CN116271223B - Preparation method and application of coupled exosome hydrogel based on biotin-avidin system - Google Patents
Preparation method and application of coupled exosome hydrogel based on biotin-avidin system Download PDFInfo
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- CN116271223B CN116271223B CN202310327414.4A CN202310327414A CN116271223B CN 116271223 B CN116271223 B CN 116271223B CN 202310327414 A CN202310327414 A CN 202310327414A CN 116271223 B CN116271223 B CN 116271223B
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- biotin
- avidin
- stem cell
- exosome
- gelatin
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Abstract
The invention discloses a preparation method and application of coupled exosome hydrogel based on a biotin-avidin system, belonging to the field of tissue engineering scaffold materials, wherein the preparation method comprises the following steps: adding the stem cell exosome and biotin into a first solvent for a first mixing reaction to obtain a biotin-marked stem cell exosome; adding gelatin and activated biotin into a second solvent for a second mixing reaction, and then dialyzing to obtain biotinylated gelatin; carrying out a third mixing reaction on the stem cell exosomes marked by biotin and avidin to obtain avidin-treated stem cell exosomes; and carrying out a fourth mixing reaction on the avidin-treated stem cell exosomes and the biotinylated gelatin to obtain the hydrogel coupled with the exosomes. According to the invention, the stem cells EXs are anchored on the methacrylamide gel, so that the residence time of the EXs in the defect part is prolonged, and the regeneration efficiency and the bioavailability of the EXs are improved.
Description
Technical Field
The invention relates to the field of tissue engineering scaffold materials, in particular to a preparation method and application of coupled exosome hydrogel based on a biotin-avidin system.
Background
Periodontitis is a chronic inflammatory disease that occurs in periodontal tissues and is characterized by progressive destruction of the tooth support structure with extremely high incidence. Periodontal tissue loss due to advanced periodontitis is a major cause of tooth loss in adults, and no effective treatment is currently available. Traditional periodontal treatment means (such as cleaning/scraping and flap turning, etc.) are used for controlling the development of inflammation and preventing the progress of diseases by removing periodontal local plaque and tartar, but these treatment methods cannot reconstruct and regenerate the periodontal tissues which have been destroyed or lost. Existing periodontal regenerative treatment strategies include guided tissue regeneration and guided bone regeneration, and although periodontal tissue regeneration effects can be obtained to some extent, the indications of clinical treatment are narrow and the predictability is poor, and the clinical treatment requirements cannot be met. The introduction of new tissue engineering technology into periodontitis treatment becomes an important point and core of periodontal regeneration research in the new century, and brings new hope for the preservation of the suffering teeth of the late periodontitis.
In recent years, stem cell exosomes (Exsosomes, EXs) are used as one of research hotspots for stem cell therapy, and a new research idea is developed for stem cell therapy of periodontitis. EXs are vesicle vesicles of about 40-160nm diameter secreted by stem cells by exocytosis and are rich in bioactive substances such as mRNA, microRNA, DNA and proteins. Stem cell EXs plays an important role in the process of tissue regeneration, and can promote the recruitment of endogenous stem cells to damaged sites, improve the efficacy of recruiting stem cells and regulate and control immune response. The prior study shows that under the condition of not implanting exogenous stem cells, the regeneration of the alveolar bone and periodontal ligament of the rat can be obviously promoted by simply using stem cells EXs.
Although stem cell EXs has unique advantages and great application prospects in tissue regeneration, the stem cell EXs have low bioavailability, difficult extraction and clinical transformation still face multiple challenges. First, stem cells of systemic injection have non-uniform EXs biodistribution and short half-life. Stem cells ex injected systemically (subcutaneously, intravenously, intraperitoneally, etc.) will soon (< 2 hours) enrich into liver, spleen, lung, gastrointestinal, etc. areas and are cleared by macrophages of spleen and kidneys, so there is a small amount of ex reaching the defective areas. Local application and injection of stem cells EXs can increase the concentration of EXs, but EXs can be diluted quickly by blood or interstitial fluid. Secondly, stem cells EXs are combined with various cells mainly through membrane fusion, the targeting effect of the combination is limited, a large amount of EXs can be taken up by tissue cells in a non-target area, and the defect repairing function is difficult to play. In addition, extraction and purification of stem cell EXs are difficult, and the application cost is high. The ultracentrifugation method is the most widely and reliable method for extracting stem cells EXs, but 1mL of cell culture solution can only extract EXs of less than 1 mug, and is difficult to meet the number requirement of 10-100 mug/time required for repairing periodontal defects of mice. Whereas for humans, the dose required for treatment is often hundreds or thousands of times that of mice. Therefore, to achieve clinical transformation of stem cells EXs, particularly for treatment of non-lethal diseases with high incidence of periodontitis, it is necessary to significantly improve regeneration efficiency of stem cells EXs, so that a small amount of stem cells EXs can efficiently repair tissue defects.
Disclosure of Invention
The invention aims to provide a preparation method and application of a coupled exosome hydrogel based on a biotin-avidin system, which are used for solving the technical problems of low exosome bioavailability and poor regeneration efficiency.
In a first aspect, embodiments of the present application provide a method for preparing a coupled exosome hydrogel based on a biotin-avidin system, the method comprising:
Adding stem cell Exosomes (EXs) and Biotin into a first solvent to perform a first mixing reaction to obtain Biotin-labeled stem cell exosomes (Biotin-EXs);
adding gelatin and activated biotin into a second solvent for a second mixing reaction, and then dialyzing to obtain biotinylated gelatin;
Performing a third mixing reaction on the biotin-labeled stem cell exosomes and Avidin to obtain Avidin-EXs;
and carrying out a fourth mixing reaction on the avidin-treated stem cell exosomes and the biotinylated gelatin to obtain the hydrogel coupled with the exosomes.
Further, the stem cell exosomes include bone marrow mesenchymal stem cell exosomes (MSC-EXs), and the Biotin includes at least one of pegylated Biotin (DSPE-PEG-Biotin), DSPE-PEG-Biotin (MW 1000), DSPE-PEG-Biotin (MW 2000), and DSPE-PEG-Biotin (MW 5000).
Further, the gelatin comprises methacrylamide gelatin (GelMA), wherein the amino substitution degree of the methacrylamide gelatin is 50-65%, and the mass concentration is 8-12%; the activated Biotin comprises at least one of Biotin-dodecapolyethylene glycol-active ester (NHS-PEG 12-Biotin)、NHS-Biotin、NHS-PEG3 -Biotin and NHS-PEG 4 -Biotin).
