A high-performance composite hydrogel, preparation method and application thereof

Abstract

The present invention belongs to the technical field of sodium hyaluronate drug preparation, and discloses a high-performance composite hydrogel, a preparation method, and its application. The method dissolves sodium hyaluronate derivative dry powder using medical injection water to obtain a transparent solution; adds an amide condensation agent to the transparent solution, mixes evenly, and allows to stand; adds milk lipid microspheres to the solution, mixes evenly, and then allows to stand for reaction; after the reaction is completed, the gel is cut into small pieces and placed in a phosphate buffer solution, and then the catalyst and milk lipid microspheres are removed by heating and dialysis; a cross-linked sodium hyaluronate gel is obtained by granulation and sterilization. After adding milk lipid microspheres in the preparation process of the present invention, the nano-volume advantage of the milk lipid microspheres is utilized. When the sodium hyaluronate modified derivative is self-crosslinked, the milk lipid microspheres are entrained in the gel; after the self-crosslinking is completed, the milk lipid microspheres are removed by an original method to obtain a sodium hyaluronate gel with uniform pore size and consistent pore wall thickness.

Description

Technical Field

[0001] The present invention belongs to the technical field of sodium hyaluronate drug preparation, and in particular relates to a high-performance composite hydrogel, a preparation method and applications thereof. Background Art

[0002] Sodium hyaluronate is a key component of human and animal skin, vitreous humor, joint lubricant, and cartilage. Composed of repetitively linked disaccharide units (1-β-4)D-glucuronic acid and (1-β-3)N-acetyl-D-glucosamine), it is widely used in reconstructive surgery, ophthalmic procedures, and as a cosmetic wrinkle filler. Despite its excellent physical and chemical properties and biocompatibility, it is susceptible to degradation by hyaluronidase in the body, resulting in a short shelf life. To address these shortcomings, cross-linked hyaluronic acid has emerged. Cross-linked sodium hyaluronate is a polymer hydrogel modified with a crosslinker without compromising biocompatibility. After rapid development and market consolidation in recent years, the current state of cross-linked sodium hyaluronate gel technology has stabilized and become more homogeneous, while market competition has intensified. However, in terms of cross-linking technologies, only two are most frequently used: divinyl sulfone (DVS) and 1,4-butanediol diglycidyl ether (BDDE). Theoretically, each molecule of cross-linker will react with two molecules of -OH on sodium hyaluronate. However, in actual production, after the cross-linker reacts with one molecule of hydroxyl, the residual functional group has high cytotoxicity and potential carcinogenic risk.

[0003] However, conventional sodium hyaluronate hydrogel preparation processes have limitations in terms of mechanical properties, stability, and the ability to control the loading and release of bioactive substances. This is primarily due to the uneven three-dimensional pore size and pore wall thickness within the hydrogel. Recent advances in nanotechnology have provided new approaches for improving the pore size uniformity of hydrogels.

[0004] Exosomes are small vesicles with a saucer-shaped structure with a diameter of 30-150nm, containing multiple components such as RNA, protein, microRNA, DNA fragments, etc. All eukaryotic cells and some prokaryotic cells can secrete them, and they are mainly distributed in various body fluids such as blood, saliva, urine, amniotic fluid and breast milk. Bovine milk exosomes have a double-layer membrane structure and physiological characteristics, which make them have good biocompatibility. Bovine milk lipid microspheres use bovine milk exosomes as raw materials and use gamma radiation generated by cobalt 60 to kill viruses in bovine milk exosomes, so as to facilitate the use of animal-derived raw materials in the medical device industry. The process of removing viruses does not affect the double-layer lipid structure, and does not affect the size and concentration of the particles. However, the existing technology rarely uses the characteristics of bovine milk lipid microspheres to prepare sodium hyaluronate gel.

