KeMA slow-release hydrogel system loaded with anti-aging exosome as well as preparation method and application of KeMA slow-release hydrogel system

Abstract

The invention discloses a KeMA slow-release hydrogel system loaded with an anti-aging exosome, a preparation method and application, the preparation method of the KeMA slow-release hydrogel system comprises the following steps: S1, preparing the anti-aging exosome, S2, synthesizing a KeMA hydrogel pre-polymerization liquid system, S3, synthesizing a KeMA slow-release hydrogel system, synthesizing the KeMA slow-release hydrogel system loaded with the anti-aging exosome from the materials of S1 and S2 according to a ratio, and S4, adding the KeMA slow-release hydrogel system loaded with the anti-aging exosome into the KeMA slow-release hydrogel system loaded with the anti-aging exosome, so as to obtain the KeMA slow-release hydrogel system loaded with the anti-aging exosome. 50 to 60 parts of the anti-aging exosome are added into every 99990 to 100010 parts of the hydrogel pre-polymerization liquid system. The KeMA hydrogel system capable of realizing efficient loading and sustained release of the BMSC-sourced anti-aging exosome is constructed for the first time, stable embedding and controllable release of the exosome are realized by optimizing physicochemical properties of the hydrogel, the problems that the exosome is easy to remove in vivo and lacks continuous action are effectively solved, and the anti-aging effect of the BMSC-sourced anti-aging exosome is improved. Meanwhile, by means of continuous release of the anti-aging exosome, aging of nucleus pulposus cells can be effectively improved, intervertebral disc degeneration is delayed, and a brand new treatment thought is provided for intervertebral disc degeneration.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine, in particular to a KeMA sustained-release hydrogel system loaded with anti-aging exosomes, a preparation method and application. BACKGROUND

[0002] With the aging of the population, lumbar and leg pain caused by intervertebral disc degeneration (IVDD) has become a global health burden. Existing therapies can only relieve symptoms or bring surgical risks, and cannot reverse degeneration. Mesenchymal stem cells (MSCs) and their exosomes have repair potential, but face challenges such as low survival rate, poor targeting, and rapid clearance in vivo, limiting clinical translation. However, the loading capacity of natural exosomes is limited, widely distributed in vivo, difficult to enrich in the lesion site, and lacks tissue or cell specificity, resulting in limited efficacy. In addition, exosomes are easily cleared in vivo, and cannot achieve sustained and stable local release, which seriously affects their clinical translation and application prospects. Therefore, how to enhance the therapeutic functional properties of natural exosomes and how to achieve safe, controllable and sustained local release in the intervertebral disc, the present application proposes a KeMA sustained-release hydrogel system loaded with anti-aging exosomes, a preparation method and application to solve the above problems. SUMMARY

[0003] The purpose of the present application is to provide a KeMA sustained-release hydrogel system loaded with anti-aging exosomes, a preparation method and application to solve the problems raised in the background.

[0004] To achieve the above purpose, the present application provides the following technical scheme: a KeMA sustained-release hydrogel system loaded with anti-aging exosomes, comprising, by mass fraction: anti-aging exosomes: 50-60 parts, keratin: 90-110 parts, and methacrylated gelatin GelMA: 99990-100010 parts.

[0005] A preparation method of a KeMA sustained-release hydrogel system loaded with anti-aging exosomes is applied to the above-mentioned KeMA sustained-release hydrogel system loaded with anti-aging exosomes, and the preparation method of the KeMA sustained-release hydrogel system comprises:

[0006] S1, preparing anti-aging exosomes;

[0007] S2, synthesizing a KeMA hydrogel pre-polymer system;

[0008] S3, synthesizing a KeMA sustained-release hydrogel system, and proportionally combining the materials of S1 and S2 into a KeMA sustained-release hydrogel system loaded with anti-aging exosomes, 50-60 parts of anti-aging exosomes are added to every 99990-100010 parts of the hydrogel pre-polymer system.

[0009] Preferably, the preparation of anti-aging exosomes in S1 comprises the following steps:

[0010] S101, cell culture and treatment, when the confluence of primary mesenchymal stem cells reaches 60-70%, replace the original culture medium with exosome-free serum culture medium containing 90-110 parts of uric stone;

[0011] S102, collect cell supernatant, after the culture is completed, collect the cell supernatant containing secretions;

[0012] S103, obtain exosomes, the collected supernatant is subjected to ultracentrifugation at a centrifugal force of 100,000 g to obtain anti-aging exosomes.

