JP2018529456A - Method for producing hollow porous microspheres - Google Patents

Abstract

The present invention relates to a production step of a polymer solution comprising dissolving a pore formation inducing substance and a hydrophobic biodegradable polymer in a volatile solvent; and dispersing the polymer solution in water or an aqueous solution containing a phase stabilizer. Forming an O / W (oil in water) type emulsion; evaporating a volatile solvent on an aqueous solution in which the polymer solution is dispersed to solidify the hydrophobic biodegradable polymer To generate a spontaneous phase separation between the hydrophobic biodegradable polymer and the pore formation inducer, and an oil phase contained in an oil in water (O / W) type emulsion (emulsion) A step of producing hollow porous microspheres comprising converting a polymer solution into O / S (Oil in Solid) microspheres; and a step of removing pore formation inducing substances contained in the hollow porous microspheres To provide a method of manufacturing a hollow porous microspheres containing.

Description

  The present invention relates to a method of manufacturing a tissue engineering scaffold that can be transferred into living tissue such as skin tissue.

  Of the medical devices for cosmetic treatment sold for the purpose of cosmetic treatment, a filler is used in such a manner that it is injected into the skin tissue of a wrinkle or depression. Most fillers are injected into the skin tissue to improve skin wrinkles and depressions in a manner that increases the volume of the skin tissue by the volume of the material itself. Currently, the most frequently used fillers are gel-like intangible substances such as hyaluronic acid and collagen. However, since they are absorbed into the skin tissue over time, the effect of the cosmetic treatment is not maintained for a long time. There is.

  In addition, in order to have a wide range of use as a medical device for cosmetic treatment, it must be transmitted by injection, but the porous material that is an already developed filler component has a size of several tens of mm or more, There is a disadvantage that transplantation into skin tissue is impossible by injection, and it must be transplanted into the skin tissue exclusively by an operation incising the skin tissue.

  As a conventional method for producing a porous material, as a porogen for forming voids in hydrophobic biodegradable polymer microspheres, water-soluble materials such as alginate, collagen, and gelatin are used. Applying a polymer material, O / W (Oil in Water), W / O (Water in Oil), W / O / W (Water in Oil in Water), or O / W / O (Oil in Water in) There is a technique for forming a multi-phase emulsion (Oil). However, it is difficult to form microspheres having large pores in the O / W type process. In the case of W / O, W / O / W and O / W / O type processes, a two-phase emulsification process is required, and both an inner-phase stabilizer and an outer-phase stabilizer for stabilizing the two-phase emulsification Required. In addition, since the specific gravity of the produced fine particles is heavier than that of water, there is a disadvantage in that a filtering step is separately required to obtain the precipitated fine particles. Furthermore, since the manufacturing process is complicated and there are many factors to control, the production is performed by a batch reaction process, so that the production efficiency is lowered and the uniformity of the voids formed in the microspheres is inferior. There is a problem that industrial application is difficult because of the high loss rate.

Korean Published Patent Publication No. 2011-0075618

  It is an object of the present invention to provide a method for manufacturing a tissue engineering scaffold that can be transferred into living tissue such as skin tissue. Specifically, since the porosity and shape of the porous microspheres are simple and the process can be easily adjusted, the porous microspheres can be injected into the skin tissue by injection, and the cells are contained in the porous microspheres. An object of the present invention is to provide a method for producing porous microspheres that can increase the volume of skin tissue by increasing the volume of the skin.

In order to solve the above-described object, one aspect of the present invention is a method for producing hollow porous microspheres,
Producing a polymer solution comprising dissolving a pore formation inducer and a hydrophobic biodegradable polymer in a volatile solvent;
Dispersing the polymer solution in water or an aqueous solution containing a phase stabilizer to form an oil in water (O / W) type emulsion;
Volatile solvent is volatilized on the aqueous solution in which the polymer solution is dispersed to solidify the hydrophobic biodegradable polymer, so that the hydrophobic biodegradable polymer and the pore formation inducing substance are between. Hollow porosity that generates spontaneous phase separation and converts oil phase polymer solution contained in the O / W (oil in water) type emulsion into O / S (oil in solid) microspheres A step of producing microspheres; and a step of removing pore formation inducers contained in the hollow porous microspheres,
A method for producing hollow porous microspheres is provided.

