primary antibody incubation: Take out the PVDF membrane from the blocking solution, wash it 3 times with TBST for 5 min each time, and transfer it to the prepared primary antibody solution. Incubate overnight on a horizontal shaker at 4 °C.

[0081] Secondary antibody incubation: Wash the membrane 3 times with TBST for 5 min each time, and transfer it to the prepared secondary antibody solution. The secondary antibody was diluted with 5% skim milk powder (prepared with TBST). Incubate at room temperature for 1.5 h.

[0082]

[0083] (4) Development and photography

[0084] (a) Washing: After secondary antibody incubation, wash the PVDF membrane 3 times with TBST for 5 min each time.

[0085] (b) Lift the PVDF membrane and drain the excess washing solution on the membrane.

[0086] (c) Place the membrane flat on the development plate, add the prepared ECL reaction solution dropwise, and put it into a chemiluminescence imager.

[0087] (d) Photography: Prepare the luminescent solution, place the strip in an exposure machine (GE company), drop the luminescent solution for exposure, and collect the image. If necessary, the Image J software can be used to analyze the gray value of the protein strip.

[0088] The results are asFigure 1 As shown, the purified exosomes are saucer-shaped or hemispherical with a concave side, with an average particle size of 121.1 nm and a concentration of 3.0*10 10 particles / mL. The WB results show that the purified exosomes express TSG101 and CD9, but do not express the endoplasmic reticulum-specific marker calnexin and the classical activation phenotype markers iNOS and Arginase of macrophages, confirming that the extracted extracellular vesicles are exosomes.

[0089] Preparation of the intermediate solution and MMP-9-responsive hydrogel in Example 3

[0090] The preparation of the intermediate solution includes the following:

[0091] 1. Synthesis of oxidized dextran (OD)

[0092] Oxidize dextran (Dex) with NaIO4 to obtain oxidized dextran OD. Dissolve 5 g of dextran in 250 mL of distilled water. Dissolve NaIO4 in 5 mL of distilled water, and then add it dropwise to the Dex solution. Then stir the mixture in the dark at room temperature for 3.5 h. Finally, add 3 mL of ethylene glycol to the mixture to react with the unreacted NaIO 4 React. Stir the mixture for another 1 h. Dialyze the resulting product against distilled water for 3 days and freeze-dry it, with an MWCO of 1400 Da to obtain OD.

[0093] 2. Prepare a 5% OD solution, a 1.5% CMC solution, and a 20 mg / mL PG-6 solution respectively with PBS solution at pH = 7.4.

[0094] (1) Prepare 10 mL of a 5% OD solution, that is, add 10 mL of PBS solution to a brown bottle containing 500 mg of OD, and dissolve it by ultrasonic / oscillation. During the process, the temperature should not exceed 25 °C;

[0095] (2) Prepare 10 mL of a 3% CMC solution, that is, add 10 mL of PBS solution to a brown bottle containing 300 mg of CMC, with a temperature not exceeding 60 °C, and dissolve it by ultrasonic / oscillation;

[0096] (3) Prepare 5 mL of a 20 mg / mL response peptide solution, that is, add 5 mL of PBS solution to a brown bottle containing 100 mg of the response peptide, and dissolve it by oscillation.

[0097] The preparation method of the MMP-9 responsive hydrogel is as follows: At room temperature, 100 μL of 5% OD is mixed evenly with 400 μL of 20 mg / mL responsive peptide to form solution A, and then it is mixed evenly with 500 μL of 3% CMC (solution B) to prepare the MMP-9 responsive hydrogel; among them, the preferred raw material ratio of the hydrogel in the present invention is: 100 mL of 5% OD + 400 mL of 2% PG-6 + 500 mL of 1.5% CMC.

