Composite PCL microspheres, preparation method and application thereof

Abstract

The application discloses composite PCL microspheres and a preparation method and application thereof. The ratio of the shell thickness of the composite PCL microspheres to the core radius is 1:1-1:3, and the particle size of the composite PCL microspheres is 20-65 mu m. The PCL polycaprolactone microspheres can make the dermal fibroblasts continuously proliferate under low inflammation reaction, make the fibroblasts grow in order around the composite microspheres, reduce the overall inflammation level, continuously produce collagen and extracellular matrix, and achieve the regeneration effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a composite PCL microsphere, a preparation method and application thereof. BACKGROUND

[0002] PCL material is widely used in tissue engineering and dermal filler due to its biocompatibility and excellent mechanical properties. However, the solid microspheres of single PCL material still face certain limitations, and its hydrophobicity may lead to poor cell adhesion. In order to improve the cell adhesion of microsphere implants, the most common method is to integrate a porous structure into the PCL microspheres. The gap between the microspheres is expected to promote cell migration and proliferation, and accelerate the formation of new tissues. The microspheres with high surface area to volume ratio and porous structure are proved to improve the space environment of cell adhesion and growth. The interconnection of the pores also provides a migration path, helps the formation of new blood vessels and improves the circulation of oxygen and nutrients. The porous structure is usually associated with uneven surfaces, which are more likely to aggregate macrophages and foreign giant cells. However, these special structures will cause persistent high inflammatory response.

[0003] Core-shell microspheres are an alternative method to overcome the limitations of PCL microspheres in dermal filler, tissue repair and other applications. Materials with core-shell structure are usually composed of two or more different materials, which have multiple functions in different applications. Therefore, core-shell microspheres show great application potential in drug delivery with dual drug release and tissue engineering of implants with dual functions of cell microenvironment and ideal mechanical stiffness. The selection of core and shell materials of microsphere implants directly affects their interaction with cells (i.e. adhesion, inflammatory response and toxicity) and the arrangement of cell growth, but this point is rarely concerned, especially in the design of such implants.

[0004] Many medical implants and regenerative medicine research and development will encounter similar wound healing processes caused by foreign body inflammation. After the inflammatory response is initiated, the release of growth factors further triggers the proliferation of fibroblasts. Collagen also plays an important role in this process, especially type I and type III collagen. Type III collagen supports early angiogenesis and cell migration, and is gradually replaced by type I collagen, increasing wound strength and helping to form mature scar tissue, ultimately ensuring the integrity of the skin and stable recovery. The orientation of fibroblasts and collagen is crucial in the last step of wound healing and extracellular matrix formation, where the arrangement of cell and collagen deposition is highly dependent on the microenvironment of the site and the characteristics of the implant. Therefore, implants with favorable properties for ordered cell adhesion and arrangement are preferred.

[0005] In addition, although the existing PCL product has a shelf life of up to 2 years, the deformation of the microspheres starts counting down from the time of preparation, and the microsphere shape at the time of injection may be completely different from that measured at the time of production. The quality of the product is uncontrollable from production to transportation and to consumer injection, especially when it is stored in a liquid or semi-liquid environment. For injection of regenerative filling stimulating material generated by foreign bodies into the dermis, the injection of irregular shapes often causes long-term inflammation. The microsphere shape at the time of injection may be completely different from that measured at the time of production. The quality is uncontrollable from production to storage, transportation, and to consumer injection, especially in a liquid environment. For injection of regenerative filling stimulating material generated by foreign bodies into the dermis, the injection of irregular shapes often causes long-term inflammation. We propose a special composite microsphere design, non-water-absorbing material wrapped PCL material combined, to delay the deformation of the product at the time of injection due to transportation, storage conditions.

[0006] At this stage, most dermal filling products have poor clinical response, mostly related to nodules. Two reasons for nodules, one is caused by uneven accumulation, and the second hidden reason is the excessive accumulation of extracellular matrix ECM. Fibroblasts are essential for wound healing in response to tissue injury and regeneration of the dermal extracellular matrix (ECM), playing a role in inflammation and immune cell recruitment. Fibroblasts respond and synthesize cytokines and chemokines to help guide the inflammatory response. They can also differentiate into myofibroblasts, producing myosin and alpha-smooth muscle actin (SMA), increasing ECM production. However, uncontrolled proliferation of fibroblasts and fibrosis are pathological features, and are also the main reason for the production of nodules in most products on the market.

[0007] Although CN114225105A mentions that the composition of the composite microsphere structure all belongs to immune stimulating regenerative material, it is inevitable to start to decompose or deform during transportation. And the person skilled in the art does not realize that the fibroblast regeneration process in the dermis is a dynamic process, and too much, too thick immune stimulating regenerative material will cause the fibroblasts in the dermis to grow disorderly, and finally cause nodules. In addition, the preparation of composite microspheres is often a challenge in large-scale production, and traditional homogeneous stirring preparation is difficult to control the thickness between each layer of the microspheres. Membrane emulsification shows significant ability in adjusting droplet size and particle size distribution. Process flexibility allows it to be applied to a variety of solid and advanced particle formulations for drug protection or controlled release. In addition, due to the mild hydrodynamic conditions, high encapsulation and loading efficiency of active pharmaceutical ingredients and biological activity of protein / peptide drugs can be maintained. Another challenge faced by micro-nano as a drug delivery system is the large-scale production of structural materials, i.e. scaling up laboratory or pilot technology to achieve consistent and reproducible production and commercialization. SUMMARY

[0008] The present application solves the clinical incompatibility problem of long-term chronic inflammation caused by dermal filler products in the prior art, and provides a composite PCL microsphere and a preparation method and application thereof.

[0009] The present application is realized by the following technical solutions:

[0010] The first object of the present application is to provide a preparation method of a composite PCL microsphere, comprising the following steps:

[0011] S1, dissolve natural polysaccharide in a first solvent as a dispersed phase W1, dissolve a first emulsifier in an organic solvent, the first emulsifier is a liposoluble emulsifier with a hydrophilic-lipophilic balance value of 2-6, as a continuous phase O1, dissolve a crosslinking agent in a second solvent, the crosslinking agent is calcium chloride or tripolyphosphate (TPP), as a crosslinking solvent, dissolve polycaprolactone in a third solvent as a dispersed phase O2, dissolve a second emulsifier in a fourth solvent, the second emulsifier is selected from two or more of PVA (polyvinyl alcohol), Tween20 (polysorbate-20) and SDS (sodium dodecyl sulfate), as a continuous phase W2;

[0012] S2, disperse the crosslinking solvent prepared in step S1 into the continuous phase O1 to obtain solution 1;

[0013] S3, place the dispersed phase W1 into a dispersed phase container, pump solution 1 obtained in step S2 into a membrane module to generate circulation, adjust the pressure of the membrane module to be stable by introducing nitrogen, and then disperse the dispersed phase W1 through the membrane as a medium to obtain an emulsion, i.e. primary emulsion W1 / O1;

[0014] S4, after standing the primary emulsion W1 / O1, evaporate the solvent by centrifugation, and freeze-dry to obtain inner microspheres;

[0015] S5, disperse the inner microspheres obtained in step S4 in the dispersed phase O2 obtained in step S1 to obtain solution 2;

[0016] S6, place solution 2 into a dispersed phase container, pump the continuous phase W2 obtained in step S1 into a membrane module to generate circulation, adjust the pressure of the membrane module to be stable by introducing nitrogen, and then disperse solution 2 through the membrane as a medium to obtain an emulsion, i.e. double emulsion W1 / O2 / W2;

[0017] S7, pour the double emulsion W1 / O2 / W2 into a curing aid solution, stir at room temperature to evaporate the solvent, centrifuge and separate, wash, and freeze-dry to obtain composite PCL microspheres.

