A collagen / hyaluronic acid composite gel for skin injection and preparation method thereof

Abstract

The present invention discloses a collagen / hyaluronic acid composite gel for skin injection and a preparation method thereof. Hyaluronic acid and collagen are cross-linked and then formed into a nanogel under high-speed stirring. The obtained nanogel has strong anti-degradation ability, a larger specific surface area, and stronger mechanical support. It can resist the shear force caused by facial muscle movement and is more suitable for deep filling.

Description

Technical Field

[0001] The invention belongs to the field of biological products and relates to the preparation of collagen / hyaluronic acid nanocomposite gel through polycarboxylic acid cross-linking and dispersion processes. Background Art

[0002] Among the injectable gels currently dominated by chemical cross-linking, the gels used for skin injection are mainly made of hyaluronic acid (HA) and collagen (RC). Filling gel products with hyaluronic acid as the matrix alone (referred to as hyaluronic acid fillers) can improve the gel's own anti-degradation ability by using chemical cross-linking agents such as 1,4-butanediol diglycidyl ether (BDDE), thereby reducing the pain of repeated injections for patients. Filling gel products with collagen as the matrix alone (referred to as collagen fillers) mostly use glutaraldehyde to achieve cross-linking, but the anti-degradation ability after cross-linking is much lower than that of hyaluronic acid fillers. Although collagen fillers have advantages that cannot be replaced by hyaluronic acid fillers, their scope of application is limited by cross-linking technology.

[0003] After mixing the two components of hyaluronic acid and collagen, three methods can be used to form a composite gel: physical crosslinking, chemical crosslinking, and biological crosslinking. However, the mechanical strength of the hyaluronic acid and collagen composite gel formed by physical crosslinking is weak, and it cannot maintain itself from the influence of environmental factors such as pH and temperature for a long time. Biological crosslinking can only occur between specific sites, so its application range is not wide. The main reaction mechanisms of chemical crosslinking include Dials-Alder reaction, Michael addition reaction, Schiff base reaction, enzyme mediation, thiol exchange / disulfide crosslinking, and click chemistry, which can be stimulated by external stimuli (temperature, pH, light, electric field / magnetic field, ultrasound, and enzyme) to form strong covalent bonds. Although the use of chemical crosslinking improves the stability (mechanical properties, physicochemical properties) and life of the composite gel, the toxicity of the crosslinking agent required in the crosslinking reaction and the harm to the environment cannot be ignored. For example, commonly used cross-linking pathways based on cross-linking agents such as BDDE, BDDA, DVS, PEGDE, adipic acid dihydrazine (ADH), divinyl sulfone (DVS), 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC), and bifunctional agents such as glutaraldehyde and diisocyanate often result in a large amount of cytotoxic or allergenic components remaining in the gel material containing unreacted monomers, and can be dissolved from the material, causing a series of inflammatory reactions, and the purification step for removing the cross-linking agent residues in the material usually takes several days to several weeks to complete. More importantly, after the above-mentioned composite gel is injected into the human body as a filler, the collagen will be quickly degraded due to enzyme degradation, resulting in the rapid disintegration of the cross-linked structure of hyaluronic acid and collagen. Its anti-degradation ability and viscoelastic properties are still lower than those of hyaluronic acid fillers, resulting in poor filling effect, and repeated injections are required to maintain the therapeutic effect. Therefore, it is not suitable for filling in deeper ranges (such as subcutaneous to periosteum) and cannot supplement collagen for deep skin.

[0004] The main methods for preparing nanogels include freeze drying, air incorporation, emulsion, solvent evaporation and thermally induced phase separation, but the process is complex, the irritating non-aqueous solvents used can lead to cytotoxicity, and the particle shape and size are not uniform, and they mainly play the role of sustained release of drug components, such as Chinese patent CN113262174A. Therefore, these methods are not suitable for preparing soft tissue fillers. Summary of the invention

[0005] The purpose of the present invention is to provide a collagen / hyaluronic acid composite gel for skin injection and a preparation method thereof. The prepared composite gel is nanoscale gel particles with uniform shape and size and is resistant to degradation, meeting the demand for soft tissue fillers that are more durable, safer, and have stronger mechanical properties.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A composite gel for skin injection is a nanogel made of a first matrix component, a second matrix component and a cross-linking auxiliary agent, wherein the first matrix component is hyaluronic acid or sodium hyaluronate, the second matrix component is natural collagen or recombinant collagen, and the cross-linking auxiliary agent includes a polycarboxylic acid substance.