Further, the method comprises the steps of, the avidin comprises streptavidin.
Further, the step of adding the stem cell exosome and biotin to a first solvent to perform a first mixing reaction to obtain a biotin-labeled stem cell exosome comprises:
Adding the stem cell exosome and the biotin into the first solvent for mixing to obtain a first mixed solution, and then reacting for 20-40 min at the temperature of 35-40 ℃ to obtain the biotin-labeled stem cell exosome;
Wherein, the mol concentration of biotin in the first mixed solution is 15-25 mu mol/L, the concentration of stem cell exosomes is 200-500ug/mL, and the particle size number is 1-4 multiplied by 10 11.
Further, the step of adding gelatin and activated biotin to a second solvent for a second mixing reaction, and then dialyzing to obtain biotinylated gelatin comprises the steps of:
Adding 0.5-1.5 mg/mL of the gelatin and 5.0-5.5 mg/mL of the activated biotin into the second solvent for mixing, reacting for 6-10 hours at room temperature, dialyzing for 24-48 hours, and freeze-drying to obtain the biotinylated gelatin;
wherein the mol ratio of the gelatin to the activated biotin is 1 (18-22).
Further, the step of performing a third mixing reaction of the biotin-labeled stem cell exosomes and avidin to obtain the avidin-labeled stem cell exosomes comprises:
Mixing the stem cell exosomes marked by biotin and the avidin, and reacting for 20-40 min at the temperature of 35-40 ℃ to obtain the avidin-treated stem cell exosomes;
Wherein the molar ratio of the stem cell exosomes labeled with biotin to the avidin is (1.5-2.5): 1.
Further, the step of subjecting the avid stem cell exosomes and the biotinylated gelatin to a fourth mixing reaction to obtain an exosome-coupled hydrogel comprises:
Mixing the stem cell exosomes subjected to avidin and the gelatin subjected to biotinylation, and reacting for 20-40 min at the temperature of 35-40 ℃ to obtain the hydrogel coupled with the exosomes.
In a second aspect, embodiments of the present application provide a coupled exosome hydrogel based on a biotin-avidin system, the coupled exosome hydrogel being made by the method of making described in the first aspect.
In a third aspect, embodiments of the present application provide a coupled exosome hydrogel prepared by the preparation method of the first aspect, and/or use of a coupled exosome hydrogel of the second aspect in the treatment and repair of bone defects.
Compared with the prior art, the scheme provided by the embodiment of the application has at least the following beneficial effects:
(1) The combination of biotin and avidin is one of the highest-intensity non-covalent combination known at present, is commonly used for qualitative and quantitative detection and positioning observation research of trace antigens and antibodies, and is covalently combined in a ratio of 1:4, and the affinity constant (K) is up to 1015mol/L, which is more than 1 ten thousand times higher than that of the general antigen-antibody reaction. And the combination of biotin and avidin has high stability, and the combination of acid, alkali, denaturant, high temperature and proteolytic enzyme and organic solvent is not interfered. In addition, the non-covalent binding based on biotin-avidin has no obvious influence on molecular activity, so that the biotin-avidin binding system can be firmly combined with materials on the premise of not reducing the activity of stem cell EVs, the retention time of EXs in the defect part is greatly prolonged, and the regeneration efficiency of EXs is improved.
(2) The binding of biotin to avidin can further enhance the crosslink density of gelatin such as GelMA, thereby improving the mechanical properties and degradability of GelMA.
(3) GelMA has good biocompatibility and photocuring performance. Collagen denatured product-gelatin in methacrylamide gelatin has better solubility and lower immunogenicity than collagen, and contains a large amount of RGD (arginine glycine aspartic acid) sequences, RGD peptide can be effectively and tightly combined with integrins on cell membranes to promote cell adhesion, is also a targeting sequence of matrix metalloproteinase, and is favorable for cell ingrowth and reconstruction to generate new tissues.
(4) The methacryloyl groups in GelMA impart photocrosslinking ability to the gel, improving the mechanical properties of gelatin. The substitution degree of different methacrylic groups can control the printing performance, degradation rate and hardness of the printing ink, and meet the requirements of different situations. The photocrosslinking property also contributes to our design of complex structures, more favorable for tissue regeneration.
In summary, the invention provides a preparation method and application of coupled exosome hydrogel based on a biotin-avidin system, which anchors stem cells EXs on, for example, methacrylamide gelatin (GelMA) to realize high-strength combination of the stem cells EXs and the gelatin, thereby achieving the purpose of integrating materials and EXs, prolonging the residence time of the EXs at the defect part and improving the regeneration efficiency and bioavailability of the EXs.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic flow chart of a preparation method of a coupled exosome hydrogel based on a biotin-avidin system and a Dialysis (Dialysis) process of Bio-GelMA according to an embodiment of the present invention; wherein, (a) schematic diagram of Bio-GelMA preparation, NHS-PEG 12 -biotin is grafted on the side chain of GelMA to synthesize biotinylated GelMA, and redundant NHS-PEG 12 -biotin is removed by dialysis. (b) During the dialysis process of Bio-GelMA, the concentration of biotin in the GelMA/NHS-PEG 12 -biotin mixture solution in the dialysis bag and in the dialysis solution outside the dialysis bag were measured over 3,6,12,24,36 hours.
FIG. 2 is a diagram showing the screening results of the synthesis conditions of Bio-GelMA; wherein (a) the biotin concentration of Bio-GelMA at different reaction times (1,6,8,10,12 hours); (b) Biotin concentrations of Bio-GelMA at different NHS-PEG 12 -biotin/GelMA molar ratios (5:1, 10:1,20:1, 40:1).
FIG. 3 is an infrared spectrum and an H 1 NMR spectrum of NHS-PEG 12 -biotin, gelMA, bio-GelMA; wherein, (a) a synthetic reaction of Bio-GelMA; (b) Infrared spectra of NHS-PEG 12 -biotin, gelMA, and Bio-GelMA; (c) GaussAmp multimodal fitting results of GelMA and Bio-GelMA infrared spectra; (d) H 1 NMR spectra of NHS-PEG 12 -biotin, gelMA, and Bio-GelMA.
FIG. 4 is a photo-curing property and a microstructure of GelMA and Bio-GelMA; (a) representative SEM pictures of GelMA and Bio-GelMA cross sections; (b) GelMA and Bio-GelMA were successfully crosslinked after 15s of blue light irradiation at 405 nm.
