Collagen inducible injectable polylactic acid microsphere filler as well as preparation method and application thereof

Abstract

The invention relates to a collagen inducible injectable polylactic acid microsphere filler as well as a preparation method and application thereof. The preparation method of the collagen induced injectable polylactic acid microsphere filler comprises the following steps: (1) preparing polylactic acid microspheres; (2) preparing polylactic acid microspheres of which the surfaces are grafted with collagen peptide; (3) preparing a composite outer water phase; (4) preparing collagen induced polylactic acid microspheres; and (5) preparing the collagen induced injectable polylactic acid microsphere filler. According to the collagen-induced injectable polylactic acid microsphere filler, fibroblasts can be specifically activated through the collagen peptide grafted on the surfaces of the polylactic acid microspheres, I-type collagen synthesis is promoted, and the collagen regeneration effect is achieved. The agarose and the PLGA in the composite outer water phase form a composite shell on the surface of the microsphere filling agent, so that the supporting stability of the microsphere filling agent is effectively improved, the degradation of a core structure is greatly delayed, long-acting filling is realized, and the subcutaneous degradation time of rats reaches 11 months or above.

Description

Technical Field

[0001] This invention relates to a collagen-inducible injectable polylactic acid microsphere filler, its preparation method and application, belonging to the field of medical aesthetic filler technology. Background Technology

[0002] Correcting soft tissue defects and repairing aging, wrinkled skin through cosmetic surgery to maintain a perfect and youthful facial appearance has become a common practice. Among various cosmetic surgery techniques, injectable cosmetic procedures account for the largest share, approximately 40% of the entire beauty market. Injectable cosmetic procedures involve localized modifications to the body through injections, achieving refined local features and overall harmony. This technique is characterized by zero downtime, rapid results, and high safety, making it the preferred choice for those seeking cosmetic enhancements.

[0003] Currently, sodium hyaluronate is the primary filler material used in injectable cosmetic procedures. However, sodium hyaluronate degrades relatively quickly, and even cross-linked sodium hyaluronate gel does not provide long-term support within the body. Furthermore, traditional sodium hyaluronate fillers are absorbent and fluid, making it difficult to ensure adequate volume changes at the injection site. To more effectively extend the support time of fillers, biodegradable polymer microspheres have emerged.

[0004] Polylactic acid (PLA), as a synthetic polymer, possesses excellent biocompatibility and biodegradability, and is widely used in the fields of biomedicine and tissue engineering. In recent years, PLA microspheres have received widespread attention as soft tissue fillers due to their advantages, including ease of use, minimal trauma, short recovery time, and significant repair effects. After injection into the skin, PLA microspheres stimulate collagen production, achieving an autologous filling effect, and are particularly effective for deeper wrinkles or folds such as crow's feet and nasolabial folds. The implanted PLA microspheres slowly degrade into carbon dioxide and water, and do not remain permanently in the dermis, with no potential adverse reactions.

[0005] However, although polylactic acid fillers can stimulate the body's own collagen regeneration and have a relatively long duration of action, pure polylactic acid materials have poor adhesion to human tissues and insufficient initial support. They are prone to displacement and deformation after filling. Furthermore, their efficiency in stimulating collagen needs to be improved, making it difficult to achieve the desired facial rejuvenation effect in a short period of time.

[0006] Therefore, there is an urgent need to develop a new type of injectable filler that can provide good dynamic support, effectively induce collagen regeneration, prolong the duration of the filling effect, and meet the higher demands of the medical aesthetics field for facial rejuvenation. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a collagen-inducible injectable polylactic acid microsphere filler, its preparation method, and its application.

[0008] The technical solution of the present invention is as follows:

[0009] A method for preparing a collagen-inducible injectable polylactic acid microsphere filler includes the following steps:

[0010] (1) Dissolve polylactic acid in dichloromethane to prepare a polylactic acid solution; then, under stirring conditions of 1000~3000 rpm, add the polylactic acid solution dropwise to a polyvinyl alcohol aqueous solution and emulsify for 20~40 min to form an oil-in-water emulsion; after vacuum evaporation, centrifugation, washing and freeze drying, the obtained oil-in-water emulsion yields polylactic acid microspheres;

[0011] (2) Disperse the polylactic acid microspheres obtained in step (1) in N,N-dimethylformamide to prepare a polylactic acid microsphere suspension; then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the polylactic acid microsphere suspension and activate the reaction at 20~30℃ for 30~60min; continue to add collagen peptides and react at 20~30℃ for 12~24h. After centrifugation, washing and freeze drying, polylactic acid microspheres with collagen peptides grafted on the surface are obtained.

[0012] (3) Add agarose, sodium hyaluronate, arginine and elastin to deionized water in sequence, heat to 90~100℃, stir until completely dissolved, and cool to 50~60℃ to obtain a composite external aqueous phase;

[0013] (4) Disperse the polylactic acid microspheres with surface-grafted collagen peptides obtained in step (2) in deionized water containing poloxamer and sonicate for 2-8 min to obtain an inner aqueous phase; then add the inner aqueous phase dropwise to an ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer and stir at 500-1000 rpm for 10-20 min to obtain a water / oil primary emulsion;

[0014] Next, the water / oil pre-emulsion was added to the composite external aqueous phase obtained in step (2), and stirred at 50-60℃ and 1200-1500rpm for 10-20min. After vacuum evaporation, gel solidification, centrifugation, washing and freeze drying, collagen-induced polylactic acid microspheres were obtained.

