Hydrogel capable of self-adaptively releasing exosome as well as preparation method and application of hydrogel

Abstract

The invention relates to the technical field of medical biological materials, in particular to hydrogel capable of self-adaptively releasing exosomes and a preparation method and application thereof.The hydrogel comprises a precursor, a buffer solution and the exosomes, the precursor is dissolved in the buffer solution to form a matrix of a three-dimensional network structure, and the exosomes are loaded in the three-dimensional network structure of the matrix; the precursor comprises HA-ADH and OHA-TK-CP, and the mass ratio of the HA-ADH to the OHA-TK-CP is (1: 5)-(1: 1); in-vivo and in-vitro research results show that the hydrogel has excellent self-repairing capability, remarkable antibacterial activity, efficient exosome release and effective ROS (reactive oxygen species) reduction capability, the wound healing of MRSA-infected diabetic rats is accelerated by the synergistic anti-inflammatory effect and tissue regeneration promotion effect of the hydrogel, and scars are relatively small after the wound healing; the preparation method is simple to operate, mild in reaction condition and beneficial to large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and in particular to a hydrogel for adaptively releasing exosomes, a preparation method and applications thereof. Background Art

[0002] As a new type of biomaterial, exosome gel has shown great potential in the fields of regenerative medicine, drug delivery and disease diagnosis in recent years. The core of this technology is to combine the biological activity of exosomes with the physical properties of hydrogels, and to achieve the regulation of cell behavior and the promotion of tissue repair by optimizing the loading and release of exosomes. Although exosome gel has a certain effect in moisturizing and promoting tissue repair, it is difficult to achieve effective removal of ROS and on-demand release of exosomes.

[0003] As a three-dimensional network structure material, hydrogel is widely used in the encapsulation and sustained release of exosomes due to its good biocompatibility, adjustable physicochemical properties and similarity to the cell microenvironment. At the same time, exosomes are nanoscale vesicles secreted by cells, containing a variety of bioactive molecules, such as proteins, lipids, RNA and DNA, and are widely involved in physiological processes such as intercellular communication, immune regulation and disease progression. Exosomes show good prospects in wound healing and anti-inflammation due to their low immunogenicity, high biocompatibility and potential therapeutic value. However, how to achieve the precise and adaptive release of exosomes at the wound site to maximize its therapeutic effect is still a technical problem that needs to be solved urgently for exosome gels.

[0004] Wound infection is a key factor affecting wound healing. Existing antibacterial hydrogels mainly inhibit bacterial infection by adding antibiotics, antimicrobial peptides or photodynamic therapy, but these methods have many limitations: First, antibiotic dependence may lead to the emergence of drug-resistant strains; second, a single antibacterial mechanism is difficult to achieve anti-inflammatory, antibacterial and synergistic regulation of exosome release; third, photodynamic therapy requires continuous intervention of external light sources, and some materials have the risk of cytotoxicity. In addition, the preparation process of existing hydrogels is complicated, and the temporal and spatial precision of exosome release is insufficient, making it difficult to adapt to the dynamically changing microenvironment of chronic infection wounds. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the first object of the present invention is to provide a hydrogel that adaptively releases exosomes; the second object of the present invention is to provide a method for preparing a hydrogel that adaptively releases exosomes; the third object of the present invention is to provide an application of a hydrogel that adaptively releases exosomes.

[0006] In order to achieve the first purpose, the technical solution adopted by the present invention is: A hydrogel for self - adaptive release of exosomes, comprising a precursor, a buffer solution, and exosomes. The precursor is dissolved in the buffer solution to form a matrix with a three - dimensional network structure, and the exosomes are loaded in the three - dimensional network structure of the matrix; Among them, the precursor includes polymer HA - ADH and polymer OHA - TK - CP, and the mass ratio of polymer HA - ADH to polymer OHA - TK - CP is 1:5 to 1:1; The polymer HA - ADH is hyaluronic acid - adipic dihydrazide; Among them, HA is hyaluronic acid, and ADH is adipic dihydrazide; The polymer OHA - TK - CP is oxidized hyaluronic acid modified with dithioacetal and choline phosphate groups; Among them, OHA is oxidized hyaluronic acid; TK is 2,2'-(propane - 2,2 - diylbis(sulfanediyl))bis(ethan - 1 - amine), and its molecular structural formula is as follows: ; CP is isopropyl 2 - (dimethyl(prop - 2 - yn - 1 - yl)ammonio)ethyl phosphate, and its molecular structural formula is as follows: .

[0007] Furthermore, in the polymer HA - ADH, the substitution degree of ADH is 65 - 75%.

[0008] Furthermore, in the polymer OHA - TK - CP, the grafting rate of TK is 30 - 35%, and the grafting rate of CP is 10 - 20%.

[0009] Furthermore, the mass content percentage of the polymer OHA - TK - CP in the hydrogel is 5 - 25%.

[0010] Furthermore, the swelling rate of the hydrogel is 115 - 128%.

[0011] Furthermore, the pH value of the buffer solution is 7 - 8.

[0012] Furthermore, the content of exosomes in the matrix is 1×10 8 ~1×10 12 per mL.

