Core-shell structure microsphere capable of stimulating collagen secretion, preparation method and use thereof

Abstract

The present invention belongs to the technical field of tissue filling materials, and specifically relates to a core-shell structure microsphere that can stimulate collagen secretion, a preparation method and a use thereof. The core microsphere of the core-shell structure microsphere adopts aliphatic polyester material as the capsule material, and the shell layer is prepared by a material mainly composed of sodium alginate sulfate-sodium alginate solution, and a porogen and / or analgesic can be added. The core microsphere of the core-shell structure microsphere is prepared by a solvent volatilization method, and the core-shell structure microsphere is prepared by a coaxial electrostatic spraying process. Studies have shown that the core-shell structure microsphere of the present invention has a round and smooth appearance, a relatively uniform shell thickness, and can still protect good dispersibility and appearance after freeze-drying and re-dissolution; in vitro and animal experiments show that the microsphere maintains a slower degradation rate and in vitro drug release rate, and can maintain the stimulating effect on collagen synthesis for a long time, and can be used for filling skin depressions or wrinkles.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue filling materials, and in particular relates to a core-shell structure microsphere capable of stimulating collagen secretion, a preparation method and a use thereof. Background Art

[0002] In recent years, as people's demand for anti-aging and beauty continues to increase, the market for injectable facial fillers made of various synthetic or natural polymers has grown exponentially. Most of the regenerative soft tissue fillers on the market are aliphatic polyester microspheres; the existing technology usually suspends the microspheres in a basic solution such as sodium carboxymethyl cellulose and sodium hyaluronate for injection. The filler has a dual filling effect: the basic solution has an immediate effect, and the microspheres have a lasting effect.

[0003] Polylactic acid microspheres can promote collagen secretion by means of the inflammatory foreign body reaction caused by the microspheres themselves. Therefore, regenerative microsphere fillers with polylactic acid as the main substrate are more common, and the specific ingredients include PLA (polylactic acid), PLLA (levorotatory polylactic acid), PDLA (dextrorotatory polylactic acid), PDLLA (racemic polylactic acid), PLGA (polylactic acid-glycolic acid copolymer), etc. However, polylactic acid microspheres generally have the following problems: 1) poor hydrophilicity, easy to agglomerate, and agglomerates are very easy to clog the needle and cause injection difficulties; 2) microsphere agglomerates cannot be accurately quantified, and after injection, excessive inflammatory stimulation due to high local concentration causes adverse reactions such as nodules and hematomas; 3) polylactic acid microspheres will accelerate degradation in the middle and late stages of the degradation process, shortening the effective action time, and causing some acidic degradation products to "suddenly increase", local lactic acid accumulation at the implantation site, causing discomfort such as soreness, and aggravating adverse reactions. Therefore, there is still a need to strike a balance between the demand for long-lasting filling materials and the comfort and potential adverse effects of the fillers. It is urgent to solve the problems of poor dispersibility and degradation regulation of existing polylactic acid microsphere suspensions, so as to retain the regeneration effect while reducing the occurrence of adverse reactions.

[0004] Patent document CN116173294A discloses a core-shell microsphere composed of a sodium alginate shell and a polylactic acid core. The hydrophilic outer layer formed by sodium alginate effectively improves the dispersibility of polylactic acid microspheres in aqueous solution, reducing the side effects such as inflammation and nodules caused or aggravated thereby. The hydrophilic shell formed by the sodium alginate layer also makes the aliphatic polyester microspheres easy to inject, without the need for re-dissolution to simplify the injection process. However, the patented microspheres have the following problems: 1) Sodium alginate is generally cross-linked with calcium and the like, and its structural stability is highly dependent on calcium ions. Therefore, it needs to be stored in inorganic salt solutions such as calcium chloride, and cannot be freeze-dried for storage. It needs to be repeatedly cleaned before injection, which is cumbersome and increases the risk of bacterial contamination; 2) Excessive introduction of calcium ions leads to adverse reactions such as muscle spasms in the skin; 3) Sodium alginate has high viscosity, and the thickness of the shell is difficult to accurately control, which is not conducive to standardized mass production; 4) There is no drug-carrying group, and drug sustained release cannot be achieved; The fine structural optimization of the hydrophilic shell layer and the performance matching of the core layer are currently one of the important means to improve the effectiveness and safety of medical beauty injection filling microspheres. Summary of the invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide a core-shell structure microsphere that can stimulate collagen secretion, a preparation method and use thereof, and specifically to provide a core-shell structure microsphere that can be used for facial soft tissue filling. Such microspheres are usually used as Class 3 medical devices to eliminate facial wrinkles.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A core-shell structure microsphere capable of stimulating collagen secretion, comprising an inner core microsphere made of aliphatic polyester material as a capsule material and an alginate shell layer covering the inner core microsphere; the alginate is a sodium alginate sulfate-alginic acid mixture; the weight ratio of the inner core microsphere to the alginate is (0.01-1): (0.05-1).