  The production method of the present invention is contained in an O / W (oil in water) type emulsion by spontaneous phase separation of a hydrophobic biodegradable polymer contained in porous microspheres and a pore formation inducer. The oil phase polymer solution can be easily realized in the structure of O / S (Oil in Solid) microspheres, and an internal phase stabilizer required for the production of two-phase emulsion type microspheres is added. Therefore, it is not necessary to consider a washing step for removing the internal phase stabilizer. Therefore, using a conventional hydrophilic pore formation inducing substance, the production process can be simplified compared to the technique of manufacturing W / O / W and O / W / O types by separate primary and secondary emulsification processes. Can do.

  The present invention can easily adjust the void ratio and the shape and size of the voids in the microsphere, and produce the micropores included in the hollow and the partition wall surrounding the hollow in which the skin tissue cells in the microsphere can grow. And is excellent in pore uniformity.

  As described above, according to the production method of the present invention, the hollow porous microsphere has a double structure including a large cavity formed in the center of the particle and a partition wall including fine pores, so that skin tissue cells in vivo Can easily move into the hollow porous microspheres using the micropores as channels, and the moved skin tissue cells can effectively proliferate in the giant cavity.

1 is a schematic diagram relating to a method of manufacturing hollow porous microspheres according to one embodiment of the present invention. FIG. It is the figure which showed the electron scanning micrograph of the particle | grain surface by the kind and content of the porogen at the time of manufacture of the hollow porous microsphere based on one Example of this invention, and a content | section. It is the figure which showed the Live / dead staining confocal microscope picture which cultured the fibroblast with the hollow porous microsphere which concerns on one Example of this invention. It is the figure which showed the cell growth rate by MTT assay after culturing the fibroblast with the hollow porous microsphere which concerns on one Example of this invention.

  In the present specification, the “average diameter” means a value obtained by averaging the diameters that are line segments connecting both ends of the cross section of the object. For example, in the case of the cavity of the present invention, the average diameter is formed in the hollow porous microsphere. It may mean an average value of the diameters of the cavities themselves when the cavities are not spherical. Or you may mean the average value of the diameter of each cavity which exists in a some microsphere. In the case of fine pores, it may mean the average value of the diameters of the fine pores themselves or the average value of the diameters of a plurality of fine pores when the fine pores themselves are not spherical.

  The present invention will be described in detail below.

  The present invention relates to a production process of a polymer solution comprising dissolving a pore formation inducer and a hydrophobic biodegradable polymer in a volatile solvent; the polymer solution is mixed with water or a phase stabilizer. Dispersing in an aqueous solution to form an O / W (oil in water) type emulsion; volatilizing a volatile solvent on the aqueous solution in which the polymer solution is dispersed to form the hydrophobic biodegradable material The polymer is solidified to generate a spontaneous phase separation between the hydrophobic biodegradable polymer and the pore formation inducer to form an oil-in-water (O / W) type emulsion. A step of producing hollow porous microspheres for converting the contained oil phase polymer solution into O / S (Oil in Solid) microspheres; and included in the hollow porous microspheres And comprising removing the pore-inducing agent, to provide a method of manufacturing a hollow porous microspheres.

  In multi-phase emulsion formation technology that has been applied as a conventional technology for producing porous microspheres, that is, multi-phase emulsification technology, an oil phase containing a hydrophobic biodegradable polymer is made into a continuous phase to form voids. The aqueous phase containing the pore formation inducer to be applied must be applied in a discontinuous phase to form a primary emulsion. In order to make the size and distribution of the discontinuous phase in the primary emulsion produced at this time uniform, continuous stirring and temperature control were essential. However, according to one embodiment of the present invention, since the hydrophobic biodegradable polymer and the void and pore formation inducer are dissolved in the same solvent, the composition uniformity and composition stability can be achieved without performing separate operations. Sex can be secured.

  In addition, in the conventional multi-phase emulsion formation technique, after the secondary emulsion is formed, stirring and temperature maintenance are possible to maintain the size and shape uniformity of the discontinuous phase in the secondary emulsion. Hydrophobic biodegradable polymer solidifies and stabilizes the size and distribution of the discontinuous phase within the discontinuous phase formed within the emulsion, that is, the aqueous solution containing pore formation inducers such as water-soluble polymers There is a problem that there is no means that can be controlled by the time. On the other hand, according to one embodiment of the present invention, a hydrophobic fluid that is not compatible with a hydrophobic biodegradable polymer and has a lower density than water is used as a pore formation inducing substance. When the volatile solvent is removed, the hydrophobic biodegradable polymer is solidified, and the hydrophobic and liquid pore-forming inducer that is not compatible with the hydrophobic biodegradable polymer spontaneously phase-separates, Voids are formed in microspheres made of a hydrophobic biodegradable polymer. Therefore, the hydrophobic biodegradable polymer can maintain the solution phase before the hydrophobic biodegradable polymer is solidified without performing a separate operation, and can ensure composition uniformity. When solidified, the uniformity and stability of the voids can be ensured by the strength of the hydrophobic biodegradable polymer.

