Engineered small extracellular vesicle hydrogel as well as preparation method and application thereof

Abstract

The invention discloses engineered small extracellular vesicle hydrogel as well as a preparation method and application thereof. The engineered small extracellular vesicle hydrogel comprises a hydrogel matrix, and GDNF-sEVs and a polyphenol compound which are loaded in the hydrogel matrix, wherein the hydrogel matrix comprises a polyacrylamide polymer, xanthan gum and carbomer, the polyacrylamide polymer comprises the following structural unit: [-CH2CH (-CONH-RX-CONH2)-], RX is C1-4 alkylene, and the polyacrylamide polymer comprises the following structural unit:-CH2CH (-CONH-RX-CONH2)-[-CH2CH (-CONH-RX-CONH2)-]. The GDNF-sEVs are small extracellular vesicles loaded with glial cell-derived neurotrophic factors (GDNF-sEVs). According to the engineered small extracellular vesicle hydrogel, through the synergistic effect of the small extracellular vesicles, the glial cell-derived neurotrophic factors and the hydrogel matrix, the injury microenvironment after spinal cord injury can be regulated and controlled, and repair of spinal cord injury is promoted.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biomedical materials, in particular to an engineered small extracellular vesicle hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Spinal cord injury (SCI) is a catastrophic injury to the central nervous system, which can cause sensory and motor dysfunction in patients, and is characterized by high morbidity and mortality. About 10.4-83 people per 100,000 people worldwide suffer from SCI each year. In addition, the incidence and mortality of SCI are increasing every year, which brings huge costs to the national society and poses a great challenge to the medical field. After SCI, a series of pathophysiological changes occur at the injury site, including inflammatory response and neuronal apoptosis, followed by the formation of cavities and scars, resulting in inhibition of axon regeneration. Due to the low plasticity and limited neuron regeneration of the central nervous system (CNS), the nerve regeneration after SCI is very weak. At present, there is no effective method to completely repair the function of the spinal cord after SCI.

[0003] Glial cell-derived neurotrophic factor (GDNF) can significantly promote the survival of motor neurons and has good therapeutic potential for motor recovery after SCI. After spinal cord injury, GDNF can be transported retrogradely along the damaged axons to the injured neurons, exerting a direct neurotrophic effect, inhibiting the death of injured neurons, and increasing the number, diameter of regenerated axons and the number of myelin sheaths, and accelerating axon regeneration. As a newly discovered neurotrophic factor with the strongest biological activity, GDNF can significantly promote the survival and regeneration of neurons and plays an extremely important role in the repair of SCI. However, natural GDNF is rapidly degraded in the body and is difficult to cross the blood-brain barrier, which greatly limits its repair effect on SCI, so it is of great significance to deliver GDNF gene protein to the injury area for sustained expression for the repair of SCI. In recent years, small extracellular vesicles (sEVs) have attracted widespread attention, which can carry a variety of biologically active molecules such as proteins, lipids and nucleic acids, and play an important role in intercellular communication. Due to its natural biocompatibility, low immunogenicity and ability to cross biological barriers, sEVs have become a new carrier in drug delivery systems. However, there is no research on sEVs for loading GDNF, and due to the flow of body fluids and rapid clearance in circulation, sEVs are difficult to achieve sustained and slow release at a specific site, thereby reducing the therapeutic effect. Therefore, it is essential to develop a hydrogel material that can deliver sEVs to a specific site of defect, so that it can be released in situ continuously. SUMMARY

[0004] Therefore, it is necessary to provide an engineered small extracellular vesicle hydrogel and a preparation method and application thereof.

[0005] The first aspect of the present application provides a use of an engineered small extracellular vesicle hydrogel in the preparation of a product for repairing or treating spinal cord injury, wherein the engineered small extracellular vesicle hydrogel comprises a hydrogel matrix and GDNF-sEVs and a polyphenol compound loaded in the hydrogel matrix; the hydrogel matrix comprises a polyacrylamide polymer, xanthan gum and carbomer, the polyacrylamide polymer comprises structural units [-CH2CH(-CONH-R X -CONH2)-], R X is a C 1-4 alkylene group;

[0006] The GDNF-sEVs are small extracellular vesicles loaded with glial cell-derived neurotrophic factor.

[0007] The second aspect of the present application provides an engineered small extracellular vesicle hydrogel, which is the engineered small extracellular vesicle hydrogel in the use of the first aspect of the present application.

[0008] The third aspect of the present application provides a preparation method of an engineered small extracellular vesicle hydrogel, comprising the following steps:

[0009] An acrylamide monomer, xanthan gum and carbomer are mixed in water, and crosslinking is carried out under the action of a photoinitiator to obtain hydrogel particles; wherein the structure of the acrylamide monomer is CH2=CH(-CONH-R X -CONH2), R X is a C 1-4 alkylene group;

[0010] The hydrogel particles are mixed with a polyphenol compound aqueous solution to obtain a functionalized hydrogel NXCT;

[0011] The functionalized hydrogel NXCT is mixed and incubated with GDNF-sEVs to prepare the engineered small extracellular vesicle hydrogel; wherein the GDNF-sEVs are small extracellular vesicles loaded with glial cell-derived neurotrophic factor.

