Preparation method of semi-fluid micro-crosslinking gel

Abstract

The invention provides a preparation method of semi-fluid micro-crosslinked gel, which comprises the following steps: dissolving a mixture of sodium hyaluronates with various different molecular weights in an alkaline solution, and centrifuging after the mixture is completely dissolved, so that the sodium hyaluronates with various different molecular weights are longitudinally arranged to realize longitudinal gradient distribution; the preparation method comprises the following steps: adding a cross-linking agent dropwise in a centrifugal state to form a layered gradient cross-linked structure, further introducing ultrasonic treatment to induce transverse rearrangement of molecules, fully utilizing the limited cross-linking agent to carry out cross-linking again, improving the utilization rate of the cross-linking agent, enhancing the mechanical property of the gel and reducing the residue of the cross-linking agent, and finally carrying out post-treatment to obtain the semi-fluid micro-cross-linked gel. The semi-fluid micro-crosslinking gel prepared by the preparation method disclosed by the invention has good mechanical properties and biological integration.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to a preparation method of a semi-fluid micro-crosslinked gel. Background Art

[0002] In recent years, crosslinked gels can be roughly divided into two categories. The first category is low-crosslinked gels with a crosslinking degree <1%, which have a relatively soft texture and good plasticity, but are prone to problems such as edema, papules, particles, and the Tyndall effect after injection. The second category is high-crosslinked gels with a crosslinking degree >2%, which can have a retention stability in the body of 12 to 24 months. However, due to the relatively high crosslinking degree of such products, there are problems such as high viscoelasticity, poor plasticity, and tissue stiffness after injection.

[0003] Chinese Patent with application publication number CN 103126975A discloses, in the preparation method of a hydrogel patch matrix with gradient drugs recorded therein, that a centrifugation method is used to achieve a gradient distribution of crosslinking agents, thereby obtaining a gradient-crosslinked hydrogel. This patent utilizes the different sedimentation speeds of calcium salt powders under the action of centrifugal force to achieve crosslinking of different gradients. Although the gradient distribution can be regulated by controlling the centrifugation temperature, speed, and time, this method cannot accurately quantify the ratio and crosslinking degree between different gradients. In addition, due to the problem of uneven contact between calcium salt powders and sodium alginate solution in this patent, the prepared gel products have poor uniformity and mechanical properties, and problems such as papules, particles, and unevenness will occur when used as injection products.

[0004] Chinese Patent CN115252895A, a unidirectional fibrous crosslinked sodium hyaluronate dermal implant and its preparation method, discloses that a mixed solution of sodium hyaluronate and a crosslinking agent is longitudinally arranged by high-speed centrifugation and crosslinked under low-speed centrifugation to obtain a gradient crosslinked gel. This patent provides shear force through centrifugation to make the long-chain molecules of hyaluronic acid, which are intertwined or in a free state and have a disordered structure, tend to be consistent, realizing the longitudinal arrangement of linear molecules of sodium hyaluronate in solution or gel state. However, this system cannot prepare gradient crosslinked gels with different density regions.

[0005] Therefore, there is a need to prepare a new type of gel to solve the problems of high viscoelasticity, poor plasticity, and too fast degradation rate of crosslinked gels in existing problems. Summary of the Invention

[0006] The purpose of the present invention is to provide a semi-fluid micro-crosslinked gel, which has both the easy plasticity of a fluid and the high cohesion and viscoelasticity brought by a network scaffold, and can solve the contradiction between viscoelasticity and degradation rate in existing gel products.

[0007] To achieve the above object, the present invention provides a method for preparing a semi-fluid micro-crosslinked gel, which is characterized by comprising the following steps:

[0008] S1. Dissolve a mixture of sodium hyaluronates with multiple molecular weights in an alkaline solution. After complete dissolution, centrifuge at a rotational speed of 3000 - 10000 r / min for 20 - 60 min at a temperature of 2 - 20 °C;

[0009] S2. Maintain the centrifugal rotational speed and uniformly drip a crosslinking agent into the centrifuge tube at a speed of 5 - 30 mm / min. After the crosslinking agent is completely dripped, continue to centrifuge for 20 - 60 min at a temperature of 2 - 20 °C to obtain Gel A;

[0010] S3. Ultrasonicate Gel A obtained in step S2 at an ultrasonic frequency of 20 - 35 KHz, a power of 100 - 360 W, an ultrasonic temperature of 30 - 50 °C, and an ultrasonic time of 1 - 5 min, and then continue to stand at 30 - 50 °C for 20 - 60 min to obtain Gel B;

[0011] S4. Neutralize, dialyze, and wash Gel B with a buffer solution to control the final concentration of sodium hyaluronate in Gel B at 20 ± 10 mg / mL;

[0012] S5. Then fill and sterilize Gel B obtained in step S4 to obtain the semi-fluid micro-crosslinked gel.

[0013] Further, the mixture of sodium hyaluronates with multiple molecular weights includes low-molecular-weight sodium hyaluronate with a molecular weight of 20 - 40 KDa, medium-molecular-weight sodium hyaluronate with a molecular weight of 80 - 120 KDa, and high-molecular-weight sodium hyaluronate with a molecular weight of 200 - 300 KDa; the corresponding mass ratio is 1:(0.5 - 5):(0.5 - 5).

[0014] Further, the alkaline solution is any one of sodium hydroxide, potassium hydroxide, disodium hydrogen phosphate, and sodium carbonate solutions with a pH of 9 - 13 and a concentration of 2 - 100 mg / mL; the mass-to-volume ratio of the alkaline solution to the sodium hyaluronate mixture is 1:5 - 1:15, where the mass unit is g and the volume unit is mL.

[0015] Further, the crosslinking agent is any one of butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), N-hydroxysuccinimide (NHS), polyethylene glycol, and polyethylene glycol derivatives;

[0016] The mass ratio of the crosslinking agent to the sodium hyaluronate mixture is 0.05 - 0.4:1.

[0017] Further, a pharmaceutical agent or a cosmetic active ingredient can also be added to the semi-fluid micro-crosslinked gel.

[0018] Further, the pharmaceutical agent is selected from any one or more of antibacterial substances, antihistamine drugs, anti-inflammatory drugs, anesthetics, analgesics, proteins / peptides, and antiviral compounds.

[0019] Further, the cosmetic active ingredient is selected from any one or more of humectants, skin beautifying agents, vitamins, proteins / amino acids, antioxidants, and particulate fillers.

[0020] Further, the mass percentage of the pharmaceutical agent or cosmetic active ingredient is 0.01% - 70%.

