Hydrogel loaded with rhizoma drynariae extracellular vesicles as well as preparation method and application of hydrogel

Abstract

The invention provides hydrogel loaded with rhizoma drynariae extracellular vesicles as well as a preparation method and application of the hydrogel, and belongs to the technical field of biological medicines. The novel bone biological material is creatively prepared by compounding rhizoma drynariae extracellular vesicles with methacryloyl gelatin, and is subjected to performance and characterization detection and research to find that the novel bone biological material is good in forming performance, high in bioavailability, good in microstructure and physical and chemical properties, and capable of repairing osteoarthritis cartilage injury, and has a good application prospect. The polypeptide is proved to have the potential of efficiently, slowly and accurately treating osteoarthritis, and can be applied to treating osteoarthritis and repairing cartilage injury.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a hydrogel loaded with extracellular vesicles of Rhizoma Drynariae, and a preparation method and application thereof. Background Art

[0002] Drynaria is the rhizome of the fern family, which can be harvested throughout the year. It has the effects of healing wounds and relieving pain, tonifying the kidney and strengthening bones. It is widely used in the clinical treatment of bone injuries and prognosis conditioning, and is the first-line drug for the clinical treatment of osteoarthritis (OA) in traditional Chinese medicine. However, it has been found in clinical practice that the main method of clinical drug administration in traditional Chinese medicine is oral decoction, which has problems such as difficulty in accurate delivery, low bioavailability, short half-life, large dosage, frequent medication and low patient compliance, which seriously limits the efficacy and clinical application of Drynaria. The slow clinical efficacy and the long-term course of OA determine the need for long-term treatment with traditional Chinese medicine. Therefore, it is of great significance to deeply explore the medicinal value of Drynaria and find bioactive substances and drug carrier systems with high bioavailability and bone targeting to break through the research bottleneck of traditional Chinese medicine treatment of OA.

[0003] In addition, sodium hyaluronate, glucocorticoids, etc. are often used for joint cavity injection in the treatment of OA. However, they have limitations such as inconsistent efficacy, low tolerance, addiction, and long-term injection of GC accelerating cartilage loss. They do not have the potential for long-term treatment of OA. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a hydrogel loaded with extracellular vesicles of Rhizoma Drynariae and its preparation method and application. The hydrogel loaded with extracellular vesicles of Rhizoma Drynariae prepared by the present invention has good bioavailability, can effectively and accurately treat osteoarthritis, and has the potential for long-term treatment of osteoarthritis.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a hydrogel loaded with extracellular vesicles of Rhizoma Drynariae, comprising the following steps:

[0007] (1) squeezing juice from Rhizoma Drynariae, removing impurities and collecting the supernatant;

[0008] (2) subjecting the supernatant to high-speed centrifugation, collecting the precipitate, and obtaining the extracellular vesicles of Rhizoma Drynariae; the centrifugal force of the high-speed centrifugation is 100,000 to 200,000 g, and the time is 60 to 120 min;

[0009] (3) Mixing the extracellular vesicles of Rhizoma Drynariae with methacrylated gelatin, a photoinitiator and a solvent, and subjecting the mixture to ultraviolet light curing reaction to obtain a hydrogel loaded with the extracellular vesicles of Rhizoma Drynariae.

[0010] Preferably, the mixing method in step (3) is:

[0011] The methacrylated gelatin, a photoinitiator and a solvent are heated and mixed to obtain a methacrylated gelatin solution;

[0012] The methacryloyl gelatin solution is mixed with the extracellular vesicles of Rhizoma Drynariae.

[0013] Preferably, the volume ratio of the Rhizoma Drynariae extracellular vesicles to the methacrylylated gelatin solution is 1:9 to 3:7;

[0014] The mass concentration of methacryloyl gelatin in the methacryloyl gelatin solution is 10-30%.

[0015] Preferably, the mass concentration of the photoinitiator in the methacrylated gelatin solution is 0.25-0.5%.

[0016] Preferably, the wavelength of the ultraviolet light curing is 405 nm, and the time of the ultraviolet light curing is 10 to 30 seconds.