[0008] Preferably, the weight ratio of sodium alginate sulfate to sodium alginate in the alginate shell layer is (2-8):(1-5).

[0009] Preferably, the aliphatic polyester material is selected from at least one of PLA (polylactic acid), PLLA (levorotatory polylactic acid), PDLA (dextrorotatory polylactic acid), PDLLA (racemic polylactic acid), and PLGA (polylactic acid-glycolic acid copolymer).

[0010] Preferably, the alginate shell layer also contains an analgesic. Further preferably, the analgesic is selected from at least one of procaine, lidocaine and tetracaine.

[0011] Preferably, the core-shell structured microspheres are porous microspheres, and the porogen used in the porous microspheres is at least one selected from sodium bicarbonate, hydrogen peroxide, and liquid paraffin.

[0012] The method for preparing the core-shell microspheres capable of stimulating collagen secretion comprises the following steps: preparing inner core microspheres, preparing shell layer solution, and preparing core-shell microspheres, wherein the core-shell microspheres are prepared by coaxial electrostatic spraying process.

[0013] Preferably, the preparation of the core microspheres is prepared by a solvent evaporation method. Further preferably, in the solvent evaporation method, the aliphatic polyester material is used as the oil phase, and the solvent used in the oil phase is dichloromethane; the PVA (polyvinyl alcohol) aqueous solution is used as the water phase. Further preferably, the concentration of the aliphatic polyester material in the oil phase is 20g / L~50g / L; the concentration of PVA in the water phase is 20g / L~50g / L.

[0014] Preferably, the concentration of sodium alginate sulfate in the shell solution is 10 g / L to 50 g / L.

[0015] Preferably, in the coaxial electrostatic spraying process, the needle specification is 22G~30G, the voltage is 8kV~35kV, the syringe push speed is 2 ml / min~15ml / min, and the receiving distance is 5cm~20cm.

[0016] Preferably, in the coaxial electrostatic spraying process, the solute of the receiving liquid is selected from at least one of CaCl2, FeCl2, ZnCl2, BaCl2, and CuCl2.

[0017] The use of the aforementioned core-shell structured microspheres capable of stimulating collagen secretion in the preparation of skin depression fillers.

[0018] The core-shell structure microspheres that can stimulate collagen secretion are used in the preparation of preparations for filling facial wrinkles.

[0019] Beneficial effects:

[0020] The present invention provides a core-shell structure microsphere with aliphatic polyester microsphere as the core layer and sodium alginate sulfate-sodium alginate as the shell layer. The advantages are:

[0021] (1) The shell layer of the core-shell microspheres of the present invention introduces sodium alginate sulfate, the shell layer thickness is relatively uniform, and the dispersibility is still good after freeze-drying and re-dissolving, and the smooth appearance can still be maintained, which is conducive to mass production and ensures stable product quality. In addition, the core-shell microspheres of the present invention can be loaded with analgesics, thereby reducing the discomfort caused by the accumulation of lactic acid during the degradation process of lactic acid and the discomfort caused by the injection of facial filling microspheres.