[0012] The foregoing provided engineered small extracellular vesicle hydrogel can regulate the injury microenvironment after spinal cord injury and promote the repair of spinal cord injury through the synergistic effect between small extracellular vesicles, glial cell-derived neurotrophic factor and the hydrogel matrix. By using the hydrogel matrix to load GDNF-sEVs, the injury microenvironment is regulated by exerting anti-inflammatory and antioxidant functions, and at the same time, the small extracellular vesicles load GDNF, which can cross the blood-brain barrier to deliver GDNF to the injury area, promote the survival of neurons, enhance axon regeneration, promote the recovery of motor function after SCI, and thus promote the repair of SCI. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments and examples of the present application, and more completely understand the present application and its beneficial effects, the drawings needed to be used in the description of the embodiments or examples will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0014] Figure 1 Morphology of CON-MSC cells and GDNF-MSC cells in an embodiment of the present application;

[0015] Figure 2 Transmission electron microscopy of CON-sEVs and GDNF-sEVs in an embodiment of the present application;

[0016] Figure 3 NTA detection particle size distribution results of CON-sEVs and GDNF-sEVs in an embodiment of the present application, wherein "Diameter" represents particle size, and "Particles" represents quantity;

[0017] Figure 4 Zeta potential diagram of CON-sEVs and GDNF-sEVs in an embodiment of the present application;

[0018] Figure 5 Western Blot detection of marker proteins in an embodiment of the present application;

[0019] Figure 6 Release curve of GDNF-sEVs in hydrogel in an embodiment of the present application, wherein A is the daily release curve, and B is the cumulative release curve;

[0020] Figure 7 Distribution of GDNF-sEVs in hydrogel in an embodiment of the present application;

[0021] Figure 8 In vitro antioxidant effect of different hydrogel groups in an embodiment of the present application; wherein A is a fluorescence image, and B is an integral optical density;

[0022] Figure 9 In vitro anti-inflammatory effect of different hydrogel groups in an embodiment of the present application; wherein A is the expression of TNF-α, B is the expression of IL-1β, and C is the expression of IL-10;

[0023] Figure 10 Staining results of different hydrogel groups for promoting axonal regeneration in an embodiment of the present application;

[0024] Figure 11 This is a graph showing motor scores after surgery in different spinal cord injury rat groups in one embodiment of the present application;

[0025] Figure 12 This is a footprint detection diagram of different spinal cord injury rat groups after surgery in one embodiment of the present application;

[0026] Figure 13 The HE staining results of spinal cord tissues of rats with different spinal cord injuries after surgery in one embodiment of the present application are shown;

[0027] Figure 14 The expression of 4-HNE in rats with different spinal cord injuries after surgery in one embodiment of the present application;

[0028] Figure 15 The expression of 8-OhdG in rats with different spinal cord injuries after surgery in one embodiment of the present application;

[0029] Figure 16 The expression of anti-inflammatory factors in different spinal cord injury rat groups after surgery in one embodiment of the present application; A represents the expression of TNF-α, B represents the expression of IL-1β, and C represents the expression of IL-10;

[0030] Figure 17 The expression of neuronal markers in different spinal cord injury rat groups after surgery in one embodiment of the present application;

[0031] Figure 18 The expression of choline acetyltransferase in rats with different spinal cord injuries after surgery in one embodiment of the present application;

[0032] Figure 19 HE staining results of the main organs of rats with different spinal cord injuries after surgery in one embodiment of the present application;

[0033] Among them, ** indicates p < 0.01, and *** indicates p < 0.001. DETAILED DESCRIPTION

[0034] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0036] In the present application, "optionally", "optional", "option" means optional, that is, selected from "have" or "no" two parallel schemes. If there are multiple "optional" in a technical solution, if there is no special description, and there is no contradiction or mutual restriction, each "optional" is independent.

[0037] In the present application, "preferably", "better", "better", "as appropriate" only describe the better effect of the implementation or embodiment, and it should be understood that it does not constitute a limitation on the scope of protection of the present application.

[0038] In the present application, the terms "have", "contain", "include" and "comprise" are synonymous, which are inclusive or open, and do not exclude additional, unmentioned members or features. Members or features, such as materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features, such as actions, conditions, timing, state, etc.

[0039] In the present application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of listed contents, and also include open technical features or technical solutions containing listed contents.

[0040] In the present application, the unit related to the data range is indicated only after the right end point with the unit, indicating that the units of the left end point and the right end point are the same.

[0041] In the present application, the steps involved in the method flow are not strictly limited in the order of execution unless otherwise specified herein. Moreover, any step can include multiple sub-steps or multiple stages, which are not necessarily executed at the same time, but can be executed at different times, and the execution order is not necessarily sequential, but can be alternated or simultaneously executed with other steps or other steps or part of sub-steps or stages.

[0042] In the present application, the exemplary description related to "in some embodiments (or examples)", "in one embodiment (or example)" and the like can cover but is not limited to the following meanings: these schemes can be combined with other schemes in a suitable manner to form new technical schemes.

