Composite gels, preparation methods and applications

Abstract

The present invention provides a composite gel, its preparation method and application, which relates to the technical field of biomedical materials. The composite gel is obtained by crosslinking two-phase microspheres composed of polylactic acid and nanoscale inorganic materials with hyaluronic acid, and the method includes crosslinking two-phase microspheres with hyaluronic acid to form a three-phase gel. The composite gel prepared by the present invention integrates the advantages of three materials and can be used for filling and repairing biological tissues.

Description

[Technical Field]

[0001] The present invention relates to the technical field of biomedical materials, in particular to composite gels, preparation methods and applications. [Background technology]

[0002] Polylactic acid (PLA), also known as polylactide, is a polyester polymer obtained by polymerizing lactic acid as the main raw material. Due to its excellent biocompatibility, biodegradability, and ease of processing, it is often used to bond with hydroxyapatite to improve its mechanical and biological properties.

[0003] Currently, regenerative injection products are widely accepted around the world, and polylactic acid serves as the main material for regenerative injection products. When polylactic acid enters human skin, it can stimulate the body's own tissue regeneration. Through the body's own tissue regeneration, it plays a role in structural reconstruction and volume filling, achieving the purpose of anti-aging. The main types of tissue that can be regenerated after stimulation are fibroblasts and collagen. In be.

[0004] Polylactic acid stimulates the body's own fibroblasts to secrete collagen, resulting in a more natural filling and repair effect, while also enhancing skin radiance and achieving overall facial rejuvenation. However, a natural drawback of polylactic acid is that it decomposes in vivo to produce lactic acid, but the metabolism of lactic acid in the body is slow, and the accumulation of large amounts of lactic acid can easily cause sterile inflammation, resulting in a series of adverse reactions.

[0005] Therefore, when using polylactic acid, it is necessary to add an alkali neutralizer to reduce sterile inflammation caused by local excessive acidity.Hydroxyapatite, which is alkaline and has good biocompatibility, is the most suitable alkali neutralizer for polylactic acid.

[0006] Composite materials formed from polylactic acid and inorganic materials have been used in biomaterials. For example, CN106139256A discloses a polylactic acid / nano-β-tricalcium phosphate composite porous scaffold, and CN111905151A discloses mesoporous bioactive glass / polylactic acid-co-glycolic acid composite microspheres.

[0007] In particular, composite materials of hydroxyapatite and polylactic acid are widely used in the field of orthopedics. Hwang Ji-hwan et al. (Journal of Composite Materials Science, 2021, 38:749-760) studied nano-HAP poly-L-lactic acid (PLLA) composite materials and found that the compressive yield strength and tensile strength of PLLA with added HAP were 9.4% and 6.6%, respectively, higher than those of pure PLLA, and the initial decomposition temperature was 1.2% lower than that of pure PLLA. 7 .4 % high It can be seen that the crystallinity increases by 6.7%.

[0008] As can be seen, PLLA materials with added HAP have clear advantages over pure PLLA materials in terms of material properties. Hydroxyapatite also reduces inflammation: compared to PLA materials with increased HAP, pure PLA elicits a stronger inflammatory response, while HAP-PLA composites only show a very mild inflammatory response in the body. It has been demonstrated that the addition of HAP neutralizes the acidic substances caused by the decomposition of PLA, reducing sterile inflammation caused by acidity.

[0009] Currently, binary composites of hydroxyapatite and polylactic acid, as well as ternary composites or composite microspheres of hydroxyapatite, polylactic acid, and other polymers, have been reported in China and overseas. Dong Yan et al. (Additive Manufacturing, 2020;34:101305) have prepared microspheres blended with polylactic acid and nanoscale hydroxyapatite, with a minimum particle size of approximately 100 μm, which are primarily used in research on microcarriers, etc.

[0010] For example, preparation method of nano-hydroxyapatite / polylactic acid composite microspheres (CN101590388A), hydroxyapatite-loaded polylactic acid porous microspheres and preparation method (CN103868658B), polylactic acid / hydroxyapatite crystal whisker composite porous scaffold for bone tissue and preparation method (CN105797215A), polylactic acid-hydroxyapatite micro-nano multi-level structure composite microsphere material and application (CN1097491 Patents such as "19A"), "Controllable Preparation Method and Application of the Surface Porous Structure of Polylactic Acid Hydroxyapatite Composite Microspheres" (CN107519536A), "Preparation Method of Hydroxyapatite / Modified Polylactic Acid Composite Microspheres" (CN109350768A), and "Preparation Method of Hydroxyapatite / Polylactic Acid / Chitosan Composite Microspheres" (CN102489231B) are mainly used for bone defect repair, filling and drug-carrying properties, but not in the field of tissue filling. Summary of the Invention

[0011] The present invention provides a composite gel, its preparation method and application, which combines the advantages of three materials, namely hyaluronic acid, polylactic acid and inorganic materials, and can be used for filling and repairing biological tissues.

[0012] The technical solutions provided by the present invention are as follows:

[0013] A composite gel ,two The biphasic microspheres are prepared by crosslinking polylactic acid / inorganic material biphasic microspheres with hyaluronic acid.

[0014] Furthermore, the inorganic material is one or more selected from calcium carbonate, calcium phosphate, and calcium tartrate; further, the calcium phosphate is preferably hydroxyapatite; and still further, the inorganic material is a nano-inorganic material.

[0015] In one embodiment, the inorganic material is nano-calcium hydroxyapatite.

[0016] Furthermore, the hyaluronic acid is crosslinked hyaluronic acid; and the crosslinked hyaluronic acid is prepared using a crosslinking agent, and the crosslinking agent is one or more selected from the group consisting of dialdehyde, disulfide, polyethylene glycol (PEG) crosslinker, divinyl sulfone, diglycidyl ether, diepoxide, diamine, and polyamine.

