Composite hydrogel as well as preparation method and application thereof

Abstract

The invention discloses composite hydrogel as well as a preparation method and application thereof. The composite hydrogel comprises a hydrogel matrix and a liposome loaded on the hydrogel matrix, the hydrogel matrix comprises a methacrylated natural protein, and the liposome is loaded with a drug; the medicine comprises a medicine for promoting cartilage differentiation and an anti-inflammatory traditional Chinese medicine monomer. According to the invention, through a liposome entrapment technology, the stability and sustained release ability of the insoluble traditional Chinese medicine monomer and the cartilage differentiation induction drug are enhanced. The particle size of the liposome is controllable, the liposome is well combined with a methacrylated natural protein network, a drug sustained release bin can be formed in the hydrogel, long-time and local precise release is achieved, the drug utilization efficiency is improved, and systematic toxic and side effects are avoided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a composite hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Cartilage tissue is difficult to self-repair, and the commonly used cartilage repair methods in clinic include bone marrow stimulation technology, autologous cartilage transplantation, etc., but these methods have problems such as limited donor source, unstable repair effect, and many postoperative complications. Therefore, developing new cartilage repair materials has become a research hotspot. Hydrogel is widely used in cartilage tissue engineering due to its similar high water content, good flexibility and controllable three-dimensional microenvironment to cartilage tissue. Silk fibroin (SF) is a natural polymer material with wide source and excellent biocompatibility. After methacrylation modification, silk fibroin introduces a double bond photosensitive group, which can be quickly photo-crosslinked to form a three-dimensional scaffold with controllable mechanical properties and shape plasticity under mild conditions, which is suitable for filling and supporting the cartilage defect site.

[0003] In the process of cartilage repair, cartilage differentiation and immune regulation are crucial. Drugs such as TGF-β1 and KGN (Kartogenin) can promote mesenchymal stem cells to differentiate into chondrocytes and enhance cartilage matrix synthesis. In addition, early inflammation and vascular ingrowth will lead to chondrocyte hypertrophy and calcification, which is not conducive to cartilage regeneration, and it is necessary to introduce anti-inflammatory drugs such as traditional Chinese medicine monomers at an early stage, but such monomers usually have poor water solubility, low bioavailability in vivo, are easily diffused or degraded, and have insufficient local maintenance time. The drug release amount is also low when loaded with nanoparticles such as silicon dioxide. Bioactive factors and polypeptides that induce cartilage differentiation have poor stability and difficult scaling-up of loading technology. SUMMARY

[0004] The present application aims to at least solve one of the above technical problems in the prior art. To this end, the purpose of the present application is to provide a composite hydrogel and a preparation method and application thereof.

[0005] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is: The first aspect of the present application provides a composite hydrogel, comprising a hydrogel matrix and a liposome loaded on the hydrogel matrix; the hydrogel matrix comprises a methacrylated natural protein, and the liposome is loaded with a drug; the drug comprises a pro-chondrogenic drug and an anti-inflammatory traditional Chinese medicine monomer.

[0006] In the present application, the methacrylated natural protein as a hydrogel skeleton has excellent mechanical properties and photo-crosslinking forming ability, can provide a three-dimensional scaffold with certain rigidity and elasticity, can provide mechanical support and low-loss drug loading, is beneficial to cell adhesion and nutrient exchange, is suitable for shape matching and in-situ injection forming repair of cartilage defect sites; through liposome loading of drugs, the stability and sustained release ability of poorly soluble drugs such as traditional Chinese medicine monomers can be enhanced, the liposome particle size is controllable, and the liposome can be well dispersed in the hydrogel matrix, not only realizing high drug loading of the composite hydrogel, but also forming a drug sustained-release warehouse in the hydrogel, realizing long-term and local precise release, improving drug utilization efficiency, and avoiding systemic toxic side effects; and the combination of chondrogenic differentiation drugs and anti-inflammatory traditional Chinese medicine monomers can create a good immune microenvironment for tissue repair, chondrogenic differentiation drugs can induce mesenchymal stem cells to differentiate into chondrocytes, promote the synthesis of type II collagen and proteoglycan, and the combination of the two can accelerate cartilage tissue regeneration.

