Poly-L-lactic acid composite porous microspheres, preparation method thereof and injection preparation

Abstract

The invention relates to the field of medical materials, and provides a poly-L-lactic acid composite porous microsphere, a preparation method thereof and an injection preparation containing the poly-L-lactic acid composite porous microsphere. The poly-L-lactic acid composite porous microspheres are prepared from a first component, a second component and optional auxiliary components through an emulsification-solvent evaporation method, the first component is poly-L-lactic acid, and the second component is a vitamin E compound or a mixture of the vitamin E compound and a vitamin C compound; the content of the first component is 63-93% of the total mass of the first component and the second component, and the content of the second component is 7-37% of the total mass of the first component and the second component. The poly-L-lactic acid composite porous microsphere disclosed by the invention has the advantages of quick response, good long-term effect, better fibroblast proliferation capacity, better collagen regeneration stimulation capacity, weaker inflammability and good safety, and the porous microsphere disclosed by the invention is larger in volume per unit mass and good in filling effect.

Description

Technical Field

[0001] The present invention relates to the field of medical materials, and in particular to poly (L-lactic acid) composite porous microspheres, a preparation method thereof, and an injectable preparation containing the poly (L-lactic acid) composite porous microspheres. Background Art

[0002] Injectable poly-L-lactic acid (PLLA) fillers are medical materials specifically used for facial augmentation and wrinkle reduction. Made from poly-L-lactic acid, a biodegradable polymer extracted from natural plants and animals, PLLA has been proven safe and effective in medical aesthetics and plastic surgery through years of research and clinical trials.

[0003] Sculptra, an injectable poly-L-lactic acid filler, was approved for marketing in the EU in 1999 and in the US in 2004. It is indicated for the reconstruction or improvement of facial volume loss in HIV patients. In 2010, it received FDA approval for injection into healthy individuals for the filling of nasolabial wrinkles. A similar domestic product, Avila, has improved its particle morphology and was approved for marketing in China in 2021. Avila's rounded, spherical particles offer a better safety profile than Sculptra's irregular, fragmented particles. However, the effects of Sculptra and Avila injections on native collagen regeneration only appear four to six weeks after use, with little wrinkle-reducing effect occurring within the first four weeks.

[0004] In 2014, AestheFill, a porous poly(dlactic acid) (PDLLA) microsphere product, received KFDA approval in South Korea as an injectable filler for the improvement of facial wrinkles and folds. PowerFill, a larger-strength version of AestheFill (500 mg / vial, 1000 mg / vial, 1500 mg / vial, and 2000 mg / vial), was also approved as an injectable filler for penis augmentation or enlargement in patients with micropenis deformity. The porous microspheres, which have a larger volume per unit mass than non-porous microspheres, significantly improve the filler effect at the injection site within the first four weeks.

[0005] A study (Superiority of poly(L-lactic acid) microspheres as dermalfillers, Chinese Chemical Letters, 32(2021)577-582) shows that PLLA has a stronger ability to stimulate collagen regeneration and is less inflammatory than PDLLA, making it a more ideal material for injectable facial fillers. Therefore, currently available PLLA and PDLLA products have their own advantages and disadvantages as injectable facial fillers.

[0006] Furthermore, when auxiliary ingredients (such as vitamins, collagen, and amino acids) need to be added, existing technologies typically first prepare porous microspheres, then mix them with the auxiliary ingredients, using the pores of the microspheres to load the auxiliary ingredients. However, this method results in low levels of the auxiliary ingredients and instability.

[0007] Therefore, there has been a need to continuously develop a poly-L-lactic acid microsphere product that is fast-acting, has good long-term effects, can stably load auxiliary ingredients, and is safe. Summary of the Invention

[0008] In order to address the deficiencies in the prior art, the present invention provides a poly-L-lactic acid composite porous microsphere with rapid onset, good long-term effect, stronger ability to stimulate collagen regeneration, weaker inflammatory properties, and good safety, as well as a preparation method thereof and an injectable preparation containing the poly-L-lactic acid composite porous microsphere.

[0009] One aspect of the present invention provides a method for preparing poly (L-lactic acid) composite porous microspheres, wherein the poly (L-lactic acid) composite porous microspheres are prepared from a first component, a second component, and an optional auxiliary component by an emulsification-solvent evaporation method. in, The first component is poly-L-lactic acid, and the second component is a vitamin E compound, or a mixture of a vitamin E compound and a vitamin C compound; The content of the first component is 63-93% by weight of the total mass of the first component and the second component, and the content of the second component is 7-37% by weight of the total mass of the first component and the second component.

[0010] Preferably, the intrinsic viscosity of the poly-L-lactic acid is 0.8-4.3 dL / g.

[0011] Preferably, the second component is a mixture of vitamin E compounds and vitamin C compounds, wherein the content of vitamin E compounds is 5-35% of the total mass of the first component and the second component, and the content of vitamin C compounds is 2-32% of the total mass of the first component and the second component.

[0012] Preferably, the vitamin E compound includes at least one selected from vitamin E, vitamin E acetate, vitamin E succinate, vitamin E palmitate, vitamin E succinate, vitamin E nicotinate, vitamin E phosphate, vitamin E linoleate, vitamin E laurate, and vitamin E retinoic acid ester.

[0013] Preferably, the vitamin C compound includes at least one selected from the group consisting of L-ascorbic acid, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, calcium ascorbyl phosphate, ascorbic acid polypeptide, ascorbic acid ethyl ether, ascorbyl dipalmitate, ascorbyl palmitate, ascorbyl glucoside and ascorbyl ethylsilanol pectate.

[0014] Preferably, the auxiliary ingredient is selected from at least one of the group consisting of vitamins or their esters, amino acids, small molecule active peptides, collagen, polyracemic lactic acid, polydextrose lactic acid, and block copolymers of polylactic acid and other components. The vitamins or their esters do not include the second ingredient, and the content of the auxiliary ingredient is 0-40% of the total mass of the first ingredient and the second ingredient.

[0015] Preferably, the emulsification-solvent evaporation method is a single emulsification-solvent evaporation method, which comprises: (A-1) dissolving the first component, the oil-soluble second component, and the optional oil-soluble auxiliary component in an organic solvent to form an oil phase; (B-1) dissolving a surfactant in water to form an aqueous phase; (C-1) mixing the oil phase and the water phase to form an emulsion, and stirring the emulsion to volatilize the organic solvent to obtain a suspension; (D-1) Screening, washing, and freeze-drying the suspension to obtain the poly (L-lactic acid) composite porous microspheres.

[0016] Preferably, the emulsification-solvent evaporation method is a double emulsification-solvent evaporation method, which comprises: (A-2) dissolving the first component, the oil-soluble second component, and the optional oil-soluble auxiliary component in an organic solvent to form an oil phase; (B-2) dissolving the water-soluble second component and optionally the water-soluble auxiliary component in water to form an internal aqueous phase; (C-2) dissolving the surfactant in water to form an external aqueous phase; (D-2) mixing the oil phase and the inner aqueous phase to form colostrum, and adding the colostrum to the outer aqueous phase to form a double emulsion; (E-2) stirring the emulsion to volatilize the organic solvent to obtain a suspension; (F-2) Screening, washing, and freeze-drying the suspension to obtain the poly (L-lactic acid) composite porous microspheres.

[0017] Preferably, in the step (D-1) or the step (F-2), the suspension is sieved and washed, and then added to a solution with suspending ability and freeze-dried to obtain the poly-L-lactic acid composite porous microspheres.