[0017] Furthermore, the crosslinking agent may be selected from the group consisting of biscarbodiimides, fatty diamines, ethylenediamine, hexamethylenediamine, endogenous polyamines (spermine or spermidine), pentaerythritol tetraglycidyl ether (PETGE), diethylenesulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), (phenylene bis-(ethyl)-carbodiimide and 1,6-hexamethylene bis(ethyl) carbodiimide), adipic dihydrazide (ADH), bis(sulfosuccinimide) suberic acid ester (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, 1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyl ether oxybutane, 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, 1,3-butadiene diepoxide, 1,2,7,8-diepoxyoctane, 1,5-hexadiene diepoxide.

[0018] Preferably, the crosslinking agent is a diamine or polyamine, and its reaction process can be found in patents such as CN99813143.1, US9907739, and CN202111326226.7.

[0019] Furthermore, the cross-linking includes cross-linking polylactic acid and hyaluronic acid in the two-phase microspheres.

[0020] Furthermore, the crosslinking between the polylactic acid and hyaluronic acid can be either physical electrostatic crosslinking or chemical crosslinking. The chemical crosslinking involves reacting the polylactic acid in the two-phase microsphere with hyaluronic acid to form an amide bond crosslinking reaction. The physical electrostatic crosslinking involves forming electrostatic crosslinks with hyaluronic acid after surface charge modification of the two-phase microsphere.

[0021] Furthermore, the chemical crosslinking comprises amino-modifying polylactic acid in the two-phase microspheres and then reacting it with hyaluronic acid to form an amide bond crosslinking reaction, and the physical electrostatic crosslinking comprises surface charge modification of the two-phase microspheres and then forming electrostatic crosslinks with crosslinked hyaluronic acid having cations.

[0022] In one embodiment, a composite gel is provided, which is obtained by cross-linking hydroxyapatite-dispersed polylactic acid biphasic microspheres composed of polylactic acid and nanoscale hydroxyapatite with hyaluronic acid.

[0023] A method for preparing a composite gel, comprising crosslinking two-phase microspheres with hyaluronic acid to form a composite gel (three-phase composite gel), wherein the two-phase microspheres are polylactic acid / inorganic two-phase microspheres.

[0024] Furthermore, the cross-linking of the two-phase microspheres with hyaluronic acid includes cross-linking of polylactic acid with hyaluronic acid in the two-phase microspheres.

[0025] Furthermore, the hyaluronic acid may be non-crosslinked or crosslinked. In one embodiment, the hyaluronic acid is crosslinked, preferably diamine-crosslinked or polyamine-crosslinked, and more preferably endogenous polyamine-crosslinked.

[0026] In one embodiment, in the hydroxyapatite-dispersed polylactic acid two-phase microspheres, the mass of hydroxyapatite accounts for 0.1 to 50% of the total mass of the microspheres, and the remainder of the microspheres, excluding hydroxyapatite, is entirely polylactic acid.

[0027] In one embodiment, a crosslinked hyaluronic acid hydrogel is prepared using a polyamine as a crosslinker, preferably comprising 0.5-20% of the total mass of hyaluronic acid, and the mass concentration of hyaluronic acid in the hydrogel is in the range of 1-35 mg / mL.

[0028] In one embodiment, the hyaluronic acid is prepared by microbial fermentation. obtained Preferably, the molecular weight range of the hyaluronic acid is 100 KDa to 3 , 000KDa, preferably 500KDa to 1 , The molecular weight of hyaluronic acid fermented by microorganisms is 500 kDa. It is a non-animal derived hyaluronic acid and has a more uniform molecular weight distribution.

[0029] In one embodiment, the crosslinks are solid-phase crosslinks; further, the solid-phase crosslinks include any one of physical electrostatic crosslinks and chemical crosslinks.

[0030] In one embodiment, the chemical crosslinking comprises amino-modifying polylactic acid in the two-phase microspheres and then reacting the modified two-phase microspheres with hyaluronic acid to form amide bond crosslinks; the amino-modification further comprises reacting the two-phase microspheres with an amine compound; and the amide bond crosslinking further comprises coupling the modified two-phase microspheres with hyaluronic acid in the presence of a carbodiimide activator.

[0031] In one embodiment, the amine compound includes a diamine, a polyamine, or a polyamino compound. Preferably, the diamine includes any one of an aliphatic diamine, an aromatic diamine, and a heteroatom diamine, for example, but not limited to, an aliphatic diamine, an ethylenediamine, and a hexamethylenediamine. The polyamine includes any one of an aliphatic polyamine, an aromatic polyamine, and a heteroatom polyamine, for example, but not limited to, an endogenous polyamine such as spermidine or spermine. The polyamino compound includes any one of a doubly aminated polyethylene glycol and a terminally aminated multi-arm polyethylene glycol. Further, the carbodiimide activator includes one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, 1,3-bis[di(methoxymethyl)methyl]carbodiimide, or salts thereof.

[0032] In one embodiment, the preparation method further comprises the step of using the carbodiimide activator in combination with an auxiliary agent, such as N-hydroxysuccinimide (NHS), tert-butyl alcohol, etc. To improve the efficiency of the crosslinking reaction, the water-soluble carbodiimide activator needs to be used in combination with the auxiliary agent, and the amount of the auxiliary agent added is 10 to 30% of the mass of the carbodiimide.

[0033] In one embodiment, the mass ratio of the two-phase microspheres to the hyaluronic acid in the chemical crosslinking is 1:0.5-20, preferably 1:1-10; Amino modification In the reaction system, the amount of the amine compound added is 30 to 200% of the mass of the two-phase microspheres; the concentration range of the amine compound is 10 to 1000 mg / mL; Furthermore, the amount of the carbodiimide activator added is 10 to 150% of the total mass of the hyaluronic acid; Furthermore, the concentration of the hyaluronic acid solution is 10 to 100 mg / mL; Furthermore, the temperature for the amino modification reaction and the amide bond crosslinking reaction is 5 to 60°C, and the reaction time is 6 to 24 hours; the pH value of the reaction system is in the range of 5.0 to 6.0.