[0005] Through the above analysis, the problems and defects of the existing technology are as follows: the sodium hyaluronate gel prepared by the existing technology has uneven pore size and inconsistent pore wall thickness. In addition, the elastic deformation of the sodium hyaluronate gel in the existing technology is poor. Summary of the Invention

[0006] To overcome the problems existing in the related art, the disclosed embodiments of the present invention provide a high-performance composite hydrogel, a preparation method and applications thereof.

[0007] The technical solution is as follows: A method for preparing a high-performance composite hydrogel, the method comprising the following steps:

[0008] S1, dissolving the sodium hyaluronate derivative dry powder in medical injection water to obtain a transparent solution;

[0009] S2, adding an amide condensation agent to the transparent solution, mixing well and allowing to stand;

[0010] S3, adding milk lipid microspheres to the solution after standing, mixing evenly and then standing to react;

[0011] S4, after the reaction is completed, the gel is cut into small pieces and placed in a phosphate buffer solution; the catalyst and milk lipid microspheres are removed by heating and dialysis;

[0012] S5, obtaining cross-linked sodium hyaluronate gel through granulation and sterilization processes.

[0013] Furthermore, in step S1, the degree of substitution of the sodium hyaluronate dry powder is 3-10%; the hyaluronic acid with a degree of substitution of 3-10% is grafted onto the carboxyl group of sodium hyaluronate using L-lysine ethyl ester hydrochloride, and the degree of substitution of the carboxyl group is 3-10%; the content of sodium hyaluronate in the transparent solution is 60 mg / mL.

[0014] In step S2, the amide condensation agent is one of 2-chloro-1-methylpyridinium iodide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride; the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC to sodium hyaluronate is (3-4):1.

[0015] In step S3, the mixture was mixed evenly and allowed to react at 4°C for 72 hours;

[0016] The bovine milk lipid microspheres are derived from milk; the bovine milk exosomes are irradiated with cobalt 60 at a dose of 10 KGy to obtain the bovine milk lipid microspheres;

[0017] Preferably, the bovine milk lipid microspheres are prepared by killing viruses from bovine milk exosomes;

[0018] Preferably, the bovine milk exosomes are obtained by size separation and purification using bovine milk as raw material;

[0019] In bovine milk lipid microspheres, the protein concentration is 100-500 μg / mL.

[0020] In step S4, the gel is cut into small pieces with a size of 1-5 cm 3 ;

[0021] The heating temperature is 50-85°C;

[0022] Heating time is 1-30min;

[0023] The dialysis temperature was room temperature, the dialysis time was 72 h, and the molecular weight cut-off of the dialysis bag used for dialysis was 3500K.

[0024] In step S5, the granulation is performed by homogenizing the gel using a homogenizer with a homogenization pressure of 5-20 Pa and a homogenization time of 1-10 min;

[0025] The sterilization temperature is 121°C and the sterilization time is 15 minutes.

[0026] Another object of the present invention is to provide a high-performance composite hydrogel prepared using the high-performance composite hydrogel preparation method, comprising a self-crosslinked L-lysine ethyl ester dihydrochloride-modified sodium hyaluronate derivative, wherein the degree of substitution of carboxyl groups in the sodium hyaluronate is 3-10%.

[0027] The high-performance composite hydrogel has a particle size of 200-600 μm;

[0028] The high-performance composite hydrogel is a sterile gel;

[0029] The pore size of the high-performance composite hydrogel is 50-150 μm;

[0030] The pore wall thickness of the high-performance composite hydrogel is 1-10 μm;

[0031] The pore walls of the high-performance composite hydrogel contain a large number of nanopores with diameters ranging from 50 to 250 nm;

[0032] The elastic modulus of the high-performance composite hydrogel is greater than 550 Pa.

[0033] Another object of the present invention is to provide an application of a cross-linked sodium hyaluronate gel prepared by the method for preparing the high-performance composite hydrogel in the preparation of preparations for repair surgery, eye surgery, or as a cosmetic product for filling wrinkles.

