Silk fibroin composite gel and preparation method thereof

Abstract

The application discloses a silk fibroin composite gel and a preparation method thereof, and relates to the technical field of tissue filling materials. The preparation method of the silk fibroin composite gel comprises the following steps: preparing silk fibroin and carrying out amino modification on the silk fibroin to obtain amino-modified silk fibroin; adopting an enzyme catalytic cross-linking process to dynamically cross-link the amino-modified silk fibroin and oxidized sodium alginate through a Schiff base bond to obtain a dynamic cross-linking network solution; adding a temperature-sensitive component into the dynamic cross-linking network solution, and mixing to obtain a temperature-responsive temperature-sensitive-dynamic double-network pre-gel solution; loading active ingredients into the temperature-sensitive-dynamic double-network pre-gel solution to obtain a composite gel solution; carrying out incubation on the composite gel solution to obtain a nascent gel; introducing a disulfide bond into the nascent gel, carrying out cross-linking reinforcement and sterilization, and obtaining the silk fibroin composite gel. Through the temperature-sensitive-dynamic double-network design, the introduction of active ingredients and the reinforcement of disulfide bond cross-linking, the application systematically solves the problems of traditional injectable gels, such as irreversibility, single function, easy collapse after filling and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tissue filling materials, in particular to a silk fibroin composite gel and a preparation method thereof. BACKGROUND

[0002] Silk fibroin is a kind of high molecular protein, mainly containing 18 kinds of amino acids. Silk fibroin has excellent mechanical properties, biocompatibility and biodegradability, and has good application prospect in the field of biological materials and tissue engineering. Silk fibroin can be used to prepare tissue filling materials. Through minimally invasive therapy of local injection, the filling materials with skin tissue repair function are injected into the local soft tissue with loose structure to improve the skin laxity, repair the tissue, and thus restore the normal function and appearance of the tissue. Although the traditional injectable filling materials (such as hyaluronic acid, polylactic acid microspheres) are widely used in clinical practice, they still have some problems, such as insufficient mechanical properties, uncontrollable degradation, and easy damage to active ingredients by chemical crosslinking agent, resulting in loss of biological activity. Although silk fibroin gel is used as a filling material in the prior art, it still has the following defects: (1) irreversible crosslinking and shaping defects, most of which rely on chemical covalent bonds (such as glutaraldehyde crosslinking), and the shape cannot be adjusted by body temperature or mechanical force after injection, so it is difficult to match the complex tissue profile (such as nasolabial sulcus and tear groove); (2) single network gel, which has low strength, resulting in easy collapse after filling and the need for repeated injection; (3) single function, the existing silk fibroin gel generally only provides mechanical filling, and lacks the functions of promoting tissue regeneration, anti-inflammatory or antibacterial, which can easily cause infection or fibrosis after operation. Based on this, the present application provides a silk fibroin composite gel and a preparation method thereof. SUMMARY

[0003] The main purpose of the present application is to provide a silk fibroin composite gel and a preparation method thereof, which aims to solve the technical problems of irreversible crosslinking, low strength and single function of the existing silk fibroin composite gel after crosslinking.

[0004] To achieve the above-mentioned purpose, the present application provides a preparation method of a silk fibroin composite gel, which comprises the following steps:

[0005] Preparation of silk fibroin and amino modification thereof to obtain amino-modified silk fibroin;

[0006] Using enzyme catalytic crosslinking process, the amino-modified silk fibroin and oxidized sodium alginate are dynamically crosslinked through Schiff base bond to obtain a dynamic crosslinking network solution;

[0007] Adding a temperature-sensitive component to the dynamic crosslinking network solution, and mixing to obtain a temperature-sensitive-dynamic double network pre-gel solution with temperature responsiveness;

[0008] Loading active ingredients into the temperature-sensitive-dynamic double network pre-gel solution to obtain a composite gel solution;

[0009] incubating the composite gel solution to obtain a nascent gel;

[0010] introducing disulfide bonds in the nascent gel, crosslinking and sterilizing to obtain a silk fibroin composite gel.

[0011] Optionally, the step of preparing silk fibroin and modifying it by amination to obtain aminated silk fibroin comprises:

[0012] dissolving silk fibroin in a lithium bromide solution, stirring and dissolving at 60-65°C for 1-2h, concentrating to a mass fraction of 6-8% after dialysis and purification, to obtain a silk fibroin solution;

[0013] adding 1,2-ethylenediamine to the silk fibroin solution, adding EDC / NHS activator, adjusting pH to 6.3-6.7, reacting for 22-26h, dialyzing, and freeze-drying to obtain aminated silk fibroin.

[0014] Optionally, the step of using an enzyme-catalyzed crosslinking process to dynamically crosslink the aminated silk fibroin and oxidized sodium alginate through a Schiff base bond to obtain a dynamic crosslinking network solution comprises:

[0015] dissolving the aminated silk fibroin and oxidized sodium alginate in a PBS buffer at 4-10°C, adding horseradish peroxidase and hydrogen peroxide, reacting for 20-30min to form a Schiff base crosslinking system, and obtaining a dynamic crosslinking network solution;

[0016] wherein the mass fraction of the aminated silk fibroin in the dynamic crosslinking network solution is 4-6wt%; the oxidation degree of the oxidized sodium alginate is 20-30%, and the mass fraction of the oxidized sodium alginate in the dynamic crosslinking network solution is 3-5wt%.