[0034] In one embodiment, the microspheres are hydroxyapatite-dispersed polylactic acid biphasic microspheres, and the chemical crosslinking includes the steps of dispersing the hydroxyapatite-dispersed polylactic acid biphasic microspheres in water, adding an amine compound (polyamine derivative), adjusting the pH, and reacting the carboxyl groups on the surface of the microspheres with the polyamine derivative to modify the surface with amino groups; and placing the surface-amino-modified hydroxyapatite-dispersed polylactic acid microspheres (PLA-HAP biphasic microspheres) in a hyaluronic acid solution, adjusting the pH to 5.0-6.0, and simultaneously adding a carbodiimide activator and an auxiliary agent to complete the coupling reaction of the PLA-HAP biphasic microspheres.

[0035] The surface modification reaction temperature is 5 to 60°C, and the reaction time is 6 to 24 hours. The coupling reaction temperature is 5 to 60°C, and the reaction time is 6 to 24 hours.

[0036] The physical electrostatic crosslinking includes modifying the surface charge of the two-phase microspheres to obtain modified two-phase microspheres carrying anions on the surface, and mixing the modified two-phase microspheres with crosslinked hyaluronic acid having cations to generate electrostatic crosslinks; the crosslinked hyaluronic acid having cations is crosslinked hyaluronic acid obtained by crosslinking with diamine or polyamine; and the surface charge modification of the two-phase microspheres includes immersing the two-phase microspheres in an alkaline buffer solution and separating them.

[0037] Furthermore, the mass ratio of the two-phase microspheres to the crosslinked hyaluronic acid in the physical crosslinking is 1:1 to 50; preferably 1:1 to 20; Furthermore, the alkaline buffer solution is an alkaline phosphate buffer solution; the pH range of the alkaline phosphate buffer solution is preferably 9.0 to 11.0.

[0038] In one embodiment, the microspheres are hydroxyapatite-dispersed polylactic acid biphasic microspheres, and the physical crosslinking includes immersing the hydroxyapatite-dispersed polylactic acid biphasic microspheres in a PBS solution of pH 9.0 to 11.0 to modify the surface charge, thereby causing the surface of the microspheres to carry a negative charge; then separating and removing the biphasic microspheres; and physically mixing them with a crosslinked hyaluronic acid gel using polyamine as a crosslinking agent, thereby electrostatically crosslinking the carboxylate anions on the surface of the polylactic acid with the ammonium cations of the crosslinked hyaluronic acid.

[0039] In one embodiment, the present invention further comprises a method for preparing hydroxyapatite-dispersed polylactic acid biphasic microspheres, said method comprising: (a) dissolving polylactic acid (PLA) in dichloromethane, stirring thoroughly to prepare a solution, and then filtering to remove insoluble matter; (b) adding hydroxyapatite, applying ultrasonic waves, and uniformly stirring to completely disperse the hydroxyapatite in the solution, thereby forming a suspension in which the hydroxyapatite is uniformly dispersed; (c) Prepare a polyvinyl alcohol solution according to the ratio of dichloromethane and stir it. During this period, add the dichloromethane suspension dropwise to the system, and mix it by stirring, shearing and emulsifying. fluid particles forming a (d) placing the emulsified solution in a water bath, stirring, and evaporating to remove the dichloromethane, and then washing and filtering the resulting solution.

[0040] In one specific embodiment, in step (a), the concentration of the PLA in dichloromethane solution is preferably 10 mg / mL to 80 mg / mL, including but not limited to 15 mg / mL, 30 mg / mL, 40 mg / mL, 60 mg / mL, and 80 mg / mL.

[0041] Preferably, the PLA comprises a mixture of one or more of poly-L-lactic acid (PLLA), polyracemic lactic acid (PDLLA) and poly-D-lactic acid (PDLA).

[0042] Preferably, the molecular weight range of the PLA is 10 KDa to 150 KDa.

[0043] In one specific embodiment, in step (b), the mass fraction of the hydroxyapatite is preferably 1% to 20%, so that it is completely incorporated into the polylactic acid microspheres. dispersion Ensure you can.

[0044] Preferably, the ultrasonic energy is 1 to 300 KJ, and more preferably, the energy is 1 to 20 KJ, and the formula for calculating the ultrasonic energy is as follows: Ultrasonic energy (KJ) = ultrasonic power (W) x ultrasonic time (sec) / 1000.

[0045] In one specific embodiment, in step (c), the concentration of the polyvinyl alcohol solution is preferably 5 mg / m L ~30mg / m L Within this concentration range, the emulsifying effect is ensured and the size of the microspheres can be controlled.

[0046] Preferably, the ratio of the dichloromethane solution to the polyvinyl alcohol solution is 1 / 2 to 1 / 10.

[0047] Preferably, the rotation speed of the stirring paddle of the stirring device is 100 to 600 rpm. m This rotation speed range ensures continuous emulsification and allows the formation of a good emulsion.

[0048] In one specific embodiment, in step (d), the water bath temperature is preferably 20-37°C, which can control the evaporation rate of dichloromethane within this temperature range, thereby optimizing the hollow effect of the microspheres and the microporous surface structure; after step (d), the obtained filter cake is dried to obtain two-phase polylactic acid hydroxyapatite nano-dispersed microspheres; preferably, the drying process includes two methods: vacuum oven drying and freeze-drying. Vacuum drying utilizes the lower boiling point of water in near-vacuum conditions to evaporate water at a lower temperature; freeze-drying utilizes the principle of ice crystal sublimation to avoid damage to the internal structure of the microspheres during the liquefaction process of the solid phase components, and can better maintain the spatial structure of the obtained polylactic acid microspheres.