[0008] Preferably, the polycarboxylic acid substance is selected from one or more of citric acid, citrate, and citric acid derivatives. The presence of these polycarboxylic acid substances enables the mixed first matrix component and the second matrix component to form a gel through cross-linking.

[0009] Preferably, the cross-linking auxiliary agent also includes a polyol. By introducing the polyol, the mixing degree of the cross-linked material (i.e., the above-mentioned matrix component) and the cross-linking agent (i.e., the above-mentioned polycarboxylic acid substance) is increased, and the corresponding cross-linking sites (cross-linking groups include hydroxyl groups contained in the above-mentioned matrix component) are also increased, and the mechanical properties of the resulting composite gel can also be improved.

[0010] Preferably, the polyol is selected from one or more of ethylene glycol, ethylene glycol derivatives, glycerol, and glycerol derivatives.

[0011] Preferably, the crosslinking is carried out in an alkaline solution, the crosslinking temperature is 30 to 50° C., and the crosslinking time is 1 to 24 hours.

[0012] Preferably, the composite gel particles are uniform in size and have a particle size of ≤100 nm.

[0013] The preparation method of the composite gel for skin injection comprises the following steps:

[0014] A first cross-linking preparatory liquid containing a first matrix component and a cross-linking auxiliary agent is prepared, and a second cross-linking preparatory liquid containing a second matrix component and a cross-linking auxiliary agent is prepared; 1 to 5 mL of the first cross-linking preparatory liquid and 1 to 5 mL of the second cross-linking preparatory liquid are mixed, added into a liquid medium and dispersed, to obtain a composite gel for skin injection dispersed in a liquid medium.

[0015] Preferably, the preparation method of the composite gel specifically comprises the following steps:

[0016] 1) 5% to 10% of 600 to 2000 kDa hyaluronic acid (or sodium hyaluronate), 0.5% to 2% of polycarboxylic acid, 0 to 20% of polyol and 68% to 94.5% of alkaline solution are mixed according to corresponding mass fractions to obtain a first cross-linking preparatory solution, and the first cross-linking preparatory solution is loaded into a first syringe, and 5% to 10% of 40 to 100 kDa recombinant collagen, 0.5% to 10% of polycarboxylic acid, 0 to 10% of polyol and 70% to 92.5% of alkaline solution are mixed according to corresponding mass fractions to obtain a second cross-linking preparatory solution, and the second cross-linking preparatory solution is loaded into a second syringe;

[0017] 2) Insert the respective injection needles of the first syringe and the second syringe into the feeding tube from two positions on the upper part of the feeding tube, and then push the first syringe (0.1-1.5 mL / min) and the second syringe (0.01-0.1 mL / min) at a certain flow rate, and add the first cross-linking preparatory liquid and the second cross-linking preparatory liquid mixed in the feeding tube (injected into the feeding tube by the first syringe and the second syringe respectively and mixed after intersection) dropwise into the low-toxic (injectable) surfactant below the feeding tube, and stir for 1-24 hours under the conditions of ≥200 rpm and heating at 30-50°C.

[0018] Preferably, in step 1, the first cross-linking preparatory liquid contains 10% to 20% of polyol, and the second cross-linking preparatory liquid contains 2% to 10% of polyol, and combined with different syringe pushing speeds (determining the flow rate), composite gels with different particle size distributions and mechanical properties can be formed.

[0019] Preferably, in step 1, the pH of the alkaline solution is 9.5 to 11.5, and the pH of the preparatory solution is adjusted by the alkaline solution to provide a suitable alkaline environment for the cross-linking reaction to activate the cross-linking groups, such as the carboxyl groups on the cross-linking agent, so that cross-linking occurs more easily.

[0020] Preferably, in step 2, the diameter of the feed tube is ≤2 mm. By changing the diameter of the feed tube (with a certain length), the dripping acceleration of the fluid (i.e., the mixture of the first cross-linking preparatory liquid and the second cross-linking preparatory liquid formed by confluence after entering the feed tube under a certain pushing speed control) can be regulated, thereby ensuring that the particle size of the composite gel can be adjusted at the nanoscale.

[0021] Preferably, in step 2, the surfactant is selected from a polyethylene glycol aqueous solution with a mass fraction of 0.5% to 3%. The low-concentration polyethylene glycol solution has a small effect on cross-linking and mainly serves to disperse the composite gel.