[0017] Optionally, the temperature-sensitive component comprises poloxamer F127 and poly-N-isopropyl acrylamide microgel.

[0018] Optionally, the step of loading the active ingredient into the temperature-sensitive-dynamic double network pre-gel solution to obtain a composite gel solution comprises:

[0019] ultrasonically dispersing the active ingredient in the temperature-sensitive-dynamic double network pre-gel solution to obtain a composite gel solution;

[0020] wherein the active ingredient comprises core-shell microspheres encapsulating growth factors and anti-inflammatory factors, graphene oxide nanosheet-anchored antibacterial peptides, and superoxide dismutase mimics.

[0021] Optionally, the core-shell microspheres are prepared by the following steps comprising:

[0022] The silk fibroin is dissolved in water, and then the vascular endothelial growth factor freeze-dried powder is added, and vortexed for 4-6 minutes to obtain an inner phase solution;

[0023] The polycaprolactone is dissolved in chloroform, stirred in a water bath at 37-42°C for 25-35 minutes, and then the interleukin-10 freeze-dried powder is added, and after ultrasonic dispersion, an outer phase solution is obtained;

[0024] The inner phase solution and the outer phase solution are respectively added dropwise into the polyvinyl alcohol aqueous solution at a speed of 450-550 rpm by using the coaxial emulsification-solvent evaporation method to form a W / O / W multiple emulsion, and then the core-shell microspheres are obtained after solidification and centrifugation.

[0025] Optionally, the graphene oxide nanosheet-anchored antibacterial peptide is prepared by the following steps comprising:

[0026] The graphene oxide powder is added into the PBS buffer solution, and after ultrasonic treatment, a graphene oxide dispersion solution is obtained;

[0027] The antibacterial peptide LL-37 is dissolved in the PBS buffer solution, and then added dropwise into the graphene oxide dispersion solution, and after stirring for 10-14 hours, centrifugation is performed at a speed of 11000-13000 rpm for 15-25 minutes, and after washing, the graphene oxide nanosheet-anchored antibacterial peptide is obtained.

[0028] Optionally, the superoxide dismutase mimic is a manganese-based metal organic framework, which has a porous structure formed by manganese ions and organic ligands, and is grafted into the temperature-sensitive-dynamic double network pre-gel solution by a covalent bond.

[0029] Optionally, after introducing the disulfide bond into the nascent gel, the step of cross-linking and strengthening and sterilization to obtain the silk fibroin composite gel comprises:

[0030] The nascent gel is immersed in a PBS buffer solution containing cystamine, the pH is adjusted to 7.0-7.5, and cross-linking is performed at 35-40°C for 2-4 hours to form a dynamic disulfide bond network, and then after sterilization by a γ-ray with a dose of 15-25 kGy, the silk fibroin composite gel is obtained.

[0031] The application also proposes a silk fibroin composite gel obtained by the above preparation method.

[0032] The application at least includes the following beneficial effects:

[0033] The application makes the silk fibroin contain amino groups by amino modification, and then crosslinks the amino silk fibroin and oxidized sodium alginate (containing aldehyde groups) through Schiff base bond (dynamic covalent bond), and introduces a temperature-sensitive component to form a temperature-responsive dynamic double network, so that the composite gel has the performance of temperature-sensitive rapid gelation; then the active ingredients are introduced, so that the composite gel can not only provide mechanical filling effect, but also has the functions of promoting growth, antibacterial and the like, and because the Schiff base bond has high dynamicity but low strength, the crosslinking is strengthened after the introduction of disulfide bond to ensure the mechanical strength of the composite gel, avoid the structure collapse after injection, so that the composite gel of the application is in a weak crosslinking state of dynamic bond (Schiff base bond) at low temperature before injection, and can be quickly solidified to form a gel after injection into the body through the body temperature (37 DEG C), which not only maintains the injectability, but also can improve the mechanical strength through the post-crosslinking of disulfide bond after implantation, and can be reshaped again after injection to match the complex tissue morphology, and has strong self-repairing ability. Therefore, the application solves the problems of irreversible, difficult to match complex tissue profile, single function, easy to collapse after filling and the like of the traditional injectable gel through the design of temperature-sensitive dynamic double network, the introduction of active ingredients and the strengthening crosslinking of disulfide bond, and realizes the integration of 'injection-gelation-reshaping'. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only some embodiments of the application, and for those skilled in the art, other drawings can be obtained without creative labor on the basis of the drawings shown.

[0035] Figure 1 The preparation method flow chart of the silk fibroin composite gel described in the embodiments of the application;

[0036] Figure 2 The performance test result graph of the silk fibroin composite gel described in the embodiments of the application.