[0017] The invention provides a hydrogel loaded with extracellular vesicles of Rhizoma Drynariae prepared by the above-mentioned preparation method, comprising a methacryloyl gelatin hydrogel matrix and extracellular vesicles of Rhizoma Drynariae loaded on the surface and inside of the methacryloyl gelatin matrix.

[0018] Preferably, in the hydrogel loaded with extracellular vesicles of Rhizoma Drynariae, the loading amount of the extracellular vesicles of Rhizoma Drynariae is 10 to 30 wt %;

[0019] The hydrogel loaded with the extracellular vesicles of Rhizoma Drynariae has a porous structure with a pore size of 200 to 500 μm.

[0020] The present invention provides application of the hydrogel loaded with extracellular vesicles of Rhizoma Drynariae in preparing osteoarthritis drugs.

[0021] The present invention provides application of the hydrogel loaded with extracellular vesicles of Rhizoma Drynariae in preparing a drug for repairing cartilage damage.

[0022] The invention provides a medicine for intra-articular injection, comprising the hydrogel loaded with extracellular vesicles of Rhizoma Drynariae.

[0023] The present invention provides a method for preparing a hydrogel loaded with extracellular vesicles of Drynariae rhizome, comprising the following steps: (1) juicing Drynariae rhizome, removing impurities and collecting the supernatant; (2) subjecting the supernatant to high-speed centrifugation, collecting the precipitate and obtaining the extracellular vesicles of Drynariae rhizome; the centrifugal force of the high-speed centrifugation is 100000-200000g, and the time is 60-120min; (3) mixing the extracellular vesicles of Drynariae rhizome with methacrylated gelatin, a photoinitiator and a solvent, and performing an ultraviolet light curing reaction to obtain a hydrogel loaded with extracellular vesicles of Drynariae rhizome (abbreviated as RD-EVLP@Gelma). The extracellular vesicles of Drynariae rhizome (RD-EVLP) exist in a nano-sized form, which can inherit the characteristics of Drynariae rhizome and promote the transfer of substances between cells, thereby improving the bioavailability of the insoluble active ingredients of Drynariae rhizome. Compared with traditional drug carrier systems, RD-EVLP has outstanding advantages in biological activity, drug delivery ability, targeting, high concentration, low toxicity and stability. It is considered to be an ideal form of biological activity. While greatly reducing the dosage of medication, it can safely, targetedly and efficiently deliver drugs to target cells. It has great application potential in disease treatment and the development of drug carriers. Methacryloyl gelatin (Gelma) as a bone tissue engineering scaffold has good biocompatibility and low immunogenicity, which can improve drug delivery, achieve sustained release of drugs and improve the stability of active molecules. The present invention creatively prepares a new type of bone biomaterial by combining extracellular vesicles of Drynaria fortunei with methacryloyl gelatin, and performs performance and characterization tests on it. The study found that it has good molding performance, high bioavailability, good micromorphology and physicochemical properties, and can repair osteoarthritis cartilage damage, proving that it has the potential to treat osteoarthritis efficiently, with sustained release and precision, and can be used to treat osteoarthritis and repair cartilage damage.

[0024] The hydrogel loaded with extracellular vesicles of Drynaria provided by the present invention can inherit the anti-inflammatory and analgesic effects of Drynaria, and has the drug delivery characteristics of a drug carrier, thereby improving the efficacy of the drug. Compared with the administration frequency of 1 day / time of oral drugs commonly used to treat OA, the administration frequency of the hydrogel loaded with extracellular vesicles of Drynaria of the present invention is 1 week / time; compared with the effect of intra-articular injection of drugs (sodium hyaluronate, etc.) that only lubricates joints to relieve symptoms and does not contain bioactive substances to treat OA, the hydrogel loaded with extracellular vesicles of Drynaria of the present invention can not only lubricate joints, but also contain definite bioactive substances to play an anti-inflammatory role. In summary, the hydrogel loaded with extracellular vesicles of Drynaria of the present invention can not only reduce the number of administrations, but also increase the efficacy, and therefore has the potential for long-term treatment.