  As an example, the pore formation inducing substance may include a hydrophobic fluid that is not compatible with a hydrophobic biodegradable polymer and has a density lower than that of water. The pore formation inducing substance is also referred to as porogen. The type of pore formation inducer is not particularly limited as long as it is not compatible with a hydrophobic biodegradable polymer and has a lower density than water. Specifically, the pore formation inducing substance may be a substance having a boiling point at 1 atm and 250 ° C. or lower, and may be a liquid substance at 1 atm and 30 to 150 ° C. For example, the pore formation inducer may be one or more selected from the group consisting of alkanes, vegetable oils, and mixtures thereof. In one embodiment, the alkane is one or more selected from the group consisting of octane, undecane, tridecane, pentadecane, and mixtures thereof, and the vegetable oil May include one or more selected from the group consisting of soybean oil, corn oil, cottonseed oil, olive oil, grape seed oil, walnut oil, sesame oil, sesame oil, and mixtures thereof. In the present invention, pores can be formed by applying other substances in addition to the specific substance. As an example, the present invention forms pores during the production of the hollow porous microspheres. Depending on the concentration and type of porogen, the size and shape of the hollow and fine pores can be adjusted, and the pore uniformity can be increased. For example, the pore formation inducing substance may be contained in an amount of 0.1 to 50% by mass, more specifically 1 to 10% by mass with respect to the total mass of the polymer solution.

  In addition, the hydrophobic biodegradable polymer according to an embodiment of the present invention includes polylactic acid (Poly-L-Lactic Acid, PLLA), polyglycolic acid (PGA), and polylactic acid-glycolic acid. Polymer (poly-co-glycic acid, PLGA), poly-ε- (caprolactone) (Polycaprolactone, PCL), polyanhydrides, polyorthoester, polyvinyl alcohol, polyvinyl, polyethylene Glycol (polyethyleneglycol), polyurethane (polyurethane), polyacrylic acid (polyacrylic acid) , Poly-N-isopropylacrylamide, poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) -polypropylene oxide-polyethylene ester ), A copolymer thereof, and one or more selected from the group consisting of a mixture thereof, as long as it is safe and degradable when injected into the skin tissue. It is not limited to.

  The step of converting the oil phase polymer solution contained in the O / W (Oil in Water) type emulsion according to an embodiment of the present invention into O / S (Oil in Solid) microspheres includes the steps of: Supplying an aqueous solution containing water or a phase stabilizer to a continuous phase and supplying the polymer solution to the aqueous solution containing the water or the phase stabilizer in a discontinuous phase. May contain. At this time, the aqueous solution containing the phase stabilizer is an aqueous solution containing a substance that stabilizes the interface. For example, polyvinyl alcohol (PVA), polyvinyl pyrrolidone, polyethylene glycol-co-polypropylene. Glycol (polyethylene glycol-co-polypropylene glycol), polyethylene glycol-co-polypropylene glycol-co-polyethylene glycol (polyethylene glycol-co-polypropylene glycol, polyethylene glycol-co-polypropylene glycol (polyethylene glycol-co-polypropylene glycol) One or more phase stabilizers selected from the group consisting of co-polylactic acid and polyethylene glycol-co-polylactic acid-polyethylene glycol were dissolved. It is an aqueous solution.

  Specifically, the step of dispersing the polymer solution in an aqueous solution containing water or a phase stabilizer, as an example, supplying the aqueous solution containing the water or phase stabilizer to a microfluidic device in a continuous phase, The method may include a step of supplying the polymer solution in a discontinuous phase to an aqueous solution containing water or a phase stabilizer supplied in the continuous phase. At this time, the step adjusts the rate at which the polymer solution is supplied in a discontinuous phase into the microconduit that transports the continuous phase, so that particles of hollow porous microspheres and hollow and micropores The method may further include adjusting the size and shape of one or more of the pores.