[0037] The implementation, functional features and advantages of the application will be further described with reference to the embodiments and the drawings. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only some of the embodiments of the application, not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0039] Embodiments of the present application provide a preparation method of silk fibroin composite gel to solve the technical problems of the prior art, as shown in the specification. Figure 1 The preparation method comprises the following steps:

[0040] S10, silk fibroin is prepared and modified by amination to obtain aminated silk fibroin.

[0041] In the specific implementation process, the silk fibroin is dissolved in a lithium bromide solution, stirred and dissolved at 60-65℃ for 1-2h, concentrated to a mass fraction of 6-8% after dialysis and purification, and a silk fibroin solution is obtained.

[0042] In the silk fibroin solution, 1,2-ethylenediamine is added, and then EDC / NHS activator is added, the pH is adjusted to 6.3-6.7, and after reaction for 22-26h, dialysis is performed, and then freeze-drying is performed to obtain aminated silk fibroin.

[0043] The present application modifies the silk fibroin by amination to make it have an amino (-NH2) group, increases the reactivity with aldehyde-containing oxidized sodium alginate, this modification improves the hydrophilicity of the silk fibroin in aqueous solution, and provides stronger reaction sites for subsequent crosslinking reaction, so that it has more activity.

[0044] S20, an enzyme-catalyzed crosslinking process is used to dynamically crosslink the aminated silk fibroin and the oxidized sodium alginate through a Schiff base bond to obtain a dynamic crosslinking network solution.

[0045] In the specific implementation process, the aminated silk fibroin and the oxidized sodium alginate are dissolved in a PBS buffer solution at 4-10℃, horseradish peroxidase and hydrogen peroxide are added, and after reaction for 20-30min, a Schiff base crosslinking system is formed to obtain a dynamic crosslinking network solution.

[0046] The mass fraction of the aminated silk fibroin in the dynamic crosslinking network solution is 4-6wt%, the oxidation degree of the oxidized sodium alginate is 20-30%, and the mass fraction of the oxidized sodium alginate in the dynamic crosslinking network solution is 3-5wt%.

[0047] Specifically, the preparation method of the oxidized sodium alginate comprises: dissolving sodium alginate in a buffer solution with a pH of 4.0-4.5, adding sodium periodate, the molar ratio of sodium alginate to sodium periodate is 1:(0.8-1.0), and avoiding light for 6-8h.

[0048] The application uses amino silk fibroin (containing amino) and oxidized sodium alginate (containing aldehyde group) to cross-link through Schiff base bond (dynamic covalent bond), and forms a reversible dynamic network through catalysis of horseradish peroxidase and hydrogen peroxide, without participation of a chemical cross-linking agent, the horseradish peroxidase is a natural enzyme, there is no residual risk, and the Schiff base bond has strong reversibility, supports shaping after injection, and avoids the problem that the gel formed by traditional chemical cross-linking (such as covalent bond) cannot be delivered through a syringe due to irreversibility.

[0049] S30, a temperature-sensitive component is added to the dynamic cross-linking network solution, and after mixing, a temperature-sensitive-dynamic double network pre-gel solution with temperature responsiveness is obtained.

[0050] In the specific implementation process, the preparation step of the poly-N-isopropyl acrylamide microgel comprises:

[0051] After the N-isopropyl acrylamide monomer, N,N-methylene bisacrylamide and ammonium persulfate are dissolved in water, the poly-N-isopropyl acrylamide (PNIPAM) microgel is obtained by reacting at 55-65 DEG C under the protection of a nitrogen atmosphere for 3.5-4.5 h; the particle size of the PNIPAM microgel is 50-100 nm.

[0052] Specifically, Pluronic F127 is a triblock copolymer (PEO-PPO-PEO), the micelles of which dissolve at low temperature (<15 DEG C), and when the temperature rises to body temperature (37 DEG C), the hydrophobic PPO segment aggregates to form a micellar network, resulting in a sharp rise in solution viscosity, realizing the phase transition from liquid to gel state, and the lower critical solution temperature of the PNIPAM microgel is about 32 DEG C, when the temperature exceeds the lower critical solution temperature, the molecular chain changes from hydrophilic to hydrophobic state, phase transition occurs and physical cross-linking points are formed, and the PNIPAM microgel is embedded in the micellar network of Pluronic F127, which can further enhance the gel strength and has temperature response reversibility, supporting local shaping after injection, and through the synergy of Pluronic F127 and PNIPAM microgel, the silk fibroin composite gel has the properties of rapid temperature-sensitive gelation, high mechanical strength and reversible plasticity, thereby adapting to complex filling requirements.

[0053] S40, the active ingredient is ultrasonically dispersed in the temperature-sensitive-dynamic double network pre-gel solution to obtain a composite gel solution.

[0054] Specifically, the active ingredient includes core-shell microspheres encapsulating growth factors and anti-inflammatory factors, graphene oxide nanosheet-anchored antibacterial peptides and superoxide dismutase mimics.