[0025] In addition, the preparation method of the hydrogel loaded with extracellular vesicles of Rhizoma Drynariae provided by the present invention is simple to operate, low in cost, and easy to realize industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the transmission electron microscopy image of RD-EVLP;

[0027] Figure 2 is the particle size distribution diagram of RD-EVLP;

[0028] Figure 3 This is the RD-EVLP@Gelma molding observation picture;

[0029] Figure 4 This is a scanning electron microscope image of RD-EVLP@Gelma;

[0030] Figure 5 This is the infrared spectrum of RD-EVLP@Gelma;

[0031] Figure 6 This is the dynamic rheological analysis diagram of RD-EVLP@Gelma;

[0032] Figure 7 Dynamic rheological analysis diagram of RD-EVLP@Gelma

[0033] Figure 8 This is a micro CT image of the knee joint of an OA rat;

[0034] Fig. 9 This is a modified Safranin O-Fast Green cartilage staining image of the knee joint of OA rats;

[0035] Fig.10 The results of toluidine blue cartilage staining of the knee joints of OA rats;

[0036] Fig.11 The results of hematoxylin and eosin cartilage staining of the knee joints of OA rats. DETAILED DESCRIPTION

[0037] The present invention provides a method for preparing a hydrogel loaded with extracellular vesicles of Rhizoma Drynariae, comprising the following steps:

[0038] (1) squeezing juice from Rhizoma Drynariae, removing impurities and collecting the supernatant;

[0039] (2) subjecting the supernatant to high-speed centrifugation, collecting the precipitate, and obtaining the extracellular vesicles of Rhizoma Drynariae; the centrifugal force of the high-speed centrifugation is 100,000 to 200,000 g, and the time is 60 to 120 min;

[0040] (3) Mixing the extracellular vesicles of Rhizoma Drynariae with methacrylated gelatin, a photoinitiator and a solvent, and subjecting the mixture to ultraviolet light curing reaction to obtain a hydrogel loaded with the extracellular vesicles of Rhizoma Drynariae.

[0041] The present invention squeezes juice from Rhizoma Drynariae, removes impurities and collects the supernatant. In the present invention, the source of Rhizoma Drynariae is preferably Rhizoma Drynariae plants or Rhizoma Drynariae decoction pieces. When the source of Rhizoma Drynariae is Rhizoma Drynariae decoction pieces, the present invention preferably adds a diluent during the juicing. In the present invention, the diluent is preferably a PBS buffer. In the present invention, the volume ratio of the mass of the Rhizoma Drynariae decoction pieces to the diluent is preferably 200g:1-2L.

[0042] The present invention has no special requirements for the juice extraction method, and any juice extraction method well known to those skilled in the art can be used.

[0043] In the present invention, the method of removing impurities preferably includes filtration and centrifugation performed in sequence. In the present invention, the number of centrifugation preferably includes three times, the speed of the first centrifugation is preferably 100-500g, specifically 100g, 150g, 200g, 250g, 300g, 350g, 400g, 450g or 500g; the time is preferably 5-30min, specifically 5min, 10min, 15min, 20min, 25min or 30min. The present invention removes floating cells by the first centrifugation.

[0044] In the present invention, the speed of the second centrifugation is preferably 1000-3000g, specifically 1000g, 1200g, 1500g, 1700g, 2000g, 2500g, 2700g or 3000g; the time is preferably 5-30min, specifically 5min, 10min, 15min, 20min, 25min or 30min. The present invention removes dead cells and shedding vesicles through the second centrifugation.

[0045] In the present invention, the speed of the third centrifugation is preferably 8000-20000g, specifically 8000g, 8500g, 9000g, 9500g, 10000g, 10500g, 11000g, 11500g or 12000g, and the time is preferably 5-30min, specifically 5min, 10min, 15min, 20min, 25min or 30min. The present invention removes dead cells, shedding vesicles and apoptotic bodies through the third centrifugation.

[0046] After obtaining the supernatant, the present invention performs high-speed centrifugation on the supernatant, collects the precipitate, and obtains the extracellular vesicles of Drynaria. In the present invention, the speed of the high-speed centrifugation is 100000-200000g, specifically 100000g, 110000g, 120000g, 130000g, 140000g, 150000g, 160000g, 170000g, 180000g, 190000g or 200000g; the time is 60-120min, specifically 60min, 65min, 70min, 75min, 80min, 85min, 90min, 95min, 100min, 105min, 110min, 115min or 120min. The present invention collects the extracellular vesicles of Drynaria by the high-speed centrifugation.