FIG. 5 is a graph of the identification result of MSC-EX; wherein, (a) representative transmission electron microscopy images of MSC-EX morphology, scale bar = 400 μm; (b) analyzing the particle size distribution of MSC-EX by a nano particle size tracker; (c) Western blot detects protein expression of HSP70, TSG101, CD63, GAPDH and beta-actin of MSC and MSC-EX; (d) The percent of EVs with diameters of 80nm,100nm,150nm,200nm and 300nm is analyzed by a nano-flow meter; (e) Nanoflow analysis of CD63 or CD81 positive MSC-EX ratios; (f) Cells take fluorescent pictures of PKH26 labeled MSC-EX, scale bar = 50 μm.
FIG. 6 is a graph showing the results of biotinylated modification of MSC-EX phospholipid bilayer membranes; wherein, (a) the MSC-EX positive rate after modified DSPE-PEG-FITC (10, 20, 50, 100 mu mol/L) with different concentrations is detected in a nano-flow mode; (b) Representative fluorescence plot of DSPE-PEG-FITC modified MSC-EX, scale bar = 10 μm; (c) And detecting the positive rate of MSC modified by DSPE-PEG-biotin (10, 20, 50 and 100 mu mol/L) with different concentrations in a nano-flow mode. (d) Representative fluorescence plot of DSPE-PEG-biotin modified MSC-EX, scale bar = 10 μm; (e) representative fluorescence maps of EX or Bio-EX uptake by cells; (f) Quantitative analysis of cell uptake of EX or Bio-EX by fluorescence intensity; results are expressed as mean ± standard deviation, ns indicating no significant difference (Student's t-test).
FIG. 7 is a graph showing the results of the retention and release rates of MSC-EX in Bio-GelMA@Bio-EX hydrogels; wherein, according to the mol ratio of biotin to avidin is 1: 1. 2:1 and 3:1, three Bio-GelMA@Bio-EX hydrogels of Bio-EX@Avidin (1:1), bio-EX@Avidin (2:1) and Bio-EX@Avidin (3:1) were synthesized and used as controls with the Bio-GelMA@EX hydrogels containing avidin and non-biotinylated modified EX. (a) Schematic representation of the synthesis of Bio-GelMA@Bio-EX hydrogels by anchoring Bio-EX to the Bio-GelMA side chains via the biotin-avidin system. (b) Representative fluorescence plots of hydrogels containing PKH-26 labeled EX after incubation in PBS for 0,1, 4 and 7 days, showing the residual exosome content in the hydrogels. (c) Fluorescence quantitative analysis of hydrogels containing PKH26 labeled EX, the ratio of fluorescence intensity was the ratio of fluorescence intensity at each time point (0, 1,4, and 7 days) to fluorescence intensity at 0d (n=5). (d) Cumulative release of EX after incubation of EX hydrogels in PBS for 1,4 and 7 days (n=6). (e) Representative in vivo imaging (f) after subcutaneous implantation of the DiR-labeled EX-containing hydrogels for days 0,1, 4,7 and 28 quantitatively analyzed EX retention in vivo by fluorescence intensity (photons/s/cm 2/sr/μw/cm 2); the ratio of fluorescence intensity is the ratio of fluorescence intensity at each time point (days 0,1, 4,7 and 28 post-operation) to the fluorescence intensity at day 0 (n=6). Data are expressed as mean ± standard deviation. * P <0.001, < P <0.01, and P <0.05 represent statistical differences between groups (one-way anova).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to specific examples.
The general idea of the technical scheme provided by the embodiment of the invention is as follows:
in a first aspect, embodiments of the present application provide a method for preparing a coupled exosome hydrogel based on a biotin-avidin system, the method comprising:
Adding stem cell Exosomes (EXs) and Biotin into a first solvent to perform a first mixing reaction to obtain Biotin-labeled stem cell exosomes (Biotin-EXs);
adding gelatin and activated biotin into a second solvent for a second mixing reaction, and then dialyzing to obtain biotinylated gelatin;
Performing a third mixing reaction on the biotin-labeled stem cell exosomes and Avidin to obtain Avidin-EXs;
and carrying out a fourth mixing reaction on the avidin-treated stem cell exosomes and the biotinylated gelatin to obtain the hydrogel coupled with the exosomes.
The good biological safety and multiple biological activities of the stem cells EXs avoid the risks brought by stem cell transplantation, and can exert the similar therapeutic effects as stem cell transplantation, so the stem cells EXs are hopefully replaced and are used for regenerating various tissues. However, due to the low extraction efficiency of stem cells EXs, short half-life and poor targeting, the stem cells EXs can be quickly cleared by macrophages in the liver, spleen and other places after being injected into a human body. Therefore, the embodiment of the application provides a preparation method of coupled exosome hydrogel based on a biotin-avidin system, which anchors stem cells EXs on, for example, methacrylamide gelatin (GelMA) and the like, so that the two are combined with each other in high strength, thereby achieving the aim of integrating materials and EXs, prolonging the residence time of the EXs at the defect part and improving the regeneration efficiency of the EXs. By designing the high-efficiency conveying system, the residence time of the stem cells EXs at the defect part is improved, the effective controlled release of the stem cells EXs at the periodontal defect part is realized, and the regeneration efficiency of the stem cells EXs is improved, so that the tissue regeneration requirement is better met.
In some embodiments, the first solvent and the second solvent may be selected from non-toxic media or solvents such as PBS, MEM, and the like.
As an implementation of the embodiment of the present application, the stem cell exosomes include bone marrow mesenchymal stem cell exosomes (MSC-EXs), and the Biotin includes at least one of pegylated Biotin (DSPE-PEG-Biotin), DSPE-PEG-Biotin (MW 1000), DSPE-PEG-Biotin (MW 2000), and DSPE-PEG-Biotin (MW 5000).
The exosome biotinylation modification strategy used in the present invention is not limited to BMMSCs source EXs, but is exemplified by BMMSCs source EXs.
The molecular formula of the selected pegylated Biotin (DSPE-PEG-Biotin) is C 275H543N4O125 P, which is saturated 18C phospholipid.
As an implementation mode of the embodiment of the application, the gelatin comprises methacrylamide gelatin, wherein the amino substitution degree of the methacrylamide gelatin is 50-65%, and the mass concentration is 8-12%; the activated Biotin comprises at least one of Biotin-dodecapolyethylene glycol-active ester (NHS-PEG 12-Biotin), NHS-Biotin, NHS-PEG3-Biotin and NHS-PEG 4-Biotin.
The biotinylation modification strategy used in the invention is not limited to GelMA, and is specifically described by taking GelMA as an example.
