Composite gel, preparation method and application

Abstract

The present invention provides a composite gel, a preparation method and an application thereof, and relates to the technical field of biomedical materials. The composite gel is prepared by cross-linking biphasic microspheres composed of polylactic acid and nano-scale inorganic materials with hyaluronic acid; the method comprises cross-linking the biphasic microspheres with hyaluronic acid to form a three-phase gel. The composite gel prepared by the present invention integrates the advantages of the 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, and in particular to a composite gel, a preparation method and an application thereof. Background Art

[0002] Polylactic acid (PLA), also known as polylactide, is a polyester polymer obtained by polymerization with lactic acid as the main raw material. It is used in combination with hydroxyapatite due to its good biocompatibility, biodegradability and easy processing to improve the mechanical and biological properties of hydroxyapatite. At present, regenerative injection products have been widely accepted worldwide, and polylactic acid serves as the main material in regenerative injection products. After entering the human skin, polylactic acid can stimulate the regeneration of the body's own tissues. Through the regeneration of the body's own tissues, it plays a role in structural remodeling and volume filling, achieving the purpose of anti-aging. The tissues that can regenerate after being stimulated are mainly fibroblasts and collagen. Polylactic acid can stimulate its own fibroblasts to secrete collagen, making the filling and repair effects more natural, while increasing the skin's glossiness and achieving the purpose of overall facial rejuvenation. However, the natural defect of polylactic acid is that polylactic acid degrades in the human body to produce lactic acid, and the body's metabolism of lactic acid is relatively slow. A large amount of lactic acid accumulation is likely to cause aseptic inflammation, thereby triggering a series of adverse reactions. Therefore, when using polylactic acid, an alkaline neutralizer needs to be added to reduce aseptic inflammation caused by excessive local acidity. Therefore, hydroxyapatite, which is alkaline and has good biocompatibility, has become the only choice for polylactic acid alkaline neutralizer.

[0003] Composite materials formed by polylactic acid and inorganic materials have been applied in biomaterials. For example, CN106139256A discloses a polylactic acid / nano β-tricalcium phosphate composite porous scaffold, and CN111905151A discloses a mesoporous bioactive glass / polylactic acid-glycolic acid copolymer composite microsphere.

[0004] In particular, composite materials of hydroxyapatite and polylactic acid have been widely used in orthopedics and other fields. Huang Chenghuan et al. (Journal of Composite Materials, 2021, 38: 749-760) studied nano-HAP-poly L-lactic acid (PLLA) composites and found that the compressive yield strength and tensile strength of PLLA with the addition of HAP were increased by 9.4% and 6.6% respectively compared with pure PLLA materials, the initial decomposition temperature was increased by 7.4% and 5.6% respectively compared with pure PLLA, and the crystallinity was increased by 6.7%. It can be seen that the PLLA material with the addition of HAP has obvious advantages over the pure PLLA material in terms of material properties. In addition, hydroxyapatite can reduce inflammation: compared with the PLA material with the addition of HAP, the inflammatory response of pure PLA is stronger, while the HAP-PLA composite material only shows a mild inflammatory response in the body. It can be proved that the addition of HAP can neutralize the acidic substances caused by the degradation of PLA and reduce the aseptic inflammation caused by acidity.

[0005] At present, there have been reports of binary composites of hydroxyapatite and polylactic acid, ternary composites of hydroxyapatite, polylactic acid and other polymers, or composite microspheres at home and abroad. Dong Yan (Additive Manufacturing, 2020; 34: 101305) et al. have prepared polylactic acid and nano-hydroxyapatite blended microspheres, with a minimum particle size of about 100 μm, which are mainly used for microcarrier research. Patents such as a method for preparing nano-hydroxyapatite / polylactic acid composite microspheres (CN101590388A), a method for preparing polylactic acid porous microspheres loaded with hydroxyapatite (CN103868658B), a polylactic acid / hydroxyapatite whisker composite porous scaffold for bone tissue and a method for preparing the same (CN105797215A), polylactic acid-hydroxyapatite micron-nano multi-level structure composite microsphere materials and applications (CN109749119 A), a method for preparing a controllable surface porous structure of polylactic acid hydroxyapatite composite microspheres and applications (CN107519536A), a method for preparing hydroxyapatite / modified polylactic acid composite microspheres (CN109350768A) and a method for preparing hydroxyapatite / polylactic acid / chitosan composite microspheres (CN102489231B) are mainly used for the repair, filling and drug loading of bone defects, but have not been applied to the field of tissue filling. Summary of the invention

[0006] The object of the present invention is to provide a composite gel, a preparation method and application thereof. The composite gel integrates the advantages of hyaluronic acid, polylactic acid and inorganic materials and can be used for filling and repairing biological tissues.

[0007] The technical solution provided by the present invention is as follows:

[0008] A composite gel is prepared by cross-linking polylactic acid and dual-phase microspheres with hyaluronic acid, wherein the dual-phase microspheres are polylactic acid / inorganic material dual-phase microspheres.

[0009] Furthermore, the inorganic material is selected from one or more of calcium carbonate, calcium phosphate or calcium tartrate; further, the calcium phosphate is preferably hydroxyapatite; further, the inorganic material is a nano inorganic material.

[0010] In one embodiment, the inorganic material is nano-calcium hydroxyapatite.

[0011] Furthermore, the hyaluronic acid is a cross-linked hyaluronic acid; further, the cross-linked hyaluronic acid is prepared by a cross-linking agent, and the cross-linking agent is selected from one or more of dialdehydes, disulfides, polyethylene glycol (PEG) cross-linkers, divinyl sulfones, diglycidyl ethers, diepoxides, diamines or polyamines.

