Monodisperse microsphere capable of rapidly promoting collagen regeneration and provided with porous structure on surface and preparation method of monodisperse microsphere

Abstract

The invention provides a preparation method of a monodisperse microsphere with a porous structure on the surface for rapidly promoting collagen regeneration, which comprises the following steps: (1) dissolving or dispersing a pore-foaming agent and a polymer in an organic solvent to obtain an internal phase fluid; the preparation method comprises the following steps: dissolving a water-soluble surfactant in water, adding a proper amount of an organic solvent, and fully mixing to obtain an external phase fluid or a collection liquid; (2) inputting the internal-phase fluid and the external-phase fluid into a microfluid device to prepare a monodisperse oil-in-water emulsion, collecting the oil-in-water emulsion by adopting a collecting solution, fully standing, and stirring to completely volatilize an organic solvent in the oil-in-water emulsion to obtain monodisperse microspheres; and (3) washing to remove the collection liquid and the pore-foaming agent on the monodisperse microspheres. The surfaces of the monodisperse microspheres prepared by the method have uniformly distributed porous structures, and the pore diameters of the porous structures are uniform. The problem that in the prior art, it is difficult to controllably prepare microspheres with uniform porous structures on the surfaces and used in the field of medical cosmetology can be solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical cosmetic materials, and relates to a monodisperse microsphere having a porous structure on the surface that rapidly promotes collagen regeneration and a preparation method thereof. Background Art

[0002] As we age, the fibroblasts in our skin gradually lose their activity, and the amount of collagen synthesized gradually decreases, leading to the appearance of skin aging phenomena such as wrinkles. The traditional way to improve wrinkles is to inject hyaluronic acid into the wrinkles to fill them, but this method is difficult to maintain the filling effect for a long time. Since microspheres can stimulate the body's immune response and secrete fibroblasts to synthesize collagen, it is expected to achieve the lasting synthesis of collagen and achieve the effect of long-term improvement of wrinkles. Therefore, it has become a research hotspot in the field of medical cosmetology in recent years.

[0003] The microspheres used in the existing medical cosmetology field have smooth surfaces, basically no porous or wrinkled structures, and the monodispersity of the microspheres is poor, and the particle size of the microspheres is not uniform enough. On the one hand, the smooth surface of the microspheres is not conducive to the adsorption, anchoring and diffusion of macrophages on the microspheres, and it is difficult to effectively attract a large number of macrophages to quickly stimulate the immune response, so its ability and speed to stimulate the production of collagen need to be improved. On the other hand, the poor monodispersity of the microspheres will lead to the inability to accurately evaluate the effect of stimulating the immune response, which will in turn cause the product dosage to be unable to be accurately calculated, and it is also easy to affect the safety of the product and reduce the effectiveness of the product. When the microspheres are injected into the body, small-sized microspheres are prone to cause granulomas, while large-sized microspheres are prone to form hard nodules due to excessive human immune response. At the same time, small-sized microspheres are easily phagocytosed by macrophages in the body and cannot play a role in stimulating collagen production. Summary of the invention

[0004] In view of the fact that the microspheres currently used in the field of medical cosmetology do not have a porous structure on their surface, the monodispersity of the microspheres is poor, the ability and speed of stimulating the production of collagen need to be improved, and they are prone to cause granulomas and nodules and other safety issues in use, the present invention provides monodisperse microspheres with a porous structure on the surface that can quickly promote collagen regeneration and a preparation method thereof. The ability of the microspheres to stimulate collagen regeneration is increased by imparting a porous structure to the surface of the microspheres, and the safety of use of the microspheres is increased by increasing the monodispersity of the microspheres.

[0005] In order to achieve the above-mentioned invention object, the technical solution provided by the present invention is as follows:

[0006] A method for preparing monodisperse microspheres with a porous surface structure that rapidly promotes collagen regeneration comprises the following steps:

[0007] (1) Preparation of internal phase, external phase fluid and collection liquid

[0008] Prepare the internal phase fluid: dissolve or disperse the porogen and the polymer in an organic solvent to obtain the internal phase fluid; in the internal phase fluid, the concentration of the polymer is 0.005-0.10 g / mL, the concentration of the porogen is 0.001-0.10 g / mL, and the organic solvent is dichloromethane or chloroform;

[0009] The external phase fluid is prepared by dissolving a water-soluble surfactant in water to form a water-soluble surfactant solution, and then adding an organic solvent in an amount of 0.02 to 0.04 times the volume of the water-soluble surfactant, and mixing them thoroughly to obtain an external phase fluid; the concentration of the water-soluble surfactant in the external phase fluid is 0.001 to 10 g / mL; the organic solvent is dichloromethane or chloroform;

[0010] Preparation of a collection solution: dissolving a water-soluble surfactant in water to form a water-soluble surfactant solution, then adding an organic solvent in an amount of 0.02 to 0.04 times the volume of the water-soluble surfactant, and mixing thoroughly to obtain a collection solution; in the collection solution, the concentration of the water-soluble surfactant is 0.001 to 10 g / mL; the organic solvent is dichloromethane or chloroform;