[0049] In one embodiment, the composite gel of the present invention has an average elastic modulus of 400 to 1000 Pa.

[0050] In one embodiment, the loss of elastic modulus of the composite gel of the present invention before and after sterilization is less than 30%, more preferably less than 20%. In another aspect, the present invention protects the composite gel produced by the above preparation method.

[0051] In a further aspect, the present invention further covers the application of the composite gel in the preparation of drug carriers, tissue fillers or tissue repair materials.

[0052] Furthermore, the drug carrier can be a biological drug, a chemical drug or a naturally extracted drug.

[0053] The beneficial effects are as follows:

[0054] (1) In this invention, first, two-phase microspheres of polylactic acid and inorganic material are prepared, and then, using solid-phase crosslinking technology, physical electrostatic crosslinking or chemical crosslinking is performed between the terminal carboxyl groups of polylactic acid in the two-phase microspheres and hyaluronic acid. The three-phase composite gel thus formed has high stability and good biocompatibility and can be used for filling and repairing biological tissues.

[0055] (2) The two-phase microspheres of the present invention further improve the hydrophilicity of polylactic acid microspheres by adding hydrophilic calcium salt inorganic materials (e.g., hydroxyapatite, calcium phosphate, calcium tartrate, etc.). In addition, the calcium-based inorganic materials in the two-phase microspheres can be introduced into the macroscopic network structure of cross-linked hyaluronic acid using micron-scale polylactic acid as a carrier (the cross-linked hyaluronic acid particles are also micron-scale). The calcium ions and the carboxyl and hydroxyl groups in hyaluronic acid form complexes that fill the network gaps between the cross-linked hyaluronic acid, thereby making the three-phase gel have a tighter network structure. At the same time, the complexes between the calcium-based inorganic materials and hyaluronic acid shorten the distance between the molecular chains of polylactic acid and hyaluronic acid, thereby improving the cross-linking reaction efficiency of both and improving the mechanical strength and thermal stability of the three-phase composite gel.

[0056] (3) The preparation method of the present invention has a simple process, is convenient to operate, and is easy to be industrialized.

[0057] (4) The composite gel prepared by the present invention can exert the immediate filling effect of hyaluronic acid, the collagen regeneration effect of polylactic acid, and the bone filling and lactic acid neutralization effects of calcium-based inorganic materials such as hydroxyapatite, thereby integrating the advantages of three materials.

[0058] In order to more clearly describe the specific embodiments of the present invention or the technical solutions of the prior art, the following will briefly introduce the drawings necessary for describing the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work. [Brief explanation of the drawings]

[0059] [Figure 1] 1 is a micrograph of the biphasic polylactic acid hydroxyapatite nano-dispersed microspheres in Example 1 of the present invention. [Figure 2] 1 is a scanning electron microscope photograph of the two-phase polylactic acid hydroxyapatite nano-dispersed microspheres in Example 1 of the present invention. [Figure 3] 1 is a locally enlarged scanning electron microscope photograph of two-phase polylactic acid hydroxyapatite nano-dispersed microspheres in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0060] The following examples are provided to clearly and completely describe the technical solutions of the present invention, and it is obvious that the described examples are only some, but not all, of the present invention. Based on the examples of the present invention, all other examples that can be obtained by those skilled in the art without creative work fall within the scope of protection of the present invention.

[0061] Example 1 Example 1. Preparation of Two-Phase Microspheres Weigh out 1.8g of polylactic acid and place it in a 50mL beaker. Then add 50mL of dichloromethane and stir with a spoon until it is completely dissolved. Then, filter it into an ultrasonic beaker using a 10mm nylon 66 syringe filter. Then, weigh out 0.2g of nanohydroxyapatite and add it to the system. Then, use 60% output power. (12KJ) The nanomaterials were allowed to act for 10 minutes at room temperature to completely disperse the nanomaterials in the dichloromethane solution of polylactic acid. During this period, 135 mL of polyvinyl alcohol solution (1.5 wt%) was added to a 300 mL three-neck flask and placed in a water bath at 30 °C. m's After the ultrasonic waves are finished, the dichloromethane suspension is stirred mechanically at high speed. Polyvinyl alcohol Slowly add to the solution and stir overnight while the dichloromethane is removed by evaporation.

[0062] The suspension obtained in the previous step was diluted to 500 mL with purified water, allowed to stand for 1 hour, and the upper suspension layer was discarded. This process was repeated twice. The precipitate was then redissolved and filtered through a 0.45 μm aqueous filter membrane to remove the liquid. The resulting solid was then dispersed in 200 mL of purified water and filtered again. This process was repeated four times. The resulting solid was then redissolved in 150 mL of purified water, placed in a Schott bottle, heated in an oven at 100°C for 30 minutes, removed, cooled, filtered, and washed three times with purified water. The solid obtained from the final wash was transferred to a culture dish and dried overnight at 40°C in a vacuum oven to obtain the final product. The particle size of the product was mainly distributed between 25 and 86 μm. A micrograph is shown in Figure 1, and scanning electron micrographs are shown in Figures 2 and 3. As can be seen from the figures, the two-phase microspheres have a regular morphology and uniform particle size.

[0063] Example 2 Example 2. Preparation of two-phase microspheres 2 Weigh out 1.6g of polylactic acid and place it in a 50mL beaker. Then add 50mL of dichloromethane and stir with a spoon until it is completely dissolved. Then, use a 10mm nylon 66 syringe filter to filter it into an ultrasonic beaker. Then, weigh out 0.2g of nanohydroxyapatite and add it to the system. Then, use 60% power. (12KJ) The nanomaterials were allowed to act for 10 minutes at room temperature to completely disperse the nanomaterials in the dichloromethane solution of polylactic acid. During this period, 90 mL of polyvinyl alcohol solution (1.5 wt%) was added to a 300 mL three-neck flask and placed in a water bath at 30 °C. m's After the ultrasonic waves are finished, the dichloromethane suspension is stirred mechanically at high speed. Polyvinyl alcohol Slowly add to the solution and stir overnight while the dichloromethane is removed by evaporation.