[0018] Membrane emulsification process has great potential in large-scale production of particles with uniform particle size distribution by increasing membrane area, successfully solving the technical bottleneck of preparing high-end uniform nanometer microspheres, and being considered as the simplest and most effective method for preparing uniform particle size. The particle size of the microspheres prepared by membrane emulsification can be controlled by controlling the pore size of the membrane medium, and the prepared microspheres have uniform particle size and controllable size components, and are monodisperse. The present application first uses the membrane emulsification method to accurately control the thickness of the core and PCL shell to prepare composite core-shell microspheres that can make the dermal fibroblasts produce intelligent repair.

[0019] The present application proposes a two-step membrane emulsification technology to prepare composite PCL microspheres, accurately controls the thickness of the core and PCL shell of the composite microspheres to prepare the timing of mechanical signal regulation of dermal fibroblast growth, and reduces chronic inflammation caused by immune stimulation due to continuous foreign body implantation. Through the multi-layer wrapping method, the deformation of the microspheres caused by transportation and storage conditions is delayed. The PCL polycaprolactone microspheres prepared by the preparation method proposed in the present application can make the dermal fibroblasts continuously proliferate under low inflammation, make the fibroblasts grow in order around the composite microspheres, reduce the overall inflammation level, and continuously produce collagen and extracellular matrix to achieve the regeneration effect.

[0020] Preferably, in step S1, the first solvent is an acidic aqueous solution, the concentration of the dispersed phase W1 is 1-3.5wt%; the organic solvent is ethyl acetate, and the dynamic viscosity of the continuous phase O1 is below 1.55-2.30mPa·s(20℃); the second solvent is water, and the concentration of the cross-linking solvent is 1%-3%g / L(solute / solvent).

[0021] The mass ratio of the cross-linking agent to the natural polysaccharide is 1:1-2.5:1.

[0022] The natural polysaccharide includes sodium alginate, chitosan, etc.; the acidic aqueous solution includes acetic acid, citric acid, etc. with pH of 3.0-5.0.

[0023] The first emulsifier is a lipid-soluble emulsifier with a hydrophilic-lipophilic balance (HLB) of 2-6, and the first emulsifier is selected from two or more of Span85, Span80, Span60 and polyglycerol polyricinoleate (PGPR).

[0024] By combining suitable composite emulsifiers, the present application can effectively avoid the dynamic reorganization of the droplet interface during the membrane extrusion process, and cause uncontrollable rupture or fusion. The prepared primary emulsion has strong stability, meets the membrane emulsion preparation of the second step, and finally emulsifies suitable composite PCL microspheres with ideal thickness

[0025] The preparation of the dispersed phase W1, the continuous phase O1, the cross-linking solvent, the dispersed phase O2 and the continuous phase W2 in step S1 is achieved by adding solutes into solvents and then heating to dissolve the solutes.

[0026] Preferably, in step S1, the third solvent is water, and the concentration of the dispersed phase O2 is 3%-8% g / L (solute / solvent); the fourth solvent is ethyl acetate, and the dynamic viscosity of the continuous phase W2 is below 1.01-1.05 mPa·s.

[0027] Preferably, the specific steps of step S2 are as follows: the cross-linking solvent prepared in step S1 is dispersed into the continuous phase O1 in a stirring manner to obtain solution 1, the stirring speed is 5000-10000 rpm, the stirring time is 3-5 min, and the particle size of the cross-linking solvent pre-dispersed in the continuous phase O1 is below 300 nm.

[0028] The preparation of the primary emulsion in step S3 or the preparation of the re-emulsion in step S6 is achieved by a membrane emulsification device (such as the one shown in FIG. 1). Figure 1 The membrane emulsification device includes a dispersed phase container provided with a dispersed phase, a continuous phase container provided with a continuous phase, and a membrane emulsification assembly. The membrane emulsification assembly is a membrane module provided with a dispersed phase and a continuous phase. The membrane module is provided with a membrane sheet that separates the dispersed phase and the continuous phase. One end of the dispersed phase container is connected to a nitrogen storage tank, and the other end is connected to the membrane module. The membrane sheet is installed into the membrane module and fixed with a nut to avoid movement and air leakage in subsequent operations. The continuous phase is pumped into the membrane module by a peristaltic pump to generate circulation. After the pressure is adjusted to be stable by introducing nitrogen from the nitrogen storage tank, the dispersed phase in the dispersed phase container is introduced into the membrane module by the pressure of the nitrogen. The dispersed phase is dispersed as a medium through the membrane sheet to obtain an emulsion.

[0029] Preferably, in step S3, the water contact angle of the membrane sheet at the end of solution 1 is 100°-115°, the average pore size is 0.08-0.20 μm, the pore density is 0.30-0.65, the ratio of the volume of the dispersed phase W1 to the membrane area is 1:1-1:10 mL / cm 2 , the pump speed is 50-100 mL / min, and the pressure is 0.05-0.10 MPa. Further preferably, in step S3, the water contact angle of the membrane sheet at the end of solution 1 is 105°-115°, the average pore size is 0.08-0.12 μm, and the pore density is 0.60-0.65.

[0030] The specific steps of step S4 are as follows: the primary emulsion W1 / O1 is placed at 15°C-35°C for a period of time, centrifuged and evaporated, washed with distilled water, and then freeze-dried to obtain inner microspheres.

[0031] In step S5, the mass ratio of the inner microspheres to polycaprolactone in the dispersed phase O2 is 1:2-1:4.

[0032] Preferably, in step S6, the membrane contacts the continuous phase W2 at a water contact angle of 20-50°, the average pore size is 0.15-0.50 μm, the pore density is 0.3-0.65, and the ratio of the volume of the dispersed phase O2 to the membrane area is 1:1-1:10 mL / cm. 2 Further preferably, in step S6, the membrane contacts the continuous phase W2 at a water contact angle of 20-30°, the average pore size is 0.32-0.45 μm, and the pore density is 0.60-0.65.

[0033] Preferably, in step S7, the coagulation aid solution is a 1-3 wt% PVA aqueous solution.

[0034] Preferably, the preparation method further comprises step S8, dispersing the composite PCL microspheres prepared in step S7 into a microsphere protective agent solution, and moving to an ice bath for cooling to obtain the composite PCL microspheres.

[0035] The microsphere protective agent solution is prepared by adding agar polysaccharide to water, heating to 75-85°C to dissolve the agar polysaccharide, and obtaining a 3-5 wt% microsphere protective agent aqueous solution. The multiple wrapping technology proposed in the present application adds non-water-absorbing material (i.e., the microsphere protective agent) to the outermost layer of the microspheres, which delays the deformation of the product caused by transportation and storage conditions when the microspheres are injected.

[0036] The second object of the present application is to provide the composite PCL microspheres obtained by the preparation method, wherein the ratio of the shell thickness to the core radius of the composite PCL microspheres is 1:1-1:3, and the particle size of the composite PCL microspheres is 20-65 μm.

[0037] The structure of the core-shell thickness of the composite microspheres proposed in the present application is more consistent with the mechanical signal timing of the dermal fibroblast proliferation and growth, controls the expression sequence and duration of alpha-smooth muscle actin SMA, programs and adjusts the microenvironment for the growth of dermal fibroblasts, thereby achieving the purpose of reducing nodules.