In some specific embodiments, the amino substitution of the methacrylamide gelatin is preferably 60% and the mass concentration is preferably 10%.
In some specific examples, the reaction concentration of Biotin-dodecapolyethylene glycol-active ester (NHS-PEG 12-Biotin) is 10. Mu. MoL.
As an embodiment of the present embodiment, the avidin comprises streptavidin.
The avidin in the present application includes streptavidin having a molecular weight of 66KD.
As an implementation manner of the embodiment of the present application, the step of adding the stem cell exosome and biotin to the first solvent to perform a first mixing reaction to obtain the biotin-labeled stem cell exosome includes:
Adding the stem cell exosome and the biotin into the first solvent for mixing to obtain a first mixed solution, and then reacting for 20-40 min at the temperature of 35-40 ℃ to obtain the biotin-labeled stem cell exosome;
Wherein, the mol concentration of biotin in the first mixed solution is 15-25 mu mol/L, the concentration of stem cell exosomes is 200-500ug/mL, and the particle size number is 1-4 multiplied by 10 11.
In some embodiments, the preferred reaction conditions for the first mixed reaction are: the temperature was 37℃and the stirring speed was 120prm, the reaction time was 30min.
In some embodiments, the molar concentration of biotin in the first mixed solution is preferably 20. Mu. Mol/L
As an embodiment of the present application, the step of adding gelatin and activated biotin to a second solvent to perform a second mixing reaction, and then performing dialysis to obtain biotinylated gelatin comprises:
Adding 0.5-1.5 mg/mL of the gelatin and 5.0-5.5 mg/mL of the activated biotin into the second solvent for mixing, reacting for 6-10 hours at room temperature, dialyzing for 24-48 hours, and freeze-drying to obtain the biotinylated gelatin;
wherein the mol ratio of the gelatin to the activated biotin is 1 (18-22).
In some embodiments, the preferred reaction conditions for the second mixed reaction are: 1mg/mL of GelMA was mixed with 5.3mg/mL of NHS-PEG12-Biotin (molar ratio: 1:20), reacted at room temperature for 8 hours, and then dialyzed for 36 hours using a dialysis bag, and the liquid in the bag was lyophilized to obtain biotinylated GelMA (Biotin-GelMA).
As an implementation manner of the embodiment of the present application, the step of performing a third mixing reaction on the biotin-labeled stem cell exosomes and avidin to obtain the avidin-labeled stem cell exosomes includes:
Mixing the stem cell exosomes marked by biotin and the avidin, and reacting for 20-40 min at the temperature of 35-40 ℃ to obtain the avidin-treated stem cell exosomes;
Wherein the molar ratio of the stem cell exosomes labeled with biotin to the avidin is (1.5-2.5): 1.
In some embodiments, the preferred reaction conditions for the third mixed reaction are: biotin-EXs and streptavidin were mixed (molar ratio 2:1) and reacted at 37℃for 30min to give Avidin-EXs (Avidin-EXs) to be reacted.
As an implementation manner of the embodiment of the present application, the step of performing a fourth mixing reaction on the avidin-converted stem cell exosomes and the biotinylated gelatin to obtain the exosome-coupled hydrogel includes:
Mixing the stem cell exosomes subjected to avidin and the gelatin subjected to biotinylation, and reacting for 20-40 min at the temperature of 35-40 ℃ to obtain the hydrogel coupled with the exosomes.
In some embodiments, the preferred reaction conditions for the fourth mixed reaction are: reacting Avidin-EXs with Biotin-GelMA at 37deg.C for 30min to obtain GelMA coupled with EXs.
It should be noted that DSPE-PEG-Biotin is a functional phospholipid-PEG derivative, and various subtypes thereof, such as DSPE-PEG-Biotin (MW 1000), DSPE-PEG-Biotin (MW 2000), and DSPE-PEG-Biotin (MW 5000), can be used in the schemes of the experiment. The NHS-PEG12-Biotin is an activated Biotin, and can be substituted with NHS-Biotin, NHS-PEG3-Biotin, NHS-PEG4-Biotin, etc.
In a second aspect, embodiments of the present application provide a coupled exosome hydrogel based on a biotin-avidin system, the coupled exosome hydrogel being made by the method of making described in the first aspect.
The biotin-avidin system is introduced into the controlled release design of stem cells EXs in the coupled exosome hydrogel based on the biotin-avidin system, the characteristics of high biotin-avidin binding strength, simplicity and convenience in modification and no influence on the biological activity of the stem cells EXs are utilized, biotin modification is respectively carried out on the stem cells EXs and GelMA gel, and then avidin is introduced, so that the biotin modified stem cells EXs and the biotin modified GelMA are combined, the integrated design of the GelMA and the stem cells EXs is realized, and periodontal tissue regeneration is promoted more efficiently. Meanwhile, since the hydrogel based on coupled exosomes of the biotin-avidin system provided by the embodiment of the present application is prepared by the preparation method described in the first aspect, it has at least the beneficial effects described in the first aspect, and the description of the present application is omitted.
In a third aspect, embodiments of the present application provide a coupled exosome hydrogel prepared by the preparation method of the first aspect, and/or use of a coupled exosome hydrogel of the second aspect in the treatment and repair of bone defects.
The GelMA coupled with EXs can be used for in-situ slow release of EXs, can be used for treating and repairing various bone defects, can also be used for mechanism research of exosomes, and has wide application prospect.
The features and capabilities of the present invention are described in further detail below in connection with the examples. The exosome biotinylation modification strategy used in the present invention is not limited to BMMSCs-derived EXs, but is exemplified by a biotinylation modification strategy such as BMMSCs-derived EXs, stem cell EXs, etc.
Unless otherwise specified, the reagents and equipment used in the examples below are all commercially available products. In addition, the statistical analyses involved in the examples below were all: the data obtained were first subjected to a normal distribution test and a variance alignment test, and the comparison between the two groups was performed using a two-tailed T-test (p=0.05).
Example 1 preparation of biotinylated GelMA and optimization of the reaction process
Synthesis of Bio-GelMA and optimization of Synthesis reaction conditions
1) GelMA was dissolved in PBS solution (1%, wt/vol), stirred continuously and heated to 60℃and cooled to room temperature after it was dissolved sufficiently. NHS-PEG12-biotin is dissolved in DMSO (5%, wt/vol) and mixed by shaking to make it fully dissolved at room temperature.