[0013] Preferably, the cell culture and treatment in S101 include the following steps:

[0014] S1011, cell culture, culture the primary mesenchymal stem cells to the second generation, and when the cell confluence reaches 60-70%, proceed to the next step;

[0015] S1012, replace the culture medium containing drugs, discard the original culture medium, and replace it with exosome-free serum culture medium containing 90-110 parts of uric stone;

[0016] S1013, re-culture, continue to culture the second generation of mesenchymal stem cells in the replaced culture medium for 2 days.

[0017] Preferably, the collection of cell supernatant in S102 includes the following steps:

[0018] S1021, terminate culture and prepare, after the culture is completed, remove the cell culture container from the incubator, terminate the culture process, and prepare to collect the supernatant;

[0019] S1022, collect and transfer, aspirate the supernatant containing cell secretions in the container and transfer it to a sterile collection tube.

[0020] Preferably, the obtaining of exosomes in S103 includes the following steps

[0021] S1031, sample preparation, aliquot the collected cell supernatant into ultracentrifuge tubes and perform balancing treatment;

[0022] S1032, ultracentrifugation, place the balanced centrifuge tubes in an ultracentrifuge and run at a centrifugal force of 100,000 g for 130-150 min to precipitate the exosomes to the bottom of the tube;

[0023] S1033, collect exosomes, discard the supernatant to obtain the anti-aging exosome precipitate at the bottom of the tube.

[0024] Preferably, the KeMA hydrogel pre-polymer system in S2 is:

[0025] S201, preparing a GelMA solution, dissolving methacrylated gelatin dry powder in double distilled water containing a photoinitiator to prepare a GelMA solution with a concentration of 10%;

[0026] S202, preparing a KeMA prepolymer solution, adding 100ug keratin to the 10% GelMA solution, then mixing to obtain a KeMA hydrogel prepolymer solution system.

[0027] Preferably, the preparation of the GelMA solution in S201 includes the following steps:

[0028] S2011, weighing and feeding, weighing methacrylated gelatin (GelMA) dry powder and adding it to a container containing double distilled water containing a photoinitiator;

[0029] S2012, dissolving and constant volume, dissolving the dry powder by stirring or oscillation to finally prepare a GelMA solution with a concentration of 10%.

[0030] Preferably, the preparation of the KeMA prepolymer solution in S202 includes the following steps:

[0031] S2021, adding functional components, adding 100ug keratin to the 10% GelMA solution prepared in S201;

[0032] S2022, obtaining a prepolymer solution, ultrasonic treatment of the mixed solution for 25-35min, after ultrasonic treatment, the keratin is fully and uniformly dispersed in the GelMA solution to obtain a uniform KeMA hydrogel prepolymer solution system.

[0033] Application of the KeMA sustained-release hydrogel system loaded with anti-aging exosomes, the KeMA sustained-release hydrogel system loaded with anti-aging exosomes is used to delay intervertebral disc degeneration.

[0034] Technical effects and advantages of the present application:

[0035] The present application first constructs a KeMA hydrogel system that can realize efficient loading and slow release of BMSC-derived anti-aging exosomes, and realizes stable embedding and controllable release of exosomes by optimizing the physical and chemical properties of the hydrogel, effectively solving the problems of easy clearance of exosomes in vivo and lack of sustained effect, and the sustained release of anti-aging exosomes can effectively improve the aging of nucleus pulposus cells and delay intervertebral disc degeneration, providing a new treatment idea for intervertebral disc degeneration. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The present application is a method implementation flowchart.

[0037] Figure 2 Anti-aging exosome electron microscope graph of the anti-aging exosome-loaded KeMA sustained-release hydrogel system of the present application.

[0038] Figure 3 Anti-aging exosome WB identification graph of the anti-aging exosome-loaded KeMA sustained-release hydrogel system of the present application.

[0039] Figure 4 Fluorescence graph of the uptake of different source exosomes by nucleus cells.

[0040] Figure 5 Anti-aging exosome anti-aging effect light microscope graph.

[0041] Figure 6 KeMA sustained-release hydrogel system SEM graph.