[0013] To achieve the second objective, the technical solution adopted by the present invention is as follows: A preparation method of a hydrogel for self - adaptive release of exosomes, which is used to prepare the hydrogel for self - adaptive release of exosomes described in any one of the above, and includes the following steps: S100. Respectively prepare an HA - ADH buffer solution and an OHA - TK - CP buffer solution containing exosomes; S200. Mix the HA-ADH buffer solution and the OHA-TK-CP buffer solution evenly, and react at 36 °C to 38 °C for 18 s to 12 min to obtain a hydrogel that adaptively releases exosomes.

[0014] To achieve the third object, the technical solution adopted by the present invention is as follows: An application of a hydrogel that adaptively releases exosomes, such as the hydrogel that adaptively releases exosomes described in any one of the above, and the application includes being used for preparing any one or several of the following: Antibacterial and / or bactericidal products, anti-inflammatory products, wound repair products, immune regulation products and drug carriers.

[0015] Further, the wound includes chronic inflammatory wounds caused by diabetes.

[0016] One or more of the above technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention provides a hydrogel that adaptively releases exosomes. The polymer HA-ADH is combined with the polymer OHA-TK-CP through a Schiff base reaction to form a three-dimensional network structure in a buffer solution and load exosomes to achieve efficient treatment. As an amphoteric ionic compound, CP has a molecular structure containing positive and negative charge regions. This property enables CP to anchor exosomes through reverse electrostatic interactions and enhance the adsorption and destruction of anionic bacterial membranes through electrostatic interactions, showing antibacterial effects.

[0017] Locally active oxygen (ROS)-induced cleavage of the thioxanthone (TK) linker triggers the adaptive release of umbilical cord mesenchymal stem cell (UC-MSC)-derived exosomes and clears ROS in the inflammatory wound microenvironment. Therefore, the synergistic combination of the matrix and exosomes significantly eliminates bacterial infections in diabetic wounds, reduces oxidative stress, regulates M2 macrophage polarization, and accelerates tissue remodeling and repair through collagen deposition, myofibroblast contraction and migration.

[0018] In vitro and in vivo studies have shown that the hydrogel has excellent self-healing ability, significant antibacterial activity, efficient exosome release and effective ROS reduction ability. Experimental results show that in a diabetic rat model, the hydrogel significantly improves the wound healing rate and reduces scar formation. Its synergistic anti-inflammatory effect and tissue regeneration promotion effect accelerate the wound healing of diabetic rats infected with methicillin-resistant Staphylococcus aureus (MRSA), and the scar is smaller after wound healing.

[0019] The preparation method of the hydrogel that adaptively releases exosomes provided by this application is simple in operation and mild in reaction conditions, which is conducive to large-scale production.

[0020] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned by practice of the present invention. Description of the Drawings

[0021] Figure 1 is the 1 1H NMR spectrum of the polymer HA-ADH provided in Example 1 of the present invention.

[0022] Figure 2 is the 1 1H NMR spectrum of the compound CP provided in Example 1 of the present invention.

[0023] Figure 3 is the 1 1H NMR spectrum of the polymer OHA provided in Example 1 of the present invention.

[0024] Figure 4 is the 1 1H NMR spectrum of the polymer OHA-TK provided in Example 1 of the present invention.

[0025] Figure 5 is the high-resolution mass spectrum of Compound III provided in Example 1 of the present invention.

[0026] Figure 6 is the 1 1H NMR spectrum of the polymer OHA-TK-CP provided in Example 1 of the present invention.

[0027] Figure 7 is the IR spectrum of different hyaluronic acids and their derivatives provided in Example 1 of the present invention.

[0028] Figure 8 is the SEM image of the self-adaptive release exosome hydrogel provided in Example 1 of the present invention.

[0029] Figure 9 is the relationship curve region of the hydrogels with different concentrations and time provided in Example 1 of the present invention.

[0030] Figure 10 is the relationship curve graph of the step time and oscillating strain of the hydrogels with different concentrations and the storage modulus and loss modulus provided in Example 2 of the present invention.

[0031] Figure 11 is the statistical chart of the bactericidal rates of three bacteria (MRSA, E. coli, and S. aureus) in different groups measured by the OD value method provided in Test Example 1 of the present invention.

[0032] Figure 12It is a statistical chart showing the bactericidal rates of three bacteria (MRSA, E. coli, and S. aureus) in different groups measured by the plate method provided in Test Example 1 of the present invention.

[0033] Figure 13 They are fluorescence images of DMAO staining, EthD-III staining, and the combination of both (Merg) in different groups (PBS, Ampicillin, Pre-Gel, and Gel) using the staining kit method provided in Test Example 1 of the present invention.

[0034] Figure 14 They are SEM images of the biofilm structures of different groups provided in Test Example 1 of the present invention.

[0035] Figure 15 It is a time-dependent ultraviolet-visible spectrogram of the hydrogel with self-adaptive release of exosomes provided in Test Example 2 of the present invention.

[0036] Figure 16 It is a curve graph showing the relationship between time and degradation rate during the treatment of the hydrogel with self-adaptive release of exosomes with different concentrations of H2O2 or PBS buffer provided in Test Example 3 of the present invention.

