Microspheres capable of being subjected to moist heat sterilization and resisting enzymolysis as well as preparation method and application of microspheres

Abstract

The invention relates to the technical field of medical beauty filling materials, and discloses microspheres capable of being subjected to moist heat sterilization and resisting enzymolysis as well as a preparation method and application of the microspheres. The preparation method of the microspheres capable of being subjected to moist heat sterilization and resisting enzymolysis comprises the following steps: dissolving a polymer in a solvent to obtain a water phase; adding the water phase into an oil phase containing an emulsifier, and emulsifying to obtain an emulsion; adding a first cross-linking agent into the emulsion, and carrying out a cross-linking reaction; then adding a second cross-linking agent, and carrying out a cross-linking reaction; and after the reaction, collecting a solid phase, washing and drying to obtain the microspheres. According to the application, the multistage interpenetrating network is constructed in the microspheres by combining crosslinking strategies of different action mechanisms, and the defect of insufficient mechanical properties of a single crosslinking mode is overcome by utilizing complementary advantages of various crosslinking modes. The microspheres are uniform in particle size, regular in morphology, higher in mechanical property, compact in structure and high in enzymolysis resistance, can be maintained in vivo for a longer time, and can be subjected to moist heat sterilization.

Description

Technical Field

[0001] This application relates to the field of medical aesthetic filler materials technology, specifically to a microsphere that can be sterilized by moist heat and is resistant to enzymatic degradation, as well as its preparation method and application. Background Technology

[0002] Injectable fillers are currently one of the most effective methods for treating facial wrinkles and depressions. Sodium hyaluronate, collagen, and polydeoxyribonucleic acid (PDRN) are clinically used in cosmetic injectable products for most wrinkles and depressions, making them ideal soft tissue fillers.

[0003] Currently, most injectable cross-linked hyaluronic acid is in gel form. It is produced by cross-linking hyaluronic acid, followed by crushing and sieving to obtain hyaluronic acid gels of the desired size. However, this process results in hyaluronic acid gels with low cross-linking degree, high swelling degree, and short duration of effect. Collagen injections (such as recombinant human collagen) and PDRN mesotherapy are widely used for cosmetic filling, injections, and tissue repair; however, they suffer from drawbacks such as rapid metabolism, short-lived effects, and weak support.

[0004] Crosslinking hyaluronic acid or collagen into microspheres can improve their stability and increase their duration and lasting effect. For example, patent application CN111848991A discloses a method for preparing crosslinked hyaluronic acid microspheres. This involves uniformly dispersing hyaluronic acid or its salts with a crosslinking agent, alkali, and water to prepare an aqueous phase. The aqueous and oil phases are then mixed to form an emulsion. After crosslinking, post-treatment is performed to obtain crosslinked hyaluronic acid microspheres. However, in this method, the crosslinking agent has difficulty penetrating into the microspheres, resulting in low crosslinking efficiency and poor resistance to enzymatic degradation. Patent application CN103848995A discloses a method for preparing hyaluronic acid nanospheres. This method first prepares two functionalized hyaluronic acids: thymine-functionalized hyaluronic acid and adenine-functionalized hyaluronic acid, and then crosslinks them to prepare microspheres. However, this method is cumbersome and unsuitable for industrial production. For example, patent N114931666B proposes a method for preparing hyaluronic acid-collagen composite cross-linked microspheres. This method uses sodium hyaluronate and recombinant collagen as raw materials, and 1,4-butanediol diglycidyl ether (BDDE) as a cross-linking agent. Microspheres are formed by cross-linking an oil-water mixture through high-speed shear emulsification. However, because the cross-linking agent BDDE poses a carcinogenic risk, even trace amounts may affect the biocompatibility and safety of the microsphere product.

[0005] Therefore, there is an urgent need to develop a new method for preparing microspheres that not only has a simple preparation process, but also produces injectable microspheres with high mechanical strength, high resistance to degradation, and high stability. Summary of the Invention

[0006] This application provides a microsphere that can be sterilized by moist heat and is resistant to enzymatic degradation, as well as its preparation method and application, aiming to solve the technical problems of existing injectable cross-linked hyaluronic acid or collagen microspheres, such as complex preparation process, poor resistance to degradation, low mechanical strength, and poor stability, which make moist heat sterilization impossible.