[0021] Further, the semi-fluid micro-crosslinked gel is applied to the preparation of biomedicines, medical devices, or cosmetics.

[0022] The pharmaceutical agents applicable to be added to the semi-fluid micro-crosslinked gel of the present invention can be water-soluble, slightly water-soluble, and water-insoluble pharmaceutical agents, including, but not limited to:

[0023] 1) Antibacterial substances, such as β-lactam antibiotics, such as cefoxitin, n-imipenem, and other thienamycin derivatives, tetracycline, chloramphenicol, neomycin, carbenicillin, polymyxin, penicillin G, polymyxin B, vancomycin, cefazolin, cephalothin II, rifamycin SV sodium, gramicidin, bacitracin, sulfonamides, aminoglycoside antibiotics, such as gentamicin, kanamycin, amikacin, sisomicin, and tobramycin; nalidixic acid and analogs such as norfloxacin and flumequine / amifloxacin (pramifloxacin) antibacterial compositions; quinocetone, and the like;

[0024] 2) Antihistamines, such as diphenhydramine, chlorpheniramine, tetrahydrozoline, antazoline, and the like;

[0025] 3) Anti-inflammatory drugs, such as cortisone, hydrocortisone, hydrocortisone acetate, betamethasone, dexamethasone, dexamethasone sodium phosphate, prednisone, methylprednisolone, medrysone, flucortolone, prednisolone, prednisolone sodium phosphate, sulindac, its salts, and its corresponding sulfides, and the like;

[0026] 4) Antiviral active ingredients, such as acyclovir, 5-iodo-2'-deoxyuridine (IDU), vidarabine (ara-cytidine A), trifluridine, and interferon and interferon inducers;

[0027] 5) Anesthetics, such as etidocaine cocaine, cinchocaine hydrochloride, dyclonine hydrochloride, naepaine, phenacaine, piprocaine, proparacaine hydrochloride, tetracaine hydrochloride, hexycaine, bupivacaine, lidocaine, mepivacaine, and prilocaine;

[0028] 6) Peptides and proteins, such as atrial natriuretic peptide, calcitonin gene-related peptide, luteinizing hormone, releasing hormone, vasoactive intestinal peptide, antidiuretic hormone, cyclosporine, botulinum toxin, interferon, substance P enkephalin, epidermal growth factor, ocular growth factor, fibronectin, insulin-like growth factor and mesodermal growth factor, recombinant collagen, recombinant elastin, recombinant mussel adhesive protein, recombinant fibronectin;

[0029] Those skilled in the art should understand that the drug compounds listed above are only illustrative.

[0030] Cosmetic active ingredients that can be added to the semi-fluid micro-crosslinked gel of the present invention can be moisturizers, skin beautifying agents, vitamins, amino acids, antioxidants, particulate fillers; cosmetic active ingredients for incorporation into the present invention include, but are not limited to:

[0031] 1) Moisturizing agents include glycerol, sorbitol, propylene glycol, dipropylene glycol, 1,3-butanediol, pentyl ethylene glycol, chondroitin sulfate, pyrrolidone carboxylate, polyoxyethylene glycoside and polyoxypropylene methyl glycoside, and the like;

[0032] 2) Skin beautifying agents include whitening agents such as placenta extract, arbutin, glutathione, ellagic acid, linoleic acid, tranexamic acid, cell activators, photosensitizers, α-hydroxy acids and β-hydroxy acids; rough and dry skin improvers such as tannic acid, vitamin E nicotinate, inositol nicotinate; skin astringents such as zinc oxide and tannic acid, and anti-seborrheic agents such as sulfur and dimethylthianthrene; and skin colorants such as α-hydroxyacetone, and the like;

[0033] 3) Vitamins include vitamin A such as vitamin A oil, retinol, retinol acetate and retinol palmitate, vitamin B2 such as riboflavin, riboflavin tetrabutyrate and flavin adenine dinucleotide, vitamin B6 such as pyridoxine hydrochloride, pyridoxine dihexanoate, pyridoxine tripalmitate, etc., vitamin B12 and its derivatives, vitamin B15 and its derivatives; vitamin C such as L-ascorbic acid, L-ascorbic acid dipalmitate, sodium (L-ascorbic acid)-2-sulfate and dipotassium L-ascorbic acid diphosphate; vitamin D, such as calciferol and vitamin D, vitamin E, such as α-tocopherol, β-tocopherol, γ-tocopherol and DL-α-tocopherol acetate and DL-α-tocopherol nicotinate and DL-α-tocopherol succinate, vitamin H, vitamin P, nicotinic acid, such as niacin, benzyl nicotinate and benzyl nicotinamide; pantothenic acid, such as calcium pantothenate, D-panthenol, panthenol ethyl ether and acetyl panthenol ethyl ether; biotin, and the like;

[0034] 4) Proteins and amino acids include glycine, valine, leucine, isoleucine, serine, threonine, lysine, aspartic acid, glutamic acid, cystine, cysteine, methionine and tryptophan; examples of nucleic acids include deoxyribonucleic acid; and examples of hormones include estradiol and vinyl estradiol, etc.; recombinant collagen, recombinant elastin, recombinant mussel mucin, recombinant fibronectin;

[0035] 5) Antioxidants include vitamin E, butylated hydroxyanisole and phytic acid;

[0036] 6) The particle size of the particulate filler is preferably from 0.02 to 100 microns. Suitable particulate fillers include spherical silica gel, silica gel powder, polymethyl methacrylate, acrylate copolymer, calcium silicate, cellulose, magnesium aluminum silicate, magnesium trisilicate, montmorillonite, or mixtures thereof.

[0037] The above particulate fillers can be treated alone or in combination with lecithin, amino acids, mineral oil, silicone oil or other various preparations on the particle surface, and the coatings used for surface treatment can be lipophilic or hydrophilic in nature.

[0038] Those skilled in the art should understand that the above-listed cosmetic active ingredients are only illustrative.

[0039] Depending on the added ingredients, the semi-fluid micro-crosslinked gel prepared by the present invention can be applied to fields such as biomedicine, medical devices and medical beauty.

[0040] Further, the semi-fluid micro-crosslinked gel provided by the present invention can be used for the preparation of skin wound repair products, soft tissue filling materials or drug sustained release products.

[0041] Beneficial effects:

[0042] By synergistically treating gradient centrifugation and ultrasound, the present invention synergistically controls molecular arrangement and crosslinking kinetics, constructs a semi-fluid micro-crosslinked gel with a three-dimensional gradient network structure, realizes multi-scale mechanical property regulation. The prepared semi-fluid micro-crosslinked gel has excellent cohesiveness and viscoelasticity, the longitudinal crosslinking density gradient adapts to the physiological characteristics of human tissues, has excellent biocompatibility, and can achieve gradient degradation.

