Porous poly-L-lactic acid microspheres containing NMN, facial fillers and preparation methods thereof

Abstract

The present invention belongs to the technical field of biomedical materials, and discloses a porous poly-L-lactic acid microsphere containing NMN, a facial filler and a preparation method thereof. The preparation method of the porous poly-L-lactic acid microsphere comprises the following steps: taking an emulsion and an aqueous solution and respectively entering a reaction vessel at a flow rate of 5-50 mL / min and 500-2500 mL / min, and performing a high-speed shearing reaction at a rotation speed of 1000-5000 rpm to obtain a microsphere suspension; performing vacuum drying on the microsphere suspension to obtain the porous poly-L-lactic acid microsphere; the emulsion comprises 2-20 wt% of poly-L-lactic acid, 3-30 wt% of polysorbate and 50-95 wt% of an organic solvent, the aqueous solution comprises 0.5-2 wt% of polyvinyl alcohol, 0.2-2 wt% of β-nicotinamide mononucleotide, 0.5-5 wt% of gelatin and 91-98.8 wt% of water, and the addition volume ratio of the emulsion to the aqueous solution is 1:(3-15). The preparation method provided by the present invention can prepare porous poly-L-lactic acid microspheres with regular shapes, narrow particle size distributions and loaded with NMN. The facial filler prepared by using the microspheres as raw materials has excellent anti-wrinkle, skin rejuvenation and whitening effects, and also has good safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a porous poly-L-lactic acid microsphere containing NMN, a facial filler, and a preparation method thereof. Background Art

[0002] In recent years, with the rapid development of the beauty industry, injectable facial fillers have developed rapidly and have become an indispensable part of the field of aesthetic medicine. Traditional injectable facial fillers will be slowly absorbed and finally disappear after injection, only playing a temporary repair role, requiring repeated re-injection, and having poor biocompatibility, and also causing risks of inflammation and other side effects. At present, there are a wide variety of facial fillers on the market, but it is still a great challenge to develop beauty products with good beauty effects, long-lasting but non-permanent curative effects, small side effects, and low incidence of adverse reactions.

[0003] Polylactic acid materials have the advantages of good biodegradability, better mechanical strength and processing performance, and good biocompatibility, and are thus widely used in the fields of tissue engineering, orthopedic repair and fixation materials, drug delivery, and surgical sutures, and are a kind of biomedical material recognized by the US Food and Drug Administration (FDA). Among them, poly-L-lactic acid can be slowly degraded into lactic acid in the body, and the lactic acid obtained by degradation can stimulate the formation of collagen, so that the collagen gradually lost with age can be supplemented, thereby achieving the purpose of improving skin quality and filling skin relaxation and depression. The curative effect can last up to two years, and poly-L-lactic acid will eventually be degraded into CO2 and H2O and excreted from the body; therefore, poly-L-lactic acid has been favored in the beauty industry in recent years.

[0004] At present, L-lactic acid in the current L-lactic acid facial filler exists in the form of microparticles or microspheres, but the preparation process of L-lactic acid microspheres or microparticles is complex, the particle size controllability is poor, and the cost is high. Moreover, if other active components are introduced during the preparation process of poly-L-lactic acid microspheres, the microspheres or microparticles will have uneven particle sizes, irregular shapes, poor redissolution, or uneven injection, etc., which will cause adverse reactions such as subcutaneous nodules or swelling, and the safety is poor. Summary of the Invention

[0005] In order to obtain a poly-L-lactic acid microsphere with a regular porous spherical structure, narrow particle size distribution, and good redissolution, and improve its safety when used as a facial filler, the present invention provides a porous poly-L-lactic acid microsphere containing NMN, a facial filler, and a preparation method thereof.