[0022] Preferably, in step 2, the stirring speed is 200-600 rpm. If polyol is not introduced into the first and second cross-linking preparatory liquids, the same effect (mainly referring to the particle size distribution when the polyol is introduced) can be achieved by further extending the stirring time or increasing the stirring intensity (speed).

[0023] Preferably, the preparation method of the composite gel further comprises the following steps: after step 2, the gel particles (i.e., nanogel) in the stirred system are separated by sieving, and then the pH of the gel particles is adjusted to 6.8-7.6 with hydrochloric acid before use for tissue filling injection.

[0024] Application of the composite gel for skin injection in the preparation of tissue fillers.

[0025] The beneficial effects of the present invention are embodied in:

[0026] The present invention uses polycarboxylic acid substances as crosslinking agents, hyaluronic acid substances (such as hyaluronic acid, sodium hyaluronate) and collagen as matrix components to construct collagen / hyaluronic acid nanocomposite gel, which has the characteristics of uniform particle size, large specific surface area, strong anti-degradation ability and low toxicity. It is easier to form in-situ gel after injection, and has good cohesion, so it can provide stronger mechanical support and resist the shear force caused by facial muscle movement, which is more suitable for deep filling. Compared with gels prepared using traditional chemical crosslinking processes, the present invention is cleaner, safer and cheaper.

[0027] Furthermore, the present invention optimizes process parameters (such as syringe pushing speed, etc.) so that the prepared nanocomposite gel has the advantages of high elastic modulus, high cohesion, degradation resistance, low pushing force and easy injection.

[0028] Furthermore, the present invention uses an alkaline cross-linking reaction system containing both polycarboxylic acid (such as citric acid) and polyol (such as ethylene glycol) to make the prepared nanocomposite gel have higher stability and flexibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are the results of the degradation experiment of nanocomposite gel fillers.

[0030] Figure 2 These are the results of cytotoxicity experiments of nanocomposite gel fillers.

[0031] Figure 3 It is a structural schematic diagram of the fluid control device; in the figure: 1-hyaluronic acid syringe, 2-feeding tube, 3-beaker, 4-magnetic stirrer, 5-recombinant collagen syringe, 6-nanocomposite gel, 7-stirring bar.

[0032] Figure 4Experimental observation of the cross-linking effect of citric acid and ethylene glycol in the formation of nanocomposite gel: (A) adding citric acid and ethylene glycol; (B) without adding citric acid and ethylene glycol.

[0033] Figure 5 This is a scanning electron microscope image of the nanogel prepared in Experimental Example 1. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are only used to explain the present invention but not to limit the protection scope of the present invention.

[0035] (I) Principle of preparing nanocomposite gel using fluid control device

[0036] See also Figure 3 The fluid control device includes a hyaluronic acid syringe 1, a recombinant collagen syringe 5, a feed tube 2 (to ensure that the injected different fluids can be mixed in the tube and flow out under the action of gravity), a beaker 3 and a magnetic stirrer 4. The feed tube 2 is fixedly placed above the beaker 3; the injection needles of the hyaluronic acid syringe 1 and the recombinant collagen syringe 5 are respectively inserted into the side walls of the feed tube 2, wherein the injection needle of the hyaluronic acid syringe 1 is slightly higher than the recombinant collagen syringe 5 in insertion position (the two injection needles can also be inserted at the same height). The hyaluronic acid syringe 1 is used to push the relatively viscous sodium hyaluronate solution into the feed tube 2 so that it falls along the feed tube 2. The recombinant collagen syringe 5 is used to push the relatively viscous recombinant collagen solution into the above-mentioned feed tube 2. When the two injected solutions are mixed in the feed tube 2, they continue to fall and form a drop-like outflowing material at the bottom of the feed tube 2. During the pushing process of the syringe, the material continuously drips into the beaker 3, and the beaker 3 contains a liquid medium (the beaker 3 is placed on the magnetic stirrer 4, and its stirring rod 7 is placed in the beaker 3). The material dripping from the feed tube 2 can be dispersed by stirring through the high-speed rotation of the stirring rod 7 and formed into smaller particles, thereby obtaining a nano-composite gel 6 under a certain cross-linking system (cross-linking agent, heating, stirring and dispersing, etc.).