[0043] In the present application, the terms "first", "second", "third" and the like in the "first aspect", "second aspect", "third aspect" and the like are only for the purpose of description, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third" and the like only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0044] In the present application, when referring to a numerical interval (i.e. a numerical range), the distribution of the optional values in the numerical interval is considered to be continuous and includes both numerical endpoints (i.e. the minimum value and the maximum value) of the numerical interval and every value between the two numerical endpoints, unless otherwise specified. When a numerical interval refers only to integers within the numerical interval, unless otherwise specified, the two endpoint integers of the numerical range and every integer between the two endpoints are equivalent to directly listing each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges encompassed therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" is intended to broadly include numerical interval types such as percentage interval, ratio interval, and the like.

[0045] Due to the lower plasticity and limited neuron regeneration of the central nervous system, the nerve regeneration after SCI is very weak. At present, there is no effective method to completely repair the function of the spinal cord after SCI.

[0046] Based on this, the embodiments of the present application at least provide a hydrogel material and a preparation method and application thereof.

[0047] In the present application, the term "small Extracellular Vesicles (sEVs)" refers to a kind of endoplasmic origin extracellular vesicles with a diameter of 30-150 nm, which are secreted by cells. Its safety, stability and ability to cross biological barriers can be used as an advantageous drug delivery carrier.

[0048] In the present application, the term "hydrogel" refers to a kind of traditional and classic biomaterial, which plays an important role in the field of tissue repair and reconstruction, has good biocompatibility and loose porous structure characteristics, can load drugs and realize drug delivery.

[0049] In the present application, the term "Glial-cell-line-derived Neurotrophic Factor (GDNF)" refers to a neurotrophic factor that has trophic activity on dopaminergic neurons, can promote nerve regeneration, axon regeneration and enhance motor function recovery after nerve injury.

[0050] In the first aspect of the present application, the use of an engineered small extracellular vesicle hydrogel in the preparation of a product for repairing or treating spinal cord injury is provided, wherein the engineered small extracellular vesicle hydrogel comprises a hydrogel matrix and GDNF-sEVs and a polyphenol compound loaded in the hydrogel matrix; wherein the hydrogel matrix comprises a polyacrylamide-based polymer, xanthan gum and carbomer, the polyacrylamide-based polymer comprises the following structural unit: [-CH2CH(-CONH-R X -CONH2)-], R X is C 1-4 alkylene; the GDNF-sEVs are small extracellular vesicles loaded with glial cell-derived neurotrophic factor.

[0051] In some embodiments, the spinal cord injury comprises one or more of complete transection spinal cord injury, contusion spinal cord injury and ischemic spinal cord injury.

[0052] In some embodiments, R X may be C1, C2, C3 or C4 alkylene.

[0053] In some embodiments, sEVs are a good drug-loaded delivery system, and by engineering sEVs to load a large amount of GDNF, the repair effect of GDNF in spinal cord injury is enhanced. The use of a hydrogel matrix to load sEVs can make up for the shortcomings of sEVs in specific applications in tissue repair, can enhance the stability of sEVs, and help deliver sEVs to the specific site of the defect for sustained in situ release. Loading the hydrogel with polyphenol compounds can endow the hydrogel with anti-inflammatory and antioxidant biological functions.

[0054] In some embodiments, the mass ratio of the polyacrylamide-based polymer, xanthan gum and carbomer is (15-25):(2-4):1. Without limitation, the mass ratio of the polyacrylamide-based polymer, xanthan gum and carbomer can be, but is not limited to, 20:2:1, 20:3:1, 20:4:1, 15:2:1, 15:3:1, 15:4:1, 25:2:1, 25:3:1 or 25:4:1.

[0055] In some embodiments, the mass ratio of the gel matrix, tannic acid, and GDNF-sEVs is (400-600):(100-200):(2-4). Without limitation, the mass ratio of the gel matrix, tannic acid, and GDNF-sEVs can be, but is not limited to, 500: 100:2, 500: 100:3, 500: 100:4, 500:200:2, 500:200:3, 500:200:4, 400: 100:3, or 400:200:3.

[0056] In some embodiments, the particle size of the GDNF-sEVs is 30-300 nm. Without limitation, the particle size of the GDNF-sEVs can be, but is not limited to, 30 nm, 60 nm, 90 nm, 120 nm, 150 nm, 180 nm, 210 nm, 240 nm, 270 nm, 300 nm, or a value or a range between any two of the aforementioned values.

[0057] In some embodiments, the molecular weight of the polyphenolic compound is 200-2000 Da. Without limitation, the molecular weight of the polyphenolic compound can be, but is not limited to, 200 Da, 400 Da, 600 Da, 800 Da, 1000 Da, 1200 Da, 1400 Da, 1600 Da, 1800 Da, 2000 Da, or a value or a range between any two of the aforementioned values.

[0058] In some embodiments, the number of phenolic hydroxyl groups in the polyphenolic compound is 5-25. Without limitation, the number of phenolic hydroxyl groups in the polyphenolic compound can be, but is not limited to, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or a value or a range between any two of the aforementioned values.