[0011] (2) Preparation of monodisperse microspheres

[0012] The inner phase fluid is introduced into the injection tube of the microfluidic device, and the outer phase fluid is introduced into the collection tube of the microfluidic device to form a monodisperse water-in-oil emulsion in the collection tube, and the water-in-oil emulsion is collected in a container containing a collection liquid, and is fully allowed to stand, and then fully stirred to completely volatilize the organic solvent in the water-in-oil emulsion to obtain monodisperse microspheres;

[0013] (3) Washing and drying

[0014] The monodisperse microspheres are washed with water to remove the collected liquid on the surface of the monodisperse microspheres, and then washed with ethanol to remove the porogen in the monodisperse microspheres to form a porous structure on the surface of the monodisperse microspheres. The monodisperse microspheres are then washed with deionized water to remove the ethanol and dried to obtain monodisperse microspheres with a porous structure on the surface.

[0015] In the technical solution of the above preparation method, the porogen is preferably 2-methylpentane, dodecane or hexadecane.

[0016] In the technical solution of the above preparation method, the water-soluble surfactant includes at least one of polyvinyl alcohol, polyglycerol ricinoleate (PGPR), block polyether F-127, Tween 80, and Tween 85.

[0017] In the technical solution of the above preparation method, the polymer is a biocompatible biodegradable polymer, and the polymer is preferably capable of being dissolved by the organic solvent described in step (1). Commonly used feasible polymers include polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), etc., but are not limited to the biocompatible biodegradable polymers listed above.

[0018] In the technical solution of the above preparation method, the injection tube and the collecting tube of the microfluidic device used in step (2) are coaxially arranged, the inner diameter of the injection tube at the outlet is preferably 40-100 μm, and the inner diameter of the receiving tube is preferably 100-400 μm.

[0019] A possible structure of a microfluidic device is as follows Figure 1 As shown, it includes an injection tube, a connecting tube and a collecting tube, which are used in conjunction with an injection pump; the injection tube is made of a cylindrical glass capillary, and its tail is pulled into a cone shape; the collecting tube is a cylindrical glass capillary; the connecting tube is a square glass tube, and a square through hole is arranged in the center of the square glass capillary; the tail of the injection tube is inserted into the head of the collecting tube and connected through the connecting tube; the injection tube, the connecting tube and the collecting tube are coaxially arranged.

[0020] In the technical solution of the above preparation method, the flow rates of the inner phase fluid and the outer phase fluid in step (2) are determined according to the channel size of the microfluidic device and the size of the monodisperse microspheres with a porous surface to be prepared. When the channel size of the microfluidic device is determined, the flow rates of the inner phase fluid and the outer phase fluid can be determined by experiments. For example, the flow rate of the inner phase fluid can be controlled to be 8 to 12 μL / min, and the flow rate of the outer phase fluid can be controlled to be 200 to 500 μL / min.

[0021] In the technical scheme of the above preparation method, the size of the oil-in-water emulsion droplets can be adjusted by adjusting the channel size of the microfluidic device in step (2), and the size of the oil-in-water emulsion droplets can also be adjusted by adjusting the flow rates of the inner phase fluid and the outer phase fluid. By adjusting the size of the oil-in-water emulsion droplets, the size of the monodisperse microspheres with a porous structure on the surface can be adjusted. The size of the monodisperse microspheres with a porous structure on the surface can also be adjusted by adjusting the concentration of the polymer in the inner phase fluid. The specific flow rates of the inner phase fluid and the outer phase fluid used are related to the channel size of the microfluidic device and the composition of the inner phase fluid and the outer phase fluid.

[0022] In the technical solution of the above preparation method, the standing time in step (2) is preferably 3 to 5 hours.

[0023] In the technical scheme of the above-mentioned preparation method, by adjusting the concentrations of the substances in the inner phase fluid, the outer phase fluid and the collected liquid in step (1), as well as the standing time in step (2), the morphology of the porous structure on the surface of the monodisperse microspheres having a porous structure can be adjusted, for example, the size of the porous structure, the porosity, the degree of interconnection between the porous structures, etc.

[0024] The present invention also provides monodisperse microspheres with a porous surface structure prepared by the above method.

[0025] Furthermore, the surface of the monodisperse microspheres with a porous structure prepared by the above method has a uniformly distributed porous structure, the pore size of the porous structure is uniform, and the porous structures are interconnected or not interconnected. The interconnection between the porous structures includes two states: partial interconnection between the porous structures or complete interconnection between the porous structures. Furthermore, the particle size of the monodisperse microspheres with a porous structure on the surface is 30 to 70 μm, and the particle size variation coefficient of the monodisperse microspheres with a porous structure on the surface does not exceed 5%.

[0026] Furthermore, the pore size of the porous structure on the surface of the monodisperse microspheres with a porous structure prepared by the above method is generally not more than 10μm, and the porous structure on the surface of the monodisperse microspheres with a porous structure prepared by the above method is usually a uniform porous structure with a pore size between 50nm and 5μm.