[0064] The suspension obtained in the previous step was diluted to 500 mL with purified water, allowed to stand for 1 hour, and the upper suspension layer was discarded. This process was repeated twice. The precipitate was then redissolved and filtered through a 0.45 μm aqueous filter membrane to remove the liquid. The resulting solid was then dispersed in 200 mL of purified water and filtered again. This process was repeated four times. The resulting solid was then redissolved in 150 mL of purified water, placed in a Schottky bottle, heated in an oven at 100°C for 30 minutes, removed, cooled, filtered, and washed three times with purified water. The solid obtained from the final wash was transferred to a culture dish and lyophilized to obtain the final product. The particle size ranged primarily from 25 to 72 μm.

[0065] Example 3 Example 3. Physical cross-linking of a three-phase gel Weigh out 1.5g of sodium hyaluronate (molecular weight 900KDa), then add 37.5mL of purified water, and after complete dissolution, the hyaluronic acid concentration at this time is 40mg / mL, and add 45mg of spermidine to the hyaluronic acid solution. On the premise of three-site cross-linking of spermidine, adjust the pH value of the hyaluronic acid solution to about 6.2 using 6mol / L hydrochloric acid solution, and then Post-stirring The mixture is stirred, 0.214 g of EDC is added, and simultaneously 0.04 g of NHS is added. The mixture is stirred uniformly, sealed, and placed in a 40°C air-blowing oven for reaction for 14 hours.

[0066] After the reaction was completed, 40 mL of absolute ethanol was added, and the mixture was ground using an IKAT25 high-shear disperser at a grinding speed of 10,000 rpm. m The gel particles were crushed for 5 minutes. After crushing, 200 mL of absolute ethanol was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and dried at a vacuum of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dried gel was collected and added to a total of 50 mL of 10 mg / mL PBS at pH 6.0. The gel was then allowed to swell completely before use.

[0067] Take 3.0g of the two-phase microspheres obtained in Example 1, immerse in 50mL of pH9.0 PBS solution to carry out surface charge modification, so that the surface of the microspheres carries negative charge; after immersion for 30 minutes, separate and take out the two-phase microspheres, drain the water, and then physically mix with 50mL of cross-linked hyaluronic acid gel, so that electrostatic cross-linking occurs between the carboxylate anion on the surface of polylactic acid and the ammonium cation of cross-linked hyaluronic acid.After uniform mixing, fill the three-phase gel into a pre-filled syringe, and sterilize under 121 ℃ for 15 minutes with moist heat to obtain the final gel product.

[0068] Example 4 Example 4. Chemical cross-linking of three-phase gels 1.5 g of the two-phase microspheres obtained in Example 1 was taken, 100 mL of purified water was added, 2.0 g of ethylenediamine was added, and the pH of the solution was adjusted to 5.5 using 1 mol / L hydrochloric acid solution. 2.0 g of EDC and 0.4 g of NHS were added for surface amino modification, and the reaction was carried out at 40°C for 16 hours. The microspheres were separated by filtration, washed with purified water to a pH range of 7.0-7.1, and dried overnight at 40°C in a vacuum drying oven to obtain surface amino-modified two-phase microspheres.

[0069] 1.5g of sodium hyaluronate was weighed, and then 37.5mL of purified water was added. After complete dissolution, the hyaluronic acid concentration was 40mg / mL. ,in obtained Surface amino modification Add 1.0 g of the two-phase microspheres. Adjust the pH of the hyaluronic acid solution to about 5.5 using 0.1 mol / L hydrochloric acid solution. Post-stirring Stir, add 0.3 g of EDC, and simultaneously add 0.06 g of NHS (20% of the mass of EDC), continue to stir uniformly, seal, and place in a 40°C air-blowing oven to react for 14 hours.

[0070] After the reaction was complete, 200 mL of absolute ethanol was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and dried at a vacuum of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dried gel was collected and a total of 50 mL of 10 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel was completely swollen, it was filled into a pre-filled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final three-phase gel.

[0071] Example 5 Example 5. Preparation of calcium carbonate biphasic microspheres Weigh out 1.6g of polylactic acid and place it in a 50mL beaker. Then add 50mL of dichloromethane and stir with a spoon until it is completely dissolved. Then, use a 10mm nylon 66 syringe filter to filter it into an ultrasonic beaker. Then, weigh out 0.2g of nano calcium carbonate and add it to the system. Then, use 60% power. (12KJ) The nanomaterials were allowed to act for 10 minutes at room temperature to completely disperse the nanomaterials in the dichloromethane solution of polylactic acid. During this period, 90 mL of polyvinyl alcohol solution (1.5 wt%) was added to a 300 mL three-neck flask and placed in a water bath at 30 °C. m's After the ultrasonic waves are finished, the dichloromethane suspension is stirred mechanically at high speed. Polyvinyl alcohol Slowly add to the solution and stir overnight while the dichloromethane is removed by evaporation.

[0072] The turbid liquid obtained in the previous step was diluted to 500 mL with purified water, allowed to stand for 1 hour, and the upper suspension layer was discarded. This process was repeated twice. The precipitate was then redissolved and filtered through a 0.45 μm aqueous filtration membrane to remove the liquid. The resulting solid was then dispersed in 200 mL of purified water and filtered again. This process was repeated four times. The resulting solid was then redissolved in 150 mL of purified water, placed in a shot bottle, heated in an oven at 100°C for 30 minutes, removed, cooled, filtered, and washed three times with purified water. The solid obtained from the final wash was transferred to a culture dish and freeze-dried to obtain the final product.

