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

Abstract

The present invention relates to a polymer matrix containing a biocompatible polymer; and a polymer matrix having a reactive functional group content of 1 mol / 10 3 The present invention relates to a microcarrier including a polymeric microparticle comprising a polypeptide having a molecular weight of 100 g or less, 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 application(s) This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0129845 filed on September 30, 2021, and all contents disclosed in the documents of that Korean patent application are incorporated herein by reference. The present invention relates to a microcarrier that can achieve high crosslinking efficiency and production yield while also achieving stability, as well as confirming drug release properties without chemical modification of protein or peptide pharmaceutical active substances, can be injected into the body immediately after 3D culture without a cell detachment process, and can provide a stable environment for adherent cells to increase cell survival rate, thereby contributing to a high in vivo transplantation rate, as well as a cell complex, medical composition, cosmetic composition, medical product, and cosmetic product containing the same. [Background technology]

[0002] A drug delivery system refers to a series of technologies that enable the delivery and release of drugs to a target site while maintaining their activity. As we enter an age of aging, the needs for health care and drug treatment are increasing, and the development of drug delivery technologies to meet these needs is actively underway.

[0003] Drug delivery systems can be classified into oral administration, transdermal administration, injection, pulmonary inhalation, and mucosal administration according to the delivery route. Oral administration is the most common drug delivery method administered orally and occupies the largest proportion in the market. Transdermal administration is a method of delivering drugs through the skin, which allows continuous administration for a long period of time and makes it easy to adjust the blood concentration. Injection types are divided into intravenous, intramuscular, and subcutaneous injections, and pulmonary inhalation and mucosal administration types have the advantage of being quickly absorbed and avoiding pain. The carriers used in the drug delivery system field must be biocompatible, and therefore attempts to utilize natural polymers are continuing. In addition, the drugs used have been expanded from existing low molecular weight synthetic drugs to high molecular weight protein and peptide drugs. The drug release model of the drug delivery system is a model for observing the drug release phenomenon according to time based on the system parameters. A drug release model based on a mathematical formula can be a tool that can determine and predict how each parameter in the drug delivery system affects the release behavior, which is very important because it allows the design of new drug delivery systems. In this process, a process is required to accurately measure and analyze the degree of drug release over time in each model.

[0004] Hyaluronic acid and gelatin, which are biocompatible polymers, are used as polymer materials for drug delivery system carriers. Hyaluronic acid, which is mainly used as a biocompatible material, is composed of N-acetyl-D-glucosamine and D-glucuronic acid, and is a biopolymer material in which the repeating units are linearly linked. It is abundant in the vitreous humor of the eyeball, synovial fluid of joints, and cockscomb. Hyaluronic acid is often used as a bioinjectable material due to its excellent biocompatibility and viscoelasticity, but its use is limited because it is easily decomposed in vivo or under acidic or alkaline conditions. On the other hand, gelatin is a polymer obtained by hydrolyzing collagen, a biological connective tissue, and is widely used as a scaffold for cell culture. Gelatin's cell adhesion ability can be used to capture and culture cells, but its strength is weak and it has phase transition properties depending on temperature, so research is being conducted to improve its physical properties by introducing functional groups using chemical methods.

[0005] Many publications on drug delivery systems using hyaluronic acid and gelatin have proposed chemical cross-linking of hyaluronic acid / gelatin to improve the physical / chemical stability of the material against the body environment, and chemical cross-linking is mainly performed through cross-linking between the hydroxyl functional group or carboxylate of hyaluronic acid and the amine or carboxylate amino acid residues of gelatin. However, in the case of deliverable proteins and peptide active substances, most of them have functional groups such as hydroxyl and amine, which are the same as the constituent materials of polymer particles, and are chemically bonded to the carrier during the cross-linking process, making it difficult to confirm the actual drug release behavior.

[0006] Therefore, there is a need to develop microcarriers or polymeric microparticles that can confirm the drug release characteristics without inducing structural changes to the protein and peptide pharmaceutical active substances to be delivered.

[0007] In addition, in the process of mass expansion of adherent cells using microcarriers, spherical microcarriers that can increase surface efficiency have been developed to overcome the limitations of existing 2D culture and are used in 3D expansion culture. 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 due to the addition of a detachment process and induction of cell damage.

[0008] Furthermore, in the case of injectable cell therapy, the cells are mainly injected into the affected area in a liquid phase in which they are suspended. In the case of adherent cells, this creates an environment that is not stable for their survival and there is a limit to the implantation rate and cell survival rate due to the immune response in the body.

[0009] 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, thereby increasing cell survival rates and contributing to a high rate of transplantation in the body. Summary of the Invention [Problem to be solved by the invention]

[0010] The object of the present invention is to provide a microcarrier which can realize high crosslinking efficiency and production yield while at the same time achieving stability, as well as confirming drug release characteristics without chemical modification of protein or peptide pharmaceutical active substances, which can be immediately injected into the body after 3D culture without a cell detachment process, and which can provide a stable environment for adherent cells, thereby increasing cell survival rate and contributing to a high in vivo transplantation rate.

[0011] Another object of the present invention is to provide a cell complex comprising the microcarrier.

[0012] Another object of the present invention is to provide a medical composition containing the above-mentioned microcarrier or cell complex.

[0013] Another object of the present invention is to provide a cosmetic composition containing the microcarrier or cell complex.