[0037] Figure 17 It is a curve graph showing the relationship between the release rate of exosomes and time in two exosome hydrogel materials (Exo-Gel and Exo-Pre-Gel) with different concentrations of H2O2 provided in Test Example 4 of the present invention.

[0038] Figure 18 They are DCFH, Hoechst, and Merge fluorescence microscope images corresponding to different substances provided in Test Example 5 of the present invention.

[0039] Figure 19 It is a statistical chart showing the amounts of TNF-α and IL-10 secreted by cells under different substance treatment conditions provided in Test Example 6 of the present invention.

[0040] Figure 20 It is the effect on cell proliferation after treatment with different substances provided in Test Example 7 of the present invention.

[0041] Figure 21 They are microscope images of the wound conditions at 0 h and 24 h after treating cells with different substances provided in Test Example 8 of the present invention.

[0042] Figure 22 They are images after crystal violet staining of cells treated with different substances provided in Test Example 8 of the present invention.

[0043] Figure 23 They are microscope images of the lumen formation after treatment with different substances provided in Test Example 9 of the present invention.

[0044] Figure 24 It is a diagram showing the growth conditions of D1 and D7 of bacterial samples after being treated with different substances provided in Test Example 10 of the present invention.

[0045] Figure 25 It is a diagram showing the wound healing conditions of rats in different groups provided in Test Example 10 of the present invention.

[0046] Figure 26 It is a hematoxylin-eosin tissue section staining diagram of different groups at different time points provided in Test Example 10 of the present invention.

[0047] Figure 27 It is a Masson tissue section staining diagram of different groups at different time points provided in Test Example 10 of the present invention. Detailed implementation manners

[0048] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with specific embodiments. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts belong to the scope protected by the present invention. The following embodiments are used to illustrate the present invention but cannot be used to limit the scope of the present invention.

[0049] A hydrogel for adaptively releasing exosomes, comprising a precursor, a buffer solution and exosomes. The precursor is dissolved in the buffer solution to form a matrix with a three-dimensional network structure, and the exosomes are loaded in the three-dimensional network structure of the matrix; Among them, the precursor includes polymer HA-ADH and polymer OHA-TK-CP, and the mass ratio of polymer HA-ADH to polymer OHA-TK-CP is 1:5 to 1:1; Polymer HA-ADH is hyaluronic acid-oxalyl dihydrazide; Among them, HA is hyaluronic acid and ADH is adipic dihydrazide; Polymer OHA-TK-CP is oxidized hyaluronic acid modified with dithioacetal and phosphocholine groups; Among them, OHA is oxidized hyaluronic acid; TK is 2,2'-(propane-2,2-diylbis(sulfanediyl))bis(ethane-1-amine), and its molecular structural formula is shown as follows: ; CP is 2-(dimethyl(prop-2-yn-1-yl)ammonio)ethyl isopropyl phosphate, and its molecular structural formula is shown as follows: .

[0050] A preparation method of a hydrogel for adaptively releasing exosomes, comprising the following steps: S100. Prepare an HA-ADH buffer solution and an OHA-TK-CP buffer solution containing exosomes respectively; S200. Mix the HA-ADH buffer solution and the OHA-TK-CP buffer solution evenly, react at 36 °C to 38 °C for 18 s to 12 min to obtain a hydrogel for adaptively releasing exosomes.

[0051] In the following examples, the experimental methods used are all conventional methods unless otherwise specified. The materials, reagents, etc. used are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, they can be obtained from commercial channels.

[0052] Example 1 Preparation of polymer HA-ADH and polymer OHA-TK-CP.

[0053] I. Preparation of polymer HA-ADH, the process is as follows: Dissolve hyaluronic acid (HA) (1.00 g) with a weight-average molecular weight (MW) of 70 kDa in 100 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer (100 mM, pH 6.5). To activate the carboxyl group on HA, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (0.96 g, 5.00 mmol) and 1-hydroxybenzotriazole (HOBt) (0.68 g, 5.00 mmol) and stir for 30 min, then add adipic dihydrazide (ADH) (4.36 g, 25.00 mmol). After reacting for 24 h, transfer the reaction solution to a dialysis membrane (MW cut-off value of 8000 Da), dialyze in 0.9 wt% NaCl aqueous solution for 3 days, and dialyze with deionized water for 2 days. Then freeze and lyophilize the obtained purified solution to obtain 0.90 g of a white flocculent solid polymer HA-ADH, whose 1 1H NMR spectrum is as Figure 1 shown. By integrating the areas of relevant peaks in the spectrum, the modification efficiency of ADH is known to be 69%.

[0054] II. Preparation of compound TK-NH2 , the process is as follows: In 100 mL of methanol, triethylamine (15.74 g, 155.3 mmol) and cysteamine (8.00 g, 103.9 mmol) were added. After stirring and dissolving, ethyl trifluoroacetate (17.67 g, 124.7 mmol) was added dropwise at room temperature, and the mixture was stirred overnight to form a reaction solution containing Compound I After that; the reaction was quenched with saturated NaCl aqueous solution. After terminating the reaction process, the reaction solution was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined and dried over anhydrous Na2SO4 to obtain the crude product of Compound I; using hexane / ethyl acetate (V / V = 10:1) as the eluent, the crude product of Compound I was purified by silica gel column chromatography, and the purified Compound I was collected and dried in vacuo to obtain 14.42 g of solid Compound I with a yield of 80%.