[0027] The formation mechanism of the monodisperse microspheres with a porous structure on the surface of the present invention is as follows:

[0028] The porogen dispersed in the oil-in-water (O / W) emulsion in the form of droplets will migrate with the interfacial tension and with the volatilization of the organic solvent (dichloromethane or chloroform). With the volatilization of the organic solvent (dichloromethane or chloroform), the polymer will continuously precipitate to form solid microspheres. The droplets formed by the porogen that migrated to the surface of the microspheres are washed and removed to obtain monodisperse microspheres with a porous structure on the surface. In the aforementioned process, the appropriate amount of organic solvent (dichloromethane or chloroform) in the external phase fluid and the collected liquid can regulate the volatilization rate of the organic solvent (dichloromethane or chloroform), and then regulate the migration rate of the porogen. The sufficient standing process in step (2) allows the porogen to migrate controllably and uniformly. The combined effect of the above technical means enables the method of the present invention to form a uniform and structurally controllable porous structure on the surface of the microspheres.

[0029] The reasons why the monodisperse microspheres with a porous surface structure described in the present invention can quickly promote collagen regeneration are mainly as follows:

[0030] Compared with polymer microspheres with smooth surfaces or basically no porous structure, the monodisperse microspheres with a porous surface described in the present invention have a larger specific surface area, which is more conducive to the adsorption and proliferation of macrophages on the monodisperse microspheres with a porous surface, and is conducive to the rapid attachment of immune cells such as macrophages to the surface of the monodisperse microspheres with a porous surface, thereby stimulating the human immune response more quickly and causing the human body to secrete collagen; at the same time, the degradation products of the monodisperse microspheres with a porous surface (such as polylactic acid, etc.) can induce myofibroblasts to produce collagen, which is also conducive to the regeneration of collagen.

[0031] Compared with the prior art, the technical solution of the present invention produces the following beneficial technical effects:

[0032] 1. The present invention provides a method for preparing monodisperse microspheres with a porous structure on the surface that can quickly promote collagen regeneration. The method uses a microfluidic device to prepare an oil-in-water emulsion with excellent size monodispersity. At the same time, by regulating the composition of each phase fluid and the collection liquid, the composition of the water-in-oil emulsion is regulated, and then the migration process of the porogen in the water-in-oil emulsion is regulated, so that the porogen can be controlled and uniformly migrated from the inside of the water-in-oil emulsion to the surface, and a uniform and structurally controllable porous structure is formed on the surface of the microsphere while achieving microsphere solidification. Compared with the existing polymer microspheres with smooth surfaces or basically no porous structure, the method of the present invention can form a large number of porous structures with uniform pore sizes on the surface of the microspheres, and the porous structures can be interconnected or not, effectively improving the surface morphology of the polymer microspheres currently used in the field of medical cosmetology, and at the same time, it can ensure that the prepared microspheres with a porous structure on the surface have excellent size but dispersibility.

[0033] 2. Compared with the existing polymer microspheres with smooth surfaces or basically no porous structure used in the field of medical cosmetology, the monodisperse microspheres with a porous surface prepared by the method of the present invention have a larger specific surface area. This structure is conducive to the adsorption and proliferation of macrophages on the monodisperse microspheres with a porous surface, and is conducive to the rapid attachment of immune cells such as macrophages to the surface of the monodisperse microspheres with a porous surface, thereby stimulating the human immune response more quickly and causing the human body to secrete collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The present invention is a schematic structural diagram of a primary capillary microfluidic device, wherein 1 is an injection tube, 2 is a connecting tube, and 3 is a collecting tube.

[0035] Figure 2 is an optical micrograph of the O / W emulsion prepared in Example 1.

[0036] Figure 3This is an optical microscopic picture of the PLLA microspheres having a porous surface structure prepared in Example 1.

[0037] Figure 4 This is a SEM image of the PLLA microspheres with a porous surface structure prepared in Example 1.

[0038] Figure 5 It is an optical microscopic picture of the O / W emulsion prepared in Comparative Example 1.

[0039] Figure 6 This is an optical microscopic picture of the PLLA microspheres prepared in Comparative Example 1.

[0040] Figure 7 Figures (A) and (B) are SEM images of the PLLA microspheres prepared in Example 1 at different magnifications.

[0041] Figure 8 This is a scanning electron microscope image of the PLLA microspheres prepared in Comparative Example 2.

[0042] Fig. 9 is an optical micrograph of the O / W emulsion prepared in Example 2.

[0043] Fig.10 This is an optical microscopic picture of the PLLA microspheres having a porous surface structure prepared in Example 2.

[0044] Fig.11 These are scanning electron microscope images of the PLLA microspheres with a porous surface structure prepared in Example 2 at different magnifications. Fig.12 is an optical micrograph of the O / W emulsion prepared in Example 3.

[0045] Fig.13 This is an optical microscopic picture of the PLLA microspheres having a porous surface structure prepared in Example 3.