[0043] In the centrifugation stage of the present invention, by utilizing the sedimentation coefficient difference of sodium hyaluronate with different molecular weights, a longitudinal gradient distribution of molecular weight is formed through centrifugation parameter regulation, laying a structural foundation for subsequent differential crosslinking; in the ultrasound stage, ultrasound treatment promotes the directional migration of unreacted crosslinking agents, promotes the orderly diffusion of crosslinking agent molecules in the transverse region, and combines with the thermal effect to activate the movement of polymer chain segments, constructing an orderly crosslinked network in the transverse dimension, thereby forming a semi-fluid micro-crosslinked gel with a three-dimensional gradient network structure.

[0044] The semi-fluid micro-crosslinked gel prepared by the present invention has a three-dimensional gradient network structure that can be highly integrated with tissues, promoting tissue repair and integration. Compared with gel products with a single molecular weight, the longitudinal crosslinking density gradient forms a bionic match with the skin tissue structure, which can improve the tissue integration degree and biocompatibility of the semi-fluid gel in vivo. On the other hand, the semi-fluid micro-crosslinked gel prepared by the present invention has a gradient crosslinking density that forms a differential enzyme cleavage site distribution, and realizes gradient degradation through the sequential control of the disentanglement-breakage of molecular chains, effectively prolonging the action time.

[0045] In addition, for the gel constructed by this solution, its longitudinal crosslinking network provides lasting support, and the transverse crosslinking network endows viscoelasticity and cohesion. The three-dimensional interpenetrating gradient structure of its longitudinal gradient crosslinking and transverse uniform crosslinking breaks through the mechanical equilibrium limit of traditional gels, overcomes the technical problem that it is difficult to have both viscoelasticity, cohesion and support in existing gel products, has both anti-displacement property and injection compliance, is not easy to displace after being injected into the human body, and is highly integrated with human tissues, avoiding the Tyndall effect, providing a new idea for the preparation of gel products.

[0046] The preparation method of the present invention reduces the overall usage amount of the crosslinking agent, but does not weaken the overall crosslinking structure. This is because this solution combines different crosslinking methods of centrifugation and ultrasound, and the two-stage crosslinking strategy improves the utilization rate of the crosslinking agent, enabling it to play a role in both longitudinal crosslinking and transverse crosslinking. Moreover, due to the relatively small total amount of the crosslinking agent used, there is less crosslinking agent residue in the prepared gel product, and the safety is higher.

[0047] Finally, the gel prepared by the present invention is a semi-fluid micro-crosslinked gel, which is easier to inject compared to gels with a higher degree of crosslinking. In addition, the gel product prepared by the present invention has longitudinal and transverse network structures, and the product prepared by the present invention has a gradually tighter crosslinking degree from top to bottom. Compared with traditional micro-crosslinked gel products, the gel product prepared by this method has stronger anti-enzymolysis ability, a slower degradation period, and a longer action time. Detailed implementation mode

[0048] Figure 1 It is a column chart of the swelling degree of Example 1, Comparative Example 1, and Comparative Examples 7-8.

[0049] Figure 2 It is a column chart of the rotational rheological data of Example 1, Comparative Example 1, Comparative Example 4, and Comparative Example 6.

[0050] Figure 3 It is a column chart of the pushing force data of Example 1, Comparative Examples 2-3, and Comparative Example 5.

[0051] Figure 4 It is a microscope photo of Examples 1-3, Comparative Example 1, and commercially available similar products.

[0052] Figure 5 These are gel pictures of Example 1, Comparative Examples 1-3, and Comparative Example 5 at extrusion times of 15 s, 75 s, and 90 s.

[0053] Figure 6 These are microscopic cell morphology pictures of the cytotoxicity test of the semi-fluid micro-crosslinked gel prepared in Example 1.

[0054] Figure 7 These are pictures of rat subcutaneous tissue sections 2 weeks after injection of the semi-fluid micro-crosslinked gels prepared in Example 1 and Example 9. Detailed implementation manners

[0056] To more fully understand the technical content of the present invention, the present invention will be further introduced and described below in conjunction with the drawings and specific embodiments; obviously, the following described embodiments are only a part of the embodiments of the present invention, rather than all embodiments; based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present invention. For those skilled in the art, the features, beneficial effects, and advantages of the present invention will become obvious by reading the content disclosed in this specification.

[0057] Unless otherwise specified, all percentages, fractions, and ratios are calculated based on the total mass of the compositions of the present invention. The term "mass content" herein can be represented by the symbol "%"

[0058] In the present invention, the proportions of each component in the composition and the cosmetic are the same.

[0059] The terms "include", "comprise", "contain", "have", or other variants herein are intended to cover non-closed inclusion, and there is no distinction between these terms. The term "comprise" means that other steps and components can be added without affecting the final result. The term "comprise" also includes the terms "consist of" and "consist essentially of". The compositions and methods / processes of the present invention can comprise, consist of, and consist essentially of the essential elements and limitations described herein and any additional or optional components, ingredients, steps, or limitations described herein.

[0060] Now, through the following multiple specific embodiments, the preparation method of the semi-fluid micro-crosslinked gel provided by the present invention is further illustrated to achieve a semi-fluid micro-crosslinked gel with different density network structures, having both the plasticity of a fluid and the high cohesion and viscoelasticity brought by the network scaffold, and also having relatively stable support. Subsequently, a certain amount of pharmaceutical preparation or cosmetic active ingredient can be added to enable it to be applied in the fields of biomedicine, medical devices, and cosmetics.

[0061] Example 1:

[0062] S1: Weigh and combine sodium hyaluronate with a molecular weight of 30KDa, sodium hyaluronate with a molecular weight of 100KDa, and sodium hyaluronate with a molecular weight of 220KDa in a mass ratio of 1:1:2 to obtain a sodium hyaluronate mixture with multiple molecular weights. The total weight is 4g, which is dissolved in 40mL of a sodium hydroxide solution with a pH of 10 and a concentration of 10mg / mL. After complete dissolution, it is added to a centrifuge tube and centrifuged at 4°C and 5000r / min for 30min.

[0063] S2: Under the conditions of maintaining the centrifugation speed and the centrifugation temperature of 4°C, 0.6g of polyethylene glycol diglycidyl ether (PEGDE) is added to the centrifuge tube at a uniform speed of 10mm / min. After the addition of PEGDE is complete, while maintaining the centrifugation temperature and speed, continue to centrifuge for 30min to obtain Gel A.

