Microcarriers, cell complexes, and medical compositions, cosmetic compositions, medical supplies, and cosmetic supplies containing the same

Abstract

According to the present invention, the present invention relates to a polymer microparticle having a core-shell structure, which comprises: a core comprising a first biocompatible polymer, a metal ion, and an organic crosslinker having one or more reactive functional groups; and a shell surrounding all or a part of the core and comprising a second biocompatible polymer, a metal ion, and an organic crosslinker having one or more reactive functional groups; and a cell attachment-inducing layer formed on the surface of the polymer microparticle; and a microcarrier, a cell complex comprising the same, a medical composition, a cosmetic composition, a medical product, and a cosmetic product.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0130366, filed on September 30, 2021, and all contents disclosed in the documents of that Korean patent application are incorporated herein by reference.

[0002] The present invention relates to microcarriers that can achieve excellent mechanical strength and stability, can be injected into the body immediately after 3D culture without a cell detachment process, and can provide a stable environment for adherent cells, contributing to high cell survival rates and a high rate of in vivo transplantation, as well as cell complexes, medical compositions, cosmetic compositions, medical supplies, and cosmetic supplies containing the same. [Background technology]

[0003] As the fields of biopharmaceuticals and regenerative medicine expand, there is an increasing demand for mass cell culture technologies that can efficiently produce cells, tissues, microorganisms, etc.

[0004] Adherent cells are cultured using microcarriers in a 3D bioreactor. Cells, culture medium, and microcarriers are placed in the bioreactor, and the culture medium is stirred to bring the cells and microcarriers into contact, allowing the cells to adhere to the microcarrier surface and be cultured. The microcarriers used in this process provide a high surface area / volume ratio for cells to attach and grow, making them suitable for mass cell culture. However, expanding adherent cells using microcarriers requires a cell detachment process after the culture is completed to recover the cells. This cell detachment process involves the use of proteolytic enzymes or temperature changes to induce cell detachment, but adding this detachment step increases production costs, reduces economic viability, and can potentially cause cell damage.

[0005] To solve this problem, the development of new materials and processes is steadily progressing, and in particular, in the case of cell therapy that involves injecting cells into the body, efforts are being made to ensure the biocompatibility of the microcarriers used to culture the cells, thereby eliminating the need for separation and purification processes. In this case, particles that are strong enough to withstand the stresses imposed by the fluid surrounding the carrier during the culture process and after infusion into the body are required.

[0006] Furthermore, in the case of transdermal drug delivery technology, in which microcarriers loaded with drugs or physiologically active substances are delivered by microneedles, the microcarriers must be made of polymers suitable for biological applications and must have sufficient strength to prevent particle deformation during the process of passing through the stratum corneum layer of the skin. Microcarriers that stably penetrate the skin can deliver the loaded drugs locally or systemically to act on the necessary lesions.

[0007] Hyaluronic acid, a commonly used biocompatible material, is a biopolymer composed of N-acetyl-D-glucosamine and D-glucuronic acid, with the repeating units linearly linked. It is abundant in the vitreous humor of the eye, synovial fluid, and cockscomb. Hyaluronic acid is often used as a bioinjectable material due to its excellent biocompatibility and viscoelasticity. However, its use is limited due to its susceptibility to degradation in vivo or under acidic or alkaline conditions. Furthermore, when applied to microcarriers, hyaluronic acid exhibits a negative charge within the biological pH range, which significantly reduces cell adhesion.

[0008] Gelatin is a polymer made by hydrolyzing collagen, a biological connective tissue, and is used as a scaffold for cell culture. Although it can be used to capture and culture cells, it is weak and temperature-sensitive, and efforts are being made to improve its strength by introducing functional groups using chemical methods.

[0009] Therefore, there is a need to develop microcarriers or polymeric microparticles that are compatible with living organisms and have excellent physical properties such as physical strength and stability against heat and enzymes.

[0010] In addition, in order to overcome the limitations of existing 2D culture in the process of mass expansion of adherent cells using microcarriers, spherical microcarriers that can increase surface efficiency have been developed and are used in 3D expansion culture. However, in the conventional technology where cells are recovered through a cell detachment process after the end of culture, there were problems such as increased manufacturing costs and the induction of cell damage due to the addition of a detachment process.

[0011] Furthermore, injectable cell therapy agents are typically injected into the affected area with cells suspended in a liquid phase, which creates an environment that is not suitable for the survival of adherent cells, and can result in low transplantation rates and cell survival rates due to immune responses in the body.

[0012] Therefore, there is a need to develop microcarriers that can be injected into the body immediately after 3D culture without a cell detachment process, and that can provide a stable environment for adherent cells, resulting in high cell survival rates and a high rate of transplantation. Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention provides a microcarrier that can achieve excellent mechanical strength and stability, can be injected into the body immediately after 3D culture without a cell detachment process, and can provide a stable environment for adherent cells, contributing to high cell survival rate and a high rate of in vivo transplantation.

[0014] The present invention also provides a cell complex comprising the microcarrier.

[0015] The present invention also provides a medical composition comprising the microcarrier or cell complex.

[0016] The present invention also provides a cosmetic composition containing the microcarrier or cell complex.

[0017] The present invention also provides a medical product containing the medical composition.

[0018] The present invention also provides a cosmetic product containing the cosmetic composition. [Means for solving the problem]

[0019] Provided herein is a microcarrier comprising: a polymeric microparticle having a core-shell structure, the core comprising a first biocompatible polymer, a metal ion, and an organic crosslinker containing one or more reactive functional groups; and a shell surrounding all or part of the core and comprising a second biocompatible polymer, a metal ion, and an organic crosslinker containing one or more reactive functional groups; and a cell attachment-inducing layer formed on the surface of the polymeric microparticle.

[0020] Also provided herein is a cell complex comprising the microcarrier; and cells attached on the surface of the microcarrier.

[0021] Also provided herein is a medical composition comprising the microcarrier or cell complex.

[0022] Also provided herein is a cosmetic composition comprising the microcarrier or cell complex.

[0023] Also provided herein are medical products comprising the medical compositions described above.

[0024] Also provided herein is a cosmetic product containing the cosmetic composition.

[0025] Microcarriers according to specific embodiments of the invention, and cell complexes, medical compositions, cosmetic compositions, medical supplies, and cosmetic supplies containing the same will be described in more detail below.

[0026] In this specification, unless expressly stated otherwise, terminology is for the purpose of referring to particular embodiments only and is not intended to limit the invention.

[0027] As used herein, the singular forms "a," "an," and "the" include the plural forms as well, unless the context clearly dictates to the contrary.

[0028] As used herein, the meaning of "comprising" embodies certain properties, regions, integers, steps, operations, elements, and / or components, and does not exclude the presence or addition of other certain properties, regions, integers, steps, operations, elements, components, and / or groups.

[0029] In this specification, terms including ordinal numbers, such as "first" and "second," are used to distinguish one component from another, and are not limited by the ordinal numbers. For example, within the scope of the present invention, a first component may be called a second component, and similarly, a second component may be called a first component.

[0030] In this specification, the term "(co)polymer" refers to both polymers and copolymers, where the term "polymer" refers to a homopolymer consisting of a single repeating unit, and the term "copolymer" refers to a composite polymer containing two or more types of repeating units.

[0031] Although the present invention can be modified in various ways and can take various forms, specific embodiments are described in detail below by way of example, but it should be understood that this is not intended to limit the present invention to the specific disclosed embodiments, but rather to include all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention.

[0032] In this specification, microparticles refer to particles whose cross section is circular or elliptical and whose minor axis / major axis ratio (sphericity) is in the range of 0.7 to 1.0. The lengths of the minor and major axes of a particle are derived by taking an optical photograph of the particle and calculating the average values ​​of 30 to 100 randomly selected particles in the optical photograph.

[0033] In this specification, diameter (Dn) refers to the diameter at n% by volume of the cumulative particle number distribution by diameter. That is, D50 is the diameter at 50% of the cumulative particle number distribution when particle diameters are accumulated in ascending order, D90 is the diameter at 90% of the cumulative particle number distribution by diameter, and D10 is the diameter at 10% of the cumulative particle number distribution by diameter.