[0055] Under a N2 atmosphere, Compound I (11.24 g, 65.0 mmol) was dissolved in 120 mL of acetonitrile to obtain an acetonitrile solution of Compound I. After adding this solution to a 200 mL Schlenk tube, acetone (2.25 mL, 32.5 mmol) was added and mixed evenly. After cooling to 0 °C, boron trifluoride diethyl etherate (11.23 mL, 90.0 mmol) was added dropwise. After the addition was complete, the reaction was stirred at 0 °C for 2 h, and then the reaction solution was slowly poured into a 15 wt% Na2CO3 solution to neutralize the excess acid. The mixture was extracted with ethyl acetate (3 × 100 mL), and the organic layers were combined and dried over anhydrous Na2SO4 overnight; the solvent was removed by rotary evaporation to obtain the crude product of Compound II The crude product was dissolved in dichloromethane (DCM), and using hexane / ethyl acetate (V / V = 15:1) as the eluent, it was purified by silica gel column chromatography to obtain the purified Compound II.

[0056] Compound II (7.60 g, 20 mmol) was dissolved in 500 mL of 6 M NaOH aqueous solution and stirred at room temperature for 4 h. After extraction with DCM (3 × 100 mL) and drying in vacuo, 2.28 g of amber-colored Compound TK-NH2 was obtained with a yield of 59%.

[0057] III. Preparation of Compound CP , the process is as follows: Under a dry N2 atmosphere, acetonitrile was redistilled with CaH2 before use to remove moisture; Dimethylprop-2-yn-1-amine was dried with CaH2 overnight and then subjected to vacuum distillation; Tetrahydrofuran (THF) was distilled and dried with lithium aluminum hydride before use to ensure that the solvent was anhydrous; Under a nitrogen atmosphere, into a 200 mL Schlenk flask, N,N-dimethylpropan-2-ylamine (16.63 g, 200 mmol), propan-2-ol (7.21 g, 120 mmol) and acetonitrile (50 mL) were successively added. After cooling to -50 °C, 2-chloro-1,3,2-dioxaphospholane 2-oxide (14.25 g, 100 mmol) was added dropwise within 2 h. After reacting for 10 h, the temperature was raised to room temperature and stirring was continued for 12 h. The precipitate was filtered under a N2 atmosphere. After removing the filtrate, the reaction was continued at 75 °C for 48 h to ensure complete reaction. The reaction product was precipitated with ether, stirred to make the paste adhere to the bottle wall, the clear liquid was removed, and it was repeatedly washed with a tetrahydrofuran solution until the supernatant was clear. The precipitate was further purified and dried in vacuo to obtain 10.73 g of a compound CP as an amber oil, with a yield of 42%. Its 1 1H NMR spectrum is as Figure 2 shown.

[0058] IV. Preparation of polymer OHA-TK-CP, the process is as follows: HA (2.00 g, MW = 340 kDa) was dissolved in 200 mL of H2O under vigorous stirring to obtain an HA solution. NaIO4 (1.08 g, 5.04 mmol) was added dropwise to this solution. After stirring in the dark at room temperature for 5 h, ethylene glycol (2 mL) was added and mixed evenly. After reacting for 1 h, the reaction solution was transferred to a dialysis membrane (MW cut-off value of 14000 Da) and dialyzed in an NaCl solution for 3 d, and then dialyzed in deionized water for 2 d. The purified solution was frozen and lyophilized to obtain 1.63 g of a white flocculent polymer OHA, whose 1 1H NMR spectrum is as Figure 3 shown. By integrating the areas of the relevant peaks in the figure, the modification efficiency of ethyl groups can be known to be 48%.

[0059] OHA (1.50 g) was dissolved in 150 mL of phosphate buffer (20 mM, pH = 7.4), EDC·HCl (2.28 g, 11.88 mmol) and NHS (1.38 g, 11.88 mmol) were added, and stirred for 30 min to activate the carboxyl groups on OHA, and then compound TK-NH2 (0.78 g, 3.96 mmol) was added, and the reaction was carried out for 24 h. The solution gradually changed from light yellow to dark brown. The reaction solution was transferred to a dialysis membrane (MW cut-off value = 8000 Da) and dialyzed in an NaCl solution for 3 days, and then dialyzed in deionized water for 2 days. The purified solution was frozen and lyophilized to obtain 1.22 g of a light brown powder of polymer OHA-TK, whose 1 1H NMR spectrum is as Figure 4As shown, by integrating the areas of the relevant peaks in the figure, it can be known that the modification efficiency of TK on OHA is 32%.

[0060] Under N2 atmosphere, compound CP (1.25 g, 5.00 mmol) and methyl azidevalerate (AVA) (0.86 g, 6.00 mmol) were dissolved in 12 mL of methanol in a 50 mL Schlenk tube; CuSO4 (25.0 mg, 0.10 mmol) and sodium ascorbate (29.7 mg, 0.15 mmol) were dissolved in 3 mL of H2O and added to the aforementioned reaction system. After stirring at room temperature for 24 h, the reaction mixture was precipitated with diethyl ether, stirred vigorously, the paste on the tube wall was collected, and after removing the supernatant, the precipitate was washed twice with tetrahydrofuran (THF) and then dried under vacuum to obtain 0.94 g of compound Ⅲ as a brown solid. , the high-resolution mass spectrum of compound Ⅲ is as Figure 5 shown.