[0018] Preferably, the mass volume ratio of the first component to the organic solvent is 0.5-3 g:10 mL.

[0019] Preferably, the particle size of the poly (L-lactic acid) composite porous microspheres is 10-150 μm.

[0020] Another aspect of the present invention provides poly (L-lactic acid) composite porous microspheres, which are prepared according to the above-mentioned preparation method.

[0021] Another aspect of the present invention provides a poly (L-lactic acid) composite porous microsphere, comprising a first component, a second component and optional auxiliary components, wherein the first component is poly (L-lactic acid), the second component is a vitamin E compound, or a vitamin E compound and a vitamin C compound, and the poly (L-lactic acid) composite porous microsphere contains a plurality of pores with a pore size of nanometer level and / or micrometer level.

[0022] Preferably, the vitamin E compound includes at least one selected from vitamin E, vitamin E acetate, vitamin E succinate, vitamin E palmitate, vitamin E succinate, vitamin E nicotinate, vitamin E phosphate, vitamin E linoleate, vitamin E laurate, and vitamin E retinoic acid ester.

[0023] Preferably, the vitamin C compound includes at least one selected from the group consisting of L-ascorbic acid, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, calcium ascorbyl phosphate, ascorbic acid polypeptide, ascorbic acid ethyl ether, ascorbyl dipalmitate, ascorbyl palmitate, ascorbyl glucoside and ascorbyl ethylsilanol pectate.

[0024] Preferably, the auxiliary ingredients are selected from at least one of the group consisting of vitamins or their esters, amino acids, small molecule active peptides, collagen, polyracemic lactic acid, polydextrose lactic acid, and block copolymers of polylactic acid and other components, and the vitamins or their esters do not include the second ingredient.

[0025] Preferably, the particle size of the poly (L-lactic acid) composite porous microspheres is 10-150 μm.

[0026] In another aspect of the present invention, an injectable preparation is provided, comprising the poly-L-lactic acid composite porous microspheres prepared according to the above preparation method, or the above-mentioned poly-L-lactic acid composite porous microspheres.

[0027] Preferably, the injectable preparation is used for medical cosmetology and plastic surgery.

[0028] The poly (L-lactic acid) composite porous microspheres of the present invention contain a plurality of nanometer-scale and / or micrometer-scale pores. Compared with the pore-free Suranya and Aiweilan, they have a faster onset of action and a better long-term effect. In addition, compared with the poly (L-lactic acid) porous microspheres, they have a stronger ability to stimulate collagen regeneration and a weaker inflammatory property, and are safer. The porous microspheres of the present invention have a better fibroblast proliferation ability than solid microspheres, and the volume per unit mass is larger, resulting in a better filling effect. In addition, in addition to containing the poly (L-lactic acid) component, the poly (L-lactic acid) composite porous microspheres of the present invention also contain at least a stable and relatively high content of vitamin E compounds, or vitamin E compounds and vitamin C compounds. While being able to stimulate collagen regeneration at the application site, they also have superior properties such as anti-oxidation, enhancing cell viability, promoting repair, and improving immunity. Moreover, as the poly (L-lactic acid) composite porous microspheres degrade, the vitamin E compounds, or vitamin E compounds and vitamin C compounds can be released stably and long-lastingly to exert their functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is an electron microscope photograph of the poly (L-lactic acid) composite porous microspheres prepared in Example 1; Figure 2 This is an electron microscope photograph of the poly (L-lactic acid) composite porous microspheres prepared in Example 2; Figure 3 This is an electron microscope photograph of the poly (L-lactic acid) composite porous microspheres prepared in Example 3; Figure 4 This is an electron microscope photograph of the poly (L-lactic acid) composite porous microspheres prepared in Example 4; Figure 5 This is an electron microscope photograph of the poly (L-lactic acid) composite porous microspheres prepared in Example 5; Figure 6 This is an electron microscope photograph of the poly (L-lactic acid) composite porous microspheres prepared in Example 6; Figure 7 This is an electron microscope photograph of the poly (L-lactic acid) composite porous microspheres prepared in Comparative Example 1; Figure 8 This is an electron microscope photograph of the poly (L-lactic acid) composite porous microspheres prepared in Comparative Example 2; Figure 9 This is the particle size distribution diagram of the poly (L-lactic acid) composite porous microspheres prepared in Example 1; Figure 10 This is the particle size distribution diagram of the poly (L-lactic acid) composite porous microspheres prepared in Example 2; Figure 11 This is the particle size distribution diagram of the poly (L-lactic acid) composite porous microspheres prepared in Example 3; Figure 12 This is the particle size distribution diagram of the poly (L-lactic acid) composite porous microspheres prepared in Example 4; Figure 13This is the in vitro release curve of vitamin E from the poly (L-lactic acid) composite porous microspheres prepared in Example 2; Figure 14 This is a volume comparison chart of four microspheres of the same mass (1 g) in Examples 1-3 and Comparative Example 1; Figure 15 This is a comparison chart of the proliferation results of fibroblasts from four microspheres in Examples 1-3 and Comparative Example 1. DETAILED DESCRIPTION

[0030] The technical solutions of the present invention are further described below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are intended only to illustrate and explain the present invention and are not intended to limit the present invention. The methods used in the present invention are conventional production methods unless otherwise specified; the raw materials used are conventional commercially available products unless otherwise specified. All percentages in the present invention are by mass unless otherwise specified.

[0031] the term Polylactic acid, also known as polylactide, is a polyester polymer obtained by polymerizing lactic acid as the main raw material. Because the lactic acid molecule contains an asymmetric carbon atom and is optically active, polylactic acid is also divided into dextrorotatory polylactic acid (also known as poly-dextrorotatory lactic acid, PDLA), levorotatory polylactic acid (also known as poly-L-lactic acid, PLLA), racemic polylactic acid (also known as poly-dl-lactic acid, PDLLA), and optically inactive polylactic acid (Meso-PLA).

[0032] In the present invention, "vitamin E compounds" is used as a general term, and vitamin E and its esters and salts are collectively referred to as vitamin E compounds. The vitamin E compounds can be selected from at least one of vitamin E, vitamin E acetate, vitamin E succinate, vitamin E palmitate, vitamin E succinate, vitamin E nicotinate, vitamin E phosphate, vitamin E linoleate, vitamin E laurate, and vitamin E retinoic acid ester, but are not limited thereto.

[0033] In the present invention, "vitamin C compounds" is used as a general term, and L-ascorbic acid and its derivatives and salts are collectively referred to as "vitamin C compounds." The vitamin C compounds can be selected from the group consisting of L-ascorbic acid (vitamin C), sodium ascorbyl phosphate, magnesium ascorbyl phosphate, calcium ascorbyl phosphate, ascorbic acid polypeptide, ascorbic acid ethyl ether, ascorbyl dipalmitate, ascorbyl palmitate, ascorbyl glucoside, and ascorbyl ethylsilanol pectate, but are not limited thereto.

[0034] In the present invention, "auxiliary ingredients" are not limited as long as they do not affect the preparation and efficacy of the poly-L-lactic acid composite porous microspheres of the present invention (such as the ability to stimulate collagen regeneration, safety, and onset time). For example, they can be at least one selected from the group consisting of vitamins or their esters, amino acids, small molecule active peptides, collagen, poly(racemic)-lactic acid, poly(D-lactic acid), and block copolymers of poly(lactic acid) and other components. When preparing the poly-L-lactic acid composite porous microspheres of the present invention, auxiliary ingredients may or may not be added, depending on actual needs.