[0006] In the first aspect, the preparation method of the porous poly-L-lactic acid microsphere provided by the present invention adopts the following technical solution:

[0007] A preparation method of a porous poly-L-lactic acid microsphere, the method specifically comprises the following steps:

[0008] S1. The emulsion and the aqueous solution are respectively fed into a reaction vessel at flow rates of 5 - 50 mL / min and 500 - 2500 mL / min, and a high-speed shearing reaction is carried out at a rotation speed of 1000 - 5000 rpm to obtain a microsphere suspension;

[0009] S2. The microsphere suspension is subjected to vacuum drying to obtain the porous poly(L-lactic acid) microspheres;

[0010] Among them, the emulsion includes 2 - 20 wt% of poly(L-lactic acid), 3 - 30 wt% of polysorbate, and 50 - 95 wt% of an organic solvent, the aqueous solution includes 0.5 - 2 wt% of polyvinyl alcohol, 0.2 - 2 wt% of β-nicotinamide mononucleotide, 0.5 - 5 wt% of gelatin, and 91 - 98.8 wt% of water, and the addition volume ratio of the emulsion to the aqueous solution is 1:(3 - 15).

[0011] In some specific embodiments, the gelatin is selected from animal gelatin and / or modified gelatin.

[0012] In some specific embodiments, the modified gelatin is selected from one or more of thiolated gelatin, phosphorylated gelatin, succinylated gelatin, and ureido-linked gelatin.

[0013] In some specific embodiments, the organic solvent is selected from one or more of chloroform, dichloromethane, and absolute ethanol.

[0014] In some specific embodiments, the temperature of the vacuum drying is 45 - 80 °C, the vacuum degree is 0.05 - 0.08 MPa, and the time is 0.5 - 4 h.

[0015] In some specific embodiments, the method for preparing the porous poly(L-lactic acid) microspheres provided by the present invention further includes centrifuging, sieving, or sedimenting the microsphere suspension before vacuum drying.

[0016] In the second aspect, the porous poly(L-lactic acid) microspheres provided by the present invention adopt the following technical solution:

[0017] A kind of porous poly(L-lactic acid) microspheres, which are prepared by the above method for preparing porous poly(L-lactic acid) microspheres.

[0018] In some specific embodiments, the porous poly(L-lactic acid) microspheres have a porous spherical skeleton structure, and β-nicotinamide mononucleotide is loaded on the porous spherical skeleton structure.

[0019] In the third aspect, the facial filler provided by the present invention adopts the following technical solution:

[0020] A facial filler, comprising 0.1-4 wt% of the porous poly-L-lactic acid microspheres according to claim 6 or 7, 0.1-4 wt% of mannitol, 0.0002-25 wt% of sodium carboxymethylcellulose, and 50-95 wt% of water.

[0021] In some specific embodiments, the molecular weight of the sodium carboxymethylcellulose is 5000-500000 Da.

[0022] Fourthly, the preparation method of the facial filler provided by the present invention adopts the following technical solution:

[0023] A preparation method of a facial filler, the method comprising: taking the porous poly-L-lactic acid microspheres, mannitol and sodium carboxymethylcellulose according to a mass ratio of (10-50):(10-50):(5-15), and continuously stirring at a rotation speed of 1000-10000 rpm for 5-45 min to obtain a mixture;

[0024] Taking the mixture and water according to a mass ratio of 1:(20-50), and continuously stirring at a rotation speed of 500-2500 rpm for 10-30 min to obtain a facial filler solution;

[0025] Freeze-drying the facial filler solution for 4-48 h to obtain the facial filler.

[0026] Beneficial effects:

[0027] (1) In the present invention, an emulsion prepared from poly-L-lactic acid and polysorbate is used as the dispersed phase, and an aqueous solution prepared from polyethylene, β-nicotinamide mononucleotide and gelatin is used as the continuous phase. The emulsion and the aqueous solution enter the reaction vessel according to a specific volume ratio and flow rate, and a porous microsphere skeleton structure is formed under the action of high-speed mechanical shear force. β-Nicotinamide mononucleotide is loaded or coated on the surface and inside of the porous microsphere skeleton structure. The porous poly-L-lactic acid microspheres are prepared by the above specific process and conditions, and the obtained porous poly-L-lactic acid microspheres have regular shapes, the particle size is between 20-60 μm, the particle size distribution is narrow, the preparation process is relatively simple, the particle size controllability is strong, and the cost is low. It has great practical application prospects in the technical field of biomedical materials;