[0012] Furthermore, the crosslinking agent is selected from biscarbodiimide, fatty diamine, ethylenediamine, hexamethylenediamine, endogenous polyamine (spermine or spermidine), pentaerythritol tetraglycidyl ether (PETGE), divinyl sulfone (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(ethylcarbodiimide), adipic acid dihydrazide (ADH), bis(sulfosuccinimide) suberate (BS ), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, 1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyl ether oxide butane, 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, 1,3-butadiene diepoxide, 1,2,7,8-diepoxyoctane, 1,5-hexadiene diepoxide. Preferably, the cross-linking agent is a diamine or a polyamine, and the reaction process can refer to patents CN99813143.1, US9907739, CN202111326226.7, etc.

[0013] Furthermore, the cross-linking includes cross-linking of polylactic acid and hyaluronic acid in the dual-phase microspheres.

[0014] Further, the crosslinking of the polylactic acid and hyaluronic acid includes any one of physical electrostatic crosslinking and chemical crosslinking. The chemical crosslinking includes reacting the polylactic acid in the biphasic microspheres with the hyaluronic acid to form an amide bond crosslinking reaction. The physical electrostatic crosslinking includes modifying the surface charge of the biphasic microspheres and then forming an electrostatic crosslinking with the hyaluronic acid. Further, the chemical crosslinking includes amino-modifying the polylactic acid in the biphasic microspheres and then reacting with the hyaluronic acid to form an amide bond crosslinking reaction, and the physical electrostatic crosslinking includes modifying the surface charge of the biphasic microspheres and then forming an electrostatic crosslinking with the crosslinked hyaluronic acid with positive ions.

[0015] In one embodiment, a composite gel is provided, which is prepared by cross-linking hyaluronic acid with biphasic polylactic acid hydroxyapatite dispersed microspheres composed of polylactic acid and nano-sized hydroxyapatite.

[0016] A method for preparing a composite gel comprises cross-linking a dual-phase microsphere and hyaluronic acid to form a composite gel (three-phase composite gel), wherein the dual-phase microsphere is a polylactic acid / inorganic material dual-phase microsphere.

[0017] Furthermore, the cross-linking of the biphasic microspheres and hyaluronic acid includes cross-linking of polylactic acid and hyaluronic acid in the biphasic microspheres.

[0018] Further, the hyaluronic acid is non-crosslinked hyaluronic acid or crosslinked hyaluronic acid. In one embodiment, the hyaluronic acid is crosslinked hyaluronic acid, preferably diamine or polyamine crosslinked hyaluronic acid, more preferably endogenous polyamine crosslinked hyaluronic acid.

[0019] In one embodiment, in the dual-phase polylactic acid hydroxyapatite dispersed microspheres, the mass of hydroxyapatite accounts for 0.1-50% of the total mass of the microspheres, and the rest of the microspheres except hydroxyapatite are polylactic acid.

[0020] In one embodiment, a cross-linked hyaluronic acid hydrogel is prepared using polyamine as a cross-linking agent. Preferably, the polyamine cross-linking agent accounts for 0.5-20% of the total mass of the hyaluronic acid. In the hydrogel, the mass concentration of hyaluronic acid ranges from 1 to 35 mg / mL.

[0021] In one embodiment, the hyaluronic acid is prepared by microbial fermentation; preferably, the molecular weight of the hyaluronic acid ranges from 100KDa to 3000KDa, preferably 500KDa to 1500KDa. The hyaluronic acid fermented by microorganisms is non-animal derived hyaluronic acid, and its molecular weight distribution is more uniform.

[0022] In one embodiment, the crosslinking is solid phase crosslinking; further, the solid phase crosslinking includes any one of physical electrostatic crosslinking and chemical crosslinking.

[0023] In one embodiment, the chemical crosslinking includes amino-modifying the polylactic acid in the biphasic microspheres, and then reacting the modified biphasic microspheres with hyaluronic acid to form amide bond crosslinks; further, the amino modification includes reacting the biphasic microspheres with amine compounds; further, the amide bond crosslinking includes coupling the modified biphasic microspheres with hyaluronic acid in the presence of a carbodiimide activator.

[0024] In one embodiment, the amine compound comprises a diamine, a polyamine or a polyamino compound; preferably, the diamine comprises any one of aliphatic diamine, aromatic diamine and heteroatom diamine; for example, but not limited to aliphatic diamine, ethylenediamine, hexamethylenediamine; the polyamine comprises any one of aliphatic polyamine, aromatic polyamine and heteroatom polyamine; for example, but not limited to endogenous polyamines such as spermidine and spermine; the polyamino compound comprises any one of double-terminated aminated polyethylene glycol and terminally aminated multi-arm polyethylene glycol;

[0025] 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 their salts.

[0026] In one embodiment, the preparation method further comprises using an auxiliary agent together with the carbodiimide activator; for example, N-hydroxysuccinimide (NHS), tert-butyl alcohol, etc. The water-soluble carbodiimide activator needs to be used in combination with the auxiliary agent to improve the efficiency of the crosslinking reaction, and the amount of the auxiliary agent added is 10-30% of the mass of the carbodiimide.

[0027] In one embodiment, the mass ratio of the biphasic microspheres to the hyaluronic acid in the chemical crosslinking is 1:0.5-20, preferably 1:1-10;

[0028] The amount of the amine compound added to the reaction system is 30-200% of the mass of the biphasic microspheres; the concentration range of the amine compound is 10-1000 mg / mL;

[0029] Furthermore, the amount of the carbodiimide activator added is 10 to 150% of the total mass of the hyaluronic acid;

[0030] Further, the concentration of the hyaluronic acid solution is 10 to 100 mg / mL;

[0031] Furthermore, the temperature of the amino modification reaction and the amide bond cross-linking reaction is 5 to 60° C., and the reaction time is 6 to 24 hours; the pH value of the reaction system ranges from 5.0 to 6.0.