[0022] (2) After sodium alginate sulfate is introduced into the shell layer of the core-shell structure microspheres of the present invention, the prepared microspheres have significantly improved collagen secretion stimulation ability (for example, stimulating the synthesis of type I and type III collagen associated with facial wrinkles) compared with microspheres without sodium alginate sulfate. While being conducive to drug loading, the core-shell structure microspheres effectively achieve controllable shell thickness, freeze-drying and re-dissolution stability, etc.; and the degradation rate is slower, thereby achieving a longer filling effect and wrinkle removal effect.

[0023] (3) The core-shell microspheres of the present invention can form pores in the shell layer using porogens. The uniform pores enable the aliphatic polyester spheres to be slowly released and degraded. In addition, the presence of the sodium alginate sulfate-sodium alginate shell layer forms a mild and lasting inflammatory response, which is beneficial for prolonging the duration of action and reducing the risk of pain, nodules and granulomas caused by local lactic acid accumulation at the implantation site. In addition, the dispersibility and uniformity of the hydrophilic core-shell microspheres in the aqueous solution are retained, reducing side effects such as nodules; making the microspheres easy to inject and improving the smoothness of the injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a light microscope photo of the SAS-PLLA microsphere sample of the present invention;

[0025] Figure 2 This is a light microscopic photograph of a freeze-dried sample of SAS-PLLA microspheres of the present invention;

[0026] Figure 3 This is a light microscopic photograph of the freeze-dried sample of SAS-PLLA microspheres of the present invention reconstituted with water;

[0027] Figure 4 Degradation curves of different microspheres;

[0028] Figure 5 This is the procaine release curve of the SAS-PLLA microspheres of the present invention;

[0029] Figure 6 The figure is a quantitative graph of the secretion of type I collagen (COL1A1) after co-culture of different microspheres with human fibroblasts CCD-986sk;

[0030] Figure 7 The figure is a quantitative graph of the secretion of type III collagen (COL3A1) after co-culture of different microspheres with human fibroblasts CCD-986sk;

[0031] Figure 8 The figure is a quantitative graph of the secretion of type I collagen (COL1A1) after different microspheres were filled into the back of New Zealand rabbits for 90 days and 180 days;

[0032] Fig. 9The quantitative graphs of type III collagen (COL3AIII) secretion after different microspheres were filled into the back of New Zealand rabbits for 90 days and 180 days;

[0033] Fig.10 HE staining of the back skin of mice filled with different microspheres 14 days after injection;

[0034] Fig.11 The MASSON staining image of the back skin filled with different microspheres 14 days after the mice were filled with different microspheres;

[0035] Fig.12 This is a picrosirius red staining image of the skin on the back of mice 14 days after the back was filled with different microspheres. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the specific embodiments are only used to explain the present invention and are not used to limit the scope of protection of the present invention.

[0037] In the following examples, sodium alginate (M / G=1:2) was purchased from MacLean's reagent (batch number S875337), sodium alginate sulfate was provided by Qingdao Marine Biomedicine Research Institute Co., Ltd. (commercial product, batch number 2023001); PLLA (batch number P875095), PLA (batch number L885075), PDLA (batch number P905786), PDLLA (batch number P905796), and PVA (batch number 767383) were purchased from MacLean's reagent. The weight average molecular weight of sodium alginate sulfate is preferably 8 kDa to 200 kDa.

[0038] Example 1 Core-shell structure microspheres and preparation

[0039] Table 1 Core-shell structure microspheres and preparation process 1

[0040]

[0041] Table 2 Core-shell structure and preparation process 2

[0042]

[0043] The preparation method of core-shell structure microspheres includes the following steps: preparation of core microspheres (aliphatic polyester microspheres, such as PLLA microspheres), preparation of sodium alginate sulfate-sodium alginate solution (shell solution, hereinafter referred to as SAS) and preparation of core-shell structure microspheres. The core microspheres are prepared by solvent evaporation method, and the core-shell structure microspheres are prepared by coaxial electrostatic spraying process.

[0044] Tables 1 and 2 show the raw materials for preparing several core-shell microspheres and the key process parameters of each preparation step. The term alginate used in the "coaxial electrostatic spraying process" in Tables 1 and 2 refers to sodium alginate and sodium alginate sulfate, rather than a single alginate.