[0073] Example 6 Example 6. Preparation of calcium tartrate biphasic microspheres Weigh out 1.6g of polylactic acid and place it in a 50mL beaker. Then add 50mL of dichloromethane and stir with a spoon until it is completely dissolved. Then, use a 10mm nylon 66 syringe filter to filter it into an ultrasonic beaker. Then, weigh out 0.2g of nano-calcium tartrate and add it to the system. 60% output power is used. (12KJ) The nanomaterials were allowed to act for 10 minutes at room temperature to completely disperse the nanomaterials in the dichloromethane solution of polylactic acid. During this period, 90 mL of polyvinyl alcohol solution (1.5 wt%) was added to a 300 mL three-neck flask and placed in a water bath at 30 °C. m's After the ultrasonic waves are finished, the dichloromethane suspension is stirred mechanically at high speed. Polyvinyl alcohol Slowly add to the solution and stir overnight while the dichloromethane is removed by evaporation.

[0074] The turbid liquid obtained in the previous step was diluted to 500 mL with purified water, allowed to stand for 1 hour, and the upper suspension layer was discarded. This process was repeated twice. The precipitate was then redissolved and filtered through a 0.45 μm aqueous filtration membrane to remove the liquid. The resulting solid was then dispersed in 200 mL of purified water and filtered again. This process was repeated four times. The resulting solid was then redissolved in 150 mL of purified water, placed in a shot bottle, heated in an oven at 100°C for 30 minutes, removed, cooled, filtered, and washed three times with purified water. The solid obtained from the final wash was transferred to a culture dish and freeze-dried to obtain the final product.

[0075] Example 7 Example 7. Chemical cross-linking of calcium carbonate three-phase gel 1.5 g of the two-phase microspheres obtained in Example 5 was taken, 100 mL of purified water was added, 2.0 g of ethylenediamine was added, and the pH of the solution was adjusted to 5.5 using 1 mol / L hydrochloric acid solution. 2.0 g of EDC and 0.4 g of NHS were added for surface amino modification, and the reaction was carried out at 40°C for 16 hours. The microspheres were separated by filtration, washed with purified water to a pH range of 7.0-7.1, and dried overnight at 40°C in a vacuum drying oven to obtain surface amino-modified two-phase microspheres.

[0076] 1.5g of sodium hyaluronate was weighed, and then 37.5mL of purified water was added. After complete dissolution, the hyaluronic acid concentration was 40mg / mL. The hyaluronic acid solution contained 1.0g of Surface amino modification Add the biphasic microspheres. Adjust the pH of the hyaluronic acid solution to about 5.5 using 0.1 mol / L hydrochloric acid solution. Post-stirring Stir, add 0.3 g of EDC, and simultaneously add 0.06 g of NHS (20% of the mass of EDC), continue to stir uniformly, seal, and place in a 40°C air-blowing oven to react for 14 hours.

[0077] After the reaction was complete, 200 mL of absolute ethanol was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and dried at a vacuum of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dried gel was collected and a total of 50 mL of 10 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel was completely swollen, it was filled into a pre-filled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final three-phase gel.

[0078] Example 8 Example 8. Chemical cross-linking of calcium tartrate triphasic gels 1.5 g of the two-phase microspheres obtained in Example 6 was taken, 100 mL of purified water was added, 2.0 g of ethylenediamine was added, and the pH of the solution was adjusted to 5.5 using 1 mol / L hydrochloric acid solution. 2.0 g of EDC and 0.4 g of NHS were added for surface amino modification, and the reaction was carried out at 40°C for 16 hours. The microspheres were separated by filtration, washed with purified water to a pH range of 7.0-7.1, and dried overnight at 40°C in a vacuum drying oven to obtain surface amino-modified two-phase microspheres.

[0079] 1.5g of sodium hyaluronate was weighed, and then 37.5mL of purified water was added. After complete dissolution, the hyaluronic acid concentration was 40mg / mL. The hyaluronic acid solution contained 1.0g of Surface amino modification Add the biphasic microspheres. Adjust the pH of the hyaluronic acid solution to about 5.5 using 0.1 mol / L hydrochloric acid solution. Post-stirring Stir, add 0.3 g of EDC, and simultaneously add 0.06 g of NHS (20% of the mass of EDC), continue to stir uniformly, seal, and place in a 40°C air-blowing oven to react for 14 hours.

[0080] After the reaction was complete, 200 mL of absolute ethanol was added to completely dehydrate the gel. The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and dried at a vacuum of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dried gel was collected and a total of 50 mL of 10 mg / mL phosphate buffer solution at pH 7.0 was added. After the gel was completely swollen, it was filled into a pre-filled syringe and sterilized with moist heat at 121°C for 15 minutes to obtain the final three-phase gel.

[0081] (Comparative Example 1) Comparative Example 1, Preparation of Non-Crosslinked Three-Phase Gel Weigh out 1.5g of sodium hyaluronate, then add 37.5mL of purified water, and after complete dissolution, the hyaluronic acid concentration is 40mg / mL. Add 1.0g of the two-phase microspheres obtained in Example 2 to the hyaluronic acid solution, and stir evenly. After stirring is completed, add 200mL of absolute ethanol to completely dehydrate the gel.

[0082] The precipitate was separated and washed five times with 200 mL of absolute ethanol. It was then placed in a vacuum oven and dried at a vacuum of -0.09 MPa and 40°C for 24 hours. After complete drying, 1.0 g of dried gel was collected and a total of 50 mL of 10 mg / mL phosphate buffer solution with a pH of 7.0 was added. After the gel was completely swollen, it was loaded into a pre-filled syringe and sterilized by moist heat at 121°C for 15 minutes to obtain a non-crosslinked three-phase gel.

[0083] (Comparative Example 2) Comparative Example 2: Preparation of a Three-Phase Non-Physically Crosslinked Blend Gel The method for preparing the spermidine-crosslinked hyaluronic acid gel was the same as in Example 3, and the overall preparation method was exactly the same.