[0059] In some embodiments, the polyphenolic compound comprises one or more of quercetin, catechin, tea polyphenol, epigallocatechin, and tannic acid. Further, the polyphenolic compound is tannic acid.

[0060] In some embodiments, the small extracellular vesicles are derived from mesenchymal stem cells, which are optionally human umbilical cord mesenchymal stem cells.

[0061] In some embodiments, the polyacrylamide-based polymer is poly(N-acryloylglycinamide).

[0062] In some embodiments, the xanthan gum with a suitable molecular weight is selected according to the need to improve the rheological properties of the hydrogel.

[0063] In some embodiments, the carbomer is selected from one or more of carbomer 940, carbomer 934, carbomer 980, and carbomer 2020. Further, the carbomer is carbomer 940.

[0064] In some embodiments, the carbomer with a suitable molecular weight is selected according to the requirement to improve the printing accuracy and mechanical properties.

[0065] In some embodiments, the hydrogel matrix comprises poly(N-acryloylglycinamide), xanthan gum and carbomer 940.

[0066] It should be noted that, in order to maintain mechanical strength and low swelling rate, N-acryloylglycinamide is selected as the main monomer to improve the photo-crosslinking ability and form a hydrogel network structure. In order to prevent diffusion after printing and maintain structural stability, xanthan gum is selected as a rheological modifier to impart shear thinning behavior and improve zero-shear viscosity (which is beneficial for shape retention). In order to improve printing accuracy and reduce layer collapse, carbomer 940 is selected to enhance storage modulus, improve gel elasticity and deformation resistance.

[0067] It should be noted that carbomer 940 is a derivative of polyacrylic acid, not just polyacrylic acid, but a modified polymer formed by crosslinking polymerization. Carbomer 940 is a crosslinked polyacrylic acid polymer, which is polymerized from acrylic acid as a monomer by using an allyl sucrose or pentaerythritol crosslinking agent. Its molecular structure is a three-dimensional network, which has stronger thickening and suspension ability compared with linear polyacrylic acid.

[0068] In some embodiments, tannic acid is used as a post-treatment agent to induce shrinkage through hydrogen bonding and hydrophobic interaction, thereby improving the resolution and mechanical properties of the hydrogel.

[0069] According to experimental exploration, if xanthan gum is omitted or replaced by other rheological modifiers (such as gelatin, sodium alginate, polyethylene glycol, etc.), the zero-shear viscosity of the hydrogel will be significantly reduced, and its shape retention ability will also be weakened, which cannot achieve the expected slow-release effect.

[0070] In a second aspect of the present application, an engineered small extracellular vesicle hydrogel is provided, which is the engineered small extracellular vesicle provided above.

[0071] In a third aspect of the present application, a preparation method of an engineered small extracellular vesicle hydrogel is provided, comprising the following steps:

[0072] S100: mixing acrylamide monomers, xanthan gum and carbomer in water, and crosslinking under the action of a photoinitiator to obtain hydrogel particles; wherein the structure of the acrylamide monomer is CH2=CH(-CONH-R X -CONH2), R X is a C 1-4 alkylene group;

[0073] S200: mixing the hydrogel particles with the polyphenol compound to obtain a functionalized hydrogel NXCT;

[0074] S300: incubating the functionalized hydrogel NXCT with GDNF-sEVs to obtain the engineered small extracellular vesicle hydrogel; wherein the GDNF-sEVs are small extracellular vesicles loaded with glial cell-derived neurotrophic factor.

[0075] In some embodiments, in step S100, the acrylamide monomer is N-acryloylglycine amide.

[0076] In some embodiments, in step S100, the carbomer is carbomer 940.

[0077] In some embodiments, in step S100, the mass ratio of the acrylamide monomer, the xanthan gum and the carbomer is (15-25):(2-4):1. Without limitation, the mass ratio of the acrylamide monomer, the xanthan gum and the carbomer can be, but is not limited to, 20:2:1, 20:3:1, 20:4:1, 15:2:1, 15:3:1, 15:4:1, 25:2:1, 25:3:1 or 25:4:1.

[0078] It should be noted that under the action of the photoinitiator and ultraviolet irradiation, NAGA forms a poly(N-acryloylglycine amide) through radical polymerization. The xanthan gum interacts with NAGA through hydrogen bonds; the carbomer 940 forms an interpenetrating network with NAGA through hydrogen bonds of carboxylate at neutral pH.

[0079] In some embodiments, the crosslinking time is 20-30 min. Without limitation, the crosslinking time can be, but is not limited to, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min or a value or range between any two of the above values.

[0080] In some embodiments, the polyphenol compound is tannic acid.

[0081] In some embodiments, the concentration of the tannic acid is 10-20 w / v%. Without limitation, the concentration of the tannic acid can be, but is not limited to, 10 w / v%, 12 w / v%, 14 w / v%, 16 w / v%, 18 w / v%, 20 w / v% or a value or range between any two of the above values.

[0082] In some embodiments, the mass ratio of the hydrogel particles to the tannic acid is (4-6):1. Without limitation, the mass ratio of the hydrogel particles to the tannic acid can be, but is not limited to, 4:1, 5:1, 6:1 or a ratio or range between any two of the above ratios.