[0015] (5) Disperse the collagen-inducible polylactic acid microspheres obtained in step (4) into deionized water containing poloxamer and sodium hyaluronate, stir and mix evenly to obtain collagen-inducible injectable polylactic acid microsphere filler.

[0016] According to a preferred embodiment of the present invention, in step (1), the concentration of the polylactic acid solution is 100~200 mg / mL.

[0017] According to a preferred embodiment of the present invention, in step (1), the mass concentration of the polyvinyl alcohol aqueous solution is 1-3%.

[0018] According to a preferred embodiment of the present invention, in step (1), the volume ratio of the polylactic acid solution to the polyvinyl alcohol aqueous solution is (1~2):10.

[0019] According to a preferred embodiment of the present invention, in step (1), the specific process of vacuum evaporation, centrifugation, washing and freeze drying is as follows: first, remove dichloromethane by vacuum evaporation at 40~60℃ and -0.08MPa for 8~12min, then centrifuge at 6000~10000rpm for 8~12min, collect the precipitate, wash with deionized water 3~5 times and then freeze dry.

[0020] According to a preferred embodiment of the present invention, in step (2), the concentration of the polylactic acid microsphere suspension is 50~100 mg / mL.

[0021] According to a preferred embodiment of the present invention, in step (2), the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to polylactic acid microspheres is (1~2):1; and the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to N-hydroxysuccinimide (NHS) is 1:(1~1.5).

[0022] According to a preferred embodiment of the present invention, in step (2), the collagen peptide and polylactic acid microspheres are added in a mass ratio of 1:(8~12).

[0023] More preferably, the collagen peptide is fish collagen peptide, porcine collagen peptide, or bovine collagen peptide.

[0024] According to a preferred embodiment of the present invention, in step (2), the specific process of centrifugation, washing and freeze-drying is as follows: centrifuge at 6000~10000 rpm for 8~12 min, collect the precipitate, wash with deionized water 3~5 times and then freeze-dry.

[0025] According to a preferred embodiment of the present invention, in step (3), the mass concentration of agarose in the composite aqueous phase is 2-4%, the mass concentration of sodium hyaluronate is 1-3%, the mass concentration of arginine is 1-3%, and the mass concentration of elastin is 0.3-0.8%.

[0026] More preferably, the elastin is fish elastin.

[0027] According to a preferred embodiment of the present invention, in step (4), the mass concentration of the deionized water containing poloxamer is 0.05~0.15%; in the ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer, the mass concentration of Span-80 is 1~2%, and the concentration of polylactic acid-glycolic acid copolymer is 100~200 mg / mL.

[0028] According to a preferred embodiment of the present invention, in step (4), the mass-to-volume ratio of the polylactic acid microspheres with surface-grafted collagen peptides, the deionized water containing poloxamer, and the ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer is 40~60 mg: 5~15 ml: (25~30) ml.

[0029] According to a preferred embodiment of the present invention, in step (4), the volume ratio of the water / oil primary emulsion to the composite external aqueous phase is (30~45):(100~120).

[0030] According to a preferred embodiment of the present invention, in step (4), the specific process of vacuum evaporation, gel solidification, centrifugation, washing and freeze-drying is as follows: first, ethyl acetate is removed by vacuum evaporation for 8-12 min at 40-60℃ and -0.08MPa, then solidified by standing at 20-30℃ for 25-35 min, then transferred to 4-10℃ for cooling for 1.5-2.5 h, then centrifuged at 2500-3500 rpm for 8-12 min, the microsphere precipitate is collected, washed with deionized water 3-5 times and then freeze-dried.

[0031] According to a preferred embodiment of the present invention, in step (5), the mass concentration of poloxamer in the deionized water containing poloxamer and sodium hyaluronate is 5-6%, and the mass concentration of sodium hyaluronate is 3-4%.

[0032] According to a preferred embodiment of the present invention, in step (5), the mass ratio of the collagen-induced polylactic acid microspheres to deionized water containing poloxamer and sodium hyaluronate is (1~2):10.

[0033] A collagen-inducible injectable polylactic acid microsphere filler is prepared according to the above method.

[0034] The above-mentioned collagen-inducible injectable polylactic acid microsphere fillers are used in the fields of biomedicine, cosmetics, and medical aesthetics.

[0035] Technical features and beneficial effects of the present invention:

[0036] 1. The collagen-inducible injectable polylactic acid (PLA) microsphere filler prepared in this invention specifically activates fibroblasts and promotes type I collagen synthesis through collagen peptides grafted onto the surface of the PLA microspheres, achieving a collagen regeneration effect. Furthermore, by combining agarose and PLGA in the aqueous phase to form a composite shell on the surface of the microsphere filler, the supporting stability of the microsphere filler is effectively improved, the degradation of the core structure is greatly delayed, and long-lasting filling is achieved, with a skin degradation time of over 11 months in rats.

[0037] 2. The collagen-inducible injectable polylactic acid microsphere filler prepared in this invention enhances the moisturizing properties and softness of the microsphere filler with sodium hyaluronate, and imparts gel elasticity to the microsphere filler through elastin, adapting to facial muscle movements and avoiding a stiff appearance. Furthermore, the microsphere filler is uniformly dispersed throughout, effectively enhancing overall mechanical stability and preventing displacement and deformation. When using a 30G needle, the pushing force is 10~15N, with no needle blockage, exhibiting good dynamic mechanical properties and injectability.