[0047] In the present invention, the particle size of the extracellular vesicles of Rhizoma Drynariae is preferably 0 to 300 nm, and is not 0, and is more preferably 100 to 200 nm. As a specific embodiment of the present invention, the concentration of the extracellular vesicles of Rhizoma Drynariae in the precipitate is preferably 3.88×10 11 ~1.43×10 12 Particles / mL.

[0048] After obtaining the extracellular vesicles of Drynariae Rhizoma, the present invention mixes the extracellular vesicles of Drynariae Rhizoma with methacrylated gelatin, a photoinitiator and a solvent, and performs a UV curing reaction to obtain a hydrogel loaded with extracellular vesicles of Drynariae Rhizoma. In the present invention, the mixing method is preferably:

[0049] The methacrylated gelatin, a photoinitiator and a solvent are heated and mixed to obtain a methacrylated gelatin solution;

[0050] The methacryloyl gelatin solution is mixed with the extracellular vesicles of Rhizoma Drynariae.

[0051] In the present invention, the photoinitiator preferably includes one or more of ethyl 2,4,6-trimethylbenzoylphenylphosphonate, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0052] In the present invention, the solvent is preferably PBS buffer. In the present invention, the temperature of the heating and mixing is preferably 40-50° C., and the time is preferably 15 min.

[0053] In the present invention, the volume ratio of the extracellular vesicles of Drynariae to the methacryloylated gelatin solution is preferably 1:9 to 3:7, specifically 1:9, 2:8 or 3:7. In the present invention, the mass concentration of methacryloylated gelatin in the methacryloylated gelatin solution is preferably 10 to 30%, specifically 10%, 15%, 20%, 25% or 30%. In the present invention, the mass concentration of the photoinitiator in the methacryloylated gelatin solution is 0.25 to 0.5%, specifically 0.25%, 0.3%, 0.4% or 0.5%.

[0054] In the present invention, the wavelength of the ultraviolet light curing is preferably 405 nm, and the time of the ultraviolet light curing is preferably 10 to 30 seconds, specifically 10 seconds, 15 seconds, 20 seconds, 25 seconds or 30 seconds.

[0055] The invention provides a hydrogel loaded with extracellular vesicles of Rhizoma Drynariae prepared by the above-mentioned preparation method, comprising a methacryloyl gelatin hydrogel matrix and extracellular vesicles of Rhizoma Drynariae loaded on the surface and inside of the methacryloyl gelatin matrix.

[0056] In the present invention, in the hydrogel loaded with extracellular vesicles of Rhizoma Drynariae, the loading amount of the extracellular vesicles of Rhizoma Drynariae is preferably 10-30wt%, specifically 10wt%, 20wt%, or 30wt%.

[0057] In the present invention, the hydrogel loaded with extracellular vesicles of Rhizoma Drynariae has a porous structure, and the pore size is preferably 200-500 μm, specifically 200 μm, 300 μm, 400 μm or 500 μm.

[0058] The present invention provides the use of the above-mentioned hydrogel loaded with extracellular vesicles of Drynariae in the preparation of osteoarthritis drugs. In the present invention, the osteoarthritis drug is preferably a drug for cartilage inflammatory infiltration. The hydrogel loaded with extracellular vesicles of Drynariae provided by the present invention can improve the cartilage inflammatory infiltration, thereby achieving the therapeutic effect of OA. In the present invention, the hydrogel loaded with extracellular vesicles of Drynariae is preferably used as an injection.

[0059] The present invention provides the use of the above-mentioned hydrogel loaded with extracellular vesicles of Drynariae in the preparation of a drug for repairing cartilage damage. The hydrogel loaded with extracellular vesicles of Drynariae provided by the present invention can significantly improve cartilage damage, and can be further applied to the preparation of joint cavity preparations to develop new dosage forms or active ingredients of characteristic traditional Chinese medicines for the treatment of cartilage damage diseases. In the present invention, the hydrogel loaded with extracellular vesicles of Drynariae is preferably used as an injection.