[0028] (2) The facial filler obtained by using the porous poly-L-lactic acid microspheres as the main active component and compounding with mannitol and sodium carboxymethylcellulose has good solubility. Among them, poly-L-lactic acid and β-nicotinamide mononucleotide can cooperate to exert excellent effects, and the porous poly-L-lactic acid microspheres are spherical and have relatively uniform particle sizes. Injecting this facial filler into the body will not cause serious inflammatory reactions. While having excellent anti-wrinkle, skin rejuvenation and whitening effects, it also has good safety. Description of the Drawings

[0029] Figure 1 SEM image (35μm) of the porous poly-L-lactic acid microspheres provided in Example 1 of the present invention;

[0030] Figure 2 Particle size dispersion diagram of the porous poly-L-lactic acid microspheres provided in Example 1 of the present invention;

[0031] Figure 3 Histological section diagram of collagen stimulation after subcutaneous injection of the facial filler into rabbits for 6 months provided in Example 5 of the present invention;

[0032] Figure 4 Histological section diagram of collagen stimulation after subcutaneous injection of the facial filler into rabbits for 6 months provided in Comparative Example 4 of the present invention. Detailed implementation manners

[0033] The inventors of the present application found in a large number of practices that the microspheres prepared from poly-L-lactic acid have non-uniform sizes and poor dispersibility; moreover, if β-nicotinamide mononucleotide (NMN) is introduced into the preparation of poly-L-lactic acid, the finally prepared microparticles will not only be spherical, but also some will be sheet-shaped, rod-shaped and irregular in shape, and when applied to facial fillers, it may cause serious inflammatory reactions in the body and have poor safety.

[0034] Through creative efforts and a large number of experiments, the inventors have creatively designed the following scheme:

[0035] Using an emulsion containing 2-20 wt% poly-L-lactic acid, 3-30 wt% polysorbate and 50-95 wt% organic solvent as the dispersed phase, and an aqueous solution containing 0.5-2 wt% polyvinyl alcohol, 0.2-2 wt% β-nicotinamide mononucleotide, 0.5-5 wt% gelatin and 91-98.8 wt% water as the dispersed phase, and the addition volume ratio of the two during the preparation process is 1:(3-15);

[0036] The emulsion and the aqueous solution enter the reaction vessel at specific flow rates of 5-50 mL / min and 500-2500 mL / min respectively, and under the action of the high-speed mechanical shear force generated by a high-speed rotor with a rotation speed of 1000-5000 rpm, a high-speed shear reaction is carried out to obtain a microsphere suspension;

[0037] The microsphere suspension is vacuum dried, and while removing the organic solvent and water, micropores are also formed on the surface and inside of the microspheres, and finally porous poly-L-lactic acid microspheres are obtained.

[0038] Using the emulsion and aqueous solution provided in the present invention as raw materials and adopting a specific preparation process, NMN is encapsulated in microspheres, and the microspheres have a regular spherical porous structure with a particle size of 20-60 μm; NMN and poly-L-lactic acid in the porous poly-L-lactic acid microspheres synergistically exert good whitening and skin rejuvenation effects, and the shape and size of the microspheres are controllable during the preparation process. The obtained microspheres have regular structures and uniform sizes, and injecting them into the body will not cause a strong inflammatory reaction, having good safety.

[0039] In some specific embodiments, the content of poly-L-lactic acid in the emulsion is within the above range values, specifically 2 wt%, 5 wt%, 8 wt%, 10 wt%, 12 wt%, 15 wt%, 18 wt%, 20 wt% or any value between them.

[0040] In some specific embodiments, the content of polysorbate in the emulsion is within the above range values, specifically 3 wt%, 5 wt%, 10 wt%, 12 wt%, 15 wt%, 18 wt%, 20 wt%, 23 wt%, 25 wt%, 27 wt%, 30 wt% or any value between them.