[0042] Figure 7 Anti-aging exosome-loaded KeMA sustained-release hydrogel system treatment effect tissue staining graph. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0044] The present application provides as shown in Figures 1-7

[0045] Embodiment one, the anti-aging exosome-loaded KeMA sustained-release hydrogel system, characterized in that, by mass parts, it comprises: anti-aging exosomes: 50-60 parts, keratin: 90-110 parts, and methylacrylated gelatin GelMA: 99990-100010 parts.

[0046] Embodiment two, the preparation method of the anti-aging exosome-loaded KeMA sustained-release hydrogel system, applied to the anti-aging exosome-loaded KeMA sustained-release hydrogel system described above, the preparation method of the KeMA sustained-release hydrogel system comprises:

[0047] S1, preparing anti-aging exosomes;

[0048] S2, synthesizing a KeMA hydrogel pre-polymer system;

[0049] ​S3, synthesis of KeMA sustained-release hydrogel system, the materials of S1 and S2 are proportioned into the KeMA sustained-release hydrogel system loaded with anti-aging exosomes, 50-60 parts of anti-aging exosomes are added into 99990-100010 parts of the hydrogel prepolymer system.

[0050] By accurately adding 50-60 parts of anti-aging exosomes per milliliter of KeMA hydrogel prepolymer system, The efficient distribution and stable loading of exosomes in the hydrogel can be achieved. This ratio not only ensures the biological activity of exosomes, but also maintains the structural integrity and mechanical properties of the hydrogel. The uniform dispersion of exosomes in the KeMA matrix helps to achieve sustained release and targeted action, thereby achieving long-lasting anti-inflammatory, antioxidant and anti-aging effects in the local microenvironment. The optimized ratio enables the hydrogel-exosome composite system to have better biological function and tissue repair potential, ensuring the stability and effectiveness of the treatment process.

[0051] Preferably, the preparation of anti-aging exosomes in S1 includes the following steps:

[0052] S101, cell culture and treatment, when the confluence of primary mesenchymal stem cells reaches 60%-70%, replace the exosome-free serum culture medium containing 90-110 parts of urolithin, and continue to culture;

[0053] S102, collect cell supernatant, after the culture is completed, collect the cell supernatant containing secretions;

[0054] S103, obtain exosomes, ultracentrifuge the collected supernatant at a centrifugal force of 100000g to obtain anti-aging exosomes.

[0055] Preferably, the cell culture and treatment in S101 include the following steps:

[0056] S1011, cell culture, culture the primary mesenchymal stem cells to the second generation, and when the cell confluence reaches 60%-70%, proceed to the next step;

[0057] S1012, replace the drug-containing culture medium, discard the original culture medium, and replace it with an exosome-free serum culture medium containing 90-110 parts of urolithin;

[0058] S1013, re-culture, continue to culture the second generation of mesenchymal stem cells in the replaced culture medium for 2 days.

[0059] Preferably, the collection of cell supernatant in S102 includes the following steps:

[0060] S1021, terminate culture and prepare, after the culture is completed, remove the cell culture container from the incubator, terminate the culture process, and prepare to collect the supernatant.

[0061] S1022, collect and transfer the supernatant containing cell secretions in the transfer container to a sterile collection tube.

[0062] Preferably, the exosomes obtained in S103 include the following steps

[0063] S1031, sample preparation, the collected cell supernatant is aliquoted into ultracentrifuge tubes and subjected to balancing treatment;

[0064] S1032, ultracentrifugation, the balanced centrifuge tube is placed in an ultracentrifuge and run at a centrifugal force of 100,000 g for 130-150 min to precipitate the exosomes at the bottom of the tube;

[0065] S1033, collect exosomes, discard the supernatant, and obtain the anti-aging exosome precipitate at the bottom of the tube.

[0066] The KeMA hydrogel pre-polymer system is prepared by mixing keratin, LAP and GelMA, which has the characteristics of being injectable and excellent mechanical support. At the same time, the RGD sequence rich on keratin can effectively anchor exosomes to achieve the purpose of sustained delivery and slow release. Urolithin-preconditioned stem cells can secrete exosomes with superior functions on the basis of their stem cell characteristics (such as self-renewal and multi-directional differentiation ability). After urolithin regulation, the expression profile of biological active molecules of stem cell-derived exosomes is optimized, which has stronger antioxidant, anti-inflammatory and anti-aging effects. This kind of exosomes can significantly improve the cell microenvironment, promote cell repair and tissue regeneration, enhance cell metabolic activity, and perform outstandingly in regulating mitochondrial function and delaying aging-related signal pathways.