[0064] S3: Place Gel A in an ultrasonic instrument, heat it to 40°C, with an ultrasonic frequency of 35KHz and a power of 360w. After ultrasonic crosslinking for 3min, then let it stand at 40°C for 30min to obtain Gel B.

[0065] S4: Gel B is neutralized with PBS, dialyzed, and washed. Control the final concentration of the sodium hyaluronate mixture in the gel to be 20mg / mL, then mix it evenly, fill it, and sterilize it to obtain a semi-fluid micro-crosslinked gel.

[0066] Example 2:

[0067] S1: Weigh and combine sodium hyaluronate with a molecular weight of 20KDa, sodium hyaluronate with a molecular weight of 80KDa, and sodium hyaluronate with a molecular weight of 200KDa in a mass ratio of 1:0.5:0.5 to obtain a sodium hyaluronate mixture with multiple molecular weights. The total weight is 4g, which is dissolved in 60mL of a disodium hydrogen phosphate solution with a pH of 9 and a concentration of 2mg / mL. After complete dissolution, it is added to a centrifuge tube and centrifuged at 2°C and 3000r / min for 20min.

[0068] S2: Under the conditions of maintaining the centrifugation temperature and speed, 0.2g of BDDE is added to the centrifuge tube at a uniform speed of 5mm / min. After the addition of BDDE is complete, while maintaining the centrifugation temperature and speed, continue to centrifuge for 20min to obtain Gel A.

[0069] S3: Place Gel A in an ultrasonic instrument, heat it to 30°C, with an ultrasonic frequency of 20KHz and a power of 100w. After ultrasonic crosslinking for 1min, then let it stand at 30°C for 20min to obtain Gel B.

[0070] S4: Gel B is neutralized, dialyzed, and washed with PBS. The final concentration of the sodium hyaluronate mixture in the gel is controlled at 10 mg / mL, and then it is mixed well, filled, and sterilized to obtain a semi-fluid micro-crosslinked gel.

[0071] Example 3:

[0072] S1: Weigh and combine 40KDa low-molecular-weight sodium hyaluronate, 120KDa medium-molecular-weight sodium hyaluronate, and 300KDa high-molecular-weight sodium hyaluronate in a mass ratio of 1:5:5 to obtain a sodium hyaluronate mixture with multiple molecular weights, with a total weight of 4 g. Dissolve it in 20 mL of a 100 mg / mL potassium hydroxide solution with a pH of 13. After complete dissolution, add it to a centrifuge tube and centrifuge at 20 °C and 10,000 r / min for 60 min.

[0073] S2: Maintain the centrifugation temperature at 20 °C, and add 1.6 g of DVS to the centrifuge tube at a constant speed of 30 mm / min. After the addition of DVS is complete, maintain the centrifugation temperature and rate and continue centrifuging for 60 min to obtain Gel A.

[0074] S3: Place Gel A in an ultrasonic instrument, heat it to 50 °C, and perform ultrasonic crosslinking at an ultrasonic frequency of 35 KHz and a power of 360 w for 5 min. Then, let it stand at 50 °C for 60 min to obtain Gel B.

[0075] S4: Gel B is neutralized, dialyzed, and washed with PBS. The final concentration of the sodium hyaluronate mixture in the gel is controlled at 30 mg / mL, and then it is mixed well, filled, and sterilized to obtain a semi-fluid micro-crosslinked gel.

[0076] Example 4:

[0077] A preparation method of a soft tissue filling material

[0078] S1: Weigh and combine 20KDa low-molecular-weight sodium hyaluronate, 120KDa medium-molecular-weight sodium hyaluronate, and 200KDa high-molecular-weight sodium hyaluronate in a mass ratio of 1:0.5:0.5 to obtain a sodium hyaluronate mixture with multiple molecular weights, with a total weight of 4 g. Dissolve it in 60 mL of a 2 mg / mL sodium hydroxide solution with a pH of 10. After complete dissolution, add it to a centrifuge tube and centrifuge at 2 °C and 5,000 r / min for 30 min.

[0079] S2: Maintain the centrifugation temperature and rate, and add 0.2 g of BDDE to the centrifuge tube at a constant speed of 5 mm / min. After the addition of BDDE is complete, maintain the centrifugation temperature and rate and continue centrifuging for 20 min to obtain Gel A.

[0080] S3: Place Gel A in an ultrasonic instrument, heat it to 30°C, carry out ultrasonic crosslinking at an ultrasonic frequency of 20 KHz and a power of 100 w for 1 min, and then let it stand at 30°C for 20 min to obtain Gel B.

[0081] S4: Gel B is neutralized, dialyzed, and washed with PBS. Control the final concentration of the sodium hyaluronate mixture in the gel to be 10 mg / mL. Then add a mixed recombinant functional protein composed of recombinant mussel adhesive protein and recombinant collagen, where the mass ratio of the mixed recombinant functional protein to the sodium hyaluronate mixture is 0.1:1. Then add mannitol and lidocaine, where the mass ratio of mannitol and lidocaine to the sodium hyaluronate mixture is 0.05:0.01:1. After mixing evenly, fill and sterilize to obtain a soft tissue filling material.

[0082] Example 5

[0083] A skin wound repair product

[0084] S1: Weigh and combine 20KDa low-molecular-weight sodium hyaluronate, 120KDa medium-molecular-weight sodium hyaluronate, and 200KDa high-molecular-weight sodium hyaluronate according to a mass ratio of 1:0.5:0.5 to obtain a sodium hyaluronate mixture with multiple molecular weights, with a total weight of 4 g. Dissolve it in 60 mL of a 2 mg / mL sodium hydroxide solution with a pH of 10. After complete dissolution, add it to a centrifuge tube and centrifuge at 2°C and 5000 r / min for 30 min.

[0085] S2: Maintain the centrifugation temperature and rate, and add 0.6 g of polyethylene glycol diglycidyl ether (PEGDE) to the centrifuge tube at a constant speed of 10 mm / min. After the polyethylene glycol diglycidyl ether is completely added dropwise, maintain the centrifugation temperature and rate and continue centrifuging for 30 min to obtain Gel A.

[0086] S3: Place Gel A in an ultrasonic instrument, heat it to 30°C, carry out ultrasonic crosslinking at an ultrasonic frequency of 20 KHz and a power of 100 w for 1 min, and then let it stand at 30°C for 20 min to obtain Gel B.