[0007] In some embodiments, the mass ratio of the hydrogel matrix to the liposome in the composite hydrogel is (0.5-30):(0.01-10); such as (0.5-10):(0.05-8), (1-25):(0.05-8), (5-20):(0.1-5), (5-18):(0.1-2), etc. In the present application, a small proportion of hydrogel matrix in the composite hydrogel can achieve high liposome loading, ensure high drug loading, and be beneficial to ensuring the effective concentration of subsequent long-acting drug release, and realizing long-acting and effective treatment effect.

[0008] In some embodiments, the liposome comprises a first liposome and a second liposome, the first liposome loaded with chondrogenic differentiation drugs; and the second liposome loaded with anti-inflammatory traditional Chinese medicine monomers. In the present application, the chondrogenic differentiation drugs and the anti-inflammatory traditional Chinese medicine monomers are loaded in different liposomes, which can effectively improve the drug loading rate; and can precisely control the amount and release trend of the two, so that they can realize different release behaviors in the body.

[0009] In some embodiments, the drug is embedded in the double-layer membrane of the liposome.

[0010] In some embodiments, the drug is wrapped in the liposome core.

[0011] In some embodiments, the drug is loaded in the liposome by electrostatic adsorption and / or chemical modification.

[0012] In some embodiments, the drug loading rate in the liposome is at least 70%; such as 70%-99%, 75%-98%, 80%-97%, 85%-95%, etc. In the present application, the drug loading rate = drug content in the liposome / drug feed amount x 100%.

[0013] In some embodiments, the drug accounts for 0.01%-30% in the liposome; such as 1%-10%, 3%-8%.

[0014] In some embodiments, the mass ratio of the chondrogenesis-promoting drug and the anti-inflammatory Chinese medicine monomer is (0.0005-1):(0.01-10), such as (0.01-1):(0.1-5), (0.1-0.8):(0.1-2), (0.3-0.7):(0.5-1.5). In the present application, the amount of the two is controlled within the range, which can achieve early release of more anti-inflammatory Chinese medicine monomers to inhibit inflammation, and long-term and local precise release of chondrogenesis-promoting drugs and anti-inflammatory Chinese medicine monomers to promote stem cell chondrogenesis, thereby improving drug utilization efficiency.

[0015] In some embodiments, the average particle size of the liposome is 10-200nm, such as 50-150nm, etc.

[0016] In some embodiments, the natural protein includes at least one of silk fibroin, sericin and collagen.

[0017] In some embodiments, the methacrylated natural protein includes natural protein modified by at least one of methacrylic acid, methacrylic anhydride and glycidyl methacrylate.

[0018] In some embodiments, the chondrogenesis-promoting drug includes at least one of TGF-β1, TGF-β1 derived polypeptide and KGN.

[0019] In some embodiments, the anti-inflammatory Chinese medicine monomer includes at least one of ginsenoside, quercetin and resveratrol.

[0020] In some embodiments, the anti-inflammatory Chinese medicine monomer includes ginsenoside Rg3; in the present application, ginsenoside Rg3 can inhibit angiogenesis and promote macrophages to polarize to M2 type, thereby creating a good immune microenvironment for tissue repair.

[0021] In some embodiments, the liposome includes cholesterol and lecithin.

[0022] In a second aspect of the present application, a preparation method of the composite hydrogel is provided, which includes the following steps: The liposome is mixed with the methacrylated natural protein, and then subjected to photo-crosslinking reaction to prepare the composite hydrogel.

[0023] In some embodiments, the method for preparing the liposome comprises the following steps: mixing the drug, cholesterol, lecithin and anhydrous ethanol, drying by evaporation, hydrating, and preparing the liposome by ultrasonic treatment.

[0024] In some embodiments, the hydrating time is 1-3h.

[0025] In some embodiments, the photo-crosslinking reaction is performed under irradiation of light at 360-450nm (e.g. 365-405nm); the reaction time of the photo-crosslinking reaction is 5s-40min; e.g. 5s-90s.

[0026] In some embodiments, the photo-crosslinking reaction is performed under a photo-crosslinking agent; the photo-crosslinking agent comprises at least one of LAP, I2959, and Eosin Y.

[0027] In a third aspect, the application provides use of the composite hydrogel in preparation of a bone repair material.

[0028] In some embodiments, the bone repair comprises repair of cartilage tissue defects.