[0038] 3. The degradation products of the collagen-inducible injectable polylactic acid microsphere filler prepared in this invention are water and carbon dioxide, with a cell survival rate of >95%, no obvious toxicity, and good biocompatibility.

[0039] 4. The preparation method of collagen-induced injectable polylactic acid microsphere filler provided by the present invention is simple to operate, has few steps, and is suitable for large-scale industrial production. Attached Figure Description

[0040] Figure 1 This is a microscope image of the collagen-inducible injectable polylactic acid microsphere filler prepared in Example 1 of the present invention. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and materials involved in the embodiments are all commercially available products.

[0042] In this embodiment, the collagen peptides are fish collagen peptides, porcine collagen peptides, or bovine collagen peptides, which are commercially available. The elastin is fish elastin, which is commercially available.

[0043] Example 1

[0044] A method for preparing a collagen-inducible injectable polylactic acid microsphere filler includes the following steps:

[0045] (1) Dissolve 150 mg of polylactic acid (molecular weight of 50000 Da) in 1 mL of dichloromethane to prepare a polylactic acid solution with a concentration of 150 mg / mL; then add it dropwise to 10 mL of 2% polyvinyl alcohol aqueous solution under stirring at 2000 rpm, emulsify for 30 min to form an oil-in-water emulsion.

[0046] The resulting oil-in-water emulsion was first evaporated under reduced pressure at 50℃ and -0.08MPa for 10 min to remove dichloromethane, then centrifuged at 8000rpm for 10 min, the precipitate was collected, washed 4 times with deionized water and then freeze-dried to obtain polylactic acid microspheres.

[0047] (2) Disperse 80 mg of polylactic acid microspheres obtained in step (1) in 1 mL of N,N-dimethylformamide to prepare a polylactic acid microsphere suspension with a concentration of 80 mg / mL; then add EDC and NHS to the polylactic acid microsphere suspension and activate the reaction at 25 °C for 45 min; continue to add 8 mg of bovine collagen peptides and react at 25 °C for 18 h. Centrifuge the reaction solution at 8000 rpm for 10 min, collect the precipitate, wash it 4 times with deionized water and freeze dry it to obtain polylactic acid microspheres with collagen peptide grafted on the surface.

[0048] The mass ratio of EDC to polylactic acid microspheres is 1.5:1, and the molar ratio of EDC to NHS is 1:1.2.

[0049] (3) Add 2g agarose, 1g sodium hyaluronate, 1g arginine and 0.5g elastin to 100ml deionized water, heat to 95℃, stir until completely dissolved, cool to 55℃ to obtain composite external aqueous phase;

[0050] (4) Disperse 40 mg of polylactic acid microspheres with surface-grafted collagen peptides obtained in step (2) in 10 mL of deionized water containing poloxamer and sonicate for 5 min to obtain an inner aqueous phase; then add the inner aqueous phase dropwise to 30 mL of ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer and stir at 800 rpm for 15 min to obtain a water / oil primary emulsion;

[0051] The deionized water containing poloxamer had a mass concentration of 0.1%; in the ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer, the mass concentration of Span-80 was 1.5% and the concentration of polylactic acid-glycolic acid copolymer was 150 mg / mL.

[0052] Next, 40 mL of water / oil pre-emulsion was added to 100 mL of the composite external aqueous phase obtained in step (3), and stirred at 55 °C and 1300 rpm for 15 min. The reaction solution was first evaporated under reduced pressure at 55 °C and -0.08 MPa for 10 min to remove ethyl acetate, and then allowed to stand and solidify at 25 °C for 30 min. After that, it was transferred to 7 °C and cooled for 2 h. Then, it was centrifuged at 3000 rpm for 10 min, and the microsphere precipitate was collected. After washing with deionized water for 4 minutes, it was freeze-dried to obtain collagen-induced polylactic acid microspheres.

[0053] (5) Disperse 15 mg of collagen-inducible polylactic acid microspheres obtained in step (4) into 100 mL of deionized water containing poloxamer and sodium hyaluronate, stir and mix evenly to obtain collagen-inducible injectable polylactic acid microsphere filler;

[0054] In the deionized water containing poloxamer and sodium hyaluronate, the mass concentration of poloxamer is 5% and the mass concentration of sodium hyaluronate is 3%.

[0055] Microscopic images of the collagen-inducible injectable polylactic acid microsphere filler prepared in this embodiment are as follows: Figure 1 As shown.

[0056] Depend on Figure 1 It can be seen that the collagen-inducible injectable polylactic acid microsphere filler has been successfully prepared and exhibits a spherical structure under a microscopic scale.

[0057] Example 2

[0058] A method for preparing a collagen-inducible injectable polylactic acid microsphere filler includes the following steps:

[0059] (1) Dissolve 200 mg of polylactic acid (molecular weight of 80000 Da) in 1 mL of dichloromethane to prepare a polylactic acid solution with a concentration of 200 mg / mL; then add it dropwise to 10 mL of 3% polyvinyl alcohol aqueous solution under stirring at 3000 rpm, emulsify for 20 min to form an oil-in-water emulsion.

[0060] The resulting oil-in-water emulsion was first evaporated under reduced pressure at 50℃ and -0.08MPa for 10 min to remove dichloromethane, then centrifuged at 8000rpm for 10 min, the precipitate was collected, washed 4 times with deionized water and then freeze-dried to obtain polylactic acid microspheres.