[0038] The second object of the present application is to provide the application of the composite PCL microspheres in preparing dermal fillers. The microspheres with a shell thickness ratio proposed in the present application can induce fibroblasts to grow around the microspheres in an orderly manner in the early stage, and can continue to induce the production of collagen through materials with low immune response in the later stage. The microspheres with this structure can program the mechanical signal timing of dermal fibroblast proliferation and growth, which is beneficial to the long-term proliferation of dermal cells and reduces the occurrence of chronic inflammation.

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] (1) This invention prepares water-in-oil-in-water (W / O / W) by a two-step membrane emulsification technology, defines the characteristics of the membrane as a fluid separation medium material, and can achieve precise control of the thickness of each layer of the hydrophilic-hydrophobic core-shell microspheres, and prepare composite PCL microspheres with a diameter of 20-100 μm and a core diameter of 5-10 μm.

[0041] (2) This invention combines HLB design, viscosity design and process of emulsifier to prepare stable primary emulsion that can withstand intra-membrane transport without breaking, realize membrane emulsification to prepare secondary emulsion, and adjust the shell and core thickness of composite microspheres.

[0042] (3) This invention, by precisely designing the thickness of the core-shell microsphere material, controls the contact and degradation time of the microsphere in the dermal microenvironment and the indirect release of the core material of the microsphere to generate a corresponding mechanical stress response, effectively stimulates and induces the continuous growth of dermal fibroblasts in an environment lacking immune stress.

[0043] (4) This invention, through multi-layer encapsulation technology, enables composite microspheres to remain undegraded and undeformed at room temperature for up to 3 months, solving the problem of uncontrollable microsphere quality from production to transportation and then to consumer injection, especially when stored in a liquid or semi-liquid environment. For injecting regenerative filler irritants generated by foreign substances into the dermis, unconventional shaped injectables are more likely to cause long-term inflammation and reduce the level of inflammatory factors. Attached Figure Description

[0044] Figure 1 : Schematic diagram of the membrane emulsification device proposed in this invention.

[0045] Figure 2 Microscopic image of composite PCL microspheres prepared in Example 1.

[0046] Figure 3 Microscopic image of composite PCL microspheres prepared in Example 2.

[0047] Figure 4 SEM image of PCL microspheres prepared in Comparative Example 1.

[0048] Figure 5 SEM image of PCL microspheres obtained in Comparative Example 2.

[0049] Figure 6 Comparative Example 3: Layering phenomenon of microspheres in continuous phase W2.

[0050] Figure 7 Comparative Example 6: Microscopic images of composite microspheres and their fluorescence micrographs.

[0051] Figure 8 Comparative Example 7: Microscopic image of composite microspheres.

[0052] Figure 9 Microscope image of core-shell chitosan microspheres of Comparative Example 8.

[0053] Figure 10 Microscope image of composite microspheres of Comparative Example 9.

[0054] Figure 11 Microscope image of composite microspheres of Comparative Example 10.

[0055] Figure 12 Simulated room temperature degradation trends of Comparative Example 11 and Example 2.

[0056] Figure 13 Microscope image of microspheres prepared in Comparative Example 13.

[0057] Figure 14 Microscope image of microspheres prepared in Comparative Example 14.

[0058] Figure 15 Fibroblast proliferation data after 24 hours for microspheres obtained in Example 2, Comparative Example 1, and Comparative Example 2.

[0059] Figure 16 24 hour cell growth for composite microspheres obtained in Example 2.

[0060] Figure 17 COX-2 expression at 24 hours, 96 hours, and 168 hours for Example 1, Comparative Example 9, and Comparative Example 10, respectively.

[0061] Figure 18 IL-6 expression at 24 hours, 96 hours, and 168 hours for Example 1, Comparative Example 9, and Comparative Example 10, respectively.

[0062] Figure 19 Wound healing test at 0h, 24h for Example 1, Comparative Example 9, and Comparative Example 10, respectively.

[0063] Figure 20 Collagen I (COL-I) expression at 24 hours, 96 hours, and 168 hours for Example 1, Comparative Example 9, and Comparative Example 10, respectively.

[0064] Figure 21 Collagen III (COL-III) expression at 24 hours, 96 hours, and 168 hours for Example 1, Comparative Example 9, and Comparative Example 10, respectively.

[0065] Figure 22 Alpha-Smooth Muscle Protein SMA expression at 24 hours, 96 hours, and 168 hours for Example 1, Comparative Example 9, and Comparative Example 10, respectively.

[0066] Explanation of reference signs: 1, dispersed phase; 2, continuous phase; 3, membrane emulsification assembly; 4, peristaltic pump; 5, pressure valve. DETAILED DESCRIPTION

[0067] The application will be further described in conjunction with the following examples. These examples are only used to illustrate the application and are not used to limit the scope of the application. The experimental methods in the following examples are not specified, which are generally in accordance with the conventional conditions in the art or in accordance with the conditions recommended by the manufacturer; the raw materials, reagents, etc. used are, unless otherwise specified, considered to be raw materials and reagents that can be obtained through conventional market or commercial channels. The solute in the dispersed phase or the continuous phase disclosed in the application is dissolved in the solvent by heating, and the heating temperature is not specifically limited, as long as the solute can be completely dissolved. The membrane disclosed in the application is prepared according to the method disclosed in ZL202310909977.4.

[0068] The membrane emulsification device comprises a dispersed phase container provided with a dispersed phase 1, a continuous phase container provided with a continuous phase 2, and a membrane emulsification assembly 3, the membrane emulsification assembly is a membrane module provided with a dispersed phase and a continuous phase, the membrane module is provided with a membrane, the dispersed phase and the continuous phase are separated by the membrane, one end of the dispersed phase container is connected to a nitrogen tank, and the other end is connected to the membrane module. The membrane is installed into the membrane module and fixed with a nut to avoid subsequent operation movement and air leakage, the continuous phase is pumped into the membrane module by a peristaltic pump 4 to generate circulation, nitrogen is introduced through the nitrogen tank, the pressure is adjusted to be stable through a pressure valve 5, and the dispersed phase in the dispersed phase container is introduced into the membrane module through the pressure of the nitrogen, and the dispersed phase is dispersed as a medium through the membrane to obtain an emulsion.

[0069] The microsphere characterization steps in the following examples and comparative examples are as follows:

[0070] Contact angle test method: cut a piece of film about 1 cm x 1 cm, with the end of the film contacting the mobile phase facing up, paste it on a glass slide and place it on the sample stage of the contact angle tester for testing. Add 1 μL of pure water to the surface of the horizontal film, capture the droplet profile by a high-resolution camera, and calculate the contact angle using the Young-Laplace algorithm with the software provided with the contact angle equipment.

[0071] Membrane average pore size and pore density test method: use a scanning electron microscope SEM to take pictures and samples of the microstructure of the membrane contacting the mobile phase. Use IMAGEJ software to automatically identify and analyze the pore size and area of the pores on the membrane surface, and the pore density calculation method is as follows: Pore density % = pore area / membrane area x 100

[0072] Microsphere size, distribution statistics: take a particle size larger microspheres, placed on a glass slide for microscopic observation. Through the microscope to take pictures of the appearance of the microspheres and its core-shell structure. Using ImageJ software to analyze the particle size of microspheres, and take digital photos to record the appearance of the microspheres.