[0060] The present invention provides a drug for intra-articular injection, comprising the above-mentioned hydrogel loaded with extracellular vesicles of Rhizoma Drynariae. In the present invention, the drug for intra-articular injection preferably also includes a pharmaceutically acceptable excipient. The present invention has no special requirements for the pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipients well known in the art can be used. In the present invention, the drug for intra-articular injection preferably also includes other active pharmaceutical ingredients, and the other active pharmaceutical ingredients are preferably sodium hyaluronate and / or glucocorticoids.

[0061] The hydrogel loaded with extracellular vesicles of Rhizoma Drynariae provided by the present invention, as well as its preparation method and application are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063] (1) Preparation of extracellular vesicles of Rhizoma Drynariae, using the following steps:

[0064] 200g of Drynaria slices were added to 1L of PBS solution precooled at 4℃ to squeeze the juice, and the supernatant was collected in a clean 50mL sterile centrifuge tube after filtration. Centrifugation at 4℃ for 10min at a speed of 300×g was used to remove floating cells; centrifugation at 2000×g for 20min was used to remove dead cells and detached vesicles; centrifugation at 10000×g for 30min was used to remove dead cells, detached vesicles, and apoptotic bodies; centrifugation at 135000×g for 70min was used to collect Drynaria extracellular vesicles, which were recorded as RD-EVLP.

[0065] The morphology and particle size of the extracted extracellular vesicles of Rhizoma Drynariae were characterized. The transmission electron microscopy images of the obtained RD-EVLP are shown in the figure below. Figure 1 As shown, it can be observed that the shape of RD-EVLP is round or oval, with a typical cup-shaped vesicle structure and a complete cell membrane structure, and the particle size is between 0 and 300 nm (scale bar is 200 nm), which is consistent with the extracellular vesicle particle size range reported in the literature, proving the successful extraction of extracellular vesicles from Rhizoma Drynariae.

[0066] The particle size distribution diagram of the obtained RD-EVLP under the nanoparticle tracer analyzer is shown in Figure 2 As shown in Figure 2, it can be observed that the particle size distribution is between 0 and 300 nm (the same as the results of transmission electron microscopy observation), and the concentration is 1.43×10 12 particles / mL, further proving the successful extraction of extracellular vesicles from Rhizoma Drynariae.

[0067] (2) Preparation of Hydrogels Loaded with Extracellular Vesicles of Rhizoma Drynariae

[0068] Take 0.05g of photoinitiator (phenyl-2,4,6-trimethylbenzoylphosphonate lithium) and add 10mL PBS buffer, dissolve in a water bath at 40-50℃ for 15min to prepare a photoinitiator standard solution. Take 300mg of Gelma and add 3mL of photoinitiator standard solution, heat and dissolve at 55℃ to obtain a Gelma blank solution, immediately add 1800μL of RD-EVLP, and add 1200μL of PBS buffer, filter to obtain RD-EVLP@Gelma precursor solution, irradiate with 405nm ultraviolet light source for 15-30s, and cross-link to obtain a hydrogel loaded with extracellular vesicles of osteopyralis, recorded as RD-EVLP@Gelma. Figure 5 to Figure 11 It is denoted as RDNVs-Gelma.

[0069] After the sample bottle was photo-crosslinked for about 30 seconds, the sample bottle was tilted to observe the state of the supramolecular material solution in the sample bottle and take photos for comparison. The results are as follows: Figure 3 shown. Figure 3 In the figure, A is the blank Gelma molding observation after UV cross-linking; B is the RD-EVLP@Gelma molding observation after UV cross-linking.

[0070] The results showed that after photo-cross-linking, the extracellular vesicles of Rhizoma Drynariae composited with methacryloyl gelatin had good molding properties.

[0071] The microstructure was observed by scanning electron microscopy. The scanning electron microscopy image of RD-EVLP@Gelma is shown in the figure below. Figure 4 The results showed that the extracellular vesicles of Drynaria fortunei complexed with methacryloyl gelatin had a porous structure with a pore size of about 200 μm.