[0014] Another object of the present invention is to provide a medical product containing the medical composition.

[0015] Another object of the present invention is to provide a cosmetic product containing the above-mentioned cosmetic composition. [Means for solving the problem]

[0016] The present invention relates to a polymer matrix containing a biocompatible polymer; and a polymer matrix having a reactive functional group content of 1 mol / 10 3 A microcarrier is provided comprising a polymeric microparticle comprising: a polypeptide having a molecular weight of 1000 g or less.

[0017] Also provided herein is a cell complex comprising the microcarrier; and a cell attached on the surface of the microcarrier.

[0018] Also provided herein is a medical composition comprising the microcarrier or cell complex described above.

[0019] Also provided herein is a cosmetic composition comprising the microcarrier or cell complex described above.

[0020] Also provided herein is a medical product comprising the medical composition described above.

[0021] Also provided herein is a cosmetic product comprising the cosmetic composition.

[0022] The microcarriers according to specific embodiments of the invention, and the cell complexes, medical compositions, cosmetic compositions, medical supplies, and cosmetic supplies containing the same will be described in more detail below.

[0023] Unless otherwise expressly stated herein, the terminology used is for the purpose of referring to particular embodiments only and is not intended to be limiting of the invention.

[0024] As used herein, the singular forms include the plural forms unless the context clearly indicates to the contrary.

[0025] As used herein, the meaning of "comprising" is to embody certain properties, regions, integers, steps, operations, elements, components and / or components, and does not exclude the presence or addition of other certain properties, regions, integers, steps, operations, elements, components and / or groups.

[0026] In this specification, terms including ordinal numbers such as "first" and "second" are used for the purpose of distinguishing one component from another component, and are not limited by the ordinal number. For example, within the scope of the present invention, a first component may be named a second component, and similarly, a second component may be named a first component.

[0027] The term "substituted or unsubstituted" as used herein means that the group is substituted or unsubstituted with one or more substituents selected from the group consisting of deuterium, halogen, cyano, nitro, hydroxy, carbonyl, ester, imide, amide, primary amino, carboxy, sulfonic acid, sulfonamide, phosphine oxide, alkoxy, aryloxy, alkylthio, arylthio, alkylsulfoxy, arylsulfoxy, silyl, boron, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, aralkenyl, alkylaryl, alkoxysilylalkyl, arylphosphine, or heterocyclic groups containing one or more of N, O, and S atoms, or that the group is substituted or unsubstituted with two or more of the above-listed substituents. For example, the "substituent with two or more substituents connected" may be a biphenyl group. In other words, the biphenyl group may be an aryl group, and may be interpreted as a substituent with two phenyl groups connected.

[0028] As used herein, the term "(co)polymer" refers to a polymer or copolymer, where the polymer refers to a homopolymer consisting of a single repeating unit, and the copolymer refers to a composite polymer containing two or more types of repeating units.

[0029] In this specification, the molecular weight of the polymer used is according to the manufacturer's measurement method, and the method does not deviate from the normal GPC measurement method. For example, in this specification, the weight average molecular weight can mean the molecular weight measured by light scattering methods or viscosity methods.

[0030] The present invention can be modified in various ways and can have various forms, and will be described in detail below by taking specific examples as examples. However, this is not intended to limit the present invention to the specific disclosed form, but it should be understood that the present invention includes all modifications, equivalents, and alternatives within the spirit and technical scope of the present invention.

[0031] As used herein, microparticles means that the particle cross section is circular or elliptical, and the ratio of the minor axis / major axis (sphericity) of the particle is in the range of 0.7 to 1.0. The lengths of the minor and major axes of the particle can be derived by taking an optical photograph of the particle and calculating the average value of any 30 to 100 particles in the optical photograph.

[0032] In this specification, emulsion means a mixed phase in which one or more of the unmixed liquids, the oil phase or the water phase, is dispersed in the other liquid (dispersion medium) in a fine particle state (dispersoid). Emulsions can be generally classified into macroemulsions, microemulsions, and nanoemulsions according to the particle size of the dispersed phase.

[0033] The present invention will now be described in more detail.

[0034] 1. Microcarriers According to one embodiment of the present invention, a polymer matrix containing a biocompatible polymer; and a polymer matrix having a reactive functional group content of 1 mol / 10 3 A microcarrier may be provided which comprises a polymeric microparticle comprising: a polypeptide having a molecular weight of 1 g or less.

[0035] Conventional polymer microparticles are prepared by adding the particles directly to an organic solvent to carry out a crosslinking reaction, and the particle morphology is greatly affected by the polymer concentration and the crosslinker content. In particular, when the polymer concentration is low or the crosslinker content is low, the particle morphology is not maintained, making it difficult to form microparticles.

[0036] Therefore, the inventors have completed the invention by performing two crosslinking reactions, as in the manufacturing method of polymeric microparticles described below, to maximize the crosslinking density with the same amount of crosslinking agent, while at the same time confirming the release behavior of the pharmacologic active substance in the microparticles.

[0037] In particular, the team confirmed through experiments that by carrying out the second crosslinking reaction in the solid phase, spherical particles can be produced even with a small amount of crosslinking agent, thereby improving production yields.