  Alternatively, the step of converting an oil phase polymer solution contained in the O / W (Oil in Water) type emulsion according to an embodiment of the present invention into O / S (Oil in Solid) microspheres may include: Supplying the aqueous solution to a membrane emulsifier in a continuous phase, and supplying the polymer solution in a discontinuous phase to an aqueous solution containing water or a phase stabilizer supplied in the continuous phase. You can leave. Instead of the microconduit used in the microfluidic device, the discontinuous phase is transferred to the conduit through which the continuous phase flows through a membrane having a constant void size. At this time, the step is formed in the membrane of the membrane emulsification apparatus. The method may further include adjusting the size of the formed voids to adjust the size and shape of one or more of the hollow porous microsphere particles and the pores, such as hollow and fine pores.

  When the microfluidic device or the membrane emulsification apparatus is used, the size and shape of the microspheres, the particle size distribution of the hollows and pores can be controlled during the production of the microspheres.

  As an example, the cavity formed in the center of the hollow porous microsphere may have an average diameter of 5 to 150 μm. If the diameter of the cavity is less than 5 μm, cell growth becomes difficult, and if it exceeds 150 μm, the strength of the microsphere is too weak and can be destroyed during in vivo injection. Specifically, the average diameter of the cavity is 5 μm or more, 10 μm or more, 13 μm or more, 15 μm or more, 17 μm or more, 20 μm or more, 23 μm or more, 25 μm or more, 27 μm or more, 30 μm or more, 33 μm or more, 35 μm or more, 37 μm or more, It may be 40 μm or more, 43 μm or more, 45 μm or more, 48 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 110 μm or more, 120 μm or more, 130 μm or more, 140 μm or more. The average diameter of the cavity is 150 μm or less, 140 μm or less, 130 μm or less, 120 μm or less, 110 μm or less, 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, 47 μm or less, 45 μm or less, 43 μm or less. 40 μm or less, 37 μm or less, 35 μm or less, 33 μm or less, 30 μm or less, 28 μm or less, 25 μm or less, 23 μm or less, 20 μm or less, 18 μm or less, 15 μm or less, 13 μm or less, 10 μm or less, or 5 μm or less. The size is not necessarily limited to the above as long as the cell can grow.

  As an example, the hollow porous microsphere particles contained in the composition according to the present invention may have an average diameter of 50 to 200 μm, but can be injected into skin tissue by injection, and If the proliferation of skin tissue cells is possible, it is not necessarily limited to the above range. Since the hollow porous microspheres have a small particle size in the above-described range, they can be administered into the skin tissue via an injection needle having an inner diameter of 300 μm or less, and existing porous materials are large. The disadvantage of having to cut and transplant skin tissue according to the particle size can be solved. When the diameter of the hollow porous microsphere is less than 50 μm, it is difficult to grow skin tissue cells, and when it exceeds 200 μm, it is not easy to perform a treatment using an injection needle. Specifically, the average diameter of the hollow porous microsphere particles is 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 110 μm or more, 120 μm or more, 130 μm or more, 140 μm or more, 150 μm or more, 160 μm. As described above, it may be 170 μm or more, 180 μm or more, 190 μm or more, or 200 μm. The average diameter of the hollow porous microsphere particles is 200 μm or less, 190 μm or less, 180 μm or less, 170 μm or less, 160 μm or less, 150 μm or less, 140 μm or less, 130 μm or less, 120 μm or less, 110 μm or less, 100 μm or less, 90 μm or less, It may be 80 μm or less, 70 μm or less, 60 μm or less, or 50 μm.

  As an example, the volume of the hollow of the hollow porous microsphere may be 20 to 80% by volume with respect to the total volume of the hollow porous microsphere. When the volume is less than 20% by volume, there is not enough space for the skin tissue cells to proliferate. When the volume exceeds 80% by volume, the partition wall thickness becomes too thin, and the shape of the microsphere cannot be maintained and collapses. obtain. As an example, the thickness of the partition may be 1/5 to 1/2 of the total diameter of the hollow porous microsphere.

  Specifically, the fine pores included in the partition walls of the hollow porous microspheres may have an average diameter of 5 to 50 μm as an example. When the average diameter is less than 5 μm, skin tissue cells cannot move into the microsphere when the hollow porous microspheres are injected into the subcutaneous tissue, and when the average diameter exceeds 50 μm, the pores in the partition walls The volume occupied by increases and the form of microspheres becomes difficult to be maintained.

  For example, the average porosity of the hollow porous microspheres may be 20 to 80%.