[0045] Taking the core-shell microsphere 1 (SAS-PLLA core-shell structure microsphere) in Table 1 of this embodiment as an example, the detailed preparation method is as follows:

[0046] 1. Preparation of core microspheres: PLLA microspheres were prepared by solvent evaporation method: 60 mL of oil phase (solvent: dichloromethane, core microsphere capsule: PLLA, concentration: 30 g / L) was dispersed in 900 mL of water phase (PVA aqueous solution, concentration: 20 g / L), stirred and mixed (stirred at 800 r / min for 8 h) and then centrifuged (centrifuged at 4000 rpm for 10 min) to obtain PLLA microspheres, which were washed 3 times with deionized water and dried for later use.

[0047] 2. Preparation of SAS: Sodium alginate and sodium alginate sulfate were weighed, dispersed in 100 mL of normal saline, heated at 70°C with stirring until dissolved to obtain a sodium alginate sulfate-sodium alginate solution, cooled to room temperature, and then sodium carbonate and procaine were added to form SAS.

[0048] 3. Preparation of SAS-PLLA core-shell microspheres: Core-shell microspheres were prepared by coaxial electrostatic spraying. 1.25 g of PLLA microspheres were suspended in 20 mL of saline to form a core layer liquid. 5 mL of core layer liquid and CS solution were drawn with a syringe, and a 22G needle was used. The syringe push speed was 15 mL / min and the voltage was 8 kV. 100 mL of 80 g / L (8%) calcium chloride solution was used as the receiving liquid, and the receiving distance was 8 cm. After cross-linking for 1 hour, the mixture was filtered with a filter to separate the core-shell microspheres, and the microspheres were washed with 0.9% NaCl solution, centrifuged at 4000 rpm for 10 min to obtain the washed core-shell microspheres, and then the core-shell microspheres were washed again with deionized water, and centrifuged again at 4000 rpm for 10 min to obtain the washed core-shell microspheres. 0.1 g of the washed core-shell microspheres were dispersed in 10 mL of 2% carboxymethyl cellulose solution, and packaged and sterilized with ultraviolet light before injection. The microspheres can also be freeze-dried and used as microsphere preparations for injection, and can be reconstituted with physiological saline before use.

[0049] Figure 1 This is a light microscope photo of the SAS-PLLA microsphere sample; Figure 2 This is a light microscopic photograph of the freeze-dried sample of the SAS-PLLA microspheres; Figure 3This is a light microscopic photograph of the freeze-dried sample of the SAS-PLLA microspheres reconstituted with water. As shown in the figure, the SAS-PLLA microsphere sample and the freeze-dried sample after water reconstitution maintain a round and smooth appearance (spherical or basically spherical), and no irregular microspheres such as flakes appear, which is beneficial to reduce tissue scars or nodules caused by irregular microsphere shapes. That is, the core-shell structure microspheres prepared by this method are resistant to freeze drying and freeze-drying reconstitution, and can still maintain good dispersibility (not easy to stick and agglomerate) and round and smooth spheres (not deformed due to freeze drying, etc.) after freeze drying and reconstitution. The shell thickness of the core-shell structure microspheres after freeze drying and reconstitution is relatively uniform, which is beneficial to reduce the degradation rate difference caused by uneven shell thickness. The particle size of the prepared SAS-PLLA core-shell structure microspheres is 40~70μm, of which the shell thickness is 10~20μm, and the microsphere particle size is within the appropriate range for facial filling injection microspheres.

[0050] The core-shell structure microspheres prepared in this embodiment are drug-loaded microspheres (such as procaine) and pore-forming agents (such as sodium bicarbonate) are added. Obviously, in the specific implementation, no drug or pore-forming agent can be added according to the needs. Obviously, in the specific implementation, the preparation solution of SAS can also use water as a solvent.