[0007] To achieve the above objectives, the present application adopts the following technical solution.

[0008] A first aspect of this application provides a method for preparing microspheres that are capable of being sterilized by moist heat and resistant to enzymatic degradation, comprising:

[0009] S1, dissolve the polymer in a solvent to obtain an aqueous phase;

[0010] S2, the aqueous phase is added to the oil phase containing the emulsifier, and emulsification is performed to obtain an emulsion;

[0011] S3, add a first crosslinking agent to the emulsion to carry out a crosslinking reaction; then add a second crosslinking agent to carry out a crosslinking reaction; after the reaction, collect the solid phase, wash and dry it to obtain microspheres.

[0012] Preferably, the polymer includes collagen, hyaluronic acid, and PDRN;

[0013] The solvent is water or an aqueous solution of sodium hydroxide;

[0014] The oil phase includes any one of vegetable oil, mineral oil, silicone oil, or liquid paraffin.

[0015] The emulsifier is dehydrated sorbitan monooleate or polysorbate-80.

[0016] More preferably, in the aqueous phase, the concentration of hyaluronic acid is 0-3 wt%, the concentration of collagen is 0.2-30 wt%, and the concentration of PDRN is 0-10 wt%.

[0017] Preferably, the first crosslinking agent is polyethylene glycol diacrylate;

[0018] The second crosslinking agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and adipic acid dihydrazide.

[0019] More preferably, in the second crosslinking agent, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to adipic acid dihydrazide is 1:(0.5-1).

[0020] Preferably, the mass ratio of the first crosslinking agent to the polymer is (1-10):1;

[0021] The mass ratio of the second crosslinking agent to the polymer is (0.1 to 1):1.

[0022] Preferably, the content of emulsifier in the oil phase is 2-10 wt%.

[0023] The volume ratio of the aqueous phase to the oil phase is 1:5 to 20.

[0024] Preferably, the temperatures for the first and second crosslinking reactions are 25–50°C.

[0025] A second aspect of this application provides microspheres prepared by the above-described preparation method.

[0026] A third aspect of this application provides the application of the aforementioned microspheres in cosmetic filling, injection, and tissue repair.

[0027] Compared with the prior art, the beneficial effects of this application are as follows:

[0028] This application constructs a multi-level interpenetrating network within microspheres by combining cross-linking strategies with different mechanisms of action. The complementary advantages of each cross-linking method overcome the limitations of single cross-linking methods in terms of mechanical properties. By introducing the cross-linking agent PEGDA into the microsphere system, PEGDA reacts with the amino groups of collagen, achieving microsphere shaping and a certain strength. Subsequently, by introducing the cross-linking agent EDC / ADH, ADH, with its strong nucleophilicity, reacts with the carboxyl groups on collagen to form amide bonds, while also acting as a molecular bridge connecting collagen molecules, constructing an irreversible, highly rigid network that significantly enhances the strength of the microspheres.

[0029] The microspheres prepared in this application have uniform particle size and regular morphology. Compared with microspheres prepared by conventional one-time cross-linking process, the microspheres of this application have higher mechanical properties, denser microsphere structure, stronger resistance to enzymatic hydrolysis, can be maintained in vivo for a longer time, and can be sterilized by moist heat. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 SEM image of the collagen microspheres prepared in Example 1;

[0032] Figure 2 SEM image of the hyaluronic acid and collagen composite microspheres prepared in Example 2;

[0033] Figure 3 SEM image of the PDRN and collagen composite microspheres prepared in Example 3;

[0034] Figure 4 SEM images of the microspheres prepared in the examples and comparative examples after wet heat sterilization performance testing. Detailed Implementation

[0035] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0036] In the following description of this embodiment, the terms "including", "comprising", "having", and "containing" are all open-ended terms, meaning that they include but are not limited to.