[0072] Fourier transform infrared spectroscopy was used for structural analysis, and the infrared spectrum of RD-EVLP@Gelma was obtained as shown in the figure. Figure 5 As shown. The results showed that RD-EVLP was successfully embedded in the composite hydrogel.

[0073] The obtained RD-EVLP@Gelma was subjected to dynamic rheological analysis. The fluidity of RD-EVLP@Gelma at different times was detected by rheological analysis. Figure 6 As shown in the figure, the rheological test of the fluidity of RD-EVLP@Gelma at different frequencies is as follows Figure 7 The results show that the elastic modulus of the composite hydrogel is greater than the viscous modulus, indicating that a structurally stable hydrogel has been formed.

[0074] Test Example 1: Study on the effect of hydrogel loaded with extracellular vesicles of osteoporosis on improving cartilage damage in OA rats

[0075] Anterior cruciate ligament transection was used to establish an OA model in rats. 100 μL of the hydrogel loaded with extracellular vesicles of Rhizoma Osteoporosis prepared in Example 1 was injected into the joint cavity for 4 weeks. The pathological changes of cartilage in OA rats were observed by micro CT, modified safranin O-fast green cartilage staining and toluidine blue staining.

[0076] Figure 8 This is a Micro CT image of the knee joint of an OA rat. Fig. 9 This is a modified Safranin O-Fast Green staining of cartilage in the knee joint of an OA rat. Figure 8 and Fig. 9 In the data, sham group represented blank control group; ACLT group represented model group; HA group represented positive control group, in which the positive drug was sodium hyaluronate; RDNVs-Gelma group was a hydrogel group loaded with extracellular vesicles of Rhizoma Drynariae.

[0077] comprehensive Figure 8 The Micro-CT results showed that compared with the model group, the knee joints of the rats in the drug-treated group had slightly improved bone reconstruction and osteophyte formation, and slightly improved cartilage and subchondral bone damage.

[0078] Depend on Fig. 9 It can be seen that the knee joint structure of the rats in the blank control group was basically intact, the knee joint surface was smooth, the matrix staining was normal, and the tide line was basically intact; the knee joint of the rats in the model group was severely damaged, the knee joint surface was irregular, the cells increased significantly, the tide line was destroyed, and a large area of ​​safranin staining was missing; the knee joint structure of the rats in the HA group was basically intact, the matrix staining was normal, and the cartilage damage was lighter than that in the model group; the knee joint structure of the rats in the RDNVs-Gelma group was basically intact, the knee joint surface was smooth, the matrix staining was normal, and the tide line was basically intact, indicating that the cartilage damage was significantly improved.

[0079] comprehensive Figure 8 , Fig. 9 It can be seen that the hydrogel loaded with extracellular vesicles of Rhizoma Osteoporosis can significantly improve cartilage damage in OA rats.

[0080] Test Example 2: Study on the effect of hydrogel loaded with extracellular vesicles of osteoporosis on improving inflammatory infiltration of cartilage in OA rats

[0081] The OA model of rats was established by anterior cruciate ligament transection. Hydrogel loaded with extracellular vesicles of Rhizoma Osteoporosis was injected into the joint cavity for 4 weeks. The pathological changes of cartilage in OA rats were observed by toluidine blue and hematoxylin and eosin staining. Fig.10 This is the result of toluidine blue cartilage staining of the knee joint of OA rats. Fig.11 This is the result of hematoxylin and eosin cartilage staining of the knee joint of OA rats. Fig.10 and Fig.11In the data, sham group represented blank control group; ACLT group represented model group; HA group represented positive control group, in which the positive drug was sodium hyaluronate; RDNVs-Gelma group was a hydrogel group loaded with extracellular vesicles of Rhizoma Drynariae.

[0082] Depend on Fig.10 It can be seen that the knee joint structure of the rats in the blank control group was basically intact, the knee joint surface was smooth, and the matrix staining was normal; the knee joint of the rats in the model group was severely damaged, the knee joint surface was irregular, and a large area of ​​toluidine blue staining was missing; the knee joint structure of the rats in the HA group was basically intact, the matrix staining was normal, and the cartilage damage was milder than that in the model group; the knee joint structure of the rats in the RDNVs-Gelma group was basically intact, the knee joint surface was smooth, and the matrix staining was normal, indicating that the cartilage damage was significantly improved.