2) NHS-PEG12-biotin solution and GelMA solution were mixed and shaken for 60 seconds and reacted at room temperature for 1,6,8, 10, 12 hours at a ratio of 1:20, respectively, to determine the optimal reaction time. Likewise, gelMA solution and NHS-PEG12-biotin solution were mixed in different molar ratios (1:5, 1:10,1:20, 1:40) to determine the optimal reaction ratio.
3) The mixture solution obtained after the reaction is transferred to a 10kd dialysis bag and then transferred to double distilled water with 100 times of volume for dialysis and purification, unreacted NHS-PEG12-biotin is removed, and the dialysis time is 36 hours. To ensure complete removal of unreacted NHS-PEG12-biotin, the dialysis process was monitored by periodically changing and collecting the dialysis fluid outside the dialysis bag 5 times (3,6,12,24,36 hours) during dialysis and determining the concentration of biotin in the GelMA/NHS-PEG12-biotin mixture solution inside the dialysis bag and in the dialysis fluid outside the dialysis bag.
4) After dialysis purification, the obtained Bio-GelMA solution was frozen in a refrigerator at-20℃for 3 hours, at-80℃for 1 hour, then transferred to a vacuum freeze dryer, dried sufficiently for 24 hours, and the obtained solid Bio-GelMA was stored in a refrigerator at-20℃for subsequent experiments.
1.2 Characterization of Bio-GelMA
Infrared spectral data
Taking 2mg of GelMA, bio-GelMA and NHS-PEG12-biotin, uniformly mixing with 200mg of potassium bromide respectively, grinding, tabletting, placing the prepared sample into an infrared spectrometer to test a peak value of 400-4000cm < -1 >, drawing a map after the test is finished, and identifying the structures of three substances. The infrared spectrum data were normalized using OMNIC 8.0 software (Thermo FISHER SCIENTIFIC, waltham, MA, USA). The normalized data were fitted using PeakFit 4.12 software (Seasolve software inc., framingham, MA, USA), with the fitted model being a GaussAmp function model.
Nuclear magnetic resonance hydrogen spectrum
30Mg of GelMA, bio-GelMA, NHS-PEG12-biotin was dissolved in 0.6ml of DMSO-d6, transferred to a nuclear magnetic resonance tube, and the samples were placed in a nuclear magnetic resonance spectrometer for identification of the chemical structures of the three substances by H1 NMR.
Inversion method
0.2Ml of 10% (wt/vol) GelMA containing 0.25% (wt/vol) LAP and Bio-GelMA were separately charged into a glass bottle, and photographed at an inclination of 60℃to the horizontal. And (3) horizontally placing the photo-cured product after photo-taking, carrying out blue light irradiation curing for 10s by using a portable curing light source of 405nm, taking a photo-taking image in an inverted mode after the photo-taking image is 60 degrees relative to the horizontal plane, and comparing the photo-curing performances of GelMA and Bio-GelMA.
Scanning Electron Microscope (SEM)
And cutting the freeze-dried GelMA and Bio-GelMA hydrogel into a sample by using a sharp blade, placing the sample on a silicon wafer, fixing the sample by using a conductive adhesive tape, and spraying gold for 90 seconds. After the end of the metal spraying, the sample was placed in a scanning chamber, and the microstructure of GelMA and Bio-GelMA was observed with SEM and photographed.
1.3 Results
1) Synthesis of Bio-GelMA
After reacting NHS-PEG12-biotin with GelMA, the excess NHS-PEG12-biotin was removed by dialysis, and the preparation procedure is shown in FIG. 1-a. The monitoring result of the dialysis process is shown in FIG. 1-b, and after 24 hours of dialysis, the biotin concentration of the GelMA/NHS-PEG12-biotin mixture solution in the dialysis bag is kept unchanged to be 215 mu mol/L, and the biotin concentration in the dialysis liquid outside the dialysis bag is hardly detected (< 10 mu mol/L).
2) Optimization of Bio-GelMA synthesis reaction conditions
To obtain the highest degree of biotinylation of Bio-GelMA, the applicant optimized the reaction conditions for synthesizing Bio-GelMA, including reaction time and molar ratio of the reaction substance NHS-PEG 12 -biotin/GelMA. As shown in FIG. 2-a, the degree of biotinylation of Bio-GelMA increased with increasing reaction time, reaching a maximum concentration of 309. Mu. Mol/L at 8 hours, and then decreased with increasing reaction time. As shown in FIG. 2-b, the biotin concentration of Bio-GelMA increased significantly with increasing NHS-PEG12-biotin/GelMA molar ratio, and when NHS-PEG12-biotin/GelMA molar ratio exceeded 20:1, the biotin concentration of Bio-GelMA stabilized at 300. Mu. Mol/L or more.
3) FTIR and H1NMR analysis of NHS-PEG12-biotin, gelMA, bio-GelMA
The chemical structure of NHS-PEG 12 -biotin, gelMA, bio-GelMA was analyzed by FTIR and H 1 NMR to further verify the successful synthesis of Bio-GelMA, the synthetic chemical reaction formula is shown in FIG. 3-a. The infrared spectrum results show (FIG. 3-b) that the absorbance is significantly reduced, the absorbance of the amide I band (1539 cm -1) is significantly increased, and the broad peak between 3100cm -1 and 3700cm -1 is significantly shifted compared to the COO-group (1396 cm -1), the amide II band (1630 cm -1) and the hydroxyl/amino group (3293 cm -1) of GelMA, bio-GelMA. Fitting results showed significant differences between the sub-peaks of GelMA and Bio-GelMA located between 3100cm -1 and 3700cm -1 (FIG. 3-c). The nuclear magnetic resonance hydrogen spectrum results showed (FIG. 3-d) that Bio-GelMA and NHS-PEG 12 -biotin had the same characteristic peaks at 6.36ppm and 6.46ppm and GelMA had the same characteristic peaks at 5.28ppm and 5.63 ppm.
4) Photocuring Properties and microstructure of GelMA and Bio-GelMA
Photocrosslinking properties and microstructure of GelMA and Bio-GelMA were examined by inversion method and SEM. As shown in FIG. 4-a, both GelMA and Bio-GelMA can be crosslinked to a gel under blue light irradiation at 405 nm. Similarly, the SEM results of FIG. 4-b show that GelMA has a similar microstructure as Bio-GelMA.
Example 2 preparation of biotinylated MSC-EXs (Bio-EXs) and optimization of related parameters
2.1 Extraction and identification step of MSC-EXs
After culturing MSCs in exosome-free serum medium for 48 hours, the supernatant was collected and the supernatant was sequentially freed from cells, cell debris and larger diameter vesicles at 200g for 5 minutes, 3000g for 10 minutes, 16000g for 30 minutes. The supernatant after centrifugation was filtered using a 0.22 μm filter, and the MSC-EX was extracted by ultracentrifugation (100000 g,75 min). To remove residual proteins in MSC-EX, MSC-EX was resuspended using PBS and purified again by ultracentrifugation. The obtained MSC-EX was stored in sub-package to-80 ℃.