[0037] In the following description of this embodiment, the term "and / or" is used to describe the association relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, B existing alone, and A and B existing simultaneously. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0038] In the following description of this embodiment, the term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0039] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0040] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0041] Those skilled in the art will understand that the numerical ranges in the embodiments of this application should be understood as each intermediate value between the upper and lower limits of the specifically disclosed range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] This application provides a method for preparing microspheres that are sterilizable by moist heat and resistant to enzymatic degradation, comprising:

[0044] S1, dissolve the polymer in a solvent to obtain an aqueous phase;

[0045] In this application, the polymer includes collagen, hyaluronic acid, and PDRN;

[0046] The solvent is water or an aqueous solution of sodium hydroxide;

[0047] In the aqueous phase, the concentration of hyaluronic acid is 0–3 wt%, the concentration of collagen is 0.2–30 wt%, and the concentration of PDRN is 0–10 wt%.

[0048] S2, the aqueous phase is added to the oil phase containing the emulsifier, and emulsification is performed to obtain an emulsion;

[0049] In this application, the oil phase includes any one of vegetable oil, mineral oil, silicone oil, or liquid paraffin, preferably liquid paraffin.

[0050] The emulsifier is selected from dehydrated sorbitan monooleate (Span 80) or polysorbate-80, preferably Span 80.

[0051] The oil phase contains 2 to 10 wt% emulsifier, preferably 2 wt%.

[0052] This application employs a reverse emulsification method, in which an aqueous phase is added to an oil phase for emulsification. The volume ratio of the aqueous phase to the oil phase is 1:5 to 20, preferably 1:5.

[0053] S3, add a first crosslinking agent to the emulsion to carry out a crosslinking reaction; then add a second crosslinking agent to carry out a crosslinking reaction; after the reaction, collect the solid phase, wash and dry it to obtain microspheres.

[0054] In this application, the first crosslinking agent is polyethylene glycol diacrylate (PEGDA), and the mass ratio of the first crosslinking agent to the polymer in step S1 is (1-10):1. The reaction temperature of the first crosslinking reaction is 25-50°C, preferably 30°C.

[0055] In this application, the second crosslinking agent comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and adipic acid dihydrazide (ADH), wherein the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to adipic acid dihydrazide is 1:(0.5-1). The mass ratio of the second crosslinking agent to the polymer is (0.1-1):1; the reaction temperature of the second crosslinking reaction is 25-50°C, preferably 30°C.

[0056] After the reaction was completed, the reaction solution was centrifuged to separate the solid phase, washed repeatedly with isopropanol by centrifugation 3-4 times, and then washed with anhydrous ethanol 3-4 times to obtain white fine particles; these particles were dried in a vacuum drying oven at 30℃ for 12 hours, and the dried microspheres were sieved to obtain microspheres with a particle size of 20-45 μm.

[0057] This application constructs a multi-level interpenetrating network within microspheres by combining cross-linking strategies with different mechanisms of action. The complementary advantages of each cross-linking method overcome the limitations of single cross-linking methods in terms of mechanical properties. By introducing the cross-linking agent PEGDA into the microsphere system, PEGDA reacts with the amino groups of collagen, achieving microsphere shaping and a certain strength. Subsequently, by introducing the cross-linking agent EDC / ADH, ADH, with its strong nucleophilicity, reacts with the carboxyl groups on collagen to form amide bonds, while also acting as a molecular bridge connecting collagen molecules, constructing an irreversible, highly rigid network that significantly enhances the strength of the microspheres.

[0058] The microspheres prepared in this application have uniform particle size and regular morphology. Compared with microspheres prepared by existing one-time cross-linking processes, the microspheres of this application have higher mechanical properties, denser structure, stronger resistance to enzymatic degradation, and can be maintained in vivo for a longer period of time. They can be used for cosmetic filling, injection and tissue repair.

[0059] The present application will be further illustrated by the following examples.

