Silk fibroin-hydroxyapatite microsphere composite filler for injection and preparation method therefor

Abstract

The present invention provides a silk fibroin-hydroxyapatite microsphere composite filler for injection and a preparation method therefor. The preparation method therefor comprises: S1, preparation of a silk fibroin solution; S2, preparation of a silk fibroin-hydroxyl cellulose gel; S3, preparation of a layer-by-layer self-assembled hydroxyapatite microsphere; and S4, preparation of the silk fibroin-hydroxyapatite microsphere composite filler for injection. The entire preparation method involves neither the addition of any chemical cross-linking agent and a photoinitiator, nor light cross-linking and radiation cross-linking. Provided is use of the described silk fibroin-hydroxyapatite microsphere composite filler for injection in medical aesthetic fillers or medical fillers, for example, in the face, trunk, limbs, etc., or in one or more of the tear troughs, crow's feet, temples, nose, chin, nasolabial fold, mouth corner juncture, frontotemporal region, and genital organ. All of the various ingredients added above have good biocompatibility, which can reduce the rejection of the filler in vivo, and avoid the skin problems of allergy, flushing, and swelling after filling.

Description

Silk protein composite hydroxyapatite microsphere filling agent for injection and preparation method thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical cosmetology and plastic surgery, and in particular relates to a preparation method of silk protein composite hydroxyapatite microsphere injection filling agent with different molecular weights. BACKGROUND

[0002] In recent years, injectable gels for medical and cosmetic fillers have developed rapidly. By increasing the cross-linking degree of hyaluronic acid, the degradation time of the injection can be prolonged, and the elastic modulus can be increased, which is suitable for different filling sites, such as tear grooves, fish tail lines, and other sites that require superficial filling, such as temples, noses, chins, and other sites that require moderate depth filling, and nasal labial folds, mouth corner junctions, and forehead temples, and other sites that require deep filling. Patent CN116284886A establishes a multiple cross-linked gel system to increase stability and viscoelasticity, but the addition of cross-linking agents can increase the toxicity of the injection and cause local tissue reactions, so there is an urgent need for a material with higher biological safety to meet different beauty needs.

[0003] Silk protein is an ideal natural polymer material with extremely low immunogenicity, good biocompatibility, and the advantage of promoting collagen regeneration, and is often used in tissue engineering, soft tissue repair, and wound repair. Different molecular weight silk proteins have different elastic moduli and complex viscosities, different degradation periods, and different filling sites and depths, but silk protein gels also have problems that need to be overcome as fillers: 1. It is difficult to accurately measure silk proteins with different molecular weights; 2. Silk protein gelation is slow, which can easily cause the initial contamination of silk protein gel to exceed the standard, so it needs to be quickly gelled to adapt to large-scale production.

[0004] Hydroxyapatite, as the main inorganic component of human and animal bones, has good biocompatibility, and its metabolites are common calcium and phosphate ions in the human body. Mixing hydroxyapatite microspheres with silk protein-cellulose gel is beneficial to achieving sustained collagen regeneration in situ. Patent CN116808290A dries the filling agent mixed after the gel and the microspheres to extend its shelf life, but it is difficult to make the microspheres uniformly dispersed in the gel matrix after reconstitution, and it is not convenient to use, the steps are relatively complicated, and it increases the difficulty of the doctor during the operation process; Patent CN117919515A mixes microspheres with a bivariate distribution with the gel matrix to make it have good injectability and shape plasticity, but the small particle size microspheres (1-10 microns) can easily cause macrophage storm and the risk of granuloma, and a safer way is needed to mix hydroxyapatite microspheres and gel matrix uniformly. SUMMARY

[0005] In view of the above, in order to overcome the defects of the prior art, the present application provides a preparation method of a silk protein composite hydroxyapatite microsphere filler with different molecular weights and a relatively narrow molecular weight distribution, in order to ensure the safety of the filler while meeting various beauty needs and being suitable for various filling sites, the present application proposes to screen silk proteins with different molecular weights by AI algorithm combined with the rheological properties of silk proteins, which have different elastic moduli and complex viscosities and different degradation periods, and are suitable for different filling sites and depths; a new type of silk protein composite hydroxyapatite microsphere filler is developed by a simple method of mixing and heating, and the hydroxy cellulose or carboxymethyl cellulose, such as hydroxypropyl methyl cellulose or hydroxypropyl cellulose or carboxymethyl cellulose, and hydroxyethyl cellulose, and the hydrophobic interaction between the silk protein molecules induces the silk protein molecular chain to change from random coil to beta-sheet, and this secondary structure transformation enables the silk protein to form small and uniform beta-sheet structures inside, and it is just the uniform distribution of the crosslinking sites throughout the hydrogel that avoids the use of chemical crosslinking agents and improves the safety of the filler; the silk protein and cellulose are wrapped on the surface of the hydroxyapatite microspheres by a layer-by-layer self-assembly method to increase the surface hydrophilicity, and the hydroxyapatite microspheres and the silk fibroin-cellulose gel are stirred and defoamed under negative pressure to prepare an injection gel with excellent mechanical properties and good microsphere dispersibility, which is easy to extrude.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: the present application proposes a preparation method of a silk protein composite hydroxyapatite microsphere filler for injection, and the mixed gel comprises the following components: silk protein-cellulose gel, modified hydroxyapatite microspheres, osmotic pressure regulator, and water for injection.

[0007] Preferably, the silk protein-cellulose gel comprises silk protein-hydroxypropyl methyl cellulose and silk protein-carboxymethyl cellulose.