[0037] (II) Preparation of Nanocomposite Gel Fillers

[0038] Experimental Example 1

[0039] A sodium hydroxide solution (pH=9.5) containing 5wt% 600kDa molecular weight sodium hyaluronate, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into a 5mL specification (filled with 5mL) syringe with a 27G injection needle (i.e., the above-mentioned hyaluronic acid syringe 1), and the pushing speed was set to 0.1mL / min; a sodium hydroxide solution (pH=9.5) containing 10wt% 40kDa molecular weight recombinant collagen, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into a 5mL specification (filled with 5mL) syringe with a 30G injection needle (i.e., the above-mentioned recombinant collagen syringe 5), and the pushing speed was set to 0.01mL / min. Then the two syringes were placed on the left and right sides of the upper part of a plastic hard tube with a diameter of 2mm and a length of 8cm, respectively. The plastic hard tube was provided with two injection holes with a vertical spacing of 4cm on the corresponding sides, and the injection needles of the two syringes were respectively inserted and fixed into the two injection holes on the plastic hard tube, and the two syringes were clamped and fixed. Then push two syringes at a set constant speed, and when the materials pushed into the plastic tube by the two syringes begin to intersect and mix, the mixed material can be dripped into a beaker containing 2.5% (w / w) polyethylene glycol solution (500mL) under the plastic tube, and stirred at 600rpm for 1h under heating conditions (50°C). After being placed at room temperature (gel particles have been deposited), pour the obtained gel particles from the beaker and pass through an 800-mesh sieve, and adjust the pH of the separated gel particles to neutral with a 5% hydrochloric acid solution (the hydrochloric acid solution is absorbed into the gel particles, and the adjustment result of the hydrochloric acid solution can be determined by the conventional method of measuring the pH of the gel), thus forming a nanogel for injection filling.

[0040] Experimental Example 2

[0041] A sodium hydroxide solution (pH=9.5) containing 10wt% 600kDa molecular weight sodium hyaluronate, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into a 5mL specification (filled with 5mL) syringe with a 27G injection needle (i.e., the above-mentioned hyaluronic acid syringe 1), and the pushing speed was set to 0.8mL / min; a sodium hydroxide solution (pH=9.5) containing 5wt% 40kDa molecular weight recombinant collagen, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into a 5mL specification (filled with 5mL) syringe with a 30G injection needle (i.e., the above-mentioned recombinant collagen syringe 5), and the pushing speed was set to 0.05mL / min. Then the two syringes were placed on the left and right sides of the upper part of a plastic hard tube with a diameter of 2mm and a length of 8cm, respectively. The plastic hard tube was provided with two injection holes with a vertical spacing of 4cm on the corresponding sides, and the injection needles of the two syringes were respectively inserted and fixed into the two injection holes on the plastic hard tube, and the two syringes were clamped and fixed. Then push the two syringes at a set constant speed, and when the materials pushed into the plastic tube by the two syringes begin to intersect and mix, the mixed material can be dripped into a beaker containing 2.5% (w / w) polyethylene glycol solution (500mL) under the plastic tube, and stirred at 400rpm for 1h under heating conditions (50°C). After being placed at room temperature (gel particles have been deposited), pour the obtained gel particles from the beaker and pass through an 800-mesh sieve, and adjust the pH of the separated gel particles to neutral using a 5% hydrochloric acid solution to form a nanogel for injection filling.

[0042] Experimental Example 3

[0043] A sodium hydroxide solution (pH=9.5) containing 10wt% 600kDa molecular weight sodium hyaluronate, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into a 5mL specification (filled with 5mL) syringe with a 27G injection needle (i.e., the above-mentioned hyaluronic acid syringe 1), and the pushing speed was set to 1.0mL / min; a sodium hydroxide solution (pH=9.5) containing 10wt% 40kDa molecular weight recombinant collagen, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into a 5mL specification (filled with 5mL) syringe with a 30G injection needle (i.e., the above-mentioned recombinant collagen syringe 5), and the pushing speed was set to 0.1mL / min. Then the two syringes were placed on the left and right sides of the upper part of a plastic hard tube with a diameter of 2mm and a length of 8cm, respectively. The plastic hard tube was provided with two injection holes with a vertical spacing of 4cm on the corresponding sides, and the injection needles of the two syringes were respectively inserted and fixed into the two injection holes on the plastic hard tube, and the two syringes were clamped and fixed. Then push two syringes at a set constant speed, and when the materials pushed into the plastic tube by the two syringes begin to intersect and mix, the mixed materials can be dripped into a beaker containing 2.5% (w / w) polyethylene glycol solution (500mL) under the plastic tube, and stirred at 200rpm for 1h under heating conditions (50°C). After being placed at room temperature (gel particles have been deposited), pour the obtained gel particles from the beaker and pass through an 800-mesh sieve, and adjust the pH of the separated gel particles to neutral using a 5% hydrochloric acid solution to form a nanogel for injection filling.