[0034] The Dn can be measured using the laser diffraction method. Specifically, the powder to be measured is dispersed in a dispersion medium and then introduced into a commercially available laser diffraction particle size analyzer (Horiba LA-960). The particle size distribution is calculated by measuring the difference in diffraction pattern depending on particle size when the particles pass through a laser beam. D10, D50, and D90 can be measured by calculating the particle diameters at 10%, 50%, and 90% of the cumulative particle number distribution by diameter measured by the analyzer. More specifically, in this specification, diameter can refer to D50.

[0035] As used herein, emulsion refers to a mixed phase in which one or more immiscible liquids, oil or water, are dispersed in the other liquid (dispersion medium) in a fine particle state (dispersoid). Emulsions are generally classified into macroemulsions, microemulsions, and nanoemulsions depending on the particle size of the dispersed phase.

[0036] The present invention will now be described in more detail.

[0037] 1. Microcarriers According to one embodiment of the invention, there is provided a microcarrier comprising: a polymeric microparticle having a core-shell structure, the core comprising a first biocompatible polymer, a metal ion, and an organic crosslinker containing one or more reactive functional groups; and a shell surrounding all or part of the core and comprising a second biocompatible polymer, a metal ion, and an organic crosslinker containing one or more reactive functional groups; and a cell attachment-inducing layer formed on the surface of the polymeric microparticle.

[0038] The present inventors have conducted research on polymeric microparticles and have confirmed through experiments that by performing an additional crosslinking reaction after crosslinking with metal ions as described above, the efficiency of the process is maximized and the mechanical strength and cell adhesion of the microparticles are significantly improved, thereby completing the invention.

[0039] Furthermore, the microcarrier of one embodiment includes a cell adhesion-inducing layer formed on the surface of the polymeric microparticles, which allows it to be injected into the body immediately after 3D culture without a cell detachment process, and the adherent cells form a complex in a stably attached state with the microcarrier, thereby achieving high cell survival rate and a high in vivo transplant rate. This was confirmed through experiments, and the invention was completed.

[0040] In one embodiment of the invention, the core comprises a polymer matrix in which a first biocompatible polymer is crosslinked via an organic crosslinker containing one or more metal ions and reactive functional groups, and the shell comprises a polymer matrix in which a second biocompatible polymer is crosslinked via an organic crosslinker containing one or more metal ions and reactive functional groups.

[0041] Specifically, the polymer matrix can include a first crosslinked region in which the biocompatible polymer is crosslinked via a metal ion; and a second crosslinked region in which the biocompatible polymer is crosslinked via an organic crosslinker containing one or more reactive functional groups.

[0042] The first crosslinked region refers to a crosslinked region formed by a crosslinking reaction between a biocompatible polymer and a metal ion, and the second crosslinked region refers to a crosslinked region formed by a crosslinking reaction between a biocompatible polymer and an organic crosslinker having one or more reactive functional groups instead of a crosslinking reaction between a biocompatible polymer and a metal ion, and a crosslinked region formed by an additional crosslinking reaction between the first crosslinked region and an organic crosslinker having one or more reactive functional groups. In other words, the polymeric microparticles of the above embodiment can be produced through a crosslinking reaction using a metal ion and an organic crosslinker having one or more reactive functional groups.

[0043] Specifically, the biocompatible polymer refers to a polymer that can be directly injected into the human body to deliver an effective substance to the human body. Specifically, the biocompatible polymer includes hyaluronic acid (HA), carboxymethyl cellulose (CMC), alginic acid, pectin, carrageenan, chondroitin sulfate, dextran sulfate, chitosan, polylysine, collagen, gelatin, carboxymethyl chitin, and the like. chitin, fibrin, agarose, pullulan, polylactide, polyglycolide (PGA), polylactide-glycolide copolymer (PLGA), polyanhydride, polyorthoester, polyetherester, polycaprolactone, polyesteramide, poly(butyric acid), poly(valeric acid), polyurethane, polyacrylate, ethylene-vinyl acetate polymer, acrylic-substituted cellulose acetate, non-degradable polyurethane, polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins The polymer may be one or more polymers selected from the group consisting of cellulose, polyolefins, polyethylene oxide, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polymethacrylate, hydroxypropylmethylcellulose (HPMC), ethylcellulose (EC), hydroxypropylcellulose (HPC), cyclodextrin, and copolymers of monomers that form such polymers.

[0044] More specifically, the biocompatible polymer may be a mixture of hyaluronic acid (HA) and gelatin.

[0045] Polymer microparticles made using only hyaluronic acid are easily degraded in vivo or under acidic or alkaline conditions, limiting their use and significantly reducing cell adhesion, while polymer microparticles made using only gelatin have significantly reduced mechanical properties.

[0046] Therefore, by using a mixture of hyaluronic acid (HA) and gelatin as a biocompatible polymer, it is possible to achieve excellent cell adhesion and mechanical properties at the same time.

[0047] Specifically, the first biocompatible polymer may include hyaluronic acid, and the second biocompatible polymer may include gelatin.

[0048] In this specification, hyaluronic acid may mean both hyaluronic acid itself and hyaluronate.Therefore, hyaluronic acid aqueous solution may also mean the concept of including the aqueous solution of hyaluronic acid, the aqueous solution of hyaluronate, and the mixed aqueous solution of hyaluronic acid and hyaluronate.The hyaluronate may be inorganic salt such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, etc., organic salt such as tetrabutylammonium hyaluronate, and their mixture.

[0049] In one embodiment of the invention, the molecular weight of hyaluronic acid is not particularly limited, but is preferably 10,000 g / mol or more and 5,000,000 g / mol or less in order to achieve diverse physical properties and biocompatibility.

[0050] As used herein, gelatin may refer to a protein obtained by treating animal-derived collagen with acid or alkali, followed by extraction.

[0051] In one embodiment of the invention, the molecular weight of gelatin is not particularly limited, but is preferably 10,000 g / mol or more and 5,000,000 g / mol or less in order to achieve a variety of physical properties and biocompatibility.

[0052] Meanwhile, the polymeric microparticles may have a core-shell structure, which can be achieved by the polymer matrix of the polymeric microparticles containing two or more biocompatible polymers, and by the difference in reactivity between the biocompatible polymers and the metal ions and the organic crosslinker containing one or more reactive functional groups.

[0053] In the core-shell structure, the core may contain a polymer matrix in which hyaluronic acid is crosslinked via a metal ion and an organic crosslinker containing one or more reactive functional groups at a ratio of more than 50 vol%, 60 vol% or more, 70 vol% or more, or 75 vol% or more relative to the total volume of the polymer matrix contained in the core. It may also contain 100 vol% or less, less than 100 vol%, 95 vol% or less, or 90 vol% or less. It may also contain 50 vol% or more but 100 vol%, 50 vol% or more but 100 vol%, 60 vol% or more but 100 vol%, 60 vol% or more but 95 vol%, 70 vol% or more but 95 vol%, 70 vol% or more but 90 vol%, or 75 vol% or more but 90 vol%. In other words, the core may contain an excess of hyaluronic acid relative to gelatin.

[0054] The fact that the core contains more than 50% by volume of a polymer matrix in which hyaluronic acid is crosslinked via a metal ion and an organic crosslinker containing one or more reactive functional groups can be determined by visually or by using a measuring device to confirm that the region is distributed over more than 50% of the total area, relative to the total volume of the polymer matrix contained in the core.

[0055] Specifically, the volume ratio of the polymer matrix in which hyaluronic acid is crosslinked via a metal ion and an organic crosslinker containing one or more reactive functional groups to the total volume of the polymer matrix contained in the core is not particularly limited, and can be calculated by a conventional measurement method. For example, the gelatin characteristic peak (1650 cm) of the prepared polymer microparticles can be calculated by the conventional measurement method. -1 ) relative to the characteristic peak of hyaluronic acid (1080 cm -1 ) can be checked by taking an IR photo.