[0060] Example 1

[0061] S1, take 0.5g of recombinant collagen with a molecular weight of 4w, dissolve it in 4.5mL of deionized water to obtain a 10% recombinant collagen solution, i.e., the aqueous phase;

[0062] S2, add 3.3g of Span 80 to a beaker, then add 165mL of liquid paraffin, and mechanically stir in a 30℃ water bath for 30min to mix it evenly to obtain the oil phase;

[0063] Add 10% recombinant collagen solution dropwise to the oil phase using a syringe, stir for 30 minutes to obtain an emulsion;

[0064] S3, add 4 mL of polyethylene glycol diacrylate to the emulsion and stir at 30°C for 12 h; then add 300 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 150 mg of adipic acid dihydrazide and stir at 30°C for 24 h.

[0065] After the reaction was complete, the reaction solution was poured into a centrifuge bottle and centrifuged at 8000 rpm for 10 min. Isopropanol was then added, and the mixture was centrifuged and washed three times, discarding the supernatant. Anhydrous ethanol was then added, and the mixture was centrifuged and washed three more times until the microspheres appeared as white, fine particles. These particles were then placed in a vacuum drying oven at 30°C and dried for 12 h. The dried microspheres were then sieved to obtain microspheres with a particle size of 20–45 μm.

[0066] Example 2

[0067] S1, take 0.25g of hyaluronic acid with a molecular weight of 141w and 0.25g of recombinant collagen with a molecular weight of 4w and dissolve them in 100mL of deionized water to obtain a mixed solution of hyaluronic acid and collagen.

[0068] S2, add 10g of Span 80 to a beaker, then add 500mL of liquid paraffin, and mechanically stir in a 30℃ water bath for 30min to mix it evenly to obtain the oil phase;

[0069] A mixed solution of hyaluronic acid and collagen was added dropwise to the oil phase using a syringe and stirred for 30 minutes to obtain an emulsion.

[0070] S3, add 4 mL of polyethylene glycol diacrylate to the emulsion and stir at 30°C for 12 h; then add 300 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 300 mg of adipic acid dihydrazide and stir at 30°C for 24 h.

[0071] After the reaction was complete, the reaction solution was poured into a centrifuge bottle and centrifuged at 8000 rpm for 10 min. Isopropanol was then added, and the mixture was centrifuged and washed three times, discarding the supernatant. Anhydrous ethanol was then added, and the mixture was centrifuged and washed three more times until the microspheres appeared as white, fine particles. These particles were then placed in a vacuum drying oven at 30°C and dried for 12 h. The dried microspheres were then sieved to obtain microspheres with a particle size of 20–45 μm.

[0072] Example 3

[0073] S1, take 0.5g of PDRN with a molecular weight of 70w and 2g of recombinant collagen with a molecular weight of 4w and dissolve them in 20ml of NaOH aqueous solution with a concentration of 0.005mol / L to obtain a mixed solution of PDRN and collagen;

[0074] S2, add 4g of Span 80 to a beaker, then add 400mL of rapeseed oil, and mechanically stir in a 30℃ water bath for 30min to mix it evenly and obtain the oil phase;

[0075] The mixed solution of PDRN and collagen was added dropwise to the oil phase using a syringe and stirred for 30 minutes to obtain an emulsion.

[0076] S3, add 8 mL of polyethylene glycol diacrylate to the emulsion and stir at 30°C for 12 h; then add 2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2 g of adipic acid dihydrazide and stir at 30°C for 24 h.

[0077] After the reaction was complete, the reaction solution was poured into a centrifuge bottle and centrifuged at 8000 rpm for 10 min. Isopropanol was then added, and the mixture was centrifuged and washed three times, discarding the supernatant. Anhydrous ethanol was then added, and the mixture was centrifuged and washed three more times until the microspheres appeared as white, fine particles. These particles were then placed in a vacuum drying oven at 30°C and dried for 12 h. The dried microspheres were then sieved to obtain microspheres with a particle size of 20–45 μm.