[0051] Example 2 In vitro degradation test of SAS-PLLA microspheres

[0052] PLLA microspheres were prepared according to step 1 (preparation of core microspheres) in Example 1; SAS-PLLA core-shell microspheres were prepared according to steps 1 to 3 in Example 1; sodium alginate-PLLA core-shell microspheres (G-PLLA core-shell microspheres) were prepared according to steps 1 to 3 in Example 1, where the difference from Example 1 was that sodium alginate sulfate was not added in step 2, and the mass ratio of fatty polyester microspheres to alginate (sodium alginate, without sodium alginate sulfate) was 0.25:0.05. In vitro degradation tests were performed on PLLA microspheres, SAS-PLLA core-shell microspheres, and G-PLLA core-shell microspheres:

[0053] Take 20mg microsphere sample and suspend it in 5ml PBS solution containing 1% elastase, and place it in an incubator at 37°C for degradation. According to the set time interval, three batches of sample solution are selected from each group of microspheres at each sampling time point, and centrifuged at 10000rpm for 5min to separate the undegraded microspheres, and collect the supernatant for lactic acid determination. The lactic acid content in the degradation solution is determined by colorimetry. Take 100µL of the sample supernatant, mix it with 1 mL of colorimetric reagent, incubate it at 37°C for 10-15min, and measure the absorbance at a wavelength of 540 nm using a spectrophotometer.

[0054]

[0055]

[0056]

[0057]

[0058] In the formula, ΔA1 is the measured OD value minus the blank OD value; ΔA2 is the standard OD value minus the blank OD value; c is the standard concentration (3 mmol / L); f is the dilution factor before the sample is added to the detection system; W0 is the initial weight of the PLLA microspheres; the molar mass of the lactic acid unit is 90 g / mol; and the molar mass of PLLA is 90 g / mol. After each sampling, the lactic acid concentration in the degradation solution is measured and multiplied by the volume of the degradation solution to obtain the amount of lactic acid at that moment (mg); based on the initial mass of the PLLA microspheres (W0) and the molar mass of lactic acid, the total amount of lactic acid that can be generated theoretically when completely degraded is calculated. The degradation rate can be defined as the percentage of the sample lactic acid amount to the total lactic acid amount. Figure 4 is the degradation curve of different microspheres. Figure 4 It can be seen that among the three types of microspheres, the degradation rate of the SAS-PLLA core-shell structure microspheres is the slowest. At the 200th day, the undegraded microspheres (remaining rate) are close to 30%, indicating that the SAS-PLLA core-shell structure microspheres of the present invention can maintain a slower degradation rate and thus exert a longer-term facial soft tissue filling effect, thereby helping to prolong the duration of the anti-wrinkle effect when injected under the dermis.

[0059] Example 3 In vitro procaine release characteristics of SAS-PLLA microspheres

[0060] The in vitro drug release characteristics of the SAS-PLLA microspheres prepared in Example 1 were investigated. 20 mg of microspheres were suspended in 5 ml of PBS solution containing 1% elastase and placed in an incubator at 37°C for degradation. Three batches of sample liquid were selected from each group of microspheres at each sampling time point at a set time interval. The samples were centrifuged at 10,000 rpm for 5 min to separate the undegraded microspheres. The supernatant was collected for procaine determination. References for procaine detection method: Huang Jingjuan. Determination of procaine hydrochloride injection by ultraviolet spectrophotometry [J]. Modern Medicine and Health, 2007, 23(5):1.

[0061] Figure 5 This is the procaine release curve of the SAS-PLLA microspheres in Example 1 of the present invention. The procaine release of the SAS-PLLA microspheres can be maintained for no less than 8 days. It can be seen that although the procaine in the core-shell structure microspheres of the present invention is located in the shell layer, it still has a good drug sustained release effect. It is beneficial to alleviate the local pain problem after the injection of facial filling microspheres.

[0062] Example 4 Effect of microspheres on collagen in human fibroblasts

[0063] Human fibroblasts (CCD-986sk purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were maintained in 2 ml of DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and cultured at 37°C in an incubator containing 5% CO2 for 48 hours. When the cell confluence reached 70%, 200 μg / mL SAS-PLLA, PLLA, and G-PLLA were added for 48 hours, respectively, and PBS was used as a blank control. After treatment, cells and supernatants were collected and centrifuged to remove floating cells. Supernatants and fibroblast lysates were collected for protein analysis. The levels of type I collagen (COL1A1) and type III collagen (COL3A1) associated with facial wrinkles were evaluated by ELISA kits (ELISA detection kits were purchased from Wuhan Finn Biotechnology Co., Ltd.). The detection method was referenced to: Oh S, Lee JH, Kim HM, Batsukh S, SungMJ, Lim TH, Lee MH, Son KH, Byun K. Poly-L-Lactic Acid Fillers Improved Dermal Collagen Synthesis by Modulating M2 Macrophage Polarization in Aged Animal Skin. Cells. 2023 May 5;12(9):1320. The results are as follows Figure 6 , 7 shown.