[0056] In the core-shell structure, the shell may contain a polymer matrix in which gelatin is crosslinked via a metal ion and an organic crosslinker containing one or more reactive functional groups at a ratio of more than 50 vol%, 60 vol% or more, 70 vol% or more, or 75 vol% or more relative to the total volume of the polymer matrix contained in the shell. It may also contain 100 vol% or less, less than 100 vol%, 95 vol% or less, or 90 vol% or less. It may also contain 50 vol% or more but 100 vol%, 50 vol% or more but 100 vol%, 60 vol% or more but 100 vol%, 60 vol% or more but 95 vol%, 70 vol% or more but 95 vol%, 70 vol% or more but 90 vol%, or 75 vol% or more but 90 vol%. In other words, the shell may contain an excess of gelatin relative to hyaluronic acid.

[0057] The fact that the polymer matrix in which gelatin is crosslinked via a metal ion and an organic crosslinking agent containing one or more reactive functional groups accounts for more than 50% by volume of the total volume of the polymer matrix contained in the shell can be determined by visually or by confirming with a measuring device that the region in question is distributed over more than 50% of the total area.

[0058] The volume ratio of the polymer matrix in which gelatin is crosslinked via a metal ion and an organic crosslinker containing one or more reactive functional groups to the total volume of the polymer matrix contained in the shell is not particularly limited and can be calculated by a conventional measurement method. For example, the volume ratio of the polymer matrix in which gelatin is crosslinked via a metal ion and an organic crosslinker containing one or more reactive functional groups to the total volume of the polymer matrix contained in the shell can be calculated by a conventional measurement method. For example, the volume ratio of the polymer matrix in which gelatin is crosslinked via a characteristic peak (1650 cm) of the prepared polymer microparticles can be calculated by a conventional measurement method.-1 ) can be checked by taking an IR photo.

[0059] The core-shell structure, which includes a core containing 50% or more by volume of a polymer matrix in which hyaluronic acid is crosslinked via an organic crosslinker containing one or more metal ions and reactive functional groups, and a shell containing 50% or more by volume of a polymer matrix in which gelatin is crosslinked via an organic crosslinker containing one or more metal ions and reactive functional groups, can be achieved by physicochemical factors such as solubility, temperature reactivity, and ionic bonding. More specifically, in the process of producing polymeric microparticles, gelatin, which has low solubility in polar solvents such as ethanol and high temperature reactivity, has reduced fluidity and can be fixed to the particle surface to form a shell, which allows hyaluronic acid to be distributed relatively more inside the particles and form a core.

[0060] In particular, in the method for producing polymeric microparticles described below, a step of forming polymeric crosslinked particles by reacting a mixture containing a biocompatible polymer and metal ions is included before the step of additionally crosslinking the polymeric crosslinked particles in a polar solvent containing an organic crosslinker having one or more reactive functional groups, so that the core-shell structure becomes more defined due to the ionic bond between the metal ions and the carboxyl groups contained in the hyaluronic acid.

[0061] Meanwhile, the polymeric microparticles may have an average diameter in distilled water of 1 μm or more, 1 μm to 450 μm, 100 μm to 450 μm, 200 μm to 400 μm, or 300 μm to 400 μm, but when the average diameter of the polymeric microparticles satisfies the above-mentioned range, they have excellent cell attachment and culture performance.

[0062] The average diameter may refer to the diameter at 50% by volume of the cumulative particle number distribution according to diameter.

[0063] The thickness of the shell of the polymeric microparticle may be 95% or less, 90% or less, 80% or less, 75% or less, 50% or less, 30% or less, 25% or less, or 20% or less of the longest diameter of the polymeric microparticle, based on the cross section having the longest diameter of the polymeric microparticle.The thickness of the shell may be 0.01% or more, 1% or more, or 5% or more of the longest diameter of the polymeric microparticle, based on the cross section having the longest diameter of the polymeric microparticle.

[0064] Furthermore, in the polymeric microparticles, the thickness of the core may be 5% or more, 10% or more, 20% or more, 25% or more, 50% or more, 70% or more, 75% or more, or 80% or more of the longest diameter of the polymeric microparticle, based on the cross section having the longest diameter of the polymeric microparticle.Furthermore, the thickness of the core may be 99.99% or less, 99% or less, or 95% or less of the longest diameter of the polymeric microparticle, based on the cross section having the longest diameter of the polymeric microparticle.

[0065] The polymeric microparticles may have a sphericity of 0.9 to 1.0, 0.93 to 1.0, 0.94 to 0.99, or 0.94 to 0.98. The sphericity can be obtained by taking an optical photograph of the polymeric microparticles and calculating the average value of 30 to 100 random particles in the optical photograph.

[0066] Furthermore, the polymeric microparticles may have an average compressive strength of 0.1 mN or more, 0.1 mN to 100 mN or less, 0.3 mN to 100 mN or less, 0.35 mN to 100 mN or less, 0.35 mN to 30 mN or less, 0.35 mN to 10 mN or less, or 0.35 mN to 3 mN or less when deformed to 25% of their average diameter after swelling in distilled water for 24 hours or more. The average compressive strength may be calculated by dividing the compressive strength of n polymeric microparticles when deformed to 25% of their average diameter by n.

[0067] For example, in this specification, the average compressive strength may be a value obtained by dividing the compressive strength when 30 polymeric microparticles are deformed to a level of 25% of the average diameter by 30.

[0068] If the average compressive strength of the polymer microparticles is less than 0.1 mN, the mechanical strength of the polymer microparticles may be poor, resulting in a technical problem of reduced stability.

[0069] Meanwhile, examples of methods for producing the polymeric microparticles contained in the microcarrier of one embodiment are not limited to these, but for example, a method for producing polymeric microparticles can be used, which includes the steps of: reacting a mixture containing a biocompatible polymer and metal ions to form polymeric crosslinked particles; and further crosslinking the polymeric crosslinked particles in a polar solvent containing an organic crosslinker having one or more reactive functional groups.

[0070] Conventional polymer microparticles require an oil washing process because they are crosslinked after forming a W / O emulsion using oil, which not only makes the process less efficient but also poses the technical problem of making it difficult to remove residual oil.

[0071] Therefore, the inventors have confirmed through experiments that, as in the above-mentioned method for producing polymeric microparticles, crosslinking with metal ions is followed by an additional crosslinking reaction using an organic crosslinker containing one or more reactive functional groups, thereby maximizing the process efficiency and significantly improving the mechanical strength and stability of the microparticles, and have completed the invention.

[0072] Specifically, the biocompatible polymer refers to a polymer that can be directly injected into the human body to deliver an effective substance to the human body. Specifically, the biocompatible polymer includes hyaluronic acid (HA), carboxymethyl cellulose (CMC), alginic acid, pectin, carrageenan, chondroitin sulfate, dextran sulfate, chitosan, polylysine, collagen, gelatin, carboxymethyl chitin, and the like. chitin, fibrin, agarose, pullulan, polylactide, polyglycolide (PGA), polylactide-glycolide copolymer (PLGA), polyanhydride, polyorthoester, polyetherester, polycaprolactone, polyesteramide, poly(butyric acid), poly(valeric acid), polyurethane, polyacrylate, ethylene-vinyl acetate polymer, acrylic-substituted cellulose acetate, non-degradable polyurethane, polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins The polymer may be one or more polymers selected from the group consisting of cellulose, polyolefins, polyethylene oxide, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polymethacrylate, hydroxypropylmethylcellulose (HPMC), ethylcellulose (EC), hydroxypropylcellulose (HPC), cyclodextrin, and copolymers of monomers that form such polymers.

[0073] More specifically, the biocompatible polymer may be a mixture of hyaluronic acid (HA) and gelatin.

[0074] Polymer microparticles made using only hyaluronic acid are easily degraded in vivo or under acidic or alkaline conditions, limiting their use and significantly reducing cell adhesion, while polymer microparticles made using only gelatin have significantly reduced mechanical properties.

[0075] Therefore, by using a mixture of hyaluronic acid (HA) and gelatin as a biocompatible polymer, it is possible to achieve excellent cell adhesion and mechanical properties at the same time.

[0076] In this specification, hyaluronic acid may mean both hyaluronic acid itself and hyaluronate.Therefore, hyaluronic acid aqueous solution may also mean the concept of including the aqueous solution of hyaluronic acid, the aqueous solution of hyaluronate, and the mixed aqueous solution of hyaluronic acid and hyaluronate.The hyaluronate may be inorganic salt such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, etc., organic salt such as tetrabutylammonium hyaluronate, and their mixture.