[0029] In some embodiments, the composite hydrogel induces cartilage regeneration.

[0030] In some embodiments, the composite hydrogel promotes chondrogenic differentiation of stem cells.

[0031] In some embodiments, the composite hydrogel promotes chondrogenic differentiation of stem cells by expression of COL2A1 gene and / or Sox9 gene.

[0032] In some embodiments, the composite hydrogel induces cartilage regeneration by inhibiting expression of M1 macrophage proteins (e.g. CD86 and / or CD11b), and / or promoting expression of M2 macrophage proteins (e.g. CD206 and / or Arg-1).

[0033] The application has the following advantages: 1) The application uses modified natural high molecular material, such as methacrylated natural protein (e.g. glycidyl methacrylate silk fibroin (SilMA)) as the hydrogel framework, which has excellent mechanical properties and photo-crosslinking forming ability, can quickly solidify to form a three-dimensional scaffold with certain rigidity and elasticity, can provide mechanical support and low-loss drug loading, is conducive to cell adhesion and nutrient exchange, and is suitable for shape matching and in-situ injection forming repair of cartilage defect sites.

[0034] 2) The liposomes of traditional Chinese medicine monomers and the liposomes of cartilage induction drugs in the hydrogel can respectively exert the dual effects of immune regulation and chondrogenic induction. Ginsenoside Rg3 can create a good immune microenvironment for tissue repair by inhibiting angiogenesis and promoting the polarization of macrophages to M2 type; KGN and other chondrogenic differentiation drugs can induce mesenchymal stem cells to differentiate into chondrocytes and promote the synthesis of type II collagen and proteoglycan, thereby accelerating the regeneration of cartilage tissue.

[0035] 3) The stability and sustained release capacity of the liposome encapsulation technology are enhanced for the poorly soluble traditional Chinese medicine monomers such as ginsenoside Rg3 and the cartilage differentiation induction drugs. The liposome particle size is controllable, and the liposome is well combined with the methacrylated natural protein such as SilMA network, so that the drug release warehouse can be formed in the hydrogel, long-term and local precise release can be realized, the drug utilization efficiency is improved, and the systemic toxic side effects are avoided.

[0036] 4) Compared with the prior art, the methacrylated natural protein such as SilMA and the double-drug liposome are used for cartilage repair for the first time. Through the controlled release mechanism of the liposome, the spatiotemporal distribution of the poorly soluble traditional Chinese medicine monomers and the cartilage induction drugs is optimized: the traditional Chinese medicine monomers exert anti-inflammatory effect in the early stage, and the sustained release of the cartilage induction drugs induces chondrogenic differentiation, thereby improving the efficiency of cartilage repair.

[0037] 5) The preparation process of the composite hydrogel of the present application is simple, the components are safe, and all raw materials can be removed through biodegradation and metabolism, which is beneficial to clinical transformation application. The system has multiple functions such as injectability, photocurable molding, drug release control and promotion of cartilage regeneration, and can be widely used in cartilage injury, joint degeneration and postoperative cartilage repair scenes, and has good clinical prospects and industrialization value. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Figure 1 is a scanning electron microscope image of the blank liposome, the ginsenoside Rg3 liposome and the KGN liposome from left to right in the embodiment 2 and the embodiment 1 of the present application.

[0039] Figure 2 Figure 2 is a drug-loaded liposome particle size and stability effect diagram in the embodiment 1 of the present application, wherein A is the particle size distribution diagram of Lipo@Rg3; B is the particle size diagram of Lipo@Rg3 and Lipo@KGN at different times; C is the particle size distribution diagram of Lipo@KGN; D is the Zeta potential of Lipo@Rg3 and Lipo@KGN.

[0040] Figure 3 Figure 3 is a real object diagram of the drug-loaded liposome composite hydrogel before crosslinking, after crosslinking, side up and upside down from left to right in the embodiment 1 of the present application.

[0041] Figure 4 Release profile of the drug-loaded liposome-composite hydrogel in Example 1 and Comparative Example 5 of the present application.

[0042] Figure 5 Cell compatibility test profile of the drug-loaded liposome-composite hydrogel in Example 1 of the present application.

[0043] Figure 6 Gene expression profile of the drug-loaded liposome-composite hydrogel in Example 1 of the present application to induce stem cells to chondrocytes.