[0032] In one embodiment, the microspheres are biphasic polylactic acid hydroxyapatite dispersed microspheres, and the chemical crosslinking includes dispersing the biphasic polylactic acid hydroxyapatite dispersed microspheres in water, adding amine compounds (polyamine derivatives), adjusting the pH value, so that the carboxyl groups on the surface of the microspheres react with the polyamine derivatives to modify the surface, and modifying the surface with amino groups; the surface amino-modified polylactic acid hydroxyapatite dispersed microspheres (PLA-HAP biphasic microspheres) are put into a hyaluronic acid solution, the pH value is adjusted to 5.0-6.0, and a carbodiimide activator and an auxiliary agent are added at the same time to complete the coupling reaction of the PLA-HAP biphasic microspheres. The surface modification reaction temperature is 5-60°C, and the reaction time is 6-24h; the coupling reaction temperature is 5-60°C, and the reaction time is 6-24h.

[0033] The physical electrostatic crosslinking includes modifying the surface charge of the biphasic microspheres to obtain modified biphasic microspheres carrying negative ions on the surface, and mixing the modified biphasic microspheres with cross-linked hyaluronic acid having positive ions to cause electrostatic crosslinking; further, the cross-linked hyaluronic acid having positive ions is a cross-linked hyaluronic acid obtained by cross-linking with diamines or polyamines; further, the surface charge modification of the biphasic microspheres includes immersing the biphasic microspheres in an alkaline buffer solution to separate them.

[0034] Furthermore, the mass ratio of the dual-phase microspheres to the cross-linked hyaluronic acid in the physical cross-linking is 1:1 to 50; preferably 1:1 to 20;

[0035] Furthermore, the alkaline buffer is an alkaline phosphate buffer; the pH range of the alkaline phosphate buffer is preferably 9.0 to 11.0.

[0036] In one embodiment, the microspheres are biphasic polylactic acid hydroxyapatite dispersed microspheres, and the physical crosslinking includes immersing the biphasic polylactic acid hydroxyapatite dispersed microspheres in a PBS solution with a pH of 9.0 to 11.0 to perform surface charge modification so that the surface of the microspheres carries a negative charge, separating and taking out the biphasic microspheres, and physically mixing them with a cross-linked hyaluronic acid gel with a polyamine as a cross-linking agent, so that the carboxylate anions on the surface of the polylactic acid and the ammonium cations in the cross-linked hyaluronic acid undergo electrostatic crosslinking.

[0037] In one embodiment, the present invention also includes a method for preparing biphasic polylactic acid hydroxyapatite dispersed microspheres, the method comprising

[0038] (a) dissolving polylactic acid (PLA) in dichloromethane and stirring to dissolve the PLA into a solution, and then filtering to remove insoluble matter;

[0039] (b) adding hydroxyapatite and performing ultrasonication, stirring evenly, so that the hydroxyapatite is completely dispersed in the solution to form a suspension in which the hydroxyapatite is evenly dispersed;

[0040] (c) preparing a polyvinyl alcohol solution according to the ratio of dichloromethane and stirring the solution, during which the dichloromethane suspension is added dropwise to the system to form microspheres under stirring, shearing and emulsification;

[0041] (d) The emulsified solution is placed in a water bath and stirred to evaporate and remove the dichloromethane, and then the resulting solution is washed and filtered.

[0042] In a specific embodiment, in step (a), preferably, the concentration of the PLA dichloromethane solution is 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.

[0043] Preferably, the PLA comprises a mixture of one or more of poly-L-lactic acid (PLLA), poly-racemic-lactic acid (PDLLA) and poly-D-lactic acid (PDLA).

[0044] Preferably, the molecular weight of the PLA is in the range of 10 KDa to 150 KDa.

[0045] In a specific embodiment, in the step (b), preferably, the mass fraction of hydroxyapatite is 1% to 20% to ensure that it can be completely incorporated into the polylactic acid microspheres.

[0046] Preferably, the ultrasonic energy is 1-300KJ, preferably 1-20KJ, and the ultrasonic energy calculation formula is as follows:

[0047] Ultrasonic energy (KJ) = ultrasonic power (W) × ultrasonic time (s) / 1000.

[0048] In a specific embodiment, in the step (c), preferably, the concentration of the polyvinyl alcohol solution is 5 mg / ml to 30 mg / ml. In this concentration range, the emulsification effect can be ensured and the size of the microspheres can be controlled.

[0049] Preferably, the ratio of the dichloromethane solution to the polyvinyl alcohol solution is 1 / 2-1 / 10.

[0050] Preferably, the rotation speed of the stirring paddle in the stirring device is 100-600 rpm / min. In this rotation speed range, continuous emulsification can be ensured to form a good emulsion.

[0051] In a specific embodiment, in step (d), preferably, the water bath temperature is 20-37°C, and the volatilization rate of dichloromethane can be controlled in this temperature range, thereby optimizing the hollow effect of the microspheres and the surface microporous structure; after step (d), the obtained filter cake is dried to obtain the dual-phase polylactic acid hydroxyapatite nano-dispersed microspheres; preferably, the drying process includes two methods: vacuum drying and freeze drying. Vacuum drying utilizes the decrease in the boiling point of water under near-vacuum conditions to evaporate water at a lower temperature. Freeze drying utilizes the principle of ice crystal sublimation, which avoids the destruction of the internal structure of the microspheres by the liquefaction process of the solid phase components, and can better maintain the spatial structure of the prepared polylactic acid microspheres.

[0052] In one embodiment, the average elastic modulus of the composite gel of the present invention is 400 to 1000 Pa.

[0053] In one embodiment, the elastic modulus loss rate 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 prepared by the preparation method.

[0054] In yet another aspect, the present invention also protects the use of the composite gel in preparing a drug carrier, a tissue filler or a tissue repair material.