[0046] Fig.14 This is a Masson staining picture of the injection site tissue 6 weeks after the injection of the freeze-dried microspheres prepared in Example 4.

[0047] Fig.15 This is a picture of Sirius red staining of the injection site tissue 6 weeks after injection of the freeze-dried microspheres prepared in Example 4.

[0048] Fig.16 This is a Masson staining picture of the injection site tissue 6 weeks after injection of the freeze-dried microspheres prepared in Comparative Example 3.

[0049] Fig.17 This is a picture of Sirius red staining of the injection site tissue 6 weeks after injection of the freeze-dried microspheres prepared in Comparative Example 3. DETAILED DESCRIPTION

[0050] The following examples further illustrate the monodisperse microspheres with a porous structure on the surface that rapidly promote collagen regeneration and the preparation method thereof provided by the present invention. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. The technical personnel in the relevant field make some non-essential improvements and adjustments to the present invention according to the above invention content for specific implementation, which still falls within the scope of protection of the present invention.

[0051] In the following embodiments, the microfluidic device used is a primary capillary microfluidic device, and its structural schematic diagram is shown in FIG. Figure 1 As shown, it includes an injection tube 1, a connecting tube 2 and a collecting tube 3, which are used in conjunction with a syringe pump. The injection tube 1 is made of a cylindrical glass capillary. The tail of the cylindrical glass capillary is pulled into a cone shape by a needle puller, and then rolled on sandpaper to a flat mouth with an inner diameter of about 60μm. The outer diameter of the circular tube section is 960μm and the inner diameter is 550μm; the collecting tube 3 is made of a cylindrical glass capillary. The two ends of the cylindrical glass capillary are polished flat. The outer diameter of the collecting tube is 960μm and the inner diameter is 200μm; the connecting tube 2 is a square glass tube. The two ends of the square glass tube are polished smooth and flat. The center part is provided with a square through hole with a through hole size of 1.0×1.0mm. After being made, the injection tube 1, the connecting tube 2 and the collecting tube 3 are ultrasonically cleaned in anhydrous ethanol and blown dry. The tail of the injection tube 1 is inserted into the head of the collecting tube 2 and connected through the connecting tube 3. The injection tube 1, the connecting tube 2 and the collecting tube 3 are coaxially arranged and fixed on the glass slide by AB glue. A steel tube can be put on the injection tube 1, and the non-inlet end is fixed by AB glue to facilitate the connection of the injection pump. The inlet end of the connecting tube 2 is fixed with a flat needle by AB glue, and the non-inlet end of the connecting tube 2 is sealed by AB glue. Each flat needle is connected to the injection pump through a pipe fitting.

[0052] Example 1

[0053] In this embodiment, monodisperse L-polylactic acid microspheres having a porous surface structure (PLLA microspheres having a porous surface structure) are prepared in the following steps:

[0054] (1) Preparation of internal phase, external phase fluid and collection liquid

[0055] Preparation of the internal phase fluid: Add poly (L-lactic acid) (PLLA) with a molecular weight of 250,000 to 350,000 to dichloromethane, then add dodecane, and stir until PLLA is completely dissolved to obtain the internal phase fluid; in the internal phase fluid, the concentration of PLLA is 0.04 g / mL, and the concentration of dodecane is 0.03 g / mL.

[0056] The external phase fluid was prepared by dissolving polyvinyl alcohol (PVA) in deionized water to obtain a PVA solution, and then adding dichloromethane in an amount 0.02 times the volume of the PVA solution and mixing thoroughly to obtain an external phase fluid; in the external phase fluid, the concentration of PVA was 0.01 g / mL.

[0057] Preparation of collecting solution: PVA was dissolved in deionized water to obtain a PVA solution, and then 0.02 times the volume of dichloromethane of the PVA solution was added and mixed thoroughly to obtain a collecting solution; in the collecting solution, the concentration of PVA was 0.01 g / mL.

[0058] (2) Preparation of monodisperse microspheres

[0059] Using structure such as Figure 1 In the microfluidic device shown, the inner phase fluid is injected into the injection tube 1 of the microfluidic device by a syringe pump, and the outer phase fluid is injected into the collecting tube 3 of the microfluidic device, a monodisperse O / W emulsion is formed in the collecting tube 3, and the monodisperse O / W emulsion is collected in a container containing a collecting liquid, allowed to stand for 3 hours, and then stirred until the dichloromethane in the monodisperse O / W emulsion is completely volatilized to obtain monodisperse microspheres.

[0060] In this step, the flow rate of the inner phase fluid was controlled at 8 μL / min and the flow rate of the outer phase fluid was controlled at 400 μL / min.

[0061] (3) Washing and drying

[0062] The monodisperse microspheres prepared in step (2) are washed with deionized water to remove the collection liquid, and then washed with ethanol to remove the dodecane in the microspheres to form a porous structure on the surface of the microspheres, and then washed with deionized water to remove the ethanol, and dried to obtain monodisperse PLLA microspheres with a porous structure on the surface.