[0038] In addition, the microcarrier of one embodiment further includes a cell attachment-inducing layer formed on the surface of the polymer microparticle, thereby enabling immediate injection into the body after 3D culture without a cell detachment process, and the adherent cells form a complex in a state where they are stably attached to the microcarrier, thereby increasing the cell survival rate and achieving a high in vivo transplantation rate. This was confirmed through experiments, and the invention was completed.

[0039] Specifically, the biocompatible polymer refers to a polymer that can be directly injected into the human body for effective drug delivery of medicines to be applied to the human body. Specifically, the biocompatible polymer may be one or more polymers selected from the group consisting of hyaluronic acid (HA), carboxymethyl cellulose (CMC), alginic acid, pectin, carrageenan, chondroitin (sulfate), dextran (sulfate), chitosan, polylysine, collagen, gelatin, carboxymethyl chitin, fibrin, agarose, pullulan, polyesteramide, poly(valeric acid), polyacrylate, cellulose acetate, poly(vinylimidazole), polyvinylpyrrolidone (PVP), polymethacrylate, hydroxypropylmethylcellulose (HPMC), ethylcellulose (EC), hydroxypropylcellulose (HPC), cyclodextrin, and polyacrylic acid copolymers, and derivatives thereof.

[0040] More specifically, the biocompatible polymer may be hyaluronic acid (HA), gelatin, or a mixture of hyaluronic acid and gelatin.

[0041] In this specification, hyaluronic acid can mean both hyaluronic acid itself and hyaluronate.Therefore, hyaluronic acid aqueous solution can mean the concept of all of the aqueous solution of hyaluronic acid, the aqueous solution of hyaluronate, and the mixed aqueous solution of hyaluronic acid and hyaluronate.The hyaluronate can be inorganic salt such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, organic salt such as tetrabutylammonium hyaluronate, and mixtures thereof.

[0042] In one embodiment of the present invention, the molecular weight of hyaluronic acid is not particularly limited, but is preferably from 500 g / mol to 5,000,000 g / mol in order to achieve diverse physical properties and biocompatibility.

[0043] As used herein, gelatin may refer to a protein obtained by treating animal-derived collagen with acid or alkali, followed by extraction.

[0044] In one embodiment of the invention, the molecular weight of gelatin is not particularly limited, but is preferably from 100,000 g / mol to 5,000,000 g / mol in order to achieve a variety of physical properties and biocompatibility.

[0045] As a result of research into polymer microparticles, the inventors have confirmed that the crosslinking density can be maximized with the same amount of crosslinker by carrying out a second crosslinking reaction in a dehydrated and aggregated solid phase after a first crosslinking reaction in a swollen emulsion, as described below, and thus completed the invention.

[0046] In particular, by carrying out the second crosslinking reaction in a more aggregated solid phase, it is possible to realize spherical particles even with a small amount of crosslinking agent, thereby improving the production yield. This was confirmed through experiments, leading to the completion of the invention.

[0047] In addition, in the case of conventional polymer microparticles, there was a technical problem that the pharmaceutical active substance participated in crosslinking during the manufacturing process, which changed the structure or eliminated or reduced the actual release characteristics. In the case of the polymer microparticles of the present invention, the pharmaceutical active substance is dispersed in the polymer matrix and the content of reactive functional groups is 1 mol / 10 3 The inventors have confirmed through experiments that excellent properties as a protein drug release model can be realized by using a polypeptide having a molecular weight of less than 1 g, and thus completed the invention.

[0048] Specifically, the polymer microparticles contained in the microcarrier of the embodiment have a reactive functional group content of 1 mol / 10 3g or less, or 0.001 mol / 10 3 g or more 1mol / 10 3 g or less, or 0.001 mol / 10 3 g or more 0.5mol / 10 3 g or less, or 0.001 mol / 10 3 g or more 0.1mol / 10 3 The polypeptide may include a polypeptide that is no more than 1 g.

[0049] That is, in the above embodiment, the content of reactive functional groups in the polypeptide is 1 mol / 10 3 g or less means that the polypeptide contained in the polymeric microparticles contained in the microcarrier of one embodiment is 3 It may mean that the reactive functional group is contained in an amount of 1 mol or less per g.

[0050] The method for measuring the reactive functional group content is not particularly limited. For example, 3 g is dissolved in an organic solvent, and an indicator such as methyl red or phenolphthalein is added, followed by neutralization titration with 0.1M hydrochloric acid (amine functional group) or sodium hydroxide (carboxyl functional group), or it can be calculated using the following formula. [Formula] Reactive amine (carboxyl) functional group content (mol / 10 3 g) = amount of hydrochloric acid added (L) (or amount of sodium hydroxide) x molar ratio (mol / L) of hydrochloric acid solution (or molar ratio of sodium hydroxide solution) / amount of polypeptide dissolved in solvent (10 3 g)

[0051] The polymeric microparticles contained in the microcarrier of one embodiment have a content of reactive functional groups of the polypeptide of 1 mol / 10 3g or less, the polypeptide has no or only a very small amount of reactive functional groups, and the reaction between the crosslinker and the reactive functional groups in the polypeptide can be minimized during the chemical crosslinking process for improving the physical and chemical stability of the polymer microparticles. As a result, not only is there no problem of loss of function of the protein drug due to crosslinking, but excellent protein drug release properties can be achieved.

[0052] In one embodiment, the hydrodynamic radius of the polypeptide may be between 1 nm and 30 nm, or between 1 nm and 20 nm, or between 1 nm and 15 nm.