[0073] Microsphere morphology observation: take the appropriate amount of microspheres, add the appropriate amount of distilled water, shake well, and make a microsphere suspension. Drop evenly dispersed on the tin foil, dry naturally for 24 h. Cut out the sample of the appropriate size, and fix the sample on the sample stage with conductive glue. Vacuum gold spraying, finally the sample stage is fixed in the scanning electron microscope (SEM), after vacuum, the sample is observed by electron beam scanning and photographed.

[0074] Composite PCL microsphere shell thickness and core radius ratio calculation method: in order to obtain the average shell thickness of the composite PCL microsphere, first, use the inverted fluorescence microscope to take pictures of the microspheres. Get the planar image, measure 200 microspheres by using ImageJ software and calculate the radius (R) of the whole microsphere and the radius (r) of the middle core microsphere.

[0075]

[0076] Example 1

[0077] A preparation method of a composite PCL microsphere, comprising the following steps:

[0078] S1, accurately weigh 1g of sodium alginate, heat and dissolve it in 100g of citric acid aqueous solution (pH=3.8) as dispersed phase 1 (W1); accurately weigh SPAN 85 and polyglycerol castor oil PGPR with a mass ratio of 1:1, heat and dissolve them in ethyl acetate, the HLB of the emulsifier combination is 2.8, and the solution viscosity is 1.95 mPa·s, as continuous phase 1 (O1); accurately weigh 1g of CaCl2, dissolve it in 100g of pure water as cross-linking solvent; accurately weigh 0.6mg of polycaprolactone PCL, ultrasonic heating and dissolve it in 20mL of ethyl acetate as dispersed phase 2 (O2); accurately weigh polyvinyl alcohol PVA and sodium dodecyl sulfate SDS with a mass ratio of 1:1, heat and dissolve them in pure water, the HLB of the emulsifier combination is 20, and the solution viscosity is 1.02 m·Pa·s, as continuous phase 2 (W2).

[0079] S2, the cross-linking solvent prepared in step S1 is quickly dispersed into the continuous phase O1 by stirring at a speed of 9000 rpm for 3 min to obtain solution 1.

[0080] S3, select a membrane with a water contact angle of 115°, the average pore size of the membrane is 0.12μm, and the pore density is 0.65. The membrane area is 12cm 2Install the membrane sheet into the membrane module and secure it with nuts to prevent movement and leakage during subsequent operations. Install and connect the membrane module to the membrane emulsification system in sequence. Figure 1 W1 is the dispersed phase, which is placed in a dispersed phase container with a volume of 9 mL. After setting a constant pump speed of 100 mL / min, the solution 1 obtained in step S2 is pumped into the membrane module through a peristaltic pump to generate circulation. The valve of the nitrogen cylinder is opened, and the pressure is adjusted to 0.07 MPa. After the dispersed phase is dispersed through the membrane as a medium, an emulsion is obtained, and the primary emulsion W1 / O1 is prepared.

[0081] S4. The colostrum W1 / O1 was left to stand at 25°C for 12 hours, centrifuged and the solvent was evaporated. After rinsing with distilled water, it was freeze-dried (vacuumed to below 10 Pa for 6 hours and dried for 6 hours) to obtain the inner microspheres.

[0082] S5. The microspheres obtained by freeze-drying in step S4 are redispersed in the dispersed phase O2, with the mass ratio of microspheres to polycaprolactone being 1:3.

[0083] S6. Select a membrane with a water contact angle of 30°, an average pore size of 0.45 μm, a pore density of 0.65, and a membrane area of ​​20 cm². 2 Install the membrane sheet into the membrane module and secure it with nuts to prevent movement and leakage during subsequent operations. Install and connect the membrane module to the membrane emulsification system in sequence. Figure 1 The dispersed phase solution O2 obtained in step S5 is the dispersed phase, and the volume of the dispersed phase O2 is 20 mL. A constant pump rate of 25 mL / min is set to pump the continuous phase W2 into the membrane module to generate circulation. The valve of the nitrogen cylinder is opened, and the pressure is adjusted to 0.12 MPa. After the dispersed phase passes through the membrane as a cutoff dispersion, an emulsion is obtained, preparing the complex emulsion W1 / O2 / W2.

[0084] S7. Pour the W1 / O2 / W2 double emulsion into a 3wt% PVA aqueous solution and stir at room temperature to evaporate the solvent. Centrifuge to separate the monodisperse polymer microspheres, wash with distilled water, and freeze-dry to obtain composite PCL microspheres.

[0085] S8. Dissolve agar polysaccharide in water at 80°C to prepare a 3wt% agar polysaccharide aqueous solution. While still hot, disperse the composite PCL microspheres prepared in step S7 into the agar polysaccharide aqueous solution by shaking, then quickly place in an ice bath to cool. Sterilize, bottle, and obtain the final composite PCL microsphere product.

[0086] The obtained microspheres were characterized, such as Figure 2 As shown, the core diameter of the composite PCL microspheres is approximately 21 μm, the outer diameter of the microspheres is approximately 42 μm, and the shell thickness to core radius ratio is 1:1.

[0087] Example 2

[0088] A preparation method of a composite PCL microsphere, comprising the following steps:

[0089] S1, precisely take 3g of chitosan, heat, and dissolve it in 100g of an acetic acid aqueous solution (pH=3.8) as a dispersed phase 1 (W1); precisely take SPAN 85 and SPAN 80 with a mass ratio of 1:1, heat, and dissolve them in ethyl acetate, the emulsifier combination HLB is 5.5, and the solution viscosity is 2.2 mPa·s, as a continuous phase 1 (O1); precisely take 1g of TPP, dissolve it in 100g of pure water as a cross-linking solvent; precisely take 0.5mg of polycaprolactone PCL, and ultrasonic heating to dissolve it in 12mL of ethyl acetate as a dispersed phase 2 (O2); precisely take polyvinyl alcohol PVA and Tween 20 with a mass ratio of 1:1, heat, and dissolve them in pure water, the emulsifier combination HLB is 16, and the solution viscosity is 1.04 m·Pa·s, as a continuous phase 2 (W2).

[0090] S2, the cross-linking solvent prepared in step S1 is quickly dispersed into the continuous phase O1 by stirring at 5000 rpm for 5 minutes to obtain solution 1;

[0091] S3, a membrane with a water contact angle of 105° is selected, the average pore size of the membrane is 0.08μm, and the pore density is 0.60. The membrane area is 12cm 2 . Install the membrane into the membrane module and fix it with a nut to avoid movement and air leakage in subsequent operations. Install and link the membrane module and the membrane emulsification system in sequence Figure 1 . W1 is the dispersed phase, which is placed in the dispersed phase container. The volume of the dispersed phase W1 is 10mL. After setting the constant pump speed to 60mL / min, solution 1 obtained in step S2 is pumped into the membrane module to generate circulation. Open the valve of the nitrogen cylinder, adjust the pressure to 0.10MPa, and then disperse the dispersed phase through the membrane as a medium to obtain an emulsion, and prepare the primary emulsion W1 / O1.

[0092] S4, the primary emulsion W1 / O1 is placed at 20℃ for 12 hours, centrifuged and evaporated, and then washed with distilled water. After freeze-drying (vacuumized to below 10Pa for 6 hours and dried for 6 hours), the inner microspheres are obtained.

[0093] S5, the inner microspheres obtained by freeze-drying in step S4 are re-dispersed in the dispersed phase O2, and the mass ratio of the inner microspheres to polycaprolactone is 1:3.