[0084] 3.0 g of the two-phase microspheres obtained in Example 1 are taken and directly physically mixed with 50 mL of cross-linked hyaluronic acid gel. After uniform mixing, the three-phase gel is filled into a pre-filled syringe and sterilized by moist heat at 121°C for 15 minutes to obtain the final gel product.

[0085] (Comparative Example 3) Comparative Example 3: Preparation of a physical blend gel of single-phase PLA microspheres and cross-linked hyaluronic acid 1.6 g of polylactic acid was weighed and placed in a 50 mL beaker, then 50 mL of dichloromethane was added and stirred with a spoon until completely dissolved, and then filtered into an ultrasonic beaker using a 10 mm nylon 66 syringe filter. During this period, 90 mL of polyvinyl alcohol solution (1.5 wt%) was added to a 300 mL three-neck flask and placed in a water bath at 30 °C, followed by 400 rp m's Stir mechanically at high speed.

[0086] Then, the dichloromethane solution Polyvinyl alcohol Slowly add to the solution and stir overnight while the dichloromethane is removed by evaporation.

[0087] The suspension obtained in the previous step was diluted to 500 mL with purified water, allowed to stand for 1 hour, and the upper suspension layer was discarded. This process was repeated twice. The precipitate was then redissolved and filtered through a 0.45 μm aqueous filtration membrane to remove the liquid. The resulting solid was then dispersed in 200 mL of purified water and filtered again. This process was repeated four times. The resulting solid was then redissolved in 150 mL of purified water, placed in a shot bottle, and heated in an oven at 100°C for 30 minutes. After cooling, it was filtered and washed three times with purified water. The solid obtained from the final wash was transferred to a culture dish and freeze-dried to obtain the final product: PLA microspheres.

[0088] 3.0 g of the obtained PLA microspheres are taken and immersed in 50 mL of pH 9.0 PBS solution to carry out surface charge modification, so that the surface of the microspheres carries a negative charge. After immersion for 30 minutes, the microspheres are separated and removed. After draining, they are physically mixed with 50 mL of the cross-linked hyaluronic acid gel obtained in Example 3, so that electrostatic cross-linking occurs between the carboxylate anions on the surface of the polylactic acid and the ammonium cations of the cross-linked hyaluronic acid. After uniform mixing, the blended gel is filled into a pre-filled syringe and sterilized with moist heat at 121 ° C for 15 minutes to obtain the final gel product.

[0089] Comparative Example 4 Comparative Example 4: Preparation of a physical blend gel of nanoscale HAP and cross-linked hyaluronic acid The method for preparing the spermidine-crosslinked hyaluronic acid gel was the same as in Example 3, and the overall preparation method was exactly the same.

[0090] 3.0 g of nanoscale HAP was taken and directly physically mixed with 50 mL of cross-linked hyaluronic acid gel. After uniform mixing, the blended gel was filled into a pre-filled syringe and sterilized by moist heat at 121°C for 15 minutes to obtain the final gel product.

[0091] (Example of effect) composite Rheological property testing of gels The results obtained in the examples and comparative examples composite The gel was divided into two types, one before sterilization and one after sterilization, and 2.0 mL of each was collected. ,flat The elastic modulus (G') of the gel is detected using a plate rheometer, and the elastic loss modulus is calculated, where G' is calculated by the following formula:

[0092] G' loss rate = (G' before sterilization - G' after sterilization) / G' before sterilization The rheometer parameters were as follows: operating gap: 1000 mm, load gap: 45000 m, operating temperature: 37°C, deformation: 1%, frequency: 0.9 Hz, operating time: 60 seconds. The rheological data of each gel are shown in Table 1.

[0093] [Table 1]

[0094] As can be seen from the data in Table 1, compared with the non-physically crosslinked three-phase blend gel in Comparative Example 2, the three-phase gel obtained by physical crosslinking in Example 3 has an improved modulus and a certain degree of improved thermal stability, indicating that physical crosslinking contributes to a certain degree to an improved modulus of the composite gel and also to a certain degree of improved thermal stability of the three-phase gel. Because Comparative Example 1 is merely a physical mixture of three materials, non-crosslinked HA, PLA, and HAP, there is no chemical bonding between the three materials, and they cannot withstand the severe hydrolysis caused by moist heat sterilization. As a result, the modulus of Comparative Example 1 after sterilization decreased by more than 97%.

[0095] In Comparative Example 3, physical crosslinking was performed on PLA and HA, but HAP was not added, so the elastic modulus of the composite gel was lower than that of Example 3. G’ After HAP and PLA form composite microspheres, nano-HAP can be introduced into the macroscopic network structure of cross-linked hyaluronic acid using the micron-scale PLA as a carrier (the cross-linked hyaluronic acid particles are also micron-scale), and the complexation of calcium ions with the carboxyl and hydroxyl groups in hyaluronic acid fills the network gaps between the cross-linked hyaluronic acid, thereby giving the three-phase gel a tighter network structure. At the same time, the complexation of HAP with HA shortens the distance between the molecular chains of PLA and HA, resulting in improved cross-linking efficiency and therefore a higher elastic modulus and stronger thermal stability.

[0096] Comparative Example 4 discloses a physical blend of nanoscale HAP and cross-linked hyaluronic acid, but does not exhibit high elastic modulus or strong thermal stability. The reason for this is that in Comparative Example 4, nanoscale HAP is directly added, and compared to micron-scale cross-linked hyaluronic acid gel, the difference in physical size between the two is large. As a result, the spatial difference between the two is large, making it difficult for HAP to fill the gaps in the cross-linked hyaluronic acid gel and preventing it from forming a denser network structure.