[0061] Compound Ⅲ (0.82 g, 2.12 mmol) was dissolved in 50 ml of phosphate buffer solution (20 mM, pH = 7.4). Subsequently, EDC·HCl (1.22 g, 6.34 mmol) and NHS (0.72 g, 6.34 mmol) were added in sequence. After stirring for 30 min, OHA-TK (0.8 g) was added, and the reaction was continued to stir for 24 h. Then the reaction solution was transferred to a dialysis membrane (MW cut-off value = 3500 Da), dialyzed in NaCl solution for 3 days first, and then dialyzed in deionized water for 2 days to obtain the dialyzed solution, which was lyophilized to obtain 0.68 g of brown polymer OHA-TK-CP, whose 1 1H NMR spectrum is as Figure 6 shown. By integrating the areas of the relevant peaks in the figure, it can be known that the modification efficiency of CP on OHA-TK is 15%; the infrared spectra (IR) of different hyaluronic acids (HA) and their derivatives are as Figure 7 shown. Compared with unmodified HA, HA-ADH shows an obvious peak at 1591 cm -1 , corresponding to the hydrazino group; for OHA, an obvious aldehyde group peak is observed at 1686 cm -1 ; the characteristic peak of OHA-TK at 1540 cm -1 is significantly enhanced; the peaks of OHA-TK-CP at 1175 cm -1 , 1354 cm -1 and 1540 cm -1 become more prominent, indicating an increase in phospholipid and methylene groups and a decrease in the carboxyl content of HA.

[0062] Preparation of hydrogel for adaptive release of exosomes in Example 2.

[0063] Weigh the polymer HA-ADH (0.5 g) prepared in Example 1 and dissolve it in PBS buffer solution (9.5 g) with a pH of 7.4. Stir at 37 °C for 30 min to obtain a clear HA-ADH buffer solution. Weigh the polymer OHA-TK-CP (1.5 g) prepared in Example 1 and dissolve it in PBS buffer solution (8.5 g) with a pH of 7.4 containing exosomes (8.5×10 11 pieces). Stir at 37 °C for 30 min to obtain a clear OHA-TK-CP buffer solution. Add the HA-ADH buffer solution and the OHA-TK-CP buffer solution into a double-chamber syringe, and inject and mix them in equal volumes to obtain a hydrogel for adaptive release of exosomes, denoted as Gel-15. Its SEM image is as Figure 8 shown. The performance test results of the Gel-15 hydrogel are as follows: in phosphate buffer solution at 37 °C and pH = 7.4, the swelling ratio is 122%; the compressive modulus is 58 Pa. Hydrogel Gel-5: Except that the addition amount of the polymer OHA-TK-CP is 0.5 g, the remaining preparation process is the same as that of Gel-15. Hydrogel Gel-10: Except that the addition amount of the polymer OHA-TK-CP is 1.0 g, the remaining preparation process is the same as that of Gel-15. Hydrogel Gel-20: Except that the addition amount of the polymer OHA-TK-CP is 2.0 g, the remaining preparation process is the same as that of Gel-15. Hydrogel Gel-25: Except that the addition amount of the polymer OHA-TK-CP is 2.5 g, the remaining preparation process is the same as that of Gel-15. The swelling ratio changes of hydrogels with different concentrations (Gel-5, Gel-10, Gel-15, Gel-20, Gel-25) at different times are as Figure 9 shown. It can be seen from the figure that hydrogels with different concentrations show certain differences in the swelling ratio. Among them, the swelling ratio of Gel-25 is relatively low, while the swelling ratios of Gel-5, Gel-10, Gel-15 and Gel-20 are relatively high and relatively close.

[0064] The mechanical property measurement results of hydrogels with different concentrations (Gel-25, Gel-20, Gel-15 and Gel-10) under two different test conditions are as Figure 10 shown; Among them, Figure A in the figure shows the changes in the storage modulus (G') and loss modulus (G") of four gels at different step times. The results show that the modulus values of all gels remained basically stable during the test, but the modulus value of Gel-25 was the highest, while the modulus value of Gel-10 was the lowest.

[0065] Figure B in the figure shows the changes in the storage modulus and loss modulus of four gels under different oscillating strains. The results show that at low strains, the modulus values of all gels were relatively stable, but as the strain increased, the modulus values decreased rapidly, especially for Gel-20 and Gel-10, whose modulus values decreased more significantly.

[0066] Comparative Example 1 Except that the polymer OHA-TK-CP was polymer OHA, the remaining preparation process was the same as that of Gel-15, denoted as Pre-Gel.

[0067] Test Example 1 Antibacterial activity in vitro of the hydrogel for adaptive release of exosomes.