[0008] Preferably, the preparation method of the silk protein-cellulose gel specifically comprises the following steps:

[0009] S1: preparation of a silk protein solution: degummed silk is prepared into silk protein through dissolution and purification, and silk proteins with different molecular weights are screened by membranes with different molecular weight cut-offs;

[0010] S2: physical gelation of silk protein: a physical crosslinking agent is dissolved in water to swell, hydroxy cellulose or carboxymethyl cellulose or the corresponding derivative aqueous solution is added to the silk protein aqueous solution prepared in step S1, the osmotic pressure is adjusted, and then the mixed solution is stirred uniformly and placed in an oven for gelation;

[0011] S3: Preparation of layer-by-layer self-assembled modified hydroxyapatite microspheres: Disperse the hydroxyapatite microspheres in the silk fibroin solution prepared in step S1, stir at room temperature, then take out the microspheres and dry; then dissolve them in a cellulose solution, stir at room temperature, then take out the microspheres and dry; repeat 3-5 times;

[0012] S4: Preparation of silk fibroin composite hydroxyapatite microsphere filler for injection: Stir the modified hydroxyapatite microspheres with the silk fibroin-cellulose gel under negative pressure to remove bubbles, then sterilize them by high-pressure steam, and aseptically fill them in pre-filled syringes to obtain the silk fibroin composite hydroxyapatite microsphere filler for injection;

[0013] Further,

[0014] S1: Preparation and determination of silk fibroin with different molecular weights: The silk fibroin is prepared by degumming, dissolving, purifying and desalting of mulberry silk, dissolved in an ionic liquid, and tested on an MCR301 rotational rheometer. The data curves at multiple test temperatures are determined, and the physical parameters required by the model are determined. Then, the non-linear model fitting is performed by AI algorithm to obtain the molecular weight data. The molecular weight of the silk fibroin is calculated from the fitting parameters.

[0015] The rheological data and viscosity data are measured according to the industry standard "Silk Fibroin for Tissue Engineering Medical Devices" Appendix B, and are imported into the Favorsun SmartMolFit "Complex Wisdom Algorithm" commercial software for biological macromolecule molecular weight calculation.

[0016] The test method is as follows:

[0017] A.1 Test procedure

[0018] A.1.1 Viscosity test

[0019] Select a suitable diameter (so that the data does not exceed the test range of the rheometer, and a stable signal can be obtained), such as a 25mm parallel plate (Parallel plate, PP25). During the test, the test is protected by nitrogen blowing of the temperature control cover (H-PTD200 hood with peltier heating / cooling); in addition, a very thin layer of low-viscosity silicone oil (viscosity about 10 mPa·s) is added at the edge of the sample and the parallel plate to prevent the sample from absorbing water vapor during the test.

[0020] Steady-state test mode is adopted: the test temperature is 30℃, and the shear rate is scanned from low to high, and the shear rate range is: 10 -3 s -1 ~ 10 3 s -1The viscosity values of the platform curve are recorded.

[0021] A.1.2 Storage modulus and loss modulus test

[0022] The sample placement and test preparation are the same as B.4.1

[0023] The linear dynamic elastic test mode, i.e. the strain amplitude control in the oscillation mode is adopted to ensure that the storage modulus (G') and the loss modulus (G") are linear within the frequency scanning range (1 x 10 2 rad / s ~ 6.81 x 10 -2 rad / s). The test is carried out under the relative humidity of 30% ~ 40%.

[0024] The frequency scanning is carried out at the following temperatures (0°C, 10°C, 20°C and 30°C) to obtain the storage modulus and the loss modulus curves of the sample at different temperatures, and the test time is guaranteed within 1.5h each time.

[0025] A.2 Calculation

[0026] B.5.1 Confirmation of the system establishment

[0027] When the AmimCl solvent system is adopted, the literature [1] validation experiment research shows that the Huggins parameter K H of the system is about 0.1, which can be judged as a good solvent for silk fibroin. The specific calculation is according to the formula (1) ~ (3).

[0028] a) The specific viscosity is calculated according to the formula (1) by measuring the viscosities of the solution and the solvent at different concentrations at the same temperature.

[0029] In the formula,

[0030] η sp - viscosity;

[0031] η - viscosity of the solution;

[0032] η s - viscosity of the solvent;

[0033] b) The value of the intrinsic viscosity is calculated according to the formula (2) according to the limit value when the concentration approaches.

[0034] In the formula,

[0035] [η] - intrinsic viscosity;

[0036] η sp - specific viscosity;

[0037] C - mass concentration of the solution.

[0038] c) Calculate Huggins parameter from viscosity of solution by formula (3): η sp = C [η] + K H (C [η]) 2 + A (C [η]) n ……… (3)

[0039] In the formula,

[0040] η sp — specific viscosity;

[0041] C — mass concentration of solution;

[0042] [η] — intrinsic viscosity;

[0043] A — viscosity of solvent;

[0044] K H — Huggins parameter.

[0045] Note 1: The document verifies that the Huggins parameter K H of AmimCl solvent system is about 0.1, which meets the requirements and does not need to be verified;

[0046] Note 2: If other solvent systems are used, verification is required.

[0047] B.5.2 Molecular weight fitting calculation

[0048] The measured rheological data and viscosity data are imported into Favorsun SmartMolFit "Fuxiangzhi algorithm" biological macromolecule molecular weight calculation commercial software to calculate the number average molecular weight and weight average molecular weight. Or calculate according to formulas (4) to (10).

[0049] a) Storage modulus and loss modulus: In the instrument parameter setting, strain ε = ε0sinωt, stress σ = σ0sin(ωt+δ), ω is the angular frequency, and δ is the stress lead angle. The stress is expanded as σ = σ0sinωtcosδ + σ0cosωtsinδ. The stress-strain relationship can be further represented by a storage modulus (G') in the same phase as the strain and a loss modulus (G") with a phase difference of π / 2 from the strain, as shown in formulas (4) to (6): σ = ε0G'sinωt + ε0G"cosωt …… (4)

[0050] G' is the storage modulus, reflecting the energy stored in the sample after applying a strain. G" is the loss modulus, reflecting the energy loss during the response. In dynamic shear method, rheometer can give the relationship of G' and G" with the alternating shear frequency ω. Storage modulus and loss modulus can be directly obtained from the instrument test results.

