A sodium hyaluronate composite injection containing lidocaine and its preparation process

Abstract

The present invention provides a sodium hyaluronate composite injection containing lidocaine and its preparation process, which belongs to the technical field of cosmetic injection preparation; its preparation process includes the following steps: preparation of modified sodium hyaluronate; preparation of lidocaine sustained-release microspheres; preparation of double-network structure gel; preparation of composite microgel injection. The present invention optimizes the formula of the composite injection and adds lidocaine, which can reduce the pain during injection and reduce the inflammatory response after injection, thereby improving the comfort and patient acceptance in clinical applications. At the same time, tripeptide-1 and phytosphingosine active ingredients are added to be compatible with sodium hyaluronate and regenerated silk fibroin. The synergistic effect can not only significantly improve the effects of skin filling, facial plastic surgery and skin moisturizing treatment, but also promote the long-term health and youthfulness of the skin, achieving a more comprehensive and lasting cosmetic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic injection preparation, and in particular to a lidocaine-containing sodium hyaluronate composite injection and a preparation process thereof. Background Art

[0002] Sodium hyaluronate, a naturally occurring high-molecular-weight polysaccharide in the human body, is widely distributed in various tissues, including the skin, joint fluid, and vitreous humor. Due to its excellent biocompatibility, moisturizing properties, and biodegradability, sodium hyaluronate has been widely used in medical cosmetology, ophthalmic surgery, and osteoarthritis treatment. In recent years, sodium hyaluronate injections have been increasingly used in skin filling, facial plastic surgery, and skin moisturizing treatments to improve skin appearance and texture and reduce wrinkles. However, traditional sodium hyaluronate injections have some problems during use, such as pain during injection and inflammatory reactions after injection. These problems limit their comfort and patient acceptance in clinical applications.

[0003] Therefore, we proposed a sodium hyaluronate composite injection containing lidocaine and its preparation process, which can reduce the pain during injection and the inflammatory response after injection, thereby improving the comfort and patient acceptance in clinical applications. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a sodium hyaluronate composite injection containing lidocaine and a preparation process thereof.

[0005] A preparation process of a sodium hyaluronate composite injection containing lidocaine comprises the following steps:

[0006] S1: Preparation of modified sodium hyaluronate

[0007] Sodium hyaluronate is activated with N-hydroxysuccinimide and 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide, followed by addition of cysteamine hydrochloride for reaction and subsequent dialysis to obtain modified sodium hyaluronate;

[0008] S2: Preparation of lidocaine sustained-release microspheres

[0009] Lidocaine sustained-release microspheres were prepared by loading lidocaine with poly-L-lactic acid and co-adding chitosan and poloxamer 407.

[0010] S3: Preparation of double network structure gel

[0011] The silk fibers were dialyzed in a lithium bromide solution to prepare a regenerated silk solution, which was then mixed with a modified sodium hyaluronate solution, and horseradish peroxidase and hydrogen peroxide were added to form a double-network structure gel after incubation.

[0012] S4: Preparation of composite microgel injections

[0013] The double-network structure gel was immersed in a phosphate buffer solution to obtain a block hydrogel. The block hydrogel, propylene glycol, tripeptide-1, phytosphingosine and lecithin were mixed and homogenized, and then a composite microgel was prepared by a liquid nitrogen quick freezing-crushing method. The composite microgel was dispersed in a physiological buffer solution and lidocaine sustained-release microspheres were added to obtain a composite microgel injection.

[0014] Furthermore, the preparation of modified sodium hyaluronate in step S1 specifically includes the following steps:

[0015] S1.1: Dissolve 4-5 parts by weight of sodium hyaluronate powder in 800-1000 parts by weight of deionized water, then add 2-3 parts by weight of N-hydroxysuccinimide and 5-7 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, mix well, and adjust the pH to 4-5 with 1.0-1.2 mol / L hydrogen chloride solution. Activate at room temperature for 2-3 hours to obtain an activated solution.

[0016] S1.2: Add 3-4 parts by weight of cysteamine hydrochloride to the activation solution, then adjust the pH to 4-5 with 1.0-1.2 mol / L sodium hydroxide solution, and react at 23-25°C in the dark for 20-24 hours to obtain a reactant;

[0017] S1.3: Transfer the reactants to a dialysis bag and dialyze with deionized water at a pH of 3.5-4 at 23-25°C in the dark for 3-4 days. Then, freeze-dry the dialyzed solution to obtain a modified sodium hyaluronate solid, which is then refrigerated at 4-5°C for later use.

[0018] Furthermore, step S2 of preparing lidocaine sustained-release microspheres specifically includes the following steps:

[0019] S2.1: Chitosan and poloxamer 407 are mixed, 1-2 wt% acetic acid is added, and the mixture is uniformly mixed. After dissolution, an in situ gel solution is obtained. 1-2 parts by weight of lidocaine hydrochloride is added to 10-15 parts by weight of the in situ gel solution to obtain a mixed solution.