[0087] S4: Gel B is neutralized, dialyzed, and washed with PBS. Control the final concentration of the sodium hyaluronate mixture in the gel to be 10 mg / mL. Then add glycerol, propylene glycol, and recombinant collagen, where the mass ratio of glycerol, propylene glycol, and recombinant collagen to the sodium hyaluronate mixture is 0.02:0.01:0.01:1. After mixing evenly, fill and sterilize to obtain a skin wound repair product.

[0088] Comparative Example 1

[0089] The difference between Comparative Example 1 and Example 1 is that the gel product prepared in Comparative Example 1 was not ultrasonically treated. The gel prepared in Comparative Example 1 was only subjected to centrifugal crosslinking treatment.

[0090] S1: Weigh and combine sodium hyaluronate with a molecular weight of 30KDa, sodium hyaluronate with a molecular weight of 100KDa, and sodium hyaluronate with a molecular weight of 220KDa in a mass ratio of 1:1:2 to obtain a sodium hyaluronate mixture with multiple molecular weights, with a total weight of 4g. Dissolve it in 40mL of a 10mg / mL sodium hydroxide solution with a pH of 10. After complete dissolution, add it to a centrifuge tube. After centrifuging at 4°C and 5000r / min for 30min, add the crosslinking agent polyethylene glycol diglycidyl ether.

[0091] S2: Maintain the centrifugation temperature at 4°C, and add 0.6g of polyethylene glycol diglycidyl ether to the centrifuge tube at a uniform speed of 10mm / min. After the addition of polyethylene glycol diglycidyl ether is complete, keep the centrifugation temperature and rate, and continue centrifuging for 30min to obtain Gel A.

[0092] S3: Place Gel A in a 40°C environment and let it stand for 33min to obtain Gel B. Then, neutralize Gel B with PBS, dialyze, and wash it to control the final concentration of the sodium hyaluronate mixture in the gel to be 20mg / mL. Then, add recombinant elastin with a mass ratio of 1:1 to the sodium hyaluronate mixture. After mixing evenly, fill and sterilize to obtain a semi-fluid micro-crosslinked gel.

[0093] Comparative Example 2

[0094] The difference between Comparative Example 2 and Example 1 is that in step S1, the centrifugation speed is 20000r / min, and the rest remains unchanged.

[0095] Comparative Example 3

[0096] The difference between Comparative Example 3 and Example 1 is that in step S1, the centrifugation time is 100min, and the rest remains unchanged.

[0097] Comparative Example 4:

[0098] The difference between Comparative Example 4 and Example 1 is that in step S1, the centrifugation temperature is 50°C, and the rest remains unchanged.

[0099] Comparative Example 5:

[0100] The difference between Comparative Example 5 and Example 1 is that in step S2, after the addition of the crosslinking agent is complete, the operation of continuing centrifugation is not carried out, and the rest remains unchanged.

[0101] Comparative Example 6:

[0102] The difference between Comparative Example 6 and Example 1 is that in step S3, ultrasonic crosslinking is carried out for 10 min, and the rest remains unchanged.

[0103] Comparative Example 7:

[0104] The difference between Comparative Example 7 and Example 1 is that after the ultrasonic crosslinking treatment in step S3, no standing operation is carried out, and the rest remains unchanged.

[0105] Comparative Example 8:

[0106] The difference between Comparative Example 8 and Example 1 is that the standing temperature after ultrasonic treatment in step S3 is 60 °C, and the rest remains unchanged.

[0107] Comparative Example 9:

[0108] The difference between Comparative Example 9 and Example 1 is that in step S1, only 4 g of 100KDa medium molecular weight sodium hyaluronate is added, and the rest remains unchanged.

[0109] Experimental process and experimental data

[0110] 1. Measurement of swelling degree

[0111] For crosslinked gels, generally, the swelling degree is an important physical parameter reflecting the hydrophilic property of the gel and is used to characterize the crosslinking degree of the gel. Under the same conditions, the higher the crosslinking degree of the gel, the smaller its swelling degree. The swelling degree of the sample was measured according to Appendix D of YY / T 0962-2021 "Sodium Hyaluronate Gel for Plastic Surgery".

[0112] Figure 1 Table 1 shows the swelling degree data of the gels prepared in Example 1, Comparative Example 1 and Comparative Examples 7-8. It can be seen from Table 1 that the swelling degree of Comparative Example 1 is higher than that of Example 1. This is because the gel prepared in Comparative Example 1 has not undergone ultrasonic crosslinking treatment and has not formed a horizontal crosslinking structure, but only a longitudinal gradient crosslinking structure. Therefore, when the longitudinal structures of the gel product prepared in Comparative Example 1 are more easily penetrated by solvent molecules, the distance between molecular chains increases, and the swelling degree increases significantly; in the process of preparing the gel in Comparative Example 7, the horizontal crosslinking reaction was not completely completed due to no standing treatment after ultrasonic crosslinking. Therefore, the swelling degree of the gel product prepared in Comparative Example 7 is higher than that of Example 1. Figure 1 The horizontal crosslinking structures of the gel products prepared in Comparative Example 1 and Comparative Example 7 are not formed or not completely formed, resulting in excessive swelling. Excessive swelling will reduce the overall strength and toughness of the gel product, which is not conducive to injection use. In addition, due to excessive swelling, the sodium hyaluronate molecular chains are easily entangled under the influence of a large amount of solvent, resulting in changes in gel properties and poor stability.

[0113] The horizontal crosslinking structure of the gel products prepared in Comparative Example 1 and Comparative Example 7 is not formed or not completely formed, resulting in excessive swelling, and excessive swelling will reduce the overall strength and toughness of the gel product, which is not conducive to injection use. In addition, due to excessive swelling, the sodium hyaluronate molecular chains are easily entangled under the influence of a large amount of solvent, resulting in changes in gel properties and poor stability.

[0114] In Comparative Example 8, during the preparation of the gel, after ultrasonic treatment, the temperature was raised to 60 °C during the standing process. However, too high a standing temperature will accelerate the degradation rate of sodium hyaluronate in the alkaline solution, resulting in a decrease in the crosslinking degree of the gel product and damaging the three-dimensional gradient crosslinking network structure of the gel product. Therefore, the swelling degree of Comparative Example 8 is relatively high compared to Example 1.

[0115] 2. Rotational rheology test

[0116] The rotational rheology test of the semi-fluid micro-crosslinked gel was measured using a rotational rheometer (NETZSCH Kinexus Lab+). The frequency range was set from 0 to 10 Hz, and the rotational rheology data graph was plotted based on the rotational rheology results at 0.1 Hz. Figure 2 Among them, G′ represents the elastic modulus, and G" represents the viscous modulus.