[0034] In combination with all the above technical solutions, the beneficial effects of the present invention are as follows:

[0035] First, the hydrogel's raw material is a modified derivative of sodium hyaluronate. The degradation products of this modified derivative of sodium hyaluronate with L-lysine ethyl ester hydrochloride are identical to those of sodium hyaluronate and L-lysine ethyl ester, thus maintaining its safety and non-toxic properties. Furthermore, it increases the solubility of sodium hyaluronate in water, facilitating subsequent self-crosslinking reactions.

[0036] Sodium hyaluronate derivatives are formed by grafting carboxyl groups of sodium hyaluronate with amino groups in L-lysine ethyl ester hydrochloride under appropriate conditions. Self-crosslinking of sodium hyaluronate derivatives utilizes the reaction of residual carboxyl groups in sodium hyaluronate with unreactive amino groups in L-lysine ethyl ester to produce hydrogels. During this process, a large number of carboxyl groups in sodium hyaluronate are occupied, resulting in the occupancy of binding sites for sodium hyaluronate hydrolase, significantly increasing the in vivo stability of the hydrogels described herein.

[0037] Second, the present invention utilizes the nanoscale volume of bovine milk lipid microspheres by incorporating them into the preparation process. Assuming uniform mixing, the bovine milk lipid microspheres can be entrained within the gel during self-crosslinking of the modified sodium hyaluronate derivative. After self-crosslinking, a unique method for removing the bovine milk lipid microspheres is employed to produce a sodium hyaluronate gel with uniform pore size and consistent pore wall thickness.

[0038] The hydrogel provided by the present invention has a large number of nanopores in its pore walls. A large number of uniform nanopores can effectively disperse pressure and enhance the elastic deformation of the gel.

[0039] Third, the hydrogel provided by the present invention contains numerous 100-micron pores and numerous nanopores within the pore walls. Furthermore, this hydrogel exhibits exceptional in vivo stability, making it a highly effective sustained-release vehicle for drug delivery. Whether it's large stem cells, immune cells, or mRNA, or smaller siRNA, small-molecule chemotherapy drugs, or natural products, these hydrogels can be used to deliver sustained-release drugs in vivo. As the hydrogel gradually degrades, the drug within is continuously released, achieving sustained-release properties.

[0040] This invention leverages the nanoscale size of milk lipid microspheres. Under the premise of uniform mixing, the modified sodium hyaluronate derivative self-crosslinks the milk lipid microspheres, entrapping them within the gel. Since the phase transition temperature of the milk lipid microspheres is much lower than that of the self-crosslinked gel, temperature fluctuations are used to remove the milk lipid microspheres, forming a structure with multiple nanopores. This type of structure has not been reported previously in China or abroad. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure;

[0042] Figure 1 This is a flow chart of a method for preparing a high-performance composite hydrogel provided by an embodiment of the present invention;

[0043] Figure 2 This is a physical picture of the injectable gel block provided by the present invention;

[0044] Figure 3 This is a picture of the homogenized product provided by the present invention;

[0045] Figure 4 The present invention provides a homogenized Figure 3 Particle size distribution diagram corresponding to the actual object;

[0046] Figure 5 This is a SEM image of the injection gel provided by the present invention;

[0047] Figure 6 The present invention provides Figure 5 Corresponding pore size statistics;

[0048] Figure 7 This is a SEM image of the hole wall provided by the present invention;

[0049] Figure 8 The present invention provides Figure 7 Corresponding nanopore size distribution in the pore wall;

[0050] Figure 9 The stress-strain curves of the gel with and without milk lipid microspheres provided by the present invention are as follows:

[0051] Figure 10 This is a transmission electron microscope (TEM) image of the milk lipid microspheres provided by the present invention;

[0052] Figure 11 This is a particle size distribution diagram of the milk lipid microspheres provided by the present invention;

[0053] Figure 12 This is a purity chart for characterizing the milk lipid microspheres provided by the present invention;

[0054] Figure 13 The present invention provides a relationship between the content of bovine milk lipid microspheres and the pore size of the injection gel, and shows SEM images of gels after adding bovine milk lipid microspheres at different concentrations;