[0077] In one embodiment of the invention, the molecular weight of hyaluronic acid is not particularly limited, but is preferably 10,000 g / mol or more and 5,000,000 g / mol or less in order to achieve diverse physical properties and biocompatibility.

[0078] As used herein, gelatin may refer to a protein obtained by treating animal-derived collagen with acid or alkali, followed by extraction.

[0079] In one embodiment of the invention, the molecular weight of gelatin is not particularly limited, but is preferably 10,000 g / mol or more and 5,000,000 g / mol or less in order to achieve a variety of physical properties and biocompatibility.

[0080] In the method for producing polymeric microparticles, the mixture of hyaluronic acid (HA) and gelatin may contain 50 to 500 parts by weight, 100 to 500 parts by weight, or 100 to 300 parts by weight of gelatin per 100 parts by weight of hyaluronic acid (HA).

[0081] If less than 50 parts by weight of gelatin is contained per 100 parts by weight of hyaluronic acid (HA), the cell adhesion of the produced polymeric microparticles will be poor, and if more than 500 parts by weight of gelatin is contained per 100 parts by weight of hyaluronic acid (HA), the mechanical properties of the produced polymeric microparticles may be reduced. In other words, by containing 50 to 500 parts by weight of gelatin per 100 parts by weight of hyaluronic acid (HA) in a mixture of hyaluronic acid (HA) and gelatin, polymeric microparticles that can achieve excellent cell adhesion and mechanical properties at the same time can be produced.

[0082] In the method for producing polymeric microparticles, the metal ions are iron ions (Fe 3+ ), aluminum ions (Al 3+ ), copper ions (Cu 2+ ), iron ions (Fe 2+ ), magnesium ions (Mg 2+ ), barium ions (Ba 2+ ), calcium ions (Ca 2+ ) etc. More specifically, the metal ion may be an iron ion, an aluminum ion, or a mixture thereof.

[0083] In the method for producing polymeric microparticles, the step of forming polymeric crosslinked particles by reacting the mixture containing the biocompatible polymer and metal ions can include the steps of: forming an aqueous solution in which the biocompatible polymer is dissolved; adding a compound containing the metal ions to a polar solvent to form a solution containing the metal ions; and mixing droplets of the aqueous solution in which the biocompatible polymer is dissolved with the solution containing the metal ions to form a mixed solution.

[0084] Specifically, in the step of forming an aqueous solution containing the biocompatible polymer, the aqueous solution containing the biocompatible polymer may contain the biocompatible polymer in an amount of 0.01 wt % to 10 wt %, 0.01 wt % to 5 wt %, 1 wt % to 5 wt %, 1 wt % to 3 wt %, 2 wt % to 3 wt %, or 2 wt % to 2.5 wt % based on the total weight of the aqueous solution containing the biocompatible polymer.

[0085] As in the method for producing polymeric microparticles, by first crosslinking with metal ions and then performing an additional crosslinking reaction with an organic crosslinker containing one or more reactive functional groups, it is possible to produce microparticles with excellent mechanical strength and stability even with a low content of biocompatible polymer, such as 0.01 wt % to 10 wt %, compared to the conventional method of forming a W / O emulsion and then performing a crosslinking reaction.

[0086] The step of adding the compound containing metal ions to a polar solvent to form a solution containing metal ions is not particularly limited, and may be, for example, formed by dispersing the compound containing metal ions in a polar solvent such as ethanol.

[0087] In addition, in the step of mixing the aqueous solution droplets containing the biocompatible polymer with the solution containing the metal ions to form a mixed solution, the particle size can be appropriately controlled using an encapsulator (BUCHI, B-390) device.

[0088] By including a step of mixing the aqueous solution droplets in which the biocompatible polymer is dissolved with the solution containing the metal ions to form a mixed solution, the metal ions are chelated to the biocompatible polymer, and the biocompatible polymer can form a crosslinked structure via the metal ions.

[0089] As in the above method for producing polymeric microparticles, by including a step of forming polymeric crosslinked particles by reacting a mixture containing a biocompatible polymer and metal ions, it is possible to produce microparticles in a polar solvent without using oil, compared to a case where a crosslinking reaction is carried out using only an organic crosslinker containing one or more reactive functional groups, and the oil washing process is omitted, thereby achieving the effect of excellent process efficiency.

[0090] In other words, as metal ions are chelated to the biocompatible polymer and the biocompatible polymer forms a crosslinked structure via the metal ions, it is possible to produce microparticles in a polar solvent without using oil, compared to when a crosslinking reaction is carried out using only an organic crosslinker containing one or more reactive functional groups, and the oil washing process is omitted, resulting in excellent process efficiency.

[0091] The compound containing the metal ion is contained in an amount of 200 to 1000 parts by weight, 300 to 1000 parts by weight, or 500 to 1000 parts by weight relative to 100 parts by weight of the biocompatible polymer.

[0092] As described above, the method for producing polymeric microparticles can produce polymeric microparticles that have sufficient mechanical strength and sphericity by performing an additional crosslinking reaction after the crosslinking reaction using metal ions, even when a small amount of a mixture containing metal ions is added in an amount of 200 to 1,000 parts by weight per 100 parts by weight of the biocompatible polymer.

[0093] If the content of the metal ion-containing compound is more than 1000 parts by weight based on 100 parts by weight of the biocompatible polymer, a technical problem may occur in that the remaining metal ions remain in the crosslinked particles.

[0094] The organic crosslinking agent containing one or more reactive functional groups can include a crosslinking agent having 1 to 30 carbon atoms and containing one or more reactive functional groups.

[0095] As described above, by carrying out an additional crosslinking reaction using a crosslinker having 1 to 30 carbon atoms and one or more reactive functional groups after crosslinking with metal ions, the process efficiency can be maximized while the mechanical strength and stability of the microparticles can be significantly improved.

[0096] The type of the reactive functional group is not particularly limited, and examples thereof include a hydroxy group, an epoxy group, a carboxy group, an amino group, a (meth)acrylate group, a nitrile group, a thiol group, an aldehyde group, and a vinyl group.

[0097] Specifically, the organic crosslinker containing one or more reactive functional groups may contain one or more formyl groups or epoxy groups, which may be crosslinkable functional groups that react with the biocompatible polymer to form crosslinked particles.

[0098] In the method for producing polymeric microparticles, examples of the organic crosslinking agent containing one or more reactive functional groups are not particularly limited. Specifically, the crosslinking agent may be glutaraldehyde, butanediol diglycidyl ether (1,4-butandiol diglycidyl ether: BDDE), ethylene glycol diglycidyl ether (ethylene glycol diglycidyl ether: EGDGE), hexanediol diglycidyl ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, or glycerol polyglycidyl ether. The polyglycidyl ether may include one selected from the group consisting of 1,2-(bis(2,3-epoxypropoxy)ethylene), pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, divinylsulfone, and epichlorohydrin.

[0099] More specifically, the organic cross-linking agent containing one or more reactive functional groups may be glutaraldehyde or 1,4-butandiol diglycidyl ether (BDDE).

[0100] Meanwhile, in the step of additionally crosslinking the polymer crosslinked particles in a polar solvent containing an organic crosslinker having one or more reactive functional groups, the organic crosslinker having one or more reactive functional groups is contained in an amount of 150 to 1,000 parts by weight, 200 to 1,000 parts by weight, 300 to 800 parts by weight, or 400 to 500 parts by weight, relative to 100 parts by weight of the biocompatible polymer.

[0101] As described above, the method for producing polymeric microparticles can produce polymeric microparticles that have sufficient mechanical strength and sphericity by performing an additional crosslinking reaction after the crosslinking reaction, even when a small amount of an organic crosslinker containing one or more reactive functional groups is added in an amount of 150 to 1,000 parts by weight per 100 parts by weight of the biocompatible polymer.

[0102] If the content of the organic crosslinker containing one or more reactive functional groups exceeds 1,000 parts by weight per 100 parts by weight of the biocompatible polymer, a technical problem may occur in that the remaining unreacted crosslinker remains in the crosslinked particles.