[0068] In the present invention, Escherichia coli (E.coli), Staphylococcus aureus (S.aureus) and MRSA were used as representatives of Gram-negative bacteria, Gram-positive bacteria and drug-resistant bacteria, and these three bacteria were used to evaluate the antibacterial ability of the hydrogel for adaptive release of exosomes provided in Example 1; In order to better evaluate the antibacterial ability of the hydrogel provided in the example, it was divided into the following groups: PBS (negative control group), 10 mg / mL ampicillin (positive control group), Pre-Gel, Gel (Gel-15); The bacterial suspension (400 μL, 1×10 8 CFU / mL) was added to a 24-well culture plate and incubated with an equal volume of the above different groups of substances at 37 °C for 4 h. The bacterial suspension was collected and centrifuged. The treated bacterial suspension was incubated in LB medium at 37 °C for 12 h, the bacterial suspension was collected and transferred to a 96-well culture plate, and the absorbance (OD) value of the bacterial suspension at 600 nm was measured using a microplate reader. The OD value of the PBS solution was labeled as N0, the OD value of the bacterial group treated with PBS was labeled as N1, and the OD value of the test bacterial group was labeled as Nt. The bactericidal rate calculation formula is as follows: Bactericidal rate (%) = [1 - (Nt - N0) / (N1 - N0)] × 100%; The bactericidal rate of the three bacteria (MRSA, E.coli and S.aureus) in different groups is as Figure 11 shown; In the colony formation experiment, the bacterial suspension (100 μL, 1×10 5Plated on LB agar plates at (CFU / mL) and incubated at 37 °C for 12 h to form visible colonies. Photos of the LB plates were taken to show the bactericidal rates of different groups of the three bacteria (MRSA, E. coli, and S. aureus), as Figure 12 shown; After co-culture with the above different groups, the bacterial suspension was stained using a live / dead staining kit. The procedure was as follows: 1 mL of the bacterial suspension was incubated with DMAO (1.5 μL) and EthD-III (1.5 μL) in the dark at room temperature for 15 min. The stained suspension was washed twice with 0.8% NaCl solution, spread on a glass slide, and fluorescence images were collected using CLSM. Fluorescence images of different groups (PBS, Ampicillin, Pre-Gel, and Gel), DMAO staining, EthD-III staining, and their combination (Merg) are shown as Figure 13 shown; The biofilm structures of different groups were observed by scanning electron microscopy. The procedure was as follows: The bacterial suspension was fixed with 2.5% glutaraldehyde for 2 h, then dispersed in deionized water. 10 μL of the sample was aliquoted onto a silicon wafer, air-dried at room temperature, and then coated with a thin layer of gold. Its SEM image is shown as Figure 14 shown.

[0069] Test Example 2 Detection of the reactive oxygen species scavenging effect of the hydrogel.

[0070] The DPPH scavenging experiment was used to detect the ROS scavenging ability of the gel. The procedure was as follows: Gel-15 (500 mg) prepared in Example 1 was incubated with a DPPH solution at a concentration of 0.04 mg / mL in ethanol in the dark. The absorbance was measured at 517 nm using a UV-visible spectrophotometer every 20 min for a total duration of 125 min. The absorbance (a) values at each time point were recorded as At. The time-dependent UV-visible spectral diagram is shown as Figure 15 shown. The seven vertical curves in the figure represent the spectral data at different time points, from 5 min to 125 min (the time interval between each curve is 20 min). It can be seen from the figure that as time increases, the absorbance of the curves gradually decreases, and there is an obvious absorption peak at a wavelength of about 500 nm, and this peak gradually weakens with time.

[0071] Test Example 3 Detection of the ROS-responsive degradation effect of the hydrogel.

[0072] Gel-15 (500 mg) prepared in Example 1 was soaked in 5 mL of PBS or H2O2 solution (50 μM or 100 μM, diluted with PBS) at room temperature, and the degradation rate and ROS response performance of Gel-15 were detected. After reaching the swelling equilibrium, the gel was removed and any surface solution was gently blotted dry with filter paper before weighing. This process was repeated every 8 hours until the gel was completely degraded. The initial mass of Gel-15 after swelling equilibrium in PBS was denoted as W0, and the residual mass of the hydrogel at different time points was denoted as Wt. The degradation rate of the gel was calculated according to the formula: degradation rate (%) = (W0 - Wt) / W0 × 100%; The relationship curve between time and degradation rate of Gel-15 under different treatment conditions is shown as Figure 16 follows. There are three curves in the figure, representing the degradation of 100 μM, 50 μM H2O2 and the PBS control group respectively. It can be seen from the figure that with the increase of time, the degradation rate of H2O2 gradually increases, and the degradation rate is the highest when the concentration of H2O2 is 100 μM, and the degradation rate of the PBS control group is the lowest. p = 0.009 indicates significant statistical significance. This result shows that there are significant differences in the degradation rates of different concentrations of H2O2.

[0073] Test Example 4 Detection of the self-adaptive release of exosomes from the hydrogel.