[0035] In the present invention, "poly-L-lactic acid composite porous microspheres" refers to a composite material containing at least poly-L-lactic acid and vitamin E compounds, or at least poly-L-lactic acid, vitamin E compounds and vitamin C compounds. The composite material is a spherical micron particle, for example, preferably, a particle size of 10-150 μm, and contains a plurality of nanometer-level and / or micrometer-level pores.

[0036] In the present invention, "nanoscale pores" refer to pores with a diameter in the nanometer range, i.e., within the range of 1-1000 nm. The cross-sectional shape of the pores is not particularly limited and may be, for example, circular, elliptical, rectangular, triangular, polygonal, or irregular. Furthermore, the pores may be through holes or blind holes, without particular limitation. The present invention also includes pores that are intertwined or interconnected.

[0037] In the present invention, "micrometer-level pores" refer to pores with a diameter in the micrometer range, preferably within the range of 1-100 μm. The cross-sectional shape of the pores is not particularly limited, and may be, for example, circular, elliptical, rectangular, triangular, polygonal, or irregular. Furthermore, the pores may be through holes or blind holes, without particular limitation. The present invention also includes pores that are intertwined or interconnected.

[0038] In the present invention, the "emulsification-solvent evaporation method" is a technology known in the art. In simple terms, it is a method of preparing an emulsion by mixing an aqueous phase containing a surfactant and an oil phase containing an organic solvent, and then volatilizing the organic solvent in the emulsion to obtain spherical particles. Preferably, the present invention uses a single emulsification-solvent evaporation method and a double emulsification-solvent evaporation method. The single emulsification-solvent evaporation method refers to the preparation of an emulsion by an aqueous phase and an oil phase, while the double emulsification-solvent evaporation method refers to the preparation of an emulsion by first mixing an inner aqueous phase and an oil phase to obtain colostrum when preparing an emulsion, and then mixing the colostrum with an outer aqueous phase to prepare an emulsion. Unless otherwise specified, in addition to the raw materials and parameters defined in the present invention, such as the first component, the second component and the auxiliary components, the reagents and experimental parameters commonly used in the art for the emulsification-solvent evaporation method can be used to prepare the poly (L-lactic acid) composite porous microspheres of the present invention.

[0039] Preparation method of poly (L-lactic acid) composite porous microspheres The poly (L-lactic acid) composite porous microspheres of the present invention are prepared from a first component, a second component and optional auxiliary components by an emulsification-solvent volatilization method, wherein the first component is poly (L-lactic acid) and the second component is a vitamin E compound or a mixture of a vitamin E compound and a vitamin C compound; the content of the first component is 63-93% of the total mass of the first and second components, and the content of the second component is 7-37% of the total mass of the first and second components.

[0040] When preparing poly (L-lactic acid) microspheres using an emulsification-solvent evaporation method, adding vitamin E compounds or vitamin E compounds and vitamin C compounds can produce composite microspheres of poly (L-lactic acid)-vitamin E compounds and poly (L-lactic acid)-vitamin E compounds-vitamin C compounds that have the ability of poly (L-lactic acid) to stimulate collagen regeneration, the antioxidant properties of vitamin E compounds to enhance cell viability, and the ability of vitamin C compounds to promote collagen formation, repair, and immunity. Compared to simple poly (L-lactic acid) microspheres, when preparing injectable preparations that require the addition of vitamin E compounds or vitamin C compounds, the composite microspheres of the present invention do not require the addition of vitamin compounds separately, and the vitamin compounds can also be slowly released as the poly (L-lactic acid) degrades, allowing the effects of the vitamin compounds to be exerted for a longer period of time. Moreover, compared to the technology of first preparing microspheres and then loading vitamin compounds, the content of vitamin compounds contained in the microspheres prepared by the method of the present invention can be adjusted according to actual conditions, and the vitamin compounds contained are not easily desorbed and are more stable. In addition, the inventors of the present invention unexpectedly discovered that the addition of vitamin compounds can make the poly-L-lactic acid microspheres have a porous structure. Compared with non-porous microspheres, the microspheres with this porous structure have a faster onset of effect and can significantly improve the filling effect of non-porous microsphere products in the four weeks before injection.

[0041] In a preferred embodiment of the present invention, the intrinsic viscosity of the poly-L-lactic acid is 0.8-4.3 dL / g. The intrinsic viscosity used in the present invention is measured by an Ubbelohde capillary viscometer.

[0042] In a preferred embodiment of the present invention, the second component is a mixture of a vitamin E compound and a vitamin C compound, wherein the vitamin E compound comprises 5-35% by weight of the combined mass of the first and second components, and the vitamin C compound comprises 2-32% by weight of the combined mass of the first and second components. By using both vitamin E and vitamin C compounds, the prepared composite porous microspheres can possess the properties of both, resulting in superior repair efficacy and safety.

[0043] In a preferred embodiment of the present invention, the vitamin E compound includes at least one selected from vitamin E, vitamin E acetate, vitamin E succinate, vitamin E palmitate, vitamin E succinate, vitamin E nicotinate, vitamin E phosphate, vitamin E linoleate, vitamin E laurate, and vitamin E retinoate; and the vitamin C compound includes at least one selected from the group consisting of L-ascorbic acid, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, calcium ascorbyl phosphate, ascorbic acid polypeptide, ascorbic acid ethyl ether, ascorbyl dipalmitate, ascorbyl palmitate, ascorbyl glucoside, and ascorbyl ethylsilanol pectinate. More preferably, the vitamin C compound is L-ascorbic acid or ascorbyl palmitate.

[0044] In a preferred embodiment of the present invention, the auxiliary component is selected from at least one of the group consisting of vitamins or their esters, amino acids, small molecule active peptides, collagen, polyracemic lactic acid, polydextrose lactic acid, and block copolymers of polylactic acid and other components, wherein the vitamins or their esters do not include the second component, and the content of the auxiliary component is 0-40% of the total mass of the first component and the second component. Preferably, the vitamin or its ester is vitamin B3. Preferably, the amino acid is histidine, arginine, or proline. Preferably, the small molecule active peptide is glutathione. When the above-mentioned auxiliary components are added during the preparation of the poly (L-lactic acid) composite porous microspheres of the present invention, the auxiliary components can be stably loaded in an adjustable content, and can be slowly released as the poly (L-lactic acid) degrades, allowing them to function more effectively.