[0051] b) Calculation of shift factor: use the viscosity curves of the sample at different temperatures in test step B.4.1 to calculate the shift factor (α T ) according to formula (7):

[0052] In the formula,

[0053] η sp - specific viscosity;

[0054] C - mass concentration of the solution;

[0055] [η] - intrinsic viscosity;

[0056] A - viscosity of the solvent;

[0057] K H - Huggins parameter.

[0058] Note 1: The Huggins parameter K H of AmimCl solvent system is about 0.1, which meets the requirements and does not need to be verified;

[0059] Note 2: If other solvent systems are used, verification is required.

[0060] B.5.2 Molecular weight fitting calculation

[0061] The measured rheological data and viscosity data are imported into Favorsun SmartMolFit "Fuxiangzhi algorithm" commercial software for biological macromolecule molecular weight calculation, and the number average molecular weight and weight average molecular weight are calculated. Or according to formula (4) ~ (10).

[0062] a) Storage modulus and loss modulus: in the instrument parameter setting, strain ε = ε0sinωt, stress σ = σ0sin(ωt+δ), ω is the angular frequency, and δ is the stress lead angle. The stress can be expanded as σ = σ0sinωtcosδ + σ0cosωtsinδ. The stress-strain relationship can be further represented by a storage modulus (G') in the same phase as the strain and a loss modulus (G") with a phase difference of π / 2 from the strain, as shown in formula (4) ~ formula (6): σ = ε0G'sinωt + ε0G"cosωt … … (4)

[0063] G' is the storage modulus, reflecting the energy stored in the sample after applying a strain. G" is the loss modulus, reflecting the energy loss during the response. In dynamic shear method, the rheometer can give the relationship between G' and G" with the alternating shear frequency ω. The storage modulus and the loss modulus can be directly obtained from the instrument test results.

[0064] b) Calculation of shift factor: The shift factor (a T ) is calculated using the viscosity curves of the sample at different temperatures in test step B.4.1, according to equation (7):

[0065] where,

[0066] a T — shift factor;

[0067] η(T) — viscosity of the sample at different temperatures, Pa-s;

[0068] η(T0) — viscosity of the sample at 30°C, Pa-s;

[0069] T — different test temperatures, °C;

[0070] T0 — 30°C;

[0071] c) Fitting of the molecular weight distribution curve:

[0072] Assuming the probability density of the silk protein molecules with molecular weight from 10 kDa to 40 kDa in the solution, the fitting curve is obtained according to equations (8) to (10) by substituting the Rouse model:

[0073] where,

[0074] G' — storage modulus;

[0075] G" — loss modulus

[0076] p — density of the solution;

[0077] R — gas constant (8.31);

[0078] T — absolute temperature at the time of test

[0079] M i — molecular weight of a silk protein molecule, here taken as 10 kDa to 40 kDa

[0080] f(i) is the probability density of the molecular weight M i

[0081] η0 — viscosity of the polymer solution; ​

[0082] τ p - relaxation time of the pth relaxation mode of the polymer chain of molecular weight M

[0083] d) Molecular weight calculation: the shift factor a obtained by using equation (7) T The test results of step B.4.2 are translated, and the data at low temperature are translated to obtain the data in the high frequency region, and the master curve is obtained; the master curve is compared with the fitting curve obtained in step B.5.2c), and the iteration is continuously iterated until the master curve and the fitting curve are optimally overlapped, that is, "a dynamic convergence factor is introduced in the optimization process, and the global optimization ability of the optimization algorithm is improved through an adaptive weight strategy. With the increase of the number of iterations, the value of the dynamic convergence factor gradually decreases to 0, and when the current iteration number reaches the maximum iteration number, the convergence process is terminated, which is the optimal overlap". This assumed value is the true probability density of the silk protein with a molecular weight of 10 kDa to 40 kDa in the solution. Further calculation from this probability density can obtain the number average molecular weight, weight average molecular weight and molecular weight distribution index of the silk protein. The calculation formula is:

[0084] The above process can also refer to the description of molecular weight testing and AI calculation in WO2023 / 024607.

[0085] S2: Preparation of silk protein-cellulose gel: Dissolve cellulose in water to swell, add cellulose aqueous solution to the silk protein aqueous solution prepared in step S1, mix and then perform ice bath ultrasonic treatment, then adjust the osmotic pressure to 250-350 mOsm / L using an osmotic pressure regulator, and then stir the mixed solution uniformly and place it in an oven for gelation.

[0086] Preferably, in S1, the molecular weight of the silk protein is 10-200KD.

[0087] Preferably, in S2, the mass ratio of the silk protein and cellulose is 1:1-10:1.

[0088] Preferably, in S2, the mass fraction of the silk protein is 0.5-5%.

[0089] Preferably, in S2, the mass fraction of the cellulose is 0.05-5%.

[0090] Preferably, in S2, the cellulose swelling time is 6-26h.

[0091] Preferably, in S2, the ultrasonic treatment conditions are an ultrasonic power of 200-500W, an ice bath temperature of 4-10℃, and an ultrasonic treatment time of 0.1-1h.

[0092] Preferably, in S2, the osmotic pressure regulator is a 0.9wt% sodium chloride solution or a phosphate buffered solution (PBS).

[0093] Preferably, in S2, the oven temperature is 70-80℃, and the gelation time is 8-24h.

[0094] The present application also provides a preparation method of a silk protein filler containing hydroxyapatite microspheres for injection, specifically comprising the following steps:

[0095] S3: dispersing the hydroxyapatite microspheres in a 2wt% silk protein solution, stirring at 200rpm at room temperature, then taking out the microspheres for drying, and then dispersing them in a 0.5wt% cellulose solution, stirring at 200rpm at room temperature, then taking out the microspheres for drying, and repeating the process several times;

[0096] Preferably, in S3, the particle size of the hydroxyapatite microspheres is 20-45 microns.