[0044] Figure 7 Macrophage immune regulation profile of the drug-loaded liposome-composite hydrogel in Example 1 of the present application. DETAILED DESCRIPTION

[0045] The present application will be further described in the following with specific examples. The raw materials, reagents or devices used in the examples and comparative examples are commercially available or can be obtained by prior art methods unless otherwise specified. The test or test method is the conventional method in the art unless otherwise specified.

[0046] In the present text, "room temperature" means 25°C.

[0047] Liposome drug encapsulation rate (=loading rate) test method: 1 mL of the drug-loaded liposome solution was added into a 10000 Da ultrafiltration tube, centrifuged at 4°C, 7500 rpm for 30 min, the upper liposome was collected, methanol was added to break the emulsion to 10 mL, filtered through a 0.22 μm microporous filter, and the amount of drug encapsulated by the liposome was determined by HPLC; another 1 mL of the drug-loaded liposome solution was added with methanol to 10 mL to break the emulsion, filtered through a 0.22 μm microporous filter, and the total amount of drug sample was determined by HPLC; the liposome drug encapsulation rate = the amount of drug encapsulated by the liposome / the total amount of drug sample × 100%. Hydrogel drug encapsulation rate (=loading rate) test method: 0.2 cm 3 The drug-loaded hydrogel was broken in 1 mL PBS, centrifuged at 4°C, 7500 rpm for 3 min, the upper liposome was collected, methanol was added to break the emulsion to 10 mL, filtered through a 0.22 μm microporous filter, and the amount of drug encapsulated by the hydrogel was determined by HPLC; another 0.2 mL of the drug-loaded hydrogel precursor solution and 0.8 mL of PBS were mixed, methanol was added to 10 mL to break the emulsion, filtered through a 0.22 μm microporous filter, and the total amount of drug sample was determined by HPLC; the hydrogel drug encapsulation rate = the amount of drug encapsulated by the hydrogel / the total amount of drug sample × 100%.

[0048] Example 1 A composite hydrogel is prepared in this example, and the specific process is as follows: (1) Dissolve silk fibroin in lithium bromide solution, then drop glycidyl methacrylate, 60°C warm bath reaction for 2h, then drop sodium hydroxide solution to continue reaction for 1h, to get glycidyl methacrylate silk fibroin (SilMA); (2) Mix 6.25 mg Rg3, 12.5 mg cholesterol, 62.5 mg lecithin with 15 mL anhydrous ethanol, evaporate to form a transparent film on the rotary evaporator, vacuum dry, add 5 mL PBS hydration for 2h, and ultrasonic to get Rg3 liposome (Lipo@Rg3), the encapsulation efficiency of Rg3 is 87.0%; (3) Mix 6.25 mg KGN, 12.5 mg cholesterol, 62.5 mg lecithin with 15 mL anhydrous ethanol, evaporate to form a transparent film on the rotary evaporator, vacuum dry, add 10 mL PBS hydration for 2h, and ultrasonic to get KGN liposome (Lipo@KGN), the encapsulation efficiency of KGN is 92.9%; (4) Dissolve SilMA in PBS, then add 4 mg / mL Rg3 liposome and 2 mg / mL KGN liposome to the SilMA solution, mix well, then drop LAP under stirring; then inject the mixed solution into a transparent glass mold, irradiate under 405 nm light for 50s, to get drug-loaded liposome composite hydrogel (denoted as SilMA@LKR). The concentration of SilMA is 80 mg / mL, the encapsulation efficiency of Rg3 liposome in SilMA is higher than 95%, and the encapsulation efficiency of KGN liposome in SilMA is higher than 96%.