[0117] It can be seen through Figure 2 that the value of the elastic modulus of Comparative Example 1 is much lower than that of Example 1, and the value of the viscous modulus is slightly lower than that of Example 1. This is because in the preparation of the gel in Comparative Example 1, only centrifugal crosslinking was carried out, resulting in only a longitudinal network structure and no transverse crosslinking structure formed by ultrasound. Therefore, the overall crosslinking strength of the gel product in Comparative Example 1 is lower than that in Example 1, and a low crosslinking degree will result in lower values of the elastic modulus and the viscous modulus.

[0118] Since in the preparation of the gel in Comparative Example 4, the temperature was too high during centrifugation in step S1, the degradation rate of sodium hyaluronate during centrifugation was too fast. On the one hand, this led to the destruction of the longitudinal crosslinking structure; on the other hand, due to the degradation of sodium hyaluronate, the transverse crosslinking structure was not able to be fully formed. Eventually, the gel product obtained had a poor crosslinking degree and it was difficult to form a three-dimensional gradient network structure. Therefore, the viscous modulus and elastic modulus of Comparative Example 4 are lower than those of Example 1.

[0119] In Comparative Example 6, the ultrasonic crosslinking time in step S3 was too long. However, if the ultrasonic treatment time is too long, the structure of molecular transverse rearrangement will be broken instead, and the structure changes from linear rearrangement to overall diffusion or dispersion, affecting the transverse crosslinking structure of the sample. Through Figure 2 the data, it can also be seen that the value of the viscous modulus of Comparative Example 6 is lower, which also proves that too long ultrasonic crosslinking treatment cannot strengthen the formation of the transverse network structure, but will break the intermolecular transverse crosslinking structure, resulting in a decrease in the viscoelasticity and flexibility of the product.

[0120] 3. Pushing force test

[0121] Using a 1.0 mL syringe from Weigao and a 30G RW needle from Terumo, the test was carried out under the body sliding mode of an intelligent electronic tensile testing machine (XLW-PC), and the test speed was selected as 30 mm / min.

[0122] According to the summary of data on similar products and clinical literature, it can be known that the ideal pushing force range for filler products that are convenient for doctors to inject is 5-40 N. From Figure 3 the pushing force data shown, it can be seen that the pushing force of Example 1 < 20 N, which is convenient for doctors to inject and use in actual operation.

[0123] During the preparation of the gel product of Comparative Example 2, the centrifugation speed was too fast and the centrifugal force became larger, resulting in uneven dispersion of sodium hyaluronate (more sodium hyaluronate was concentrated on the outside, while less sodium hyaluronate was in the area near the center). After crosslinking, the prepared gel product had poor uniformity and orderliness, resulting in uneven force application during the pushing process and the need to apply force locally during pushing. Therefore, its pushing force data increased compared with Example 1.

[0124] In the preparation process of Comparative Example 3, the centrifugation time was too long, and long-term centrifugation would increase the risk of its adhesion to the tube wall and also make the distribution of the crosslinking agent uneven, resulting in different particle sizes and hardness in the gel product. Therefore, the pushing force of the gel product of Comparative Example 3 increased compared with Example 1.

[0125] If no centrifugation was carried out after adding the crosslinking agent in Comparative Example 5, the crosslinking agent would be mainly concentrated in the central part, while the content of the crosslinking agent on both sides was less, resulting in uneven distribution of the crosslinking agent and too large a difference in the crosslinking degree in the prepared gel product. Therefore, the pushing force of the gel sample prepared in Comparative Example 5 was too large and not convenient for injection compared with Example 1.

[0126] It was found by comparing Example 1 with Comparative Example 2, Comparative Example 3, and Comparative Example 5 that when the uniformity of the gel was poor, its pushing force would increase.

[0127] 4. Microscope photos

[0128] The microscope photos of the semi-fluid micro-crosslinked gel were taken by dispersing a small amount of the sample in methylene blue solution, placing it on a glass slide, covering it with a coverslip, and observing and photographing it under an Olympus (Olmpus CX 33).

[0129] Observing Examples 1-3, Comparative Example 1, and the commercially available similar product Restylane Defyne (product name: Restylane Defyne, model specification 1 mL; production batch number: 21545; production manufacturer: Q-med AB, Seminariegatan 21 SE-75228) under the microscope, it can be seen thatFigure 4 The network structures of the gel products prepared in Examples 1-3 are compact and complex. However, the commercially available product Restylane Defyne is a granular gel under a microscope. The gel sample prepared in Comparative Example 1 lacks a transverse cross-linked vein structure under a microscope, and the longitudinal cross-linked structure formed is looser and has fewer cross-linking points compared with that in Example 1. This further shows that the microstructure of the semi-fluid micro-cross-linked gel prepared in Examples 1-3 has a flexible cross-linked structure with linear, reticular, and different density regions, and the interlacing of multiple structures can enhance the cohesiveness of the gel and result in a slower degradation rate.

[0130] 5. Cohesiveness evaluation

[0131] At room temperature, 1 mL of the semi-fluid micro-cross-linked gel was mixed with 0.1 mg of toluidine blue until the dye was evenly distributed in the semi-fluid micro-cross-linked gel matrix for about 3 min. Subsequently, the colored semi-fluid micro-cross-linked gel was drawn into a 1 mL glass syringe and then injected into water at a constant rate of 50 mm / min using an automatic extrusion device, and a magnetic stirrer was started. Photos were taken and recorded at extrusion times of 15 s, 75 s, and 90 s, respectively. The experimental results are as Figure 5 shown. It can also be seen from the Figure 5 cohesiveness results that the gel product prepared in Example 1 did not show dispersion in water at extrusion times of 15 s, 75 s, and 90 s.

[0132] However, dispersion occurred in Comparative Example 1 at an extrusion time of 15 s, and by an extrusion time of 75 s, the gel product prepared in Comparative Example 1 had completely disintegrated, indicating that without the step of ultrasonic cross-linking, the gel prepared lacked cross-linking in the transverse structure and did not form a three-dimensional gradient cross-linked network. Therefore, when an external stress occurred, the gel prepared in Comparative Example 1 was prone to spreading and losing its shape.

[0133] The gel product prepared in Comparative Example 2 started to show dispersion at an extrusion time of 75 s. This is because during the centrifugal cross-linking process in Comparative Example 2, the centrifugal rate was too fast, resulting in uneven distribution of sodium hyaluronate, and the internal cross-linking degree of the gel product prepared after cross-linking was uneven, thus affecting the cohesiveness of the product.