[0041] In the present invention, the organic solvent used in the emulsion has a certain influence on the morphology and size of the finally prepared porous poly-L-lactic acid microspheres. Specifically, it can be one or more of chloroform, dichloromethane, and absolute ethanol; and the actual amount of the organic solvent is adaptively adjusted according to the amounts of poly-L-lactic acid and polysorbate used.

[0042] In some specific embodiments, the content of polyvinyl alcohol in the aqueous solution is within the above range values, specifically 0.5 wt%, 0.7 wt%, 0.9 wt%, 1.0 wt%, 1.2 wt%, 1.5 wt%, 1.8 wt%, 2.0 wt% or any value between them.

[0043] In some specific embodiments, the content of β-nicotinamide mononucleotide in the aqueous solution is within the above range values, specifically 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.7 wt%, 1.0 wt%, 1.5 wt%, 1.7 wt%, 2.0 wt% or any value between them.

[0044] In some specific embodiments, the content of gelatin in the aqueous solution is within the above range, specifically 0.5 wt%, 0.8 wt%, 1.2 wt%, 1.5 wt%, 2.0 wt%, 3.0 wt%, 4.0 wt%, 5.0 wt% or any value between them.

[0045] In the present invention, the addition volume ratio of the emulsion and the aqueous solution during the preparation process affects the three-dimensional spatial structure of the poly-L-lactic acid microspheres; when the addition amount of the emulsion is too small, the number of poly-L-lactic acid microspheres formed is small, and more sheet-like or coronal particles will be formed; while when the addition amount of the aqueous solution is too small, the formed porous polylactic acid microspheres are prone to agglomeration and have poor uniformity. Therefore, only by using the emulsion and the aqueous solution disclosed in the present invention, in two specific proportions and combined with a specific preparation process, can porous poly-L-lactic acid microspheres with a spherical shape, uniform particle size and good dispersibility be prepared.

[0046] In some specific embodiments, the addition volume ratio of the emulsion and the aqueous solution is 1:3, 1:4, 1:5, 1:7, 1:10, 1:12, 1:13, 1:15 or any value therebetween. In the present invention, the high-speed homogenizer in the reaction vessel rotates at a specific speed, and the generated high-speed mechanical shear force acts on the emulsion and the aqueous solution that enter the reaction vessel at a specific flow rate, and poly-L-lactic acid coats β-nicotinamide mononucleotide while agglomerating to form microspheres with regular shapes and uniform particle sizes.

[0047] In some specific embodiments, the rotation speed of the high-speed homogenizer in the reaction vessel is 1000 rpm, 1100 rpm, 1300 rpm, 1500 rpm, 2000 rpm, 2300 rpm, 2500 rpm, 2700 rpm, 3000 rpm, 3200 rpm, 3700 rpm, 4000 rpm, 4300 rpm, 4500 rpm, 4700 rpm, 5000 rpm or any value therebetween.

[0048] In some specific embodiments, the flow rate of the emulsion entering the reaction vessel is 5 mL / min, 8 mL / min, 10 mL / min, 12 mL / min, 15 mL / min, 18 mL / min, 20 mL / min, 25 mL / min, 30 mL / min, 35 mL / min, 40 mL / min, 50 mL / min or any value therebetween.

[0049] In some specific embodiments, the flow rate of the aqueous solution entering the reaction vessel is 500 mL / min, 800 mL / min, 900 mL / min, 1000 mL / min, 1200 mL / min, 1500 mL / min, 1800 mL / min, 2000 mL / min, 2500 mL / min or any value therebetween.

[0050] Adding gelatin during the preparation of porous poly-L-lactic acid microspheres is beneficial to the formation of microspheres with uniform particle sizes. At the same time, during the vacuum drying process, gelatin acts as a pore-forming agent to form pores inside and on the surface of the microspheres, and finally microsphere particles with a porous spherical structure can be prepared.

[0051] The gelatin used in the present invention can be animal gelatin, modified gelatin or a mixture of both. Among them, animal gelatin refers to a macromolecular hydrophilic colloid obtained by degrading collagen in connective tissues such as animal skin, bone, and myofascial membranes.