[0045] In a preferred embodiment of the present invention, the emulsification-solvent evaporation method of the present invention is a single emulsification-solvent evaporation method, comprising: (A-1) dissolving the first component, the oil-soluble second component and the optional oil-soluble auxiliary component in an organic solvent to form an oil phase; (B-1) dissolving the surfactant, the water-soluble second component and the optional water-soluble auxiliary component in water to form an aqueous phase; (C-1) mixing the oil phase and the aqueous phase to form an emulsion, stirring the emulsion to evaporate the organic solvent, and obtaining a suspension; and (D-1) screening, washing, and freeze-drying the suspension to obtain the poly-L-lactic acid composite porous microspheres. In another preferred embodiment of the present invention, the emulsification-solvent evaporation method is a double emulsification-solvent evaporation method, comprising: (A-2) dissolving the first component, the oil-soluble second component, and any oil-soluble auxiliary components in an organic solvent to form an oil phase; (B-2) dissolving the water-soluble second component and any water-soluble auxiliary components in water to form an internal aqueous phase; (C-2) dissolving a surfactant in water to form an external aqueous phase; (D-2) mixing the oil phase and the internal aqueous phase to form colostrum, which is then added to the external aqueous phase to form a double emulsion; (E-2) stirring the double emulsion to evaporate the organic solvent to obtain a suspension; and (F-2) sieving, washing, and freeze-drying the suspension to obtain poly(L-lactic acid) composite porous microspheres. The above method can simply prepare the poly(L-lactic acid) composite porous microspheres of the present invention. In a preferred embodiment of the present invention, in step (D-1) or step (F-2), the suspension is sieved and washed, then added to a suspending solution and freeze-dried to obtain the poly(L-lactic acid) composite porous microspheres. As mentioned above, the emulsification-solution volatilization method is a technology known in the art. In addition to the first component, the second component and the auxiliary component of the present invention, the reagents and experimental parameters commonly used in the emulsification-solvent volatilization method in the art can be used to prepare the poly (L-lactic acid) composite porous microspheres of the present invention. In a preferred embodiment of the present invention, the mass volume ratio of the first component to the organic solvent is 0.5-3g:10mL. At this mass volume ratio, the morphology of the prepared microspheres is better. In a preferred embodiment of the present invention, the organic solvent is selected from at least one of the group consisting of dichloromethane and methanol. In a preferred embodiment of the present invention, the surfactant is selected from polyvinyl alcohol. In a preferred embodiment of the present invention, the mass concentration of the surfactant in the aqueous phase or the external aqueous phase is 0.25-1%. In a preferred embodiment of the present invention, the volume ratio of the oil phase to the aqueous phase, or the oil phase to the internal aqueous phase and the external aqueous phase is 1:50-320. In a preferred embodiment of the present invention, in step (D-2), the oil phase and the inner aqueous phase are mixed to form colostrum, with a stirring speed of 10,000-20,000 rpm and a stirring time of 90-150 s; and the colostrum is added to the outer aqueous phase and mixed to form a double emulsion, with a stirring speed of 3,000-6,000 rpm and a stirring time of 2-3 min.In a preferred embodiment of the present invention, in step (D-2), colostrum is added to the external aqueous phase through a microporous membrane and mixed to form a double emulsion. In a preferred embodiment of the present invention, in step (C-1) or step (E-2), the stirring speed is 200-300 rpm and the stirring time is 5 hours.

[0046] The particle size of the poly (L-lactic acid) composite porous microspheres prepared by the method of the present invention is not particularly limited and can be controlled according to actual needs by the sieve aperture used in the screening step. In a preferred embodiment of the present invention, the particle size of the poly (L-lactic acid) composite porous microspheres is 10-150 μm. Poly (L-lactic acid) composite porous microspheres within this particle size range are suitable for preparing injectable preparations.

[0047] Poly (L-lactic acid) composite porous microspheres The poly (L-lactic acid) composite porous microspheres of the present invention include a first component, a second component, and optional auxiliary components, wherein the first component is poly (L-lactic acid), the second component is a vitamin E compound, or a vitamin E compound and a vitamin C compound, and the poly (L-lactic acid) composite porous microspheres contain a plurality of pores with nanometer and / or micrometer pore sizes. Preferably, the content of the second component in the poly (L-lactic acid) composite porous microspheres is 5.29-34.1% by mass of the poly (L-lactic acid) composite porous microspheres.

[0048] Injectable preparations The poly-L-lactic acid composite porous microspheres of the present invention are mixed with an injectable agent and optional medical excipients to prepare an injectable preparation, which can be used for medical cosmetology and plastic surgery. Example 1

[0049] Weigh 1.86g PLLA (intrinsic viscosity 0.8dL / g) and 140mg vitamin E acetate and dissolve them in 6.2mL dichloromethane (CH2Cl2) to form an oil phase (PLLA:CH2Cl2=3g:10mL; the content of vitamin E acetate is 7% of the total mass of PLLA and vitamin E acetate). The oil phase is added to 500mL of 0.5% (w / v) polyvinyl alcohol (PVA) aqueous solution at 10°C under homogenization at 4000rpm, homogenized and emulsified for 2min to obtain an emulsion. The emulsion is transferred to a cantilever stirrer at a speed of 200 rpm and stirred for 5h to evaporate and remove the organic solvent. Filter through a sieve, rinse with deionized water, and freeze-dry to obtain powdered microspheres. The particle size distribution of the obtained poly (L-lactic acid) composite porous microspheres is 10-150μm. As Figure 1 As shown in Figure 3, the microspheres were observed to be porous under a scanning electron microscope (Hitachi SU8010). Example 2

[0050] Weigh 1.26g PLLA (intrinsic viscosity 4.3dL / g) and 740mg vitamin E and dissolve them in 25.2mL dichloromethane to form an oil phase (PLLA:CH2Cl2=0.5g:10mL; vitamin E content is 37% of the total mass of PLLA and vitamin E). The oil phase is added to 2000mL 1% (w / v) PVA aqueous solution at 10°C under homogenization at 4000rpm, homogenized and emulsified for 4min to obtain an emulsion. Transfer the emulsion to a cantilever stirrer at a speed of 200 rpm and stir for 5h to evaporate and remove the organic solvent. Filter through a sieve, rinse with deionized water, and freeze-dry to obtain powdered microspheres. The particle size distribution of the obtained poly (L-lactic acid) composite porous microspheres is 10-150μm. As Figure 2 As shown in Figure 3, the microspheres were observed to be porous under a scanning electron microscope (Hitachi SU8010). Example 3

[0051] 1.8 g of poly(vinyl lysine) (intrinsic viscosity 1.1 dL / g) and 100 mg of vitamin E were dissolved in 9 mL of dichloromethane to form an oil phase (PLLA:CH2Cl2 = 2 g:10 mL, with a vitamin E content of 5% of the combined mass of PLLA, vitamin E, and vitamin C). 100 mg of vitamin C was dissolved in 2 mL of water to form an internal aqueous phase (with a vitamin C content of 5% of the combined mass of PLLA, vitamin E, and vitamin C, and a combined vitamin E and vitamin C content of 10% of the combined mass of PLLA, vitamin E, and vitamin C). The oil phase was added to the internal aqueous phase and emulsified at 13,400 rpm for 90 seconds to obtain colostrum. The colostrum was then added to 1000 mL of a 0.5% (w / v) aqueous solution of polyvinyl alcohol (PVA) at 8°C and homogenized for 3 minutes to obtain a double emulsion. The emulsion was transferred to a cantilever stirrer at 260 rpm and stirred for 5 h to evaporate and remove the organic solvent; the mixture was filtered through a mesh, rinsed with deionized water, and freeze-dried to obtain powdered microspheres. The particle size distribution of the obtained poly (L-lactic acid) composite porous microspheres was 10-150 μm. Figure 3 As shown in Figure 3, the microspheres were observed to be porous under a scanning electron microscope (Hitachi SU8010). Example 4