[0049] Example 2 A composite hydrogel is prepared in this example, and the specific process is as follows: (1) Dissolve silk fibroin in lithium bromide solution, then drop glycidyl methacrylate, 60°C warm bath reaction for 2h, then drop sodium hydroxide solution to continue reaction for 1h, to get glycidyl methacrylate silk fibroin (SilMA); (2) Mix 6.25 mg Rg3, 12.5 mg cholesterol, 62.5 mg lecithin with 15 mL anhydrous ethanol, evaporate to form a transparent film on the rotary evaporator, vacuum dry, add 5 mL PBS hydration for 2h, and ultrasonic to get Rg3 liposome (Lipo@Rg3), the encapsulation efficiency of Rg3 is 87.0%; (3) 12.5 mg of cholesterol, 62.5 mg of lecithin and 15 mL of anhydrous ethanol were mixed, evaporated to form a transparent film on a rotary evaporator, vacuum dried, hydrated with 10 mL of PBS for 2 h, and broken by ultrasonic to obtain blank liposomes (Lipo); then 50 μg of TGF-β1 was added to the Lipo solution, and the mixture was gently shaken in the dark for 4 h, and then stood for 3 h to obtain TGF-β1 liposomes (Lipo@TGF-β1), and the encapsulation rate of TGF-β1 was 76.7%; (4) SerMA was dissolved in PBS, and then 5 mg / mL of Rg3 liposomes and 0.5 mg / mL of TGF-β1 liposomes were added to the SerMA solution, which was mixed uniformly, and then LAP was added dropwise under stirring; then the mixture was injected into a transparent glass mold, and irradiated under 405 nm light for 50 s to obtain a drug-loaded liposome composite hydrogel (denoted as SerMA@LRT). The concentration of SerMA was 40 mg / mL, the encapsulation rate of Rg3 liposomes in SilMA was higher than 96%, and the encapsulation rate of TGF-β1 liposomes in SilMA was higher than 97%.

[0050] Example 3 In this example, a composite hydrogel was prepared, and the specific process was as follows: (1) Fibroin was dissolved in a lithium bromide solution, and then glycidyl methacrylate was added dropwise, and the mixture was reacted at 60°C for 2 h, and then sodium hydroxide solution was added dropwise and the reaction was continued for 1 h to obtain glycidyl methacrylate-modified fibroin (SilMA); (2) 4.167 mg of Rg3, 2.083 mg of KGN, 12.5 mg of cholesterol, 62.5 mg of lecithin and 15 mL of anhydrous ethanol were mixed, evaporated to form a transparent film on a rotary evaporator, vacuum dried, hydrated with 3.33 mL of PBS for 2 h, and then ultrasonically broken to obtain Rg3 / KGN liposomes (Lipo@Rg3 / KGN), and the encapsulation rates of Rg3 and KGN were 70.2% and 70.7%, respectively; (4) SilMA was dissolved in PBS, and then 6 mg / mL of Lipo@Rg3 / KGN was added to the SilMA solution, which was mixed uniformly, and then LAP was added dropwise under stirring; then the mixture was injected into a transparent glass mold, and irradiated under 405 nm light for 50 s to obtain a drug-loaded liposome composite hydrogel (denoted as SilMA@LK / R). The concentration of SilMA was 80 mg / mL, and the encapsulation rate of Lipo@Rg3 / KGN in SilMA was higher than 95%.

[0051] Comparative Example 1 In this comparative example, a composite hydrogel was prepared, and the specific process was as follows: (1) Dissolve silk fibroin in lithium bromide solution, then drop glycidyl methacrylate, warm bath reaction for 2h, then drop sodium hydroxide solution to continue reaction for 1h, to obtain glycidyl methacrylate silk fibroin (SilMA); (2) Dissolve SilMA in PBS, drop LAP under stirring, mix uniformly, then inject into transparent glass mold, irradiate under 405nm light for 50s, to obtain composite hydrogel (recorded as SilMA). The concentration of SilMA is 80mg / mL.

[0052] Comparative Example 2 This comparative example prepared a composite hydrogel, the specific process is as follows: (1) Dissolve silk fibroin in lithium bromide solution, then drop glycidyl methacrylate, warm bath reaction for 2h, then drop sodium hydroxide solution to continue reaction for 1h, to obtain glycidyl methacrylate silk fibroin (SilMA); (2) Mix KGN, cholesterol, lecithin with anhydrous ethanol, evaporate to form transparent film on rotary evaporator, vacuum dry, hydrate for 2h, ultrasonic crushing to obtain KGN liposome (Lipo@KGN), the encapsulation efficiency of KGN is 92.9%; (3) Dissolve SilMA in PBS, then add 2mg / mL KGN liposome to the aqueous solution of SilMA; mix uniformly, then drop LAP under stirring; then inject the mixture into transparent glass mold, irradiate under 405nm light for 50s, to obtain composite hydrogel (recorded as SilMA@LK). The concentration of SilMA is 80mg / mL, the encapsulation efficiency of KGN liposome in SilMA is higher than 98%.