[0063] Figure 2 is an optical micrograph of the O / W emulsion prepared in this example. Figure 2 It can be seen that the O / W emulsion prepared in this example has good monodispersity. Figure 3 is an optical microscopic picture of the PLLA microspheres with a porous surface structure prepared in this embodiment. Figure 3 It can be seen that the diameter of the PLLA microspheres with a porous surface structure prepared in this embodiment is between 51 and 58 μm, mainly distributed between 53 and 54 μm. The coefficient of variation (CV value) of the diameter of the PLLA microspheres with a porous surface structure prepared in this embodiment was calculated based on the optical microscopic image of the PLLA microspheres with a porous surface structure. The results show that the CV value does not exceed 3%, indicating that the PLLA microspheres with a porous surface structure are uniform in size and have good monodispersity. Figure 4 is a scanning electron microscope image of the PLLA microspheres with a porous surface structure prepared in this embodiment. Figure 4It can be seen that the size and morphology of the PLLA microspheres with porous structures on the surface are uniform, and the surface has a large number of evenly distributed and uniformly sized porous structures. Carefully observing the surface pore structure of the PLLA microspheres with porous structures on the surface, it can be found that these surface pore structures are basically separated by pore walls, that is, there is basically no mutual penetration between the pores.

[0064] Comparative Example 1

[0065] In this comparative example, monodisperse PLLA microspheres without a porous structure on the surface were prepared by the following steps:

[0066] (1) Preparation of internal phase, external phase fluid and collection liquid

[0067] Preparation of the internal phase fluid: Add PLLA with a molecular weight of 250,000 to 350,000 into dichloromethane, and stir until the PLLA is completely dissolved to obtain the internal phase fluid; in the internal phase fluid, the PLLA concentration is 0.04 g / mL.

[0068] Prepare the external phase fluid: dissolve PVA in deionized water to obtain a PVA solution, then add dichloromethane 0.02 times the volume of the PVA solution and mix thoroughly to obtain an external phase fluid; in the external phase fluid, the concentration of PVA is 0.01 g / mL.

[0069] Prepare the collecting solution: dissolve PVA in deionized water to obtain a PVA solution to obtain the collecting solution; in the collecting solution, the concentration of PVA is 0.01 g / mL.

[0070] (2) Preparation of monodisperse microspheres

[0071] Using structure such as Figure 1 In the microfluidic device shown, the inner phase fluid is injected into the injection tube 1 of the microfluidic device by a syringe pump, and the outer phase fluid is injected into the collecting tube 3 of the microfluidic device, a monodisperse O / W emulsion is formed in the collecting tube 3, and the monodisperse O / W emulsion is collected in a container containing a collecting liquid, and stirred until the dichloromethane in the monodisperse O / W emulsion is completely volatilized to obtain monodisperse microspheres.

[0072] In this step, the flow rate of the inner phase fluid was controlled at 10 μL / min and the flow rate of the outer phase fluid was controlled at 200 μL / min.

[0073] (3) Washing and drying

[0074] The monodisperse microspheres prepared in step (2) are washed with deionized water to remove the collection liquid, and are naturally air-dried to obtain monodisperse PLLA microspheres.

[0075] Figure 5 is an optical micrograph of the O / W emulsion prepared in this comparative example. Figure 5 It can be seen that the monodispersity of the O / W emulsion is good. Figure 6 is an optical microscopic picture of the PLLA microspheres prepared in this comparative example. Figure 6 It can be seen that the diameter of PLLA microspheres is between 48 and 53 μm, mainly distributed between 52 and 53 μm. The coefficient of variation (CV value) of the diameter is calculated based on the optical microscopic image of the PLLA microspheres. The results show that the CV value does not exceed 3%, indicating that the PLLA microspheres are uniform in size and have good monodispersity. Figure 7 Figures (A) and (B) are scanning electron microscope images of the PLLA microspheres prepared in this comparative example at different magnifications. Figure 7 As shown in Figure (A), the size and morphology of PLLA microspheres are uniform. Figure 7 Figure (B) shows that there are only a few nanopore structures on the surface of the PLLA microspheres, and this small amount of nanopore structure has little effect on the surface properties of the PLLA microspheres.

[0076] Combining Example 1 and Comparative Example 1, it can be seen that the porogen contained in the internal phase fluid and the appropriate amount of dichloromethane contained in the collection liquid have a significant effect on the surface morphology (surface pore structure) of the PLLA microspheres, because the amount of dichloromethane in the collection liquid will affect the migration of the porogen during the microsphere formation process. At the same time, the static operation after collecting the monodisperse O / W emulsion in the container containing the collection liquid in step (2) will also affect the surface morphology of the PLLA microspheres.

[0077] Comparative Example 2

[0078] In this comparative example, whether the external phase fluid and the collected liquid contain dichloromethane, and whether the microspheres are left to stand in step (2) are investigated for their effects on the microsphere morphology. The steps are as follows:

[0079] (1) Preparation of internal phase, external phase fluid and collection liquid

[0080] Preparation of the internal phase fluid: Add PLLA with a molecular weight of 250,000 to 350,000 into dichloromethane, then add dodecane, and stir until PLLA is completely dissolved to obtain the internal phase fluid; in the internal phase fluid, the concentration of PLLA is 0.04 g / mL, and the concentration of dodecane is 0.03 g / mL.