[0052] 1.5 g of poly(vinyl ester) laminar emulsion (PLLA) (intrinsic viscosity 1.9 dL / g) and 200 mg of vitamin E were dissolved in 15 mL of dichloromethane to form an oil phase (PLLA:CH2Cl2 = 1 g:10 mL, with a vitamin E content of 10% of the combined mass of PLLA, vitamin E, and vitamin C). 300 mg of vitamin C was dissolved in 2 mL of water to form an internal aqueous phase (with a vitamin C content of 15% of the combined mass of PLLA, vitamin E, and vitamin C, and a combined vitamin E and vitamin C content of 25% of the combined mass of PLLA, vitamin E, and vitamin C). The oil phase was added to the internal aqueous phase and emulsified at 19,900 rpm for 90 seconds to obtain colostrum. The colostrum was then added to 1000 mL of a 0.25% (w / v) aqueous solution of polyvinyl alcohol (PVA) at 8°C at 6,000 rpm and homogenized for 2 minutes to obtain a double emulsion. The double emulsion was transferred to an overhead stirrer at 260 rpm and stirred for 5 hours to evaporate and remove the organic solvent. The obtained poly (L-lactic acid) composite porous microspheres have a particle size distribution of 10-150 μm. Figure 4 As shown in the figure, the microspheres were observed to be porous under a scanning electron microscope (Hitachi SU8010). Example 5

[0053] 1.3 g of PLLA (intrinsic viscosity 1.1 dL / g), 560 mg of PDLLA (intrinsic viscosity 0.4 dL / g), and 100 mg of vitamin E were dissolved in 13 mL of dichloromethane to form an oil phase (PLLA:CH2Cl2 = 1 g:10 mL, with a vitamin E content of 6.9% of the combined mass of PLLA, vitamin E, and vitamin C). 40 mg of vitamin C was dissolved in 2 mL of water to form an aqueous phase (with a vitamin C content of 2.8% of the combined mass of PLLA, vitamin E, and vitamin C, and a combined vitamin E and vitamin C content of 9.7% of the combined mass of PLLA, vitamin E, and vitamin C). The oil phase was added to the aqueous phase and emulsified at 13,400 rpm for 90 seconds to obtain colostrum. The colostrum was then added to 1000 mL of a 0.25% (w / v) PVA aqueous solution at 8°C at 3000 rpm and homogenized for 2 minutes to obtain a double emulsion. The emulsion was transferred to a cantilever stirrer at 280 rpm and stirred for 5 h to evaporate and remove the organic solvent. The mixture was filtered through a sieve, rinsed with deionized water, and freeze-dried to obtain powdered microspheres. The particle size distribution of the obtained poly (L-lactic acid) composite porous microspheres was 10-150 μm. Figure 5 As shown in Figure 3, the microspheres were observed to be porous under a scanning electron microscope (Hitachi SU8010). Example 6

[0054] 800 mg of PLLA (intrinsic viscosity 1.9 dL / g), 460 mg of PLLA (intrinsic viscosity 1.1 dL / g), and 400 mg of vitamin E were dissolved in 20 mL of dichloromethane to form an oil phase (PLLA:CH2Cl2 = 0.63 g:10 mL, with a vitamin E content of 20% of the combined mass of PLLA, vitamin E, and vitamin C). 340 mg of vitamin C was dissolved in 2 mL of water to form an aqueous phase (with a vitamin C content of 17% of the combined mass of PLLA, vitamin E, and vitamin C, and a combined vitamin E and vitamin C content of 37% of the combined mass of PLLA, vitamin E, and vitamin C). The oil phase was added to the aqueous phase and emulsified at 19,900 rpm for 90 seconds to obtain colostrum. The colostrum was then homogenized at 6,000 rpm and added to 2,000 mL of a 1% (w / v) PVA aqueous solution at 8°C for 2 minutes to obtain a double emulsion. The emulsion was transferred to a cantilever stirrer at 260 rpm and stirred for 5 h to evaporate and remove the organic solvent. The mixture was filtered through a sieve, rinsed with deionized water, and freeze-dried to obtain powdered microspheres. The particle size distribution of the obtained poly (L-lactic acid) composite porous microspheres was 10-150 μm. Figure 6 As shown, the porous morphology was observed under a scanning electron microscope (Hitachi SU8010). Example 7

[0055] 1.26 g of PLLA (intrinsic viscosity 1.9 dL / g) and 700 mg of vitamin E were dissolved in 12.6 mL of dichloromethane to form an oil phase (PLLA:CH2Cl2 = 1 g:10 mL, with a vitamin E content of 35% of the combined mass of PLLA, vitamin E, and vitamin C). 40 mg of vitamin C was dissolved in 1 mL of water to form an aqueous phase (vitamin C content of 2% of the combined mass of PLLA, vitamin E, and vitamin C, and the total content of vitamin E and vitamin C was 37% of the combined mass of PLLA, vitamin E, and vitamin C). The oil phase was added to the aqueous phase and emulsified at 19,900 rpm for 90 seconds to obtain colostrum. The colostrum was then homogenized at 6,000 rpm into 2,000 mL of a 1% (w / v) PVA aqueous solution at 8°C and homogenized for 2 minutes to obtain a double emulsion. The double emulsion was transferred to an overhead stirrer at 260 rpm and stirred for 5 hours to evaporate and remove the organic solvent. The mixture was filtered through a mesh, rinsed with deionized water, and freeze-dried to obtain powdered microspheres. The obtained poly (L-lactic acid) composite porous microspheres had a particle size distribution of 10-150 μm and were porous under a scanning electron microscope (Hitachi SU8010). Example 8

[0056] 1.26 g of PLLA (intrinsic viscosity 1.1 dL / g) and 100 mg of vitamin E were dissolved in 6.3 mL of dichloromethane to form an oil phase (PLLA:CH2Cl2 = 2 g:10 mL, with a vitamin E content of 5% of the combined mass of PLLA, vitamin E, and vitamin C). 640 mg of vitamin C was dissolved in 2 mL of water to form an aqueous phase (with a vitamin C content of 32% of the combined mass of PLLA, vitamin E, and vitamin C, and a combined vitamin E and vitamin C content of 37% of the combined mass of PLLA, vitamin E, and vitamin C). The oil phase was added to the aqueous phase and emulsified at 19,900 rpm for 90 seconds to obtain colostrum. The colostrum was then homogenized at 6,000 rpm into 2,000 mL of a 1% (w / v) PVA aqueous solution at 8°C and homogenized for 2 minutes to obtain a double emulsion. The double emulsion was transferred to an overhead stirrer at 260 rpm and stirred for 5 hours to evaporate and remove the organic solvent. The mixture was filtered through a mesh, rinsed with deionized water, and freeze-dried to obtain powdered microspheres. The obtained poly (L-lactic acid) composite porous microspheres had a particle size distribution of 10-150 μm and were porous under a scanning electron microscope (Hitachi SU8010). Example 9

[0057] 1.6 g of PLLA (intrinsic viscosity 1.9 dL / g), 500 mg of PDLA (intrinsic viscosity 0.6 dL / g), and 200 mg of vitamin E were dissolved in 20 mL of dichloromethane to form an oil phase (PLLA:CH2Cl2 = 0.8 g:10 mL, with a vitamin E content of 10.3% of the combined mass of PLLA, vitamin E, and vitamin C). 150 mg of vitamin C and 50 mg of glutathione were dissolved in 2 mL of water to form an aqueous phase (vitamin C content of 7.7% of the combined mass of PLLA, vitamin E, and vitamin C, and the combined vitamin C and vitamin E content of 18% of the combined mass of PLLA, vitamin E, and vitamin C). The oil phase was added to the aqueous phase and emulsified at 13,400 rpm for 120 s to obtain colostrum. The colostrum was then added to 1000 mL of a 0.5% (w / v) PVA aqueous solution at 6°C at 5000 rpm and homogenized for 2 minutes to obtain a double emulsion. The emulsion was transferred to an overhead mixer at 240 rpm and stirred for 5 hours to evaporate the organic solvent. The mixture was then filtered through a mesh, rinsed with deionized water, and freeze-dried to obtain powdered microspheres. The resulting poly(L-lactic acid) composite porous microspheres had a particle size distribution of 10-150 μm. Scanning electron microscopy (Hitachi SU8010) revealed a porous morphology. Example 10