[0083] In some embodiments, the time for mixing the hydrogel particles with tannic acid is 1 h to 1.5 h. Without limitation, the time for mixing the hydrogel particles with tannic acid can be, but is not limited to, 1 h, 1.3 h, 1.5 h, or a value or a range between any two of the above values.

[0084] In some embodiments, the time for incubating the functionalized hydrogel NXCT with GDNF-sEVs is 22 h to 26 h. Without limitation, the time for incubating the functionalized hydrogel NXCT with GDNF-sEVs can be, but is not limited to, 22 h, 23 h, 24 h, 25 h, 26 h, or a value or a range between any two of the above values.

[0085] In some embodiments, the method for preparing GDNF-sEVs comprises:

[0086] S100': After culturing the mesenchymal stem cells to the 2nd generation, adding a recombinant lentivirus overexpressing glial cell-derived neurotrophic factor for co-culturing to the 3rd to 6th generation, and collecting the culture solution;

[0087] S200': Performing ultracentrifugal separation and purification on the culture solution to obtain the GDNF-sEVs.

[0088] Some embodiments are provided below.

[0089] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods not specified in the following examples are preferably referred to the instructions given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0090] In the following examples, the measurement parameters of the raw material components may, without specific instructions, have slight deviations within the weighing accuracy range. With respect to the temperature and time parameters, acceptable deviations caused by the instrument testing accuracy or operation accuracy are allowed.

[0091] In the following examples, N-acryloylglycine amide is purchased from Zhengzhou Alpha Chemical Co., Ltd., with a molecular weight of 128.13; xanthan gum is purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., with a CAS number of 11138-66-2 and a molecular weight of 241.11496; carbomer 940 is purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., with a CAS number of 9007-20-9 and a molecular weight of 280.747.

[0092] Example 1

[0093] 1. Preparation of GDNF-sEVs

[0094] (1) Primary culture of umbilical cord mesenchymal stem cells (MSC): Human umbilical cord tissue was minced into 1-3 mm pieces within 5 hours after collection. 3 Tissue fragments were cultured in α-MEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin at 37°C and 5% CO2. The obtained cells were used for subsequent experiments at passages 2 to 6.

[0095] (2) The MSC cells obtained by primary culture in step (1) were cultured to the P2 generation, and the recombinant lentivirus (from Shanghai Hanheng Biotechnology) overexpressing GDNF was co-cultured with them to obtain GDNF-MSC overexpressing GDNF. The GDNF-MSC were then passaged to obtain the P3 generation, and then serum-free α-MEM was added to collect the culture supernatant. The collection time was 48 hours, and the cell density of the collected supernatant was above 70%; the supernatant collection was stopped after the cells were cultured to the P6 generation.

[0096] (3) The collected supernatant was centrifuged at 500g (4°C) and 2000g (4°C) for 10 min to remove dead cells and cell debris; the supernatant was centrifuged at 10,000g at 4°C for 30 min to remove organelles. Then, the supernatant was transferred to a 100 kDa MWCO ultrafiltration centrifuge tube and centrifuged at 2000g for 30 min. Then, the concentrate in the upper tube was collected and ultracentrifuged at 100,000g at 4°C for 70 min to collect the precipitate (i.e., GDNF-sEVs particles). The GDNF-sEVs particles were then resuspended with phosphate-buffered saline (PBS) and centrifuged again at 100,000g for 70 min to collect the precipitate. Finally, the GDNF-sEVs pellet was resuspended with phosphate-buffered saline (PBS), and the purified GDNF-sEVs were collected and filtered on a 0.22 μm pore filter to obtain a GDNF-sEVs suspension. The solution was stored in a -80°C freezer for future use.

[0097] (4) The cultured MSC cells without GDNF (CON-MSC) and GDNF-MSC cells were observed under an optical microscope. The results are as follows: Figure 1 As shown, it can be seen that GDNF-MSC cells were successfully prepared.

[0098] 2. Identification of GDNF-sEVs

[0099] (1) Transmission electron microscopy (TEM) observation of the morphology of small extracellular vesicles: Take an appropriate amount of sEVs suspension without GDNF (CON-sEVs) and GDNF-sEVs suspension onto a 2 mm diameter sample-loaded copper grid, and let it stand at room temperature for 5 min. Then, gently absorb the residual liquid on the edge of the copper grid with filter paper, and then place the copper grid upside down on a 30 g / L phosphotungstic acid (pH 6.8) droplet at room temperature for 5 min. Finally, dry the copper grid under an incandescent lamp, and observe and take pictures under a transmission electron microscope. The results are shown in Figure 2 .

[0100] (2) Nanoparticle tracking analysis (NTA) detection of particle size: First, dilute the sEVs suspension without GDNF (CON-sEVs) and the GDNF-sEVs suspension to the optimal concentration for instrument detection using PBS, mix thoroughly, and then use a disposable syringe to inject 1 mL of the sample into the sample chamber at a constant speed. The particle size and concentration of the small extracellular vesicles will be presented by the software provided with the instrument. The results are shown in Figure 3 . It can be seen that the particle size of GDNF-sEVs does not change significantly compared with CON-sEVs, and the particle size is mainly distributed in the range of 30 nm to 300 nm.