[0067] Preferably, the KeMA hydrogel pre-polymer system in S2 is:

[0068] S201, preparing a GelMA solution, dissolving methacrylated gelatin dry powder in double distilled water containing a photoinitiator to prepare a GelMA solution with a concentration of 10%;

[0069] S202, preparing a KeMA pre-polymer solution, adding 100 μg of keratin to the 10% GelMA solution, then mixing to finally obtain a KeMA hydrogel pre-polymer system.

[0070] Preferably, the preparation of the GelMA solution in S201 includes the following steps:

[0071] S2011, weighing and feeding, weighing methacrylated gelatin (GelMA) dry powder and adding it to a container containing double distilled water containing a photoinitiator;

[0072] S2012 Dissolution and constant volume, the dry powder is dissolved by stirring or oscillation, and finally a 10% GelMA solution is prepared.

[0073] Preferably, the preparation of the KeMA prepolymer solution in S202 includes the following steps:

[0074] S2021, adding functional components, 100 μg keratin is added to the 10% GelMA solution prepared in S201;

[0075] S2022, obtaining a prepolymer solution, the mixed solution is ultrasonically treated for 25-35 min, after ultrasonic treatment, the keratin is fully and uniformly dispersed in the GelMA solution, and a uniform KeMA hydrogel prepolymer solution system is obtained.

[0076] The KeMA hydrogel prepolymer solution system is composed of methacrylated gelatin GelMA and keratin containing RGD sequence. Among them, GelMA has excellent biocompatibility and biodegradability, can provide a support environment similar to natural extracellular matrix, promote cell adhesion, proliferation and tissue regeneration; the RGD specific recognition sequence contained in keratin can bind to the integrin receptor on the cell surface, realize the effective anchoring and directional fixation of exosomes, and enhance the local retention and sustained release effect of exosomes in the hydrogel. The system forms a three-dimensional cross-linked network through light-induced polymerization, while considering the mechanical strength and biological activity, it can realize the precise action of exosomes in the microenvironment, thereby significantly improving the bioavailability and therapeutic effect of exosomes; the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP), which is a high-efficiency, low-toxicity photoinitiator with excellent water solubility and biocompatibility. It can rapidly initiate the crosslinking reaction of methacryl group under mild light conditions, forming a stable three-dimensional hydrogel network structure. Compared with traditional photoinitiators, LAP has higher initiation efficiency and more uniform reaction, and can react in the visible or near ultraviolet region, avoiding damage to biological active substances and cells caused by strong ultraviolet light. Therefore, the selection of LAP as a photoinitiator not only improves the crosslinking efficiency and controllability of the system, but also ensures the safety and biological friendliness of the material preparation process.

[0077] Extraction of anti-aging exosomes: Take the primary mesenchymal stem cells, and subculture them to P2 generation by the conventional method. When the cell density reaches about 60%-70%, replace the serum culture medium containing urolithin (100 μm) and remove the exosomes, and continue to culture for 2 days. After the culture ends, collect the cell culture supernatant, and sequentially perform the treatment centrifugation to remove impurities: first centrifuge at 300 g for 10 min to remove dead cells, then centrifuge at 2,000 g for 10 min to remove cell debris, and then centrifuge at 10,000 g for 30 min to further remove larger particles. Filter the obtained supernatant through a 0.22 μm microporous filter to remove larger vesicles and impurities. Then perform ultracentrifugation at 100,000 g for 70 min twice, discard the supernatant each time, carefully aspirate the precipitate, and finally gently resuspend the precipitate with 1 mL of sterile normal saline to obtain the urolithin-treated anti-aging exosome sample, which is stored at -80 ℃ for standby use.

[0078] Electron microscopy and WB identification of anti-aging exosomes: Take the urolithin-treated anti-aging exosome sample obtained by the foregoing extraction, drop a proper amount of exosome suspension onto a carbon film copper grid (Formvar / carbon-coated copper grid), and place it at room temperature for 10 min to allow the exosomes to adhere. After discarding the excess liquid, use 2% uranyl acetate to negatively stain for 30 s-1 min, use filter paper to absorb the excess staining liquid, and naturally dry it at room temperature. Use a transmission electron microscope (TEM) to observe it. As shown in Figure 2 , the exosomes are typically round or tea tray-like vesicle structures, with complete membranes and uniform sizes, and the morphology is consistent with the characteristics of typical exosomes.