[0020] S2.2: Dissolve 0.25-0.5 parts by weight of poly-L-lactic acid in 2-3 parts by weight of dichloromethane to obtain an oil phase. Add 0.2-0.3 parts by weight of the mixed solution to the oil phase and mix with ultrasound at 470-500W in an ice bath for 1-2 minutes to obtain a primary emulsion.

[0021] S2.3: Add the primary emulsion to 5-8 parts by weight of a 1-2% (w / v) polyvinyl alcohol solution, stir at 3400-3500 rpm using a high-speed disperser for 1-2 minutes to obtain a double emulsion, add the double emulsion to 60-80 parts by weight of a 0.2-0.5% (w / v) polyvinyl alcohol solution, and magnetically stir at 37-38°C for 4-5 hours to obtain a solidified microsphere solution;

[0022] S2.4: Centrifuge the solidified microsphere solution at 3500-4000 rpm for 10-12 minutes, and then wash the precipitate with distilled water 2-3 times to obtain lidocaine sustained-release microspheres.

[0023] Furthermore, step S3 of preparing the double network structure gel specifically includes the following steps:

[0024] S3.1: Dissolve 10-20 parts by weight of silk fibroin in 100-200 parts by weight of a 9-10 mol / L lithium bromide solution at 60-62°C with stirring for 60-70 minutes. After cooling, place the solution in a dialysis bag and dialyze against deionized water for 2-3 days. Filter the solution through absorbent cotton and centrifuge at 10,000-11,000 rpm for 5-10 minutes to obtain a regenerated silk fibroin solution.

[0025] S3.2: Dissolving the modified sodium hyaluronate solid in deionized water to form a 1-2% by mass modified sodium hyaluronate solution, adjusting the pH to 7.4-7.6 with sodium hydroxide solution, and then adding an equal amount of regenerated silk fibroin solution and mixing in an ice bath to obtain a mixed solution;

[0026] S3.3: Add horseradish peroxidase to the mixed solution, then add hydrogen peroxide, stir evenly, and incubate in a biochemical incubator at 37-38°C for 3-5 hours to obtain a double network structure gel.

[0027] Furthermore, step S4 of preparing the composite microgel injection specifically includes the following steps:

[0028] S4.1: Soak the double-network structure gel in 0.01-0.02 mol / L phosphate buffer solution for 3-4 days to obtain a block hydrogel;

[0029] S4.2: Add 10-20 parts by weight of the block hydrogel, 0.2-0.3 parts by weight of propylene glycol, and 1-2 parts by weight of tripeptide-1 to 50-80 parts by weight of pure water, and heat in a water bath at 45-48°C to dissolve to obtain a mixed solution;

[0030] S4.3: Add 1-2 parts by weight of phytosphingosine to 2-3 parts by weight of lecithin, dissolve by heating in a water bath at 45-48°C, then add to the mixture, ultrasonically disperse for 10-20 minutes, and homogenize using a microfluidizer to obtain a mixed emulsified gel.

[0031] S4.4: Quickly freeze the mixed emulsified gel solution in liquid nitrogen for 5-8 minutes, then crush and grind it in a mortar with a pestle. Sieve it through 400-500 μm and 300-400 μm stainless steel sieves, respectively, to obtain a 300-500 μm composite microgel.

[0032] S4.5: The composite microgel is uniformly dispersed in 30-50 parts by weight of physiological buffer, and then 2-3 parts by weight of lidocaine sustained-release microspheres are added, ultrasonically assisted oscillation is performed for 10-20 minutes, and vacuum-packed after sterilization to obtain a composite microgel injection.

[0033] Furthermore, the concentration of chitosan in the mixed solution of step S2.1 is 3.5-4%.

[0034] Furthermore, the concentration of Poloxamer 407 in the mixed solution of step S2.1 is 2.5-3%.

[0035] Furthermore, the concentration of horseradish peroxidase in the mixed solution of step S3.3 is 10-12 U / mL.

[0036] Furthermore, the concentration of hydrogen peroxide in the mixed solution in step S3.3 is 3-4 mmol / L.

[0037] A sodium hyaluronate compound injection containing lidocaine is prepared by any of the above-mentioned processes for preparing a sodium hyaluronate compound injection containing lidocaine.

[0038] Compared with the prior art, the present invention has at least the following beneficial effects:

[0039] 1. The present invention optimizes the formula of the composite injection and adds lidocaine, which can reduce the pain during injection and the inflammatory response after injection, thereby improving the comfort and patient acceptance in clinical applications. At the same time, the addition of tripeptide-1 and phytosphingosine active ingredients are combined with sodium hyaluronate and regenerated silk fibroin. The synergistic effect can not only significantly improve the effects of skin filling, facial plastic surgery and skin moisturizing treatment, but also promote the long-term health and youthfulness of the skin, achieving a more comprehensive and lasting cosmetic effect.