[0134] Through Figure 5It can be seen that the gel prepared in Comparative Example 3 started to disperse at an extrusion time of 15 s, and the phenomenon of wall sticking occurred. By the extrusion time of 90 s, most of the structure of Comparative Example 3 had dispersed. This shows that too long a centrifugation time does not make the crosslinked structure tighter, but instead the prepared gel product has poor cohesiveness. This may be because excessive centrifugation causes excessive sedimentation of sodium hyaluronate, affecting the penetration of the subsequent crosslinking agent, resulting in poor uniformity of the crosslinking density. On the other hand, too long a centrifugation time causes a thick deposition layer of sodium hyaluronate macromolecules to form on the tube wall, affecting the effect of subsequent ultrasonic treatment, resulting in defects in both the longitudinal crosslinking network and the transverse crosslinking network existing in the system, thus leading to poor support and cohesiveness.

[0135] It can be seen that Figure 5 when the gel prepared in Comparative Example 5 reached an extrusion time of 90 s, although it was different from the state of almost completely dispersing shown in Comparative Examples 1-3, partial dispersion of the gel in Comparative Example 5 also occurred. This shows that although a longitudinal and transverse cross-linked network structure can still be formed without continuing the centrifugation operation after adding the crosslinking agent, this is only by relying on the diffusion of crosslinking agent molecules to crosslink with sodium hyaluronate. However, within a certain period of time, the crosslinking agent molecules cannot diffuse evenly, so the crosslinking agent is unevenly distributed, and the crosslinking uniformity of the prepared gel is poor, especially there are defects in the longitudinal crosslinking structure, resulting in limited formation of the three-dimensional gradient crosslinking network and poor support and cohesiveness.

[0136] 6. Enzymatic resistance

[0137] Take 1 g of the semi-fluid micro-crosslinked gel, add 1 mL of 20 U / mL HA enzyme solution, and shake on a shaker at 37 °C and 150 r / min for 0 min, 30 min, 90 min, 150 min, 5 h, 8 h, and 24 h (sampling is carried out separately for each node). Heat the sample to 100 °C and react for 10 min to stop the enzymatic reaction. Take 1 g of the sample at the 0 min node, add 10 mL of 0.5 mol / L sulfuric acid solution, place it in a constant temperature oven at (95 ± 5) °C, heat for 2 h to completely dissolve it, add 10 mL of 1 mol / L sodium hydroxide solution to neutralize the sulfuric acid, transfer the solution to a 100 mL volumetric flask, add water to make up the volume and shake well for standby. Take 0.2 g of the sample at the remaining nodes and dilute it to 15 mL, 20 mL, and 25 mL with purified water respectively. Filter the diluted solution with quantitative slow filter paper as the test solution, and test and calculate the sodium hyaluronate content according to the method for determining the sodium hyaluronate content in Appendix C of "Crosslinked Sodium Hyaluronate Gel for Plastic Surgery" (YY / T 0962-2021). Taking the 0 min node as a control, investigate the degradation of the samples at each time point. The specific results are shown in Table 1. Table 1 is a record table of the in vitro degradation rate values of Example 1, Comparative Example 1, Comparative Example 5, and Comparative Examples 7-8 at different time periods.

[0138] Table 1 Record of in vitro degradation rate values of Example 1, Comparative Example 1, Comparative Example 5, and Comparative Examples 7 - 8 at different time periods

[0139] Degradation rate / % Example 1 Comparative Example 1 Comparative Example 5 Comparative Example 7 Comparative Example 8 30 min 22.46 60.04 42.19 62.12 38.78 90 min 30.05 103.05 73.24 85.08 77.88 160 min 48.48 103.05 96.47 101.45 92.95 300 min 73.92 103.05 102.38 101.45 102.21 480 min 103.41 103.05 103.11 101.45 100.04

[0140] It can be seen from the data in Table 1 that the gel product prepared in Example 1 degraded by less than 50% after being placed for 160 min, while in Comparative Example 1, Comparative Example 5, Comparative Example 7, and Comparative Example 8, the degradation rate was > 90% when placed for 160 min. Among them, Comparative Example 1 had completely degraded at 160 min, indicating that the anti - degradation property of the gel product prepared in Comparative Example 1 was much lower than that of Example 1. This shows that the anti - degradation property of the longitudinally cross - linked network structure in the gel product of Comparative Example 1 is much lower than that of the three - dimensional gradient cross - linked network structure in Example 1.

[0141] In Comparative Example 5, it had degraded by 96.47% at 160 min, indicating that without centrifugation after adding the cross - linker, the distribution of the cross - linker would be uneven, resulting in an uneven overall cross - linked structure of the gel, and even unreacted cross - linkers being wrapped and unable to participate in subsequent cross - linking reactions, thus making it difficult to form a three - dimensional gradient cross - linked network structure and leading to a faster degradation rate. In Comparative Example 7, it had completely degraded at 160 min, indicating that without standing after ultrasonic treatment and directly proceeding to the next step of operation, the horizontal cross - linked structure of the product was not fully formed, the overall cross - linking degree was weak, resulting in defects or even difficulty in forming a three - dimensional gradient cross - linked network structure, and the degradation rate of the gel was also fast.

[0142] In Comparative Example 8, it had degraded by 92.95% at 160 min, indicating that during the cross - linking and standing process after ultrasonic treatment, the temperature was too high, which would accelerate the degradation rate of sodium hyaluronate in the alkaline solution, resulting in a weakened overall cross - linking strength of the gel, an unstable structure, and thus a faster enzymatic hydrolysis rate compared to Example 1.

[0143] 7. Cytotoxicity

[0144] Mouse fibroblasts (NCTC Clone 929, L cell, L - 929, derivative of Strain L) were cultured in MEM medium containing antibiotics and 10% serum in an incubator at 37°C and 5% CO2. When the cell density reached 80%, they were digested with trypsin and collected for counting. The cell concentration was adjusted to 1×10 5 cells / mL; the above cell suspension was added to a 96 - well plate, 100 μL per well, for a total of 1×10 4 cells / well, and cultured at 37°C and 5% CO2 for 24 h. The test samples were prepared at a concentration of 0.2 g / mL in serum - containing medium and extracted in a shaker for 24 h at a temperature of 37°C and a rotation speed of 100 r / min.