[0055] Figure 14 The relationship between the content of bovine milk lipid microspheres and the pore size of the injection gel provided by the present invention is a relationship diagram between the content of bovine milk lipid microspheres and the pore size of the injection gel;

[0056] Figure 15 This is a graph showing the in vivo stability of the injectable gel provided by the present invention;

[0057] Figure 16 This is a SEM electron microscope image of the milk lipid microspheres provided by the present invention. DETAILED DESCRIPTION

[0058] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0059] The innovation of this invention lies in leveraging the advantages of bovine milk lipid microspheres, such as their nanoscale size and excellent biosafety, by adding them to the self-crosslinking of a modified sodium hyaluronate derivative. After the crosslinking is complete, the bovine milk lipid microspheres are encapsulated in the gel. Temperature changes are then used to remove the bovine milk lipid microspheres, forming a structure with multiple nanopores.

[0060] Example 1: The high-performance composite hydrogel provided by the present invention has a particle size of 200-600 μm.

[0061] The high-performance composite hydrogel is a sterile gel.

[0062] The pore size of the high-performance composite hydrogel is between 50-150 μm, preferably 100 μm.

[0063] The pore wall thickness of the high-performance composite hydrogel is between 1 and 10 μm.

[0064] The pore walls of the high-performance composite hydrogel contain a large number of nanopores with a diameter of 50-250 nm, preferably 170 nm.

[0065] The elastic modulus of the high-performance composite hydrogel is greater than 550 Pa.

[0066] The high-performance composite hydrogel is mainly composed of a self-crosslinked sodium hyaluronate derivative modified with L-lysine ethyl ester dihydrochloride. The degree of substitution of carboxyl groups in the sodium hyaluronate is 3-10%, preferably 5%.

[0067] like Figure 1 As shown, the embodiment of the present invention provides a method for preparing a high-performance composite hydrogel, comprising the following steps:

[0068] S1, dissolving the sodium hyaluronate derivative dry powder in medical injection water to obtain a transparent solution;

[0069] The degree of substitution of sodium hyaluronate dry powder is 3-10%. Hyaluronic acid with a degree of substitution of 3-10% is obtained by grafting L-lysine ethyl ester hydrochloride onto the carboxyl group of sodium hyaluronate, and the degree of substitution of the carboxyl group is 3-10%.

[0070] Preferably, the degree of substitution of sodium hyaluronate dry powder is 5%; the hyaluronic acid with a substitution degree of 5% is obtained by grafting L-lysine ethyl ester hydrochloride onto the carboxyl group of sodium hyaluronate, and the degree of substitution of the carboxyl group is 5%.

[0071] The molecular weight of the sodium hyaluronate is 1-1.5 million.

[0072] The content of sodium hyaluronate in the transparent solution is 60 mg / mL.

[0073] S2, adding an amide condensation agent to the transparent solution, mixing well and allowing to stand;

[0074] The standing time is 2h;

[0075] The catalyst is one of 2-chloro-1-methylpyridinium iodide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is preferred. The mass ratio of EDC to sodium hyaluronate is (3-4):1, preferably 3.5:1.

[0076] S3, adding milk lipid microspheres to the solution after standing, mixing evenly and then standing to react;

[0077] Milk exosomes were isolated and purified from fresh milk through acid precipitation (pH 4.0), ultracentrifugation, and column chromatography. Milk lipid microspheres were then sterilized by cobalt-60 irradiation. The particle size distribution of the milk lipid microspheres was characterized by nanoflow cytometry, and the exosome morphology was observed by transmission electron microscopy.