[0078] Comparative Example 1

[0079] S1, take 0.5g of recombinant collagen with a molecular weight of 4w, dissolve it in 4.5mL of deionized water to obtain a 10% recombinant collagen solution, i.e., the aqueous phase;

[0080] S2, add 3.3g of Span 80 to a beaker, then add 165mL of liquid paraffin, and mechanically stir in a 30℃ water bath for 30min to mix it evenly to obtain the oil phase;

[0081] Add 10% recombinant collagen solution dropwise to the oil phase using a syringe, stir for 30 minutes to obtain an emulsion;

[0082] S3, add 4 mL of polyethylene glycol diacrylate, 300 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 150 mg of adipic acid dihydrazide to the emulsion, and stir at 30 °C for 24 h.

[0083] After the reaction was complete, the reaction solution was poured into a centrifuge bottle and centrifuged at 8000 rpm for 10 min. Isopropanol was then added, resulting in the precipitation of a gel-like flocculent substance; no well-dispersed white particles were observed. Comparative Example 1 failed to successfully prepare microspheres.

[0084] Comparative Example 2

[0085] S1, take 0.5g of recombinant collagen with a molecular weight of 4w, dissolve it in 4.5mL of deionized water to obtain a 10% recombinant collagen solution, i.e., the aqueous phase;

[0086] S2, add 3.3g of Span 80 to a beaker, then add 165mL of liquid paraffin, and mechanically stir in a 30℃ water bath for 30min to mix it evenly to obtain the oil phase;

[0087] Add 10% recombinant collagen solution dropwise to the oil phase using a syringe, stir for 30 minutes to obtain an emulsion;

[0088] S3, add 4 mL of polyethylene glycol diacrylate to the emulsion and stir at 30°C for 12 h; then add another 4 mL of polyethylene glycol diacrylate and stir at 30°C for 24 h.

[0089] After the reaction was complete, the reaction solution was poured into a centrifuge bottle and centrifuged at 8000 rpm for 10 min. Isopropanol was then added, and the mixture was centrifuged and washed three times, discarding the supernatant. Anhydrous ethanol was then added, and the mixture was centrifuged and washed three more times until the microspheres appeared as white, fine particles. These particles were then placed in a vacuum drying oven at 30°C and dried for 12 h. The dried microspheres were then sieved to obtain microspheres with a particle size of 20–45 μm.

[0090] Comparative Example 3

[0091] S1, take 0.5g of recombinant collagen with a molecular weight of 4w, dissolve it in 4.5mL of deionized water to obtain a 10% recombinant collagen solution, i.e., the aqueous phase;

[0092] S2, add 3.3g of Span 80 to a beaker, then add 165mL of liquid paraffin, and mechanically stir in a 30℃ water bath for 30min to mix it evenly to obtain the oil phase;

[0093] Add 10% recombinant collagen solution dropwise to the oil phase using a syringe, stir for 30 minutes to obtain an emulsion;

[0094] S3, add 300 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 150 mg of adipic acid dihydrazide to the emulsion, and stir at 30 °C for 12 h; then add 300 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 150 mg of adipic acid dihydrazide, and stir at 30 °C for 24 h.

[0095] After the reaction was complete, the reaction solution was poured into a centrifuge bottle and centrifuged at 8000 rpm for 10 min. Isopropanol was then added, and the mixture was centrifuged and washed three times, discarding the supernatant. Anhydrous ethanol was then added, and the mixture was centrifuged and washed three more times until the microspheres appeared as white, fine particles. These particles were then placed in a vacuum drying oven at 30°C and dried for 12 h. The dried microspheres were then sieved to obtain microspheres with a particle size of 20–45 μm.

[0096] Comparative Example 4

[0097] S1, take 0.25g of hyaluronic acid with a molecular weight of 141w and 0.25g of recombinant collagen with a molecular weight of 4w and dissolve them in 100mL of deionized water to obtain a mixed solution of hyaluronic acid and collagen.

[0098] S2, add 10g of Span 80 to a beaker, then add 500mL of liquid paraffin, and mechanically stir in a 30℃ water bath for 30min to mix it evenly to obtain the oil phase;

[0099] A mixed solution of hyaluronic acid and collagen was added dropwise to the oil phase using a syringe and stirred for 30 minutes to obtain an emulsion.