[0053] Since the hydrodynamic radius of the polypeptide is between 1 nm and 30 nm, the technical effect of being able to adjust the release rate of the polypeptide in the same polymeric microparticle can be realized.

[0054] Specifically, the hydrodynamic radius of the polypeptide can be measured by placing 3 mL of a 1 mg / mL aqueous polypeptide solution in a plastic square cuvette at 37°C using a NanoBrook 173 plus (Brookhaven Instruments Corporation) device.

[0055] On the other hand, in the above embodiment, the content of the reactive functional group is 1 mol / 10 3 g or less can include polypeptides in which 90 mol% or more, or 90 mol% or more to 100 mol% or 95 mol% or more to 100 mol% or 99 mol% or more to 100 mol% or less of reactive functional groups on the surface are substituted with a blocking compound.

[0056] Examples of the blocking compound are not particularly limited, and any compound having a functional group that can react with the reactive functional group contained in the polypeptide to form a chemical bond and a blocking group with weak reactivity can be used without limitation.

[0057] Examples of functional groups capable of reacting with reactive functional groups contained in the polypeptide to form chemical bonds include isocyanate groups, carboxy groups, halogen groups, etc. Examples of the blocking groups having low reactivity include alkyl groups having 1 to 50 carbon atoms, cycloalkyl groups having 2 to 50 carbon atoms, aryl groups having 6 to 50 carbon atoms, heteroaryl groups having 3 to 50 carbon atoms, etc.

[0058] In this specification, an alkyl group is a monovalent functional group derived from an alkane and may be linear or branched, but the number of carbon atoms in the linear alkyl group is not particularly limited and is preferably 1 to 20. In addition, the number of carbon atoms in the branched alkyl group is 3 to 20. Specific examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, sec-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl, heptyl, n-heptyl, 1-methylhexyl, octyl, n-octyl, tert-octyl, 1-methylheptyl, 2-ethylhexyl, 2-propylpentyl, n-nonyl, 2,2-dimethylheptyl, 1-ethyl-propyl, 1,1-dimethyl-propyl, isohexyl, 2-methylpentyl, 4-methylhexyl, 5-methylhexyl, 2,6-dimethylheptan-4-yl, and the like. The alkyl group may be substituted or unsubstituted, and when substituted, examples of the substituents are as described above.

[0059] In the present specification, the cycloalkyl group is a monovalent functional group derived from a cycloalkane, and may be monocyclic or polycyclic, but is not particularly limited, and has 3 to 20 carbon atoms. In another embodiment, the cycloalkyl group has 3 to 10 carbon atoms. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 3-methylcyclopentyl, 2,3-dimethylcyclopentyl, cyclohexyl, 3-methylcyclohexyl, 4-methylcyclohexyl, 2,3-dimethylcyclohexyl, 3,4,5-trimethylcyclohexyl, 4-tert-butylcyclohexyl, cycloheptyl, cyclooctyl, and bicyclo[2,2,1]heptyl. The cycloalkyl group may be substituted or unsubstituted, and if substituted, examples of the substituent are as described above.

[0060] In the present specification, the aryl group is a monovalent functional group derived from arene, and is not particularly limited, and preferably has 6 to 20 carbon atoms, and may be a monocyclic aryl group or a polycyclic aryl group. The aryl group may be a monocyclic aryl group such as a phenyl group, a biphenyl group, a terphenyl group, etc., but is not limited thereto. The polycyclic aryl group may be a naphthyl group, an anthracenyl group, a phenanthryl group, a pyrenyl group, a perylenyl group, a chrysenyl group, a fluorenyl group, etc., but is not limited thereto. The aryl group may be substituted or unsubstituted, and if substituted, examples of the substituent are as described above.

[0061] In the present specification, a heteroaryl group includes one or more non-carbon atoms, i.e., heteroatoms, and specifically, the heteroatoms may include one or more atoms selected from the group consisting of O, N, Se, S, etc. The number of carbon atoms is not particularly limited, but is preferably 4 to 20, and the heteroaryl group may be monocyclic or polycyclic. Examples of the heterocyclic group include a thiophene group, a furanyl group, a pyrrole group, an imidazolyl group, a thiazolyl group, an oxazolyl group, an oxadiazolyl group, a pyridyl group, a bipyridyl group, a pyrimidyl group, a triazinyl group, a triazolyl group, an acridyl group, a pyridazinyl group, a pyrazinyl group, a quinolinyl group, a quinazolinyl group, a quinoxalinyl group, a phthalazinyl group, a pyridopyrimidyl group, a pyridopyrazinyl group, a pyrazinopyrazinyl group, an isoquinolinyl group, an indolyl group, a carbazolyl group, a benzoxazolyl group, a benzimidazolyl group, a benzothiazolyl group, a benzocarbazolyl group, a benzothiophene group, a di ... Examples of heteroaryl groups include, but are not limited to, an aryl group, a benzofuranyl group, a phenanthrolinyl group, a thiazolyl group, an isoxazolyl group, an oxadiazolyl group, a thiadiazolyl group, a benzothiazolyl group, a phenothiazinyl group, an aziridyl group, an azaindolyl group, an isoindolyl group, an indazolyl group, a purine group, a pteridine group, a β-carbolyl group, a naphthyridine group, a ter-pyridyl group, a phenazinyl group, an imidazopyridyl group, a pyropyridyl group, an azepine group, a pyrazolyl group, and a dibenzofuranyl group. The heteroaryl group may be substituted or unsubstituted, and when substituted, examples of the substituent are as described above.