[0074] The enzyme-linked immunosorbent assay was used to evaluate the self-adaptive release of ROS levels by Gel-15 and Exo-Pre-Gel. The process is as follows: Exo-Gel-15 (denoted as Exo-Gel in the figure) was prepared using dio-labeled exosomes according to the preparation method provided in Example 1, and Exo-Pre-Gel was prepared using dio-labeled exosomes according to the preparation method provided in Comparative Example 1; Exo-Gel and Exo-Pre-Gel were respectively immersed in 5 mL of PBS or H2O2 solution (50 μM or 100 μM, diluted with PBS). After reaching the swelling equilibrium, 100 μL of the solution was collected every 8 h, and an equal volume of the corresponding solution was added to maintain the total volume. The fluorescence intensity at an emission wavelength of 507 nm (excitation wavelength of 488 nm) was measured to quantify the released exosomes. The initial mass of exosomes in Exo-gel was denoted as m0, and the cumulative mass of exosomes released at different times was denoted as m. The cumulative exosome release rate was calculated as: Exo release amount (%) = m / (m0) × 100%; The relationship between the release rate of exosomes and time in two exosome hydrogel materials (Exo-Gel and Exo-Pre-Gel) with different concentrations of hydrogen peroxide (H2O2) is shown as Figure 17 follows.

[0075] Test Example 5: Test on the scavenging of reactive oxygen species by hydrogel in RAW264.7 macrophages.

[0076] DCFH-DA staining was used to detect intracellular oxidative stress; RAW264.7 macrophages were cultured in a 24-well plate (1×10 5 cells per well), pre-cultured in an incubator for 24 h, and then stimulated with serum-free medium containing 100 ng / mL LPS for 12 h. Subsequently, the cells were treated with different substances, PBS, Gel, Exo, and Exo-Gel (the same as in Test Example 4). After 24 h, DCFH-DA and Hoechst 33342 were stained for 20 min in the dark, and observed under a fluorescence microscope. The results are as Figure 18 shown. In the figure, DCFH, Hoechst, and Merge correspond to the images of ROS detection, nuclear staining, and the superposition of the two, respectively.

[0077] Test Example 6: Enzyme-linked immunosorbent assay of macrophages.

[0078] RAW264.7 macrophages were seeded in a 24-well plate (1×10 5 cells per well) and pre-cultured in an incubator for 24 h. Subsequently, the cells were stimulated with serum-free medium containing LPS (100 ng / mL) for 12 h. Then, the cells were treated with different substances, PBS, Gel (Gel-15), Exo, or Exo-Gel (the same as in Test Example 4). After 24 h, the supernatant was collected and measured using an ELISA kit to detect the secretion of immune regulatory factors secreted by cells under different treatment conditions. As Figure 19 shown, Figure A in the figure shows the effect on TNF-α under different treatment conditions of different groups, and Figure B in the figure shows the effect on IL-10 under different treatment conditions of different groups.

[0079] Test Example 7 The CCK-8 method was used to detect the effect on cell proliferation after treatment with different substances. The results are as Figure 20 shown; Among them, Figure A in the figure shows the effect of different substances, PBS, Gel (Gel-15), Exo, and Exo-Gel, on the cell proliferation rate. The Exo-Gel treatment group showed a significant proliferation effect on the third day (P<0.0001). Figure B in the figure shows the effect of different substances, PBS, Gel, Exo, and Exo-Gel, on the proportion of each stage of the cell cycle (G1, S, G2 / M). The Exo-Gel treatment group showed significant changes in the S phase and G2 / M phase (P = 0.0148 and P = 0.0107), while the change in the G1 phase was not significant (P = 0.0918).

[0080] Test Example 8 Detect the effect of the hydrogel on cell migration.

[0081] The effect of different treatments on cell migration was evaluated by cell scratch assay and transwell assay, and the process was as follows: Cell scratch assay: HUVECs were seeded in 12-well culture plates (5×10 5 cells per well). After culturing until 100% confluence, a "wound" was made by scratching the monolayer cells with the tip of a plastic pipette. After washing the monolayer with PBS, the cells were treated with different substances PBS, Gel, Exo, and Exo-Gel respectively. After incubation for 24 h, the wound condition was observed under an optical microscope, as Figure 21 shown; In the transwell assay, HUVECs were resuspended in 200 μL of complete medium (about 5×10 4 cells) and then incubated with the medium treated with different substances PBS, Gel, Exo, and Exo-Gel respectively. After incubation for 24 h, the upper chamber membrane was carefully removed with a cotton swab. At room temperature, the cells in the lower chamber were fixed in 4% paraformaldehyde for 30 min. After crystal violet staining, the migrated HUVECs were observed under an inverted microscope, as Figure 22 shown.

[0082] Test Example 9 The effect of the hydrogel on the formation of tubules in HUVECs.

[0083] The Matrigel solution was thawed overnight at 4 °C and then added to 96-well plates. Subsequently, it was incubated at 37 °C for 1 h to solidify, ensuring no air bubbles were formed. After the Matrigel solidified, HUVECs were collected and resuspended in 200 μL of complete medium (about 5×10 4 cells) and then treated with different substances PBS, Gel, Exo, and Exo-Gel respectively on the Matrigel. After incubation for 4 h, the formation of lumens was observed under an optical microscope, and the results were as Figure 23 shown.

[0084] Test Example 10 The effect of the hydrogel on the healing of diabetic wounds infected with MRSA in rats.