[0081] Preparation of external phase fluid: PVA was dissolved in deionized water to obtain an external phase fluid, and the concentration of PVA in the external phase fluid was 0.01 g / mL.

[0082] Preparation of collecting solution: PVA was dissolved in deionized water to obtain collecting solution, and the concentration of PVA in the collecting solution was 0.01 g / mL.

[0083] (2) Preparation of monodisperse microspheres

[0084] Using structure such as Figure 1In the microfluidic device shown, the inner phase fluid is injected into the injection tube 1 of the microfluidic device by a syringe pump, and the outer phase fluid is injected into the collecting tube 3 of the microfluidic device, a monodisperse O / W emulsion is formed in the collecting tube 3, the monodisperse O / W emulsion is collected by a container containing a collecting liquid, and then stirred until the dichloromethane in the monodisperse O / W emulsion is completely volatilized to obtain monodisperse microspheres.

[0085] In this step, the flow rate of the inner phase fluid was controlled at 400 μL / h and the flow rate of the outer phase fluid was controlled at 6 mL / h.

[0086] (3) Washing and drying

[0087] The monodisperse microspheres prepared in step (2) are washed with deionized water to remove the collection liquid, and then washed with ethanol to remove dodecane in the microspheres to form a porous structure on the surface of the microspheres, and then washed with deionized water to remove the ethanol, and dried to obtain monodisperse PLLA microspheres.

[0088] Figure 8 is a scanning electron microscope image of the PLLA microspheres prepared in this comparative example, Figure 8 It can be seen that the surface morphology of the PLLA microspheres prepared in this comparative example under the same process conditions varies greatly. Some PLLA microspheres have a larger pore structure on the surface, while some PLLA microspheres have a smaller pore structure on the surface, and even some PLLA microspheres have almost no pore structure on the surface. This shows that when preparing microspheres, the method of this comparative example has very poor controllability over the surface structure of the microspheres, and microspheres with uniform surface pore structure cannot be obtained. This may be due to the uncontrollable migration speed of the porogen during the preparation of the microspheres.

[0089] Combining Example 1 and Comparative Example 2, it can be seen that when the internal phase fluid contains a porogen, although a porous structure can be formed on the surface of the microsphere, the controllability of the porous structure on the surface of the microsphere is very poor, and it is impossible to ensure that all microspheres form a surface porous structure, and it is also impossible to ensure that the microspheres prepared under the same process conditions have a consistent or similar surface morphology. It is explained that adding an appropriate amount of dichloromethane when preparing the external phase fluid and the collection liquid, and performing a suitable standing operation after collecting the monodisperse O / W emulsion in a container containing the collection liquid in step (2) are necessary to increase the controllability of the morphology of the surface pore structure of the microspheres and increase the uniformity of the morphology of the surface pore structure.

[0090] Example 2

[0091] In this embodiment, monodisperse PLLA microspheres with a porous surface structure (PLLA microspheres with a porous surface structure) are prepared in the following steps:

[0092] (1) Preparation of internal phase, external phase fluid and collection liquid

[0093] Preparation of the internal phase fluid: Add PLLA with a molecular weight of 250,000 to 350,000 into dichloromethane, then add dodecane, and stir until PLLA is completely dissolved to obtain the internal phase fluid; in the internal phase fluid, the concentration of PLLA is 0.04 g / mL, and the concentration of dodecane is 0.05 g / mL.

[0094] Prepare the external phase fluid: dissolve PVA in deionized water to obtain a PVA solution, then add dichloromethane 0.04 times the volume of the PVA solution and mix thoroughly to obtain an external phase fluid; in the external phase fluid, the concentration of PVA is 0.01 g / mL.

[0095] Preparation of collecting solution: PVA was dissolved in deionized water to obtain a PVA solution, and then 0.04 times the volume of dichloromethane of the PVA solution was added and mixed thoroughly to obtain a collecting solution; in the collecting solution, the concentration of PVA was 0.01 g / mL.

[0096] (2) Preparation of monodisperse microspheres

[0097] Using structure such as Figure 1 In the microfluidic device shown, the inner phase fluid is injected into the injection tube 1 of the microfluidic device by a syringe pump, and the outer phase fluid is injected into the collecting tube 3 of the microfluidic device, a monodisperse O / W emulsion is formed in the collecting tube 3, and the monodisperse O / W emulsion is collected in a container containing a collecting liquid, allowed to stand for 3 hours, and then stirred until the dichloromethane in the monodisperse O / W emulsion is completely volatilized to obtain monodisperse microspheres.

[0098] In this step, the flow rate of the inner phase fluid was controlled at 8 μL / min and the flow rate of the outer phase fluid was controlled at 500 μL / min.