[0055] The preparation step of the core-shell microspheres comprises:

[0056] The silk fibroin is dissolved in water, and then the freeze-dried vascular endothelial growth factor powder is added and vortexed for 4-6 minutes to obtain an inner phase solution;

[0057] The polycaprolactone is dissolved in chloroform, stirred in a 37-42°C water bath for 25-35 minutes, and then the freeze-dried interleukin-10 powder is added and ultrasonically dispersed to obtain an outer phase solution;

[0058] The inner phase solution and the outer phase solution are respectively added dropwise into a polyvinyl alcohol aqueous solution at a speed of 450-550 rpm by using the coaxial emulsification-solvent evaporation method to form a W / O / W multiple emulsion, and then the multiple emulsion is solidified and centrifuged to obtain the core-shell microspheres.

[0059] The present application introduces active ingredients of vascular endothelial growth factor (VEGF) and interleukin-10 (IL-10) into the core-shell microspheres as a carrier, and the VEGF is wrapped by silk fibroin. The silk fibroin is a natural high polymer material, which can provide excellent biocompatibility and degradability, and avoid the inflammatory reaction caused by artificial synthetic materials. The VEGF is a water-soluble growth factor, and the silk fibroin aqueous solution forms a hydrophilic core layer to wrap the VEGF through hydrogen bonding and electrostatic interaction, which can prevent the inactivation of the VEGF. Meanwhile, the beta-sheet structure of the silk fibroin can form a dense network to delay the diffusion of the VEGF, thereby achieving the slow release of the VEGF. The polycaprolactone is a hydrophobic material, which is dissolved in chloroform to form a continuous phase to wrap the inner phase droplets, thereby forming a physical barrier. Since the IL-10 is easily degraded by proteases, the polycaprolactone shell layer can block the external enzyme environment to maintain the activity of the IL-10. The present application precisely designs the structure of the core-shell microspheres through the hydrophilicity of the silk fibroin and the hydrophobicity of the polycaprolactone and the coaxial emulsification layering, thereby realizing the regulation of the release time sequence of the VEGF and the IL-10 (slow release of VEGF + fast release of IL-10). The present application not only ensures the dual functions of growth promotion and anti-inflammation, but also makes the growth promotion and anti-inflammation orderly connected to avoid functional antagonism.

[0060] The preparation steps of the graphene oxide nanoplatelet-anchored antibacterial peptide include:

[0061] The graphene oxide powder is added into a PBS buffer solution, and then ultrasonically treated to obtain a graphene oxide dispersion liquid;

[0062] The antibacterial peptide LL-37 is dissolved in a PBS buffer solution, and then added dropwise into the graphene oxide dispersion liquid. After stirring for 10-14 hours, the mixture is centrifuged at a speed of 11000-13000 rpm for 15-25 minutes, and then washed to obtain the graphene oxide nanoplatelet-anchored antibacterial peptide.

[0063] Specifically, the superoxide dismutase mimetic is a manganese-based metal organic framework (Mn-MOF) having a porous structure formed by manganese ions and organic ligands and grafted into the temperature-sensitive-dynamic double network pre-gel solution through a covalent bond. The Mn-MOF can mimic the catalytic function of natural SOD and has an antioxidant capacity of scavenging free radicals, with an in vitro superoxide radical scavenging rate of 85% or more. Covalent connection of the Mn-MOF with the silk fibroin enables uniform dispersion of the Mn-MOF in the gel while retaining the antioxidant activity of the Mn-MOF.

[0064] The application anchors the antibacterial peptide LL-37 to graphene oxide as a carrier to form an antibacterial complex GO / LL-37 to inhibit bacterial infection. Bacterial infection can induce a local reactive oxygen species (ROS) burst, and the Mn-MOF can neutralize ROS to protect the tissue from oxidative damage, while the GO / LL-37 inhibits bacterial proliferation, forming a double protection to block the vicious cycle of “infection-inflammation-oxidative stress” and jointly exert the antibacterial-antioxidant function.

[0065] S50, after incubation of the composite gel solution, a nascent gel is obtained.

[0066] After injection of the composite gel solution into a mold, the nascent gel is obtained by incubation at 35-40℃ for 25-35 min.

[0067] S60, after introduction of a disulfide bond in the nascent gel, crosslinking reinforcement and sterilization are performed to obtain a silk fibroin composite gel.

[0068] Specifically, the nascent gel is immersed in a PBS buffer containing cystamine, the pH is adjusted to 7.0-7.5, and the crosslinking is performed at 35-40℃ for 2-4 h to form a dynamic disulfide bond network. After sterilization by 15-25 kGy dose of gamma rays, the silk fibroin composite gel is obtained.

[0069] The application first forms an injectable nascent gel through a mild Schiff base bond, and then performs post-crosslinking through a disulfide bond to enhance the long-term stability of the composite gel. The Schiff base bond (dynamic but low strength) and the disulfide bond (high strength and controllable degradation) synergize to enable the composite gel to maintain injectability and, after implantation, to enhance the mechanical strength through post-crosslinking of the disulfide bond, so that the composite gel can be reshaped after injection to match complex tissue morphology and has a strong self-repairing ability.