[0097] After S3, the surface of the microspheres is covered with a layer of polymer composite film, and the surface micropores disappear, equivalent to layer-by-layer self-assembly.

[0098] S4: deaerating the modified microspheres with negative pressure stirring in a silk protein-cellulose gel, then autoclaving them, and filling them into pre-filled syringes to obtain a silk protein mixed gel containing hydroxyapatite microspheres for injection.

[0099] Preferably, in S4, the mass of the hydroxyapatite microspheres and the volume of the silk protein solution are in a ratio of 1-20g:100mL.

[0100] Preferably, in S4, the mass of the hydroxyapatite microspheres and the volume of the cellulose solution are in a ratio of 1-20g:100mL.

[0101] Preferably, in S4, the mass ratio of the hydroxyapatite microspheres and the silk protein-cellulose gel is 1:10-1:1.

[0102] Preferably, in S4, the stirring conditions of the hydroxyapatite microspheres and the silk protein-cellulose gel are 2-10kPa, 500-2500rpm / min, and 0.1-2h.

[0103] Preferably, in S4, the temperature of the autoclaving is 115-125℃, and the time is 15-30min.

[0104] The hydroxyl cellulose or carboxyl cellulose is selected from one or more of hydroxypropyl cellulose, or hydroxypropyl methyl cellulose, carboxymethyl cellulose, and hydroxyethyl cellulose.

[0105] A silk protein composite hydroxyapatite microsphere filler for injection is obtained by the above preparation method.

[0106] The use of the above-mentioned silk protein composite hydroxyapatite microsphere filling agent for medical and cosmetic fillers, such as for one or more of the face or for tear grooves, crow's feet, temples, nose, chin, nasolabial folds, corner of the mouth, forehead-temples.

[0107] The beneficial effects obtained by the present application are as follows:

[0108] The present application provides a preparation method of silk protein composite hydroxyapatite filling agent with different molecular weights. The hydrophobic interaction between the hydroxypropyl methylcellulose molecules or carboxymethyl cellulose or hydroxyethyl cellulose and the silk protein molecules after heating induces the silk protein molecular chain to change from random coil to β-sheet. This secondary structure transformation can form small and uniform β-sheet structures inside the silk protein, and the uniform distribution of the crosslinking sites throughout the gel system forms a three-dimensional fiber network structure, thereby avoiding the skin allergy problem caused by the addition of chemical crosslinking agents. By layer-by-layer self-assembly, a layer of silk fibroin-cellulose film is first covered on the surface of the hydroxyapatite microspheres to increase the hydrophilicity of the microsphere surface, and then the silk protein-cellulose gel is stirred uniformly, which can make the hydroxyapatite microspheres disperse uniformly in the gel system, avoiding the problems of facial redness, nodules, etc. caused by the aggregation of the filling agent. The molecular weight of the silk protein is determined by AI algorithm combined with the rheological properties of the silk protein. Different molecular weights of silk protein have different degrees of elastic modulus and complex viscosity. The silk protein mixed gel is applied to different filling sites and levels to meet different beauty needs. The above-mentioned various components have good biocompatibility, which can reduce the rejection of the filling material in the body and avoid the skin problems of allergy and redness after filling. BRIEF DESCRIPTION OF DRAWINGS

[0109] Figure 1 is the rheological property of the product prepared in Examples 1-3 at 0.7 Hz.

[0110] Figure 2 is the in vitro degradation performance result of the silk protein-cellulose gel in Examples 1-3.

[0111] Figure 3 is a comparison of the gel state of the silk protein-cellulose gel prepared in Example 1 (left) and the silk protein gel prepared in Comparative Example 1 placed in an oven for 8 h (right).

[0112] Figure 4 is a particle size distribution graph of the hydroxyapatite microspheres in Example 1.

[0113] Figure 5 is a comparison of the scanning electron microscope of the microspheres after layer-by-layer self-assembly in Example 1 (left) and the microspheres in Comparative Example 2 (right).

[0114] Figure 6 is a comparison of the pushing force of the products prepared in Example 1 and Comparative Example 2.

[0115] Figure 7 is a hematoxylin-eosin staining chart of the product prepared in Examples 1-3 one month after implantation.

[0116] Figure 8 is a type I collagen and type III collagen immunohistochemical staining chart of the product prepared in Examples 1-3 one month after implantation. DETAILED DESCRIPTION

[0117] The application is further described below in conjunction with specific examples. It should be understood that the examples are used only for illustrating the application and not intended to limit the scope of protection of the application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the disclosure, and these equivalent forms also fall within the scope of protection defined by the application.

[0118] Example 1

[0119] S1: Preparation of silk protein solution: mulberry silk is subjected to degumming, dissolution, clarification filtration, purification desalination and concentration to obtain silk protein, which is dissolved in an ionic liquid and tested on an MCR301 rotational rheometer to determine data curves at multiple test temperatures, then the data is modeled using the rouse model, and the physical parameters required by the model are determined, and then the AI algorithm is used for nonlinear model fitting to screen out silk proteins with a molecular weight of 10-50KD, and the mass fraction of the silk protein aqueous solution is 10%.

[0120] S2: Preparation of silk protein-hydroxypropyl methyl cellulose gel:

[0121] a) 1g of hydroxypropyl methyl cellulose (Anhui Shanhe Pharmaceutical Auxiliary Co., Ltd., substitution type: 1828) is dissolved in 20mL of water, fully swelled for 24h to obtain a 5% hydroxypropyl methyl cellulose aqueous solution;

[0122] b) 1.01g of disodium hydrogen phosphate and 0.25g of sodium dihydrogen phosphate are dissolved in 100mL of water to obtain a 0.1M PBS solution;

[0123] c) 20g of the solution prepared in step S1, 10g of the 5% hydroxypropyl methyl cellulose aqueous solution and 10g of the 0.1M PBS solution are mixed with 60g of water, and then ultrasonically treated at 300W power in an ice bath for 0.5h, and then placed in a 70℃ oven for 8h.

