Double-layer microsphere as well as preparation method and application thereof

Abstract

The invention discloses a double-layer microsphere as well as a preparation method and application thereof. The double-layer microsphere comprises an inner core and a coating outer layer coating the inner core, an inner core comprises astaxanthin and PLGA or PLA wrapping the astaxanthin, an outer layer comprises collagen and a collagen-loaded nano-hydroxyapatite and vitamin c mixture, through cooperation and position distribution of all the raw materials, the double-layer microspheres are formed, the synergistic effect of immediate filling and long-acting regeneration is achieved, and the effect of immediate filling and long-acting regeneration is achieved. The outer layer of the double-layer microsphere is rich in collagen, nano-hydroxyapatite and vitamin C, so that instant filling after injection can be realized, endogenous collagen synthesis is promoted, inflammatory reaction is inhibited, and the mechanical property of the microsphere is enhanced; the inner core is formed by encapsulating fat-soluble astaxanthin by PLGA or PLA, and the expression of matrix metalloproteinase-1 is continuously inhibited through a slow release mechanism, so that the collagen degradation is reduced, and the filling effect is prolonged.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, and in particular to a bilayer microsphere, its preparation method, and its application. Background Technology

[0002] The main external signs of skin aging are roughness, dryness, pigmentation, and sagging. Injections of fillers can make the skin look plump and smooth.

[0003] Commonly used filler materials include hyaluronic acid, collagen, polycaprolactone, polylactic acid, polylactic-co-glycolic acid copolymer, and hydroxyapatite. Collagen is a major component of the extracellular matrix, so using collagen materials can directly improve sagging skin. However, collagen has the drawback of a rapid degradation rate and inability to maintain its effects long-term.

[0004] Therefore, it is essential to provide a solution that can prolong the filling effect of collagen. Summary of the Invention

[0005] In view of this, this application provides a bilayer microsphere, its preparation method and application, to solve the problem of how to improve the durability of collagen-filled bilayer microspheres.

[0006] To achieve the above technical objectives, this application adopts the following technical solution: In a first aspect, this application provides a bilayer microsphere comprising a core and an outer coating covering the core; the core comprises astaxanthin and PLGA or PLA coating the astaxanthin, and the outer coating comprises collagen and a mixture of collagen-loaded nano-hydroxyapatite and vitamin C.

[0007] Preferably, the molecular weight of PLGA is 30-100 kDa, wherein the mass ratio of lactic acid to glycolic acid in PLGA is 70-75:25-30; and the molecular weight of PLA is 100-500 kDa.

[0008] Preferably, the mass ratio of astaxanthin to PLGA or PLA is 1:5-10.

[0009] Preferably, the particle size of the nano-hydroxyapatite is 100-200 nm; the particle size of the core is ≤20 µm; and the particle size of the bilayer microspheres is 30-100 µm.

[0010] Secondly, this application provides a method for preparing bilayer microspheres, comprising the following steps: S1. Add astaxanthin and organic solutions of PLGA or PLA to an aqueous solution of polyvinyl alcohol for coating treatment to obtain the kernel; S2. Nano-hydroxyapatite, vitamin C, and the core are added to an acetic acid solution of collagen to obtain a mixture; the mixture is emulsified with liquid paraffin, and then glutaraldehyde is added to carry out a cross-linking reaction to obtain bilayer microspheres.

[0011] Preferably, the concentration of the organic solution of PLGA or PLA is 20-100 mg / ml.

[0012] Preferably, the volume ratio of the mixture to liquid paraffin is 1:5-15.

[0013] Preferably, the concentration of the acetic acid solution of collagen is 5-15 mg / ml.

[0014] Preferably, the mass ratio of nano-hydroxyapatite to vitamin C is 2:0.1-0.5.

[0015] Thirdly, this application provides the application of bilayer microspheres in the preparation of injectable skin tissue filling biomaterials.

[0016] The beneficial effects of this application are as follows: By constructing bilayer microspheres with specific structures and component distributions, this application achieves a synergistic effect of immediate filling and long-term regeneration: the outer layer is rich in collagen, nano-hydroxyapatite, and vitamin C, enabling immediate filling after injection, promoting endogenous collagen synthesis, inhibiting inflammatory responses, and enhancing the mechanical properties of the microspheres; the core consists of PLGA or PLA encapsulating fat-soluble astaxanthin, which continuously inhibits matrix metalloproteinase-1 expression through a sustained-release mechanism, reducing collagen degradation and prolonging the filling effect. The spatial distribution and functional complementarity of each component achieve a sustained collagen filling effect, significantly improving the durability, safety, and biocompatibility of skin tissue repair. Attached Figure Description

[0017] Figure 1 The degradation rate test results are for the bilayer microspheres; Figure 2 The results show the in vitro release rate of astaxanthin from bilayer microspheres. Figure 3 The results show the in vitro release rate of bilayer microsphere nano-hydroxyapatite. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0019] This application provides a bilayer microsphere comprising a core and an outer coating layer covering the core; the core comprises astaxanthin and PLGA or PLA coating the astaxanthin, and the outer layer comprises collagen and a mixture of collagen-loaded nano-hydroxyapatite and vitamin C.