The particle size distribution of MSC-EX was analyzed using a nanoparticle size analyzer. MSC-EX was diluted with PBS to the appropriate concentration (100-500 particles/. Mu.L) and then run on-machine, and the data collected was analyzed using a nanoparticle size analyzer with software ZetaView PMX (Particle Metrix, meerbusch, north Rhine-WESTPHALIA, germany) to obtain the Particle size distribution of MSC-EX.
MSC-EX surface topography was examined using Transmission Electron Microscopy (TEM). After dropping 20. Mu. LMSC-EX suspension on copper mesh, it was counterstained with 2% phosphotungstic acid solution for 10 minutes, after which it was dried, it was observed and photographed under 120kV using TEM.
Western blot was used to detect expression of MSC-EX exosome characteristic proteins. RIPA extracts total protein, 20. Mu.g protein was added to each loading well of 4-12% gradient pre-mix, run for 35 min at 150V constant pressure, and flow-through membrane at 300 mA for 2 hours. 5% nonfat milk powder was blocked at room temperature for 2 hours, TBST was used to wash the membrane, followed by overnight incubation with exosome-characterized proteins and reference primary antibodies (CD 63, HSP70, TSG101, β -Actin and GAPDH) at 4℃and membrane washing was performed after incubation. After the film washing is finished, the film is incubated with the corresponding HRP-conjugated secondary antibody for two hours at room temperature, and after the film washing is performed again, the film is incubated with the luminous solution for 5 minutes, and the film is placed in an imager for exposure and photographing.
The particle size distribution of MSC-EX and the surface protein markers were detected using a nanofluidic instrument. According to the literature related to analysis of exosomes by the early-stage nanoflow instrument, the line was washed with a dedicated wash solution prior to each test, the VSSC-H threshold was manually set to 1500 to reduce background interference, and the acquisition signal threshold was set to 10. To improve the accuracy of the detection, the flow rate of the sample is manually set to be 10 mu L/min, the concentration of the sample is diluted to be less than 10000 particles/mu L by using particle-free (less than 100 particles/mu L) PBS, and then the data is acquired, wherein the acquisition amount is not less than 100000. Calibration was performed using 80, 100, 150, 200, 300 and 500nm nano fluorescent microsphere standards with particle size analysis range of 60-220nm. The percent fluorescence of Phycoerythrin (PE) was measured using a 585/42nm filter channel, the percent fluorescence of Allophycocyanin (APC) was measured using a 660/20nm filter channel, and the percent Fluorescence of Isothiocyanate (FITC) was measured using a 525/40nm filter channel.
After the machine parameters were set, 100. Mu.L of MSC-EX was incubated with 1. Mu.g of the APC-labeled CD63 antibody or PE-labeled CD81 antibody at room temperature for 15min in the absence of light, and the ratio and particle size distribution of CD63 or CD81 positive MSC-EX were detected using a nanofluidic instrument with undyed MSC-EX as a negative control. The collected data was analyzed using software CytExpert 2.0.0.
2.2 Biotinylation modification of MSC-EXs (Bio-EXs) and optimization of the parameters of preparation
First, the applicant uses DSPE-PEG-FITC or DSPE-PEG-biotin to modify MSC-EX phospholipid bilayer membranes. DSPE-PEG-FITC/DSPE-PEG-biotin was dissolved in absolute ethanol (10 mmol/L) to prepare a mother liquor which was stored to-80 ℃. Working solutions (0, 10, 20, 50 and 100. Mu. Mol/L) of different concentrations were then prepared by dilution of DSPE-PEG-FITC/DSPE-PEG-biotin with PBS and incubated with MSC-EXs at room temperature for 30 minutes, respectively. Ultracentrifugation (100000 g,75 min) removed excess unbound DSPE-PEG-FITC/DSPE-PEG-biotin. The DSPE-PEG-biotin modified exosomes still need to be incubated with SA-PE for 30 minutes at room temperature in the absence of light, and excessive SA-PE is removed by overspeed dissociation washing after incubation. The positive labelling rate of exosomes DSPE-PEG-FITC/DSPE-PEG-biotin was detected using a nanofluidic instrument.
The DSPE-PEG-FITC/DSPE-PEG-biotin modified MSC-EX was examined using a laser confocal microscope, incubated with 20. Mu. Mol/L DSPE-PEG-FITC/DSPE-PEG-biotin for 30min at room temperature protected from light, and washed by ultracentrifugation. DSPE-PEG-biotin labeled MSC-EX still requires additional incubation with Alexar 568 conjugated streptavidin for 30 minutes at room temperature followed by ultracentrifugation washing. The DSPE-PEG-FITC/DSPE-PEG-biotin marked MSC-EX is placed under a laser confocal microscope for observation and photographing.
2 Mu L of PKH26 (Ex 551nm/Em 567 nm) dye liquor and 1ml of diluent C are mixed to prepare exosome dye liquor, 100 mu L of MSC-EX extracted and exosome dye liquor are fully mixed and placed at room temperature for incubation for 10 minutes in dark place, and after dyeing, the residual free dye is removed by centrifugal washing by using a 100kD ultrafiltration tube. PKH26 labeled MSC-EX was incubated with macrophages in incubator, after 12 hours, the specimens were fixed with 4% paraformaldehyde, stained with DAPI, observed under confocal microscope and photographed.
2.3 Correlation results
1) Identification of MSC-EX:
TEM images show that the extracted MSC-EX has a typical cup-like structure. The nanoparticle size tracker results showed that MSC-EX particle size was mainly distributed between 60 and 220nm with a peak at 120 nm. Western blot results showed that MSC-EX highly expressed the exosome-associated proteins (CD 63, TSG101 and HSP 70) compared to MSC (FIGS. 5 a-c). The nanofluidic results showed that MSC-EX particle size was mainly distributed between 60-200nm with a peak at 70nm (FIG. 5-d), CD63 positive rate of 56.99% and CD81 positive rate of 66.12% (FIG. 5-e). The confocal fluorescence image showed that PKH 26-labeled MSC-EX was phagocytosed by cells after 24 hours incubation with cells (FIG. 5-f).