[0085] After 2 months of high-fat feeding, SD rats were fasted for 8 h and then intraperitoneally injected with 1% streptozotocin (STZ, 65 mg / kg) to establish a type 2 diabetes model. The blood glucose levels of the rats were continuously monitored throughout the process using a SureStep blood glucose monitor. Rats with blood glucose levels below 16.7 mM were considered to have failed in modeling and received additional STZ injections. Rats with blood glucose levels exceeding 16.7 mM for 1 week were classified as diabetic. A circular (15 mm in diameter) full-thickness skin defect wound was made on the back of diabetic SD rats, and then an MRSA suspension (100 μL, 107 CFU / mL). To verify MRSA infection and evaluate the antibacterial effect of the hydrogel, on the 1st day and the 7th day after infection, bacterial samples were collected from the wound surfaces of SD rats treated with different substances (PBS, Gel, Exo, and Exo-Gel) on the back using sterile cotton swabs. The swabs were placed in 1 ml of physiological saline, and the viable MRSA load in the diluted suspension was evaluated through a colony formation assay by culturing on LB agar plates at 37°C for 24 h. The results are as follows Figure 24 shown. As can be seen from the figure, on the first day (D1), the colonies under all treatment conditions were relatively dense, but the colony distribution in the Exo- and Exo-Gel-treated groups was slightly sparser. On the 7th day (D7), the colonies in the PBS-treated group were still dense, but the colonies in the Gel-, Exo-, and Exo-Gel-treated groups were significantly reduced. In particular, almost no colonies could be seen on the culture media treated with Gel and Exo-Gel, indicating that these treatments may have an inhibitory effect on bacterial growth; Diabetic rats with mrsa-infected wounds were randomly divided into a PBS group, a Gel group, an Exo group, and an Exo-Gel group, with 3 rats in each group. The wound surfaces were covered with 3M Tegaderm film, and the hydrogel dressings were fixed. Wound debridement and dressing changes were performed at the scheduled time, and the wound sites were photographed. The results are as follows Figure 25 shown; Hematoxylin and eosin (H&E) and Masson's trichrome staining were used to evaluate the healing status, as shown in Figure 26 and Figure 27 shown.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: They can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; And these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A hydrogel for adaptively releasing exosomes, characterized in that, It includes a precursor, a buffer solution, and exosomes. The precursor is dissolved in the buffer solution to form a matrix with a three-dimensional network structure, and the exosomes are loaded in the three-dimensional network structure of the matrix; Among them, the precursor includes polymer HA-ADH and polymer OHA-TK-CP, and the mass ratio of polymer HA-ADH to polymer OHA-TK-CP is 1:5 to 1:1; Polymer HA-ADH is hyaluronic acid - adipic dihydrazide; Among them, HA is hyaluronic acid, and ADH is adipic dihydrazide; Polymer OHA-TK-CP is oxidized hyaluronic acid modified with dithioacetal and phosphocholine groups; Among them, OHA is oxidized hyaluronic acid; TK is 2,2'-(propane-2,2-diylbis(sulfanediyl))bis(ethane-1-amine), and its molecular structural formula is as shown below: ； CP is 2-(dimethyl(prop-2-yn-1-yl)ammonio)ethyl isopropyl phosphate, and its molecular structural formula is as shown below: 。 2. The hydrogel for adaptively releasing exosomes according to claim 1, wherein, In polymer HA-ADH, the substitution degree of ADH is 65% to 75%.

3. The hydrogel for adaptively releasing exosomes according to claim 1, wherein In polymer OHA-TK-CP, the grafting rate of TK is 30% to 35%, and the grafting rate of CP is 10% to 20%.

4. The hydrogel for adaptively releasing exosomes according to claim 1, wherein The mass content percentage of polymer OHA-TK-CP in the hydrogel is 5% to 25%.

5. The hydrogel for adaptively releasing exosomes according to claim 1, wherein, Under physiological conditions, the swelling rate of the hydrogel is 115% to 128%.

6. The hydrogel for adaptively releasing exosomes according to claim 1, wherein The pH value of the buffer solution is 7 to 8.

7. The hydrogel for adaptively releasing exosomes according to claim 1, wherein, The content of exosomes in the matrix is 1×10 8 ~1×10 12 per mL.

8. A method for preparing a hydrogel that adaptively releases exosomes, characterized in that, A hydrogel for preparing the exosome self-adaptive release as described in any one of claims 1 to 7 includes the following steps: S100. Prepare HA-ADH buffer solution and OHA-TK-CP buffer solution containing exosomes respectively; S200. Mix the HA-ADH buffer solution and the OHA-TK-CP buffer solution evenly, and react at 36°C to 38°C for 18 s to 12 min to obtain the exosome self-adaptive release hydrogel.

9. Application of a hydrogel for adaptively releasing exosomes, characterized in that, The hydrogel for exosome self-adaptive release as described in any one of claims 1 to 7, the applications include preparing any one or several of the following: Antibacterial and / or bactericidal products, anti-inflammatory products, wound repair products, immunomodulatory products, and drug carriers.

10. Use of the hydrogel for adaptively releasing exosomes according to claim 9, characterized in that, The wound includes chronic inflammatory wounds caused by diabetes.

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