[0044] Experimental Example 4

[0045] A sodium hydroxide solution (pH=9.5) containing 5wt% 600kDa molecular weight sodium hyaluronate and 0.5wt% citric acid was loaded into a 5mL specification (filled with 5mL) syringe with a 27G injection needle (i.e., the above-mentioned hyaluronic acid syringe 1); a sodium hydroxide solution (pH=9.5) containing 10wt% 40kDa molecular weight recombinant collagen and 0.5wt% citric acid was loaded into a 5mL specification (filled with 5mL) syringe with a 30G injection needle (i.e., the above-mentioned recombinant collagen syringe 5). Others were the same as in Experimental Example 1.

[0046] Comparative Example 1

[0047] A sodium hydroxide solution (pH=9.5) containing 5wt% 600kDa molecular weight sodium hyaluronate, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into a 5mL specification (filled with 5mL) syringe with a 27G injection needle (i.e., the above-mentioned hyaluronic acid syringe 1), and the pushing speed was set to 2mL / min; a sodium hydroxide solution (pH=9.5) containing 10wt% 40kDa molecular weight recombinant collagen, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into a 5mL specification (filled with 5mL) syringe with a 30G injection needle (i.e., the above-mentioned recombinant collagen syringe 5), and the pushing speed was set to 0.01mL / min. Then the two syringes were placed on the left and right sides of the upper part of a plastic hard tube with a diameter of 2mm and a length of 8cm, respectively. The plastic hard tube was provided with two injection holes with a vertical spacing of 4cm on the corresponding sides, and the injection needles of the two syringes were respectively inserted and fixed into the two injection holes on the plastic hard tube, and the two syringes were clamped and fixed. Then push the two syringes at a set constant speed, and when the materials pushed into the plastic tube by the two syringes begin to intersect and mix, they can be dripped into the beaker containing 2.5% (w / w) polyethylene glycol solution (500mL) under the plastic tube, and stirred at 600rpm for 1h under heating conditions (50°C). After being placed at room temperature (gel particles have been deposited), pour the obtained gel particles from the beaker and pass through an 800-mesh sieve, and adjust the pH of the separated gel particles to neutral using 5% hydrochloric acid solution.

[0048] Comparative Example 2

[0049] A sodium hydroxide solution (pH=9.5) containing 5wt% 600kDa molecular weight sodium hyaluronate, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into a 5mL specification (filled with 5mL) syringe with a 27G injection needle (i.e., the above-mentioned hyaluronic acid syringe 1), and the pushing speed was set to 0.1mL / min; a sodium hydroxide solution (pH=9.5) containing 10wt% 40kDa molecular weight recombinant collagen, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into another 5mL specification (filled with 5mL) syringe with a 27G injection needle (i.e., the above-mentioned recombinant collagen syringe 5), and the pushing speed was set to 0.2mL / min. Then the two syringes were placed on the left and right sides of the upper part of a plastic hard tube with a diameter of 2mm and a length of 8cm, respectively. The plastic hard tube was provided with two injection holes with a vertical spacing of 4cm on the corresponding sides, and the injection needles of the two syringes were respectively inserted and fixed into the two injection holes on the plastic hard tube, and the two syringes were clamped and fixed. Then push the two syringes at a set constant speed, and when the materials pushed into the plastic tube by the two syringes begin to intersect and mix, the mixed material can be dripped into the beaker containing 2.5% (w / w) polyethylene glycol solution (500mL) under the plastic tube, and stirred at 600rpm for 1h under heating conditions (50°C). After it is placed at room temperature (gel particles have been deposited), pour the obtained gel particles from the beaker and pass through an 800-mesh sieve, and adjust the pH of the separated gel particles to neutral using 5% hydrochloric acid solution.