[0052] In some specific embodiments, the modified gelatin can be, but is not limited to, one or more of thiolated gelatin, phosphorylated gelatin, succinylated gelatin, and ureido-linked gelatin.

[0053] Among them, thiolated gelatin refers to a compound obtained by modifying the carboxyl groups in the macromolecular chain of animal gelatin with disulfide, phosphorylated gelatin refers to a compound obtained by phosphorylating animal gelatin, succinylated gelatin refers to a compound obtained by succinamide-modifying animal gelatin, and ureido-linked gelatin refers to a compound obtained by cross-linking hexamethylene diisocyanate and animal gelatin through a ureido bridge bond.

[0054] In some preferred embodiments, thiolated gelatin or ureido-linked gelatin is selected and added to an aqueous solution to prepare porous poly-L-lactic acid microspheres. Thiolated gelatin and ureido-linked gelatin can be completely degraded and absorbed in vivo and have good biosafety.

[0055] In some specific embodiments, the temperature of vacuum drying is 45°C, 48°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C or any value between them, the vacuum degree of vacuum drying is 0.05 - 0.08 MPa, specifically 0.05 MPa, 0.055 MPa, 0.06 MPa, 0.072 MPa, 0.08 MPa or any value between them, and the time is 0.5 - 4 h, specifically 0.5 h, 0.6 h, 0.9 h, 1.0 h, 1.3 h, 1.5 h, 1.8 h, 2.0 h, 2.2 h, 2.5 h, 3.0 h, 3.5 h, 4.0 h or any value between them.

[0056] In some preferred embodiments, before vacuum drying, the microsphere suspension is centrifuged, sieved or sedimented to remove most of the organic solvents and water, which can effectively shorten the time required for vacuum drying, greatly improve production efficiency and save production costs.

[0057] The present invention also provides porous poly-L-lactic acid microspheres prepared by the above method. It has a porous spherical skeleton structure, and β-nicotinamide mononucleotide is loaded on the porous spherical skeleton structure. Through the porous spherical skeleton structure, two immiscible substances (poly-L-lactic acid and NMN) are fully combined, and the synergistic effects of poly-L-lactic acid and NMN are exerted. Moreover, the prepared porous poly-L-lactic acid microspheres have stable properties, are easy to industrialize, and have good application prospects.

[0058] In the facial filler provided by the present invention, the above-mentioned porous poly-L-lactic acid microspheres are used as the main active ingredient, supplemented by mannitol and sodium carboxymethylcellulose. In the facial filler, the specific contents of each component are as follows:

[0059]

[0060] In some specific embodiments, the content of the porous poly-L-lactic acid microspheres in the facial filler is 0.1 wt%, 0.5 wt%, 0.7 wt%, 1.0 wt%, 1.3 wt%, 1.5 wt%, 1.8 wt%, 2.0 wt%, 2.4 wt%, 2.8 wt%, 3.0 wt%, 3.3 wt%, 3.5 wt%, 3.8 wt%, 4.0 wt% or any value between them.

[0061] In some specific embodiments, the content of mannitol in the facial filler is 0.1 wt%, 0.5 wt%, 0.7 wt%, 1.0 wt%, 1.3 wt%, 1.5 wt%, 1.8 wt%, 2.0 wt%, 2.4 wt%, 2.8 wt%, 3.0 wt%, 3.3 wt%, 3.5 wt%, 3.8 wt%, 4.0 wt% or any value between them.

[0062] In some specific embodiments, the content of sodium carboxymethylcellulose in the facial filler is 0.0002 wt%, 0.001 wt%, 0.005 wt%, 0.01 wt%, 0.05 wt%, 0.1 wt%, 1.0 wt%, 2.4 wt%, 3.0 wt%, 5.0 wt%, 8.0 wt%, 10.5 wt%, 15.0 wt%, 20 wt%, 23 wt%, 25 wt% or any value between them.