[0061] (2) Disperse 100 mg of polylactic acid microspheres obtained in step (1) in 1 mL of N,N-dimethylformamide to prepare a polylactic acid microsphere suspension with a concentration of 100 mg / mL; then add EDC and NHS to the polylactic acid microsphere suspension and activate the reaction at 25 °C for 60 min; continue to add 12.5 mg of fish collagen peptide and react at 25 °C for 24 h. Centrifuge the reaction solution at 8000 rpm for 10 min, collect the precipitate, wash it 4 times with deionized water and freeze dry it to obtain polylactic acid microspheres with collagen peptide grafted on the surface.

[0062] The mass ratio of EDC to polylactic acid microspheres is 2:1, and the molar ratio of EDC to NHS is 1:1.5.

[0063] (3) Add 4g agarose, 2g sodium hyaluronate, 2g arginine and 0.8g elastin to 120ml deionized water, heat to 100℃, stir until completely dissolved, cool to 60℃ to obtain composite external aqueous phase;

[0064] (4) Disperse 50 mg of polylactic acid microspheres with surface-grafted collagen peptides obtained in step (2) in 10 mL of deionized water containing poloxamer and sonicate for 5 min to obtain an inner aqueous phase; then add the inner aqueous phase dropwise to 30 mL of ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer and stir at 800 rpm for 15 min to obtain a water / oil primary emulsion;

[0065] The deionized water containing poloxamer had a mass concentration of 0.1%; in the ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer, the mass concentration of Span-80 was 1.5% and the concentration of polylactic acid-glycolic acid copolymer was 150 mg / mL.

[0066] Next, 40 mL of water / oil pre-emulsion was added to 120 mL of the composite external aqueous phase obtained in step (3), and stirred at 55℃ and 1500 rpm for 10 min. The reaction solution was first evaporated under reduced pressure at 55℃ and -0.08 MPa for 10 min to remove ethyl acetate, and then allowed to stand and solidify at 25℃ for 30 min. Then it was transferred to 10℃ and cooled for 2 h. Then it was centrifuged at 3000 rpm for 10 min, and the microsphere precipitate was collected. After washing with deionized water for 4 minutes, it was freeze-dried to obtain collagen-induced polylactic acid microspheres.

[0067] (5) Disperse 20 mg of collagen-inducible polylactic acid microspheres obtained in step (4) into 100 mL of deionized water containing poloxamer and sodium hyaluronate, stir and mix evenly to obtain collagen-inducible injectable polylactic acid microsphere filler;

[0068] In the deionized water containing poloxamer and sodium hyaluronate, the mass concentration of poloxamer is 6% and the mass concentration of sodium hyaluronate is 4%.

[0069] Example 3

[0070] A method for preparing a collagen-inducible injectable polylactic acid microsphere filler includes the following steps:

[0071] (1) Dissolve 100 mg of polylactic acid (molecular weight of 50000 Da) in 1 mL of dichloromethane to prepare a polylactic acid solution with a concentration of 100 mg / mL; then add it dropwise to 10 mL of 1% polyvinyl alcohol aqueous solution under stirring at 1000 rpm, emulsify for 40 min to form an oil-in-water emulsion;

[0072] The resulting oil-in-water emulsion was first evaporated under reduced pressure at 50℃ and -0.08MPa for 10 min to remove dichloromethane, then centrifuged at 8000rpm for 10 min, the precipitate was collected, washed 4 times with deionized water and then freeze-dried to obtain polylactic acid microspheres.

[0073] (2) Disperse 50 mg of polylactic acid microspheres obtained in step (1) in 1 mL of N,N-dimethylformamide to prepare a polylactic acid microsphere suspension with a concentration of 50 mg / mL; then add EDC and NHS to the polylactic acid microsphere suspension and activate the reaction at 25 °C for 30 min; continue to add 5 mg of porcine collagen peptide and react at 25 °C for 12 h. Centrifuge the reaction solution at 8000 rpm for 10 min, collect the precipitate, wash it 4 times with deionized water and freeze dry it to obtain polylactic acid microspheres with collagen peptide grafted on the surface.

[0074] The mass ratio of EDC to polylactic acid microspheres is 1:1, and the molar ratio of EDC to NHS is 1:1.

[0075] (3) Add 3g agarose, 1.5g sodium hyaluronate, 1.5g arginine and 0.6g elastin to 100ml deionized water, heat to 95℃, stir until completely dissolved, and cool to 55℃ to obtain a composite external aqueous phase;

[0076] (4) Disperse 60 mg of polylactic acid microspheres with surface-grafted collagen peptides obtained in step (2) in 10 mL of deionized water containing poloxamer and sonicate for 5 min to obtain an inner aqueous phase; then add the inner aqueous phase dropwise to 30 mL of ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer and stir at 800 rpm for 15 min to obtain a water / oil primary emulsion;

[0077] The deionized water containing poloxamer had a mass concentration of 0.1%; in the ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer, the mass concentration of Span-80 was 1.5% and the concentration of polylactic acid-glycolic acid copolymer was 150 mg / mL.

[0078] Next, 40 mL of water / oil pre-emulsion was added to 100 mL of the composite external aqueous phase obtained in step (3), and stirred at 55℃ and 1500 rpm for 10 min. The reaction solution was first evaporated under reduced pressure at 55℃ and -0.08 MPa for 10 min to remove ethyl acetate, and then allowed to stand and solidify at 25℃ for 30 min. Then it was transferred to 10℃ and cooled for 2 h. Then it was centrifuged at 3000 rpm for 10 min, and the microsphere precipitate was collected. After washing with deionized water for 4 minutes, it was freeze-dried to obtain collagen-induced polylactic acid microspheres.