[0078] Among them, it is preferred to mix evenly and then let it react at 4°C for 72 hours;

[0079] The bovine milk lipid microspheres are derived from milk; the bovine milk exosomes are irradiated with cobalt 60 at a dose of 10 KGy to obtain the bovine milk lipid microspheres;

[0080] Preferably, the bovine milk lipid microspheres are prepared by killing viruses from bovine milk exosomes;

[0081] Preferably, the bovine milk exosomes are obtained by size separation and purification using bovine milk as raw material;

[0082] In the bovine milk lipid microspheres, the protein concentration is 100-500 μg / mL, preferably 300 μg / mL.

[0083] Among them, bovine milk lipid microspheres have inherent negative charge, high mechanical strength, uniform size (50-100nm), and poor thermal stability. When heated, they disintegrate into phospholipids and proteins, releasing a small amount of nucleic acid inside. Bovine milk lipid microspheres occupy space during the preparation of sodium hyaluronate gels, and are then removed through heating and dialysis, thereby achieving the goal of producing sodium hyaluronate gels with uniform pore size and consistent pore wall thickness.

[0084] S4, after the reaction is completed, the gel is cut into small pieces and placed in a phosphate buffer solution; the catalyst and milk lipid microspheres are removed by heating and dialysis;

[0085] The gel is cut into small pieces with a size of 1-5 cm 3 , preferably 1cm 3 .

[0086] The heating temperature is 50-85°C, preferably 65°C.

[0087] The heating time is 1-30 min, preferably 10 min.

[0088] The dialysis temperature was room temperature, the dialysis time was 72 h, and the molecular weight cut-off of the dialysis bag used for the dialysis was 3500K.

[0089] S5, cross-linked sodium hyaluronate gel is obtained through granulation and sterilization processes;

[0090] The granulation is performed by homogenizing the gel using a homogenizer, with a homogenization pressure of 5-20 Pa, preferably 10 Pa, and a homogenization time of 1-10 min, preferably 3 min.

[0091] The sterilization is conventional moist heat sterilization of sodium hyaluronate, that is, high temperature and high pressure sterilization at 121° C. for 15 minutes.

[0092] Exemplarily, the present invention also provides a use of a cross-linked sodium hyaluronate gel prepared by the method for preparing a high-performance composite hydrogel in the preparation of a wrinkle filling preparation for repair surgery, eye surgery, or as a cosmetic product.

[0093] Example 2: 3 g of homemade sodium hyaluronate lysine derivative powder was dissolved in 50 mL of medical injection water to obtain a 60 mg / mL transparent solution. 2 mL of LEDC solution (6 g / mL) was added to the solution, mixed evenly, and allowed to stand for 2 hours. Milk lipid microspheres were added, mixed evenly, and allowed to stand at 4°C for 72 hours. The final concentration of milk lipid microspheres was 100 ug / mL. After the reaction, the gel was cut into small pieces of 1 cm 3The solution was placed in phosphate buffer, heated at 65°C for 10 minutes and dialyzed for 72 hours to remove EDC and milk lipid microspheres. Cross-linked sodium hyaluronate gel was obtained through granulation and sterilization.

[0094] Example 3: Take 3g of homemade sodium hyaluronate lysine derivative dry powder and dissolve it in 50mL of medical injection water to obtain a 60mg / mL transparent solution. Add 2mL of LEDC solution (6g / mL) to the solution, mix well and let it stand for 2h. Add bovine milk lipid microspheres, mix well and let it stand at 4℃ for 72h. The final concentration of bovine milk lipid microspheres is 200ug / mL. After the reaction is completed, cut the gel into small pieces of 1cm 3 The solution was placed in a phosphate buffer solution, then heated at 65°C for 10 minutes and dialyzed for 72 hours to remove EDC and milk lipid microspheres. Granulation and sterilization were then performed to obtain a cross-linked sodium hyaluronate gel.