[0099] (3) Washing and drying

[0100] The monodisperse microspheres prepared in step (2) are washed with deionized water to remove the collection liquid, and then washed with ethanol to remove the dodecane in the microspheres to form a porous structure on the surface of the microspheres, and then washed with deionized water to remove the ethanol, and dried to obtain monodisperse PLLA microspheres with a porous structure on the surface.

[0101] Fig. 9 is an optical micrograph of the O / W emulsion prepared in this example. Fig. 9 It can be seen that the O / W emulsion prepared in this example has good monodispersity. Fig.10 is an optical microscopic picture of the PLLA microspheres with a porous surface structure prepared in this embodiment. Fig.10It can be seen that the diameter of the PLLA microspheres with a porous surface structure prepared in this embodiment is between 55 and 58 μm. The coefficient of variation (CV value) of the diameter of the PLLA microspheres with a porous surface structure prepared in this embodiment was calculated based on the optical microscopic image of the PLLA microspheres with a porous surface structure. The results showed that the CV value did not exceed 3%, indicating that the PLLA microspheres with a porous surface structure were uniform in size and had good monodispersity. Fig.11 is a scanning electron microscope image of the PLLA microspheres with a porous surface structure prepared in this example at different magnifications. Fig.11 It can be seen that the size and morphology of the PLLA microspheres with porous surface structures are uniform, and the surface has a large number of evenly distributed and uniformly sized pore structures. Carefully observing the surface pore structure of the PLLA microspheres with porous surface structures, it can be found that most of the pores in these surface pore structures are not completely isolated by the pore walls, and these surface pore structures have high penetration, and the surface of the microspheres presents a wrinkle-like structure.

[0102] It can be seen from Example 1 and Example 2 that the surface pore structure of the prepared microspheres having a porous surface can be adjusted by regulating the process parameters in the method of the present invention.

[0103] Example 3

[0104] In this embodiment, monodisperse PLLA microspheres with a porous surface structure (PLLA microspheres with a porous surface structure) are prepared. The operation of this embodiment is basically the same as that of Example 1, with the only difference being that in step (2), the flow rate of the inner phase fluid is controlled to be 10 μL / min and the flow rate of the outer phase fluid is controlled to be 200 μL / min.

[0105] Fig.12 is an optical micrograph of the O / W emulsion prepared in this example. Fig.12 It can be seen that the O / W emulsion prepared in this example has good monodispersity. Fig.13 is an optical microscopic picture of the PLLA microspheres with a porous surface structure prepared in this embodiment. Fig.13It can be seen that the diameter of the PLLA microspheres with a porous structure on the surface prepared in this embodiment is between 63 and 66 μm. The coefficient of variation (CV value) of the diameter of the PLLA microspheres with a porous structure on the surface prepared in this embodiment is calculated based on the optical microscopic image of the PLLA microspheres with a porous structure on the surface. The results show that the CV value does not exceed 3%, indicating that the size of the PLLA microspheres with a porous structure on the surface is uniform and has good monodispersity. By observing the scanning electron microscope image of the PLLA microspheres with a porous structure on the surface prepared in this embodiment, it is found that the surface morphology of the PLLA microspheres with a porous structure on the surface prepared in this embodiment is very close to the surface morphology of the PLLA microspheres with a porous structure on the surface prepared in Example 1. In combination with Example 1, it can be seen that by adjusting the process parameters in the method described in the present invention, the diameter of the prepared monodisperse microspheres with a porous structure on the surface can also be adjusted.

[0106] Example 4

[0107] 1 g of sodium carboxymethylcellulose and 3 g of mannitol were dissolved in deionized water and the volume was adjusted to 100 mL to obtain a mixed solution. 3 g of PLLA microspheres with a porous surface structure prepared in Example 1 were added to 100 mL of the mixed solution, fully stirred and evenly dispersed in the mixed solution, and freeze-dried to obtain freeze-dried microspheres.

[0108] Comparative Example 3

[0109] 1 g of sodium carboxymethylcellulose and 3 g of mannitol were dissolved in deionized water and the volume was adjusted to 100 mL to obtain a mixed solution. 3 g of monodisperse PLLA microspheres without a porous surface structure prepared in Comparative Example 1 were added to 100 mL of the mixed solution, fully stirred and evenly dispersed in the mixed solution, and freeze-dried to obtain freeze-dried microspheres.

[0110] Example 5

[0111] Using SD rats as animal models, the freeze-dried microspheres prepared in Example 4 and Comparative Example 3 were dispersed in physiological saline, respectively, and then subcutaneously injected into SD rats, and continued to be fed for 6 weeks after subcutaneous injection to compare the difference in the filling effect of the freeze-dried microspheres prepared in Example 4 and Comparative Example 3 in SD rats. After 3 days, 1 week, 3 weeks, and 6 weeks of subcutaneous injection, the tissues at the subcutaneous injection site were taken for tissue sectioning, dewaxing and rehydration, and then Masson staining and Sirius red staining were performed respectively.