[0098] Example 4

[0099] The MMP-9 responsive hydrogel obtained in Example 3 was characterized and analyzed, including the following contents:

[0100] 1. Scanning / transmission microscope (SEM)

[0101] (1) Sample preparation:

[0102] Gel sample preparation: The freshly prepared gel sample was cut into a square gel block of 1*1*1 cm 3 , a certain amount of distilled water was added, and it was left standing in a 4°C refrigerator overnight. The sample after swelling equilibrium was quickly frozen in liquid nitrogen for 30 min, and then immediately placed in a freeze dryer for drying for 24 h. The bulk gel sample was directly adhered to the conductive adhesive.

[0103] (2) Gold spraying test:

[0104] Use an Oxford Quorum SC7620 sputtering coater to spray gold for 45 s with a gold spraying current of 10 mA; then use a TESCAN MIRA LMS scanning electron microscope to take pictures of the sample morphology, and the acceleration voltage is 3 kV during the morphology shooting.

[0105] The results are as Figure 3 shown, and the hydrogel shows a cross-linked three-dimensional network structure.

[0106] 2. Gel degradation analysis

[0107] Phosphate buffer solution (PBS) and 10, 20, 50 ng / mL MMP-9 responsive peptide solutions were respectively added to the hydrogel samples of constant weight, and they were placed in a 37°C incubator for soaking. The change in the weight of the hydrogel samples after soaking for different times was measured, 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:

[0108] Degradation rate (%) = (W 0 - W t ) / W 0 ×100%

[0109] where W 0 is the initial weight of the hydrogel, and Wt is the weight of the hydrogel measured at different soaking times.

[0110] The preferred raw material ratio of the hydrogel in the present invention is: 100 mL of 5% OD + 400 mL of 2% PG-6 + 500 mL of 1.5% CMC.

[0111] The responsive degradation of the MMP-9 responsive hydrogel is as Figure 4 shown. In PBS solution, the state of the hydrogel is basically stable within 7 days. After adding MMP-9 on the 7th day, the degradation rate of the hydrogel significantly increases. Especially during the 7th - 10th day, the degradation rate of the 50 ng / mL MMP-9 concentration group is significantly faster than other groups. At 21 d, the degradation rates of the hydrogels in the PBS, 10 ng / mL, 20 ng / mL, and 50 ng / mL MMP-9 groups are 40%, 47%, 49%, and 60% respectively.

[0112] Preparation of the MMP-9 Responsive Hydrogel Loaded with M2 Macrophage Exosomes in Example 5

[0113] Based on the method described in Example 3, the preferred raw material ratio of the hydrogel in this example is: 100 mL of 5% OD + 400 mL of 2% PG-6 + 500 mL of 1.5% CMC. In this example, when preparing the responsive peptide solution, it is dissolved with M2 macrophage exosomes. The specific steps are as follows:

[0114] (1) Prepare a 1.2 mL mixture of the responsive peptide and M2 macrophage exosomes, that is, add 0.7 mL of PBS solution and 0.5 mL of M2 macrophage exosome solution with a concentration of 3×10 10 particles / mL to the brown bottle containing 24 mg of the responsive peptide, and shake to dissolve. Among them, there are 1.5×10 10 particles of exosomes, and the concentration of the responsive peptide is still 20 mg / mL.

[0115] (2) Take 300 mL of 5% OD and mix it evenly with the 1.2 mL mixture of the responsive peptide and exosomes, and then mix it evenly with 1.5 mL of 3% CMC. At room temperature, 3 mL of the MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes can be crosslinked, and each milliliter of the hydrogel contains 5×10 9 particles of exosomes.

[0116] Evaluation of the Responsive Degradation Function of the MMP-9 Responsive Hydrogel Loaded with M2 Macrophage Exosomes in Example 6

[0117] 1. In vitro evaluation:

[0118] Exosomes containing fluorescent labels were prepared by Dil staining. Dil-labeled M2 macrophage exosomes were spread on the bottom of a 24-well plate. Medium without exosomes and different concentrations of MMP-9 were added, and after co-incubation for 24 h, the supernatant was extracted. After filtration through a 0.22 mm filter, the supernatant was added to M1 macrophages and cultured for another 24 h to observe the internalization of exosomes and the polarization of M1 under different concentrations of MMP-9. NTA was performed on the collected supernatant, and the results showed that the exosome content was the highest in the 50 ng / mL group ( Figure 5 a). Meanwhile, immunofluorescence showed that the internalization of exosomes was most significant in the group with an MMP-9 concentration of 50 ng / mL, with significant differences compared to the control group and the 10 ng / mL group ( Figure 5 b).