[0020] This application achieves the effect of continuous collagen filling in bilayer microspheres through the synergy and positional distribution of various raw materials. The specific mechanism is as follows: In this application, the collagen is located on the outer layer and can directly contact the skin, providing an immediate skin-filling effect. Nano-hydroxyapatite and vitamin C are loaded onto the collagen through cross-linking. Nano-hydroxyapatite is a water-soluble and biodegradable material located on the outer layer and in direct contact with the body, which can promote the production of the body's own endogenous collagen and utilize its own biodegradation to achieve sustained release. At the same time, it can improve the mechanical properties of the bilayer microspheres to maintain the release of collagen. The bilayer microspheres in this application are an injectable reagent. Vitamin C is located on the outside and can directly contact pro-inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, and reduce the inflammatory response caused by the injected microspheres by inhibiting pro-inflammatory cytokines.

[0021] Polylactic acid-glycolic acid or polylactic acid is located in the core to reduce the degradation rate of collagen and maintain the long-term filling effect of collagen. The astaxanthin in this application is a fat-soluble substance located in the core. It is continuously released in vitro through the outer cross-linking network and the inner polymer. It can reduce the expression of matrix metalloproteinase-1 in skin fibroblasts to promote the production of endogenous collagen in the body, while avoiding the inflammatory response caused by the burst release of astaxanthin.

[0022] This application utilizes the different solubilities of astaxanthin and nano-hydroxyapatite to enable both to achieve sustained release when used as skin tissue filling injection preparations, thereby promoting collagen regeneration. However, due to the different solubilities of astaxanthin and nano-hydroxyapatite, if the positions of the inner and outer layers are reversed, the effect of simultaneous sustained release cannot be achieved.

[0023] In some embodiments, the molecular weight of PLGA is 30-100 kDa, wherein the mass ratio of lactic acid to glycolic acid in PLGA is 70-75:25-30; and the molecular weight of PLA is 100-500 kDa.

[0024] In this embodiment, the collagen degradation rate is adjusted by regulating the molecular weight of PLGA (polylactic-co-glycolic acid) / PLA or the ratio of lactic acid to glycolic acid in PLGA, making the degradation time controllable within 2 months to 2 years and maintaining a long-term filling effect. At this ratio, the higher proportion of lactic acid units results in a copolymer with higher hydrophobicity and a slower hydrolysis rate, which is beneficial for extending the sustained-release period of the core. Simultaneously, this molecular weight range ensures good processing performance and mechanical strength, preventing premature rupture of microspheres in vivo and subsequent burst release of active ingredients. For PLA, a high molecular weight of 100-500 kDa is used, further enhancing its structural stability and significantly delaying the degradation of astaxanthin and the inner collagen protective layer, achieving a collagen filling effect lasting for more than 2 months, meeting the clinical need for long-lasting filling.

[0025] In some embodiments, the mass ratio of astaxanthin to PLGA or PLA is 1:5-10.

[0026] In this embodiment, within a defined range, a dense and stable polymer coating layer can be formed while ensuring sufficient astaxanthin loading. An excessively high astaxanthin ratio can lead to phase separation or microsphere structural defects, causing burst release; while an excessively low ratio will result in minimal effect on promoting endogenous collagen production.

[0027] In some embodiments, the particle size of nano-hydroxyapatite is 100-200 nm; the particle size of the core is ≤20 µm; and the particle size of the bilayer microspheres is 30-100 µm.

[0028] In this embodiment, controlling the particle size of nano-hydroxyapatite facilitates its uniform loading within the collagen network and enhances its interaction with the extracellular matrix; a core particle size ≤200 µm ensures that it can be effectively encapsulated within the outer collagen system, preventing structural instability during preparation; the bilayer microsphere particle size is controlled at 30-100 µm, meeting the safe particle size standard for subcutaneous or dermal injection, avoiding the risk of embolism, while also facilitating uniform distribution in the interstitial spaces, achieving a smooth and natural filling effect of collagen.