  In the manufacturing method according to an embodiment of the present invention, the step of removing the pore formation inducing substance contained in the porous microspheres may include, as one embodiment, the pores by washing the microspheres with water and then freeze-drying them. A step of removing the formation inducer may be included. The lyophilization may be vacuum drying. The above-mentioned pore formation inducing substance used in the present invention maintains a liquid state at 1 atm and 30 to 150 ° C. and has a boiling point at 1 atm and 250 ° C. or less, and uses the hydrophobic biodegradable polymer of the present invention. The porous microspheres remain in a state where the voids are filled without being volatilized or evaporated during the manufacturing process of the porous microspheres. Therefore, until the pore formation inducing substance is removed through freeze-drying as described above, the void stability can be maximized while the pore formation inducing substance having voids formed therein is retained.

  The hollow porous microspheres manufactured by the above-described manufacturing method according to an embodiment of the present invention may include a cavity formed in the center; and a partition wall including fine pores surrounding the cavity. In addition, an embodiment of the present invention can provide a composition for injecting skin tissue or increasing volume of skin tissue, which includes hollow porous microspheres. When hollow porous microspheres according to one embodiment of the present invention are included in an injectable composition and injected into skin tissue, in vivo skin tissue cells migrate into the hollow porous microspheres through the micropores. In the central cavity, the migrated skin tissue cells can grow and regenerate or increase the volume of the skin tissue. That is, a cell existing in a living tissue can penetrate into the hollow porous microsphere, and can grow and divide to form a new living tissue. According to an embodiment of the present invention, by adjusting the mixing ratio of the void and pore formation inducing substance and the hydrophobic biodegradable polymer, the micropores of the cavity and the partition formed in the center of the microsphere The size and shape of the microspheres can be adjusted, and the fibroblasts, adipocytes, etc. existing outside the hollow porous microspheres can penetrate into the porous microspheres and then grow and divide Can be given.

  Further, according to the present invention, since the newly grown skin tissue cells retain their volume even after the hollow porous microspheres are biodegraded in the skin after a certain period of time, the skin wrinkle or skin depression site In addition to increasing the volume enhancement effect of the skin tissue, the effect can be maintained safely and for a long time. Accordingly, the present invention is a technique for manufacturing a tissue engineering scaffold that can be transmitted into a living tissue such as a skin tissue, and the hollow porous microspheres manufactured according to the present invention can be obtained through skin tissue regeneration. It can be applied not only to the improvement of skin wrinkles or depressions, but also to the pharmaceutical industry such as bone tissue regeneration. More specifically, the hollow porous microspheres of the present invention are used for the purpose of improving aging including skin wrinkles, improving skin color, or filler or lifting for improving skin elasticity. However, the present invention is not necessarily limited thereto as long as it is for tissue regeneration of the skin. For example, it may be used for artificial skin, artificial cartilage, bone filler, orthopedic prosthesis and the like.

  Hereinafter, the present invention will be described in more detail through examples. It is to be understood that these examples are merely illustrative of the present invention and that the scope of the present invention should not be construed as being limited by these examples. It will be obvious to the person.

[Example 1] Production 1 of hollow porous microspheres
Hollow porous microspheres according to an example of the present invention were manufactured by the following method (see FIG. 1).
Step 1: Fabrication of a simple fluidic device A 30-gauge injection needle bent at 90 ° is placed in a PVC tube, and a microfluidic device is fabricated by inserting a fine glass tube between the injection needle and the PVC tube. did. The manufactured microfluidic device was embedded with fine gaps using epoxy adhesive.

Step 2: Production of PLLA solution containing alkanes PLLA (RESOMER LR 704S, Evonik Industries AG) 0.1 g as a hydrophobic biodegradable polymer, Dichloromethane (34355-0350, Junsei) as a volatile solvent 10 g, alkane substances as pore formation inducers, specifically octane (Octane, 41236, Sigma-aldrich) or undecane (Undecane, U407, Sigma-aldrich) or tridecane (Tridecane, T57401, Sigma-aldrichene or pentadecane) , 76510, Sigma-aldrich) 0.1 g, 0.3 g and 0.6 g, respectively. In this way, a hydrophobic biodegradable polymer (PLLA) solution was produced.

Step 3: Production of a homogeneous PLLA emulsion A microfluidic device is fed with 2% PVA solution in a continuous phase with a flow rate of 1.5 ml per minute, and the PVA solution is fed with the alkane obtained in step 2 above. The PLLA solution containing the species was fed in a discontinuous phase with a flow rate of 0.1 ml per minute using a 30 cage syringe needle to form an emulsion with a constant size.