[0094] S6, a membrane with a water contact angle of 20° is selected, the average pore size of the membrane is 0.32μm, and the pore density is 0.60. The membrane area is 20cm 2 . Install the membrane into the membrane module and fix it with a nut to avoid movement and air leakage in subsequent operations. Install and link the membrane module and the membrane emulsification system in sequence Figure 1). The dispersion phase solution O2 obtained in step S5 is the dispersion phase. The volume of the dispersion phase O2 is 20 mL. The constant pump rate is set to 40 mL / min, and the continuous phase W2 is pumped into the membrane module to generate circulation. After the valve of the nitrogen cylinder is opened and the pressure is adjusted to 0.09 MPa, the dispersion phase passes through the membrane as a cutoff dispersion to obtain an emulsion, and the multiple emulsion W1 / O2 / W2 is prepared.

[0095] S7, the W1 / O2 / W2 multiple emulsion is poured into a 1wt% PVA aqueous solution, and the solvent is evaporated at room temperature under stirring. The monodisperse polymer microspheres are separated by centrifugation, washed with distilled water, and freeze-dried to obtain the composite PCL microspheres.

[0096] S8, the agar polysaccharide is added to water and heated to 80°C until the agar polysaccharide is dissolved to prepare a 1wt% agar polysaccharide aqueous solution. The composite PCL microspheres prepared in step S7 are dispersed into the agar polysaccharide aqueous solution while hot, and then quickly placed in an ice bath for cooling. After sterilization, the product is bottled to obtain the final product of the composite PCL microspheres.

[0097] The obtained microspheres are characterized, as shown in Figure 3 The inner core diameter of the composite PCL microspheres is about 32 μm, the outer diameter of the microspheres is about 48 μm, and the shell thickness is 16:32 = 1:2.

[0098] Comparative Example 1

[0099] The membrane emulsification method for preparing smooth single PCL microspheres includes the following steps:

[0100] S1, 0.5 mg of polycaprolactone PCL is precisely weighed and dissolved in 12 mL of ethyl acetate by ultrasonic heating as the dispersion phase; polyvinyl alcohol PVA and Tween 20 with a mass ratio of 1:1 are precisely weighed, heated, and dissolved in pure water, and the emulsifier combination HLB is 16, and the solution viscosity is 1.04 m·Pas, as the continuous phase.

[0101] S2, a membrane sheet with a water contact angle of 20° is selected, the average pore size of the membrane sheet is 0.32 μm, and the pore density is 0.60. The membrane area is 20 cm 2 . The membrane sheet is installed into the membrane module and fixed with nuts to avoid movement and air leakage during subsequent operations. The membrane module is sequentially installed and linked with the membrane emulsification system Figure 1 . The volume of the dispersion phase is 20 mL. The constant pump rate is set to 40 mL / min, and the continuous phase is pumped into the membrane module to generate circulation. After the valve of the nitrogen cylinder is opened and the pressure is adjusted to 0.09 MPa, the dispersion phase passes through the membrane as a cutoff dispersion to obtain an emulsion, and the O / W is prepared.

[0102] S3, pour the O / W into 1 wt% PVA aqueous solution, stir at room temperature to evaporate the solvent. Centrifugal separation of monodisperse polymer microspheres, rinsed with distilled water, freeze-dried and dried to obtain smooth single PCL microspheres.

[0103] The obtained microspheres were characterized as shown in Figure 4

[0104] Comparative Example 2

[0105] Porous single PCL microspheres were prepared by membrane emulsification, including the following steps:

[0106] S1, precisely weigh 0.5 mg of polycaprolactone PCL and 0.01 g of NaCl, and ultrasonic heating to dissolve in 12 mL of ethyl acetate as the dispersed phase; precisely weigh the mass ratio of 1:1 of polyvinyl alcohol PVA and Tween 20, heat and dissolve in pure water, the combined HLB of the emulsifier is 16, and the solution viscosity is 1.04 m Pa s, as the continuous phase.

[0107] S2, select a film with a water contact angle of 20°, the average pore size of the film is 0.32 μm, and the pore density is 0.60. The film area is 20 cm 2 . Install the film into the membrane module and fix it with nuts to avoid movement and air leakage during subsequent operations. Install and link the membrane module with the membrane emulsification system in sequence Figure 1 . The volume of the dispersed phase is 20 mL. Set the constant pump speed to 40 mL / min, pump the continuous phase into the membrane module to generate circulation. Open the valve of the nitrogen cylinder, adjust the pressure to 0.09 MPa, and then the dispersed phase passes through the membrane as a cutoff dispersion to obtain the emulsion, and prepare O / W.

[0108] S3, pour the O / W into 1 wt% PVA aqueous solution, stir at room temperature to evaporate the solvent. Centrifugal separation of monodisperse polymer microspheres, rinsed with distilled water, freeze-dried and dried to obtain porous single PCL microspheres.

[0109] The obtained PCL microspheres were characterized as shown in Figure 5

[0110] Comparative Example 3

[0111] The continuous phase 1 (O1) did not use a combined emulsifier, but used polyglycerol polyricinoleate PGPR, which was the same as Example 1, including the following steps:

[0112] ​​S1, precisely take 1 g of sodium alginate, heat and dissolve it in 100 g of a citric acid solution (pH = 3.8) as a dispersed phase 1 (W1); precisely take polyglycerol ricinoleate PGPR, the amount of which is the sum of the mass of SPAN 85 and polyglycerol ricinoleate PGPR in Example 1, heat and dissolve it in ethyl acetate, the HLB is 4, the overall solution viscosity is 2.45 mPa-s, as a continuous phase 1 (01); precisely take 1 g of CaCl2, dissolve it in 100 g of pure water as a cross-linking solvent; precisely take 0.6 mg of polycaprolactone PCL, ultrasonic heating and dissolve it in 20 mL of ethyl acetate as a dispersed phase 2 (02); precisely take polyvinyl alcohol PVA and sodium dodecyl sulfate SDS with a mass ratio of 1:1, heat and dissolve them in pure water, the emulsifier combination HLB is 20, the solution viscosity is 1.02 mPa-s, as a continuous phase 2 (W2).

[0113] S2, the cross-linking solvent prepared in step S1 is quickly dispersed into the continuous phase O1 by stirring at 9000 rpm for 3 min to obtain solution 1.

[0114] S3, select a membrane with a water contact angle of 115°, the average pore size of the membrane is 0.12 μm, and the pore density is 0.65. The membrane area is 12 cm 2 . Install the membrane into the membrane module and fix it with nuts to avoid movement and air leakage during subsequent operations. Install and link the membrane emulsification system in sequence with the membrane module Figure 1 ). W1 is the dispersed phase, which is placed in the dispersed phase container. The volume of the dispersed phase W1 is 9 mL. After setting the constant pump speed to 100 mL / min, solution 1 obtained in step S2 is pumped into the membrane module to generate circulation. After adjusting the pressure to 0.07 MPa by opening the valve of the nitrogen cylinder, the dispersed phase is dispersed through the membrane as a medium to obtain an emulsion, and the primary emulsion W1 / O1 is prepared.

[0115] S4, place the primary emulsion W1 / O1 at 25°C for 12 hours, centrifuge and evaporate the solvent, and rinse with distilled water. After freeze-drying, the inner microspheres are obtained.

[0116] S5, disperse the inner microspheres obtained by freeze-drying in step S4 in the dispersed phase O2.