[0029] This application provides a method for preparing bilayer microspheres, comprising the following steps: S1. Add astaxanthin and organic solutions of PLGA or PLA to an aqueous solution of polyvinyl alcohol for coating treatment to obtain the kernel; S2. Nano-hydroxyapatite, vitamin C, and the core are added to an acetic acid solution of collagen to obtain a mixture; the mixture is emulsified with liquid paraffin, and then glutaraldehyde is added to carry out a cross-linking reaction to obtain bilayer microspheres.

[0030] Specifically, in step S1, astaxanthin, PLGA (polylactic acid-glycolic acid copolymer), or PLA (polylactic acid) are dissolved in dichloromethane to obtain an organic solution of astaxanthin and PLGA or PLA. The organic solution is slowly added to a polyvinyl alcohol aqueous solution while stirring. After the addition is complete, stirring is continued for 6 hours to evaporate the organic solvent. The lower layer precipitate is collected by centrifugation, washed three times with purified water, and filtered through a microporous membrane. Microspheres with a particle size of less than 20 micrometers are taken and freeze-dried to obtain the core (monolayer microspheres). In step S2, collagen is dissolved in an acetic acid solution, and nano-hydroxyapatite, vitamin C, and monolayer microspheres are added and mixed evenly to obtain a mixture. The mixture is slowly added to liquid paraffin containing Span 80, and after stirring and emulsifying for 2 hours, glutaraldehyde is slowly added for crosslinking for 1 hour. The lower layer precipitate is collected by centrifugation, washed three times each with petroleum ether, isopropanol, and purified water, and filtered through a microporous membrane. Microspheres with a particle size of 30-100 micrometers are taken and freeze-dried to obtain bilayer microspheres.

[0031] In some embodiments, the concentration of the polyvinyl alcohol aqueous solution is 0.5-2% (V / V), the ratio of the organic solution of astaxanthin and PLGA or PLA to the polyvinyl alcohol aqueous solution is 1:3-1:10 (V / V), the concentration of the acetic acid solution is 2-4% (V / V), the amount of monolayer microspheres added is 10 mg / mL, and the ratio of Span 80 to liquid paraffin is 3:100 (V / V).

[0032] In some embodiments, the concentration of the organic solution of PLGA or PLA is 20-100 mg / ml.

[0033] In this embodiment, the concentration of the organic solution of PLGA or PLA is limited. Too high a concentration will result in a large solution viscosity, making it difficult to disperse, resulting in excessively large particle size, poor uniformity and sphericity, while too low a concentration will result in the inability to form spheres.

[0034] In some embodiments, the volume ratio of the mixture to liquid paraffin is 1:5-15.

[0035] In this embodiment, the formation of microspheres through emulsification and crosslinking requires a suitable oil-water ratio. If the oil-water ratio is too high, the emulsion droplets will easily come into contact with each other and polymerize into large droplets, resulting in excessively large microsphere particle size. When the oil-water ratio reaches a certain level, the emulsion droplets can be evenly dispersed, and the effect on particle size is no longer significant. However, excessive use of the oil phase can easily lead to waste.

[0036] In some embodiments, the concentration of the acetic acid solution of collagen is 5-15 mg / ml.

[0037] In this embodiment, if the collagen concentration is too high, it will result in high viscosity, making it difficult to prepare bilayer microspheres.

[0038] In some embodiments, the mass ratio of nano-hydroxyapatite to vitamin C is 2:0.1-0.5; In this embodiment, controlling the amount of nano-hydroxyapatite and astaxanthin is beneficial to further enhance the effect of promoting endogenous collagen production.

[0039] This application provides the use of bilayer microspheres in the preparation of medical biomaterials for skin tissue filling.

[0040] The following specific embodiments further illustrate this solution.

[0041] Example 1 A bilayer microsphere comprises a core and an outer coating layer covering the core; the core comprises astaxanthin and PLGA coated with astaxanthin, and the outer layer comprises collagen and a mixture of collagen-loaded nano-hydroxyapatite and vitamin C; the PLGA has a molecular weight of 100 kDa, and the mass ratio of lactic acid to glycolic acid in the PLGA is 75:25; the mass ratio of astaxanthin to PLGA is 1:10; the nano-hydroxyapatite has a particle size of 100 nm; the core has a particle size ≤20 µm; and the bilayer microsphere has a particle size of 30-100 µm.