Step 4: Preparation of uniform hollow porous microspheres The emulsion obtained in Step 3 above was dispersed in a 2% PVA collection phase and stirred at 150 rpm, and then dichloromethane was sufficiently volatilized.

Step 5: Removal of pore formation inducing substance to provide porosity After washing the PLLA beads obtained in Step 4 several times with distilled water (DW), the alkanes are sublimated using a freeze dryer. A porous PLLA bead of hollow porous microspheres of uniform size was manufactured.

  The produced hollow porous microspheres are shown in FIG. It can be seen that the cavity is more effectively produced in the center of the microsphere as the content of pore formation inducer increases or as the length of the hydrocarbon chain increases.

[Example 2] Production of hollow porous microspheres 2
Hollow porous microspheres according to an example of the present invention were manufactured by the following method.

Step 1: Fabrication of a simple fluidic device A 30-cage injection needle bent at 90 ° is inserted into a PVC tube, and a microfluidic device is fabricated by inserting a fine glass tube between the injection needle and the PVC tube. did. The fabricated microfluidic device filled in the fine gaps using epoxy adhesive.

Step 2: Production of PLLA solution containing vegetable oil PLLA (RESOMER LR 704S, Evonik Industries AG) 0.1 g as hydrophobic biodegradable polymer, Dichloromethane (34355-0350, Junsei) 10 g, pore formation induction A hydrophobic biodegradable polymer (PLLA) solution was prepared by mixing 0.5 g of cottonseed oil or soybean oil as a substance.

Step 3: Production of a uniform PLLA emulsion A microfluidic device is fed with 2% PVA solution in a continuous phase with a flow rate of 1.5 ml per minute, and the PVA solution is fed to the plant obtained in Step 2 above. The PLLA solution containing the essential oil was supplied in a discontinuous phase with a flow rate of 0.1 ml per minute using a 30-cage syringe needle to form an emulsion of a certain size. .

Step 4: Preparation of uniform PLLA beads The emulsion obtained in Step 3 above was dispersed in 2% PVA collection phase and stirred at 150 rpm, and then dichloromethane was fully volatilized.

Step 5: Removal of internal pore formation inducing substance to provide porosity After the PLLA bead obtained in Step 4 is washed several times with distilled water (DW), vegetable oil is added using a freeze dryer. A hollow PLLA bead having hollow porous microspheres of uniform size was produced by removing.

[Test Example 1] Culture of skin tissue cells An experiment for comparing cell mobility and proliferation efficacy depending on the presence or absence of hollow formation of hollow porous microspheres according to an example of the present invention was performed as follows.

As comparative examples, porous microspheres without hollow (small pores: tridecane 3 mass%) and porous microspheres with hollow according to Example 1 (large pores: alkanes containing 6 mass% of tridecane) Each was sterilized with 70% ethanol, washed thoroughly with PBS, and NIH3T3 fibroblasts were transplanted to observe the behavior of cell proliferation. NIH3T3 fibroblasts are dispersed in a culture medium at a concentration of 1 × 10 ³ cells / mL and stirred for 6 hours using a spinner flask, and then the porous particles are transferred to a culture plate (culture plate) and cultured. did. After culturing the cells, the cultured porous microsphere particles were stained with LIVE / DEAD on the first, third, seventh, and tenth days, and the presence or absence of cell attachment and proliferation was confirmed by a confocal microscope and MTT assay. As shown in FIGS. 3 and 4, the hollow porous microspheres including the hollow are larger than the porous microspheres (small pores) that do not include the hollow and include only the fine pores. It can be confirmed that the penetration and proliferation of NIH3T3 fibroblasts is successful by the 10th day. This means that the hollow contained in the porous microspheres has a great effect on cell penetration and proliferation.

  The present invention can provide the following embodiment as an example.

  The first embodiment is a method for producing hollow porous microspheres, comprising: producing a polymer solution comprising dissolving a pore formation inducing substance and a hydrophobic biodegradable polymer in a volatile solvent; A step of dispersing a molecular solution in water or an aqueous solution containing a phase stabilizer to form an oil in water (O / W) type emulsion; a volatile solvent is added to the aqueous solution in which the polymer solution is dispersed; Volatilizes and solidifies the hydrophobic biodegradable polymer to generate spontaneous phase separation between the hydrophobic biodegradable polymer and the pore formation inducing substance, and O / W (Oil in A process for producing hollow porous microspheres comprising converting an oil phase polymer solution contained in a water-type emulsion into O / S (oil in solid) microspheres; and said hollow porosity Comprising removing the pore forming inducing substance contained in globules, it is possible to provide a method for producing a hollow porous microspheres.