[0103] Meanwhile, in the step of additionally crosslinking the polymer crosslinked particles in a polar solvent containing an organic crosslinker having one or more reactive functional groups, the polar solvent is not particularly limited, and examples thereof include ethanol, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylcaprolactam, 2-pyrrolidone, N-ethylpyrrolidone, N-vinylpyrrolidone, dimethyl sulfoxide, tetramethylurea, pyridine, dimethyl sulfone, hexamethyl sulfoxide, gamma-butyrolactone, 3-methoxy-N,N-dimethylpropanamide, 3-ethoxy-N,N-dimethylpropanamide, 3-butoxy-N,N-dimethylpropanamide, 1,3-dimethyl-imidazolidinone, ethyl amyl ketone, methyl nonyl ketone, The alkyl ether may be one selected from the group consisting of methyl ethyl ketone, methyl isoamyl ketone, methyl isopropyl ketone, cyclohexanone, ethylene carbonate, propylene carbonate, diglyme, 4-hydroxy-4-methyl-2-pentanone, ethylene glycol monomethyl ether, ethylene glycol monomethyl ether acetate, ethylene glycol monoethyl ether, ethylene glycol monoethyl ether acetate, ethylene glycol monopropyl ether, ethylene glycol monopropyl ether acetate, ethylene glycol monoisopropyl ether, ethylene glycol monoisopropyl ether acetate, ethylene glycol monobutyl ether, and ethylene glycol monobutyl ether acetate.

[0104] In addition, in the step of additionally crosslinking the polymeric crosslinked particles in a polar solvent containing an organic crosslinker having one or more reactive functional groups, the polar solvent containing the organic crosslinker having one or more reactive functional groups may be an alkaline mixed solvent.

[0105] That is, the additional crosslinking reaction of the present invention is carried out in an alkaline mixed solvent obtained by mixing a polar solvent with an alkaline aqueous solution. Examples of the alkaline aqueous solution are not particularly limited, but may be, for example, an aqueous sodium hydroxide solution.

[0106] The polar solvent containing the organic crosslinking agent having one or more reactive functional groups is an alkaline mixed solvent, so that the nucleophilic substitution reaction (S N This can create a favorable environment for the cross-linking reaction, thereby increasing the efficiency of the cross-linking reaction.

[0107] Meanwhile, the microcarrier of one embodiment may include a cell attachment-inducing layer formed on the surface of the polymeric microparticles. The cell attachment-inducing layer is made of a cell adhesive material, allowing adherent cells to stably attach, spread, and culture.

[0108] The cell attachment-inducing layer may comprise one or more cell adhesive substances selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polydopamine, poly-L-lysine, vitronectin, RGD-containing peptides, acrylic polymers containing RGD, lignin, cationic dextran, and derivatives thereof. That is, the cell attachment-inducing layer may comprise one or a mixture of two or more cell adhesive substances selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polydopamine, poly-L-lysine, vitronectin, RGD-containing peptides, acrylic polymers containing RGD, lignin, cationic dextran, and derivatives thereof.

[0109] The cell adhesion-inducing layer is formed on the surface of the polymeric microparticles, i.e., the cell adhesion-inducing layer may be in direct contact with the surface of the polymeric microparticles, or may be in contact with the surface of another layer that is in contact with the surface of the polymeric microparticles.

[0110] For example, the microcarrier includes a cell attachment-inducing layer formed so as to be in direct contact with the surface of the polymeric microparticles. By introducing the cell attachment-inducing layer onto the surface of the microcarrier, the degree of buoyancy of the microcarrier in the culture medium can be adjusted, thereby achieving the effect of stably attaching and culturing cells.

[0111] The cell adhesion-inducing layer may have a thickness of 1 nm to 10,000 nm, 10 nm to 1,000 nm, 50 nm to 500 nm, or 80 nm to 200 nm. The thickness of the cell adhesion-inducing layer is calculated by subtracting the radius of the internal polymeric microparticles from the radius of the entire microcarrier. The radius refers to half the diameter. Methods for measuring the diameter are not particularly limited, but include, for example, confocal fluorescence microscopy, electron transmission microscopy (TEM), cross-sectional IR, and cross-sectional SEM imaging.

[0112] The microcarriers may have an average diameter of 1 μm to 1,000 μm, or 10 μm to 1,000 μm, or 100 μm to 1,000 μm, or 100 μm to 800 μm. Although the method for measuring the diameter is not particularly limited, one example is that it can be measured using an optical microscope.

[0113] Meanwhile, the ratio of the radius of the polymeric microparticle to the thickness of the cell adhesion-inducing layer may be 1:0.00001 to 1:0.1, or 1:0.0001 to 1:0.01.

[0114] If the ratio of the radius of the polymeric microparticles to the thickness of the cell adhesion-inducing layer is less than 1:0.00001, the cell adhesion-inducing layer will be too thin compared to the polymeric microparticles, which may reduce the adhesion between cells and microcarriers during cell culture. If the ratio exceeds 1:0.1, the cell adhesion-inducing layer will be too thick compared to the polymeric microparticles, which may change the physical properties of the polymeric microparticles, such as their hydrophilicity, thereby reducing their dispersibility.

[0115] The microcarriers may have a cell adhesion property calculated by the following formula of 2000% or more, or 2500% or more, or 2900% or more, or 4000% or less, or 2000% to 4000%, or 2500% to 4000%, or 2900% to 4000%.

[0116] Formula Cell adhesion = (number of cells after incubating microcarriers in cell culture medium at 37°C for 7 days / number of cells initially contained in the cell culture medium) x 100

[0117] The cell culture medium, cells, and culture conditions are not particularly limited, and various conventionally known cell culture mediums, cells, and culture conditions can be applied without limitation.Furthermore, the method for measuring the cell number is also not particularly limited, and various conventionally known methods for measuring the cell number can be applied without limitation.

[0118] The microcarriers have a cell adhesion property calculated by the following formula of 2000% or more, or 2500% or more, or 2900% or more, or 4000% or less, or 2000% to 4000%, or 2500% to 4000%, or 2900% to 4000%, thereby improving the cell adhesion of the microcarriers. This has the advantage of increasing the surface efficiency through three-dimensional expansion culture, enabling mass expansion of adherent cells.

[0119] In contrast, when the cell adhesion of the microcarriers calculated by the following formula is not within the above-mentioned range, the cell adhesion force on the surface of the microcarriers is weak, making it difficult to perform three-dimensional expansion culture sufficiently.

[0120] Meanwhile, the microcarrier may further include a primer polymer layer formed on the surface of the polymer microparticle.

[0121] That is, the surface of the polymeric microparticle further comprises a mixed layer of one primer polymer layer and one cell adhesion-inducing layer. The order of lamination of the mixed layer of one primer polymer layer and one cell adhesion-inducing layer is not particularly limited, and any structure in which the cell adhesion-inducing layer is laminated on the primer polymer layer, or a structure in which the primer polymer layer is laminated on the cell adhesion-inducing layer, is applicable.

[0122] Meanwhile, the primer polymer layer acts as an adhesive layer that can introduce functional polymers onto the surface of the polymer microparticles, thereby effectively introducing a polymer layer for cell attachment onto the surface of the microcarrier and maintaining it stably during culture.

[0123] The primer polymer layer may include, but is not limited to, any one or more catechol derivatives capable of inducing aqueous phase adhesion selected from the group consisting of L-dihydroxyphenylalanine (L-DOPA), dopamine, polydopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof.

[0124] The microcarrier may be a microcarrier for cell culture.

[0125] 2.Cell complex According to another embodiment of the present invention, there is provided a cell complex comprising the microcarrier of the above embodiment and cells attached to the surface of the microcarrier. The content relating to the microcarrier may include all of the content described above in the above embodiment.

[0126] The cell complex of the other embodiment can achieve high cell survival rate and high in vivo transplantation rate by forming a complex in which cells are stably attached to the microcarrier.

[0127] The cells are not limited to adherent animal cells, but may be, for example, fibroblasts, epithelial cells, osteoblasts, chondrocytes, hepatocytes, human umbilical cord blood cells, human bone marrow-derived mesenchymal stem cells, CHO (Chinese hamster ovary) cells, kidney cells (HEK293, BHK21, MDCK, Vero cells, etc.), or a mixture of two or more of these.