[0042] The method for preparing bilayer microspheres includes the following steps: S1.0.2g of polylactic acid-glycolic acid copolymer and 0.02g of astaxanthin were dissolved in 10mL of dichloromethane to obtain an organic solution of astaxanthin and PLGA. Then, it was added to 100mL of 2% polyvinyl alcohol aqueous solution while stirring. After the addition was completed, the mixture was stirred for 6 hours to evaporate the organic solvent. The precipitate was collected by centrifugation, washed three times with purified water, and filtered through a microporous membrane. Microspheres with a particle size of less than 20 micrometers were freeze-dried to obtain monolayer microspheres, which are the core. S2. Dissolve 0.05g of collagen in 10mL of 2% acetic acid solution, add 0.2g of nano-hydroxyapatite, 0.01g of vitamin C, and 0.1g of monolayer microspheres and mix well to obtain a mixture. Slowly add the mixture to 150mL of liquid paraffin containing Span 80, stir and emulsify for 2 hours, then slowly add glutaraldehyde for crosslinking for 1 hour. Centrifuge and take the lower precipitate, wash three times each with petroleum ether, isopropanol, and purified water, filter through a microporous membrane, take microspheres with a particle size of 30-100 micrometers, freeze-dry to obtain bilayer microspheres.

[0043] Example 2 A bilayer microsphere comprises a core and an outer coating layer covering the core; the core comprises astaxanthin and PLGA coated with astaxanthin, and the outer layer comprises collagen and a mixture of collagen-loaded nano-hydroxyapatite and vitamin C; the PLGA has a molecular weight of 30-100 kDa, and the mass ratio of lactic acid to glycolic acid in the PLGA is 75:25; the mass ratio of astaxanthin to PLGA is 0.9:6; the nano-hydroxyapatite has a particle size of 100 nm; the core has a particle size ≤20 µm; and the bilayer microsphere has a particle size of 30-100 µm.

[0044] The method for preparing bilayer microspheres includes the following steps: S1.0.6g of polylactic acid-glycolic acid copolymer and 0.09g of astaxanthin were dissolved in 10mL of dichloromethane to obtain an organic solution of astaxanthin and PLGA. Then, it was added to 50mL of 1% polyvinyl alcohol aqueous solution while stirring. After the addition was completed, the mixture was stirred for 6 hours to evaporate the organic solvent. The precipitate was collected by centrifugation, washed three times with purified water, and filtered through a microporous membrane. Microspheres with a particle size of less than 20 micrometers were freeze-dried to obtain monolayer microspheres, which are the core. S2. Dissolve 0.1g of collagen in 10mL of 3% acetic acid solution, add 0.2g of nano-hydroxyapatite, 0.01g of vitamin C, and 0.1g of monolayer microspheres and mix well to obtain a mixture. Slowly add the mixture to 100mL of liquid paraffin containing Span 80, stir and emulsify for 2 hours, then slowly add glutaraldehyde for crosslinking for 1 hour. Centrifuge and take the lower precipitate, wash three times each with petroleum ether, isopropanol, and purified water, filter through a microporous membrane, take microspheres with a particle size of 30-100 micrometers, freeze-dry to obtain bilayer microspheres.

[0045] Example 3 A bilayer microsphere comprises a core and an outer coating layer covering the core; the core comprises astaxanthin and PLGA coated with astaxanthin, and the outer layer comprises collagen and a mixture of collagen-loaded nano-hydroxyapatite and vitamin C; the PLGA has a molecular weight of 30-100 kDa, and the mass ratio of lactic acid to glycolic acid in the PLGA is 75:25; the mass ratio of astaxanthin to PLGA is 1:5; the nano-hydroxyapatite has a particle size of 100 nm; the core has a particle size ≤20 µm; and the bilayer microsphere has a particle size of 30-100 µm.

[0046] The method for preparing bilayer microspheres includes the following steps: S1.1g of polylactic acid-glycolic acid copolymer and 0.2g of astaxanthin were dissolved in 10mL of dichloromethane to obtain an organic solution of astaxanthin and PLGA. Then, it was added to 30mL of 0.5% polyvinyl alcohol aqueous solution while stirring. After the addition was completed, the mixture was stirred for 6 hours to evaporate the organic solvent. The lower layer precipitate was collected by centrifugation, washed three times with purified water, and filtered through a microporous membrane. Microspheres with a particle size of less than 20 micrometers were freeze-dried to obtain monolayer microspheres, which are the core. S2. Dissolve 0.15g of collagen in 10mL of 4% acetic acid solution, add 0.2g of nano-hydroxyapatite, 0.01g of vitamin C, and 0.1g of monolayer microspheres and mix well to obtain a mixture. Slowly add the mixture to 50mL of liquid paraffin containing Span 80, stir and emulsify for 2 hours, then slowly add glutaraldehyde for crosslinking for 1 hour. Centrifuge and take the lower precipitate, wash three times each with petroleum ether, isopropanol, and purified water, filter through a microporous membrane, take microspheres with a particle size of 30-100 micrometers, freeze-dry to obtain bilayer microspheres.