[0040] 2. The present invention obtains thiol-modified sodium hyaluronate by thiol-modifying sodium hyaluronate, and then utilizes the dual-mediating effect of horseradish peroxidase on the thiol groups on the side chains of the modified sodium hyaluronate and the phenolic hydroxyl groups on the side chains of the regenerated silk fibroin to generate disulfide bonds between the modified sodium hyaluronate macromolecules and dityrosine bonds between the tyrosine residues on the side chains of the regenerated silk fibroin macromolecules, thereby preparing a gel with a double network structure. The double network structure in the gel produces a mutual protection effect on the two components of the modified sodium hyaluronate and the regenerated silk fibroin fiber, enabling them to basically maintain the original particle morphology. The ability of the double network gel to resist enzymatic degradation is significantly enhanced, and the degradation rate is significantly reduced. Therefore, the use effect of the composite injection can be improved to achieve a lasting cosmetic effect.

[0041] 3. The present invention adds chitosan and poloxamer 407 to the preparation of lidocaine sustained-release microspheres. The addition of chitosan and poloxamer 407 greatly improves the drug encapsulation rate and drug loading of the microspheres, effectively improving the anesthetic and analgesic effects. In addition, poly-L-lactic acid is used as the encapsulation material to achieve sustained release of lidocaine. At the same time, poly-L-lactic acid can increase the expression of type III collagen, promote skin collagen regeneration, increase the expression of IL-10 and TIMP1, reduce inflammatory responses, and regulate the expression of matrix metalloproteinases MMP-2 and MMP-3, thereby reducing the destruction of collagen and elastin fibers in the extracellular matrix of aged skin. Poly-L-lactic acid has a significant promoting effect on fibrous tissue regeneration, thereby improving the cosmetic effect of the composite injection.

[0042] 4. The present invention prepares composite microgels with particle sizes mainly distributed in the range of 300-500 μm through the "liquid nitrogen quick freezing-crushing method". This method does not significantly damage the pore structure inside the microgel and can maintain a double-network structure. The regenerated silk component and high internal porosity in the composite microgel are more conducive to cell adhesion and spreading, and guide the uniform distribution of cells inside the gel. The composite microgel with a double-network structure can provide physical protection for cells, significantly maintain cell proliferation vitality, accelerate the regeneration and repair of soft tissues in the body, and improve its filling and cosmetic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, further serve to explain the principles of the present disclosure and to enable one skilled in the relevant art to make and use the present disclosure.

[0044] Figure 1 This is a flow chart of the preparation process of a sodium hyaluronate composite injection containing lidocaine used in an embodiment of the present invention. DETAILED DESCRIPTION

[0045] The following describes in detail the preparation process of a lidocaine-containing sodium hyaluronate composite injection provided by the present invention, in conjunction with the accompanying drawings and specific examples. It is also noted that, to provide a more detailed description, the following examples are best and preferred embodiments, and those skilled in the art may employ alternative methods for implementing certain known techniques. Furthermore, the accompanying drawings are intended only to provide a more detailed description of the embodiments and are not intended to limit the present invention.

[0046] Example 1

[0047] A preparation process of a sodium hyaluronate composite injection containing lidocaine, such as Figure 1 As shown, the following steps are included:

[0048] S1: Preparation of modified sodium hyaluronate

[0049] S1.1: Dissolve 4 parts by weight of sodium hyaluronate powder in 800 parts by weight of deionized water, then add 2 parts by weight of N-hydroxysuccinimide and 5 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, mix thoroughly, and adjust the pH to 4 with 1.0 mol / L hydrogen chloride solution. Activate at room temperature for 2 hours to obtain an activated solution.

[0050] S1.2: Add 3 parts by weight of cysteamine hydrochloride to the activation solution, adjust the pH to 4 with 1.0 mol / L sodium hydroxide solution, and react in the dark at 23°C for 20 h to obtain a reactant;

[0051] S1.3: Transfer the reactants to a dialysis bag and dialyze against deionized water (pH 3.5) at 23°C in the dark for 3 days. Freeze-dry the dialyzed solution to obtain a modified sodium hyaluronate solid. Refrigerate at 4°C until ready for use.

[0052] S2: Preparation of lidocaine sustained-release microspheres

[0053] S2.1: Chitosan and poloxamer 407 were mixed, and 1 wt% acetic acid was added. The mixture was mixed and dissolved to obtain an in situ gel solution. 1 part by weight of lidocaine hydrochloride was added to 10 parts by weight of the in situ gel solution to obtain a mixed solution. The chitosan concentration in the mixed solution was 3.5%, and the poloxamer 407 concentration was 2.5%.