2) Preparation and identification of biotinylated MSCs:
The nanoflow results showed that DSPE-PEG-FITC self-assembled into the MSC-EX phospholipid bilayer membrane structure and that after incubation with gradient concentrations of DSPE-PEG-FITC (10, 20, 50, 100 μmol/L), FITC positive rate of MSC-EX increased with increasing DSPE-PEG-FITC concentration, 63.82%, 77.03%, 82.73% and 85.56%, respectively (fig. 6-a). Representative fluorescence plots also showed that PKH26 labeled MSC-EX was co-localized with PKH26 after co-incubation with 20. Mu. Mol/L DSPE-PEG-FITC, and that almost all PKH26 labeled MSC-EX was modified by DSPE-PEG-FITC (FIG. 6-b). Similar to DSPE-PEG-FITC, the nanofluidic results and representative fluorescence plots show that DSPE-PEG-biotin can self-assemble into phospholipid bilayer membrane structures of MSC-EX. Nanofluidic results showed MSC-EX biotin positivity of 60.94%, 80.25%, 82.56% and 84.21% after incubation with gradient concentrations of DSPE-PEG-biotin (10, 20, 50, 100. Mu. Mol/L), respectively (FIG. 6-c). Representative fluorescence plots also showed that after PKH 26-labeled MSC-EX was incubated with 20. Mu. Mol/L DSPE-PEG-biotin, DSPE-PEG-biotin was co-localized with PKH67 (Ex 490nm/Em 502 nm), and almost all PKH 67-labeled MSC-EX was modified by DSPE-PEG-biotin (FIG. 6-d). Fluorescence plots showed that both EX and Bio-EX were taken up by cells after 24 hours of co-incubation with no significant difference in uptake rate (fig. 6-e, f). These results demonstrate that 20. Mu. Mol/L DSPE-PEG-biotin can be used for subsequent biotinylated modification of MSC-EXs, and that the biotinylated modification of MSC-EXs does not affect its ability to be taken up by cells.
Example 3 preparation of GelMA hydrogels based on biotin-avidin System coupled MSC-EXs and optimization of related parameters
3.1 Preparation of GelMA hydrogels based on biotin-avidin System coupled MSC-EXs
By biotin-avidin-biotin system "bridging", applicants intend to graft MSC-EX to GelMA side chains. Based on the theoretical basis that one avidin can be combined with four biotins at most, the applicant designs the biotin-avidin molar ratio with different proportions for exploring the influence of a biotin-avidin system on the slow/controlled release of exosomes. Specifically, different molar ratios of Avidin@Bio-EX (1:1, 1:2, 1:3) were prepared by first incubating Bio-EX with different molar ratios of streptavidin (Avidin) for 1 hour at 4deg.C. 2X 1010 Avidin@Bio-EX and Avidin@EX without biotinylation modification were resuspended in 50. Mu.L PBS and then mixed with 50. Mu.L of 20% GelMA (wt/vol) containing 0.5% LAP (wt/vol) to prepare Bio-GelMA@Bio-EX or Bio-GelMA@EX, and the mixture was gelled by irradiation with 405nm blue light for 15 seconds for the subsequent experiments.
3.2 Retention and Release of MSC-EX in Bio-GelMA@Bio-EX hydrogel
1) MSC-EX retention (in vitro) by fluorescence intensity analysis
To monitor the retention of EX in Bio-GelMA@Bio-EX (in vitro), avidin@Bio-EX and EX without biotin modification were labeled with PKH26, respectively. Bio-EX was loaded into Bio-GelMA via a biotin avidin biotin "bridge" and light crosslinked into gel, which was immersed in 1.5ml PBS. The new PBS is replaced at 0, 1, 4 and 7 days, and the fluorescence microscope is used for photographing, so that parameters such as exposure degree, contrast and the like of the microscope are kept consistent during photographing. Images were analyzed for fluorescence intensity using ImageJ 1.53k software and EX in vitro retention was calculated using the following formula:
Retention of MSC-EX (in vitro) =fluorescence intensity Each time point /fluorescence intensity Initial time point ×100%
2) Analysis of release rate of MSC-EX by exosome ELISA (in vitro)
To monitor the release rate of Bio-GelMA@Bio-EX (in vivo), the Bio-GelMA@Bio-EX hydrogel was immersed in 1.5ml PBS, the PBS was collected at 1,4, 7 days and replaced with fresh PBS. The collected PBS (containing gel release EX) was concentrated using an ultrafiltration tube and resuspended to 100 μl PBS, and the EX concentration was determined using an exosome ELISA kit according to manufacturer's instructions, and the EX release rate was calculated according to the following formula:
Release rate of EX (total) = (concentration First, the 1 Tiantian (Chinese character of 'Tian') ×pbs volume First, the 1 Tiantian (Chinese character of 'Tian') + … concentration First, the X Tiantian (Chinese character of 'Tian') ×pbs volume First, the X Tiantian (Chinese character of 'Tian') )/(concentration First, the 1 Tiantian (Chinese character of 'Tian') ×pbs volume First, the 1 Tiantian (Chinese character of 'Tian') +concentration First, the 4 Tiantian (Chinese character of 'Tian') ×pbs volume First, the 4 Tiantian (Chinese character of 'Tian') +concentration First, the 7 Tiantian (Chinese character of 'Tian') ×pbs volume First, the 7 Tiantian (Chinese character of 'Tian') ) ×100%
3) Analysis of MSC-EX retention by in vivo imaging (in vivo)
EX retention in vivo was analyzed by in vivo imaging. Avidin@Bio-EX and EX which is not modified by biotin are loaded into hydrogel after being marked by DiR and are subjected to light crosslinking to form a Bio-GelMA@Bio-EX or Bio-GelMA@EX hydrogel cylinder with the diameter of 0.5 cm and the height of 0.3 cm. Animal experiments were approved by the ethical committee for animal experiments at university of army medicine, 48C 57/BL6 mice at 8 weeks of age were purchased from the animal experiment center at university of army medicine. The operation steps are as follows: after induction of anesthesia in mice with 2% isoflurane and a small animal anesthesia machine, anesthesia was maintained at 1% concentration and surgery was performed. A1 cm incision was made in the skin on both sides of the spinal column on the back of the mice, and hydrogel cylinders were implanted. The fluorescence intensity of DiR-labeled EX in hydrogels was detected with a small animal biopsy imager at 0, 1, 4, 7 days post-surgery, analyzed using LIVING IMAGE v4.3.1 software and the in vivo retention of EX was calculated using the following formula:
Retention of MSC-EX (in vivo) =fluorescence intensity Each time point /fluorescence intensity Initial time point ×100%
3.3 Results
3.1 Retention and Release Rate of MSC-EX in Bio-GelMA@Bio-EX hydrogel
Retention Rate of MSC-EX (in vitro)
GelMA hydrogel construction strategy based on biotin-avidin system coupling MSC-EXs is shown in the following figure. The fluorescence plot shows that on day 1, little fluorescence of Bio-GelMA@EX has been detected (FIG. 7-b), and statistical analysis also shows that Bio-GelMA@EX is significantly lower than that of the other three groups of Bio-GelMA@Bio-EX hydrogels (FIG. 7-c). On day 4, a clear fluorescent signal was still observed in the three groups of Bio-GelMA@Bio-EX hydrogels, and the fluorescent intensity of the control group of Bio-GelMA@EX hydrogels was still the lowest (FIG. 7-b). On day 7, the fluorescence intensities of Bio-EX@Avidin (2:1) and Bio-EX@Avidin (3:1) in the three groups of Bio-GelMA@Bio-EX hydrogels were still significantly higher than those of the control group (FIG. 7-c).