[0055] Furthermore, the drug carrier can be a drug carrier of a biological drug, a chemical drug or a naturally extracted drug.

[0056] Beneficial effects:

[0057] (1) The present invention first prepares polylactic acid-inorganic material biphasic microspheres, and then uses solid-phase crosslinking technology to physically electrostatically crosslink or chemically crosslink the carboxyl groups at the ends of the polylactic acid in the biphasic microspheres with hyaluronic acid to form a three-phase composite gel with high stability and good biocompatibility, which can be used for filling and repairing biological tissues.

[0058] (2) The biphasic microspheres of the present invention further improve the hydrophilicity of the polylactic acid microspheres by adding hydrophilic calcium salt inorganic materials (such as hydroxyapatite, calcium phosphate, calcium tartrate, etc.). In addition, the calcium-based inorganic materials in the biphasic microspheres can be introduced into the network structure of the macroscopic cross-linked hyaluronic acid (the particles of the cross-linked hyaluronic acid are also micron-level) with micron-level polylactic acid as a carrier, and the network gaps between the cross-linked hyaluronic acids are filled through the complexation of calcium ions with the carboxyl and hydroxyl groups in the hyaluronic acid, so that the three-phase gel has a tighter network structure; at the same time, with the help of the complexation of the calcium-based inorganic materials and the hyaluronic acid, the distance between the polylactic acid and the hyaluronic acid molecular chains is shortened, thereby improving the cross-linking reaction efficiency of the two, and improving the mechanical strength and thermal stability of the three-phase composite gel.

[0059] (3) The preparation method of the present invention has simple process, convenient operation and is easy to realize industrialization.

[0060] (4) The composite gel prepared by the present invention can exert the instant filling effect of hyaluronic acid, the collagen regeneration effect of polylactic acid, and the bone filling and neutralization effect of calcium-based inorganic materials such as hydroxyapatite, integrating the advantages of the three materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0062] Figure 1 This is a microscope photo of the dual-phase polylactic acid hydroxyapatite nano-dispersed microspheres in Example 1 of the present invention;

[0063] Figure 2 This is a scanning electron microscope photograph of the dual-phase polylactic acid hydroxyapatite nanodispersed microspheres in Example 1 of the present invention;

[0064] Figure 3 This is a partial magnified scanning electron microscope photograph of the dual-phase polylactic acid hydroxyapatite nano-dispersed microspheres in Example 1 of the present invention. DETAILED DESCRIPTION

[0065] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0066] Example 1: Preparation of biphasic microspheres 1

[0067] Weigh 1.8g of polylactic acid and place it in a 50mL beaker, then add 50mL of dichloromethane, stir with a spoon to dissolve it completely, then filter it into a special ultrasonic beaker with a 10mm nylon 66 material syringe filter, then weigh 0.2g of nano hydroxyapatite and add it to the system, 60% power for 10min to fully disperse the nano material in the dichloromethane solution of polylactic acid, during this period, 135mL of polyvinyl alcohol solution (1.5wt%) was added to a 300mL three-necked flask and placed in a 30°C water bath, followed by mechanical stirring at a rate of 400rpm / min. After the ultrasonic wave is finished, the dichloromethane turbid liquid is slowly added to the polyethanol solution, and stirred overnight to volatilize and remove the dichloromethane.

[0068] Add purified water to dilute the turbid solution obtained in the previous step to 500mL, then let it stand for 1h, pour off the upper suspension, repeat twice, then redissolve the precipitate, filter with a 0.45μm water filter membrane, remove the liquid, then disperse the obtained solid with 200mL of purified water, filter again, repeat four times, then redissolve the obtained solid with 150mL of purified water, add it to a Schott bottle, put it in a 100℃ oven and heat for 30min, take it out, wait for it to cool and filter, wash it with purified water three times, transfer the solid obtained from the last wash to a culture dish, put it in a vacuum drying oven at 40℃ and dry it overnight to obtain the final product. The particle size of the product is mainly distributed between 25-86μm. Microscope photos are shown in Figure 1 , SEM photos are shown in Figure 2-3 As can be seen from the figure, the dual-phase microspheres have regular morphology and uniform particle size.

[0069] Example 2: Preparation of biphasic microspheres 2

[0070] Weigh 1.6g of polylactic acid and place it in a 50mL beaker, then add 50mL of dichloromethane, stir with a spoon to dissolve it completely, then filter it into a special ultrasonic beaker with a 10mm nylon 66 material syringe filter, then weigh 0.2g of nano hydroxyapatite and add it to the system, 60% power for 10min to fully disperse the nano material in the dichloromethane solution of polylactic acid, during this period, 90mL of polyvinyl alcohol solution (1.5wt%) was added to a 300mL three-necked flask and placed in a 30°C water bath, followed by mechanical stirring at a rate of 400rpm / min. After the ultrasonic wave is finished, the dichloromethane turbid liquid is slowly added to the polyethanol solution, and stirred overnight to volatilize and remove the dichloromethane.

[0071] The turbid solution obtained in the previous step was diluted to 500mL with purified water, then allowed to stand for 1h, the upper suspension was discarded, repeated twice, and then the precipitate was redissolved, filtered with a 0.45μm water filter membrane, the liquid was removed, and then the solid was dispersed with 200mL of purified water, filtered again, repeated four times, and then the solid was redissolved with 150mL of purified water, added to a Schott bottle, placed in a 100℃ oven for 30min, taken out, cooled and filtered, washed with purified water three times, and the solid obtained from the last washing was transferred to a culture dish and freeze-dried to obtain the final product. The particle size is mainly distributed between 25-72μm.