[0054] S2.2: Dissolve 0.25 parts by weight of poly (L-lactic acid) in 2 parts by weight of dichloromethane to obtain an oil phase. Add 0.2 parts by weight of the mixed solution to the oil phase and mix with ultrasound at 470W for 1 minute in an ice bath to obtain a primary emulsion.

[0055] S2.3: The primary emulsion was added to 5 parts by weight of a 1% (w / v) polyvinyl alcohol solution, and stirred at 3400 rpm for 1 minute using a high-speed disperser to obtain a double emulsion. The double emulsion was then added to 60 parts by weight of a 0.2% (w / v) polyvinyl alcohol solution, and magnetically stirred at 37°C for 4 hours to obtain a solidified microsphere solution.

[0056] S2.4: Centrifuge the solidified microsphere solution at 3500 rpm for 10 min, and then wash the precipitate twice with distilled water to obtain lidocaine sustained-release microspheres;

[0057] S3: Preparation of double network structure gel

[0058] S3.1: Dissolve 10 parts by weight of silk fibroin in 100 parts by weight of a 9 mol / L lithium bromide solution at 60°C with stirring for 60 minutes. After cooling, place the solution in a dialysis bag and dialyze against deionized water for 2 days. Filter the solution through absorbent cotton and centrifuge at 10,000 rpm for 5 minutes to obtain a regenerated silk fibroin solution.

[0059] S3.2: Dissolving the modified sodium hyaluronate solid in deionized water to form a 1% (mass fraction) modified sodium hyaluronate solution, adjusting the pH to 7.4 with sodium hydroxide solution, and then adding an equal mass of the regenerated silk fibroin solution and mixing in an ice bath to obtain a mixed solution;

[0060] S3.3: Add horseradish peroxidase to the mixed solution to a concentration of 10 U / mL, then add hydrogen peroxide to a concentration of 3 mmol / L. After stirring, incubate in a biochemical incubator at 37°C for 3 h to obtain a double-network gel.

[0061] S4: Preparation of composite microgel injections

[0062] S4.1: Soak the double-network structured gel in 0.01 mol / L phosphate buffer solution for 3 days to obtain a block hydrogel;

[0063] S4.2: Add 10 parts by weight of the block hydrogel, 0.2 parts by weight of propylene glycol, and 1 part by weight of tripeptide-1 to 50 parts by weight of pure water, and heat in a 45°C water bath to dissolve to obtain a mixed solution;

[0064] S4.3: Add 1 part by weight of phytosphingosine to 2 parts by weight of lecithin, dissolve by heating in a 45°C water bath, and then add the mixture. Ultrasonic dispersion is performed for 10 minutes, and homogenization is performed using a microfluidizer to obtain a mixed emulsified gel solution.

[0065] S4.4: The mixed emulsified gel solution was quickly frozen in liquid nitrogen for 5 minutes, then crushed and repeatedly ground in a mortar with a pestle. The mixture was then sieved through 400 μm and 300 μm stainless steel meshes to obtain composite microgels of 300-400 μm in size.

[0066] S4.5: The composite microgel is uniformly dispersed in 30 parts by weight of physiological buffer, and then 2 parts by weight of lidocaine sustained-release microspheres are added, ultrasonically assisted oscillation is performed for 10 minutes, and vacuum packaging is performed after sterilization to obtain a composite microgel injection.

[0067] Example 2

[0068] A preparation process of a sodium hyaluronate composite injection containing lidocaine, such as Figure 1 As shown, the following steps are included:

[0069] S1: Preparation of modified sodium hyaluronate

[0070] S1.1: Dissolve 5 parts by weight of sodium hyaluronate powder in 1000 parts by weight of deionized water, then add 3 parts by weight of N-hydroxysuccinimide and 7 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, mix thoroughly, and adjust the pH to 5 with 1.2 mol / L hydrogen chloride solution. Activate at room temperature for 2 hours to obtain an activated solution.

[0071] S1.2: Add 4 parts by weight of cysteamine hydrochloride to the activation solution, adjust the pH to 5 with 1.2 mol / L sodium hydroxide solution, and react in the dark at 23°C for 20 h to obtain a reactant;

[0072] S1.3: Transfer the reactants to a dialysis bag and dialyze against deionized water (pH 4) at 23°C in the dark for 3 days. Freeze-dry the dialyzed solution to obtain a modified sodium hyaluronate solid. Refrigerate at 4°C until ready to use.

[0073] S2: Preparation of lidocaine sustained-release microspheres

[0074] S2.1: Chitosan and poloxamer 407 were mixed, and 2 wt% acetic acid was added. The mixture was uniformly mixed and dissolved to obtain an in situ gel solution. 2 parts by weight of lidocaine hydrochloride was added to 15 parts by weight of the in situ gel solution to obtain a mixed solution. The chitosan concentration in the mixed solution was 4%, and the poloxamer 407 concentration was 3%.