[0053] Comparative Example 3 This comparative example prepared a composite hydrogel, the specific process is as follows: (1) Dissolve silk fibroin in lithium bromide solution, then drop glycidyl methacrylate, warm bath reaction for 2h, then drop sodium hydroxide solution to continue reaction for 1h, to obtain glycidyl methacrylate silk fibroin (SilMA); (2) Mix Rg3, cholesterol, lecithin with anhydrous ethanol, evaporate to form transparent film on rotary evaporator, vacuum dry, hydrate for 2h, ultrasonic crushing to obtain Rg3 liposome (Lipo@Rg3), the encapsulation efficiency of Rg3 is 87.0%; (3) SilMA was dissolved in PBS, and then 4 mg / mL of Rg3 liposome was added to the aqueous solution of SilMA. After mixing evenly, LAP was added dropwise under stirring. The mixture was injected into a transparent glass mold and irradiated under 405 nm light for 50 s to obtain a composite hydrogel (denoted as SilMA@LR). The concentration of SilMA was 80 mg / mL, and the encapsulation efficiency of Rg3 liposome in SilMA was higher than 97%.

[0054] Comparative Example 4 In this comparative example, a drug-loaded liposome composite microsphere was prepared, and the specific process was as follows: (1) Fibroin was dissolved in lithium bromide solution, and then glycidyl methacrylate was added dropwise. After warm bath reaction for 2 h, sodium hydroxide solution was added dropwise for further reaction for 1 h to obtain glycidyl methacrylate-modified fibroin (SilMA); (2) Rg3, cholesterol, and lecithin were mixed with anhydrous ethanol, evaporated on a rotary evaporator to form a transparent film, vacuum dried, hydrated for 2 h, and ultrasonically broken to obtain Rg3 liposome (Lipo@Rg3). The encapsulation efficiency of Rg3 was 87.0%; (3) KGN, cholesterol, and lecithin were mixed with anhydrous ethanol, evaporated on a rotary evaporator to form a transparent film, vacuum dried, hydrated for 2 h, and ultrasonically broken to obtain KGN liposome (Lipo@KGN). The encapsulation efficiency of KGN was 92.9%; (4) SilMA was dissolved in PBS, and then 4 mg / mL of Rg3 liposome and 2 mg / mL of KGN liposome were added to the SilMA solution. After mixing evenly, LAP was added dropwise under stirring. Then the mixture was injected into a microfluidic channel and irradiated under 405 nm light for 50 s, and then washed with PBS for 3 times to obtain a drug-loaded liposome composite microsphere (denoted as SilMA@LKR-MP). The concentration of SilMA was 80 mg / mL, the encapsulation efficiency of Rg3 liposome in SilMA was lower than 8%, and the encapsulation efficiency of KGN liposome in SilMA was lower than 2%.

[0055] Comparative Example 5 In this comparative example, a composite hydrogel was prepared, and the specific process was as follows: (1) Fibroin was dissolved in lithium bromide solution, and then glycidyl methacrylate was added dropwise. After warm bath reaction for 2 h, sodium hydroxide solution was added dropwise for further reaction for 1 h to obtain glycidyl methacrylate-modified fibroin (SilMA); (2) Rg3 was dissolved in anhydrous ethanol, and then mesoporous silica (MSN) nanoparticles were added dropwise. After adsorption for 24 h at room temperature in the dark, Rg3-loaded nanoparticles (MSN-Rg3) were obtained. The encapsulation efficiency of Rg3 was 82.4%. (3) KGN was dissolved in anhydrous ethanol, and then mesoporous silica (MSN) nanoparticles were added dropwise. The mixture was adsorbed at room temperature for 24 hours in the dark to obtain KGN-loaded nanoparticles (MSN-KGN), and the encapsulation efficiency of KGN was 85.8%; (4) SilMA was dissolved in PBS, and then 4 mg / mL of MSN-Rg3 and 2 mg / mL of MSN-KGN were added to the SilMA solution. After mixing, LAP was added dropwise under stirring. Then the mixture was injected into a mold and irradiated at 405 nm for 50 s to obtain a drug-loaded composite hydrogel (denoted as SilMA@MKR). The concentration of SilMA was 80 mg / mL, the encapsulation efficiency of MSN-Rg3 in SilMA was higher than 93%, and the encapsulation efficiency of MSN-KGN in SilMA was higher than 94%.