[0128] The density of the cells is 1.02 g / cm 3 More than 1.1g / cm 3 It may be less than.

[0129] The cells can be attached onto the surface of the microcarrier, that is, they can be in direct contact with the surface of the microcarrier, or they can be in contact with the surface of another layer that is in contact with the surface of the microcarrier.

[0130] 3.Medicinal compositions According to yet another embodiment of the present invention, there is provided a medical composition comprising the microcarrier of the one embodiment or the cell complex of the other embodiment. The details regarding the microcarrier may include all of the details described above in the one embodiment. The details regarding the cell complex may include all of the details described above in the other embodiments.

[0131] The pharmaceutical active substance may be present in a state contained within the polymeric microparticles.

[0132] Examples of the pharmaceutically effective substance are not particularly limited, and depending on the intended use of the polymeric microparticles of the embodiment, any effective substance suitable for the intended use can be applied without limitation.That is, specific examples of the pharmaceutically active substance are not limited, and include amphetaminil, arecoline, atropine, bupranolol, buprenorphine, capsaicin, carisoprodol, chlorpromazine, ciclopirox olamine, cocaine, desipramine, dyclonine, epinephrine, ethosuximide, fluoxetine, hydromorphine, imipramine, lidocaine, methamphetamine, melproic acid, and the like. a drug selected from the group consisting of methylphenidate, morphine, oxybutynin, nadolol, nicotine, nitroglycerin, pindolol, prilocaine, procaine, propanolol, rivastigmine, scopolamine, selegiline, tulobuterol, valproic acid, donepezil, and the like, as well as erythropoietin (EPO), human growth hormone (hGH), exenatide, glucagon-like peptide-1 (GLP-1), insulin, granulocyte colony-stimulating factor (CSF), and the like. These include peptide or protein drugs selected from the group consisting of steroid hormones, estrogen, progesterone, and parathyroid hormone (PTH), and all pharmacological substances with proven pharmacological effects can be applied without any restrictions.

[0133] The amount of the pharmaceutically active substance added is not particularly limited, and can be used without limitation depending on the intended use and target. For example, the active substance can be added in an amount of 0.0001 to 1,000,000 parts by weight per 100 parts by weight of the polymeric microparticles, and can be added in a small or excessive amount relative to the polymeric microparticles.

[0134] 4. Beauty composition According to yet another embodiment of the present invention, there is provided a cosmetic composition comprising the microcarrier of the one embodiment or the cell complex of the other embodiment. The details regarding the microcarrier may include all of the details described above in the one embodiment. The details regarding the cell complex may include all of the details described above in the other embodiments.

[0135] The cosmetic benefit agent may be present contained within the polymeric microparticles.

[0136] Examples of the cosmetically effective substance are not particularly limited, and any effective substance suitable for the intended use of the polymeric microparticles of the present embodiment can be used without limitation. That is, specific examples of the cosmetically effective substance are not limited, and include natural extracts, proteins, vitamins, enzymes, antioxidants, etc., and any substance with proven cosmetic effects can be used without limitation.

[0137] The amount of the cosmetic active substance added is not particularly limited, and can be used without limitation depending on the intended use and target. For example, the cosmetic active substance can be added in an amount of 0.0001 to 1,000,000 parts by weight per 100 parts by weight of the polymeric microparticles, and can be added in a small or excessive amount relative to the polymeric microparticles.

[0138] 5. Medical supplies According to yet another embodiment of the present invention, there is provided a medical product comprising the medical composition of the other embodiment. The content relating to the medical composition may include all of the content described above in the other embodiment.

[0139] Examples of the medical supplies are not particularly limited, but are suitable for cases where the properties of the present invention are to be realized by being inserted into the body or where strength must be maintained for a long period of time, and include, for example, internal prostheses, internally inserted drug delivery devices, transdermal patches, wound healing agents, etc.

[0140] 6.Beauty supplies According to yet another embodiment of the present invention, a beauty product including the cosmetic composition of the other embodiment is provided. The content related to the cosmetic composition may include all of the content described above in the other embodiment.

[0141] Examples of the beauty products are not particularly limited, but include beauty creams, lotions, hair gels, packs, etc., in order to realize the characteristics of the present invention.

[0142] The structure of the cosmetic pack is not particularly limited, but may include, for example, a support and a cosmetic active substance delivery layer formed on the support and containing the polymeric microparticles of the other embodiment. Examples of the support include woven fabric, nonwoven fabric, silicone, polyethylene terephthalate, polyethylene, polypropylene, polyurethane, metal net, polyester, etc. [Effects of the Invention]

[0143] According to the present invention, there are provided microcarriers that can achieve excellent mechanical strength and stability, can be injected into the body immediately after 3D culture without a cell detachment process, and can provide a stable environment for adherent cells, thereby contributing to high cell survival rates and a high in vivo transplantation rate, as well as cell complexes, medical compositions, cosmetic compositions, medical supplies, and cosmetic supplies containing the same. [Brief explanation of the drawings]

[0144] [Figure 1] 1 is an optical microscope (OM) photograph of the polymeric microparticles of Example 1. [Figure 2] 1 is an IR photograph of the characteristic peak (1650 cm −1 ) of gelatin in the polymeric microparticles of Example 1. [Figure 3] 1 is an IR photograph of the characteristic peak of hyaluronic acid (1080 cm −1 ) relative to the characteristic peak of gelatin (1650 cm −1 ) of the polymer microparticles of Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0145] The present invention will be described in more detail in the following examples, but the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0146] <Example: Production of polymeric microparticles and microcarriers for cell culture> Example 1 (1) Production of polymer microparticles 200 mg of hyaluronic acid salt (weight average molecular weight: 500 kDa) was dissolved in 0.1 N NaOH aqueous solution at 2 wt.%, and 250 mg of gelatin (gel strength: 300 g Bloom) was dissolved in distilled water at 2.5 wt.%. 10 mL of each solution was mixed to make 20 mL, and then this was treated with iron ions (Fe 3+ Droplets formed using an encapsulator were added to 80 mL of ethanol solution containing 4 g of FeCl3, a compound containing methyl ...

[0147] 2.2 g of 1,4-butandiol diglycidyl ether (BDDE) was mixed with an 80% ethanol solution containing 20% ​​0.1N NaOH aqueous solution, and the crosslinked particles were added. The crosslinking reaction was carried out at room temperature for 3 days to produce polymeric microparticles. The produced particles were washed with ethanol and then distilled water, and the produced crosslinked particles were collected using a 45 μm mesh sieve. The collected crosslinked particles were then filtered through a 500 μm mesh sieve, and the remaining crosslinked particles were analyzed.

[0148] An optical microscope (OM) photograph of the prepared polymeric microparticles is shown in FIG.

[0149] The characteristic peak of gelatin (1650 cm) of the prepared polymer microparticles -1 ) is shown in Figure 2. The characteristic peak of gelatin (1650 cm -1 The areas containing gelatin were brightened due to the difference in intensity of the light, confirming that gelatin was distributed in the shell of the polymer microparticles.

[0150] The characteristic peak of gelatin (1650 cm) of the prepared polymer microparticles -1 ) relative to the characteristic peak of hyaluronic acid (1080 cm -1 ) is shown in Figure 3. Areas where hyaluronic acid is present in a higher relative amount to gelatin appear brighter, confirming that hyaluronic acid is distributed in a higher relative amount compared to gelatin in the core of the prepared polymer microparticles.

[0151] (2) Manufacturing of microcarriers for cell culture The collected particles were immersed in Tris buffer (pH 8.0) containing 1 mg / mL dopamine and coated for 2 hours at room temperature under stirring. After washing off excess coating material with ethanol, the particles were filtered through a 45 μm sieve and used as microcarriers for cell culture.

[0152] Example 2 Iron ions (Fe 3+ Instead of an ethanol solution containing 4 g of FeCl3, a compound containing aluminum ions (Al 3+ Polymeric microparticles and microcarriers for cell culture were produced in the same manner as in Example 1, except that an ethanol solution containing 4 g of AlCl3, a compound containing methyl ...