[0072] Example 3: Physical crosslinking of three-phase gel

[0073] Weigh 1.5g sodium hyaluronate (molecular weight 900KDa), then add 37.5mL of purified water, after complete dissolution, the concentration of hyaluronic acid is 40mg / mL, add 45mg of spermidine to the hyaluronic acid solution. Under the premise of spermidine three-point cross-linking, adjust the pH value of the hyaluronic acid solution to about 6.2 with 6mol / L hydrochloric acid solution, then add and stir well, add 0.214g EDC, add 0.04g NHS and continue to stir evenly, seal and place in a 40℃ blast drying oven for reaction for 14h. After the reaction is completed, add 40mL of anhydrous ethanol, and use IKA T25 high shear disperser to crush the gel particles, the crushing speed is 10000 rpm, and last for 5min. After the crushing is completed, continue to add 200mL of anhydrous ethanol to completely dehydrate the gel. Separate the precipitate, wash it with 200 mL of anhydrous ethanol for 5 times, and then put it into a vacuum oven and dry it at -0.09 MPa at 40 °C for 24 h. After complete drying, take 1.0 g of dry gel, add 50 mL of pH 6.0 PBS with a concentration of 10 mg / mL, and wait for the gel to swell completely before use.

[0074] Take 3.0g of the biphasic microspheres obtained in Example 1, soak them in 50mL of PBS solution with pH 9.0 for surface charge modification, so that the surface of the microspheres carries negative charge, and after soaking for 30 minutes, separate and take out the biphasic microspheres, drain and physically mix them with 50mL of cross-linked hyaluronic acid gel, so that the carboxylate anions on the surface of polylactic acid and the ammonium cations in the cross-linked hyaluronic acid are electrostatically cross-linked. After mixing evenly, fill the three-phase gel into a prefilled syringe, and sterilize it with wet heat at 121°C for 15min to obtain the final product gel.

[0075] Example 4: Chemical crosslinking of three-phase gel

[0076] Take 1.5 g of the biphasic microspheres obtained in Example 1, add 100 mL of purified water, add 2.0 g of ethylenediamine, adjust the pH of the solution to 5.5 using 1 mol / L hydrochloric acid solution, add 2.0 g of EDC and 0.4 g of NHS for surface amino modification reaction, and react at 40°C for 16 h. Filter and separate the microspheres, wash the microspheres with purified water to a pH range of 7.0-7.1, and dry them in a vacuum drying oven at 40°C overnight to obtain biphasic microspheres with surface amino modification.

[0077] Weigh 1.5g sodium hyaluronate, then add 37.5mL of purified water, after complete dissolution, the concentration of hyaluronic acid is 40mg / mL, add Example 2 to the hyaluronic acid solution to obtain 1.0g of biphasic microspheres. Adjust the pH value of the hyaluronic acid solution to about 5.5 with 0.1mol / L hydrochloric acid solution, then add and stir well, add 0.3g EDC and add 0.06g NHS (20% of the mass of EDC) and continue to stir well, seal and place in a 40°C forced drying oven for reaction for 14h. After the reaction is completed, add 200mL of anhydrous ethanol to completely dehydrate the gel. Separate the precipitate, wash the precipitate with 200mL of anhydrous ethanol for 5 times continuously, then put the precipitate into a vacuum oven, and vacuum dry it at a vacuum degree of -0.09MPa at 40°C for 24h. After complete drying, take 1.0 g of the dry gel and add 50 mL of 10 mg / mL pH 7.0 phosphate buffer. After the gel is completely swollen, fill the gel into a prefilled syringe and sterilize it with wet heat at 121°C for 15 min to obtain the final three-phase gel.

[0078] Example 5: Preparation of calcium carbonate dual-phase microspheres

[0079] Weigh 1.6g of polylactic acid and place it in a 50mL beaker, then add 50mL of dichloromethane, stir with a spoon to dissolve it completely, then filter it into a special ultrasonic beaker with a 10mm nylon 66 material syringe filter, then weigh 0.2g of nano calcium carbonate and add it to the system, 60% power for 10min to fully disperse the nano material in the dichloromethane solution of polylactic acid, during this period, 90mL of polyvinyl alcohol solution (1.5wt%) is added to a 300mL three-necked flask and placed in a 30°C water bath, followed by mechanical stirring at a rate of 400rpm / min. After the ultrasonic wave is finished, the dichloromethane turbid liquid is slowly added to the polyethanol solution, and the dichloromethane is removed by stirring overnight and volatilization.

[0080] The turbid solution obtained in the previous step was diluted to 500 mL by adding purified water, and then allowed to stand for 1 h, the upper suspension was poured off, and the process was repeated twice. The precipitate was then redissolved, filtered with a 0.45 μm water filter membrane, the liquid was removed, and the solid was then dispersed with 200 mL of purified water, filtered again, and repeated four times. The solid was then redissolved with 150 mL of purified water, added to a Schott bottle, placed in an oven at 100 ° C and heated for 30 min, taken out, cooled and filtered, and washed three times with purified water. The solid obtained from the last wash was transferred to a culture dish and freeze-dried to obtain the final product.

[0081] Example 6: Preparation of calcium tartrate biphasic microspheres

[0082] Weigh 1.6g of polylactic acid and place it in a 50mL beaker, then add 50mL of dichloromethane, stir with a spoon to dissolve it completely, then filter it into a special ultrasonic beaker with a 10mm nylon 66 material syringe filter, then weigh 0.2g of nano calcium tartrate and add it to the system, 60% power for 10min to fully disperse the nano material in the dichloromethane solution of polylactic acid, during this period, 90mL of polyvinyl alcohol solution (1.5wt%) was added to a 300mL three-necked flask and placed in a 30°C water bath, followed by mechanical stirring at a rate of 400rpm / min. After the ultrasonic wave is finished, the dichloromethane turbid liquid is slowly added to the polyethanol solution, and the dichloromethane is removed by stirring overnight and volatilization.