[0124] S3: Preparation of layer-by-layer self-assembled hydroxyapatite microspheres:

[0125] a) 3g of hydroxyapatite microspheres (particle size 20-45um, Sigma) are dissolved in 100mL of 2wt% silk protein solution, stirred at 200rpm for 3h at room temperature, and the reaction mixture is concentrated and dried;

[0126] b) Dissolve the concentrated and dried sample in 100 mL of 0.5 wt% cellulose solution, stir at 200 rpm for 1 hour at room temperature, concentrate and dry the reactants; repeat steps a) and b) 3 times.

[0127] S4: Preparation of a facial filler made from silk protein composite hydroxyapatite microspheres for injection:

[0128] The modified hydroxyapatite microspheres were added to 7g of the gel prepared in step S2, and stirred for 30 minutes at a speed of 2300 rpm and a vacuum of 2 kPa. The mixture was then aseptically filled into 1mL pre-filled syringes and sterilized by high-pressure steam at 121℃ for 30 minutes to obtain the silk protein filler containing hydroxyapatite microspheres.

[0129] Example 2

[0130] S1: Preparation of silk fibroin solution: Same as step S1 in Example 1. Silk fibroin with a molecular weight of 50KD to 100KD was screened using an AI algorithm combined with the rheological properties of silk fibroin. The mass fraction of the silk fibroin aqueous solution was 10%.

[0131] S2: Preparation of silk fibroin-carboxymethyl cellulose gel:

[0132] a) Weigh 0.8g of carboxymethyl cellulose and hydroxypropyl methyl cellulose and dissolve it in 16mL of water. Allow it to swell completely for 24h to obtain a 5% (w / w) aqueous solution of hydroxypropyl methyl cellulose.

[0133] b) Weigh 0.808 g of disodium hydrogen phosphate and 0.2 g of sodium dihydrogen phosphate and dissolve them in 80 mL of water. After complete dissolution, a 0.1 M PBS solution is obtained.

[0134] c) Weigh 10g of the solution prepared in step S1, 5g of 5% carboxymethyl cellulose hydroxypropyl methyl cellulose aqueous solution and 5g of 0.1M PBS solution, mix them with 30g of water, sonicate in an ice bath at 280W power for 0.5h, and place in a 70℃ oven for 8h.

[0135] S3: Preparation of layer-by-layer self-assembled hydroxyapatite microspheres:

[0136] a) Weigh 3g of hydroxyapatite microspheres (particle size 20-45um), dissolve them in 100mL of 2wt% silk protein solution, stir at 200 rpm for 2h at room temperature, and concentrate and dry the reactants;

[0137] b) Dissolve the concentrated and dried sample in 100 mL of 0.5 wt% cellulose solution, stir at 200 rpm for 0.5 h at room temperature, concentrate and dry the reactants; repeat steps a) and b) 3 times.

[0138] S4: Preparation of silk protein composite hydroxyapatite microsphere filler for injection:

[0139] The modified hydroxyapatite microspheres were added to 7 g of the gel prepared in step S2, stirred at a speed of 2000 revolutions per minute and a vacuum degree of 2 kilopascals for 25 minutes, aseptically filled in 1 mL pre-filled syringes, and then sterilized with high-pressure steam at a temperature of 121°C for 30 minutes to obtain a silk protein filler containing hydroxyapatite microspheres.

[0140] Example 3

[0141] S1: Preparation of a silk protein solution: same as step S1 in Example 1. Silk proteins with a molecular weight of 100 KD to 150 KD were screened by AI algorithm combined with the rheological properties of silk proteins, and the mass fraction of the silk protein aqueous solution was 10%.

[0142] S2: Preparation of a silk-hydroxypropyl methyl cellulose gel:

[0143] a) 1 g of hydroxypropyl methyl cellulose was weighed into 20 mL of water, swelled for 24 h to obtain a 5% hydroxypropyl methyl cellulose aqueous solution;

[0144] b) 0.808 g of disodium hydrogen phosphate and 0.2 g of sodium dihydrogen phosphate were weighed into 80 mL of water, and after being fully dissolved, a 0.1 M PBS solution was obtained;

[0145] c) 40 g of the solution prepared in step S1, 10 g of the 5% hydroxypropyl methyl cellulose aqueous solution, and 10 g of the 0.1 M PBS solution were mixed with 40 g of water, and then ultrasonically treated at a power of 280 W in an ice bath for 0.5 h and placed in a 70°C oven for 8 h.

[0146] S3: Preparation of layer-by-layer self-assembled hydroxyapatite microspheres:

[0147] a) 3 g of hydroxyapatite microspheres (particle size 20-45 um) were weighed into 100 mL of a 2 wt% silk protein solution, stirred at 200 revolutions per minute at room temperature for 4 h, and the reaction was concentrated and dried;

[0148] b) The concentrated and dried sample was dissolved in 100 mL of a 0.5 wt% cellulose solution, stirred at 200 revolutions per minute at room temperature for 2 h, and the reaction was concentrated and dried; steps a) and b) were repeated 3 times.

[0149] S4: Preparation of silk protein composite hydroxyapatite microsphere filler for injection:

[0150] The concentrated hydroxyapatite microspheres were added to 7 g of the gel prepared in step S2, stirred at a speed of 1800 rpm and a vacuum degree of 5 kPa for 35 min, aseptically filled in 1 mL pre-filled syringes, and then sterilized by high-pressure steam at a temperature of 121 °C for 30 min to obtain a silk protein filler containing hydroxyapatite microspheres.