[0153] Example 3 Polymeric microparticles and microcarriers for cell culture were prepared in the same manner as in Example 1, except that 2.2 g of 50% glutaraldehyde was added instead of 2.2 g of 1,4-butandiol diglycidyl ether (BDDE).

[0154] <Comparative Example> Comparative Example 1: Production of polymeric microparticles Hyaluronate (weight-average molecular weight: 500 kDa) and gelatin (gel strength: 300 g Bloom) were dissolved in distilled water at 2 wt.% and 20 wt.% concentrations, respectively, to prepare 5 ml of each solution. These two solutions were then mixed with liquid paraffin solution to prepare a microemulsion mixture. 2.2 g of 1,4-butandiol diglycidyl ether (BDDE) was added as a crosslinker to the mixture, and the crosslinking reaction was carried out at room temperature for 5 days to produce polymeric microparticles. The resulting particles were washed sequentially with acetone, dichloromethane, and distilled water, and the resulting crosslinked particles were collected using a 45 μm mesh sieve. The collected crosslinked particles were then filtered through a 500 μm mesh sieve, and the remaining crosslinked particles were analyzed.

[0155] Comparative Example 2: Production of polymeric microparticles Hyaluronic acid salt (weight average molecular weight: 500 kDa) was dissolved in 0.1 N NaOH aqueous solution at 2 wt.%. Gelatin (gel strength: 300 g Bloom) was dissolved in distilled water at 2.5 wt.%. 10 mL of each solution was mixed to make 20 mL. Then, iron ions (Fe 3+ Droplets formed using an encapsulator were added to 80 mL of ethanol solution containing 4 g of FeCl3, a compound containing methyl methyl ketone (Methyl ketone), and the crosslinking reaction was carried out at 4°C for 2 hours. After that, the crosslinked particles were washed with ethanol and distilled water to produce crosslinked particles. The crosslinked particles were collected using a 45 μm mesh sieve. The collected crosslinked particles were then filtered through a 500 μm mesh sieve, and the remaining crosslinked particles were analyzed.

[0156] Comparative Example 3: Production of polymeric microparticles 200 mg of hyaluronate (weight-average molecular weight: 500 kDa) was dissolved in 0.1 N NaOH solution at a concentration of 2 wt.% and 250 mg of gelatin (gel strength: 300 g Bloom) was dissolved in distilled water at a concentration of 2.5 wt.% to prepare 10 ml of each solution. These two solutions were then mixed with liquid paraffin solution to prepare a microemulsion mixture. 2.2 g of 1,4-butanediol diglycidyl ether (BDDE) was then added to the mixture as a crosslinker and allowed to crosslink for 5 days at room temperature. The mixture was washed sequentially with acetone, dichloromethane, and distilled water, and the resulting crosslinked particles were collected through a 45 μm mesh sieve. The collected crosslinked particles were then filtered through a 500 μm mesh sieve and analyzed.

[0157] Comparative Example 4: Production of polymeric microparticles Hyaluronate (weight-average molecular weight: 500 kDa) was dissolved in 0.1 N NaOH solution at a concentration of 2 wt.% and gelatin (gel strength: 300 g Bloom) was dissolved in distilled water at a concentration of 2.5 wt.% to prepare 5 ml each. These two solutions were then mixed with liquid paraffin solution to prepare a microemulsion mixture. 2.2 g of 1,4-butandiol diglycidyl ether (BDDE) was then added to the mixture as a crosslinker and allowed to crosslink for 5 days at room temperature. The mixture was washed sequentially with acetone, dichloromethane, and distilled water, and the resulting crosslinked particles were collected through a 45 μm mesh sieve. The collected crosslinked particles were then filtered through a 500 μm mesh sieve and analyzed.

[0158] Comparative Example 5: Production of polymeric microparticles Hyaluronate (weight-average molecular weight: 500 kDa) and gelatin (gel strength: 300 g Bloom) were dissolved in distilled water at 2 wt.% and 2.5 wt.%, respectively, to prepare 5 ml of each solution. These two solutions were then mixed with liquid paraffin solution to prepare a microemulsion-containing mixture. 2.2 g of 1,4-butandiol diglycidyl ether (BDDE) was added as a crosslinker to the mixture, and the crosslinking reaction was carried out at room temperature for 5 days. The mixture was washed sequentially with acetone, dichloromethane, and distilled water, and the resulting crosslinked particles were recovered through a 45 μm mesh sieve. The recovered crosslinked particles were then filtered through a 500 μm mesh sieve, and the remaining crosslinked particles were analyzed.

[0159] Comparative Example 6: Production of polymeric microparticles Alginate and cellulose were dissolved in distilled water at 2.5 wt.% each, and 10 mL of each solution was mixed to prepare 20 mL of solution. This was then diluted with calcium ions (Ca 2+The droplets formed using an encapsulator were added to 80 mL of ethanol solution containing 4 g of CaCl2, a compound containing methyl methyl acrylate. The droplets were then subjected to a crosslinking reaction at room temperature for 2 hours, and washed with ethanol to produce crosslinked particles.

[0160] 2.2 g of 1,4-butandiol diglycidyl ether (BDDE) was mixed with an 80% ethanol solution containing 20% ​​0.1N NaOH aqueous solution, and the crosslinked particles were added. The crosslinking reaction was carried out at room temperature for 3 days to produce polymeric microparticles. The produced particles were washed with ethanol and then distilled water, and the produced crosslinked particles were collected using a 45 μm mesh sieve. The collected crosslinked particles were then filtered through a 500 μm mesh sieve, and the remaining crosslinked particles were analyzed.

[0161] Comparative Example 7: Production of polymeric microparticles 200 mg of hyaluronic acid salt (weight average molecular weight: 500 kDa) was dissolved in 0.1 N NaOH solution at a concentration of 2 wt.% and 250 mg of gelatin (gel strength: 300 g Bloom) was dissolved in distilled water at a concentration of 2.5 wt.%. 10 mL of each solution was mixed to make 20 mL, and then this was added with iron ions (Fe 3+ Droplets formed using an encapsulator (BUCHI, B-390) were added to 80 mL of ethanol solution containing 4 g of FeCl3, a compound containing methyl ...

[0162] The cross-linked particles are then treated with calcium ions (Ca 2+ The polymeric microparticles were prepared by adding 4 g of CaCl2, a compound containing methylcellulose, to 80 mL of ethanol solution and crosslinking at 4°C for 2 hours. The resulting particles were washed with ethanol and then distilled water, and the crosslinked particles were collected using a 45 μm mesh sieve. The collected crosslinked particles were then filtered through a 500 μm mesh sieve, and the remaining crosslinked particles were analyzed.

[0163] Comparative Example 8: Production of microcarriers for cell culture The polymeric microparticles obtained in Example 1(1) were used as microcarriers for cell culture.

[0164] <Experimental Example 1> The polymeric microparticles prepared in the above examples and comparative examples were evaluated for average diameter, sphericity, strength, cell culture compatibility, and stability by the following methods.

[0165] 1. Average diameter The average diameter of the polymeric microparticles in distilled water of the examples and comparative examples was measured using a laser particle size analyzer (Horiba, Partica LA-960).

[0166] 2. Spheroidization degree Optical photographs (Olympus, BX53) of the polymeric microparticles of the examples and comparative examples were taken, and the sphericity was calculated from the photographs.

[0167] The sphericity according to the present invention was calculated as the average value of the ratio of the shortest diameter to the longest diameter (longest diameter ratio) of 30 particles randomly selected in an optical photograph.

[0168] In this case, the closer the sphericity value is to 1, the closer the particle is to a sphere.

[0169] 3.Strength The strength of the polymeric microparticles of the examples and comparative examples was measured using a texture analyzer. Thirty microparticles swollen in distilled water for 24 hours were placed as a monolayer under a flat cylindrical probe equipped with a 5 N load cell. The initial trigger force was set to 1 mN, and the particles were compressed at a rate of 1 mm / s. The compressive force was determined as the force required to deform the particles to 25% of their average diameter.

[0170] The average compressive strength was calculated by dividing the compressive force by 30, which is the number of microparticles measured.