[0062] Examples of halogen groups are fluorine, chlorine, bromine or iodine.

[0063] More specifically, examples of the blocking compound include blocking compounds having a functional group containing an electrophilic carbon, such as fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RBITC), tetramethylrhodamine B isothiocyanate, N-hydroxy-succinimidyl-ester linked fluorescein (NHS-fluorescein), 6-[fluorescein-5(6)-carboxamido]hexanoic acid N-hydroxysuccinimide ester, fluorescein-5-EX N-hydroxysuccinimide ester, Alexa Fluor (trade name) 350 NHS Ester, Alexa Fluor (trade name) 430 NHS Ester, Alexa Fluor (trade name) 488 NHS Ester, and Rhodamine Green (trade name) Carboxylic Acid, Succinimidyl Ester, Hydrochloride (5(6)-CR110, SE), Rhodamine Red (trade name)-X, Succinimidyl Ester, 5-isomer, and Alexa Fluor (trade name) 647 NHS Ester.

[0064] In one embodiment, the reactive functional groups of the polypeptide are reacted with the blocking compound to be substituted by 90 mol % or more, so that the content of the reactive functional groups of the polypeptide contained in the polymer microparticles contained in the microcarrier of the one embodiment described above is 1 mol / 10 3 g or less.

[0065] That is, in the embodiment, the reactive functional groups of the polypeptide are substituted by 90 mol % or more by reacting with the blocking compound, and the polypeptide has no or only a very small amount of reactive functional groups, so that the reaction between the crosslinker and the reactive functional groups can be minimized during the chemical crosslinking process for improving the physical and chemical stability of the polymer microparticles. As a result, not only is there no problem of loss of function of the protein drug due to crosslinking, but excellent protein drug release properties can be achieved.

[0066] In the embodiment, the reactive functional group may be one or more selected from the group consisting of an amine group, a hydroxyl group, a phenylhydroxyl group, and an imide group.

[0067] The reactive functional group may be a reactive functional group contained in a polypeptide contained in a polymeric microparticle.

[0068] When the reactive functional group reacts with a crosslinking agent during the chemical crosslinking process for improving physical and chemical stability, problems may arise such as loss of function of the protein drug due to crosslinking, as well as failure to achieve the release properties of the protein drug.

[0069] In one embodiment, the polypeptide may include a polypeptide having a functional group that is a bond between a blocking compound and a reactive functional group. That is, the polypeptide may have a functional group that is a bond between a blocking compound and a reactive functional group, and a reactive functional group that does not react with the blocking compound. The content of the reactive functional group that does not react with the blocking compound in the polypeptide is 1 mol / 10 as described above. 3 g or less can be satisfied.

[0070] The examples of the functional group which is a combination of the blocking compound and the reactive functional group are not limited, and any functional group contained in the reaction product obtained by the reaction of the above-mentioned various blocking compounds and the reactive functional group can be applied without limitation. Examples of the functional group which is a combination of the blocking compound and the reactive functional group include urethane, thiourethane, urea, thiourea, amide, ether, thioether, amine, etc.

[0071] In a more specific example, the polypeptide may include a polypeptide having a thiourea or thiourethane functional group, which is a bond between a blocking compound containing an isothiocyanate group and a reactive functional group.

[0072] Specifically, the polymeric microparticles may comprise the reaction product of a blocking compound including an isothiocyanate and a polypeptide including a reactive functional group.

[0073] In other words, the isothiocyanate group (-N=C=S) contained in the blocking compound containing isothiocyanate reacts with the reactive functional group contained in the polypeptide containing a reactive functional group to form a thiourea functional group, thereby allowing the polypeptide to include a thiourea functional group.

[0074] In addition, examples of the polypeptide containing the reactive functional group are not limited, but any polypeptide suitable for the application of the polymeric microparticle of the embodiment can be applied without limitation. That is, specific examples of the polypeptide are not limited, and can contain a primary amine at the N-terminus. For example, the polypeptide can be polyglycine, polyalanine, polyvaline, polyleucine, polyisoleucine, polyphenylalanine, polytryptophan, polyglutamic acid, polyproline, or polyaspartic acid.

[0075] The polypeptide has a nucleophilic functional group other than a primary amine at 1 mol / 10 3 If the amount is more than g, not all of the reactive functional groups will be protected even in the reaction with the blocking compound, and the drug release properties of the polymer microparticles may be reduced due to an addition reaction between the chemical crosslinker and the unprotected reactive functional groups.

[0076] The amount of the polypeptide to be added is not particularly limited, and the amount can be freely adjusted depending on the application and subject. For example, the polypeptide can be added in an amount of 0.0001 to 1,000 parts by weight based on 100 parts by weight of the polymeric microparticles, and can be added in a small amount without any excessive amount based on the polymeric microparticles.

[0077] Meanwhile, the polymer matrix may include a first crosslinked region in which the biocompatible polymer is crosslinked via a first crosslinking agent; and a second crosslinked region in which the biocompatible polymer is crosslinked via a second crosslinking agent.