[0064] The levels of COL1A1 and COL3A1 in fibroblasts treated with SAS-PLLA were significantly higher than those in fibroblasts treated with PBS, indicating that SAS-PLLA can effectively stimulate the regeneration of the two collagens. In addition, the levels of COL1A1 and COL3A1 in fibroblasts treated with SAS-PLLA were also significantly higher than those in the PLLA and G-PLLA groups, suggesting that SAS-PLLA has a better facial filling or anti-wrinkle effect.

[0065] Example 5 Investigation of the in vivo effects of microspheres (rabbit experiment)

[0066] New Zealand rabbits (weight about 2kg, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., bred in the Animal Laboratory of Qingdao Marine Biomedical Research Institute, animal experiments were approved by the Animal Experiment Ethics Committee of Qingdao Marine Biomedical Research Institute.) were depilated on their backs and divided into four groups: saline, PLLA, SAS-PLLA, and G-PLLA, with 6 rabbits in each group, half male and half female. 10 mg / mL (400 μL) of different microspheres were injected into the dermis of six different areas (5 cm × 5 cm) of the back hair removal site. The injection site was depilated again on the 90th and 180th day after injection, and the skin tissue was collected under isoflurane anesthesia. The content of type I collagen (COL1A1) and type III collagen (COL3A1) in the skin tissue was determined by ELISA kit. Reference: Oh, S.; Seo, SB; Kim, G.; Batsukh, S.; Park, C.-H.; Son, KH; Byun, K. Poly-D,L-Lactic Acid Filler Increases Extracellular Matrix by Modulating Macrophages and Adipose-Derived Stem Cells in AgedAnimal Skin. Antioxidants 2023, 12 , 1204.

[0067] like Figure 8 and 9 As shown, at 90 days, the expression levels of the two proteins in the skin of the PLLA group, SAS-PLLA group and G-PLLA group were significantly higher than those of the saline group, among which the SAS-PLLA group was significantly higher than the PLLA group and the G-PLLA group; at 180 days, the results were similar, but the expression levels of the two proteins in the SAS-PLLA group and the G-PLLA group were higher than those in the PLLA group, and the expression levels of the two proteins in the SAS-PLLA group were higher than those in the other groups. The above results show that the addition of sodium alginate sulfate and the porous structure of the SAS-PLLA group can enable the slow and lasting degradation of the PLLA core, thereby producing a mild and lasting inflammatory response, promoting the continuous secretion of collagen, and maintaining higher collagen secretion.

[0068] Example 6 Investigation of the in vivo effects of microspheres (mouse experiment)

[0069] Mice (6-week-old C57BL / 6 purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., and raised in the animal laboratory of Qingdao Marine Biomedical Research Institute. Animal experiments were approved by the Animal Experiment Ethics Committee of Qingdao Marine Biomedical Research Institute.) were depilated on the back and divided into four groups: saline, PLLA, SAS-PLLA, and G-PLLA, with 6 mice in each group, half male and half female. 10 mg / mL (200 μL) of different microspheres were injected into the dermis of six different areas (3 cm × 3 cm) of the back depilatory site. On the 14th day after injection, the injection site was depilated again, and the skin tissue was collected under isoflurane anesthesia for picrosirius red staining, MASSON staining, and HE staining. The results are shown in Figures 10 to 12 As shown,

[0070] HE staining results showed that obvious inflammatory response was detected in the PLLA group 14 days after treatment, with some nodules; both the SAS-PLLA group and the G-PLLA group had mild inflammatory response, but no nodules were produced. This indicates that the SAS-PLLA group can maintain inflammatory stimulation, which is beneficial to collagen synthesis, but has better safety and does not cause nodules.