[0100] S3, add 150 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 150 mg of adipic acid dihydrazide to the emulsion, and stir at 30 °C for 12 h; then add 300 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 150 mg of adipic acid dihydrazide, and stir at 30 °C for 24 h.

[0101] After the reaction was complete, the reaction solution was poured into a centrifuge bottle and centrifuged at 8000 rpm for 10 min. Isopropanol was then added, and the mixture was centrifuged and washed three times, discarding the supernatant. Anhydrous ethanol was then added, and the mixture was centrifuged and washed three more times until the microspheres appeared as white, fine particles. These particles were then placed in a vacuum drying oven at 30°C and dried for 12 h. The dried microspheres were then sieved to obtain microspheres with a particle size of 20–45 μm.

[0102] Comparative Example 5

[0103] S1, take 0.5g of PDRN with a molecular weight of 70w and 2g of recombinant collagen with a molecular weight of 4w and dissolve them in 20ml of NaOH aqueous solution with a concentration of 0.005mol / L to obtain a mixed solution of PDRN and collagen;

[0104] S2, add 4g of Span 80 to a beaker, then add 400mL of rapeseed oil, and mechanically stir in a 30℃ water bath for 30min to mix it evenly and obtain the oil phase;

[0105] The mixed solution of PDRN and collagen was added dropwise to the oil phase using a syringe and stirred for 30 minutes to obtain an emulsion.

[0106] S3, add 8 mL of polyethylene glycol diacrylate to the emulsion and stir at 30°C for 24 h.

[0107] After the reaction was complete, the reaction solution was poured into a centrifuge bottle and centrifuged at 8000 rpm for 10 min. Isopropanol was then added, and the mixture was centrifuged and washed three times, discarding the supernatant. Anhydrous ethanol was then added, and the mixture was centrifuged and washed three more times until the microspheres appeared as white, fine particles. These particles were then placed in a vacuum drying oven at 30°C and dried for 12 h. The dried microspheres were then sieved to obtain microspheres with a particle size of 20–45 μm.

[0108] The microstructure of the microspheres prepared in Examples 1-3 was tested. The SEM image of the collagen microspheres prepared in Example 1 is shown below. Figure 1 As shown; SEM image of the hyaluronic acid and collagen composite microspheres prepared in Example 2 is shown. Figure 2 As shown; SEM image of the PDRN and collagen composite microspheres prepared in Example 3 is shown. Figure 3 As shown. From Figures 1-3 As can be seen, the microspheres prepared in this application exhibit a regular spherical morphology with a smooth and flat surface.

[0109] The anti-degradation properties of the microspheres prepared in the examples and comparative examples were tested, and the specific methods are as follows:

[0110] Take 0.01 g (1 mg / mL) of sample into a vial, add 10 mL of collagenase III solution (collagenase III concentration is 1 mg / mL) to the vial, seal the vial and place it in a constant temperature water bath shaker at 37℃ and 120 rpm for 24 h. After 24 h, take it out, add protease inhibitor, transfer the mixture to a centrifuge tube and centrifuge at 10000 rpm and 4℃ for 20 min. After centrifugation, take 1 mL of supernatant into a stoppered test tube for testing.

[0111] Collagen content was tested according to the BCA method in General Chapter 0731, Part IV, of the 2020 edition of the Chinese Pharmacopoeia. A lower enzymatic hydrolysis rate indicates better resistance to degradation and a longer duration of filling effect in the human body. The test results are shown in Table 1. The collagen degradation rate was calculated as follows:

[0112]

[0113] Table 1. Collagen degradation rate of microspheres in the examples and comparative examples.

[0114] sample Degradation rate Example 1 24％ Example 2 10％ Example 3 34％ Comparative Example 2 93％ Comparative Example 3 65％ Comparative Example 4 55％ Comparative Example 5 90％

[0115] As shown in Table 1, compared with the microspheres prepared in this application, the microspheres prepared by the strategy of two cross-linking with the same cross-linking agent in Comparative Examples 2-4 and the single cross-linking strategy in Comparative Example 5 all had significantly higher collagen degradation rates. This application constructs a multi-level interpenetrating network within the microspheres through a two-cross-linking strategy using PEGDA and EDC / ADH, leveraging the complementary advantages of each cross-linking method to overcome the shortcomings of insufficient mechanical properties in single cross-linking methods, thereby improving the enzymatic resistance of the microspheres. Among them, the microspheres of Example 2 exhibited the lowest collagen degradation rate.