  The second embodiment is a hollow porous microporous material according to the first embodiment, wherein the pore formation inducing substance is a hydrophobic fluid that is not compatible with a hydrophobic biodegradable polymer and has a density lower than that of water. A method of manufacturing a sphere can be provided.

  According to a third embodiment, in any one or more of the first embodiment and the second embodiment, the pore formation inducing substance is selected from the group consisting of alkanes, vegetable oils, and mixtures thereof. One or more methods of producing hollow porous microspheres can be provided.

  In a fourth embodiment, in the third embodiment, the alkane is one or more selected from the group consisting of octane, undecane, tridecane, pentadecane, and mixtures thereof. The vegetable oil is one or more selected from the group consisting of soybean oil, corn oil, cottonseed oil, olive oil, grape seed oil, walnut oil, sesame oil, sesame oil, and mixtures thereof, hollow porosity A method for producing microspheres can be provided.

  In the fifth embodiment, in any one or more of the first to fourth embodiments, an oil phase polymer solution contained in the O / W (oil in water) type emulsion is added to the O / W. The step of converting into S (Oil in Solid) microspheres includes supplying an aqueous solution containing the water or phase stabilizer to the microfluidic device in a continuous phase, and including the water or phase stabilizer supplied in the continuous phase. A method for producing hollow porous microspheres comprising the step of supplying the polymer solution to an aqueous solution in a discontinuous phase can be provided.

  The sixth embodiment is a method according to any one or more of the first to fifth embodiments, in which the rate of supplying the polymer solution in a discontinuous phase is adjusted, and the particles and pores of the hollow porous microspheres are adjusted. It is possible to provide a method for producing hollow porous microspheres, the method further comprising adjusting one or more of the sizes and forms.

  In a seventh embodiment, the oil phase polymer solution contained in the O / W (Oil in Water) type emulsion is added to the O / W (oil in water) type emulsion in any one or more of the first to sixth embodiments. The step of converting into S (Oil in Solid) microspheres includes supplying an aqueous solution containing the water or phase stabilizer to the membrane emulsifying device in a continuous phase, and containing the water or phase stabilizer supplied in the continuous phase. A method for producing hollow porous microspheres comprising the step of supplying the polymer solution to an aqueous solution in a discontinuous phase can be provided.

  The eighth embodiment is the seventh embodiment, wherein the size of one or more of the hollow porous microsphere particles and pores is adjusted by adjusting the size of the voids formed in the membrane of the membrane emulsifying device. A method for producing hollow porous microspheres can further be provided, further comprising the step of adjusting the morphology.

  In the ninth embodiment, in any one or more of the first embodiment to the eighth embodiment, the step of removing the pore formation inducing substance contained in the microsphere is performed by freezing the microsphere after washing with water. A method for producing hollow porous microspheres comprising the step of drying to remove pore formation inducing substances can be provided.

  In a tenth embodiment according to any one or more of the first to ninth embodiments, the hydrophobic biodegradable polymer is polylactic acid (Poly-L-Lactic Acid, PLLA) or polyglycolic acid. (Polyglycolic acid, PGA), polylactic acid-glycolic acid copolymer (poly (lactic-co-glycolic acid), PLGA), poly-ε- (caprolactone) (Polycaprolactone, PCL), polyanhydrides (polyhydrides) Orthoester, poly (vinyl alcohol), polyethylene glycol, polyurethane, poly (polyethane) Polyacrylic acid, poly-N-isopropylacrylamide, poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) copolymer (polypropylene oxide) -polypropylene It is possible to provide a method for producing hollow porous microspheres, which is at least one selected from the group consisting of oxide-polyethylene oxide copolymer), copolymers thereof, and mixtures thereof.

  In an eleventh embodiment according to any one or more of the first to tenth embodiments, the produced hollow porous microsphere includes a cavity formed in the center; and micropores surrounding the cavity. A method for producing hollow porous microspheres including partition walls can be provided.

  In a twelfth embodiment, there is provided a composition for injecting a skin tissue or a volume-enhancing injection of a skin tissue, comprising hollow porous microspheres produced by any one or more of the production methods of the first to tenth embodiments. Can be provided.