[0058] 1.6 g of poly(vinyl lysate) laminar emulsion (PLLA) (intrinsic viscosity 1.9 dL / g) and 200 mg of vitamin E palmitate were dissolved in 20 mL of dichloromethane to form an oil phase (PLLA:CH2Cl2 = 0.8 g:10 mL, vitamin E palmitate content 10.1% of the combined mass of PLLA, vitamin E palmitate, and vitamin C). 180 mg of vitamin C and 20 mg of vitamin B3 were dissolved in 2 mL of water to form an aqueous phase (vitamin C content 9.1% of the combined mass of PLLA, vitamin E palmitate, and vitamin C, and the combined mass of vitamin E palmitate and vitamin C was 19.2% of the combined mass of PLLA, vitamin E palmitate, and vitamin C). The oil phase was added to the aqueous phase and emulsified at 13,400 rpm for 90 s to obtain colostrum. The colostrum was then added to 1000 mL of a 0.5% (w / v) aqueous solution of poly(vinyl lysate) amide (PVA) at 6°C and homogenized for 2 minutes to obtain a double emulsion. The emulsion was transferred to an overhead mixer at 240 rpm and stirred for 5 hours to evaporate the organic solvent. The mixture was then filtered through a mesh, rinsed with deionized water, and freeze-dried to obtain powdered microspheres. The resulting poly(L-lactic acid) composite porous microspheres had a particle size distribution of 10-150 μm. Scanning electron microscopy (Hitachi SU8010) revealed a porous morphology. Example 11

[0059] 3.2 g of poly(vinyl alcohol) ester (PLLA) (intrinsic viscosity 1.9 dL / g), 600 mg of vitamin E, and 200 mg of ascorbic acid palmitate were dissolved in a mixture of 32 mL of dichloromethane and 2 mL of methanol to form an oil phase (PLLA:organic solvent = 0.9 g:10 mL; vitamin E content was 15% of the combined mass of PLLA, vitamin E, and ascorbic acid palmitate; ascorbic acid palmitate content was 5% of the combined mass of PLLA, vitamin E, and ascorbic acid palmitate; and the total content of vitamin E and ascorbic acid palmitate was 20% of the combined mass of PLLA, vitamin E, and ascorbic acid palmitate). The oil phase was then passed through a microporous membrane into 2000 mL of a 1% (w / v) aqueous solution of polyvinyl alcohol (PVA) to form an emulsion. The emulsion was transferred to an overhead stirrer at 300 rpm and stirred for 5 hours to evaporate the organic solvent. The mixture was then filtered through a mesh, rinsed with deionized water, and freeze-dried to obtain powdered microspheres. The particle size distribution of the obtained poly (L-lactic acid) composite porous microspheres was 10-150 μm. The microspheres were observed to be round and porous under a scanning electron microscope (Hitachi SU8010). Example 12

[0060] 2.5 g of poly(DL-lactide-co-glycolide) (PDLGA, intrinsic viscosity 0.4 dL / g), and 440 mg of vitamin E were dissolved in 20 mL of dichloromethane to form an oil phase (PLLA:CH2Cl2 = 1.25 g:10 mL; vitamin E content was 15% of the combined mass of PLLA and vitamin E). 60 mg of amino acids (20 mg each of histidine, arginine, and proline) were dissolved in 3 mL of water to form an aqueous phase. The oil phase was added to the aqueous phase and emulsified at 10,000 rpm for 150 s to obtain colostrum. The colostrum passed through a microporous membrane into 1500 mL of 0.5% (w / v) PVA aqueous solution to form a double emulsion. The double emulsion was transferred to an overhead stirrer at 240 rpm and stirred for 5 h to evaporate the organic solvent. The mixture was then sieved, rinsed with deionized water, and freeze-dried to obtain powdered microspheres. The particle size distribution of the obtained poly (L-lactic acid) composite porous microspheres was 10-150 μm, and the microspheres were porous when observed under a scanning electron microscope (Hitachi SU8010). Example 13

[0061] 3.3 g of poly(vinyl lysine) (intrinsic viscosity 3.1 dL / g), 580 mg of vitamin E, and 100 mg of ascorbic acid palmitate were dissolved in 30 mL of dichloromethane to form an oil phase (PLLA:CH2Cl2 = 1.1 g:10 mL; vitamin E content was 14.6% of the combined mass of PLLA, vitamin E, and ascorbic acid palmitate; ascorbic acid palmitate content was 2.5%; and vitamin E and ascorbic acid palmitate content was 17.1% of the combined mass of PLLA, vitamin E, and ascorbic acid palmitate). 20 mg of collagen (recombinant human collagen, molecular weight 44 kDa) was dissolved in 3 mL of water to form an aqueous phase. The oil phase was added to the aqueous phase and emulsified at 10,000 rpm for 90 s to obtain colostrum. The colostrum then passed through a microporous membrane into 3,000 mL of a 0.5% (w / v) PVA aqueous solution to form a double emulsion. The emulsion was transferred to an overhead mixer at 240 rpm and stirred for 5 hours to evaporate the organic solvent. The mixture was then filtered through a mesh, rinsed with deionized water, and freeze-dried to obtain powdered microspheres. The resulting poly(L-lactic acid) composite porous microspheres had a particle size distribution of 10-150 μm. Scanning electron microscopy (Hitachi SU8010) revealed a porous morphology.

[0062] Comparative Example 1 Weigh 1.5g PLLA (intrinsic viscosity 1.1dL / g) and dissolve it in 10mL dichloromethane to form an oil phase. Add the oil phase to 1000mL 0.5% (w / v) PVA aqueous solution at 6°C at 7000rpm and homogenize and emulsify for 2min to obtain an emulsion. Transfer the emulsion to a cantilever stirrer at a speed of 240 rpm and stir for 5h to evaporate and remove the organic solvent. Filter through a sieve, rinse with deionized water, and freeze-dry to obtain powdered microspheres with a particle size distribution of 10-150μm. Figure 7 As shown in the figure, the microspheres were observed to be round and dense under a scanning electron microscope (Hitachi SU8010).

[0063] Comparative Example 2 PLLA microspheres were prepared by the emulsification-solvent evaporation method. Referring to the amount of NaCl added in the method for preparing PDLLA porous microspheres in CN102369234A, NaCl was added to the external aqueous phase to a NaCl concentration of 25% (w / v). Scanning electron microscopy showed that the prepared microspheres had a round morphology with superficial indentations and no porous appearance.

[0064] Weigh 1.5g PLLA (intrinsic viscosity 1.1dL / g) and dissolve it in 10mL dichloromethane to form an oil phase. Add the oil phase to 1000mL of 0.5% (w / v) PVA and 25% (w / v) NaCl aqueous solution at 6°C at 7000rpm, homogenize and emulsify for 2min to obtain an emulsion. Transfer the emulsion to a cantilever stirrer at a speed of 240 rpm and stir for 5h to evaporate and remove the organic solvent. Filter through a sieve, rinse with deionized water, and freeze-dry to obtain powdered microspheres with a particle size distribution of 10-150μm. Figure 8 As shown in Figure 2, the morphology of the microspheres was observed under a scanning electron microscope (Hitachi SU8010). It can be seen that the microspheres are round with shallow indentations and no porous appearance.