[0075] S2.2: Dissolve 0.5 parts by weight of poly (L-lactic acid) in 3 parts by weight of dichloromethane to obtain an oil phase. Add 0.3 parts by weight of the mixed solution to the oil phase and mix with ultrasound at 470W for 1 minute in an ice bath to obtain a primary emulsion.

[0076] S2.3: The primary emulsion was added to 8 parts by weight of a 2% (w / v) polyvinyl alcohol solution, and stirred at 3400 rpm for 1 minute using a high-speed disperser to obtain a double emulsion. The double emulsion was then added to 80 parts by weight of a 0.5% (w / v) polyvinyl alcohol solution, and magnetically stirred at 37°C for 4 hours to obtain a solidified microsphere solution.

[0077] S2.4: Centrifuge the solidified microsphere solution at 3500 rpm for 10 min, and then wash the precipitate twice with distilled water to obtain lidocaine sustained-release microspheres;

[0078] S3: Preparation of double network structure gel

[0079] S3.1: Dissolve 20 parts by weight of silk fibroin in 200 parts by weight of a 10 mol / L lithium bromide solution at 60°C with stirring for 60 minutes. After cooling, place the solution in a dialysis bag and dialyze against deionized water for 2 days. Filter the solution through absorbent cotton and centrifuge at 10,000 rpm for 5 minutes to obtain a regenerated silk fibroin solution.

[0080] S3.2: Dissolving the modified sodium hyaluronate solid in deionized water to form a 2% (mass fraction) modified sodium hyaluronate solution, adjusting the pH to 7.6 with sodium hydroxide solution, and then adding an equal mass of the regenerated silk fibroin solution and mixing in an ice bath to obtain a mixed solution;

[0081] S3.3: Add horseradish peroxidase to the mixed solution to a concentration of 12 U / mL, then add hydrogen peroxide to a concentration of 4 mmol / L. After stirring, incubate in a biochemical incubator at 37°C for 3 h to obtain a double-network gel.

[0082] S4: Preparation of composite microgel injections

[0083] S4.1: Soak the double-network structured gel in 0.02 mol / L phosphate buffer solution for 3 days to obtain a block hydrogel;

[0084] S4.2: Add 20 parts by weight of the block hydrogel, 0.3 parts by weight of propylene glycol, and 2 parts by weight of tripeptide-1 to 80 parts by weight of pure water, and heat in a water bath at 45°C to dissolve to obtain a mixed solution;

[0085] S4.3: Add 2 parts by weight of phytosphingosine to 3 parts by weight of lecithin, dissolve by heating in a 45°C water bath, and then add the mixture. Ultrasonic dispersion is performed for 10 minutes, and homogenization is performed using a microfluidizer to obtain a mixed emulsified gel solution.

[0086] S4.4: The mixed emulsified gel solution was quickly frozen in liquid nitrogen for 5 minutes, then crushed and repeatedly ground in a mortar with a pestle. The mixture was then sieved through 400 μm and 300 μm stainless steel meshes to obtain composite microgels of 300-400 μm in size.

[0087] S4.5: The composite microgel is uniformly dispersed in 50 parts by weight of physiological buffer, and then 3 parts by weight of lidocaine sustained-release microspheres are added, ultrasonically assisted oscillation is performed for 10 minutes, and vacuum packaging is performed after sterilization to obtain a composite microgel injection.

[0088] Example 3

[0089] A preparation process of a sodium hyaluronate composite injection containing lidocaine, such as Figure 1 As shown, the following steps are included:

[0090] S1: Preparation of modified sodium hyaluronate

[0091] S1.1: Dissolve 4 parts by weight of sodium hyaluronate powder in 800 parts by weight of deionized water, then add 2 parts by weight of N-hydroxysuccinimide and 5 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, mix thoroughly, and adjust the pH to 4 with 1.0 mol / L hydrogen chloride solution. Activate at room temperature for 3 hours to obtain an activated solution.

[0092] S1.2: Add 3 parts by weight of cysteamine hydrochloride to the activation solution, adjust the pH to 4 with 1.0 mol / L sodium hydroxide solution, and react at 25°C in the dark for 24 hours to obtain a reactant;

[0093] S1.3: Transfer the reactants to a dialysis bag and dialyze against deionized water (pH 3.5) at 25°C in the dark for 4 days. Freeze-dry the dialyzed solution to obtain a modified sodium hyaluronate solid. Refrigerate at 5°C until ready for use.

[0094] S2: Preparation of lidocaine sustained-release microspheres

[0095] S2.1: Chitosan and poloxamer 407 were mixed, and 1 wt% acetic acid was added. The mixture was uniformly mixed and dissolved to obtain an in situ gel solution. 1 part by weight of lidocaine hydrochloride was added to 10 parts by weight of the in situ gel solution to obtain a mixed solution. The chitosan concentration in the mixed solution was 3.5%, and the poloxamer 407 concentration was 2.5%.