[0063] The facial filler is formulated with the above contents to improve the yield of the porous poly-L-lactic acid microspheres and reduce the weight loss, so that the porous poly-L-lactic acid microspheres can be stably released after the facial filler is injected into the body, improving the safety of using the facial filler.

[0064] When the weight-average molecular weight of sodium carboxymethylcellulose is 5000 - 500000 Da, it can assist the porous poly-L-lactic acid microspheres to exert the best biological activity. In some specific embodiments, the weight-average molecular weight of sodium carboxymethylcellulose is 5000 Da, 10000 Da, 50000 Da, 100000 Da, 150000 Da, 200000 Da, 250000 Da, 300000 Da, 350000 Da, 400000 Da, 450000 Da, 500000 Da or any value therebetween.

[0065] The present invention also provides a method for preparing the above facial filler, which specifically includes:

[0066] Taking the porous poly-L-lactic acid microspheres, mannitol and sodium carboxymethylcellulose according to the mass ratio of (10 - 50):(10 - 50):(5 - 15), and stirring and mixing uniformly at 1000 - 10000 rpm to obtain a mixture;

[0067] Taking the mixture and water according to the mass ratio of 1:(20 - 50), and continuously stirring at a rotation speed of 500 - 2500 rpm for 10 - 30 min to obtain a facial filler solution;

[0068] Freeze-drying the facial filler solution for 4 - 48 h to obtain the facial filler-like product.

[0069] In some specific embodiments, the facial filler solution is freeze-dried for 4 - 48 h under the conditions of a temperature range of -60°C to 40°C and a pressure of 5 - 30 Pa to obtain the facial filler-like product.

[0070] The facial filler solution is freeze-dried during the process of continuous cooling, heating, and then cooling, and the air pressure changes with the change of temperature.

[0071] In some specific embodiments, the temperature range during the lyophilization process can specifically be -60 to 40 °C, -60 to 30 °C, -60 to 10 °C, -50 to 40 °C, -50 to 20 °C, -20 to 40 °C, -10 to 40 °C, or any range therebetween; the pressure is 5 Pa, 6 Pa, 8 Pa, 10 Pa, 14 Pa, 16 Pa, 18 Pa, 20 Pa, 23 Pa, 25 Pa, 27 Pa, 29 Pa, 30 Pa, or any value therebetween; the time of lyophilization is adaptively changed with the change of temperature and pressure, specifically being 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 18 h, 20 h, 25 h, 30 h, 36 h, 42 h, 48 h, or any value therebetween. In the present invention, the specific added mass ratio of each component in the facial filler and the specific mixing method enable the porous poly-L-lactic acid microspheres in the facial filler to have good dispersibility, and combined with the good redissolvability of the porous poly-L-lactic acid microspheres themselves, injecting this facial filler into the body will not cause serious inflammatory reactions. While having excellent anti-wrinkle, skin rejuvenation and whitening effects, it also has good safety.

[0072] The embodiments of the present invention will be described in detail below. The examples are intended to explain the present invention and should not be construed as a limitation to the present invention. For those without specific technical or conditions noted in the examples, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For the reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0073] In the following examples and comparative examples, the parts of each raw material refer to parts by weight.

[0074] Example 1.

[0075] This example is used to illustrate the preparation of porous poly-L-lactic acid microspheres, which specifically includes the following steps:

[0076] S1. Take 10 parts of poly-L-lactic acid and 20 parts of polysorbate 60 and dissolve them in 70 parts of chloroform, and stir and mix evenly to obtain an emulsion; take 1 part of NMN, 1 part of polyvinyl alcohol and 3 parts of animal gelatin and dissolve them in 95 parts of deionized water, and stir and mix evenly to obtain an aqueous solution.

[0077] S2. Using the emulsion as the dispersed phase and the aqueous solution as the continuous phase, add the emulsion and the aqueous solution to the emulsification reactor at a flow rate of 10 mL / min and 1500 mL / min respectively and in a volume ratio of 1:3 through a vacuum magnetic pump. The rotation speed of the high-speed homogenizer in the emulsification reactor is 1000 rpm. The emulsion and the aqueous solution undergo a high-speed shear reaction under strong shear force to obtain a microsphere suspension.