[0079] (5) Disperse 10 mg of collagen-inducible polylactic acid microspheres obtained in step (4) into 100 mL of deionized water containing poloxamer and sodium hyaluronate, stir and mix evenly to obtain collagen-inducible injectable polylactic acid microsphere filler;

[0080] In the deionized water containing poloxamer and sodium hyaluronate, the mass concentration of poloxamer is 5.5% and the mass concentration of sodium hyaluronate is 3.5%.

[0081] Comparative Example 1

[0082] A method for preparing a polylactic acid microsphere filler without a composite external aqueous phase includes the following steps:

[0083] (1) Dissolve 150 mg of polylactic acid (molecular weight of 50000 Da) in 1 mL of dichloromethane to prepare a polylactic acid solution with a concentration of 150 mg / mL; then add it dropwise to 10 mL of 2% polyvinyl alcohol aqueous solution under stirring at 2000 rpm, emulsify for 30 min to form an oil-in-water emulsion.

[0084] The resulting oil-in-water emulsion was first evaporated under reduced pressure at 50℃ and -0.08MPa for 10 min to remove dichloromethane, then centrifuged at 8000rpm for 10 min, the precipitate was collected, washed 4 times with deionized water and then freeze-dried to obtain polylactic acid microspheres.

[0085] (2) Disperse 80 mg of polylactic acid microspheres obtained in step (1) in 1 mL of N,N-dimethylformamide to prepare a polylactic acid microsphere suspension with a concentration of 80 mg / mL; then add EDC and NHS to the polylactic acid microsphere suspension and activate the reaction at 25 °C for 45 min; continue to add 8 mg of bovine collagen peptides and react at 25 °C for 18 h. Centrifuge the reaction solution at 8000 rpm for 10 min, collect the precipitate, wash it 4 times with deionized water and freeze dry it to obtain polylactic acid microspheres with collagen peptide grafted on the surface.

[0086] The mass ratio of EDC to polylactic acid microspheres is 1.5:1, and the molar ratio of EDC to NHS is 1:1.2.

[0087] (5) Disperse 10 mg of collagen-induced polylactic acid microspheres obtained in step (2) into 100 mL of deionized water containing poloxamer and sodium hyaluronate, stir and mix evenly to obtain a filler without a composite external aqueous phase.

[0088] Compared with Example 1, the filler without a composite external aqueous phase prepared in this comparative example only retains the structure of polylactic acid microspheres + collagen peptides + basic carrier, lacks the agarose-PLGA composite shell, and lacks the functional modifications of elastin and arginine.

[0089] Comparative Example 2

[0090] A method for preparing a collagen peptide-free polylactic acid microsphere filler includes the following steps:

[0091] (1) Dissolve 150 mg of polylactic acid (molecular weight of 50000 Da) in 1 mL of dichloromethane to prepare a polylactic acid solution with a concentration of 150 mg / mL; then add it dropwise to 10 mL of 2% polyvinyl alcohol aqueous solution under stirring at 2000 rpm, emulsify for 30 min to form an oil-in-water emulsion.

[0092] The resulting oil-in-water emulsion was first evaporated under reduced pressure at 50℃ and -0.08MPa for 10 min to remove dichloromethane, then centrifuged at 8000rpm for 10 min, the precipitate was collected, washed 4 times with deionized water and then freeze-dried to obtain polylactic acid microspheres.

[0093] (2) Add 2g agarose, 1g sodium hyaluronate, 1g arginine and 0.5g elastin to 100ml deionized water, heat to 95℃, stir until completely dissolved, and cool to 55℃ to obtain a composite external aqueous phase;

[0094] (3) Disperse 40 mg of polylactic acid microspheres obtained in step (1) in 5 mL of deionized water containing poloxamer and sonicate for 5 min to obtain an inner aqueous phase; then add the inner aqueous phase dropwise to 25 mL of ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer and stir at 800 rpm for 15 min to obtain a water / oil primary emulsion.

[0095] The deionized water containing poloxamer had a mass concentration of 0.1%; in the ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer, the mass concentration of Span-80 was 1.5% and the concentration of polylactic acid-glycolic acid copolymer was 150 mg / mL.

[0096] Next, 30 mL of water / oil pre-emulsion was added to 100 mL of the composite external aqueous phase obtained in step (2), and stirred at 55 °C and 1300 rpm for 15 min. The reaction solution was first evaporated under reduced pressure at 55 °C and -0.08 MPa for 10 min to remove ethyl acetate, and then allowed to stand and solidify at 25 °C for 30 min. Then it was transferred to 7 °C and cooled for 2 h. Next, it was centrifuged at 3000 rpm for 10 min, and the microsphere precipitate was collected. After washing with deionized water for 4 minutes, it was freeze-dried to obtain collagen-free polylactic acid microspheres.

[0097] (5) Disperse 15 mg of the microspheres obtained in step (4) into 100 mL of deionized water containing poloxamer and sodium hyaluronate, stir and mix evenly to obtain polylactic acid microsphere filler without collagen peptide grafting.

[0098] In the deionized water containing poloxamer and sodium hyaluronate, the mass concentration of poloxamer is 5% and the mass concentration of sodium hyaluronate is 3%.