[0056] Test Example 1 In this test example, the performance of the composite hydrogel or composite microspheres of the examples and comparative examples was characterized, and the specific process was as follows: Micro-morphology test: The liposomes Lipo in Example 2, the drug-loaded liposomes Lipo@Rg3 and Lipo@KGN in Example 1 were ultrasonically dispersed for 10 min, 10 μL of 0.1% solution was dropped on the front of a common copper mesh, and it was left to dry naturally overnight. The next day, the dried copper mesh was inverted on a droplet of 3wt% phosphotungstic acid to negatively stain the liposomes, and it was stained at room temperature for 3 min. Then the copper mesh was taken out and naturally dried, and then it was placed in a transmission electron microscope (TEM) to take pictures and obtain the micro-morphology images of the liposomes.

[0057] As Figure 1 shown in the TEM results, the liposomes in Example 1 showed a classic vesicular structure, indicating that the liposomes were successfully constructed.

[0058] Particle size measurement and stability evaluation: The drug-loaded liposomes Lipo@Rg3 and Lipo@KGN solutions in Example 1 were diluted with ultrapure water (volume ratio 1:5), and vortexed thoroughly to ensure uniform dispersion of the samples. 1 mL of the suspension was placed in a special container cup for particle size and zeta potential measurement, and the particle size and zeta potential of the liposomes were measured at room temperature. The measurement was repeated on the 1st day, the 7th day and the 14th day to evaluate the changes in the stability performance of the liposomes.

[0059] As Figure 2 shown in the measurement results, the particle size of the Rg3 liposomes was about 107.6 nm, and the zeta potential was about -9 mV; the particle size of the KGN liposomes was about 75 nm, and the zeta potential was about -18 mV. Both of them maintained good stability within 14 days.

[0060] Figure 3From left to right, the pictures show the state of the composite hydrogel of Example 1 before crosslinking, after crosslinking, after crosslinking and being placed on the side, and after crosslinking and being inverted.

[0061] Release performance: 0.5 cm 3 The hydrogel samples of Example 1 (SilMA@LKR) and Comparative Example 5 (SilMA@MKR) were placed in a dialysis bag with a cut-off of 3500 Da, and then immersed in 1.5 mL of a PBS solution in a constant-temperature shaking incubator at 37°C. At the specified time points (4 h, 1 d, 3 d, 7 d, and 14 d), 1 mL of old solution was collected (divided into two parts and stored at -20°C), and 1 mL of new solution was added. The release concentration of the drug was detected using HPLC or HPLC-MS.

[0062] The measurement results are shown in Table 2. Figure 4 As shown, the SilMA@LKR can slowly release the two drugs, and the amounts of Rg3 and KGN released in 14 days are 31.7% and 26.5%, respectively. The SilMA@MKR releases a lower concentration of Rg3 and KGN, which cannot be detected by HPLC. After using HPLC-MS for detection, the results show that Rg3 is almost not released, and the amount of KGN released by the SilMA@MKR is 13.2%.

[0063] In vitro cell compatibility test: After the drug-loaded liposome composite hydrogel of the example and comparative example was freeze-dried and sterilized, it was immersed in a 37°C incubator for 24 h (0.2 cm 3 / 1 mL), and diluted to 1 / 4 using culture medium; 1000 mouse bone marrow mesenchymal stem cells were then planted in 1 / 4 of the hydrogel immersion solution, and 10% CCK-8 working solution was added after 7 days of culture. After incubation in a cell incubator for 1 h, the absorbance value (OD value) of each well was detected using a microplate reader at a wavelength of 450 nm.

[0064] The obtained cell compatibility performance test graph is shown in Figure 2. Figure 5 As shown, compared with the culture medium control group (CON), the materials of each group have good proliferation effect and good cell viability.