[0101] (3) Potential measurement: Use a Zeta potential analyzer to detect CON-sEVs and GDNF-sEVs, respectively. The results are shown in Figure 4 . It can be seen that the absolute value of the potential of GDNF-sEVs is smaller.

[0102] (4) Western blot detection of surface marker proteins: After the extracted small extracellular vesicles are fully lysed, add 1 / 4 volume of 5x SDS loading buffer, boil for 5 min, load 200 μg of total protein, perform electrophoresis, transfer the membrane, and then block it with 50 g / L skim milk in TBS / T at room temperature for 1 h. React with rabbit anti-human TSG101 antibody, rabbit anti-human CD81 antibody, rabbit anti-human calnexin antibody, and rabbit anti-human Alix (1:500) at 4°C overnight. The next day, wash the membrane with TBST 3 times, incubate with HRP-labeled goat anti-rabbit IgG secondary antibody at 37°C for 1 h, wash the membrane with TBST 3 times, add the pre-mixed HRP chemiluminescence substrate, and detect it by a chemiluminescence gel imaging system. The results are shown in Figure 5 . It can be seen that the marker proteins TSG101, CD81, and GDNF of GDNF-sEVs are positively expressed, and Calnexin is negatively expressed.

[0103] 3. Preparation of GDNF-sEVs@NXCT hydrogel

[0104] N-acryloylglycineamide NAGA (2 g), xanthan gum XG (0.3 g) and carbomer 940 CBP940 (0.1 g) were mixed in deionized water (10 mL) with constant stirring for 30 min. Then, a photoinitiator Irgacure 1173 (10 μL) was added to the above solution to form a hydrogel precursor solution. Pouring into a mold, using ultraviolet irradiation crosslinking for 25 min, the curing was completed. Then tannic acid TA was dissolved in deionized water at a ratio of 1:10, the obtained hydrogel particles after curing were immersed in the tannic acid solution for one hour, and then the functionalized hydrogel NXCT was obtained.

[0105] The obtained hydrogel was sterilized overnight outside, and washed with 70% ethanol and PBS for sterilization, then the obtained GDNF-sEVs suspension was injected into the NXCT hydrogel, and then the obtained GDNF-sEVs@NXCT was incubated at 4°C for 24 hours to obtain the multifunctional hydrogel GDNF-sEVs@NXCT of sEVs.

[0106] 4. Performance detection of GDNF-sEVs@NXCT

[0107] 4.1 Release profile

[0108] The hydrogel was incubated in 1 mL PBS solution in vitro. At intervals of 1 day, 500 μL of supernatant was collected and replaced with an equal volume of PBS solution. Then the collected samples were analyzed by bicinchoninic acid (BCA) detection method to determine the amount of free sEVs in the supernatant. The daily release curve and the cumulative release curve over time were calculated and plotted, as shown in A and B of 6, it can be seen that the GDNF-sEVs in GDNF-sEVs@NXCT can be released for about a week, and more than 80% of GDNF-sEVs can be released from the NXCT hydrogel.

[0109] 4.2 Distribution of GDNF-sEVs in hydrogel

[0110] GDNF-sEVs were labeled with Dil dye, and then the labeled GDNF-sEVs were wrapped in NXCT hydrogel, and the adhesion and distribution of GDNF-sEVs in NXCT hydrogel were detected by laser confocal microscope, as shown in Figure 7 , it can be seen that more GDNF-sEVs adhere to the porous structure, and these GDNF-sEVs are uniformly distributed in the hydrogel.

[0111] 5. In vitro functional detection of GDNF-sEVs@NXCT

[0112] The hydrogels were grouped into a hydrogel group (NXCT), a sEVs-containing hydrogel group (CON-sEVs@NXCT), and a GDNF-sEVs-containing hydrogel group (GDNF-sEVs@NXCT) for in vitro functional detection.

[0113] 5.1 Antioxidant capacity

[0114] The hydrogels were co-cultured with PC12 cells for 24 h, and then the culture medium was replaced with peroxidation culture medium containing 100 μM H2O2 for 24 h. After peroxidation culture, ROS assay kit (purchased from Beijing Solabio Technology Co., Ltd.) was used to detect, samples were observed by fluorescence microscope, and relative fluorescence was analyzed by Image J and GraphPad Prism, results as shown in A and B of Figure 8 , it can be seen that under the fluorescence microscope, the fluorescence intensity of DCF is weakened under the action of GDNF-sEVs@NXCT hydrogel, and it has strong antioxidant capacity.

[0115] 5.2 Anti-inflammatory capacity

[0116] The hydrogels were co-cultured with PC12 cells for 24 h, and then the culture medium was replaced with culture medium containing 1% LPS for 24 h, and then the supernatant was collected, and the expression levels of TNF-α, IL-1β and IL-10 in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA) kit (purchased from Hangzhou Link Biological Technology Co., Ltd.) according to the manufacturer's instructions. The absorbance was measured at 450 nm wavelength using a microplate reader, and the results are shown in A, B and C of Figure 9 , it can be seen that GDNF-sEVs@NXCT can better inhibit the production of pro-inflammatory factors and promote the production of anti-inflammatory factors, thereby having better anti-inflammatory effect.