[0116] The microspheres prepared in the examples and comparative examples were tested for their moist heat sterilization performance. The specific methods are as follows:

[0117] Take 0.1 g (20 mg / mL) of the experimental sample into a vial, place the vial in an autoclave for sterilization at 121℃ for 30 min; after sterilization, freeze-dry the sample and then perform SEM imaging. Figure 4 As shown. Among them, Figure 4 Figure A shows the microspheres of Example 1, Figure B shows the microspheres of Example 2, Figure C shows the microspheres of Example 3, Figure D shows the microspheres of Comparative Example 2, Figure E shows the microspheres of Comparative Example 3, Figure F shows the microspheres of Comparative Example 4, and Figure G shows the microspheres of Comparative Example 5.

[0118] from Figure 4 It can be seen that the microspheres in Examples 1, 2, and 3 retained their spherical structure intact after moist heat sterilization, thus achieving successful moist heat sterilization. However, the microspheres in Comparative Examples 2, 3, 4, and 5 all showed varying degrees of damage after moist heat sterilization, failing to achieve the desired results. Specifically, in Comparative Example 2, the intact spherical structure was barely visible; in Comparative Example 3, the microsphere structure was severely damaged, with the microspheres agglomerating into sheet-like aggregates and exhibiting poor monodispersity; in Comparative Example 4, the microsphere structure collapsed, showing good monodispersity; and in Comparative Example 5, the microspheres were essentially broken into fragments.

[0119] Although this application has been described in detail in this specification with general descriptions and specific embodiments, some modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of this application are all within the scope of protection claimed in this application.

Claims

1. A method for preparing microspheres that are sterilizable by moist heat and resistant to enzymatic degradation, characterized in that, include: S1, dissolve the polymer in a solvent to obtain an aqueous phase; S2, the aqueous phase is added to the oil phase containing the emulsifier, and emulsification is performed to obtain an emulsion; S3, add a first crosslinking agent to the emulsion to carry out a crosslinking reaction; then add a second crosslinking agent to carry out a crosslinking reaction; after the reaction, collect the solid phase, wash and dry it to obtain microspheres.

2. The preparation method according to claim 1, characterized in that, The polymer includes collagen, hyaluronic acid, and PDRN; The solvent is water or an aqueous solution of sodium hydroxide; The oil phase includes any one of vegetable oil, mineral oil, silicone oil, or liquid paraffin. The emulsifier is dehydrated sorbitan monooleate or polysorbate-80.

3. The preparation method according to claim 2, characterized in that, In the aqueous phase, the concentration of hyaluronic acid is 0–3 wt%, the concentration of collagen is 0.2–30 wt%, and the concentration of PDRN is 0–10 wt%.

4. The preparation method according to claim 1, characterized in that, The first crosslinking agent is polyethylene glycol diacrylate; The second crosslinking agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and adipic acid dihydrazide.

5. The preparation method according to claim 4, characterized in that, In the second crosslinking agent, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to adipic acid dihydrazide is 1:(0.5-1).

6. The preparation method according to claim 1, characterized in that, The mass ratio of the first crosslinking agent to the polymer is (1-10):1; The mass ratio of the second crosslinking agent to the polymer is (0.1 to 1):

1.

7. The preparation method according to claim 1, characterized in that, The oil phase contains 2-10 wt% emulsifier; The volume ratio of the aqueous phase to the oil phase is 1:5 to 20.

8. The preparation method according to claim 1, characterized in that, The temperatures for the first and second crosslinking reactions are 25–50°C.

9. Microspheres prepared by the preparation method according to any one of claims 1-8.

10. The application of the microspheres according to claim 9 in cosmetic filling, injection and tissue repair.

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