[0112] Fig.14 , 15 These are Masson's staining and Sirius red staining pictures of the injection site tissue 6 weeks after the injection of the freeze-dried microspheres prepared in Example 4. Fig.16 , 17The Masson staining and Sirius red staining pictures of the tissue at the injection site 6 weeks after the injection of the freeze-dried microspheres prepared in Comparative Example 3. The pixel area percentage data of Sirius red stained polarized light type I collagen fibers and type III collagen fibers of the tissue at the injection site are shown in the following table, in which "new collagen" refers to the pixel area percentage of the aforementioned type I collagen fibers and type III collagen fibers.

[0113]

[0114] Combined with Figures 14 to 17 As can be seen from the above table, compared with the freeze-dried microspheres prepared in Comparative Example 3, the freeze-dried microspheres prepared in Example 1 can stimulate the production of collagen faster after subcutaneous injection into the body, especially after 6 weeks of subcutaneous injection into the body, the rate of stimulating collagen production between the two showed a more obvious difference.

Claims

1. A method for preparing monodisperse microspheres with a porous surface structure that rapidly promotes collagen regeneration, characterized in that: The following steps are involved: (1) Preparation of internal phase, external phase fluid and collection liquid Preparing the internal phase fluid: dissolving or dispersing the porogen and the polymer in an organic solvent to obtain the internal phase fluid; In the internal phase fluid, the concentration of the polymer is 0.005-0.10 g / mL, the concentration of the porogen is 0.001-0.10 g / mL, and the organic solvent is dichloromethane or chloroform; The external phase fluid is prepared by dissolving a water-soluble surfactant in water to form a water-soluble surfactant solution, and then adding an organic solvent in an amount of 0.02 to 0.04 times the volume of the water-soluble surfactant, and mixing them thoroughly to obtain an external phase fluid; the concentration of the water-soluble surfactant in the external phase fluid is 0.001 to 10 g / mL; the organic solvent is dichloromethane or chloroform; Preparation of a collection solution: dissolving a water-soluble surfactant in water to form a water-soluble surfactant solution, then adding an organic solvent in an amount of 0.02 to 0.04 times the volume of the water-soluble surfactant, and mixing thoroughly to obtain a collection solution; in the collection solution, the concentration of the water-soluble surfactant is 0.001 to 10 g / mL; the organic solvent is dichloromethane or chloroform; (2) Preparation of monodisperse microspheres The inner phase fluid is introduced into the injection tube of the microfluidic device, and the outer phase fluid is introduced into the collection tube of the microfluidic device to form a monodisperse water-in-oil emulsion in the collection tube, and the water-in-oil emulsion is collected in a container containing a collection liquid, and is fully allowed to stand, and then fully stirred to completely volatilize the organic solvent in the water-in-oil emulsion to obtain monodisperse microspheres; (3) Washing and drying The monodisperse microspheres are washed with water to remove the collected liquid on the surface of the monodisperse microspheres, and then washed with ethanol to remove the porogen in the monodisperse microspheres to form a porous structure on the surface of the monodisperse microspheres. The monodisperse microspheres are then washed with deionized water to remove the ethanol and dried to obtain monodisperse microspheres with a porous structure on the surface.

2. The method for preparing monodisperse microspheres having a porous surface structure that rapidly promotes collagen regeneration according to claim 1, characterized in that: The porogen is 2-methylpentane, dodecane or hexadecane.

3. The method for preparing monodisperse microspheres with a porous surface structure that rapidly promotes collagen regeneration according to claim 1, characterized in that: The water-soluble surfactant includes at least one of polyvinyl alcohol, polyglycerol ricinoleate, block polyether F-127, Tween 80 and Tween 85.

4. The method for preparing monodisperse microspheres having a porous surface structure that rapidly promotes collagen regeneration according to claim 1, characterized in that: The polymer is a biocompatible biodegradable polymer including polylactic acid, polylactic acid-co-glycolic acid, and polycaprolactone.

5. The method for preparing monodisperse microspheres with a porous surface structure that rapidly promotes collagen regeneration according to any one of claims 1 to 4, characterized in that: The injection tube and the collecting tube of the microfluidic device used in step (2) are coaxially arranged, the inner diameter of the injection tube at the outlet is 40 to 100 μm, and the inner diameter of the receiving tube is 100 to 400 μm.

6. The method for preparing monodisperse microspheres with a porous surface structure for rapidly promoting collagen regeneration according to any one of claims 1 to 4, characterized in that: The standing time in step (2) is 3 to 5 hours.

7. Monodisperse microspheres having a porous surface structure prepared by the method according to any one of claims 1 to 6.

8. The monodisperse microspheres having a porous surface structure according to claim 7, characterized in that: The surface of the monodisperse microsphere has a uniformly distributed porous structure, the pore size of the porous structure is uniform, and the porous structures are interconnected or not interconnected.

9. The monodisperse microspheres having a porous surface structure according to claim 8, characterized in that: The particle size of the monodisperse microspheres is 30-70 μm, and the particle size variation coefficient of the monodisperse microspheres does not exceed 5%.

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