[0099] Compared with Example 1, the collagen peptide-free polylactic acid microsphere filler prepared in this comparative example only retains the structure of polylactic acid microspheres + composite external aqueous phase + basic carrier, and there is no collagen peptide grafting on the surface of the microspheres.

[0100] Test case

[0101] 1. Collagen induction experiment

[0102] Cell preparation: Dermal fibroblasts from SD rats were cultured in DMEM medium containing 10% fetal bovine serum to passage 3, and the cell concentration was adjusted to 5 × 10⁶ cells / year. 4 Inoculate 1 mL per well into a 24-well plate and incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0103] Sample preparation: The polylactic acid microsphere packing materials prepared in Examples 1-3 and Comparative Examples 1-2 were diluted to 1 mg / mL with sterile PBS buffer, filtered through a 0.22 μm filter membrane, and 100 μL was added to each culture well. A blank control group (with only an equal amount of PBS buffer added) was set up, and each group had 3 replicates.

[0104] Culture and sampling: Cells were cultured for 1 month, 3 months and 6 months respectively. After the expiration, the cell culture supernatant was collected, and the cells were also digested with trypsin to collect the cells.

[0105] Type I collagen content detection: Enzyme-linked immunosorbent assay (ELISA) was used, and the operation was strictly followed according to the instructions of the Type I collagen detection kit. The concentration of Type I collagen in the supernatant of each well was measured, and the mean and standard deviation were calculated.

[0106] Fibroblast counting: The collected cells were washed twice with PBS, resuspended in 1 mL of culture medium, and 10 μL of cell suspension was mixed with 10 μL of trypan blue staining solution. The number of live cells was counted under a microscope. Three fields of view were counted for each sample, and the average value was taken.

[0107] The results of type I collagen content detection and fibroblast counting are shown in Table 1.

[0108] Table 1

[0109]

[0110] As shown in Table 1, the collagen-inducible injectable polylactic acid (PLA) microsphere fillers prepared in Examples 1-3 showed significantly higher levels of type I collagen and fibroblasts after 6 months compared to the PLA microsphere filler prepared in Comparative Example 2 without collagen peptide grafting. This indicates that the collagen peptides grafted onto the surface of PLA microspheres in this invention can specifically activate fibroblasts, promote type I collagen synthesis, and achieve collagen regeneration.

[0111] 2. Long-term support performance test

[0112] Animal model establishment: Fifteen healthy SD rats weighing 200-250g were selected and divided into five groups of three. After hair removal on the back, the rats were disinfected with 75% alcohol. Four points were symmetrically injected into the back of each rat, and 0.5mL of the test filler was injected into each point. The five groups were filled with polylactic acid microsphere fillers prepared in Examples 1-3 and Comparative Examples 1-2, respectively, and the injection sites were marked.

[0113] Volume Measurement: At 1 month, 3 months, 6 months, and 11 months post-injection, the volume of each injection site was measured using the water displacement method. Specifically, after anesthetizing the rats, fluid surrounding the injection site was aspirated using a sterile syringe and placed into a measuring cup of known volume. The volume of water displaced was recorded as the volume of the filling site, and the volume maintenance rate was calculated.

[0114] Volume retention rate = current volume / initial injection volume × 100%.

[0115] Observation of microsphere degradation status: Rats were sacrificed after 11 months, and tissue blocks from the injection site were taken, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under an optical microscope. The degree of degradation (no degradation, mild degradation, moderate degradation, and extensive degradation) was recorded.

[0116] The results of volume retention rate measurement and microsphere degradation state observation are shown in Table 2.

[0117] Table 2

[0118]

[0119] As shown in Table 2, and as shown in Table 1, the collagen-inducible injectable polylactic acid (PLA) microsphere fillers prepared in Examples 1-3 achieved an 11-month volume retention rate of 33-42%, with intact microsphere morphology. In contrast, the PLA microsphere filler prepared in Comparative Example 1 without a composite external aqueous phase only achieved an 11-month volume retention rate of 18%, with significant microsphere degradation. This is because the present invention effectively improves the supporting stability of the microsphere filler and greatly delays the degradation of the core structure through the composite external aqueous phase (agarose-PLGA shell). The PLA microsphere filler prepared in Comparative Example 2 without collagen peptide grafting also did not exceed 30% in 11 months. This is because Comparative Example 2 lacked collagen regeneration assistance, and its supporting performance was still weaker than that of Examples 1-3, fully demonstrating the synergistic effect of collagen regeneration + composite shell.

[0120] 3. Dynamic mechanical property test

[0121] Sample preparation: The polylactic acid microsphere fillers prepared in Examples 1-3 and Comparative Examples 1-2 were injected into a sterile circular mold (20 mm in diameter and 2 mm in thickness), and left to stand for 24 h at 37°C and 60% humidity to form a uniform gel sample. Three parallel samples were prepared for each group.

[0122] Elastic modulus (G') testing: A rotational rheometer was used with a flat plate fixture (20 mm in diameter) and a plate spacing of 1.5 mm. The temperature was kept constant at 37°C (simulating the human body environment). The gel sample was then placed in the center of the fixture, excess gel sample was scraped off, and the test began after equilibration for 10 minutes.

[0123] First, a strain scan (strain range 0.1%~10%, angular frequency 10 rad / s) is performed to determine the linear viscoelastic region of the gel sample; then, a frequency scan (frequency range 0.1~100 rad / s, strain 1%) is performed within the linear viscoelastic region to record the average value of the storage modulus (G', i.e., elastic modulus) of the gel sample, which is used as an evaluation index of the gel sample's elasticity.