[0079] To further verify that the obtained vesicles are exosomes, extract the total protein of the exosomes, and quantify it by the BCA method. Take an equal amount of protein sample to perform SDS-PAGE electrophoresis separation, transfer it to a membrane, and use specific antibodies against exosome marker proteins to perform immunodetection. The main marker proteins include HSP70, CD63, TSG101, and Calnexin.

[0080] It is found that positive bands of the above marker proteins are detected at the corresponding molecular weights, while intracellular proteins such as Calnexin are not detected, indicating that there is no obvious cell contamination in the sample.

[0081] As shown in Figure 3 , it is indicated that the vesicles secreted by the urolithin-treated mesenchymal stem cells have typical exosome characteristics, and the anti-aging exosomes are successfully obtained.

[0082] Anti-aging exosome uptake and anti-aging effect verification: To verify the uptake ability of nucleus pulposus cells to anti-aging exosomes, fluorescence labeling method was used for observation. The extracted exosomes were labeled with fluorescent dye PKH26 (red fluorescence), and the operation was carried out according to the reagent instruction book. After removing the free dye, resuspend in PBS. Add the labeled exosomes to the plated nucleus pulposus cell culture system at a final concentration of the appropriate concentration, and culture for 6h and 12h. Remove the unbound exosomes with PBS, fix the cells, and stain the nucleus with DAPI (blue) and F-actin (green).

[0083] The observation results under laser confocal microscope are shown in Figure 4 It can be seen that the red fluorescence signal is distributed in the cytoplasm of nucleus pulposus cells, and the fluorescence signal in the negative control group (untreated or ordinary exosome treatment group) is very small, indicating that nucleus pulposus cells can effectively uptake anti-aging exosomes.

[0084] The nucleus pulposus cells were divided into two groups, conventional exosome plus aging stimulation group and anti-aging exosome plus aging stimulation group. After culturing each group of cells under conventional conditions for 48h, the cell aging degree was detected by β-galactosidase staining. The results showed that the proportion of blue staining positive cells in the anti-aging exosome group was significantly lower than that in the conventional exosome group, indicating that the uric acid treatment of stem cell derived exosomes can effectively delay the aging process of nucleus pulposus cells, and has a clear anti-aging biological effect.

[0085] Preparation of KeMA hydrogel pre-polymer system: Weigh the methacrylated gelatin (GelMA) dry powder, add it to double distilled water (DDH2O) containing a photoinitiator, and prepare a GelMA solution with a mass fraction of 10%. Stir in a 37°C water bath until completely dissolved. Then add 100μg keratin to the above GelMA solution and ultrasonic treatment for 30min to make the keratin fully dispersed and uniformly mixed with the GelMA molecules. The resulting homogeneous solution is the KeMA hydrogel pre-polymer system.

[0086] Formation of KeMA sustained-release hydrogel: Transfer the above KeMA pre-polymer solution to the predetermined mold and irradiate under ultraviolet light conditions (wavelength 365nm, power 10mW / cm²) for 30s to make the methacryl group crosslinking reaction occur, forming a three-dimensional network of KeMA hydrogel. The obtained hydrogel is washed with PBS to remove unreacted substances, and the shaped KeMA sustained-release hydrogel system is obtained.

[0087] SEM characterization of KeMA sustained-release hydrogel: To observe the microstructure of the KeMA sustained-release hydrogel, the prepared KeMA hydrogel sample was subjected to freeze-drying treatment for 24 h to remove the internal moisture and obtain a stable porous solid sample. The dried sample was cut into small pieces of appropriate size and fixed on the SEM sample stage using conductive glue. Subsequently, the sample surface was treated with a metal sputter coater (about 10 nm thickness) to enhance the conductivity and imaging clarity.

[0088] The surface morphology of the gold-coated sample was observed using a scanning electron microscope (SEM), and its microstructure characteristics were recorded, as shown in FIG. 6. Figure 6 The results show that the KeMA sustained-release hydrogel exhibits a typical three-dimensional porous network structure with uniform pore size distribution and complete pore walls, and internal interconnected microporous channels, which are conducive to the loading and sustained release of exosomes and other effective components.