[0096] S2.2: Dissolve 0.25 parts by weight of poly (L-lactic acid) in 2 parts by weight of dichloromethane to obtain an oil phase. Add 0.2 parts by weight of the mixed solution to the oil phase and mix with ultrasound at 500W in an ice bath for 2 minutes to obtain a primary emulsion.

[0097] S2.3: The primary emulsion was added to 5 parts by weight of a 1% (w / v) polyvinyl alcohol solution, and stirred at 3500 rpm for 2 minutes using a high-speed disperser to obtain a double emulsion. The double emulsion was then added to 60 parts by weight of a 0.2% (w / v) polyvinyl alcohol solution, and magnetically stirred at 38°C for 5 hours to obtain a solidified microsphere solution.

[0098] S2.4: Centrifuge the solidified microsphere solution at 4000 rpm for 12 minutes, and then wash the precipitate three times with distilled water to obtain lidocaine sustained-release microspheres.

[0099] S3: Preparation of double network structure gel

[0100] S3.1: Dissolve 10 parts by weight of silk fibroin in 100 parts by weight of 9 mol / L lithium bromide solution at 62°C with stirring for 70 minutes. After cooling, place the solution in a dialysis bag and dialyze against deionized water for 3 days. Filter the solution through absorbent cotton and centrifuge at 11,000 rpm for 10 minutes to obtain a regenerated silk fibroin solution.

[0101] S3.2: Dissolving the modified sodium hyaluronate solid in deionized water to form a 1% (mass fraction) modified sodium hyaluronate solution, adjusting the pH to 7.4 with sodium hydroxide solution, and then adding an equal mass of the regenerated silk fibroin solution and mixing in an ice bath to obtain a mixed solution;

[0102] S3.3: Add horseradish peroxidase to the mixed solution to a concentration of 10 U / mL, then add hydrogen peroxide to a concentration of 3 mmol / L. After stirring, incubate in a biochemical incubator at 38°C for 5 h to obtain a double-network gel.

[0103] S4: Preparation of composite microgel injections

[0104] S4.1: Soak the double-network structure gel in 0.01 mol / L phosphate buffer solution for 4 days to obtain a block hydrogel;

[0105] S4.2: Add 10 parts by weight of the block hydrogel, 0.2 parts by weight of propylene glycol, and 2 parts by weight of tripeptide-1 to 50 parts by weight of pure water, and heat in a water bath at 48°C to dissolve to obtain a mixed solution;

[0106] S4.3: Add 1 part by weight of phytosphingosine to 2 parts by weight of lecithin, dissolve by heating in a 48°C water bath, and then add the mixture. Ultrasonic dispersion is performed for 20 minutes, and homogenization is performed using a microfluidizer to obtain a mixed emulsified gel solution.

[0107] S4.4: The mixed emulsified gel solution was quickly frozen in liquid nitrogen for 8 minutes, then crushed and repeatedly ground in a mortar with a pestle. The mixture was then sieved through 500 μm and 400 μm stainless steel meshes to obtain composite microgels of 400-500 μm in size.

[0108] S4.5: The composite microgel is uniformly dispersed in 30 parts by weight of physiological buffer, and then 2 parts by weight of lidocaine sustained-release microspheres are added, ultrasonically assisted oscillation is performed for 20 minutes, and vacuum packaging is performed after sterilization to obtain a composite microgel injection.

[0109] Comparative Example 1

[0110] Compared with Example 1, the difference of Comparative Example 1 is that Comparative Example 1 removes step S3.3 and step S4.1, replaces the block hydrogel in step S4.2 with an equal weight portion of the mixed solution of step S3.2, and the other steps remain unchanged to prepare a composite microgel injection, which is recorded as Comparative Example 1.

[0111] Comparative Example 2

[0112] Compared with Example 1, the difference of Comparative Example 2 is that Comparative Example 2 removes step S1, step S3.3 and step S4.1, replaces the modified sodium hyaluronate solid in step S3.2 with an equal mass of sodium hyaluronate, and replaces the block hydrogel in step S4.2 with an equal weight of the mixed solution of step S3.2. The other steps remain unchanged to prepare a composite microgel injection, which is recorded as Comparative Example 2.

[0113] Evaluation of anti-enzymatic performance

[0114] Accurately weigh 0.5 g of the composite microgel injection of Examples 1-3 and Comparative Example 1, add 2 mL of phosphate buffer (0.1 mol / L, pH 7.0) and 2 mL of hyaluronidase solution (600 U / mL), mix well, and place in a 42 ° C water bath. At different enzymatic hydrolysis time points, 50 μL of the mixed solution was diluted to 3 mL, and the absorbance at 232 nm was measured using a Thermo Evolution 201 UV spectrophotometer. The time when the absorbance no longer changes is the enzymatic hydrolysis time.