[0097] This also shows that by forming two-phase microspheres with HAP and PLA and supporting the HAP on a micron-scale PLA carrier, a tighter network structure can be formed with the cross-linked hyaluronic acid gel, thus providing a higher elastic modulus and stronger thermal stability. Therefore, the composite gel can improve the elastic modulus and thermal stability of the three-phase gel through the synergistic effect between the cross-linked hyaluronic acid and the PLA-HAP two-phase microspheres, and all three components are essential.

[0098] The elastic modulus of the three-phase composite gels obtained by chemical crosslinking in Examples 4, 7, and 8 was significantly improved. This indicates that when physical crosslinking is replaced by chemical crosslinking, the bond strength formed by chemical crosslinking is much stronger than the interaction strength of physical crosslinking, so the interaction between the crosslinked hyaluronic acid, HAP (or other nanoscale calcium salts), and PLA is further strengthened, and the elastic modulus and thermal stability of the chemically crosslinked three-phase gel are further improved compared to the physically crosslinked three-phase gel.

[0099] Finally, it should be noted that the above embodiments are only used to describe the technical solutions of the present invention, and are not intended to limit them; the present invention will be described in more detail with reference to the above embodiments, but those skilled in the art should understand that they can modify the technical solutions described in the above embodiments or make equivalent substitutions for some or all of the technical features therein; these modifications or equivalent substitutions will not cause the essence of the corresponding technical solutions to depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite gel obtained by crosslinking two-phase microspheres with hyaluronic acid, wherein the two-phase microspheres are polylactic acid / inorganic material two-phase microspheres; The polylactic acid is micron-scale, the crosslinking comprises crosslinking polylactic acid and hyaluronic acid in the two-phase microspheres; The inorganic material is one or more selected from calcium carbonate, calcium phosphate, and calcium tartrate, and the inorganic material is a nano-inorganic material. Composite gel.

2. The calcium phosphate is preferably hydroxyapatite. The composite gel of claim 1 .

3. The crosslinking between the polylactic acid and the hyaluronic acid includes any one of physical electrostatic crosslinking and chemical crosslinking; Preferably, the hyaluronic acid is cross-linked hyaluronic acid; further, the cross-linked hyaluronic acid is prepared using a cross-linking agent, the cross-linking agent being one or more selected from dialdehydes, disulfides, polyethylene glycol (PEG) cross-linkers, divinyl sulfones, diglycidyl ethers, diepoxides, and polyamines; and further, the cross-linking agent is an endogenous polyamine. The composite gel of claim 2.

4. A method for preparing a composite gel as described in any one of claims 1 to 3, comprising a step of cross-linking two-phase microspheres with hyaluronic acid to form a composite gel, wherein the two-phase microspheres are polylactic acid / inorganic material two-phase microspheres, and the cross-linking includes cross-linking between polylactic acid and hyaluronic acid in the two-phase microspheres.

5. The crosslinking is a solid-phase crosslinking; and the solid-phase crosslinking includes any one of a physical electrostatic crosslinking and a chemical crosslinking; Preferably, the chemical crosslinking comprises the step of amino-modifying polylactic acid in the two-phase microspheres and then reacting the modified two-phase microspheres with hyaluronic acid to form amide bond crosslinks; further, the amino-modification comprises reacting the two-phase microspheres with an amine compound; and further, the amide bond crosslinking comprises coupling the modified two-phase microspheres with hyaluronic acid in the presence of a carbodiimide activator; Preferably, the physical electrostatic crosslinking comprises: surface charge modification of the two-phase microspheres to obtain modified two-phase microspheres carrying anions on the surface; and mixing the modified two-phase microspheres with crosslinked hyaluronic acid having cations to generate electrostatic crosslinks; and further, the surface charge modification of the two-phase microspheres comprises immersing the two-phase microspheres in an alkaline buffer solution and separating them. The preparation method according to claim 4.

6. The hyaluronic acid is crosslinked hyaluronic acid, and the crosslinked hyaluronic acid is prepared using a crosslinking agent, and the crosslinking agent is one or more selected from the group consisting of dialdehyde, disulfide, polyethylene glycol (PEG) crosslinker, divinyl sulfone, diglycidyl ether, diepoxide, and polyamine. The preparation method according to claim 5.

7. The amine compound includes a polyamine or a polyamino compound, Preferably, the polyamine includes any one of an aliphatic polyamine, an aromatic polyamine, and a heteroatom polyamine; and the polyamino compound includes any one of a doubly aminated polyethylene glycol and a terminally aminated multi-arm polyethylene glycol; Further, the carbodiimide activator comprises one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, 1,3-bis[di(methoxymethyl)methyl]carbodiimide, or a salt thereof; The preparation method according to claim 5.

8. The mass ratio of the two-phase microspheres to the hyaluronic acid in the chemical crosslinking is 1:0.5-20, preferably 1:1-10; Furthermore, the amount of the amine compound added is 30% to 200% of the mass of the two-phase microspheres, and the concentration range of the amine compound is 10 mg / mL to 1000 mg / mL; Furthermore, the amount of the carbodiimide activator added is 10% to 150% of the total mass of the hyaluronic acid; Furthermore, the concentration of the hyaluronic acid solution is 10 mg / mL to 100 mg / mL; Furthermore, the temperature for the amino modification reaction and the amide bond crosslinking reaction is 5°C to 60°C, and the reaction time is 6 hours to 24 hours.

8. The preparation method according to claim 5 or 7.

9. The mass ratio of the two-phase microspheres to the cross-linked hyaluronic acid in the physical electrostatic cross-linking is 1:1-50; preferably 1:1-20; Furthermore, the cation-containing crosslinked hyaluronic acid is crosslinked hyaluronic acid obtained by crosslinking with a polyamine; Furthermore, the alkaline buffer solution is an alkaline phosphate buffer solution; the pH range of the alkaline phosphate buffer solution is preferably 9.0 to 11.

0. The preparation method according to claim 5.

10. Application of the composite gel according to any one of claims 1 to 3 in the manufacture of a drug carrier, a filler or a tissue repair material.

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