[0047] Example 4 A bilayer microsphere, otherwise identical to Example 1, except that PLGA is replaced with PLA of equal molecular weight.

[0048] Comparative Example 1 A bilayer microsphere, otherwise identical to Example 1, except that the mass ratio of lactic acid to glycolic acid in PLGA is 50:50.

[0049] Comparative Example 2 A bilayer microsphere, otherwise identical to Example 1, except that it does not include PLGA.

[0050] Comparative Example 3 A bilayer microsphere, otherwise identical to Example 1, except that it does not include nano-hydroxyapatite.

[0051] Testing and Evaluation Different examples and comparative samples (1g each) were dissolved in 100 mL of PBS buffer solution, dispersed evenly, and then placed in a constant temperature (37℃) shaker with uniform shaking (60 rpm). The samples were removed at specific time points, centrifuged to collect the precipitate, and freeze-dried. The degradation rate of the bilayer microspheres was calculated based on the remaining mass ratio after degradation, and the graph is shown below. Figure 1 As shown.

[0052] The in vitro release rate of astaxanthin was determined by high-performance liquid chromatography (HPLC). 1 g samples from different examples and comparative examples were dissolved in 100 mL of PBS buffer solution, dispersed evenly, and then placed in a constant temperature (37°C) shaker with uniform shaking at 60 rpm. At specific time points, 1 mL of the release solution was collected, filtered, and 10 μL was injected into the HPLC system. The astaxanthin content was calculated based on peak area using the external standard method, and the resulting graph is shown below. Figure 2 As shown.

[0053] Dissolve 1 g of each of the samples from different examples and comparative examples in 100 mL of PBS buffer solution. After dispersing evenly, place the samples in a constant temperature (37°C) shaker and oscillate at a constant speed (60 rpm). Remove the samples at specific time points, centrifuge to collect the precipitate, ignite at 600°C for 30 minutes, weigh the residue after ignition, calculate the remaining mass ratio of nano-hydroxyapatite, and plot the graph as shown below. Figure 3 As shown.

[0054] The results show that this application can instantly fill collagen and continuously release astaxanthin and hydroxyapatite. The combined effect of the two can continuously stimulate the production of endogenous collagen to achieve long-term effects.

[0055] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A double-layered microsphere, characterized in that, It includes a core and an outer coating covering the core; the core includes astaxanthin and PLGA or PLA coating the astaxanthin, and the outer coating includes collagen and a mixture of collagen-loaded nano-hydroxyapatite and vitamin C.

2. The bilayer microspheres according to claim 1, characterized in that, The PLGA has a molecular weight of 30-100 kDa, wherein the mass ratio of lactic acid to glycolic acid in the PLGA is 70-75:25-30; the PLA has a molecular weight of 100-500 kDa.

3. The bilayer microspheres according to claim 1, characterized in that, The mass ratio of astaxanthin to PLGA or PLA is 1:5-10.

4. The bilayer microspheres according to claim 1, characterized in that, The nano-hydroxyapatite has a particle size of 100-200 nm; the core has a particle size of ≤20 µm; and the bilayer microspheres have a particle size of 30-100 µm.

5. A method for preparing bilayer microspheres as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Add astaxanthin and organic solutions of PLGA or PLA to an aqueous solution of polyvinyl alcohol for coating treatment to obtain the core. S2. Nano-hydroxyapatite, vitamin C, and the core are added to an acetic acid solution of collagen to obtain a mixture; the mixture is emulsified with liquid paraffin, and then glutaraldehyde is added to carry out a cross-linking reaction to obtain the bilayer microspheres.

6. The preparation method according to claim 4, characterized in that, The concentration of the organic solution of PLGA or PLA is 20-100 mg / ml.

7. The preparation method according to claim 4, characterized in that, The volume ratio of the mixture to liquid paraffin is 1:5-15.

8. The preparation method according to claim 4, characterized in that, The concentration of the acetic acid solution containing collagen is 5-15 mg / ml.

9. The preparation method according to claim 4, characterized in that, The mass ratio of nano-hydroxyapatite to vitamin C is 2:0.1-0.

5.

10. The use of the bilayer microspheres as described in any one of claims 1-4 in the preparation of injectable skin tissue filling biomaterials.

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