[0078] The first crosslinked region may refer to a crosslinked region formed by a first crosslinking reaction between an emulsion containing a biocompatible polymer and a first crosslinking agent, and the second crosslinked region may refer to a crosslinked region formed by a second crosslinking reaction between the emulsion containing a biocompatible polymer and a second crosslinking agent without a first crosslinking reaction between the emulsion and the first crosslinking agent, and a crosslinked region formed through an additional second crosslinking reaction between the first crosslinked region and the second crosslinking agent.

[0079] In one embodiment of the present invention, the first crosslinking agent is not particularly limited, but may include one of iron chloride (III) and aluminum chloride.

[0080] In one embodiment of the present invention, examples of the second crosslinking agent are not particularly limited. Specifically, the second crosslinking agent may be 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, ... The second crosslinking agent may include one or more selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), trimethylpropane polyglycidyl ether, bis(2,3-epoxypropoxy)ethylene, pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, divinylsulfone, and glutaraldehyde. More specifically, the second crosslinking agent may be 1,4-butandiol diglycidyl ether (BDDE).

[0081] Meanwhile, the polymer microparticles contained in the microcarrier may have an average diameter in distilled water of 1 μm to 1,000 μm, 100 μm to 1,000 μm, 100 μm to 800 μm, 100 μm to 750 μm, 100 μm to 600 μm, or 150 μm to 600 μm. When the average diameter of the polymer microparticles is within the above range, the cell attachment and culture performance are excellent.

[0082] In addition, the polymeric microparticles contained in the microcarrier may have a swelling degree of 10 or less, 0.1 to 10, 0.1 to 5, 0.5 to 5, or 0.9 to 4.5, according to the following mathematical formula 1. [Formula 1] Swelling degree = {(average diameter in distilled water) 3 - (average diameter of dry particles) 3} / (average diameter of dry particles) 3

[0083] If the swelling degree of the polymer microparticles exceeds 10, the crosslinking density is low and the mechanical strength is poor, which may cause a technical problem of reduced stability.

[0084] In addition, the polymeric microparticles contained in the microcarrier of one embodiment may release 20% to 100%, 50% to 100%, 75% to 100%, or 90% to 100% of a pharmacologic active substance under release conditions of pH 5.0 or higher and 30°C to 40°C after incubation for 150 hours or more.

[0085] Specifically, the release conditions may be pH 5.0 or higher, pH 5.0 or higher and pH 14.0 or lower, pH 5.0 or higher and pH 11.0 or lower, or pH 5.6 or higher and pH 11.0 or lower.

[0086] Moreover, the release conditions can be specifically 30°C or higher and 40°C or lower, 35°C or higher and 40°C or lower, or 36°C or higher and 38°C or lower.

[0087] Since the release amount of the polymeric microparticles contained in the microcarrier of the embodiment is 20% or more and 100% or less, polymeric microparticles with excellent active pharmaceutical ingredient release properties can be realized by using a crosslinking agent.

[0088] Meanwhile, examples of methods for producing polymeric microparticles contained in the microcarrier of one embodiment of the present invention are not limited to these, but may include, for example, a method for producing polymeric microparticles, which includes a step of subjecting a mixture containing a conjugate of a polypeptide and a blocking compound, a biocompatible polymer, and a first crosslinking agent to a first crosslinking reaction to form crosslinked particles; and a step of extracting the crosslinked particles and subjecting them to a second crosslinking reaction in an organic solvent containing a second crosslinking agent.

[0089] Conventional polymer microparticles are prepared by adding the particles directly to an organic solvent to carry out a crosslinking reaction. Therefore, the particle morphology is greatly affected by the polymer concentration and the crosslinker content. In particular, when the polymer concentration is low or the crosslinker content is low, the particle morphology is not maintained, making it difficult to form microparticles.

[0090] Therefore, the inventors performed two crosslinking reactions as in the manufacturing method of the polymeric microparticles, thereby maximizing the crosslinking density with the same amount of crosslinker, while simultaneously confirming the release behavior of the pharmacologic active substance.

[0091] In particular, the team confirmed through experiments that by carrying out the second crosslinking reaction in the solid phase, spherical particles can be produced even with a small amount of crosslinking agent, thereby improving production yields.

[0092] Specifically, the biocompatible polymer refers to a polymer that can be directly injected into the human body for effective drug delivery of medicines to be applied 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(butyl acid), poly(valeric acid), polyurethane, polyacrylate, ethylene-vinyl acetate polymer, acrylic substituted cellulose acetate, non-degradable polyurethane, polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulphonate polyolefins The polymer may be one or more polymers selected from the group consisting of polyolefins, polyethylene oxide, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polymethacrylate, hydroxypropyl methylcellulose (HPMC), ethyl cellulose (EC), hydroxypropyl cellulose (HPC), cyclodextrin, poly(N-isopropylamide) (PNIPAam), poloxamer, and polyacrylic acid copolymers, and derivatives thereof.

[0093] More specifically, the biocompatible polymer may be hyaluronic acid (HA), gelatin, or a mixture of hyaluronic acid and gelatin.

[0094] In this specification, hyaluronic acid can mean both hyaluronic acid itself and hyaluronate.Therefore, hyaluronic acid aqueous solution can mean the concept of all of the aqueous solution of hyaluronic acid, the aqueous solution of hyaluronate, and the mixed aqueous solution of hyaluronic acid and hyaluronate.The hyaluronate can be inorganic salt such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, organic salt such as tetrabutylammonium hyaluronate, and mixtures thereof.