[0083] Depend on Fig.11 It can be seen that the knee joint surfaces of rats in the blank control group were regular and complete, the chondrocytes were normal, and the tide lines were complete; the knee joints of rats in the model group were severely injured, the knee joint surfaces were irregular, and pannus was visible; the knee joint structure of rats in the HA group was basically intact, and the cartilage damage was less severe than that in the model group; the knee joint surfaces of rats in the RDNVs-Gelma group were regular and complete, and the tide lines were basically intact, indicating that the cartilage damage was significantly improved.

[0084] comprehensive Fig.10 , Fig.11 It can be seen that the hydrogel loaded with extracellular vesicles of Rhizoma Osteoporosis can significantly improve the inflammatory infiltration of cartilage in OA rats.

[0085] This invention is the first to conduct in-depth research on the hydrogel loaded with extracellular vesicles of Rhizoma Drynariae, integrating drug delivery, sustained release, and promotion of tissue regeneration. It cross-integrates and innovates new technologies and methods of traditional Chinese medicine with the fields of bone biomaterials and tissue engineering, and provides a new strategy for efficient, sustained-release, and precise clinical treatment of osteoarthritis.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a hydrogel loaded with extracellular vesicles of Rhizoma Drynariae, comprising the following steps: (1) squeezing juice from Rhizoma Drynariae, removing impurities and collecting the supernatant; (2) subjecting the supernatant to high-speed centrifugation, collecting the precipitate, and obtaining the extracellular vesicles of Rhizoma Drynariae; the centrifugal force of the high-speed centrifugation is 100,000 to 200,000 g, and the time is 60 to 120 min; (3) Mixing the extracellular vesicles of Rhizoma Drynariae with methacrylated gelatin, a photoinitiator and a solvent, and subjecting the mixture to ultraviolet light curing reaction to obtain a hydrogel loaded with the extracellular vesicles of Rhizoma Drynariae.

2. The preparation method according to claim 1, characterized in that: The mixing method in step (3) is: The methacrylated gelatin, a photoinitiator and a solvent are heated and mixed to obtain a methacrylated gelatin solution; The methacryloyl gelatin solution is mixed with the extracellular vesicles of Rhizoma Drynariae.

3. The preparation method according to claim 2, characterized in that: The volume ratio of the Rhizoma Drynariae extracellular vesicles to the methacrylylated gelatin solution is 1:9 to 3:7; The mass concentration of methacryloyl gelatin in the methacryloyl gelatin solution is 10-30%.

4. The preparation method according to claim 2, characterized in that: The mass concentration of the photoinitiator in the methacrylated gelatin solution is 0.25-0.5%.

5. The preparation method according to claim 1, characterized in that: The wavelength of the ultraviolet light curing is 405 nm, and the time of the ultraviolet light curing is 10 to 30 seconds.

6. The hydrogel loaded with extracellular vesicles of Rhizoma Drynariae prepared by the preparation method according to any one of claims 1 to 5, comprising a methacryloyl gelatin hydrogel matrix and extracellular vesicles of Rhizoma Drynariae loaded on the surface and inside of the methacryloyl gelatin matrix.

7. The hydrogel loaded with extracellular vesicles of Rhizoma Drynariae according to claim 6, characterized in that: In the hydrogel loaded with extracellular vesicles of Rhizoma Drynariae, the loading amount of the extracellular vesicles of Rhizoma Drynariae is 10 to 30 wt %; The hydrogel loaded with the extracellular vesicles of Rhizoma Drynariae has a porous structure with a pore size of 200 to 500 μm.

8. Use of the hydrogel loaded with extracellular vesicles of Rhizoma Drynariae according to claim 6 or 7 in the preparation of osteoarthritis drugs.

9. Use of the hydrogel loaded with extracellular vesicles of Rhizoma Drynariae according to claim 6 or 7 in the preparation of a drug for repairing cartilage damage.

10. A drug for intra-articular injection, characterized in that: A hydrogel comprising the extracellular vesicles loaded with Rhizoma Drynariae as described in claim 6 or 7.

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