[0078] S3. Place the microsphere suspension in a vacuum dryer and conduct vacuum drying for 4 h under the conditions of a temperature of 45 °C and a vacuum degree of 0.05 MPa to obtain porous poly(L-lactic acid) microspheres.

[0079] It can be seen from Figure 1 and Figure 2 that the porous poly(L-lactic acid) microspheres have regular shapes, a porous spherical skeleton structure, and the particle size is in the range of 15 - 50 μm, with good particle size uniformity.

[0080] Examples 2 - 4.

[0081] In Examples 2 - 4, the porous poly(L-lactic acid) microspheres were prepared according to the method provided in Example 1, with the differences shown in Table 1 and other conditions being the same.

[0082] Table 1.

[0083]

[0084]

[0085] Comparative Examples 1 - 3.

[0086] In Comparative Examples 1 - 3, the porous poly(L-lactic acid) microspheres were prepared according to the method provided in Example 1, with the differences shown in Table 2 and other conditions being the same.

[0087] Table 2.

[0088]

[0089]

[0090] Test Example 1.

[0091] The particle sizes of the porous poly(L-lactic acid) microspheres provided in Examples 1 - 4 and Comparative Examples 1 - 3 were measured using a BT-9300ST laser particle size analyzer, and the average particle size and particle size polydispersity index were statistically calculated based on the test results. The results are shown in Table 3.

[0092] Table 3.

[0093]

[0094] It can be seen from the test results that compared with Comparative Examples 1 - 3, the microspheres prepared by using the methods provided in Examples 1 - 4 of the present invention have good dispersibility and uniformity in particle size, indicating that only by adopting the specific flow rates of the emulsion and aqueous solution entering the reaction vessel, specific raw materials and input amounts disclosed in the present invention can microspheres with good dispersibility and uniformity in particle size, regular structure, and uniform size be prepared, and the preparation reproducibility of this preparation method is good.

[0095] Example 5

[0096] This example is used to illustrate the preparation of a facial filler, which specifically includes the following steps:

[0097] S1. Take 10 parts of the porous poly-L-lactic acid microspheres provided in Example 1, 10 parts of mannitol, and 5 parts of sodium carboxymethylcellulose, and stir evenly at a rotation speed of 1000 rpm to obtain a mixture;

[0098] S2. Take 10 parts of the mixture and 200 parts of water, and stir and mix evenly at a rotation speed of 500 rpm for 20 min to obtain a filler solution;

[0099] S3. Freeze-dry the filler solution for 24 h under the conditions of a temperature range of -50 to 20 °C and a pressure of 10 to 25 Pa to obtain a facial filler.

[0100] The weight-average molecular weight of the sodium carboxymethylcellulose used in this example is 5000 Da.

[0101] Examples 6-8

[0102] Examples 6-8 prepare facial fillers according to the method provided in Example 5, with the differences shown in Table 4, and other conditions being the same.

[0103] Table 4

[0104]

[0105] Comparative Examples 4-6

[0106] Comparative Examples 4-6 prepare facial fillers according to the method provided in Example 5, with the differences shown in Table 5, and other conditions being the same.

[0107] Table 5

[0108]

[0109] Test Example 2

[0110] Perform in vitro bioequivalence experiments using the facial fillers provided in Examples 5-8 and Comparative Examples 4-6: Subcutaneously inject the facial fillers provided in Examples 5-8 and Comparative Examples 4-6 into the abdomen of rabbits, and at the 8th week, 16th week, and 24th week, respectively, prepare tissue sections to observe the stimulation of collagen proliferation and the change in collagen thickness, and perform collagen thickness scoring according to the microscopic light microscopy measurement method. The test results are as Figure 3 and 4 and Table 6 show.