[0050] Comparative Example 3

[0051] A sodium hydroxide solution (adjusting pH=9.5) containing 5wt% 600kDa molecular weight sodium hyaluronate, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into a 5mL specification (filled with 5mL) syringe with a 27G injection needle (i.e., the above-mentioned hyaluronic acid syringe 1), and the pushing speed was set to 0.1mL / min; a sodium hydroxide solution (adjusting pH=9.5) containing 10wt% 40kDa molecular weight recombinant collagen, 0.5wt% citric acid and 10wt% ethylene glycol was loaded into a 5mL specification (filled with 5mL) syringe with a 30G injection needle (i.e., the above-mentioned recombinant collagen syringe 5), and the pushing speed was set to 0.01mL / min. Then the two syringes were placed on the left and right sides of the upper part of a plastic hard tube with a diameter of 2mm and a length of 8cm, respectively. The plastic hard tube was provided with two injection holes with a vertical spacing of 4cm on the corresponding sides, and the injection needles of the two syringes were respectively inserted and fixed into the two injection holes on the plastic hard tube, and the syringes were clamped and fixed. Then push the two syringes at a set constant speed, and when the materials pushed into the plastic straw by the two syringes begin to intersect and mix, the mixed material can be dripped into the beaker containing 2.5% (w / w) polyethylene glycol solution (500mL) under the plastic hard tube, and stirred at 100rpm for 1h under heating conditions (50°C). After it is placed at room temperature (gel particles have been deposited), pour the obtained gel particles from the beaker and pass through an 800-mesh sieve, and adjust the pH of the separated gel particles to neutral using 5% hydrochloric acid solution.

[0052] Comparative Example 4

[0053] Gel group experiment (adding citric acid and ethylene glycol):

[0054] Without using the above-mentioned fluid control device; add citric acid and 10wt% ethylene glycol with an initial concentration of 0.5wt% to a sodium hydroxide solution (pH=9.5) containing 5wt% 600kDa molecular weight sodium hyaluronate and 10wt% 40kDa molecular weight recombinant collagen, react at 50°C for 1h (without stirring), and then cool to room temperature after the reaction to observe the gel formed by cross-linking ( Figure 4 A), and then the remaining solution was removed through an 800-mesh sieve to separate the entire gel.

[0055] Solution group experiment (without citric acid and ethylene glycol):

[0056] Without using the above-mentioned fluid control device; a sodium hydroxide solution (pH=9.5) containing 5wt% 600kDa molecular weight sodium hyaluronate and 10wt% 40kDa molecular weight recombinant collagen was heated for 1h (50°C without stirring), and then the solution was allowed to cool to room temperature after the heating was stopped. No gel formation was observed, and the solution was still in the form of solution ( Figure 4 B).

[0057] Comparative Example 5

[0058] Without using the above-mentioned fluid control device; polyethylene glycol diglycidyl ether (PEGDE) with an initial concentration of 10wt% is added to a sodium hydroxide solution (pH adjusted to 9.5) containing 5wt% 600kDa molecular weight hyaluronic acid and 10wt% 40kDa molecular weight recombinant collagen, and cross-linked at 50°C for 1h (without stirring). After cross-linking, it is cooled to room temperature and passed through an 800-mesh sieve to separate the entire gel.

[0059] In the above preparation cases, recombinant collagen can use humanized type III collagen to avoid the occurrence of immune rejection reaction; the mixture (i.e., material) contained in the hyaluronic acid syringe 1 and the recombinant collagen syringe 5 is not cross-linked and can be pushed out of the injection needle; the purpose of screening is to separate the gel, and it has no effect of granulation and particle size limitation.

[0060] (III) Performance test of nanocomposite gel filler

[0061] 1. Appearance observation and particle size distribution detection

[0062] Using a cryo-scanning electron microscope, the gel samples obtained in Experimental Examples 1, 2, 3, 4 and Comparative Examples 1, 2, 3 were directly dispersed and observed, 10 fields of view were randomly selected, and more than 10 particles were measured in each field of view to obtain the particle size data (Table 1). The results can be seen in Experimental Example 1 ( Figure 5 ) and the gels obtained in Experimental Examples 2, 3, and 4 were spherical and uniform in size.

[0063] Table 1. Particle size analysis data

[0064]

[0065]

[0066] At the same time, the above results also show that in Experimental Examples 1, 2, 3, and 4, larger particle gels are formed by cross-linking (under the action of citric acid or citric acid and ethylene glycol) and nanogels with corresponding particle sizes are obtained after stirring and dispersion. After adjusting the pH, the nanogels meet the basic requirements for direct subcutaneous injection as tissue fillers.