[0124] Skin mobility test: Ten healthy New Zealand rabbits were selected. After hair removal and disinfection of their backs, polylactic acid microsphere fillers prepared in Examples 1-3 and Comparative Examples 1-2 were injected into five different locations on the back of each rabbit. 0.3 mL was injected at each injection point. One month after injection, the flexibility of the rabbit's back skin during spontaneous activities (such as turning the head and bending over) was observed. The number of injection points without stiffness and with natural movement was recorded, and the qualified rate was calculated.

[0125] Pass rate = (Number of pass points / Total number of injection points) × 100%.

[0126] The results of the elastic modulus (G') test and the skin mobility test are shown in Table 3.

[0127] Table 3

[0128]

[0129] As shown in Table 3, the elastic modulus of the collagen-inducible injectable polylactic acid (PLA) microsphere fillers prepared in Examples 1-3 is 76-85 kPa, which matches the natural elasticity of the skin, and the qualified skin mobility ratio is 80-100%, with virtually no stiffness. The elastic moduli of the PLA microsphere fillers without a composite aqueous phase prepared in Comparative Example 1 and without collagen peptide grafting prepared in Comparative Example 2 are 115 kPa and 105 kPa, respectively, with the highest qualified skin mobility ratio being 70%, while Comparative Example 1 only has 30%, resulting in stiffness. This indicates that the elastin in the composite aqueous phase of the collagen-inducible injectable PLA microsphere fillers prepared in this invention is key to regulating the material's elasticity and adapting to muscle movement.

[0130] 4. Injectability test

[0131] Pushing force test: Using a 30G injection needle, 1 mL of polylactic acid microsphere filler prepared in Examples 1-3 and Comparative Examples 1-2 was drawn and installed on a tensile testing machine. The pushing speed was set to 10 mm / min. The maximum force required to completely push the filler out of the needle was recorded as the pushing force. Each group was tested 5 times and the average value was taken.

[0132] Observation of needle blockage: Take 10 30G sterile injection needles, draw 1mL of polylactic acid microsphere filler prepared in Examples 1-3 and Comparative Examples 1-2 into each needle, manually inject at a uniform speed, and observe whether needle blockage or poor injection occurs during the injection process. Count the number of blocked needles.

[0133] Particle size distribution measurement: Using a laser particle size analyzer, the polylactic acid microsphere fillers prepared in Examples 1-3 and Comparative Examples 1-2 were diluted 10 times with deionized water, ultrasonically dispersed for 5 minutes, and then injected into the detection cell to measure the microsphere particle size distribution and record the particle size distribution. Each group was tested 3 times and the average value was taken.

[0134] Particle size span = (D90 - D10) / D50.

[0135] The results of the injectability test are shown in Table 4.

[0136] Table 4

[0137]

[0138] Table 4 shows that the collagen-inducible injectable polylactic acid (PLA) microsphere fillers prepared in Examples 1-3 have a pushing force of 10-15 N, no plugging, a particle size range of 0.8-0.9 mm, and uniform microspheres. In contrast, the PLA microsphere fillers without a composite external aqueous phase prepared in Comparative Example 1 and without collagen peptide grafting prepared in Comparative Example 2 have pushing forces of 25 N and 20 N respectively, both exhibiting plugging, and a particle size range of 1.0-1.4 mm. This indicates that the composite external aqueous phase in the collagen-inducible injectable PLA microsphere fillers prepared in this invention can improve the dispersion stability of the microspheres, reduce the pushing force, and optimize injectability.

[0139] 5. Biocompatibility test

[0140] Inflammatory response observation: Ten healthy SD rats were selected. After hair removal and disinfection of the back, 0.5 mL of polylactic acid microsphere filler prepared in Examples 1-3 and Comparative Examples 1-2 was injected into 5 different locations on the back of each rat. A blank control group was set up (injected with an equal volume of sterile PBS). For one week after injection, the injection sites were observed daily for whether redness, swelling, exudation and other inflammatory reactions appeared, and the number of rats with redness and swelling was recorded.

[0141] Cell viability assay: SD rat fibroblasts were cultured to passage 3, and then the concentration was adjusted to 1×10⁻⁶. 5Cells were seeded at 100 μL per well in a 96-well plate. After incubation for 24 h, 10 μL of polylactic acid microspheres prepared in Examples 1-3 and Comparative Examples 1-2 (1 mg / mL, aseptically treated) were added. Cells were cultured for another month. Cell viability was detected using a CCK-8 assay kit. The cell viability of each group was calculated according to the kit instructions (OD value of experimental group / OD value of control group × 100%).

[0142] Liver and kidney function indicators were tested: Fifteen healthy SD rats were selected and divided into 5 groups of 3 rats each. The rats were injected intraperitoneally with polylactic acid microspheres prepared in Examples 1-3 and Comparative Examples 1-2 (dose of 1 mL / kg). One month later, blood was collected from the abdominal aorta, and the serum was separated by centrifugation. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Scr) in the serum were detected using a fully automated biochemical analyzer, and the results were recorded.

[0143] The results of the biocompatibility test are shown in Table 5.

[0144] Table 5

[0145]

[0146] As shown in Table 5, Examples 1-3 and Comparative Examples 1-2 showed no significant toxicity. However, Example 1 showed less inflammatory response and higher cell survival rate compared to the comparative examples, demonstrating that collagen peptides and the composite external aqueous phase (arginine regulating pH) synergistically enhance biocompatibility and avoid the slight irritation that may be caused by a single component.