[0119] 2. In vivo evaluation:

[0120] Establishment of a diabetic wound model in mice:

[0121] (1) Anesthesia: Ten 8-week-old male diabetic mice were intraperitoneally injected with 1% sodium pentobarbital solution for anesthesia (administered at a dose of 0.1 mL / 20 g);

[0122] (2) Surgery: After anesthesia, the hair on the back of the mice was removed. Using a trephine punch with a diameter of 10 mm, marks were made on both sides of the back of the mice at a position 1 cm from the spinal midline, and the skin at the marked sites was excised full-thickness;

[0123] Postoperative drug administration for diabetic wounds in mice: On the day of modeling, drugs were administered to the wounds on both sides of the back of each mouse. On one side, 25 μL of Dil-labeled M2-Exos was subcutaneously injected around the wound margin at 3, 6, 9, and 12 o'clock using a microsyringe, for a total of 100 μL; on the other side, the wound was covered with 100 μL of the synthesized hydrogel. The hydrogel was loaded with an equal amount of Dil-labeled M2 macrophage exosomes.

[0124] Evaluation of the sustained-release effect of M2 macrophage exosomes on diabetic wounds in mice: Selecting 1 day, 3 days, 7 days, and 10 days after surgery as time points, the mice were anesthetized again and placed in a small animal in vivo imager to detect the red fluorescence distribution and intensity on the wounds on both sides of the back.

[0125] The results showed that after four-point injection of free M2-Exos, the fluorescence signal was gradually cleared within 3 days, but a large amount of M2-Exos released from the MMP-9-responsive hydrogel loaded with M2 macrophage exosomes was still retained on the 7th day ( Figure 6 a). This result indicates that the 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.

[0126] Example 7 In Vivo Application and Evaluation of Therapeutic Effect of MMP-9 Responsive Hydrogel Loaded with M2 Macrophage Exosomes

[0127] In this example, male diabetic db / db mice for scientific research were used, purchased from Weishang Lide (Beijing), and approved by the Animal Ethics Committee.

[0128] 1. Model establishment: Seventy-two 7-week-old male diabetic mice were divided into 4 groups, namely the PBS group, the simple exosome injection group (Exo group), the simple MRH use group (MRH group), and the hydrogel-loaded exosome group (Exo@MRH group), with 18 mice in each group.

[0129] 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 mice was shaved, and a circular punch with a diameter of 10 mm was used to mark at a position 1 cm from the spinal midline on both sides of the back of the mice, and the skin at the marked area was subjected to full-thickness resection.

[0130] 2. Postoperative administration: The PBS group was injected with a total of 100 μL of PBS around the wound by subcutaneous four-point injection method;

[0131] The Exo group was injected with 100 μL of M2-exo in the same way; the MRH group and the Exo@MRH group used 100 μL of the synthesized hydrogel to cover the wound, and the Exo@MRH group with 100 μL loaded the same amount of Exo as the Exo group.

[0132] The wounds of all four groups were covered with sterile transparent films to keep the wounds moist.

[0133] 3. Photograph and record: On the 1st, 3rd, 7th, 10th, 14th, and 21st days after surgery, the wound healing conditions of all wounds of the mice in each group were observed and photographed; the initial area of the surgical wound was recorded as A 0 , and the actual wound area on the nth day was A t . According to the formula:

[0134] Wound healing rate = (A 0 - A t ) / A 0 × 100%

[0135] The wound area was calculated using Image J software, and the wound healing curves of the mice in each group were plotted ( Figure 7 b);

[0136] 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, while the wound healing rate 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 the faster healing of diabetic wounds( Figure 7 a), and the difference was statistically significant (P < 0.05).