[0095] In one embodiment of the invention, the molecular weight of hyaluronic acid is not particularly limited, but is preferably 500 g / mol or more and 5,000,000 g / mol or less in order to achieve diverse physical properties and biocompatibility.

[0096] As used herein, gelatin may refer to a protein obtained by treating animal-derived collagen with acid or alkali, followed by extraction.

[0097] In one embodiment of the invention, the molecular weight of gelatin is not particularly limited, but is preferably from 100,000 g / mol to 5,000,000 g / mol in order to achieve a variety of physical properties and biocompatibility.

[0098] In the conjugate of the polypeptide and the blocking compound, the examples of the blocking compound are not limited to a specific one, and any compound having a functional group that can react with the reactive functional group contained in the polypeptide to form a chemical bond and a blocking group with weak reactivity can be used without limitation.

[0099] Examples of the functional group capable of reacting with the reactive functional group contained in the polypeptide to form a chemical bond include an isocyanate group, a carboxy group, a halogen group, etc. Examples of the blocking group having low reactivity include an alkyl group having 1 to 50 carbon atoms, a cycloalkyl group having 2 to 50 carbon atoms, an aryl group having 6 to 50 carbon atoms, a heteroaryl group having 3 to 50 carbon atoms, etc.

[0100] More specifically, examples of the blocking compound include blocking compounds having a functional group containing an electrophilic carbon, such as fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RBITC), tetramethylrhodamine B isothiocyanate, N-hydroxy-succinimidyl-ester linked fluorescein (NHS-fluorescein), 6-[fluorescein-5(6)-carboxamido]hexanoic acid N-hydroxysuccinimide ester, fluorescein-5-EX N-hydroxysuccinimide ester, Alexa Fluor (trade name) 350 NHS Ester, Alexa Fluor (trade name) 430 NHS Ester, Alexa Fluor (trade name) 488 NHS Ester, and Rhodamine Green (trade name) Carboxylic Acid, Succinimidyl Ester, Hydrochloride (5(6)-CR110, SE), Rhodamine Red (trade name)-X, Succinimidyl Ester, 5-isomer, and Alexa Fluor (trade name) 647 NHS Ester.

[0101] In addition, the examples of the polypeptide are not particularly limited, but any polypeptide suitable for the application of the polymeric microparticle of the embodiment can be applied without limitation. That is, the specific examples of the polypeptide are not limited, and can include one or less primary amines. For example, the polypeptide can be polyglycine, polyalanine, polyvaline, polyleucine, polyisoleucine, polyphenylalanine, polytryptophan, polyglutamic acid, polyproline, or polyaspartic acid.

[0102] When the polypeptide contains two or more types of primary amines, not all of the reactive functional groups are protected with thiourea-based functional groups even in the reaction with a blocking compound containing isothiocyanate, resulting in the inclusion of reactive functional groups that are not protected with thiourea-based functional groups, and the drug release properties of the polymer microparticles may be reduced due to the addition reaction of the unprotected reactive functional groups.

[0103] In a more specific example, the conjugate of the polypeptide and the blocking compound may include a conjugate of a blocking compound containing an isothiocyanate group and a polypeptide having a thiourea or thiourethane functional group, which is a conjugate of a reactive functional group.

[0104] Specifically, the polymeric microparticles may comprise the reaction product of a blocking compound including an isothiocyanate and a polypeptide including a reactive functional group.

[0105] In other words, the isothiocyanate group (-N=C=S) contained in the blocking compound containing isothiocyanate reacts with the reactive functional group contained in the polypeptide containing a reactive functional group to form a thiourea functional group, thereby including a polypeptide having a thiourea functional group.

[0106] The first crosslinking agent is not particularly limited, but may include one of iron chloride (III) and aluminum chloride.

[0107] Meanwhile, the step of forming crosslinked particles by subjecting a mixture containing a conjugate of the polypeptide and the blocking compound, a biocompatible polymer, and a first crosslinking agent to a first crosslinking reaction may include the steps of: forming a mixed solution in which the polypeptide, the blocking compound, and the biocompatible polymer are dissolved; and ejecting the mixed solution and adding it to the first crosslinking agent.

[0108] That is, the first crosslinking reaction may be a liquid phase reaction carried out by adding a first crosslinker to a mixed solution containing the conjugate of the polypeptide and the blocking compound and the biocompatible polymer.

[0109] In addition, in the step of forming crosslinked particles by subjecting a mixture including the conjugate of the polypeptide and the blocking compound, the biocompatible polymer, and the first crosslinking agent to a first crosslinking reaction, the first crosslinking reaction may be an ionic crosslinking reaction.

[0110] That is, in the step of forming crosslinked particles by a first crosslinking reaction of a mixture including the conjugate of the polypeptide and the blocking compound, the biocompatible polymer, and the first crosslinker, the first crosslinker may be an ionic compound. In one embodiment of the present invention, the first crosslinker is not particularly limited. Specifically, the first crosslinker may include one of iron(III) chloride and aluminum chloride.

[0111] Meanwhile, in the step of extracting the crosslinked particles and subjecting them to a second crosslinking reaction in an organic solvent containing a second crosslinker, the crosslinked particles and the second crosslinker may be crosslinked in a solid phase.