[0151] Example 4

[0152] S1: Preparation of a silk protein solution: same as step S1 in Example 1. Silk proteins with a molecular weight of 100 KD-150 KD were screened by AI algorithm combined with the rheological properties of silk proteins, and the mass fraction of the silk protein aqueous solution was 10%.

[0153] S2: Preparation of a silk-hydroxypropyl methyl cellulose gel:

[0154] a) 1 g of hydroxypropyl methyl cellulose was weighed into 20 mL of water, swelled for 24 h to obtain a 5% hydroxypropyl methyl cellulose aqueous solution;

[0155] b) 0.808 g of disodium hydrogen phosphate and 0.2 g of sodium dihydrogen phosphate were weighed into 80 mL of water, and a 0.1 M PBS solution was obtained after complete dissolution;

[0156] c) 40 g of the solution prepared in step S1, 10 g of the 5% hydroxypropyl methyl cellulose aqueous solution, and 10 g of the 0.1 M PBS solution were mixed with 40 g of water, and then ultrasonically treated at a power of 280 W in an ice bath for 0.5 h and placed in a 70 °C oven for 8 h.

[0157] S3: Preparation of layer-by-layer self-assembled hydroxyapatite microspheres:

[0158] a) 3 g of hydroxyapatite microspheres (particle size 40-60 um) were weighed into 100 mL of a 2 wt% silk protein solution, stirred at a speed of 200 rpm at room temperature for 4 h, and the reaction was concentrated and dried;

[0159] b) The concentrated and dried sample was dissolved in 100 mL of a 0.5 wt% cellulose solution, stirred at a speed of 200 rpm at room temperature for 2 h, and the reaction was concentrated and dried; steps a) and b) were repeated 5 times.

[0160] S4: Preparation of a silk protein composite hydroxyapatite microsphere filler for injection:

[0161] The concentrated hydroxyapatite microspheres were added to 7 g of the gel prepared in step S2, stirred at a speed of 1800 rpm and a vacuum degree of 5 kPa for 35 min, aseptically filled in 1 mL pre-filled syringes, and then sterilized by high-pressure steam at a temperature of 121 °C for 30 min to obtain a silk protein filler containing hydroxyapatite microspheres.

[0162] Comparative Example 1 - without cellulose composite filler

[0163] The gel prepared without cellulose has a slower rate and is loose, with more surface water. As shown in Figure 3, the filler preparation process is as follows:

[0164] S1: Preparation of silk fibroin solution: same as step S1 in Example 1. Silk fibroin with a molecular weight of 10-50KD was selected by AI algorithm combined with the rheological properties of silk fibroin, and the mass fraction of silk fibroin aqueous solution was 10%.

[0165] S2: Preparation of silk fibroin gel:

[0166] a) Weigh 0.808g of disodium hydrogen phosphate, 0.2g of sodium dihydrogen phosphate into 80mL of water, and dissolve thoroughly to obtain a 0.1M PBS solution;

[0167] b) Weigh 10g of the solution prepared in step S1 and 5g of 0.1M PBS solution, mix with 35g of water, and then ultrasonic in ice bath at 300W power for 0.5h, and place in a 70℃ oven for 24h.

[0168] S3: Preparation of silk fibroin filler containing hydroxyapatite microspheres for injection:

[0169] a) Weigh 3g of hydroxyapatite microspheres and dissolve in 100mL of 2wt% silk fibroin solution, stir at 200rpm at room temperature overnight, and concentrate the reactants;

[0170] b) Dissolve the concentrated sample in 100mL of 0.5wt% cellulose solution, stir at 200rpm at room temperature overnight, and concentrate the reactants; repeat 3 times as above.

[0171] S4: Add the concentrated hydroxyapatite microspheres to 7g of the gel prepared in step S2, stir at 2300rpm under a vacuum of 2kPa for 30min, sterilely fill in 1mL pre-filled syringes, and then sterilize with high-pressure steam at a temperature of 121℃ for 30min to obtain a silk fibroin mixed gel containing hydroxyapatite microspheres.

[0172] Comparative Example 2 - without surface modification of hydroxyapatite

[0173] As can be seen from FIG. 3, the push force fluctuates greatly for the filler prepared from hydroxyapatite without layer-by-layer self-assembly modification, which is mainly because the hydroxyapatite is not uniformly dispersed and is prone to agglomeration and precipitation, so the local hydroxyapatite concentration of the filler is inconsistent, resulting in a large fluctuation in the push force. After the hydroxyapatite microspheres are subjected to layer-by-layer self-assembly and then mixed with the silk fibroin-cellulose gel, the mixed gel has a smaller and more uniform push force, which indicates that the hydroxyapatite microspheres are well dispersed in the gel.

[0174] The filler preparation test process is as follows:

[0175] S1: Preparation of silk fibroin solution: The mulberry silk is subjected to degumming, dissolution, clarification filtration, purification desalination and concentration to obtain silk fibroin, which is dissolved in an ionic liquid and subjected to testing on an MCR301 rotary rheometer. The data curves at multiple test temperatures are determined, and the data are modeled by using the rouse model. The physical parameters required by the model are determined, and the nonlinear model fitting is performed by using an AI algorithm. The silk fibroin with a molecular weight of 10-50 KD is screened, and the mass fraction of the silk fibroin aqueous solution is 10%.

[0176] S2: Preparation of silk fibroin gel:

[0177] a) 0.808 g of disodium hydrogen phosphate and 0.2 g of sodium dihydrogen phosphate are weighed and dissolved in 80 mL of water to obtain a 0.1 M PBS solution;

[0178] b) 10 g of the solution prepared in step S1 and 5 g of the 0.1 M PBS solution are weighed and mixed uniformly with 35 g of water. After being subjected to ice bath ultrasonic treatment at a power of 300 W for 0.5 h, the mixture is placed in a 70°C oven for 24 h.