[0117] S6, select a membrane with a water contact angle of 30°, the average pore size of the membrane is 0.45 μm, and the pore density is 0.65. The membrane area is 20 cm 2 . Install the membrane into the membrane module and fix it with nuts to avoid movement and air leakage during subsequent operations. Install and link the membrane emulsification system in sequence with the membrane module Figure 1). The solution O2 obtained in step S5 is the dispersed phase. The volume of the dispersed phase O2 is 20 mL. The constant pump rate is set to 25 mL / min, and the continuous phase W2 is pumped into the membrane module to generate circulation. Before this step (after the valve of the nitrogen cylinder is opened and the pressure is adjusted to 0.12 MPa, the dispersed phase passes through the membrane as a cutoff dispersion to obtain an emulsion), delamination occurs (as shown in Figure 6 , and the W1 / O2 / W2 multiple emulsion cannot be prepared.

[0118] Comparative Example 4

[0119] The same as Example 1, except that the continuous phase 1 (O1) does not use a combined emulsifier, and SPAN 85 is used, and the amount of SPAN 85 added is the sum of the mass of SPAN 85 and polyglycerol castor oil PGPR in Example 1. The W1 / O2 / W2 multiple emulsion cannot be prepared.

[0120] Comparative Example 5

[0121] The same as Example 2, except that the membrane selected in step S6 has a contact angle of 65°, and the other steps are consistent. Delamination also occurs, and the W1 / O2 / W2 multiple emulsion cannot be prepared.

[0122] Comparative Example 6

[0123] In comparison with Example 2, only one emulsifier is used in the continuous phase W2 in step S1, and no combined emulsifier is used.

[0124] S1, precisely weigh 3 g of chitosan, heat, and dissolve it in 100 g of an acetic acid aqueous solution (pH = 3.8) as the dispersed phase 1 (W1); precisely weigh SPAN 85 and SPAN 80, heat, and dissolve them in ethyl acetate, the emulsifier combination HLB is 5.5, and the solution viscosity is 2.2 mPa·s, as the continuous phase 1 (O1); precisely weigh 1 g of TPP, dissolve it in 100 g of pure water, as the cross-linking solvent; precisely weigh 0.5 mg of polycaprolactone PCL, and ultrasonically heat and dissolve it in 12 mL of ethyl acetate as the dispersed phase 2 (O2). Precisely weigh the polyvinyl alcohol PVA, heat, and dissolve it in pure water, the emulsifier combination HLB is 16, and the solution viscosity is 1.04 m·Pa·s, as the continuous phase 2 (W2).

[0125] S2, the cross-linking solvent prepared in step S1 is quickly dispersed into the continuous phase O1 by stirring at 5000 rpm for 5 min to obtain solution 1;

[0126] S3, a membrane sheet with a water contact angle of 105° is selected, the average pore size of the membrane sheet is 0.08 μm, and the pore density is 0.60. The membrane area is 12 cm 2The membrane is installed into the membrane module and fixed with nuts to avoid moving and air leakage in the following operation. The membrane module is installed and linked with the membrane emulsification system in sequence. Figure 1 W1 is the dispersed phase, which is put into the dispersed phase container. The volume of the dispersed phase W1 is 10 mL. After setting the constant pump speed at 60 mL / min, the solution 1 obtained in step S2 is pumped into the membrane module to generate circulation. After opening the valve of the nitrogen cylinder and adjusting the pressure to 0.10 MPa, the dispersed phase is dispersed through the membrane as a medium to obtain an emulsion, and a primary emulsion W1 / O1 is prepared.

[0127] S4, the primary emulsion W1 / O1 is left at 20°C for a period of time, centrifuged and the solvent is evaporated, and then washed with distilled water. After freeze-drying, the inner microspheres are obtained.

[0128] S5, the inner microspheres obtained by freeze-drying in step S4 are re-dispersed in the dispersed phase O2.

[0129] S6, a membrane with a water contact angle of 20° is selected, the average pore size of the membrane is 0.32 μm, and the pore density is 0.60. The membrane area is 20 cm 2 The membrane is installed into the membrane module and fixed with nuts to avoid moving and air leakage in the following operation. The membrane module is installed and linked with the membrane emulsification system in sequence. Figure 1 The dispersed phase solution O2 obtained in step S5 is the dispersed phase. The volume of the dispersed phase O2 is 20 mL. After setting the constant pump speed at 40 mL / min, the continuous phase W2 is pumped into the membrane module to generate circulation. After opening the valve of the nitrogen cylinder and adjusting the pressure to 0.09 MPa, the dispersed phase is dispersed through the membrane as a cutoff to obtain an emulsion, and a re-emulsion W1 / O2 / W2 is prepared.

[0130] S7, the W1 / O2 / W2 re-emulsion is poured into a 1 wt% PVA aqueous solution at a certain speed, and the solvent is evaporated by stirring at room temperature. The monodisperse polymer microspheres are separated by centrifugation, washed with distilled water, and freeze-dried to obtain composite microspheres.

[0131] The composite microspheres are dyed with a fat-soluble dye. Figure 7 The left image is a microscopic image, and the right image is a fluorescence microscopic image. In this system, only the PCL material can be dyed red, and the chitosan cannot be dyed red with the fat-soluble dye, Figure 7 Almost all the microspheres are red solid spheres in the middle right, indicating that the PCL-chitosan composite microsphere structure is not formed. And the size of all the microspheres is below 15 μm.

[0132] Comparative Example 7

[0133] In comparison with Example 2, only one emulsifier is used in the continuous phase W2 in step S1, and no combination of emulsifiers is used.

[0134] Compared with Comparative Example 6, Tween 20 was used in the continuous phase in step S1, and the rest of the steps were the same. The PCL-chitosan composite microsphere structure could not be effectively formed (as shown in Figure 8 ), and the oil emulsion was layered.

[0135] Comparative Example 8

[0136] The same as Example 2, except that the temperature in step S4 was 40°C. Chitosan began to degrade and became strips or other irregular shapes, as shown in Figure 9 .

[0137] Comparative Example 9

[0138] The same as Example 1, except that the membrane area in S3 was 12 cm 2 , the volume of the dispersed phase was 36 mL, and the ratio of the dispersed phase to the membrane area was 3:1 (mL:cm 2 ).

[0139] As shown in Figure 10 , the prepared PCL-sodium alginate core had a radius of about 252 μm, and the shell had a thickness of about 100 μm, with a shell thickness:core radius ratio of 1:2.52.

[0140] Comparative Example 10

[0141] The same as Example 1, except that the membrane area in S6 was 20 cm 2 , the volume of the dispersed phase was 40 mL, and the ratio of the dispersed phase to the membrane area was 2:1 (mL:cm 2 ).

[0142] As shown in Figure 11 , the prepared PCL-sodium alginate core had a radius of 12 μm, and the shell had a thickness of about 80 μm, with a shell thickness:core radius ratio of 20:3 (mL:cm 2 ).

[0143] Comparative Example 11

[0144] The same as Example 2, except that the concentration of agarose in S8 was 0.01 wt%.

[0145] As shown in Figure 12 , the degradation rate increased significantly, which was not conducive to long-term storage.

[0146] Comparative Example 12

[0147] The same as Example 2, except that the concentration of agarose in S8 was 5 wt%, which was too high, causing the needle to be blocked and difficult to advance.