MSC-EX release rate (in vitro)
Over time, the total EX release was continuously increasing, and the total EX release was significantly lower for the Bio-GelMA@Avidin (2:1) group on days 1 and 4 than for the control group, bio-GelMA@EX, with no significant differences between the other groups (FIG. 7-d).
Retention Rate of MSC-EX (in vivo)
In vivo imaging results showed that the fluorescence intensities of the four sets of hydrogels decreased with time. The fluorescence of the control Bio-GelMA@EX hydrogel was barely detectable 7 days after subcutaneous implantation, whereas the Bio-GelMA@Bio-EX hydrogel was still detectable 28 days after subcutaneous implantation (FIG. 7-e). The quantitative analysis results show that the fluorescence intensity of the Bio-GelMA@EX hydrogel of the control group on the 1 st day is greatly reduced to be 23.37% of that of the initial group, and the fluorescence of the hydrogel of the Bio-GelMA@Avidin (2:1) group still remains 51.58%. Similarly, on day 4,7,28, the fluorescence intensity of the hydrogel was significantly higher for the Bio-GelMA@Avidin (2:1) group than for the Bio-GelMA@EX group, with no significant differences between the other groups (FIG. 7-f).
These results demonstrate that the biotin-avidin system released MSC-EXs most efficiently at a 1:2 molar ratio of Avidin@Bio-EX.
Various embodiments of the application may exist in a range of forms; it should be understood that the description in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the application; it is therefore to be understood that the range description has specifically disclosed all possible sub-ranges and individual values within that range. For example, it should be considered that a description of a range from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1,2, 3, 4, 5, and 6, wherever applicable. In addition, whenever a numerical range is referred to herein, it is meant to include any reference number (fractional or integer) within the indicated range.
In addition, in the description of the present specification, the terms "include", "comprising" and the like mean "including but not limited to". Relational terms such as "first" and "second", and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Claims (9)
1. A method for preparing a coupled exosome hydrogel based on a biotin-avidin system, the method comprising:
adding the stem cell exosome and biotin into a first solvent for a first mixing reaction to obtain a biotin-marked stem cell exosome;
adding gelatin and activated biotin into a second solvent for a second mixing reaction, and then dialyzing to obtain biotinylated gelatin;
Carrying out a third mixing reaction on the stem cell exosomes marked by biotin and avidin to obtain avidin-treated stem cell exosomes;
Performing a fourth mixing reaction on the avidin-modified stem cell exosome and the biotinylated gelatin to obtain the coupled exosome hydrogel based on a biotin-avidin system;
Wherein the gelatin is methacrylamide gelatin when gelatin and activated biotin are added into a second solvent for a second mixing reaction.
2. The method of claim 1, wherein the stem cell exosomes comprise bone marrow mesenchymal stem cell exosomes and the biotin comprises pegylated biotin.
3. The preparation method of claim 1, wherein the amino substitution degree of the methacrylamide gelatin is 50-65%, and the mass concentration is 8-12%; the activated Biotin comprises at least one of Biotin-dodecapolyethylene glycol-active ester, NHS-Biotin, NHS-PEG 3 -Biotin and NHS-PEG 4 -Biotin.
4. The method according to claim 1, wherein, the avidin comprises streptavidin.
5. The method of claim 1 or 2, wherein the step of adding the stem cell exosomes and biotin to the first solvent to perform a first mixing reaction, to obtain biotin-labeled stem cell exosomes comprises:
Adding the stem cell exosome and the biotin into the first solvent for mixing to obtain a first mixed solution, and then reacting for 20-40 min at the temperature of 35-40 ℃ to obtain the biotin-labeled stem cell exosome;
The molar concentration of biotin in the first mixed solution is 15-25 mu mol/L, the concentration of stem cell exosomes is 200-500 ug/mL, and the particle size number is 1-4 multiplied by 10 11.
6. A method according to claim 1 or 3, wherein the step of adding gelatin and activated biotin to a second solvent for a second mixing reaction, and then dialyzing to obtain biotinylated gelatin comprises:
Adding 0.5-1.5 mg/mL of the gelatin and 5.0-5.5 mg/mL of the activated biotin into the second solvent, mixing, reacting for 6-10 hours at room temperature, dialyzing for 24-48 hours, and freeze-drying to obtain the biotinylated gelatin;
Wherein the mol ratio of the gelatin to the activated biotin is 1 (18-22).
7. The method of claim 1 or 4, wherein the step of subjecting the biotin-labeled stem cell exosomes and avidin to a third mixing reaction, to obtain an avidin-labeled stem cell exosomes comprises:
mixing the stem cell exosomes marked by biotin and the avidin, and reacting for 20-40 min at the temperature of 35-40 ℃ to obtain the avidin-treated stem cell exosomes;
Wherein the molar ratio of the stem cell exosomes labeled with biotin to the avidin is (1.5-2.5): 1.
8. The method according to claim 1, wherein the step of subjecting the avidin-labeled stem cell exosomes and the biotinylated gelatin to a fourth mixing reaction to obtain the biotin-avidin system-based exosome-coupled hydrogel comprises:
Mixing the avidin-modified stem cell exosome and the biotinylated gelatin, and reacting for 20-40 min at 35-40 ℃ to obtain the hydrogel based on the coupling exosome of the biotin-avidin system.
9. A biotin-avidin system-based coupled exosome hydrogel, characterized in that the biotin-avidin system-based coupled exosome hydrogel is prepared by the preparation method according to any one of claims 1 to 8.
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