[0070] The embodiments of the application also provide a silk fibroin composite gel obtained by the preparation method of the silk fibroin composite gel.

[0071] The above technical solutions of the application will be described in detail below in conjunction with specific embodiments.

[0072] Embodiment 1

[0073] A preparation method of a silk fibroin composite gel, comprising the following steps:

[0074] 1. Preparation of silk fibroin solution

[0075] According to a solid-liquid ratio of 1:10, silk fibers are dissolved in a 9.3 mol / L LiBr solution, stirred and dissolved at 60°C for 2h, injected into a dialysis bag with a molecular weight cut-off of 12kDa, and dialyzed in deionized water for 72h (water change every 4h) until the conductivity is <5μS / cm; then concentrated to a mass fraction of 8% at 40°C by rotary evaporation, and stored at 4°C for standby;

[0076] 2. Preparation of aminoated silk fibroin

[0077] Take 50mL silk fibroin solution, add 1,2-ethylenediamine (molar ratio of silk fibroin to 1,2-ethylenediamine is 1:6), 50mM EDC, 60mM NHS, adjust pH to 6.5, and magnetically stir at 25°C for 24h; then inject into a dialysis bag with a molecular weight cut-off of 3.5kDa, and dialyze in an acetic acid buffer with pH of 5.0 for 48h; after freeze-drying, white powder of aminoated silk fibroin is obtained, and stored at -20°C in a sealed state;

[0078] 3. Preparation of oxidized sodium alginate

[0079] Dissolve 5g of sodium alginate in 500mL of acetic acid buffer with pH of 4.2, add sodium periodate (molar ratio of sodium alginate to sodium periodate is 1:0.9), stir in the dark for 7h; then add 10mL of ethylene glycol to terminate the reaction, and dialyze in a dialysis bag with a molecular weight cut-off of 7kDa for 48h to remove unreacted substances; after freeze-drying, oxidized sodium alginate with an oxidation degree of 25% is obtained, and stored at 4°C for dry preservation;

[0080] 4. Preparation of temperature-sensitive-dynamic double network pre-gel solution

[0081] Dissolve 5w% of aminoated silk fibroin and 4wt% of oxidized sodium alginate in PBS buffer at 4°C, adjust pH to 7.0, add 12U / mL of horseradish peroxidase and 4mmol / L of hydrogen peroxide, stir at 25°C for 25min to obtain a dynamic cross-linked network solution;

[0082] Dissolve N-isopropyl acrylamide monomer, N,N-methylene bisacrylamide and ammonium persulfate in water, and then react at 55-65°C for 3.5-4.5h under nitrogen atmosphere to obtain poly-N-isopropyl acrylamide (PNIPAM) microgel with a particle size of 80nm;

[0083] In the dynamic crosslinking network solution, 18wt% of poloxamer F127 (Pluronic F127) and 4wt% of PNIPAM microgel were added, and homogenized at a speed of 1000 rpm for 10 min to obtain a temperature-sensitive-dynamic double network pre-gel solution, which was temporarily stored at 4℃;

[0084] 5. Preparation of active ingredients

[0085] 8 g of silk fibroin powder was dissolved in 92 mL of deionized water, and stirred at 4℃ and 500 rpm for 2 h until completely dissolved to obtain a 8wt% silk fibroin solution; VEGF lyophilized powder was added to the silk fibroin solution at a ratio of 1:10 (w / w), and vortexed for 5 min to obtain an internal phase solution;

[0086] 5 g of polycaprolactone (molecular weight 50 kDa) was dissolved in 95 mL of chloroform, and stirred in a 40℃ water bath for 30 min, then IL-10 lyophilized powder (mass ratio of polycaprolactone to IL-10 20:1) was added, and ultrasonically dispersed at a power of 200 W for 10 min to obtain an external phase solution;

[0087] A coaxial needle (inner diameter 0.4 mm, outer diameter 1.2 mm) was used, the internal phase flow rate was set at 0.5 mL / min, and the external phase flow rate was set at 2 mL / min. The internal phase solution and the external phase solution were injected into the inner and outer channels of the coaxial needle respectively, and then added dropwise into 1% (w / v) polyvinyl alcohol aqueous solution at a speed of 500 rpm to form a W / O / W multiple emulsion. The chloroform was volatilized by stirring for 4 h, and the shell was solidified. After centrifugation at a speed of 3000 rpm for 10 min, the microspheres were collected, washed with deionized water for 3 times, and then lyophilized to obtain core-shell microspheres;

[0088] 10 mg of graphene oxide powder was added to 10 mL of PBS buffer (pH 7.4), and ultrasonically treated (power 300 W, frequency 40 kHz) for 1 h to obtain a graphene oxide dispersion of 1 mg / mL;

[0089] 1 mg of antibacterial peptide LL-37 was dissolved in 1 mL of PBS buffer, and slowly added dropwise into the graphene oxide dispersion (mass ratio of graphene oxide to LL-37 1:1). After stirring for 12 h, the unbound LL-37 was removed by centrifugation at a speed of 12000 rpm for 20 min. After washing the precipitate with PBS buffer for 3 times, graphene oxide nanosheet-anchored antibacterial peptide (GO / LL-37) was obtained;