[0148] Comparative Example 13

[0149] The same as example 1, except that: S6 chooses the commonly used homogenization stirring method emulsification, the speed is 1000 rpm, and the time is 6 min. The microspheres obtained by this method form a multi-core-single-shell microsphere structure, and the outer shell is more than 100 μm, as shown in Figure 13 The 168-hour IL-6 expression amount is 110.36 pg / mL, which is much higher than the IL-6 expression amount of example 1 Figure 18 ) at any test period, indicating a high degree of inflammation.

[0150] Comparative example 14

[0151] The same as example 1, except that: S6 chooses the commonly used homogenization stirring method emulsification, the speed is 10000 rpm, and the time is 6 min. The average microsphere shell thickness-core radius of the microspheres prepared by this method is about 1:6, and the shell-core structure is as shown in Figure 14 The shell is only 2 μm thick, and the 24-hour fibroblast migration rate is only 2.3%, which cannot effectively stimulate the migration and growth of fibroblasts.

[0152] Example 3

[0153] The same as example 1, except that:

[0154] In step S1, the concentration of the dispersed phase W1 is 1 wt%; the dynamic viscosity of the continuous phase O1 is below 1.55 mPa·s (20°C); the concentration of the cross-linking solvent is 1% g / L (solute / solvent); the first emulsifier is a liposoluble emulsifier with a hydrophilic-lipophilic balance (HLB) of 2, and the first emulsifier is Span85 and Span80 with a mass ratio of 1:1; the concentration of the dispersed phase O2 is 3% g / L (solute / solvent); and the dynamic viscosity of the continuous phase W2 is 1.01 mPa·s. The mass ratio of the cross-linking agent to the natural polysaccharide is 2.5:1.

[0155] In step S3, the water contact angle of the membrane sheet contacting solution 1 end is 100°, the average pore size is 0.08 μm, the pore density is 0.30, and the ratio of the volume of the dispersed phase W1 to the membrane area is 1:1 mL / cm 2 , the pump speed is 50 mL / min, and the pressure is 0.05 MPa.

[0156] In step S4, the primary emulsion W1 / O1 is placed at 15°C.

[0157] In step S5, the mass ratio of the inner microspheres to polycaprolactone in the dispersed phase O2 is 1:2.

[0158] In step S6, the water contact angle of the membrane sheet contacting the continuous phase W2 end is 30°, the average pore size is 0.15 μm, the pore density is 0.3, and the ratio of the volume of the dispersed phase O2 to the membrane area is 1:1 mL / cm 2pump speed was 25 mL / min and the pressure was 0.08 MPa.

[0159] In step S7, the coagulation aid solution was a 1wt% PVA aqueous solution.

[0160] In step S8, the concentration of the agar polysaccharide was 3wt%.

[0161] Example 4

[0162] The same as Example 1, except that:

[0163] In step S1, the concentration of the dispersed phase W1 was 3.5% g / L (solute / solvent); the dynamic viscosity of the continuous phase O1 was 2.3 mPa-s (20°C) or less; the concentration of the cross-linking solvent was 3% g / L (solute / solvent); the first emulsifier was a liposoluble emulsifier with a hydrophilic-lipophilic balance (HLB) of 6, and the first emulsifier was Span 80 and Span 60 at a mass ratio of 1:1; the concentration of the dispersed phase O2 was 8% g / L (solute / solvent); and the dynamic viscosity of the continuous phase W2 was 1.05 mPa-s. The mass ratio of the cross-linking agent to the natural polysaccharide was 1:1.

[0164] In step S3, the water contact angle of the membrane contact solution 1 end was 115°, the average pore size was 0.20 μm, the pore density was 0.65, and the ratio of the volume of the dispersed phase W1 to the membrane area was 1:10 mL / cm 2 The pump speed was 100 mL / min and the pressure was 0.10 MPa.

[0165] In step S4, the primary emulsion W1 / O1 was left to stand at 35°C.

[0166] In step S5, the mass ratio of the inner microsphere to the polycaprolactone in the dispersed phase O2 was 1:4.

[0167] In step S6, the water contact angle of the membrane contact continuous phase W2 end was 50°, the average pore size was 0.50 μm, the pore density was 0.65, and the ratio of the volume of the dispersed phase O2 to the membrane area was 1:10 mL / cm 2 The pump speed was 50 mL / min and the pressure was 0.15 MPa.

[0168] In step S7, the coagulation aid solution was a 3wt% PVA aqueous solution.

[0169] In step S8, the concentration of the agar polysaccharide was 5wt%.

[0170] Test Example:

[0171] 1. The microspheres obtained in Example 2, Comparative Example 1, and Comparative Example 2 were subjected to a fibroblast proliferation test.

[0172] 96-well plates were set up for control and drug groups, and 100 μL of cell suspension (about 5000 cells per well) and 100 μL of complete medium were added to each well, and the cells were cultured for 24 h to adhere completely. The required 4 groups of microspheres were weighed and placed in a clean bench for ultraviolet irradiation for 24 h. The various microspheres were diluted to 800 μg / ml of microsphere suspension using DEME high-sugar medium (containing 3% antiserum, without serum). The old culture medium in the 96-well plate was discarded, and the control wells, sample wells, and blank wells were sequentially added, with 3 parallel repeated wells. The plate was placed in a cell culture incubator for culture. CCK-8 working solution was prepared, and the absorbance of the liquid in the wells at 450 nm was detected using a microplate reader, and the cell survival rate was calculated according to the formula.

[0173]

[0174] Example 2, Comparative Example 1, 2 were compared, as shown in Figure 15 Although most products recommend porous microspheres to stimulate collagen regeneration and cell adhesion, the side effects they produce can reduce cell viability and proliferation rate. In addition, it was observed that fibroblasts had already grown in an orderly manner around the microspheres of Example 2 after 24 hours Figure 16 ).

[0175] 2. The microspheres obtained from Example 1, Comparative Example 9 and Comparative Example 10 were subjected to fibroblast inflammation test, fibroblast wound healing test, fibroblast collagen test, and complex PCL microsphere simulation room temperature degradation experiment.

[0176] Fibroblast inflammation test: control and drug groups were set up, 100 μL of cell suspension was added to the 96-well plate, and the control and experimental drug groups were added to the experimental group, respectively. The control group was added with 100 μL of high-sugar medium (containing 4% antiserum, without serum), and was placed in a 37°C, 5% CO2 incubator for culture. First, the supernatant of the cell culture fluid was collected.

[0177] Subsequently, the sample and standard were added to the ELISA plate well, the corresponding antibody and enzyme substrate were added, and incubation and color development were performed. Finally, the absorbance was read at 450 nm wavelength using a microplate reader, and the concentrations of COX-2 and IL-6 were calculated by combining the standard curve.

[0178] Subsequently, the sample and standard were added to the ELISA plate well, the corresponding antibody and enzyme substrate were added, and incubation and color development were performed. Finally, the absorbance was read at 450 nm wavelength using a microplate reader, and the concentrations of COX-2 and IL-6 were calculated by combining the standard curve.

[0179] Fibroblast wound healing test: Scratch inserts were placed in 6-well plates, 100 μL of cell suspension was added to each well of the scratch insert, and incubated at 37 °C, 5% CO2 incubator for 24 h to form a uniform monolayer of cells scratch, simulating artificial wounds. The control group was set up, and the drug group was treated with high glucose medium (containing 4% triantigen, without serum). The initial width of the scratch was recorded, and images were taken at the set time points to observe and record the process of cell scratch. The wound closure degree at different time points was quantified by percentage of wound area, and analyzed using ImageJ software. The calculation formula is as follows:

[0180]

[0181] Fibroblast collagen and alpha smooth muscle actin test: The levels of type I and type III collagen and alpha smooth muscle actin were determined by ELISA method, the samples and standard were added to the ELISA plate wells, the corresponding antibody and enzyme substrate were added, incubated and developed. Finally, the absorbance was read at 450 nm wavelength using a microplate reader, and the total amount of type I and type III collagen and the expression amount of alpha smooth muscle actin were calculated by combining the standard curve.