[0147] The embodiments described above are merely preferred implementations of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a collagen-inducible injectable polylactic acid microsphere filler, characterized in that, The steps include the following: (1) Dissolve polylactic acid in dichloromethane to prepare a polylactic acid solution; then, under stirring conditions of 1000~3000 rpm, add the polylactic acid solution dropwise to a polyvinyl alcohol aqueous solution and emulsify for 20~40 min to form an oil-in-water emulsion; after vacuum evaporation, centrifugation, washing and freeze drying, the obtained oil-in-water emulsion yields polylactic acid microspheres; (2) Disperse the polylactic acid microspheres obtained in step (1) in N,N-dimethylformamide to prepare a polylactic acid microsphere suspension; then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the polylactic acid microsphere suspension, and activate the reaction at 20~30℃ for 30~60min; continue to add collagen peptides, and react at 20~30℃ for 12~24h. After centrifugation, washing and freeze drying, polylactic acid microspheres with collagen peptides grafted on the surface are obtained. (3) Add agarose, sodium hyaluronate, arginine and elastin to deionized water in sequence, heat to 90~100℃, stir until completely dissolved, and cool to 50~60℃ to obtain a composite external aqueous phase; (4) Disperse the polylactic acid microspheres with surface-grafted collagen peptides obtained in step (2) in deionized water containing poloxamer and sonicate for 2-8 min to obtain an inner aqueous phase; then add the inner aqueous phase dropwise to an ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer and stir at 500-1000 rpm for 10-20 min to obtain a water / oil primary emulsion; Next, the water / oil pre-emulsion was added to the composite external aqueous phase obtained in step (2), and stirred at 50-60℃ and 1200-1500rpm for 10-20min. After vacuum evaporation, gel solidification, centrifugation, washing and freeze drying, collagen-induced polylactic acid microspheres were obtained. (5) Disperse the collagen-inducible polylactic acid microspheres obtained in step (4) into deionized water containing poloxamer and sodium hyaluronate, stir and mix evenly to obtain collagen-inducible injectable polylactic acid microsphere filler.

2. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the polylactic acid solution is 100~200 mg / mL; the mass concentration of the polyvinyl alcohol aqueous solution is 1~3%; and the volume ratio of the polylactic acid solution to the polyvinyl alcohol aqueous solution is (1~2):

10. The specific process of vacuum evaporation, centrifugation, washing and freeze drying is as follows: first, remove dichloromethane by vacuum evaporation at 40~60℃ and -0.08MPa for 8~12min, then centrifuge at 6000~10000rpm for 8~12min, collect the precipitate, wash it with deionized water 3~5 times and then freeze dry it.

3. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the polylactic acid microsphere suspension is 50~100 mg / mL; The mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to polylactic acid microspheres is (1~2):1; the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to N-hydroxysuccinimide (NHS) is 1:(1~1.5); the mass ratio of collagen peptides to polylactic acid microspheres is 1:(8~12). The specific process of centrifugation, washing and freeze-drying is as follows: centrifuge at 6000~10000rpm for 8~12min, collect the precipitate, wash with deionized water 3~5 times and then freeze-dry.

4. The preparation method according to claim 1, characterized in that, In step (3), the mass concentration of agarose in the composite external aqueous phase is 2-4%, the mass concentration of sodium hyaluronate is 1-3%, the mass concentration of arginine is 1-3%, and the mass concentration of elastin is 0.3-0.8%.

5. The preparation method according to claim 1, characterized in that, In step (4), the mass concentration of the deionized water containing poloxamer is 0.05~0.15%; in the ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer, the mass concentration of Span-80 is 1~2%, and the concentration of polylactic acid-glycolic acid copolymer is 100~200 mg / mL; The mass-to-volume ratio of the polylactic acid microspheres with surface-grafted collagen peptides, the deionized water containing poloxamer, and the ethyl acetate oil phase containing Span-80 and polylactic acid-glycolic acid copolymer is 40~60 mg: 5~15 ml: (25~30) ml.

6. The preparation method according to claim 1, characterized in that, In step (4), the volume ratio of the water / oil primary emulsion to the composite external aqueous phase is (30~45):(100~120). The specific process of vacuum evaporation, gel solidification, centrifugation, washing and freeze-drying is as follows: First, ethyl acetate is removed by vacuum evaporation at 40~60℃ and -0.08MPa for 8~12 min. Then, the mixture is allowed to stand and solidify at 20~30℃ for 25~35 min. Next, it is transferred to 4~10℃ and cooled for 1.5~2.5 h. Then, it is centrifuged at 2500~3500 rpm for 8~12 min, the microsphere precipitate is collected, washed with deionized water 3~5 times and then freeze-dried.

7. The preparation method according to claim 1, characterized in that, In step (4) and step (5), the mass concentration of poloxamer in the deionized water containing poloxamer and sodium hyaluronate is 5-6%, and the mass concentration of sodium hyaluronate is 3-4%; the mass ratio of collagen-induced polylactic acid microspheres to deionized water containing poloxamer and sodium hyaluronate is (1-2):

10.

8. A collagen-inducible injectable polylactic acid microsphere filler, characterized in that, It is prepared according to the method described in any one of claims 1 to 7.

9. The application of the collagen-inducible injectable polylactic acid microsphere filler according to claim 8 in the fields of biomedicine, cosmetics, and medical aesthetics.

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