[0090] Mn-MOF was prepared by mixing 2 mmol MnCl2·4H2O and 4 mmol 2-methyl imidazole in 20 mL methanol, and then ultrasonic treatment for 10 min, and then transferred to a 50 mL polytetrafluoroethylene autoclave and reacted at 100℃ for 12 h, and then the precipitate was collected by centrifugation and washed with methanol for 3 times, and then dried at 60℃ under vacuum to obtain Mn-MOF;

[0091] 6. Primary gel forming

[0092] The core-shell microspheres, GO / LL-37 and Mn-MOF were ultrasonically dispersed (ultrasonic power was 100 W, and ultrasonic time was 5 min) in the temperature-sensitive-dynamic double network pre-gel solution to obtain a composite gel solution;

[0093] The composite gel solution was injected into a silica gel mold, and then incubated at 37℃ for 30 min to obtain a primary gel;

[0094] 7. Post-crosslinking reinforcement

[0095] The primary gel was soaked in a PBS buffer containing 0.2 mol% cystamine, and the pH was adjusted to 7.2, and then crosslinked at 37℃ for 3 h to form a dynamic disulfide bond network, and then sterilized by 20 kGy dose of γ-ray irradiation, and then sterile packaged at 4℃ to obtain a silk fibroin composite gel.

[0096] Example 2

[0097] Compared with Example 1, the parameters were adjusted as follows:

[0098] The dynamic crosslinking time in step 4 was 20 min;

[0099] The temperature-sensitive component ratio in step 4 was Pluronic F127 20 wt%, and PNIPAM microgel 5%;

[0100] In step 5, the VEGF freeze-dried powder was added into the silk fibroin solution at a mass ratio of 1:8, and the mass ratio of polycaprolactone to IL-10 was 15:1;

[0101] The post-crosslinking time in step 7 was 4 h.

[0102] Comparative Example 1: single dynamic network gel (without temperature-sensitive component)

[0103] Compared with Example 1, Pluronic F127 and PNIPAM microgel were not added in step 4, and the other steps were the same.

[0104] Comparative Example 2: without core-shell microspheres

[0105] Compared with Example 1, no core-shell microspheres were prepared in Step 5, and VEGF and IL-10 were directly ultrasonically dispersed in the temperature-sensitive-dynamic double network pre-gel solution in Step 6, and the remaining steps were the same.

[0106] Comparative Example 3 without graphene oxide nanosheets

[0107] Compared with Example 1, the preparation of GO / LL-37 in Step 5 was omitted, and free LL-37 was directly ultrasonically dispersed in the temperature-sensitive-dynamic double network pre-gel solution in Step 6, and the remaining steps were the same.

[0108] Comparative Example 4 static covalent crosslinking (glutaraldehyde crosslinking)

[0109] Compared with Example 1, the preparation process of the dynamic crosslinking network solution in Step 4 was replaced by glutaraldehyde crosslinking, that is:

[0110] 5w% of aminated silk fibroin and 4wt% of oxidized sodium alginate were dissolved in a PBS buffer at 4°C, the pH was adjusted to 8.0, 1wt% of glutaraldehyde was added, and the mixture was stirred at 25°C for 24h to allow the glutaraldehyde to fully react with the amino groups of the aminated silk fibroin to form covalent imine bonds; then 0.1M glycine was added to block the unreacted aldehyde groups and terminate the crosslinking reaction, and the mixture was dialyzed in a dialysis bag with a molecular weight cut-off of 7kDa for 48h to remove residual glutaraldehyde, thereby obtaining a static covalent crosslinking network solution; the remaining steps were the same as those of Example 1.

[0111] Comparative Example 5 without cystamine post-crosslinking reinforcement

[0112] Compared with Example 1, the nascent gel in Step 7 was not soaked in a PBS buffer containing cystamine, but was directly sterilized by irradiation, and the remaining steps were the same.

[0113] Experimental Example 1

[0114] The rheological properties of the composite gels in Example 1 and Comparative Examples 1-5 were tested using a TA Instruments DHR-3 rheometer with the following settings: flat-plate mode (diameter 20mm, gap 1mm), temperature sweep (25°C→45°C, rate 2°C / min), frequency 1Hz, strain 1%. The test indicators included: 25°C viscosity, 37°C gelation time, elastic modulus (G'), 45°C modulus reversible adjustment rate, and self-repairing rate (i.e. the ratio of the recovered modulus to the original value after shear damage). The test results are shown in Table 1 and Figure 2

[0115] Table 1

[0116]