[0065] It can be seen from the above examples and comparative examples that by adding vitamin E compounds, or a mixture of vitamin E compounds and vitamin C compounds, poly-L-lactic acid composite porous microspheres with a porous structure can be prepared, while without adding the above-mentioned vitamin E compounds, or a mixture of vitamin E compounds and vitamin C compounds, or adding other substances, poly-L-lactic acid composite porous microspheres with a porous structure cannot be obtained. Example 14

[0066] In this example, the content and particle size distribution of the second component in the microspheres prepared in Examples 1-13 and Comparative Examples 1-2 were tested.

[0067] Particle size distribution: The test was performed using a Malvern 3000 laser particle size analyzer in accordance with the third method (light scattering method) of the fourth part of the General Chapter 0982 of the Chinese Pharmacopoeia (2020 Edition) on particle size and particle size distribution.

[0068] Content of the second component in the microspheres (the percentage of the second component in the total mass of the microspheres): Vitamin C and vitamin C compounds Test solution: Accurately weigh 30 mg of microspheres and place them in a glass test tube. Add 1 mL of dichloromethane to dissolve them. Add 10 mL of diluent and shake to mix. Let stand to separate the layers. Centrifuge the supernatant as the test solution.

[0069] The mobile phase, diluent, and reference solution preparations, as well as the chromatographic method, were performed according to USP-NF 580 Vitamin C Content - Method 2 - Procedure 1. Blank solutions (diluent), reference solutions, and test solutions were analyzed sequentially using a Waters Alliance E2695 high-performance liquid chromatograph.

[0070] Vitamin E and vitamin E compounds Test solution: Accurately weigh 30 mg of microspheres, place them in a 10 mL volumetric flask, add n-hexane, sonicate, dissolve and dilute to the scale, filter, and use as the test solution.

[0071] Reference solution: Weigh about 20 mg of vitamin E or vitamin E-like compound reference substance, place it in a 50 mL volumetric flask, dissolve it with n-hexane and dilute to the scale as the reference solution.

[0072] The chromatographic method was set up in accordance with the inspection items for related substances of vitamin E in Part II of the Chinese Pharmacopoeia (2020 edition). A Shimadzu 2030 gas chromatograph was used to perform chromatographic analysis of the blank solution (n-hexane), the reference solution, and the test solution.

[0073] Table 1 summarizes the first component, the second component and their amounts used in Examples 1-13 and Comparative Examples 1-2, the content of the second component in the prepared microspheres, and the particle size distribution. Figure 9-12 These are the particle size distribution diagrams of Examples 1-4 respectively.

[0074] Table 1

[0075] As can be seen from Table 1 above, when the poly-L-lactic acid composite porous microspheres are prepared by the method of the present invention, less vitamin E compounds are lost during the preparation process, and most of the vitamin E compounds can be retained in the prepared microspheres. Moreover, as the poly-L-lactic acid composite porous microspheres degrade, the vitamin E compounds can exert their effects for a longer period of time. Example 15

[0076] In this example, the poly (L-lactic acid) composite porous microspheres prepared in Example 2 were used as an example to test the in vitro release of vitamin E.

[0077] Solution preparation: Release medium / blank solution: Take 1.36 g of potassium dihydrogen phosphate, 1.42 g of disodium hydrogen phosphate, and 50 g of Tween 80, add 1000 mL of ultrapure water, adjust the pH to 7.4 ± 0.1 with sodium hydroxide, and degas by ultrasonication. Reference solution: Accurately weigh 20 mg of vitamin E and place it in a 50 mL volumetric flask. Add methanol to dissolve and dilute to the mark. Shake well to prepare the reference stock solution. Accurately weigh 1 mL of the reference stock solution and place it in a 10 mL volumetric flask. Add methanol to dilute to the mark. Shake well to prepare the reference solution. Test solution: Accurately weigh 30 mg of the poly (L-lactic acid) composite porous microspheres prepared by the method of Example 2 and place them in a stoppered ground-mouth glass bottle. Add 200 mL of release medium preheated to 55°C. Place the stoppered ground-mouth glass bottle in a constant temperature water bath at 55°C ± 0.5°C. Prepare three parallel samples in the same manner.

[0078] Detection: Sampling: Sample the test solution on days 1, 2, 3, 7, 10, 14, 21, 28, and 35. Shake the flask 20 minutes before sampling to homogenize the solution, then allow the poly (L-lactic acid) composite porous microspheres to settle. Accurately pipette 4 mL of the upper layer of solution and, after sampling, add an equal volume of release medium at the same temperature. Detection method: The blank solution, reference solution, and three parallel test solutions prepared above were analyzed separately using a high performance liquid chromatograph (Waters Alliance E2695). The chromatographic conditions were as follows: chromatographic column: Waters Symmetry Shield RP18 (3.5 μm, 4.6 mm × 100 mm); mobile phase: 97% methanol solution; flow rate: 1.0 mL / min, isocratic elution, elution time: 10 min; column temperature: 30°C; detector: UV detector; detection wavelength: 292 nm; injection volume: 20 μL.

[0079] result: The cumulative release of vitamin E was calculated using the following formula:

[0080] Wherein, C is the test sample concentration, mg / mL; Cs is the concentration of the reference substance, mg / mL; and are the main peak areas of the test solution and the reference solution, respectively; Cn and Cn-1 are the concentrations of the test solution calculated for this and last sampling, respectively, in mg / mL; m s is the mass of vitamin E in the microspheres, mg; D is the degree of release; Dn and Dn-1 are the cumulative release rates of the test sample at this and last sampling respectively.

[0081] The cumulative release of vitamin E from poly-L-lactic acid composite porous microspheres at different times is shown in Table 2, and the release curve is shown in Figure 13 .

[0082] Table 2

[0083] From Table 2 and Figure 13 It can be seen that at 55°C, the cumulative release of vitamin E from the porous microspheres over 35 days was >80%, the RSD between samples was <5%, and the release behavior was stable. Example 16

[0084] Take 1g of each microsphere prepared according to the method of Examples 1-3 and Comparative Example 1, and place them in a measuring cylinder to obtain the volume of the microsphere. The test results are as follows: Figure 14 As shown in the figure, at the same mass, the volume of the microspheres prepared according to the method of the present invention is significantly larger than that of the microspheres prepared in Comparative Example 1, and is even nearly twice that of Comparative Example 1 (Example 2). The poly-L-lactic acid composite porous microspheres of the present invention have a larger volume per unit mass and a better filling effect. Example 17

[0085] This example uses the microspheres of Examples 1-3 and Comparative Example 1 as examples to test the proliferation ability of the microspheres on fibroblasts.