[0067] The above comparative examples show that when the pushing speed of the hyaluronic acid syringe 1 is increased to more than 1.5 mL / min, or when the pushing speed of the recombinant collagen syringe 5 is increased to more than 0.1 mL / min, or when the stirring speed of the polyethylene glycol solution in the beaker is lower than 200 rpm, the gel particle size range will be significantly increased.

[0068] 2. Mechanical properties

[0069] Elastic modulus test method: perform strain scanning on the gel sample at 25°C, select the shear strain of 0.1%, the oscillation frequency of 10-0.1 Hz, and the sample relaxation time of 30 min in the linear viscoelastic region, perform oscillation frequency scanning, obtain the viscoelastic curve, and read the elastic modulus value at 0.1 Hz.

[0070] Pushing force test method: refer to Appendix A of YY T 0962-2021 Cross-linked Sodium Hyaluronate Gel for Plastic Surgery.

[0071] The test results are shown in Table 2.

[0072] Table 2. Elastic modulus and pushing force test data

[0073] Case Elastic modulus / Pa(0.1Hz) Pushing force / N Experimental Example 1 278 10 Experimental Example 2 349 15 Experimental Example 3 303 12 Experimental Example 4 253 11 Comparative Example 1 102 20 Comparative Example 2 78 18 Comparative Example 3 90 23 Comparative Example 4 (Gel Group) 87 30 Comparative Example 5 109 35

[0074] It can be seen from Table 2 that compared with the gels prepared in the comparative examples, the elastic modulus of the nanogels prepared in Experimental Examples 1, 2, 3, and 4 is significantly improved, and the pushing force is significantly reduced, that is, the nanogels prepared in the experimental examples have obvious advantages when used as injectable fillers.

[0075] 3. Degradation performance

[0076] Take 0.2g (initial weight) of the gel sample prepared in the above case, add 2mL enzyme solution (collagenase 0.5mg / mL and hyaluronidase 50U / mL). Place in a 37.0℃ incubator, and after 1, 2, 3, 4, 5, and 6 weeks, separate the gel sample from the degradation buffer (solvent for preparing the enzyme solution), drain, and weigh again. Calculate the degradation rate by the following formula:

[0077] Degradation rate (%) = 100-(W t / W0)×100

[0078] Among them, W0 and W t are the initial weight and re-weighing weight of the sample, respectively.

[0079] Depend on Figure 1 It can be seen that compared with the gel prepared in Comparative Example 4 (gel group) and Comparative Example 5, the nanogels prepared in Experimental Examples 1, 2, 3, and 4 are more resistant to degradation, that is, the nanogels prepared in the experimental examples have obvious advantages when used as injectable fillers.

[0080] 4. Cytotoxicity

[0081] Cytotoxicity test process: 0.2g / mL gel samples were grouped (experimental examples and comparative examples) and heat-extracted at 37°C for 24 hours (the extraction reagent was MEM medium) to obtain the extract. L929 mouse fibroblast suspension was added to a 96-well plate, 100 μL per well, for a total of 1×10 4cells / well, cultured at 37°C, 5% CO2 for 24 hours. After the culture was completed, the culture medium in the wells was discarded, and the extract was added in groups, with 6 wells in each group. A blank control group (L929 mouse fibroblast culture medium) was set up, and the wells were placed in a 37°C, 5% CO2 saturated humidity incubator. After 24 hours of culture, the 96-well plate was taken out, the liquid in the wells was discarded, 50 μL (1 mg / mL) of MTT stain was added to each well, and the wells were placed in a 37°C, 5% CO2 saturated humidity incubator for 2 hours. After the culture was completed, the liquid in the wells was discarded, 100 μL of isopropanol was added to each well, the mixture was shaken, and the plate was placed in an ELISA reader, and the reading was read at 570nm (reference wavelength 650nm).

[0082] Quantitative evaluation: OD value was measured and relative cell survival rate was calculated according to the following formula. A decrease of cell activity (relative cell survival rate) greater than 30% was considered to be a cytotoxic reaction. The evaluation of the reaction degree was referred to Table 3.