[0083] The turbid solution obtained in the previous step was diluted to 500 mL by adding purified water, and then allowed to stand for 1 h, the upper suspension was poured off, and the process was repeated twice. The precipitate was then redissolved, filtered with a 0.45 μm water filter membrane, the liquid was removed, and the solid was then dispersed with 200 mL of purified water, filtered again, and repeated four times. The solid was then redissolved with 150 mL of purified water, added to a Schott bottle, placed in an oven at 100 ° C and heated for 30 min, taken out, cooled and filtered, and washed three times with purified water. The solid obtained from the last wash was transferred to a culture dish and freeze-dried to obtain the final product.

[0084] Example 7: Chemical crosslinking of calcium carbonate three-phase gel

[0085] Take 1.5 g of the biphasic microspheres obtained in Example 5, add 100 mL of purified water, add 2.0 g of ethylenediamine, adjust the pH of the solution to 5.5 using 1 mol / L hydrochloric acid solution, add 2.0 g of EDC and 0.4 g of NHS for surface amino modification reaction, and react at 40°C for 16 h. Filter and separate the microspheres, wash the microspheres with purified water to a pH range of 7.0-7.1, and dry them in a vacuum drying oven at 40°C overnight to obtain biphasic microspheres with surface amino modification.

[0086] Weigh 1.5g sodium hyaluronate, then add 37.5mL of purified water, and after complete dissolution, the concentration of hyaluronic acid is 40mg / mL. Add 1.0g of biphasic microspheres to the hyaluronic acid solution. Adjust the pH value of the hyaluronic acid solution to about 5.5 with 0.1mol / L hydrochloric acid solution, then add and stir well, add 0.3g EDC and add 0.06g NHS (20% of the mass of EDC) and continue to stir evenly, seal and place in a 40℃ forced air drying oven to react for 14h. After the reaction is completed, add 200mL of anhydrous ethanol to completely dehydrate the gel. Separate the precipitate, wash the precipitate with 200mL of anhydrous ethanol for 5 times continuously, then put the precipitate into a vacuum oven, and vacuum dry it at -0.09MPa at 40℃ for 24h. After complete drying, take 1.0 g of the dry gel and add 50 mL of 10 mg / mL pH 7.0 phosphate buffer. After the gel is completely swollen, fill the gel into a prefilled syringe and sterilize it with wet heat at 121°C for 15 min to obtain the final three-phase gel.

[0087] Example 8: Chemical crosslinking of calcium tartrate three-phase gel

[0088] Take 1.5 g of the biphasic microspheres obtained in Example 6, add 100 mL of purified water, add 2.0 g of ethylenediamine, adjust the pH of the solution to 5.5 using 1 mol / L hydrochloric acid solution, add 2.0 g of EDC and 0.4 g of NHS for surface amino modification reaction, and react at 40°C for 16 h. Filter and separate the microspheres, wash the microspheres with purified water to a pH range of 7.0-7.1, and dry them in a vacuum drying oven at 40°C overnight to obtain biphasic microspheres with surface amino modification.

[0089] Weigh 1.5g sodium hyaluronate, then add 37.5mL of purified water, and after complete dissolution, the concentration of hyaluronic acid is 40mg / mL. Add 1.0g of biphasic microspheres to the hyaluronic acid solution. Adjust the pH value of the hyaluronic acid solution to about 5.5 with 0.1mol / L hydrochloric acid solution, then add and stir well, add 0.3g EDC and add 0.06g NHS (20% of the mass of EDC) and continue to stir evenly, seal and place in a 40℃ forced air drying oven to react for 14h. After the reaction is completed, add 200mL of anhydrous ethanol to completely dehydrate the gel. Separate the precipitate, wash the precipitate with 200mL of anhydrous ethanol for 5 times continuously, then put the precipitate into a vacuum oven, and vacuum dry it at -0.09MPa at 40℃ for 24h. After complete drying, take 1.0 g of the dry gel and add 50 mL of 10 mg / mL pH 7.0 phosphate buffer. After the gel is completely swollen, fill the gel into a prefilled syringe and sterilize it with wet heat at 121°C for 15 min to obtain the final three-phase gel.

[0090] Comparative Example 1. Preparation of non-crosslinked three-phase gel

[0091] Weigh 1.5g of sodium hyaluronate, then add 37.5mL of purified water, and after complete dissolution, the concentration of hyaluronic acid is 40mg / mL. Add 1.0g of the biphasic microspheres obtained in Example 2 to the hyaluronic acid solution and stir evenly. After stirring, add 200mL of anhydrous ethanol to completely dehydrate the gel. Separate the precipitate, wash the precipitate with 200mL of anhydrous ethanol for 5 consecutive times, and then put the precipitate into a vacuum oven and vacuum dry it at a vacuum degree of -0.09MPa at 40°C for 24h. After complete drying, take 1.0g of dry gel, add 50mL of pH7.0 phosphate buffer at a concentration of 10mg / mL, and after the gel is completely swollen, fill the gel into a prefilled syringe and sterilize it with wet heat at 121°C for 15min to obtain a non-crosslinked three-phase gel.

[0092] Comparative Example 2. Preparation of three-phase non-physical crosslinked blended gel

[0093] The preparation method of spermidine cross-linked hyaluronic acid gel is the same as that in Example 3, and the overall preparation method is completely consistent.

[0094] 3.0 g of the two-phase microspheres obtained in Example 1 were taken and directly physically mixed with 50 mL of the cross-linked hyaluronic acid gel. After uniform mixing, the three-phase gel was filled into a prefilled syringe and sterilized by wet heat at 121° C. for 15 min to obtain the final product gel.