[0179] S3: Preparation of silk fibroin mixed gel containing hydroxyapatite microspheres for injection:

[0180] 3 g of hydroxyapatite microspheres are weighed and added to 7 g of the gel prepared in step S2. The mixture is stirred at a rotation speed of 2300 rpm and a vacuum degree of 2 kPa for 30 min, and then is aseptically filled in a 1 mL pre-filled syringe. The syringe is subjected to high-pressure steam sterilization at a temperature of 121°C for 30 min to obtain the silk fibroin mixed gel containing hydroxyapatite microspheres.

[0181] Rheological property test

[0182] The mixed gels prepared in Examples 1-3 were subjected to rheological testing according to the following procedure: a 25mm parallel plate (PP25) was selected, and the sample was extruded onto the plate, and a very thin layer of low viscosity silicone oil was added to the edge of the sample and the parallel plate to prevent water vapor absorption during the test. Frequency sweep was performed at 25°C under a deformation pressure of 0.1%, and the modulus and viscosity of each sample at 0.7Hz were obtained, and each sample was tested in triplicate.

[0183] Figure 1 shows the elastic modulus and complex viscosity of the mixed gels prepared in Examples 1-3 at 0.7Hz, and the results show that the greater the molecular weight of the silk protein, the higher the elastic modulus and complex viscosity of the filler, the better the support and plasticity of the filler, and the more excellent the mechanical properties, and the less likely to displace, and the more suitable for deeper filling.

[0184] Degradation rate test

[0185] The in vitro degradation performance of the mixed gels prepared in Examples 1-3 was determined according to the following procedure: a citric acid buffer solution was prepared by dissolving 21g of citric acid monohydrate in 500mL of water, adding 200mL of 1mol / L sodium hydroxide solution, and then diluting with water to the full scale. 40.4mL of the solution was mixed with 59.6mL of 0.1mol / L hydrochloric acid solution to prepare a citric acid buffer solution. 20mL of the solution was added to 1g of the mixed gel prepared in Examples 1-3, and the mixture was placed in a 37°C water bath environment. The mass loss of the filler was measured at 1 day, 7 days, 4 weeks, 8 weeks, and 12 weeks, and the degradation rate of the filler was calculated based on the mass loss.

[0186] Figure 2 shows the in vitro degradation curve of the mixed gels prepared in Examples 1-3, and the results show that the greater the molecular weight of the silk protein, the longer the degradation period of the filler, and the slower the degradation rate.

[0187] Pushing force test

[0188] The pushing force of the mixed gels prepared in Example 1 and Comparative Example 2 was determined according to the following procedure: a 1mL syringe containing the mixed gel was placed on a pushing force measuring instrument, and the pushing rod was pushed at a speed of 30mm / min, and the pushing force curve within a displacement of 2mm was recorded.

[0189] Figure 3 shows the pushing force curve of the mixed gels prepared in Example 1 and Comparative Example 2, and the results show that the hydroxyapatite microspheres are well dispersed in the gel after hierarchical self-assembly, and the mixed gel prepared by mixing the silk protein-cellulose gel with the hydroxyapatite microspheres has a smaller and more uniform pushing force.

[0190] Experimental Example 4 - Animal Experiment

[0191] The mixed gels obtained in Examples 1 to 3 were implanted subcutaneously in the back of New Zealand white rabbits, 0.1 mL per implantation site, and the skin reaction at the implantation site was observed at 1 week, 2 weeks, 1 month, and 3 months after implantation. The rabbits were sacrificed at 3 months, and the samples were sectioned, stained with hematoxylin-eosin, and immunohistochemically stained for collagen types I and III.

[0192] The results showed that there were no abnormal phenomena such as redness and erythema in the appearance of the skin and the subcutaneous side of the white rabbits within 3 months, no inflammatory cell infiltration, and no abnormal phenomena such as fibrous cysts were observed by hematoxylin-eosin staining, proving that the filler had good biocompatibility. Immunohistochemical staining and quantitative results showed that the higher the molecular weight of the silk protein, the more collagen types I and III were secreted, as shown in Table 1. High molecular weight is conducive to deep filling and promotes collagen regeneration and tissue reconstruction.

[0193] Table 1 Quantitative table of collagen types I and III immunohistochemical staining of mixed gels of Examples 1 to 3 after 1 month

[0194] In addition, Figure 4 shows the particle size analysis of the hydroxyapatite microspheres used, and the results show that the median particle size is about 34 microns, the proportion of microspheres with a particle size of 20-45 microns is as high as 94.6% or more, and the proportion of microspheres with a particle size of less than 20 microns is less than 2.2%, which avoids the risk of small particle size microspheres easily triggering a macrophage storm.

[0195] Figure 5 shows that, by scanning electron microscopy, the microspheres are covered with a layer of silk protein-cellulose film after layer-by-layer self-assembly, making the surface of the microspheres smoother and avoiding the formation of granulomas after injection.

[0196] Figure 6 shows that, compared with the microspheres that have not been assembled, the microspheres after layer-by-layer self-assembly have a smoother pushing force curve after mixing with the gel, which also proves that the microspheres are more uniformly dispersed in the gel matrix after layer-by-layer self-assembly.

[0197] Figure 7 shows that the silk protein composite hydroxyapatite microsphere filler for injection has good biological safety after implantation, with no tissue necrosis and only a small amount of macrophage infiltration, and visible neovascularization with fibroblast support.

[0198] Figure 8 illustrates that the composite implants generate partial collagen fibers after being implanted subcutaneously for 1 month, and the collagen fibers are distributed in the gel gaps and the surface of the microspheres, and the type I and III collagens are arranged in parallel between the microsphere gaps. The higher the molecular weight of the silk fibroin, the greater the collagen area (brown area) around the implant after the silk fibroin composite hydroxyapatite microsphere facial filler for injection is implanted. The above results prove that the silk fibroin composite hydroxyapatite microsphere facial filler for injection can be used as a soft tissue filler and implanted subcutaneously, and has good biocompatibility and the ability to promote collagen regeneration.