[0117] From Table 1 and Figure 2 ​It can be seen that the viscosity of the composite gel in Comparative Example 1 at 25℃ is obviously higher than that in Example 1, and the gelation time at 37℃ is >300s, while that in Example 1 is <10s, and the elastic modulus G' in Comparative Example 1 is only 0.2kPa, and the self-repairing rate is 72%, indicating that the single dynamic crosslinking network in Comparative Example 1 has a greater impact on the rheological properties of the composite gel, and the injectability is poorer, and the self-repairing rate is also reduced; the 25℃ viscosity of the composite gel in Comparative Example 4 is the highest, and the gelation time at 37℃ is >1800s, the modulus adjustment rate at 45℃ is 0, and the self-repairing rate is 0, indicating that the composite gel formed by covalent imine bond static crosslinking in Comparative Example 4 is completely irreversible, cannot trigger secondary shaping, and has poor injectability; in Comparative Example 5, no disulfide bond is introduced for post-crosslinking strengthening, resulting in a certain degree of decrease in the elastic modulus G' of the composite gel, the modulus adjustment rate at 45℃ and the self-repairing rate, indicating that disulfide bond crosslinking strengthening is beneficial to improving the mechanical strength of the composite gel. This shows that the temperature-sensitive-dynamic double network in the application is beneficial to realizing rapid gelation, and the dynamic bond supports reversible shaping, and the disulfide bond strengthening can provide mechanical support.

[0118] Experimental Example 2

[0119] The composite gels in Example 1 and Comparative Example 2 were respectively immersed in a PBS buffer solution at 37℃, and the pH was 7.4, and the samples were taken at regular time intervals, and the release amount of VEGF was detected by HPLC, and the release amount of IL-10 was detected by ELISA, and the cumulative release rate of VEGF and IL-10 was calculated;

[0120] Among them, the cumulative release rate = release amount / total drug loading amount x 100%.

[0121] The test results are shown in Table 2 below.

[0122] Table 2

[0123] Group VEGF release rate (24h) VEGF release rate (28 days) IL-10 release rate (5 days) Example 1 8 75 85 Comparative Example 2 85 95 20

[0124] As can be seen from Table 2, after 24h, the VEGF release of the composite gel in Comparative Example 2 is 85%, while the VEGF release of the composite gel in Example 1 is <10%, and after 28 days, the cumulative release rate of VEGF in the composite gel in Example 1 is 75%, which is still not completely released, while the VEGF in the composite gel in Comparative Example 2 has been basically exhausted; after 5 days, the IL-10 release rate of the composite gel in Example 1 is 85%, and the IL-10 release rate of the composite gel in Comparative Example 2 is 20%. It shows that by wrapping VEGF and IL-10 with core-shell microspheres in the application, the release time sequence is controlled (VEGF slow release+IL-10 fast release), so that the angiogenesis and anti-inflammation are orderly connected, avoiding the release confusion and functional antagonism caused by directly adding VEGF and IL-10 in Comparative Example 2.

[0125] In summary, the aminoated silk fibroin and oxidized sodium alginate are crosslinked by dynamic Schiff base bond in the present application, supporting reversible plasticity, the Pluronic F127 / PNIPAM microgel provides temperature-sensitive rapid gelation, and a phased crosslinking strategy is adopted, first crosslinked by dynamic Schiff base bond for rapid forming, and then strengthened by disulfide bond to improve long-term stability, so that the composite gel of the present application is in a weak crosslinking state of dynamic bond (Schiff base bond) before injection (at low temperature) to maintain fluidity, after injection into the body, the dynamic bond is triggered to reorganize by body temperature (37℃), which can quickly solidify to form a gel, which not only maintains injectability, but also can improve the mechanical strength through post-crosslinking of disulfide bond after implantation, and can be reshaped after injection to match complex tissue morphology, and has strong self-repairing ability; the present application uses a core-shell microsphere gradient drug delivery system to release VEGF (long-acting pro-angiogenic) and IL-10 (fast-acting anti-inflammatory) in time sequence, avoiding drug antagonism and improving treatment synergy; at the same time, the synergistic effect of antibacterial-antioxidant compound blocks the vicious cycle of "infection-inflammation-oxidative stress". The present application solves the problems of irreversible, difficulty in matching complex tissue profile, single function, easy to collapse after filling and other problems of traditional injectable gels by the design of temperature-sensitive-dynamic double network, core-shell microsphere gradient drug delivery and antibacterial-antioxidant compound integration, realizes the integration of "injection-gelation-shaping", and is suitable for soft tissue repair and medical beauty filling.

[0126] The above is only an optional embodiment of the present application, and does not limit the patent scope of the present application, and any equivalent structural transformation made by using the content of the present application specification and drawings, or directly / indirectly applied in other related technical fields under the inventive concept of the present application is included in the patent protection scope of the present application.

Claims

1. A method for preparing a silk fibroin composite gel, characterized by, The method comprises the following steps: Preparation of silk fibroin and amino-modification thereof to obtain amino-modified silk fibroin; Dynamic cross-linking of the amino-modified silk fibroin and oxidized sodium alginate through a Schiff base bond by using an enzyme catalytic cross-linking process to obtain a dynamic cross-linking network solution; Addition of a temperature-sensitive component to the dynamic cross-linking network solution, and mixing to obtain a temperature-responsive temperature-sensitive-dynamic double network pre-gel solution; Loading of an active ingredient into the temperature-sensitive-dynamic double network pre-gel solution to obtain a composite gel solution; Incubation of the composite gel solution to obtain a nascent gel; Introduction of a disulfide bond into the nascent gel, cross-linking reinforcement and sterilization to obtain a silk fibroin composite gel.