[0182] Composite PCL microspheres room temperature degradation experiment: Samples of similar size and weight (50 mg) were used. First, the initial dry weight (W0) of the sample was determined, and the weight (Wt) at each time interval was recorded. The remaining weight percentage was calculated using the following formula:

[0183]

[0184] Test results:

[0185] COX-2 significantly affects the proliferation, survival and fibrotic function of fibroblasts through the interaction of inflammatory mediators and other pro-fibrotic signaling pathways. As shown in Figure 17 PCL composite microspheres obtained in Example 1 have a specific shell thickness, which can up-regulate the expression of COX-2 at an early stage (24 hours) to stimulate the growth of fibroblasts and collagen, and the expression of COX-2 is significantly maintained at a low level at a later stage (96 and 168 hours). The overall microsphere size meets the size requirements of injectable microspheres. A too thick PCL shell leads to a long-term high level of COX-2 expression (Comparative Examples 9 and 10). The composite PCL microspheres of Example 1 can regulate the expression level of inflammatory factors of fibroblast proliferation in a time sequence.

[0186] IL-6 is a typical inflammatory factor of inflammatory response. As shown in Figure 18 PCL composite microspheres prepared in Example 1 have low inflammation and can effectively inhibit the high level expression of IL-6 (Comparative Examples 9 and 10).

[0187] As shown in Figure 19 , the cell repair rate of Example 1 is significantly higher than that of Comparative Examples 9 and 10.

[0188] As shown in Figure 20 and 21 , the amount of collagen produced is comparable or even better, but the inflammatory factors produced are lower. This indicates that even under low levels of inflammatory expression, collagen production is still continuing.

[0189] The composite PCL microsphere structure obtained in Example 1 is more conducive to regulating the production of fibroblasts and extracellular matrix in a low inflammatory environment. Therefore, under a low inflammatory state, the PCL composite microspheres obtained in Example 1 can still produce collagen at a high level. As shown in Figure 22 , the alpha smooth muscle actin has been significantly increased within 24 hours after early treatment, and compared with Comparative Examples 9 and 10, the growth rate of alpha smooth muscle actin in Example 1 is slowed down in the later stage, which can reduce the disordered growth in the later stage after use.

[0190] The above description of the embodiments is only used to help understand the technical solutions of the present application and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A composite PCL microsphere, characterized in that, The composite PCL microspheres have a shell thickness to core radius ratio of 1:1 to 1:3, and a particle size of 20-65 µm. The preparation method of the composite PCL microspheres includes the following steps: S1. Natural polysaccharides are dissolved in a first solvent as the dispersed phase W1; a first emulsifier is dissolved in an organic solvent, wherein the first emulsifier is a lipid-soluble emulsifier with a hydrophilic-lipophilic balance value of 2-6, and is selected from two or more of Span85, Span80, Span60, and polyglycerol ricinoleate, as the continuous phase O1; a crosslinking agent is dissolved in a second solvent, wherein the crosslinking agent is calcium chloride or tripolyphosphate, as the crosslinking solvent; polycaprolactone is dissolved in a third solvent as the dispersed phase O2; a second emulsifier is dissolved in a fourth solvent, wherein the second emulsifier is selected from two or more of PVA, Tween20, and SDS, as the continuous phase W2; S2. Disperse the crosslinking solvent prepared in step S1 into the continuous phase O1 to obtain solution 1; S3. Place the dispersed phase W1 into the dispersed phase container, pump the solution 1 obtained in step S2 into the membrane module to generate circulation, introduce nitrogen gas to adjust the pressure of the membrane module to a stable state, and then disperse the dispersed phase W1 through the membrane as a medium to obtain an emulsion, namely the primary emulsion W1 / O1. S4. After the colostrum W1 / O1 is allowed to stand at 15°C-35°C, the solvent is evaporated by centrifugation and then freeze-dried to obtain the inner microspheres. S5. Disperse the microspheres obtained in step S4 in the dispersed phase O2 obtained in step S1 to obtain solution 2. S6. Place solution 2 into the dispersion phase container. The continuous phase W2 obtained in step S1 is pumped into the membrane module to generate circulation. After nitrogen gas is introduced to adjust the pressure of the membrane module to a stable state, solution 2 is dispersed through the membrane as a medium to obtain an emulsion, namely, a double emulsion W1 / O2 / W2. The water contact angle at the end of the membrane in contact with the continuous phase W2 is 20°-50°. S7. Pour the composite emulsion W1 / O2 / W2 into the curing aid solution, stir at room temperature to evaporate the solvent, centrifuge, wash, freeze dry, and obtain composite PCL microspheres.

2. The composite PCL microspheres according to claim 1, characterized in that, In step S1, the first solvent is an acidic aqueous solution, and the concentration of the dispersed phase W1 is 1-3.5 wt%; the organic solvent is ethyl acetate, and the dynamic viscosity of the continuous phase O1 is 1.55-2.30 mPa·s; the second solvent is water, and the concentration of the crosslinking solvent is 1%-3% g / L.

3. The composite PCL microspheres according to claim 1 or 2, characterized in that, In step S1, the third solvent is water, the concentration of the dispersed phase O2 is 3%-8% g / L; the second emulsifier is selected from two or more of PVA, Tween 20 and SDS; the fourth solvent is ethyl acetate; and the dynamic viscosity of the continuous phase W2 is 1.01-1.05 mPa·s.

4. The composite PCL microspheres according to claim 1 or 2, characterized in that, The specific steps of step S2 are as follows: the crosslinking solvent prepared in step S1 is dispersed into the continuous phase O1 by stirring to obtain solution 1. The stirring speed is 5000-10000 rpm and the stirring time is 3-5 min. The particle size of the crosslinking solvent pre-dispersed in the continuous phase O1 is less than 300 nm.

5. The composite PCL microspheres according to claim 1 or 2, characterized in that, In step S3, the water contact angle at the membrane contacting solution end is 100°-115°, the average pore size is 0.08-0.20 µm, the pore density is 0.30-0.65, and the volume ratio of the dispersed phase W1 to the membrane area is 1:1-1:10 mL / cm². 2 The pump speed is 50-100 mL / min, and the pressure is 0.05-0.10 MPa.

6. The composite PCL microspheres according to claim 1 or 2, characterized in that, In step S6, the average pore size is 0.15-0.50 µm, the pore density is 0.3-0.65, and the volume ratio of the dispersed phase O2 to the membrane area is 1:1 to 1:10 mL / cm². 2 The pump speed is 25-50 mL / min, and the pressure is 0.08-0.15 MPa.

7. The composite PCL microspheres according to claim 1 or 2, characterized in that, In step S7, the curing aid solution is a 1-3 wt% PVA aqueous solution.

8. The composite PCL microspheres according to claim 1 or 2, characterized in that, The method also includes step S8, dispersing the composite PCL microspheres prepared in step S7 into a microsphere protectant solution, and then transferring them to an ice bath to cool them down, thereby obtaining composite PCL microspheres.

9. The application of the composite PCL microspheres according to claim 1 in the preparation of dermal fillers.

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