[0117] 5.3 Promoting axon regeneration capacity

[0118] After co-culturing GDNF-sEVs@NXCT with dorsal root ganglion (DRG) for 48 h, it was fixed with 4% formaldehyde, 1:200 TUJ1 antibody fluorescence staining, and samples were observed by fluorescence microscope, results as shown in Figure 10 , by co-culturing DRG cells with different groups of hydrogels, it can be found that CON-sEVs@NXCT and GDNF-sEVs@NXCT can both promote axon regeneration, and GDNF-sEVs@NXCT has better effect.

[0119] 6, GDNF-sEVs@NXCT promotes improvement of motor function of spinal cord injury rats

[0120] 6.1 Spinal cord injury hemisection model

[0121] After the rats were weighed and anesthetized, they were placed on the operating table in a prone position. The fur on the back of the mouse was prepared. After the skin was disinfected with iodophor, a midline incision was made on the back, the skin and muscle were separated, the spine was fully exposed, and the T9-T11 laminectomy was performed after accurate positioning. The operation was as gentle as possible to avoid damage to the spinal cord. The lamina was removed to fully expose the spinal cord. The T9-T11 segment was exposed, and a wedge-shaped incision was made on the left side of the median spinal cord vein to form a defect of about 2 mm in size on the left half of the spinal cord (the sham operation group did not perform this operation). Different groups of hydrogels were placed, and after hemostasis, the wound was sutured layer by layer. The rats were assisted in urination 3 times a day until they recovered their independent urination function.

[0122] 6.2 Motor score

[0123] The SD rat spinal cord injury hemisection model was established and the corresponding groups were implanted with hydrogels. Different rats were divided into sham operation group (Sham), spinal cord injury group (SCI), hydrogel group (NXCT), sEVs-containing hydrogel group (CON-sEVs@NXCT), and GDNF-sEVs-containing hydrogel group (GDNF-sEVs@NXCT). The Basso-Beattie-Bresnahan (BBB) score was detected on days 1, 7, 14, 21, 28, 35, 42, 49, and 56 after the operation, and the results are shown in Figure 11 It can be seen that the score of the GDNF-sEVs@NXCT group is higher.

[0124] 6.3 Footprint detection

[0125] On day 56 after spinal cord injury in SD rats, dye was added to the front and hind limbs of the rats, and they were allowed to crawl on paper to leave marks. The length of the front and rear steps on the injured side was measured, and the results were statistically analyzed, as shown in Figure 12 It can be seen that the three groups of implanted hydrogels can promote the recovery of the motor function of the rat hind limbs. In comparison, GDNF-sEVs@NXCT has a more obvious recovery effect, and the improvement in step length is more effective, achieving the best recovery effect of motor function.

[0126] 6.4 HE staining of spinal cord tissue

[0127] On day 56 after spinal cord injury in SD rats, the spinal cord tissue specimens of the rats in each group were obtained by heart perfusion with formaldehyde, and the sections were prepared and stained using hematoxylin-eosin staining (HE). The results are shown in Figure 13 It indicates that the GDNF-sEVs@NXCT hydrogel can better improve the pathological changes after spinal cord injury.

[0128] 7. In vivo functional detection

[0129] 7.1 Antioxidant capacity

[0130] After 7 days of spinal cord injury surgery, the spinal cord tissues of rats in each group were taken to make sections and perform immunofluorescence staining. 4-hydroxy nonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OHdG) were selected to observe the fluorescence expression of the sections, and the results are shown in Figure 14 and Figure 15 4-HNE and 8-OHdG are the products of lipid peroxidation and DNA oxidative damage caused by oxidative stress, respectively, and GDNF-sEVs@NXCT can significantly reduce oxidative stress damage after spinal cord injury in rats.

[0131] 7.2 Anti-inflammatory ability

[0132] After 7 days of spinal cord injury surgery, the plasma samples of rats in each group were taken and subjected to enzyme-linked immunosorbent assay (ELISA) kit to determine the expression levels of TNF-α, IL-1β and IL-10 in the supernatant. The determination was performed according to the manufacturer's instructions. The absorbance was measured at 450 nm wavelength using a microplate reader, and the results are shown in A, B and C of Figure 16 It can be seen that GDNF-sEVs@NXCT can better inhibit the production of pro-inflammatory factors and promote the production of anti-inflammatory factors, thereby having better anti-inflammatory effect.

[0133] 7.3 Promote nerve regeneration ability

[0134] After 56 days of spinal cord injury surgery, the spinal cord tissues of rats in each group were taken to make sections and perform immunofluorescence staining. Neuronal marker (NF) and glial cell marker (GFAP) immunofluorescence staining were selected to observe the fluorescence expression of the sections.