[0095] Example 4: 3 g of homemade sodium hyaluronate lysine derivative powder was dissolved in 50 mL of medical injection water to obtain a 60 mg / mL transparent solution. 2 mL of LEDC solution (6 g / mL) was added to the solution, mixed evenly, and allowed to stand for 2 hours. Milk lipid microspheres were added, mixed evenly, and allowed to stand at 4°C for 72 hours. The final concentration of milk lipid microspheres was 300 ug / mL. After the reaction, the gel was cut into small pieces of 1 cm 3 The solution was placed in a phosphate buffer solution, then heated at 65°C for 10 minutes and dialyzed for 72 hours to remove EDC and milk lipid microspheres. Granulation and sterilization were then performed to obtain a cross-linked sodium hyaluronate gel.

[0096] Example 5: Take 3g of homemade sodium hyaluronate lysine derivative dry powder and dissolve it in 50mL of medical injection water to obtain a 60mg / mL transparent solution. Add 2mL of LEDC solution (6g / mL) to the solution, mix well and let it stand for 2h. Add bovine milk lipid microspheres, mix well and let it stand at 4℃ for 72h. The final concentration of bovine milk lipid microspheres is 400ug / mL. After the reaction is completed, cut the gel into small pieces of 1cm 3 The solution was placed in a phosphate buffer solution, then heated at 65°C for 10 minutes and dialyzed for 72 hours to remove EDC and milk lipid microspheres. Granulation and sterilization were then performed to obtain a cross-linked sodium hyaluronate gel.

[0097] Example 6: 3 g of homemade sodium hyaluronate lysine derivative powder was dissolved in 50 mL of medical injection water to obtain a 60 mg / mL transparent solution. 2 mL of LEDC solution (6 g / mL) was added to the solution, mixed evenly, and allowed to stand for 2 hours. Milk lipid microspheres were added, mixed evenly, and allowed to stand at 4°C for 72 hours. The final concentration of milk lipid microspheres was 500 ug / mL. After the reaction, the gel was cut into small pieces of 1 cm 3The solution was placed in a phosphate buffer solution, then heated at 65°C for 10 minutes and dialyzed for 72 hours to remove EDC and milk lipid microspheres. Granulation and sterilization were then performed to obtain a cross-linked sodium hyaluronate gel.

[0098] Example 7: The sample obtained in Example 2 was steam sterilized (121°C, 15 minutes). 200 μl of the sample was injected subcutaneously into mice via syringe and observed for one month. The retention rate was calculated by measuring the size of the injection site. Helix was injected into the other side of the mouse as a control.

[0099] Among them, in the subcutaneous experiment of mice, Figure 2 This is a physical picture of the injectable gel block provided by the present invention; Figure 3 This is a picture of the homogenized product provided by the present invention; Figure 4 The present invention provides a homogenized Figure 3 Particle size distribution diagram corresponding to the actual object; Figure 5 This is a SEM image of the injection gel provided by the present invention; Figure 6 The present invention provides Figure 5 Corresponding pore size statistics; Figure 7 This is a SEM image of the hole wall provided by the present invention; Figure 8 The present invention provides Figure 7 Corresponding nanopore size distribution in the pore wall; Figure 9 The stress-strain curves of the gel with and without milk lipid microspheres provided by the present invention are as follows: Figure 10 This is a transmission electron microscope (TEM) image of the milk lipid microspheres provided by the present invention; Figure 11 This is a particle size distribution diagram of the milk lipid microspheres provided by the present invention; Figure 12 This is a purity chart for characterizing the milk lipid microspheres provided by the present invention; Figure 13 The present invention provides a relationship between the content of bovine milk lipid microspheres and the pore size of the injection gel, and shows SEM images of gels after adding bovine milk lipid microspheres at different concentrations; Figure 14 The relationship between the content of bovine milk lipid microspheres and the pore size of the injection gel provided by the present invention is a relationship diagram between the content of bovine milk lipid microspheres and the pore size of the injection gel; Figure 16 This is a SEM electron microscope image of the milk lipid microspheres provided by the present invention.