[0137] 4. Histological analysis:

[0138] On the 1st, 3rd, 7th, 10th, 14th, and 21st days after surgery, 3 mice in each group were sacrificed by cervical dislocation. A circular incision was made with the wound center as the center at 1 cm from the wound edge to cut off the full-thickness skin of the mice. The tissues were fixed with 4% paraformaldehyde, embedded, and sectioned. HE staining and Masson staining were performed.

[0139] The HE results showed that the narrowest scar width was observed in the Exo@MRH group on the 7th, 10th, and 14th days after injury. On the 14th day, the wounds in the Exo@MRH group were all closed. Compared with the control group, the newly formed epithelium in the Exo@MRH group was arranged neatly, and keratinocytes were evenly distributed throughout the epithelium, achieving epithelial reformation. The results of Masson staining showed that compared with the control group, the dermis in the Exo@MRH group was thinner and flatter, and the density of collagen fibers was lower and the arrangement was more regular( Figure 8 a).

[0140] 5. Immunofluorescence of wound tissues: Tissue sections at each time point were obtained according to step 4 described above, and CD86 / CD206 immunofluorescence co-staining of the tissues 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 expression of CD86 was significantly reduced on the 14th day, indicating that Exo@MRH could effectively intervene M1 macrophages into M2 cells, reverse the excessive inflammatory reaction in diabetic wounds, and promote the smooth progress of the wound repair process( Figure 8 b and Figure 8 c).

[0141] The working principle of the present invention is to mix oxidized dextran (OD), short peptide Pro-Val-Gly-Leu-Iso-Gly (PG-6), and carboxymethyl chitosan (CMC) in a certain proportion and crosslink them to obtain a Schiff base gel responsive to MMP-9. Load M2-Exos into the hydrogel to obtain an MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes, and prepare an intelligent hydrogel dressing that can intelligently respond to the inflammatory reaction process according to the change in the MMP-9 content in the wound surface. This dressing can remain stable in the low MMP-9 environment of the wound surface in the early stage of inflammation, while rapidly degrading and releasing its content M2-Exos when MMP-9 increases significantly in the late stage of inflammation, achieving precise regulation of the inflammatory reaction 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.

[0142] The above description of the present invention and its implementation manners is not restrictive. What is shown in the drawings is only one of the implementation manners of the present invention, and the actual structure is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar structural manners and embodiments without creative efforts without departing from the spirit of the present invention, they shall fall within 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. Select oxidized dextran (OD), carboxymethyl chitosan (CMC) and response peptide Pro-Val-Gly-Leu-Iso-Gly (PG-6), respectively, and prepare OD, CMC and PG-6 solutions respectively for use; S3. Preparation of MMP-9 responsive hydrogel loaded with M2 macrophage-derived exosomes, specifically: S301. Preparation of 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 1.2 mL of a mixed solution of response peptide and M2 macrophage exosome; 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 S301, 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.

2. 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 5 g of dextran in 250 mL 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, the mixture was stirred for another 1 h, and the resulting product was dialyzed against distilled water for 3 days and lyophilized, wherein the MWCO was 1400 Da, to obtain synthetic oxidized dextran (OD); S202. Select oxidized dextran (OD), carboxymethyl chitosan (CMC) and Pro-Val-Gly-Leu-Iso-Gly (PG-6) respectively, and use phosphate buffer solution (PBS) with pH=7.4 to prepare 5% OD solution, 3% CMC solution and 20 mg / mL PG-6 solution respectively. 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 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 CMC and dissolve it by ultrasonication / oscillation. (3) Prepare 5 mL of the response peptide at 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.

3. The preparation method according to claim 2, 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.

4. The preparation method according to claim 1, 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.

5. An MMP-9 responsive hydrogel loaded with M2 macrophage exosomes prepared according to the preparation method according to any one of claims 1 to 4.

6. Use of the MMP-9 responsive hydrogel loaded with M2 macrophage exosomes according to claim 5 in the preparation of a drug for regulating the inflammatory response process of diabetic wounds and promoting the healing of diabetic wounds.

Images