[0135] After 56 days of spinal cord injury surgery, the spinal cord tissues of rats in each group were taken to make sections and perform immunofluorescence staining. Choline acetyltransferase (ChAT) immunofluorescence staining was selected to observe the fluorescence expression of the sections, and the results are shown in Figure 17 and Figure 18 It can be seen that compared with the injury group and other groups, after treatment with GDNF-sEVs@NXCT hydrogel, the neuronal marker (NF) and choline acetyltransferase (ChAT) significantly increased, and the glial cell marker (GFAP) significantly decreased. It is shown that after spinal cord injury, GDNF-sEVs@NXCT has stronger function of promoting nerve repair and regeneration.

[0136] 8. Biological safety detection of GDNF-sEVs@NXCT

[0137] At 56 days after the spinal cord injury operation, the main organs of the rats in each group were taken, sections were made, hematoxylin-eosin staining (HE staining) was performed, and the safety of GDNF-sEVs@NXCT treatment was evaluated, and the results are shown in FIG. 8. Figure 19 As can be seen from the comparison of the HE staining of the whole body tissues of the rats treated with GDNF-sEVs@NXCT, including the heart, liver, spleen, lung and kidney, with the control group, the injury group and other groups, no toxic effect of the hydrogel treatment on the whole body tissues was observed, indicating the safety of GDNF-sEVs@NXCT.

[0138] The technical features of the above-described embodiments can be combined in any manner. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict, they should be considered as falling within the scope of the present disclosure.

[0139] The above-described embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Use of an engineered small extracellular vesicle hydrogel in the preparation of a product for repairing or treating spinal cord injury, wherein: The engineered small extracellular vesicle hydrogel comprises a hydrogel matrix and GDNF-sEVs and polyphenol compounds loaded in the hydrogel matrix; the hydrogel matrix comprises a polyacrylamide polymer, xanthan gum and carbomer, and the polyacrylamide polymer comprises the following structural units: [-CH2CH(-CONH-R X -CONH2)-],R X C 1-4 alkylene; The GDNF-sEVs are small extracellular vesicles loaded with glial cell line-derived neurotrophic factor.

2. The use according to claim 1, characterized in that The mass ratio of the polyacrylamide polymer, xanthan gum and carbomer is (15-25): (2-4):

1.

3. The use according to claim 1, characterized in that The mass ratio of the hydrogel matrix, tannic acid and GDNF-sEVs is (400-600): (100-200): (2-4).

4. The use according to any one of claims 1 to 3, wherein Meet one or more of the following conditions: The molecular weight of the polyphenol compound is 200Da~2000Da; The number of phenolic hydroxyl groups in the polyphenol compound is 5 to 25; The polyphenol compound includes one or more of quercetin, catechin, tea polyphenols, epigallocatechin and tannic acid; The small extracellular vesicles are derived from mesenchymal stem cells, and optionally, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells; The polyacrylamide polymer is poly(N-acryloylglycineamide); The carbomer is selected from one or more of the following: carbomer 940, carbomer 934, carbomer 980 and carbomer 2020.

5. An engineered small extracellular vesicle hydrogel, characterized in that: The engineered small extracellular vesicle hydrogel is the engineered small extracellular vesicle hydrogel in the use according to any one of claims 1 to 4.

6. A method for preparing an engineered small extracellular vesicle hydrogel, characterized in that: The following steps are involved: Acrylamide monomers, xanthan gum and carbomer are mixed in water and cross-linked under the action of a photoinitiator to obtain hydrogel particles; wherein the structure of the acrylamide monomer is CH2=CH(-CONH-R X -CONH2), R X C 1-4 alkylene; mixing the hydrogel particles with a polyphenol compound to obtain a functionalized hydrogel NXCT; The functionalized hydrogel NXCT is mixed and incubated with GDNF-sEVs to prepare the engineered small extracellular vesicle hydrogel; wherein the GDNF-sEVs are small extracellular vesicles loaded with glial cell line-derived neurotrophic factor.

7. The preparation method according to claim 6, wherein Meet one or more of the following conditions: The acrylamide monomer is N-acryloyl glycinamide; The carbomer is carbomer 940; The mass ratio of the acrylamide monomer, xanthan gum and carbomer is (15-25): (2-4): 1; The cross-linking time is 20 min to 30 min.

8. The preparation method according to claim 6, wherein The step of mixing the hydrogel particles with the polyphenol compound satisfies one or more of the following conditions: The polyphenol compound is tannic acid; The concentration of the polyphenol compound is 10w / v%~20w / v%; The mass ratio of the hydrogel particles to the polyphenol compound is (4-6):1; The time for mixing the hydrogel particles and the polyphenol compound is 1 hour to 1.5 hours.

9. The preparation method according to claim 6, wherein The functionalized hydrogel NXCT and GDNF-sEVs were mixed and incubated for 22 h to 26 h.

10. The preparation method according to any one of claims 6 to 9, characterized in that The preparation method of the GDNF-sEVs comprises: After culturing mesenchymal stem cells to the second passage, recombinant lentivirus overexpressing glial cell line-derived neurotrophic factor was added and co-cultured to the third to sixth passages, and the culture medium was collected; The culture fluid is subjected to ultraspeed separation and purification to obtain the GDNF-sEVs.

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