[0115] The measurement results are shown in Table 1.

[0116] Table 1. Results of enzymatic hydrolysis time determination in Examples 1-3 and Comparative Example 1

[0117] Enzymatic hydrolysis time (h) Example 1 62 Example 2 60 Example 3 61 Comparative Example 1 21 Comparative Example 2 10.5

[0118] As can be seen from the data in Table 1, the enzymatic hydrolysis time of the embodiment is significantly higher than that of comparative examples 1-2, indicating that the ability of the prepared double network gel to resist enzymatic degradation is significantly enhanced and the degradation rate is significantly reduced. Therefore, the use effect of the composite injection can be improved, so that the composite injection can be retained in the body for a longer time, achieving a lasting cosmetic effect.

[0119] Comparative Example 3

[0120] Compared with Example 1, the difference of Comparative Example 3 is that step S2.1 is removed in Comparative Example 3, and the mixed solution in S2.2 is replaced with an equal weight portion of lidocaine hydrochloride. The other steps remain unchanged to prepare a composite microgel injection, which is recorded as Comparative Example 3.

[0121] The drug loading and encapsulation efficiency of the lidocaine sustained-release microspheres of Examples 1-3 and Comparative Example 3 were determined by photometry. The determination results are shown in Table 2.

[0122] Table 2. Drug loading and encapsulation efficiency test results of Examples 1-3 and Comparative Example 3

[0123] Drug loading (%) Encapsulation efficiency (%) Example 1 23.17 42.34 Example 2 22.89 41.28 Example 3 22.91 41.82 Comparative Example 3 13.68 26.87

[0124] From the data in Table 2, it can be seen that the drug loading and encapsulation efficiency of the embodiment are higher than those of the comparative example, indicating that the addition of chitosan and poloxamer 407 greatly improves the drug encapsulation efficiency and drug loading of the microspheres, effectively improving and prolonging the anesthetic and analgesic effects.

[0125] Comparative Example 4

[0126] Compared with Example 1, Comparative Example 4 is different in that step S2 is removed and the lidocaine sustained-release microspheres in step S4.5 are replaced with lidocaine hydrochloride in equal parts by weight, which is recorded as Comparative Example 4.

[0127] Evaluation of collagen content secreted by cells

[0128] Cells from Examples 1-3 and Comparative Example 4 were cultured for 7 days, and the total collagen content was determined using a collagen ELISA kit. The results are shown in Table 3.

[0129] Table 3. Collagen content determination results of Examples 1-3 and Comparative Example 4

[0130] Collagen content pg / mL Example 1 39.2 Example 2 38.7 Example 3 38.9 Comparative Example 4 30.4

[0131] It can be seen from the data in Table 3 that the data in Example 3 are all higher than those in Comparative Example 4, indicating that the use of poly-L-lactic acid as an encapsulating material can achieve sustained release of lidocaine while promoting autologous collagen secretion, thereby achieving a long-lasting and natural beauty effect.

[0132] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A process for preparing a sodium hyaluronate composite injection containing lidocaine, characterized in that: The steps include: S1: Preparation of modified sodium hyaluronate Sodium hyaluronate is activated with N-hydroxysuccinimide and 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide, and then cysteamine hydrochloride is added for reaction and dialyzed to obtain modified sodium hyaluronate; S2: Preparation of lidocaine sustained-release microspheres S2.1: Chitosan and poloxamer 407 are mixed, 1-2 wt% acetic acid is added, and the mixture is uniformly mixed. After dissolution, an in situ gel solution is obtained. 1-2 parts by weight of lidocaine hydrochloride is added to 10-15 parts by weight of the in situ gel solution to obtain a mixed solution. S2.2: Dissolve 0.25-0.5 parts by weight of poly-L-lactic acid in 2-3 parts by weight of dichloromethane to obtain an oil phase. Add 0.2-0.3 parts by weight of the mixed solution to the oil phase and mix with ultrasound at 470-500W in an ice bath for 1-2 minutes to obtain a primary emulsion. S2.3: Add the primary emulsion to 5-8 parts by weight of a 1-2% (w / v) polyvinyl alcohol solution, stir using a high-speed disperser at 3400-3500 rpm for 1-2 minutes to obtain a double emulsion, add the double emulsion to 60-80 parts by weight of a 0.2-0.5% (w / v) polyvinyl alcohol solution, and magnetically stir at 37-38°C for 4-5 hours to obtain a solidified microsphere solution; S2.4: Centrifuge the solidified microsphere solution at 3500-4000 rpm for 10-12 minutes, and then wash the precipitate with distilled water 2-3 times to obtain lidocaine sustained-release microspheres; S3: Preparation of double network structure gel The silk fibers were dialyzed in a lithium bromide solution to prepare a regenerated silk fibroin solution, which was then mixed with a modified sodium hyaluronate solution, and horseradish peroxidase and hydrogen peroxide were added to form a double-network structure gel after incubation. S4: Preparation of composite microgel injections S4.1: Soak the double-network structure gel in 0.01-0.02 mol / L phosphate buffer solution for 3-4 days to obtain a block hydrogel; S4.2: Add 10-20 parts by weight of the block hydrogel, 0.2-0.3 parts by weight of propylene glycol, and 1-2 parts by weight of tripeptide-1 to 50-80 parts by weight of pure water, and heat in a water bath at 45-48°C to dissolve to obtain a mixed solution; S4.3: Add 1-2 parts by weight of phytosphingosine to 2-3 parts by weight of lecithin, dissolve by heating in a water bath at 45-48°C, then add to the mixture, ultrasonically disperse for 10-20 minutes, and homogenize using a microfluidizer to obtain a mixed emulsified gel. S4.4: Quickly freeze the mixed emulsified gel solution in liquid nitrogen for 5-8 minutes, then crush and grind it in a mortar with a pestle. Sieve it through 400-500 μm and 300-400 μm stainless steel sieves, respectively, to obtain a 300-500 μm composite microgel. S4.5: The composite microgel is uniformly dispersed in 30-50 parts by weight of physiological buffer, and then 2-3 parts by weight of lidocaine sustained-release microspheres are added, ultrasonically assisted oscillation is performed for 10-20 minutes, and vacuum-packed after sterilization to obtain a composite microgel injection.