[0083] Relative cell survival rate (%) = (average OD value of the test group / average OD value of the blank control group) × 100%

[0084] Table 3. Cytotoxicity rating

[0085]

[0086]

[0087] Depend on Figure 2 It can be seen that the cell survival rates of Experimental Examples 1, 2, 3, and 4 are higher than those of Comparative Example 4 (gel group) and Comparative Example 5, and the cytotoxicity of Experimental Examples 1, 2, 3, and 4 is graded as Grade 0, while the cytotoxicity of Comparative Example 5 is Grade 2, that is, the cross-linking process for preparing nanogels of the present invention is lower in toxicity than the traditional method (see Comparative Example 5). Therefore, the preparation process of the nanogel of the present invention avoids the safety problems such as cytotoxicity existing in the preparation of gels using commonly used chemical cross-linking agents.

[0088] In short, the present invention forms nanogel by crosslinking hyaluronic acid (sodium) and collagen under high-speed stirring by means of a fluid control device. This hyaluronic acid / collagen composite gel has strong anti-degradation ability, wherein hyaluronic acid provides an immediate effect for deep filling, and protects the collagen compounded therewith, and with the degradation of hyaluronic acid, collagen is slowly released, and collagen is supplemented in the deep layer of the skin to stimulate collagen regeneration. More importantly, the composite gel has a larger specific surface area and stronger mechanical support, and can resist the shear force caused by facial muscle movement, thereby being more suitable for deep filling.

Claims

1. A method for preparing a composite gel for skin injection, characterized in that: The preparation method of the composite gel comprises the following steps: A first cross-linking preparatory liquid containing a first matrix component and a cross-linking auxiliary agent is prepared, and a second cross-linking preparatory liquid containing a second matrix component and a cross-linking auxiliary agent is prepared; 1-5 mL of the first cross-linking preparatory liquid and 1-5 mL of the second cross-linking preparatory liquid are mixed and then stirred and dispersed in a heated liquid medium to obtain a composite gel; The preparation method of the composite gel specifically comprises the following steps: 1) 600-2000 kDa hyaluronic acid or sodium hyaluronate, polycarboxylic acid substances, polyols and alkaline solutions are mixed at corresponding mass fractions of 5%-10%, 0.5%-2%, 0-20% and 68%-94.5% to obtain a first cross-linking preparatory solution, and the first cross-linking preparatory solution is loaded into a first syringe; 40-100 kDa recombinant collagen, polycarboxylic acid substances, polyols and alkaline solutions are mixed at corresponding mass fractions of 5%-10%, 0.5%-10%, 0-10% and 70%-92.5% to obtain a second cross-linking preparatory solution, and the second cross-linking preparatory solution is loaded into a second syringe; the polycarboxylic acid substances are selected from one or more of citric acid and citric acid derivatives; 2) inserting the first syringe and the second syringe into the upper part of the feeding tube (2), and then pushing the first syringe and the second syringe at 0.1-1.0 mL / min and 0.01-0.1 mL / min respectively, adding the first cross-linking preparatory liquid and the second cross-linking preparatory liquid mixed in the feeding tube (2) to form a larger particle gel through cross-linking, and then adding it to the surfactant, and stirring and dispersing at ≥200 rpm and 30-50° C. for 1-24 hours to obtain a nanogel with a particle size of ≤100 nm, and the surfactant is selected from a polyethylene glycol aqueous solution with a mass fraction of 0.5%-3%.

2. The method for preparing the composite gel according to claim 1, characterized in that: After step 2), the gel particles in the stirred system are separated by sieving, and then the pH of the gel particles is adjusted to 6.8-7.

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3. A composite gel obtained by the method for preparing a composite gel for skin injection according to claim 1, characterized in that: The composite gel is a nanogel made of a first matrix component, a second matrix component and a cross-linking auxiliary agent, wherein the first matrix component is hyaluronic acid or sodium hyaluronate, the second matrix component is recombinant collagen, and the cross-linking auxiliary agent includes a polycarboxylic acid substance; The composite gel is a collagen / hyaluronic acid nanocomposite gel formed by heating and stirring to increase the mixing degree of the first matrix component, the second matrix component and the cross-linking agent polycarboxylic acid substance and cross-linking the mixed first matrix component and the second matrix component under alkaline conditions, and the hyaluronic acid in the composite gel protects the collagen compounded therewith; The cross-linking auxiliary agent also includes polyols.

4. The composite gel according to claim 3, characterized in that: The polyol is selected from one or more of ethylene glycol, ethylene glycol derivatives, glycerol, and glycerol derivatives.

5. Use of the composite gel according to claim 3 in preparing a tissue filler.

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