[0095] Comparative Example 3. Preparation of single-phase PLA microspheres and cross-linked hyaluronic acid physical blend gel

[0096] Weigh 1.6g of polylactic acid and place it in a 50mL beaker, then add 50mL of dichloromethane, stir with a spatula to completely dissolve it, and then filter it into a special ultrasonic beaker with a 10mm nylon 66 syringe filter. During this period, add 90mL of polyvinyl alcohol solution (1.5wt) into a 300mL three-necked flask and place it in a 30℃ water bath, then stir it mechanically at a rate of 400rpm / min. Then slowly add the dichloromethane solution to the polyethanol solution and stir overnight to evaporate and remove the dichloromethane. The turbid solution obtained in the previous step was diluted to 500 mL by adding purified water, and then allowed to stand for 1 h, the upper suspension was poured off, and the process was repeated twice. The precipitate was then redissolved, filtered with a 0.45 μm water filter membrane, the liquid was removed, and the solid was then dispersed with 200 mL of purified water, filtered again, and repeated four times. The solid was then redissolved with 150 mL of purified water, added to a Schott bottle, placed in an oven at 100 ° C and heated for 30 min, taken out, cooled and filtered, and washed three times with purified water. The solid obtained from the last wash was transferred to a culture dish and freeze-dried to obtain the final product, PLA microspheres.

[0097] 3.0 g of the obtained PLA microspheres were taken and soaked in 50 mL of a pH 9.0 PBS solution for surface charge modification, so that the surface of the microspheres carried negative charges. After soaking for 30 minutes, the microspheres were separated and taken out, and after being drained, they were physically mixed with 50 mL of the cross-linked hyaluronic acid gel obtained in Example 3, so that the carboxylate anions on the surface of the polylactic acid and the ammonium cations in the cross-linked hyaluronic acid were electrostatically cross-linked. After uniform mixing, the blended gel was filled into a prefilled syringe and sterilized with wet heat at 121° C. for 15 min to obtain the final product gel.

[0098] Comparative Example 4. Preparation of Physically Blended Gel of Nanoscale HAP and Cross-linked Hyaluronic Acid

[0099] The preparation method of spermidine cross-linked hyaluronic acid gel is the same as that in Example 3, and the overall preparation method is completely consistent.

[0100] Take 3.0g of nano-HAP and mix it directly with 50mL of cross-linked hyaluronic acid gel. After mixing evenly, fill the blended gel into a pre-filled syringe and sterilize it with wet heat at 121°C for 15min to obtain the final product gel.

[0101] Effect example:

[0102] Rheological properties of three-phase gel

[0103] The three-phase gel obtained in the examples and comparative examples was divided into two types, before sterilization and after sterilization, and 2.0 mL was taken out from each type. The elastic modulus (G') of the gel was measured using a TA DHR-2 plate rheometer, and the elastic modulus loss rate was calculated. The G' loss rate was calculated by the following formula:

[0104] G' loss rate = (G' before sterilization - G' after sterilization) / G' before sterilization

[0105] The rheometer parameters are: operating gap: 1000 μm, loading gap: 45000 μm, operating temperature: 37°C, deformation: 1%, frequency: 0.9 Hz, and operating time: 60 s. The rheological data of each gel are shown in Table 1:

[0106] Table 1. Rheological data of three-phase gel

[0107] Example Before sterilization G'(Pa) After sterilization G'(Pa) G' loss rate Example 3 473 344 27.3％ Example 4 867 698 19.5％ Example 7 813 615 24.4％ Example 8 837 618 26.2％ Comparative Example 1 372 10 97.3％ Comparative Example 2 403 256 36.5％ Comparative Example 3 275 166 39.6％ Comparative Example 4 266 153 42.5％

[0108] It can be seen from the data in Table 1 that the three-phase gel obtained by physical crosslinking in Example 3 has an improved elastic modulus and a certain improvement in thermal stability compared to the three-phase non-physical crosslinked blended gel in Comparative Example 2, indicating that physical crosslinking has a certain contribution to the improvement of the elastic modulus of the composite gel and also improves the thermal stability of the three-phase gel to a certain extent. However, since Comparative Example 1 is just a physical mixture of non-crosslinked HA, PLA, and HAP, there is no chemical bond between the three substances, and they cannot resist the violent hydrolysis during wet heat sterilization, resulting in a decrease in the elastic modulus of Comparative Example 1 of more than 97% after sterilization.

[0109] Although PLA and HA are physically cross-linked in Comparative Example 3, HAP is not added, and the elastic modulus of the composite gel is lower than that of Example 3, and the heat loss rate is high. This shows that after HAP forms composite microspheres with PLA, nano-HAP is introduced into the network structure of macroscopic cross-linked hyaluronic acid (the particles of cross-linked hyaluronic acid are also micron-level) with micron-level PLA as a carrier, and the network gaps between the cross-linked hyaluronic acids are filled through the complexation of calcium ions with the carboxyl and hydroxyl groups in the hyaluronic acid, so that the three-phase gel has a tighter network structure; at the same time, with the help of the complexation of HAP and HA, the distance between the PLA and HA molecular chains is shortened, thereby improving the cross-linking reaction efficiency of the two, and thus having a higher elastic modulus and stronger thermal stability.

[0110] Comparative Example 4 discloses that nano-scale HAP is physically blended with cross-linked hyaluronic acid, but does not show higher elastic modulus and stronger thermal stability. This is because Comparative Example 4 is directly added with nano-scale HAP, compared with micron-scale cross-linked hyaluronic acid gel, the physical size difference between the two is large, resulting in a large spatial difference between the two, and HAP is difficult to fill the gap of cross-linked hyaluronic acid gel, so that a tighter network structure cannot be formed. This also further illustrates that by forming a biphasic microsphere with HAP and PLA, HAP is loaded on the micron-scale carrier of PLA, and a tighter network structure can be further formed with the cross-linked hyaluronic acid gel, so as to have a higher elastic modulus and stronger thermal stability. Therefore, the composite gel is the mutual synergy between cross-linked hyaluronic acid, PLA-HAP biphasic microspheres, and the elastic modulus and thermal stability in the three-phase gel can be improved, and the three components in the three-phase gel are indispensable.