2. The method for preparing the silk fibroin composite gel according to claim 1, characterized in that, The step of preparing silk fibroin and amino-modification thereof to obtain amino-modified silk fibroin comprises: Dissolution of silk fibroin in a lithium bromide solution, stirring and dissolving at 60-65 DEG C for 1-2 hours, concentration to a mass fraction of 6-8% after dialysis and purification, and obtaining a silk fibroin solution; Addition of 1,2-ethylenediamine to the silk fibroin solution, addition of an EDC / NHS activator, adjustment of pH to 6.3-6.7, reaction for 22-26 hours, dialysis, and freeze-drying to obtain amino-modified silk fibroin.

3. The method for preparing the silk fibroin composite gel according to claim 1, characterized in that, The step of dynamic cross-linking of the amino-modified silk fibroin and oxidized sodium alginate through a Schiff base bond by using an enzyme catalytic cross-linking process to obtain a dynamic cross-linking network solution comprises: Dissolution of the amino-modified silk fibroin and oxidized sodium alginate in a PBS buffer solution at 4-10 DEG C, addition of horseradish peroxidase and hydrogen peroxide, reaction for 20-30 minutes, formation of a Schiff base cross-linking system, and obtaining a dynamic cross-linking network solution; The mass fraction of the amino-modified silk fibroin in the dynamic cross-linking network solution is 4-6 wt%; the oxidation degree of the oxidized sodium alginate is 20-30%, and the mass fraction of the oxidized sodium alginate in the dynamic cross-linking network solution is 3-5 wt%.

4. The method for preparing the silk fibroin composite gel according to claim 1, characterized in that, The temperature-sensitive component comprises poloxamer F127 and poly-N-isopropyl acrylamide microgels.

5. The method for preparing the silk fibroin composite gel according to claim 1, characterized in that, The step of loading of an active ingredient into the temperature-sensitive-dynamic double network pre-gel solution to obtain a composite gel solution comprises: Ultrasonic dispersion of the active ingredient in the temperature-sensitive-dynamic double network pre-gel solution to obtain a composite gel solution; The active ingredient comprises core-shell microspheres encapsulating growth factors and anti-inflammatory factors, graphene oxide nanosheet-anchored antibacterial peptides and superoxide dismutase mimics.

6. The method for preparing the silk fibroin composite gel according to claim 5, characterized in that, The core-shell microspheres are prepared by the following steps: Dissolution of silk fibroin in water, vortex mixing for 4-6 minutes after addition of a vascular endothelial growth factor freeze-dried powder to obtain an internal phase solution; Dissolution of polycaprolactone in chloroform, stirring in a 37-42 DEG C water bath for 25-35 minutes, and ultrasonic dispersion after addition of an interleukin-10 freeze-dried powder to obtain an external phase solution; The inner phase solution and the outer phase solution are respectively added into a polyvinyl alcohol aqueous solution at a rotating speed of 450 rpm-550 rpm by using a coaxial emulsification-solvent evaporation method to form a W / O / W multiple emulsion, and then the core-shell microspheres are obtained after solidification and centrifugation.

7. The method for preparing the silk fibroin composite gel according to claim 5, characterized in that, The graphene oxide nanoplatelet-anchored antibacterial peptide is prepared by the following steps, comprising: The graphene oxide powder is added into a PBS buffer solution, and after ultrasonic treatment, a graphene oxide dispersion solution is obtained; The antibacterial peptide LL-37 is dissolved in a PBS buffer solution, and then added dropwise into the graphene oxide dispersion solution, and after stirring for 10 h-14 h, centrifugation is performed at a speed of 11000 rpm-13000 rpm for 15 min-25 min, and after washing, the graphene oxide nanoplatelet-anchored antibacterial peptide is obtained.

8. The method for preparing the silk fibroin composite gel according to claim 5, characterized in that, The superoxide dismutase mimic is a manganese-based metal organic framework, which has a porous structure formed by manganese ions and organic ligands, and is grafted into the temperature-sensitive-dynamic double network pre-gel solution by a covalent bond.

9. The method for preparing the silk fibroin composite gel according to claim 1, characterized in that, The step of introducing a disulfide bond into the nascent gel, cross-linking and strengthening, and sterilization to obtain the silk fibroin composite gel comprises: The nascent gel is immersed in a PBS buffer solution containing cystamine, the pH is adjusted to 7.0-7.5, and cross-linking is performed at 35℃-40℃ for 2 h-4 h to form a dynamic disulfide bond network, and after sterilization by using a 15 kGy-25 kGy dose of γ-rays, the silk fibroin composite gel is obtained.

10. A silk fibroin composite gel, characterized by, The silk fibroin composite gel is obtained by using the preparation method of the silk fibroin composite gel according to any one of claims 1-9.

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