[0199] The above describes the embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a silk fibroin complex hydroxyapatite microsphere filler for injection, characterized by, The method comprises the following steps: S1: Preparation of silk fibroin solution: degummed silk is prepared into silk fibroin through dissolution and purification, and silk fibroin with different molecular weights is screened through membranes with different molecular weight cut-offs; S2: Physical gelation of silk fibroin: a physical crosslinking agent, which is hydroxyl cellulose or carboxyl cellulose or corresponding derivatives, is dissolved in water and swelled, and the silk fibroin aqueous solution prepared in step S1 is added with the hydroxyl cellulose or carboxyl cellulose or corresponding derivatives aqueous solution, the osmotic pressure is adjusted, the mixed solution is stirred uniformly, and is placed in an oven for gelation to obtain a silk fibroin-cellulose gel; S3: Preparation of layer-by-layer self-assembly modified hydroxyapatite microspheres: the hydroxyapatite microspheres are dispersed in the silk fibroin solution prepared in step S1, stirred at room temperature, and then taken out for drying; and then the microspheres are dissolved in a cellulose solution, stirred at room temperature, and then taken out for drying; the above steps are repeated for 3-5 times; S4: Preparation of silk fibroin composite hydroxyapatite microsphere filler for injection: the modified hydroxyapatite microspheres and the silk fibroin-cellulose gel prepared in step S2 are stirred under negative pressure to remove bubbles, and then are sterilized by high-pressure steam, and are aseptically filled in pre-filled syringes to obtain a silk fibroin composite hydroxyapatite microsphere filler for injection. No additional chemical crosslinking agent or photo initiator is needed in the whole preparation process, and no photo crosslinking or irradiation crosslinking is needed.

2. The method for preparing silk protein complex hydroxyapatite microsphere filler for injection according to claim 1, characterized in that, The step S1 comprises the following steps: The degummed silk is dissolved in lithium bromide aqueous solution at a mass / volume ratio of less than or equal to 1:1, and is fully dissolved in water at 25-100℃ for 10-120 minutes to obtain a mixed solution containing silk fibroin and a small amount of insoluble particles; the mixed solution is filtered through a filter bag to remove the insoluble particle impurities to obtain a clear lithium bromide protein solution; the clear solution is diluted with one time of purified water, and is subjected to desalination through a purification system; the purified silk fibroin aqueous solution is dried in a flat dish, and the mass concentration of the silk fibroin aqueous solution is determined to be 1-10% by the mass difference method; and the silk fibroin with different molecular weights is screened through membranes with different molecular weight cut-offs.

3. The preparation method of the injectable silk protein composite hydroxyapatite microfiller as described in claim 1, characterized in that, The step S2 comprises the following steps: hydroxyl cellulose is weighed and dissolved in water to be fully swelled for 10-30 hours; sodium dihydrogen phosphate and disodium hydrogen phosphate are weighed and dissolved in water; the silk fibroin solution, the hydroxyl cellulose aqueous solution, the sodium dihydrogen phosphate and disodium hydrogen phosphate solution, and a certain amount of water are mixed and stirred uniformly; and the mixture is placed in an oven at 70-80℃ for gelation for 6-30 hours; finally, the mass concentration of the silk fibroin aqueous solution is 0.5-10%, the mass concentration of the hydroxyl cellulose aqueous solution is 0.1-10%, and the molar mass concentration of the sodium dihydrogen phosphate and disodium hydrogen phosphate is 0.2-1M. The step S3 comprises the following steps: the hydroxyapatite microspheres are dispersed in 0.5-5wt% silk fibroin solution, stirred at room temperature for 1-5 hours, and then the microspheres are taken out and dried at 50-90℃ for 0.5-3 hours; and then the microspheres are dissolved in 0.2-1wt% cellulose solution, stirred at room temperature for 1-5 hours, and then the microspheres are taken out and dried at 50-90℃ for 0.5-3 hours; the above steps are repeated for 3-5 times.

4. The preparation method of the injectable silk protein composite hydroxyapatite microsphere filler as described in claim 1, characterized in that, ​ 5. The method of claim 1-4, wherein the silk fibroin composite hydroxyapatite microsphere filler for injection is prepared by the steps of, The hydroxyl cellulose or carboxyl cellulose is selected from one or more of hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose; the degummed silk is selected from one or more of mulberry silk, tussah silk, spider silk.

6. The method for preparing the silk protein composite hydroxyapatite microsphere filler for injection according to any one of claims 1-4, wherein the adjusting the osmotic pressure is adjusting the osmotic pressure to 250-350 mOsm / L by using an osmotic pressure adjusting agent.

7. The method for preparing the silk protein composite hydroxyapatite microsphere filler for injection according to claim 5, wherein the adjusting the osmotic pressure is adjusting the osmotic pressure to 250-350 mOsm / L by using an osmotic pressure adjusting agent.

8. A silk protein complexed hydroxyapatite microsphere filler for injection, characterized by: obtained by the method for preparing according to any one of claims 1-7.

9. Use of the silk protein composite hydroxyapatite microsphere filler for injection as claimed in claim 8, characterized by: for preparing a medical filler.

10. The use according to claim 9, characterized in that, for preparing a medical and aesthetic filler.

11. Use according to claim 9, characterized in that, The medical filler is used for one or more of tear trough, crow's feet, stretch marks, temple, nose, chin, nasolabial folds, vermilion junction, frontotemporal, neck, genital.

12. The use according to claim 9, characterized in that, The medical filler is used for skin tissue or soft tissue filling and repair.

13. The use according to claim 9, characterized in that, The medical filler is used for face, trunk or extremity filling and repair. The medical filler is used for face, trunk or extremity filling and repair.

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