[0086] Solution preparation: Sterile culture medium: DMEM medium (Gibco, USA) was used to prepare 10% fetal bovine serum (Gibco, USA); Microsphere solutions: For the microspheres of Examples 1-3 and Comparative Example 1, each microsphere was prepared using sterile culture medium to prepare five solutions with different gradient concentrations of 0.20% (w / v), 0.40% (w / v), 0.80% (w / v), 0.16% (w / v), and 0.32% (w / v); Detection: Fibroblasts (human dermal fibroblasts (Guangdong Boxi Biotechnology Co., Ltd.)) were seeded at a density of 10,000 cells / well in each of the Example, Comparative, and Control groups. Six replicates were set up for each concentration. The plates were incubated in a 37°C, 5% CO2 incubator for 24 hours. The original culture medium was then aspirated, and 100 μL of microsphere solution of varying concentrations was added to the Example and Comparative groups, respectively. 100 μL of sterile culture medium was added to the control group, and the cells were incubated for a further 48 hours. After adding 10 μL of 5 mg / mL MTT solution, the cells were incubated for a further 4 hours. Then, 200 μL of dimethyl sulfoxide was added and the cells were shaken for 10 minutes. The absorbance (A) at a wavelength of 490 nm was measured using a microplate reader (SPARK, Tecan Austria GmbH, Austria).

[0087] The fibroblast proliferation experimental data obtained by detection are shown in Table 3. Figure 15 A comparison of the cell proliferation results of four microspheres is shown.

[0088] Table 3

[0089] From Table 3 and Figure 15 It can be seen that the poly (L-lactic acid) composite porous microspheres of the present invention have stronger fibroblast proliferation ability than the solid microspheres of Comparative Example 1.

[0090] The present invention may also have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art may make various corresponding changes and modifications based on the present invention, but these corresponding changes and modifications should all fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing poly (L-lactic acid) composite porous microspheres, characterized by: The poly (L-lactic acid) composite porous microspheres are prepared from the first component, the second component and optional auxiliary components by an emulsification-solvent evaporation method. in, The first component is poly-L-lactic acid, and the second component is a vitamin E compound, or a mixture of a vitamin E compound and a vitamin C compound; The content of the first component is 63-93% by weight of the total mass of the first component and the second component, and the content of the second component is 7-37% by weight of the total mass of the first component and the second component.

2. The preparation method according to claim 1, wherein: The intrinsic viscosity of the poly (L-lactic acid) is 0.8-4.3 dL / g.

3. The preparation method according to claim 1, wherein: The second component is a mixture of vitamin E compounds and vitamin C compounds, wherein the content of the vitamin E compounds is 5-35% of the total mass of the first component and the second component, and the content of the vitamin C compounds is 2-32% of the total mass of the first component and the second component.

4. The preparation method according to claim 1, wherein: The vitamin E compound includes at least one selected from vitamin E, vitamin E acetate, vitamin E succinate, vitamin E palmitate, vitamin E succinate, vitamin E nicotinate, vitamin E phosphate, vitamin E linoleate, vitamin E laurate, and vitamin E retinoic acid ester.

5. The preparation method according to claim 1, wherein: The vitamin C compound includes at least one selected from the group consisting of L-ascorbic acid, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, calcium ascorbyl phosphate, ascorbic acid polypeptide, ascorbic acid ethyl ether, ascorbyl dipalmitate, ascorbyl palmitate, ascorbyl glucoside and ascorbyl ethylsilanol pectate.

6. The preparation method according to claim 1, wherein: The auxiliary component is selected from at least one of the group consisting of vitamins or their esters, amino acids, small molecule active peptides, collagen, polyracemic lactic acid, polydextrose lactic acid, and block copolymers of polylactic acid and other components. The vitamins or their esters do not include the second component, and the content of the auxiliary component is 0-40% of the total mass of the first component and the second component.

7. The preparation method according to claim 1, wherein: The emulsification-solvent evaporation method is a single emulsification-solvent evaporation method, which includes: (A-1) dissolving the first component, the oil-soluble second component, and the optional oil-soluble auxiliary component in an organic solvent to form an oil phase; (B-1) dissolving a surfactant in water to form an aqueous phase; (C-1) mixing the oil phase and the water phase to form an emulsion, and stirring the emulsion to volatilize the organic solvent to obtain a suspension; (D-1) Screening, washing, and freeze-drying the suspension to obtain the poly (L-lactic acid) composite porous microspheres.

8. The preparation method according to claim 1, wherein: The emulsification-solvent evaporation method is a double emulsification-solvent evaporation method, which comprises: (A-2) dissolving the first component, the oil-soluble second component, and the optional oil-soluble auxiliary component in an organic solvent to form an oil phase; (B-2) dissolving the water-soluble second component and optionally the water-soluble auxiliary component in water to form an internal aqueous phase; (C-2) dissolving the surfactant in water to form an external aqueous phase; (D-2) mixing the oil phase and the inner aqueous phase to form colostrum, and adding the colostrum to the outer aqueous phase to form a double emulsion; (E-2) stirring the emulsion to volatilize the organic solvent to obtain a suspension; (F-2) Screening, washing, and freeze-drying the suspension to obtain the poly (L-lactic acid) composite porous microspheres.

9. The preparation method according to claim 7 or 8, characterized in that: In the step (D-1) or the step (F-2), the suspension is sieved and washed, and then added to a solution with suspending ability and freeze-dried to obtain the poly (L-lactic acid) composite porous microspheres.

10. The preparation method according to claim 7 or 8, characterized in that: The mass volume ratio of the first component to the organic solvent is 0.5-3 g:10 mL.

11. The preparation method according to claim 1, characterized in that: The particle size of the poly (L-lactic acid) composite porous microspheres is 10-150 μm.

12. A poly (L-lactic acid) composite porous microsphere, characterized by: Prepared according to the preparation method according to any one of claims 1 to 11.

13. A poly (L-lactic acid) composite porous microsphere, characterized by: The invention comprises a first component, a second component and optional auxiliary components, wherein the first component is poly (L-lactic acid), the second component is a vitamin E compound, or a vitamin E compound and a vitamin C compound, and the poly (L-lactic acid) composite porous microspheres contain a plurality of pores with a pore size of nanometer level and / or micrometer level.

14. The poly (L-lactic acid) composite porous microspheres according to claim 13, characterized in that: The vitamin E compound includes at least one selected from vitamin E, vitamin E acetate, vitamin E succinate, vitamin E palmitate, vitamin E succinate, vitamin E nicotinate, vitamin E phosphate, vitamin E linoleate, vitamin E laurate, and vitamin E retinoic acid ester.

15. The poly (L-lactic acid) composite porous microspheres according to claim 13, characterized in that: The vitamin C compound includes at least one selected from the group consisting of L-ascorbic acid, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, calcium ascorbyl phosphate, ascorbic acid polypeptide, ascorbic acid ethyl ether, ascorbyl dipalmitate, ascorbyl palmitate, ascorbyl glucoside and ascorbyl ethylsilanol pectate.

16. The poly (L-lactic acid) composite porous microspheres according to claim 13, characterized in that: The auxiliary ingredients are selected from at least one of the group consisting of vitamins or their esters, amino acids, small molecule active peptides, collagen, polyracemic lactic acid, polydextrose lactic acid, block copolymers of polylactic acid and other components, and the vitamins or their esters do not include the second ingredient.

17. The poly (L-lactic acid) composite porous microspheres according to any one of claims 13 to 16, characterized in that: The particle size of the poly (L-lactic acid) composite porous microspheres is 10-150 μm.

18. A preparation for injection, characterized in that: The invention comprises the poly (L-lactic acid) composite porous microspheres prepared by the preparation method according to any one of claims 1 to 10, or the poly (L-lactic acid) composite porous microspheres according to any one of claims 13 to 17.

19. The injection preparation according to claim 18, characterized in that: The injection preparation is used for medical cosmetology and plastic surgery.

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