[0145] After the culture was completed, the culture medium in the culture plate was discarded, and the leaching solution, negative control, positive control, and blank control were added to each group, with 5 wells in each group. The plate was placed in an incubator at 37°C with 5% CO2 for 24 h. Subsequently, the cell culture plate was taken out, the culture medium in it was discarded, 50 μL of MTT staining agent (1 mg / mL) was added to each well, and the plate was placed in an incubator at 37°C with 5% CO2 and saturated humidity for 2 h;

[0146] Finally, the liquid in the culture plate was discarded, 100 μL of isopropanol was added to each well, shaken and mixed evenly, and then placed in an enzyme-linked immunosorbent assay (ELISA) reader for reading at 570 nm (reference wavelength 620 nm). For each concentration group and the positive control group, the relative cell survival rate was calculated based on the OD value of the blank control group;

[0147] Relative cell survival rate (%) = (average OD value of the test group / average OD value of the blank control group), and the blank control group was recorded as 100%; the results were as Figure 6 shown. The relative cell survival rates of Example 1, the negative control group, and the positive control group were 100%, 98%, and 0%, respectively.

[0148] The cell morphology of each group was observed under an inverted microscope (XL Core), and the results were as Figure 6 shown. The cell morphology of the negative control was similar to that of Example 1, without cell lysis or decreased cell proliferation. Considering the cell proliferation rate and cell morphology, it can be determined that the semi-fluid micro-crosslinked gel has no cytotoxicity. This indicates its high biosafety.

[0149] 8. Tissue compatibility experiment

[0150] SD rats (250 - 300 g) were divided into two groups, A and B, and anesthetized with 2% isoflurane. In group A, 0.2 mL of the gel product prepared in Example 1 was subcutaneously injected into each SD rat; in group B, 0.2 mL of the gel product prepared in Comparative Example 9 was subcutaneously injected into each SD rat. Two weeks after the injection, the rats in groups A and B were sacrificed, and the tissues at the injection sites were taken. Then the obtained tissue samples were fixed in formalin solution, and after dehydration, treatment with a clearing agent, wax infiltration, embedding, etc., tissue sections were made. The sections were then dewaxed in xylene, hydrated, stained with hematoxylin for 5 minutes, rinsed with water, differentiated with hydrochloric acid ethanol for several seconds, then rinsed with running water to turn blue for 10 - 15 minutes, and finally stained with eosin (HE) for 1 minute. Finally, after dehydration, treatment with xylene for transparency, and mounting, they were observed under a microscope.

[0151] Through Figure 7Under the microscope, the section results can intuitively show that the gel prepared in Example 1 has good compatibility with various parts of the tissue (epidermis, dermis, subcutaneous tissue), forming fibrous interweaving, and new blood vessels can be seen growing in; while the gel product prepared in Comparative Example 9 only remains at the injection site and forms an obvious boundary with the tissue. It can be seen from this that the gel product with a three-dimensional gradient cross-linked network structure prepared in Example 1 can better mimic the natural tissue hierarchy, be highly compatible with the tissue, reduce the body's immune rejection reaction, and have good biocompatibility; while the gel prepared in Comparative Example 9, due to only using sodium hyaluronate with a single molecular weight and not forming a three-dimensional cross-linked network structure with a gradient distribution of cross-linking density, has poor structural matching with the surrounding tissue, resulting in obvious foreign body reactions and immune rejection reactions.

Claims

1. A method for preparing a semi-fluid micro-crosslinked gel, characterized in that, It includes the following steps: S1. Dissolve the sodium hyaluronate mixture with multiple molecular weights in an alkaline solution. After complete dissolution, centrifuge at a rotational speed of 3000 - 10000 r / min for 20 - 60 min at a temperature of 2 - 20 °C; S2. Maintain the centrifugal rotational speed and uniformly add a crosslinking agent to the centrifuge tube at a speed of 5 - 30 mm / min. After the crosslinking agent is completely added, continue to centrifuge for 20 - 60 min at a temperature of 2 - 20 °C to obtain Gel A; S3. Ultrasonicate Gel A obtained in step S2 at an ultrasonic frequency of 20 - 35 KHz, a power of 100 - 360 w, an ultrasonic temperature of 30 - 50 °C, and an ultrasonic time of 1 - 5 min. Then continue to stand at 30 - 50 °C for 20 - 60 min to obtain Gel B; S4. Neutralize, dialyze, and wash Gel B, and control the final concentration of sodium hyaluronate in Gel B to be 20 ± 10 mg / mL; S5. Then fill and sterilize Gel B obtained in step S4 to obtain the semi-fluid micro-crosslinked gel.

2. The preparation method according to claim 1, characterized in that, The sodium hyaluronate mixture with multiple molecular weights includes low-molecular-weight sodium hyaluronate with a molecular weight of 20 - 40 KDa, medium-molecular-weight sodium hyaluronate with a molecular weight of 80 - 120 KDa, and high-molecular-weight sodium hyaluronate with a molecular weight of 200 - 300 KDa; the corresponding mass ratio is 1:(0.5 - 5):(0.5 - 5).

3. The preparation method according to claim 1 or 2, characterized in that, The alkaline solution is any one of sodium hydroxide, potassium hydroxide, disodium hydrogen phosphate, and sodium carbonate solutions with a pH of 9 - 13 and a concentration of 2 - 100 mg / mL; The mass-to-volume ratio of the alkaline solution to the sodium hyaluronate mixture is 1:5 - 1:15, where the mass unit is g and the volume unit is mL.

4. The preparation method according to claim 1 or 2, characterized in that, The crosslinking agent is any one of butanediol diglycidyl ether (BDDE), divinyl sulfone (DVS), N-hydroxysuccinimide (NHS), polyethylene glycol, and polyethylene glycol derivatives; The mass ratio of the crosslinking agent to the sodium hyaluronate mixture is 0.05 - 0.4:

1.

5. The preparation method according to claim 1 or 2, characterized in that, Pharmaceutical agents or cosmetic active ingredients can also be added to the semi-fluid micro-crosslinked gel.

6. The preparation method according to claim 5, wherein The pharmaceutical agents are selected from any one or more of antibacterial substances, antihistamine drugs, anti-inflammatory drugs, anesthetics, analgesics, proteins / peptides, and antiviral compounds.

7. The preparation method according to claim 5, wherein The cosmetic active ingredients are selected from any one or more of moisturizers, skin beautifying agents, vitamins, proteins / amino acids, antioxidants, and particulate fillers.

8. The preparation method according to claim 5, characterized in that, The mass percentage of the pharmaceutical agents or cosmetic active ingredients is 0.01% - 70%.

9. The preparation method according to claim 6, characterized in that, The semi-fluid micro-crosslinked gel is applied to the preparation of biomedicines, medical devices, or cosmetics.

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