[0111] The elastic modulus of the three-phase composite gel obtained by chemical crosslinking in Examples 4, 7, and 8 is significantly improved, indicating that when physical crosslinking is replaced by chemical crosslinking, the interaction between crosslinked hyaluronic acid, HAP (or other nanoscale calcium salts) and PLA is further enhanced because the bond strength formed by chemical crosslinking is much higher than the interaction strength of physical crosslinking. Compared with the physically crosslinked three-phase gel, the elastic modulus and thermal stability of the chemically crosslinked three-phase gel are further improved.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite gel, characterized in that: The biphasic microspheres are prepared by cross-linking biphasic microspheres with hyaluronic acid, wherein the biphasic microspheres are polylactic acid / inorganic material biphasic microspheres; The cross-linking includes cross-linking of polylactic acid and hyaluronic acid in the dual-phase microspheres; The cross-linking of the polylactic acid and the hyaluronic acid includes any one of physical electrostatic cross-linking and chemical cross-linking; The physical electrostatic crosslinking includes modifying the surface charge of the biphasic microspheres to obtain modified biphasic microspheres with negative ions on the surface, and mixing the modified biphasic microspheres with crosslinked hyaluronic acid having positive ions to generate electrostatic crosslinking; The cross-linked hyaluronic acid with positive ions is cross-linked hyaluronic acid obtained by cross-linking with endogenous polyamines; The chemical cross-linking comprises modifying the polylactic acid in the biphasic microspheres with polyamines or polyamino compounds, and reacting the modified biphasic microspheres with hyaluronic acid to form amide bond cross-linking; The inorganic material is selected from one or more of calcium carbonate, calcium phosphate or calcium tartrate.

2. The composite gel according to claim 1, characterized in that The calcium phosphate is hydroxyapatite.

3. The composite gel according to claim 1 or 2, characterized in that: The inorganic material is a nano inorganic material.

4. The composite gel according to claim 1, characterized in that The endogenous polyamine is spermine or spermidine.

5. A method for preparing a composite gel, characterized in that: The method comprises cross-linking a biphasic microsphere with hyaluronic acid to form a composite gel, wherein the biphasic microsphere is a polylactic acid / inorganic material biphasic microsphere, and the cross-linking comprises cross-linking of the polylactic acid and the hyaluronic acid in the biphasic microsphere; The crosslinking includes any one of physical electrostatic crosslinking and chemical crosslinking; The chemical cross-linking comprises modifying the polylactic acid in the biphasic microspheres with polyamines or polyamino compounds, and reacting the modified biphasic microspheres with hyaluronic acid to form amide bond cross-linking; The physical electrostatic crosslinking includes modifying the surface charge of the biphasic microspheres to obtain modified biphasic microspheres with negative ions on the surface, and mixing the modified biphasic microspheres with crosslinked hyaluronic acid having positive ions to generate electrostatic crosslinking; The cross-linked hyaluronic acid with positive ions is cross-linked hyaluronic acid obtained by cross-linking with endogenous polyamines; The inorganic material is selected from one or more of calcium carbonate, calcium phosphate or calcium tartrate.

6. The preparation method according to claim 5, characterized in that: The amide bond cross-linking comprises coupling the modified biphasic microspheres with hyaluronic acid in the presence of a carbodiimide activator.

7. The preparation method according to claim 5, characterized in that: The polyamine includes any one of aliphatic polyamine, aromatic polyamine and heteroatom polyamine; the polyamino compound includes any one of double-terminated aminated polyethylene glycol and terminally aminated multi-arm polyethylene glycol.

8. The preparation method according to claim 6, characterized in that: The carbodiimide activator includes 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[bis(methoxymethyl)methyl]carbodiimide or salts thereof.

9. The preparation method according to claim 5, characterized in that: The mass ratio of the biphasic microspheres to the hyaluronic acid in the chemical crosslinking is 1:0.5-20.

10. The preparation method according to claim 9, characterized in that: The mass ratio of the biphasic microspheres to the hyaluronic acid in the chemical crosslinking is 1:1-10.

11. The preparation method according to claim 5, characterized in that: The added amount of the polyamine or polyamino compound is 30-200% of the mass of the biphasic microspheres; the concentration range of the amine compound is 10-1000 mg / mL.

12. The preparation method according to claim 6, characterized in that: The added amount of the carbodiimide activator is 10-150% of the total mass of the hyaluronic acid.

13. The preparation method according to claim 5, characterized in that: The temperature of the amino modification reaction and the amide bond cross-linking reaction is 5-60° C., and the reaction time is 6-24 hours.

14. The preparation method according to claim 5, characterized in that: The surface charge modification of the biphasic microspheres comprises immersing the biphasic microspheres in an alkaline buffer solution and separating them.

15. The preparation method according to claim 5, characterized in that: The mass ratio of the biphasic microspheres to the cross-linked hyaluronic acid in the physical electrostatic cross-linking is 1:1-50.

16. The preparation method according to claim 15, characterized in that: The mass ratio of the biphasic microspheres to the cross-linked hyaluronic acid in the physical electrostatic cross-linking is 1:1-20.

17. The preparation method according to claim 14, characterized in that: The alkaline buffer is an alkaline phosphate buffer.

18. The preparation method according to claim 17, characterized in that: The pH range of the alkaline phosphate buffer is 9.0-11.

0.

19. Use of the composite gel according to any one of claims 1 to 4 or the composite gel prepared by the preparation method according to any one of claims 5 to 18 in preparing drug carriers, fillers or tissue repair materials.

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