[0111] Table 6

[0112]

[0113]

[0114] From Figure 3 and Figure 4 and the test results in Table 6, it can be seen that, compared with Comparative Examples 4 to 6, when the facial fillers provided in Examples 5 to 8 of the present invention are subcutaneously injected into rabbits, good collagen stimulation effects are achieved, and obvious collagen hyperplasia and anti-wrinkle effects are obtained. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention.

Claims

1. A method for preparing porous poly-L-lactic acid microspheres, characterized in that, The method specifically includes the following steps: S1. Take the emulsion and the aqueous solution and enter the reaction vessel at flow rates of 5 - 50 mL / min and 500 - 2500 mL / min respectively, and carry out high-speed shearing reaction at a rotation speed of 1000 - 5000 rpm to obtain a microsphere suspension; S2. Vacuum-dry the microsphere suspension to obtain the porous poly-L-lactic acid microspheres; Wherein, the emulsion includes 2 - 20 wt% of poly-L-lactic acid, 3 - 30 wt% of polysorbate, and 50 - 95 wt% of an organic solvent, the aqueous solution includes 0.5 - 2 wt% of polyvinyl alcohol, 0.2 - 2 wt% of β-nicotinamide mononucleotide, 0.5 - 5 wt% of gelatin, and 91 - 98.8 wt% of water, and the added volume ratio of the emulsion and the aqueous solution is 1:(3 - 15); The porous poly-L-lactic acid microspheres have a porous spherical skeleton structure, and β-nicotinamide mononucleotide is loaded on the porous spherical skeleton structure.

2. The preparation method of the porous poly-L-lactic acid microspheres according to claim 1, wherein The gelatin is selected from animal gelatin and / or modified gelatin.

3. The preparation method of the porous poly-L-lactic acid microspheres according to claim 2, wherein, The modified gelatin is selected from one or more of thiolated gelatin, phosphorylated gelatin, succinylated gelatin, and ureido-linked gelatin.

4. The preparation method of the porous poly-L-lactic acid microspheres according to claim 1, wherein, The organic solvent is selected from one or more of chloroform, dichloromethane, and absolute ethanol.

5. The preparation method of the porous poly-L-lactic acid microspheres according to claim 1, wherein, The temperature of the vacuum drying is 45 - 80 °C, the vacuum degree is 0.05 - 0.08 MPa, and the time is 0.5 - 4 h.

6. The preparation method of the porous poly(L-lactic acid) microspheres according to claim 1, characterized in that, This method also includes centrifuging, sieving, or sedimenting the microsphere suspension before vacuum drying.

7. A porous poly(L-lactic acid) microsphere, characterized in that, The porous poly-L-lactic acid microspheres are prepared by the preparation method of the porous poly-L-lactic acid microspheres according to any one of claims 1 - 6.

8. The porous poly(L-lactic acid) microspheres according to claim 7, wherein, The porous poly-L-lactic acid microspheres have a porous spherical skeleton structure, and β-nicotinamide mononucleotide is loaded on the porous spherical skeleton structure.

9. A facial filler, characterized in that, The facial filler includes 0.1 - 4 wt% of the porous poly-L-lactic acid microspheres according to claim 7 or 8, 0.1 - 4 wt% of mannitol, 0.0002 - 25 wt% of sodium carboxymethylcellulose, and 50 - 95 wt% of water.

10. The facial filler according to claim 9, wherein The weight-average molecular weight of the sodium carboxymethylcellulose is 5000 - 500000 Da.

11. The preparation method of the facial filler according to claim 9 or 10, characterized in that, This method includes: taking the porous poly-L-lactic acid microspheres, mannitol, and sodium carboxymethylcellulose according to a mass ratio of (10 - 50):(10 - 50):(5 - 15), and continuously stirring at a rotation speed of 1000 - 10000 rpm for 5 - 45 min to obtain a mixture; Taking the mixture and water according to a mass ratio of 1:(20 - 50), and continuously stirring at a rotation speed of 500 - 2500 rpm for 10 - 30 min to obtain a facial filler solution; Freeze-drying the facial filler solution for 4 - 48 h to obtain the facial filler.

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