[0065] Example 4 In this example, the drug-loaded liposome composite hydrogel of Example 1 was used for chondrogenic differentiation research, and the specific process was as follows: More than 80-90% of the mBMSCs cells were digested using 0.25% trypsin, centrifuged, counted, and resuspended. 5 μL of 1×10 5Cell / cone (5 μL) mBMSCs were seeded on 48-well plates, and after 2-3 h, the medium was replaced with drug-loaded liposome complex hydrogel extract (diluted to 1 / 4 according to the in vitro cell compatibility test described above) in Example 1 or Comparative Examples 1-3 and a pure culture medium control group (CON). Cultured for 7 d, PCR was used to detect the expression of stem cell chondrogenic differentiation genes (collagen type II COL2A1 and Sox9).

[0066] The test results are shown in Table 1. Figure 6 As shown, compared with Comparative Examples 1-3, the drug-loaded liposome complex hydrogel in Example 1 can promote the expression of COL2A1 (chondrocyte matrix composition gene) and Sox9 (chondrocyte differentiation characteristic gene) by stem cells, indicating that it is conducive to chondrogenic differentiation of stem cells.

[0067] Example 5 This example uses the drug-loaded liposome complex hydrogel of Example 1 for anti-inflammatory, and the specific process is as follows: The mouse macrophage cell line RAW264.7 was seeded at 1×10 4 cells per well on 48-well plate cell slides. 100 ng / mL LPS was used to intervene the cells for 12 h, and then the liquid in the plate was removed and replaced with the drug-loaded liposome complex hydrogel extract in Example 1 and a pure culture medium control group (CON). After 3 d of culture, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, washed with PBS three times, permeated with 0.1% Triton for 30 min, washed with PBS three times, then blocked with 0.5% BSA PBS solution, and after blocking, the primary antibody was added for incubation at 4°C overnight. The next day, the antibody was recovered, and the secondary antibody was added for incubation in the dark for 1-2 h, DAPI staining for 3 min, and mounting with an anti-fluorescence quencher for imaging.

[0068] The test results are shown in Table 2. Figure 7 As shown, the drug-loaded liposome complex hydrogel in Example 1 can inhibit the expression of M1 macrophage marker proteins (CD86 and CD11b) and promote the expression of M2 macrophage marker proteins (CD206 and Arg-1), that is, it can create a microenvironment conducive to chondrocyte regeneration through immune regulation.

[0069] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A composite hydrogel, characterized by: The composite hydrogel comprises a hydrogel matrix and liposomes loaded on the hydrogel matrix; the hydrogel matrix comprises methacrylated natural protein, and the liposomes are loaded with drugs; the drugs comprise chondrogenesis-promoting drugs and anti-inflammatory traditional Chinese medicine monomers.

2. The composite hydrogel of claim 1, wherein: In the composite hydrogel, the mass ratio of the hydrogel matrix to the liposomes is (0.5-30):(0.01-10).

3. The composite hydrogel of claim 1, wherein: The liposomes comprise first liposomes and second liposomes, the first liposomes are loaded with chondrogenesis-promoting drugs, and the second liposomes are loaded with anti-inflammatory traditional Chinese medicine monomers.

4. The composite hydrogel of claim 1, wherein: The drugs are embedded in the double membrane of the liposomes; and / or, the drugs are wrapped in the inner core of the liposomes, and / or, the drugs are loaded in the liposomes by electrostatic adsorption and / or chemical modification.

5. The composite hydrogel of claim 1, wherein: In the liposomes, the loading rate of the drugs is at least 70%.

6. The composite hydrogel of claim 1, wherein: In the liposomes, the proportion of the drugs is 0.01%-30%; and / or, the average particle size of the liposomes is 10-200 nm.

7. The composite hydrogel of claim 1, wherein: The natural protein comprises at least one of silk fibroin, sericin and collagen; and / or, the methacrylated natural protein comprises natural protein modified by at least one of methacrylic acid, methacrylic anhydride and glycidyl methacrylate.

8. The composite hydrogel of claim 1, wherein: The chondrogenesis-promoting drugs comprise at least one of TGF-β1, TGF-β1 derived polypeptide and KGN; and the anti-inflammatory traditional Chinese medicine monomers comprise at least one of ginsenoside, quercetin and resveratrol.

9. A method of preparing a composite hydrogel according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: The liposomes are mixed with the methacrylated natural protein, and then subjected to photo-crosslinking reaction to prepare the composite hydrogel.

10. Use of the composite hydrogel according to any one of claims 1-8 in preparation of bone repair materials.

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