[0089] In vivo treatment effect verification of KeMA sustained-release hydrogel system loaded with anti-aging exosomes:

[0090] Construction of IVDD model induced by lumbar vertebrae acupuncture: 6-week-old healthy SPF male SD rats were selected as experimental animals, with a body weight range of 250-300 g. Before the experiment, the rats were acclimated to the constant temperature and humidity conditions for 1 week. Isoflurane inhalation anesthesia was used for anesthesia, and after sufficient anesthesia, the rats were fixed on the operating table and positioned at the 5 / 6 intervertebral disc of the tail vertebrae (Co5 / Co6). A 21G injection needle was used to puncture the tail vertebrae intervertebral disc in the middle direction, and the needle was inserted to a depth of about 3 mm. After insertion, the needle was rotated 360° and held for 30 s, and then slowly pulled out. After the operation was completed, the puncture area was immediately disinfected with iodophor to prevent infection, and postoperative care was given for three days to help the rats recover. This method successfully established a rat model of intervertebral disc degeneration (IVDD) induced by lumbar vertebrae acupuncture.

[0091] Experimental grouping and treatment: After the model was successfully established, the experimental rats were randomly divided into 3 groups: Con group, IVDD group, and Antig@Exo-KeMA group. Only the IVDD group and the Antig@Exo-KeMA group were injected with 20 μL of the corresponding components using a microsyringe, and the entire operation was completed under sterile conditions. After treatment, the rats continued to be routinely fed, and their general state and activity were observed regularly.

[0092] At 8 weeks after the operation, the rats in each group were sacrificed, and the tail intervertebral disc tissues were taken for histological staining analysis. The specimens were fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded, and sectioned. The obtained sections were subjected to Safranin O-Fast Green (SO-FG) staining to observe the integrity of the nucleus pulposus and annulus fibrosus structure and the changes in the matrix composition.

[0093] As shown in FIG. 7,Figure 7 As shown, the structure of the intervertebral disc of the control group (Con) was clear, the nucleus pulposus cells were uniformly distributed, the matrix was rich and the proteoglycan staining was obvious; the nucleus pulposus region of the model group (IVDD) was collapsed, the number of cells was significantly reduced, the annulus fibrosus was broken and disordered, and the proteoglycan staining was significantly weakened; while the intervertebral disc tissue structure of the Exo-KeMA group was well restored, the nucleus pulposus morphology and matrix composition were significantly improved, the annulus fibrosus was arranged in order, and the staining intensity was significantly higher than that of the IVDD group.

[0094] The histological observation results show that the KeMA sustained-release hydrogel loaded with anti-aging exosomes can stably release exosomes in vivo, effectively maintain the local anti-aging activity, significantly reduce the degree of intervertebral disc degeneration, and promote the repair of nucleus pulposus tissue structure and matrix composition, indicating that the system has good tissue repair and anti-aging protection effect in the treatment of degenerative intervertebral disc (IVDD).

[0095] Example Three: Application of KeMA Sustained-Release Hydrogel System Loaded with Anti-aging Exosomes

[0096] Finally, it should be noted that: the above only for the preferred embodiments of the present application, and not for the limitation of the present application, although the foregoing detailed description of the present application is made with reference to the foregoing embodiments, for the skilled in the art, it still can be modified, or part of the technical features of the equivalent replacement, within the spirit and principles of the present application, any modification, equivalent replacement, improvement, etc., should be included in the scope of protection of the present application.

Claims

1. A KeMA sustained-release hydrogel system loaded with anti-aging exosomes, characterized in that, The product comprises, by weight, 50-60 parts of anti-aging exosomes, 90-110 parts of keratin, and 99,990-100,010 parts of methacryloyl gelatin (GelMA).

2. A method for preparing a KeMA sustained-release hydrogel system loaded with anti-aging exosomes, applicable to the KeMA sustained-release hydrogel system loaded with anti-aging exosomes as described in claim 1, characterized in that, The preparation method of the KeMA sustained-release hydrogel system includes: S1, Preparation of anti-aging exosomes; S2, Synthesizing the KeMA hydrogel prepolymer system; S3, synthesize the KeMA sustained-release hydrogel system. The materials of S1 and S2 are synthesized in proportion to form a KeMA sustained-release hydrogel system loaded with anti-aging exosomes. 50 to 60 parts of anti-aging exosomes are added to every 99,990 to 100,010 parts of hydrogel prepolymer system.