2. The preparation process of a sodium hyaluronate composite injection containing lidocaine according to claim 1, characterized in that: Step S1: Preparation of modified sodium hyaluronate, specifically comprising the following steps: S1.1: Dissolve 4-5 parts by weight of sodium hyaluronate powder in 800-1000 parts by weight of deionized water, then add 2-3 parts by weight of N-hydroxysuccinimide and 5-7 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, mix thoroughly, and adjust the pH to 4-5 with 1.0-1.2 mol / L hydrogen chloride solution. Activate at room temperature for 2-3 hours to obtain an activated solution. S1.2: Add 3-4 parts by weight of cysteamine hydrochloride to the activation solution, then adjust the pH to 4-5 with 1.0-1.2 mol / L sodium hydroxide solution, and react at 23-25°C in the dark for 20-24 hours to obtain a reactant; S1.3: Transfer the reactants to a dialysis bag and dialyze with deionized water at a pH of 3.5-4 at 23-25°C in the dark for 3-4 days. Then, freeze-dry the dialyzed solution to obtain a modified sodium hyaluronate solid, which is then refrigerated at 4-5°C for later use.

3. The preparation process of a lidocaine-containing sodium hyaluronate composite injection according to claim 2, characterized in that: Step S3: Preparation of the double network structure gel, specifically comprising the following steps: S3.1: Dissolve 10-20 parts by weight of silk fibroin in 100-200 parts by weight of a 9-10 mol / L lithium bromide solution at 60-62°C with stirring for 60-70 minutes. After cooling, place the solution in a dialysis bag and dialyze against deionized water for 2-3 days. Filter the solution through absorbent cotton and centrifuge at 10,000-11,000 rpm for 5-10 minutes to obtain a regenerated silk fibroin solution. S3.2: Dissolve the modified sodium hyaluronate solid in deionized water to form a 1-2% by mass modified sodium hyaluronate solution, then adjust the pH to 7.4-7.6 with sodium hydroxide solution, then add an equal amount of regenerated silk fibroin solution and mix in an ice bath to obtain a mixed solution; S3.3: Add horseradish peroxidase to the mixed solution, then add hydrogen peroxide, stir evenly, and incubate in a biochemical incubator at 37-38°C for 3-5 hours to obtain a double network structure gel.

4. The preparation process of a lidocaine-containing sodium hyaluronate composite injection according to claim 1, characterized in that: The concentration of chitosan in the mixed solution of step S2.1 is 3.5-4%.

5. The preparation process of a lidocaine-containing sodium hyaluronate composite injection according to claim 1, characterized in that: The concentration of Poloxamer 407 in the mixed solution of step S2.1 is 2.5-3%.

6. The preparation process of a lidocaine-containing sodium hyaluronate composite injection according to claim 3, characterized in that: The concentration of horseradish peroxidase in the mixed solution of step S3.3 is 10-12 U / mL.

7. The process for preparing a lidocaine-containing sodium hyaluronate composite injection according to claim 3, wherein: The concentration of hydrogen peroxide in the mixed solution in step S3.3 is 3-4 mmol / L.

8. A sodium hyaluronate composite injection containing lidocaine, characterized in that: The hyaluronate compound injection is prepared by the preparation process of the lidocaine-containing sodium hyaluronate compound injection according to any one of claims 1 to 7.

Images