[0071] Masson staining results showed that 14 days after microsphere injection, the collagen fibers in the SAS-PLLA and PLLA groups were neatly arranged and dense, while the collagen fibers in the saline group were messy and sparse. Among the three types of microspheres, the SAS-PLLA group showed a better effect in stimulating collagen regeneration.

[0072] By using picrosirius red staining, different types of collagen fibers can be distinguished based on their specific form and different colors under polarized light. Type I collagen fibers are densely packed and appear red and yellow, while type III collagen fibers appear as green thread-like structures.

[0073] In the optical photographs of tissue sections stained with picrosirius red, a large number of type I collagen fibers and type III collagen fibers were observed in the SAS-PLLA group, which were significantly more than those in the other groups; among the three microspheres, SAS-PLLA showed a more excellent ability to stimulate collagen regeneration slowly and persistently.

[0074] In the above experiments on rabbits and mice, no adverse reactions of SAS-PLLA on the blood system were observed. No toxic reactions of sodium alginate sulfate were found in MTT cytotoxicity test, skin sensitization test and acute toxicity test.

Claims

1. A core-shell microsphere capable of stimulating collagen secretion, characterized in that: The core-shell structure microspheres are composed of a core microsphere with aliphatic polyester material as the capsule material and an alginate shell layer covering the core microspheres; the alginate is a sodium alginate sulfate-sodium alginate mixture; the weight ratio of the core microspheres to the alginate is (0.01-1):(0.05-1); the weight ratio of sodium alginate sulfate to sodium alginate in the alginate shell layer is (2-8): (1~5); The preparation method of the core-shell structure microspheres comprises the following steps: preparation of inner core microspheres, preparation of shell solution, and preparation of core-shell structure microspheres, wherein the preparation of the core-shell structure microspheres adopts a coaxial electrostatic spraying process; the preparation of the inner core microspheres adopts a solvent evaporation method; in the solvent evaporation method, aliphatic polyester material is used as the oil phase, and the solvent used in the oil phase is dichloromethane; polyvinyl alcohol aqueous solution is used as the water phase; the concentration of the aliphatic polyester material in the oil phase is 20g / L~50g / L; the concentration of polyvinyl alcohol in the water phase is 20g / L~50g / L; the concentration of sodium alginate sulfate in the shell solution is 10g / L~50g / L; in the coaxial electrostatic spraying process, the needle specification is 22G~30G, the voltage is 8kV~35kV, the syringe push speed is 2ml / min~15ml / min, and the receiving distance is 5cm~20cm.

2. The core-shell structure microsphere capable of stimulating collagen secretion according to claim 1, characterized in that: The aliphatic polyester material is selected from at least one of L-polylactic acid, D-polylactic acid, racemic polylactic acid, and polylactic acid-glycolic acid copolymer.

3. The core-shell structure microsphere capable of stimulating collagen secretion according to claim 1, characterized in that: The alginate shell also contains an analgesic.

4. The core-shell structure microsphere capable of stimulating collagen secretion according to claim 3, characterized in that: The analgesic is selected from at least one of procaine, lidocaine and tetracaine.

5. The core-shell structure microsphere capable of stimulating collagen secretion according to claim 1, characterized in that: The core-shell structure microspheres are porous microspheres, and the porogen used in the porous microspheres is at least one selected from sodium bicarbonate, hydrogen peroxide, and liquid paraffin.

6. The core-shell structure microsphere capable of stimulating collagen secretion according to claim 1, characterized in that: In the coaxial electrostatic spraying process, the solute of the receiving liquid is selected from at least one of CaCl2, FeCl2, ZnCl2, BaCl2, and CuCl2.

7. Use of the core-shell structure microsphere capable of stimulating collagen secretion according to any one of claims 1 to 6 in the preparation of a skin depression filler.

8. Use of the core-shell structure microspheres capable of stimulating collagen secretion as claimed in any one of claims 1 to 6 in the preparation of a preparation for filling facial wrinkles.

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