3. The method for preparing the KeMA sustained-release hydrogel system loaded with anti-aging exosomes according to claim 2, characterized in that, The preparation of anti-aging exosomes in S1 includes the following steps: S101, Cell Culture and Treatment: When the primary mesenchymal stem cells reach a confluence of 60%–70%, replace the culture medium with exosome-free serum containing 90–110 parts of urolithin and continue culturing. S102, collect cell supernatant. After the culture is completed, collect the cell supernatant containing secretions. S103, obtain exosomes by ultracentrifuging the collected supernatant at 100,000g to obtain anti-aging exosomes.

4. The method for preparing the KeMA sustained-release hydrogel system loaded with anti-aging exosomes according to claim 3, characterized in that, The cell culture and processing in S101 includes the following steps: S1011, Cell culture: Primary mesenchymal stem cells are cultured to the second generation. When the cell confluence reaches 60% to 70%, the next step is carried out. S1012, replace the drug-containing culture medium, discard the original culture medium, and replace it with exosome-free serum culture medium containing 90-110 parts of urolithin; S1013 was cultured again, and the second-generation mesenchymal stem cells were cultured in the replaced culture medium for 2 days.

5. The method for preparing the KeMA sustained-release hydrogel system loaded with anti-aging exosomes according to claim 3, characterized in that, The collection of cell supernatant in S102 includes the following steps: S1021, Termination of culture and preparation: After the culture is completed, remove the cell culture container from the incubator, terminate the culture process, and prepare to collect the supernatant. S1022, Collection and Transfer: Aspirate the supernatant containing cell secretions from the container and transfer it to a sterile collection tube.

6. The method for preparing the KeMA sustained-release hydrogel system loaded with anti-aging exosomes according to claim 3, characterized in that, The process of obtaining exosomes in step S103 includes the following steps. S1031, Sample preparation: Aliquot the collected cell supernatant into ultracentrifuge tubes and perform equilibration. S1032, ultracentrifugation: Place the balanced centrifuge tube in an ultracentrifuge and run it under a centrifugal force of 100,000g for 130-150 minutes to allow the exosomes to precipitate to the bottom of the tube. S1033, collect exosomes, discard the supernatant, and obtain the anti-aging exosome precipitate at the bottom of the tube.

7. The method for preparing the KeMA sustained-release hydrogel system loaded with anti-aging exosomes according to claim 2, characterized in that, The KeMA hydrogel prepolymer system in S2 is as follows: S201, Prepare GelMA solution by dissolving methacrylamide gelatin powder in double-distilled water containing a photoinitiator to prepare a 10% GelMA solution. S202, prepare KeMA prepolymer solution by adding 100 μg keratin to a 10% GelMA solution and mixing well to obtain the KeMA hydrogel prepolymer solution system.

8. The method for preparing the KeMA sustained-release hydrogel system loaded with anti-aging exosomes according to claim 7, characterized in that, The preparation of the GelMA solution in S201 includes the following steps: S2011, Weighing and Feeding: Weigh out the dry powder of methacrylamide gelatin (GelMA) and add it to a container containing double-distilled water containing a photoinitiator. S2012 is dissolved and brought to a constant volume by stirring or shaking to dissolve the dry powder and finally prepare a 10% GelMA solution.

9. The method for preparing the KeMA sustained-release hydrogel system loaded with anti-aging exosomes according to claim 7, characterized in that, The preparation of the KeMA prepolymer solution in S202 includes the following steps: S2021, add functional components, add 100 μg of keratin to the 10% GelMA solution prepared in S201; S2022, obtain the prepolymer solution, and sonicate the mixed solution for 25-35 minutes. After sonication, the keratin is fully and uniformly dispersed in the GelMA solution to obtain a homogeneous KeMA hydrogel prepolymer solution system.

10. Application of the KeMA sustained-release hydrogel system for loading anti-aging exosomes, wherein the KeMA sustained-release hydrogel system for loading anti-aging exosomes as described in claim 1 is characterized in that, The KeMA sustained-release hydrogel system loaded with anti-aging exosomes is used to delay intervertebral disc degeneration.

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