  The embodiments have been disclosed for the purpose of explaining the present invention, and the above description does not limit the scope of the present invention. Accordingly, various modifications, variations, and alternatives can be made by those skilled in the art without departing from the meaning and scope of the present invention.

Claims (12)

A method for producing hollow porous microspheres, comprising:
Producing a polymer solution comprising dissolving a pore formation inducer and a hydrophobic biodegradable polymer in a volatile solvent;
Dispersing the polymer solution in water or an aqueous solution containing a phase stabilizer to form an oil in water (O / W) type emulsion;
Volatile solvent is volatilized on the aqueous solution in which the polymer solution is dispersed to solidify the hydrophobic biodegradable polymer, so that the hydrophobic biodegradable polymer and the pore formation inducing substance are between. Hollow including generating spontaneous phase separation to convert oil phase polymer solution contained in O / W (Oil in Water) type emulsion into O / S (Oil in Solid) microspheres Production stage of porous microspheres;
Removing a pore formation inducing substance contained in the hollow porous microsphere,
Method for producing hollow porous microspheres.   The method for producing hollow porous microspheres according to claim 1, wherein the pore formation inducing substance is a hydrophobic fluid that is not compatible with a hydrophobic biodegradable polymer and has a density lower than that of water.   The method for producing hollow porous microspheres according to claim 2, wherein the pore formation inducing substance is at least one selected from the group consisting of alkanes, vegetable oils, and mixtures thereof. The alkane is at least one selected from the group consisting of octane, undecane, tridecane, pentadecane, and mixtures thereof.
The vegetable oil is one or more selected from the group consisting of soybean oil, corn oil, cottonseed oil, olive oil, grape seed oil, walnut oil, sesame oil, sesame oil, and mixtures thereof. Method for producing hollow porous microspheres.   The step of converting the oil phase polymer solution contained in the O / W (Oil in Water) type emulsion into O / S (Oil in Solid) microspheres is performed by the microfluidic device in the water or phase stability. 2. The method according to claim 1, comprising: supplying an aqueous solution containing an agent in a continuous phase, and supplying the polymer solution in a discontinuous phase to water supplied in the continuous phase or an aqueous solution containing a phase stabilizer. Method for producing hollow porous microspheres.   The method of claim 5, further comprising adjusting a rate at which the polymer solution is supplied in a discontinuous phase to adjust the size and shape of one or more of the particles and pores of the hollow porous microsphere. Of manufacturing hollow porous microspheres.   The step of converting the oil-phase polymer solution contained in the O / W (Oil in Water) type emulsion into O / S (Oil in Solid) microspheres is performed by the membrane emulsifying device. 2. The method according to claim 1, comprising: supplying an aqueous solution containing an agent in a continuous phase, and supplying the polymer solution in a discontinuous phase to water supplied in the continuous phase or an aqueous solution containing a phase stabilizer. Method for producing hollow porous microspheres.   The method according to claim 7, further comprising adjusting a size of a void formed in the membrane of the membrane emulsifying device to adjust the size and shape of one or more of the particles and pores of the hollow porous microsphere. The manufacturing method of hollow porous microsphere of description.   2. The hollow porosity according to claim 1, wherein the step of removing pore formation inducing substances contained in the microspheres comprises the step of lyophilizing the microspheres and then lyophilizing to remove the pore formation inducing materials. A method for producing microspheres.   The hydrophobic biodegradable polymer includes polylactic acid (Poly-L-Lactic Acid, PLLA), polyglycolic acid (Polyglycolic acid, PGA), and polylactic acid-glycolic acid copolymer (poly (lactic-co-glycolic acid). acid), PLGA), poly-ε- (caprolactone) (Polycaprolactone, PCL), polyanhydrides, polyorthoesters, polyvinyl alcohol (polyvinylpropylene), polyethylene glycol (polyethylenepolyurethane). Polyacrylic acid, poly-N-isopropyl acetate Polyamide of poly-N-isopropyl acrylate, poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) -polypropylene oxide-polyethylene oxide, and polypropylene oxide-polyethylene oxide The method for producing hollow porous microspheres according to claim 1, wherein the method is one or more selected from the group consisting of a coalescence and a mixture thereof. The produced hollow porous microsphere is:
The manufacturing method of the hollow porous microsphere as described in any one of Claims 1-10 including the cavity formed in the center; and the partition containing the micropores surrounding the said cavity.   A composition for regenerating skin tissue or increasing volume of skin tissue, comprising hollow porous microspheres produced by the production method according to any one of claims 1 to 10.