[0171] 4.Cell culture compatibility A 6-well plate was filled with cell culture medium, and polymeric microparticles and cells were added and cultured using the plate-rocking method. The temperature of the culture medium was maintained at 37°C, and the cells were cultured for 3 days, after which the number of cells cultured with polymeric microparticles was counted.

[0172] At this time, the compatibility with cell culture was evaluated according to the following criteria.

[0173] Suitable: The number of cells cultured is 100% or more of the number of cells input. Unsuitable: The number of cells cultured is less than 100% of the number of cells added, or the microparticles are degraded during culture.

[0174] 5. Stability The stability of polymeric microparticles contained in phosphate-buffered saline solution during sterilization using a high-temperature, high-pressure sterilizer (autoclave) and particle stability during long-term culture were evaluated according to the following criteria.

[0175] Conforms to: Weight loss of dried polymer microparticles before and after use in an autoclave is 20% or less Non-compliant: Weight loss of dried polymeric microparticles before and after use in an autoclave exceeds 20%

[0176] [Table 1]

[0177] As shown in Table 1, the polymeric microparticles of the Examples not only demonstrated a cell culture rate of over 100% relative to the number of cells introduced, making them suitable for cell culture, but also demonstrated a weight loss of less than 20% for the dried polymeric microparticles before and after autoclave treatment, confirming their suitability for sterilization and long-term culture. Furthermore, the polymeric microparticles of the Examples exhibited excellent mechanical properties, with an average compressive strength of 0.37 mN or greater, and a high proportion of particles exhibiting high crosslink density. In other words, the polymeric microparticles of the Examples were confirmed to be suitable for cell culture, sterilization, and long-term culture, while also exhibiting excellent crosslink density and mechanical properties. In contrast, the polymeric microparticles of Comparative Example 1 not only demonstrated a weight loss of over 20% for the dried polymeric microparticles before and after autoclave treatment, making them unsuitable for sterilization and long-term culture, but also demonstrated poor mechanical properties, with an average compressive strength of 0.23 mN, confirming a high proportion of particles exhibiting low crosslink density.

[0178] Furthermore, it was observed that the polymer microparticles of Comparative Example 2 shrank due to dissolution of the non-crosslinked polymer during the distilled water washing step. Furthermore, the microparticles were decomposed during cell culture, resulting in less than 100% of the cultured cells compared to the number of cells introduced, making them unsuitable for cell culture. Furthermore, the weight loss rate of the dried polymer microparticles before and after autoclave treatment exceeded 20%, making them unsuitable for sterilization and long-term culture. Furthermore, the average compressive strength was 0.1 mN, indicating poor mechanical properties.

[0179] Furthermore, unlike Comparative Example 1, the polymeric microparticles of Comparative Examples 3 to 5 were not formed by adjusting the concentration of the aqueous solution containing the biocompatible polymer to the same level as in Example 1, and it was confirmed that in the examples, polymeric microparticles could be formed even at low biocompatible polymer concentrations.

[0180] The polymer microparticles of Comparative Example 6 showed an average compressive strength of 0.29 mN in distilled water due to chemical crosslinking with a crosslinking agent. However, it was confirmed that they were not suitable for cell culture because they used alginate and cellulose, which do not have cell adhesion properties, as biocompatible polymers. In addition, calcium ions (Ca 2+ ) can be cross-linked to react reversibly with calcium ions present in cell culture medium, reducing particle strength during cell culture.

[0181] It was confirmed that the polymeric microparticles of Comparative Example 7 were manufactured only through ionic crosslinking, and some of the microparticles were decomposed during sterilization and cell culture, making them unsuitable for cell culture.

[0182] <Experimental Example 2> The physical properties of the microcarriers for cell culture prepared in Example 1 and Comparative Example 8 were measured by the following methods and are shown in Table 2.

[0183] 6. Coating layer thickness The thickness of the coating layer formed on the surface of the polymer microparticles in the microcarriers for cell culture was measured by cross-sectional TEM images.

[0184] 7.Cell adhesion Mesenchymal stem cells (density: 1.05 g / cm) were cultured in a 100 mL vertical wheel bioreactor (PBS). 3 The cells were cultured at 37°C for 7 days, and the number of cells cultured on the microcarriers was counted. The cell adhesion of the microcarriers was evaluated by comparing the number of cells initially added with the number of cells according to the following formula:

[0185] Cell adhesion = (number of cells after incubating microcarriers in cell culture medium at 37°C for 7 days / number of cells initially contained in the cell culture medium) x 100

[0186] [Table 2]

[0187] As shown in Table 2, the cell culture microcarrier of Example 1 was found to have significantly improved cell adhesion compared to Comparative Example 8 by including a 0.1 μm cell adhesion material coating layer on the surface of the polymeric microparticles.

Claims

1. A polymeric microparticle having a core-shell structure, comprising: a core comprising a first biocompatible polymer, a metal ion, and an organic crosslinker comprising two or more reactive functional groups; and a shell surrounding all or a portion of the core, the shell comprising a second biocompatible polymer, a metal ion, and an organic crosslinker comprising two or more reactive functional groups; and a cell attachment-inducing layer formed on the surface of the polymeric microparticles; the first biocompatible polymer includes hyaluronic acid; the second biocompatible polymer includes gelatin; The metal ions are iron ions (Fe ３＋ ), aluminum ions (Al ３＋ ), the cell attachment-inducing layer comprises one or more cell adhesive substances selected from the group consisting of collagen, fibronectin, chitosan, polydopamine, poly-L-lysine, vitronectin, RGD-containing peptides, acrylic polymers containing RGD, lignin, and cationic dextran; Microcarriers having a cell adhesion of 2000% or more as calculated by the following formula: [Formula] Cell adhesion = (number of cells after incubating microcarriers in cell culture medium at 37°C for 7 days / number of cells initially contained in the cell culture medium) x 100.

2. the core comprises a polymer matrix in which a first biocompatible polymer is crosslinked via a metal ion and an organic crosslinker having two or more reactive functional groups; The microcarrier of claim 1 , wherein the shell comprises a polymer matrix in which a second biocompatible polymer is crosslinked via a metal ion and an organic crosslinker containing two or more reactive functional groups.

3. The core has a volume of:

3. The microcarrier of claim 2, comprising a polymer matrix in which hyaluronic acid is crosslinked via a metal ion and an organic crosslinker containing two or more reactive functional groups at a volume ratio of more than 50%.

4. The shell has a volume of:

3. The microcarrier of claim 2, comprising a polymer matrix of gelatin crosslinked at a ratio of more than 50% by volume via a metal ion and an organic crosslinker containing two or more reactive functional groups.

5. The microcarrier of claim 1 , wherein the polymeric microparticles have an average diameter in distilled water of 1 μm or more.

6. Based on the cross section having the longest diameter of the polymeric microparticle, The microcarrier of claim 1, wherein the shell thickness is 95% or less of the longest diameter of the polymeric microparticle.

7. The organic crosslinking agent containing two or more reactive functional groups is The microcarrier according to claim 1, which comprises a crosslinking agent having 1 to 30 carbon atoms and containing two or more reactive functional groups.

8. The polymeric microparticles are 2. The microcarrier according to claim 1, which has an average compressive strength of 0.1 mN or more when deformed to a level of 25% of the average particle diameter.

9. The polymeric microparticles are 2. The microcarrier according to claim 1, wherein the sphericity, which is the ratio of the shortest diameter to the longest diameter of any particle in an optical photograph (long diameter ratio), is 0.9 or more and 1.0 or less.

10. The microcarrier according to claim 1, wherein the cell attachment-inducing layer has a layer thickness of 1 nm to 10,000 nm.

11. 10. The microcarrier of claim 1, wherein the microcarrier has an average diameter of 1 μm to 1000 μm.

12. The microcarrier of claim 1 , wherein the microcarrier is a microcarrier for cell culture.

13. The microcarrier of claim 1; and cells attached on the surface of the microcarriers.

14. A medical composition comprising any one of the microcarriers of claim 1 or the cell complexes of claim 13.

15. A cosmetic composition comprising any one of the microcarriers of claim 1 or the cell complexes of claim 13.

16. A medical product comprising the medical composition of claim 14.

17. A cosmetic product comprising the cosmetic composition of claim 15.

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