[0100] The results showed that adding different concentrations of milk lipid microspheres during the preparation of the injection gel can lead to different microscopic pore sizes of the gel. The more milk lipid microspheres added, the larger the gel pore size ( Figure 10-12 ). At the same time, it can also lead to the presence of a large number of nano-sized holes in the pore walls of the injection gel, which can effectively disperse the pressure and enhance the elastic deformation of the gel. According to the in vivo results analysis, the injection gel is relatively stable in vivo. Figure 15This is a graph showing the in vivo stability of the injectable gel provided by the present invention. Each mouse received an injection of 200 μl of the injectable gel on the left side of its back and 200 μl of Hedwigia lactis on the right side. The size of the raised spot was observed after the injections. The raised spot was observed for 28 days, and the in vivo stability of the injectable gel was calculated. A comparison of the homemade injectable gel of the present invention with commercially available Hedwigia lactis revealed that the homemade injectable gel exhibited superior stability 28 days after subcutaneous injection in mice, with the injection site volume remaining virtually unchanged. The characteristic data (such as viral content) that distinguishes bovine milk lipid microspheres from milk exosomes are shown in the following table:

[0101]

[0102] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant description of other embodiments.

[0103] The above description is only a preferred specific implementation method of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing a high-performance composite hydrogel, characterized in that: The method comprises the following steps: S1, dissolving the sodium hyaluronate derivative dry powder in medical injection water to obtain a transparent solution; S2, adding an amide condensation agent to the transparent solution, mixing well and allowing to stand; S3, adding milk lipid microspheres to the solution after standing, mixing evenly and then standing to react; S4, after the reaction is completed, the gel is cut into small pieces and placed in a phosphate buffer solution; the catalyst and milk lipid microspheres are removed by heating and dialysis; S5, cross-linked sodium hyaluronate gel is obtained through granulation and sterilization processes; In step S1, L-lysine ethyl ester hydrochloride is grafted onto the carboxyl group of sodium hyaluronate, and the degree of substitution of the carboxyl group is 3-10%; the content of sodium hyaluronate in the transparent solution is 60 mg / mL; In step S2, the amide condensation agent is one of 2-chloro-1-methylpyridinium iodide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride; The pore wall thickness of the high-performance composite hydrogel is 1-10 μm.

2. The method for preparing a high-performance composite hydrogel according to claim 1, wherein: In step S3, the mixture was mixed evenly and allowed to react at 4°C for 72 hours; The milk exosomes were irradiated with cobalt-60 at a dose of 10 KGy to obtain milk lipid microspheres; The milk exosomes are obtained by using milk as a raw material and being purified through size separation; In bovine milk lipid microspheres, the protein concentration is 100-500 μg / mL.

3. The method for preparing the high-performance composite hydrogel according to claim 1, characterized in that: In step S4, the gel is cut into small pieces with a size of 1-5 cm 3 ; The heating temperature is 50-85°C; Heating time is 1-30min; The dialysis temperature was room temperature, the dialysis time was 72 h, and the molecular weight cut-off of the dialysis bag used for dialysis was 3500K.

4. The method for preparing the high-performance composite hydrogel according to claim 1, characterized in that: In step S5, the granulation is performed by homogenizing the gel using a homogenizer, with a homogenization pressure of 5-20 Pa and a homogenization time of 1-10 min; the sterilization temperature is 121° C. and the sterilization time is 15 min.

5. The method for preparing the high-performance composite hydrogel according to claim 1, wherein: The high-performance composite hydrogel has a particle size of 200-600 μm; The high-performance composite hydrogel is a sterile gel; The pore size of the high-performance composite hydrogel is 50-150 μm.

6. The method for preparing the high-performance composite hydrogel according to claim 1, characterized in that: The pore walls of the high-performance composite hydrogel contain a large number of nanopores with diameters ranging from 50 to 250 nm; The elastic modulus of the high-performance composite hydrogel is greater than 550 Pa.

7. Use of a cross-linked sodium hyaluronate gel prepared by the method for preparing a high-performance composite hydrogel according to any one of claims 1 to 4 in preparing a preparation for repair surgery, eye surgery, or as a cosmetic product for wrinkle filling.

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