[0112] That is, the method for producing polymeric microparticles may include a second crosslinking reaction performed in a solid phase in an alkaline organic solvent. Also, the second crosslinking reaction performed in a solid phase may mean a second crosslinking reaction performed in a more aggregated state by dehydrating swollen polymer particles. In one embodiment of the invention, when the second crosslinking reaction performed in a solid phase in an organic solvent is included, that is, when the second crosslinking reaction performed in a more aggregated state by dehydrating swollen polymer particles is included, a superior crosslinking efficiency is achieved compared to the case where the second crosslinking reaction is not performed, and microparticles having high strength can be obtained.

[0113] The crosslinked particles can be collected using a mesh sieve with a mesh size of 30 μm or more. After the crosslinked particles formed through the first crosslinking reaction are collected, they are redispersed in an organic solvent, and a second crosslinking agent is added to the particles, so that the second crosslinking reaction can be carried out in a more aggregated solid phase.

[0114] The examples of the second crosslinking agent are not particularly limited. Specifically, the second crosslinking agent may be 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, ... The second crosslinking agent may include one or more selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), trimethylpropane polyglycidyl ether, bis(2,3-epoxypropoxy)ethylene, pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, divinylsulfone, and glutaraldehyde. More specifically, the second crosslinking agent may be 1,4-butandiol diglycidyl ether (BDDE).

[0115] The first crosslinking agent and the second crosslinking agent may each independently comprise one or more selected from the group consisting of iron(III) chloride, aluminum chloride, 1,4-butanediol diglycidyl ether, glutaraldehyde, ethylene glycol diglycidyl ether, hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, bisepoxypropoxyethylene, pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, and divinyl sulfone.

[0116] In addition, the second crosslinking agent may be included in an amount of 30 to 300 parts by weight, 40 to 250 parts by weight, or 50 to 200 parts by weight, based on 100 parts by weight of the biocompatible polymer. According to an embodiment of the present invention, the method for producing polymeric microparticles includes a first crosslinking reaction followed by a second crosslinking reaction as described above, and thereby 30 to 300 parts by weight of the second crosslinking agent is added based on 100 parts by weight of the biocompatible polymer to produce polymeric microparticles having sufficient mechanical strength and sphericity.

[0117] If the second crosslinker is added in an amount of less than 30 parts by weight per 100 parts by weight of the biocompatible polymer, the degree of crosslinking will be low and it will be difficult to form particles. If the second crosslinker is added in an amount of more than 300 parts by weight, a technical problem may occur in that an excess amount of unreacted crosslinker is not removed and remains in the crosslinked particles.

[0118] Meanwhile, in the step of extracting the crosslinked particles and subjecting them to a second crosslinking reaction in an organic solvent containing a second crosslinking agent, the organic solvent may be ethanol, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylcaprolactam, 2-pyrrolidone, N-ethylpyrrolidone, N-vinylpyrrolidone, dimethylsulfoxide, tetramethylurea, pyridine, dimethylsulfone, hexamethylsulfoxide, γ-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, methyl ethyl ketone, methyl isobutane, methyl ethyl ketone ... The alkyl ether may be one or more selected from the group consisting of milketone, 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.

[0119] Meanwhile, the microcarrier of one embodiment may further include a cell attachment-inducing layer formed on the surface of the polymeric microparticle. The cell attachment-inducing layer is made of a cell adhesive material and allows adherent cells to stably attach, spread, and be cultured.

[0120] The cell attachment-inducing layer may include one or more cell adhesive substances selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polydopamine, poly-L-lysine, vitronectin, peptides containing RGD, acrylic polymers containing RGD, lignin, cationic dextran, and derivatives thereof. That is, the cell attachment-inducing layer may include a cell adhesive substance including one or a mixture of two or more of the cell adhesive substances selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polydopamine, poly-L-lysine, vitronectin, peptides containing RGD, acrylic polymers containing RGD, lignin, cationic dextran, and derivatives thereof.

[0121] The cell adhesion-inducing layer may be 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 the cell adhesion-inducing layer may be in contact with the surface of another layer that is in contact with the surface of the polymeric microparticles.

[0122] As an example, the microcarrier includes a cell attachment-inducing layer formed so as to be in direct contact with the surface of the polymeric microparticle. By introducing the cell attachment-inducing layer onto the surface of the microcarrier, the buoyancy of the microcarrier in the culture medium can be adjusted, and the effect of stably attaching and culturing cells can be obtained.

[0123] The cell adhesion-inducing layer may have a layer thickness of 1 nm to 10,000 nm, or 10 nm to 1,000 nm, or 50 nm to 500 nm, or 80 nm to 200 nm, and the layer thickness of the cell adhesion-inducing layer may be calculated by subtracting the radius of the internal polymer microparticle from the radius of the entire microcarrier. The radius means half the diameter. Examples of methods for measuring the diameter are not particularly limited, but may be measured using a confocal fluorescence microscope, a transmission electron microscope (TEM), or cross-sectional IR or cross-sectional SEM images.

[0124] The microcarriers can 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. Examples of methods for measuring the diameter are not particularly limited, but one example is that it can be measured through an optical microscope.

[0125] Meanwhile, the ratio of the radius of the polymer microparticle to the thickness of the cell attachment-inducing layer may be 1:0.00001 to 1:0.1, or 1:0.0001 to 1:0.01.

[0126] If the ratio of the radius of the polymer microparticles to the thickness of the cell adhesion-inducing layer is less than 1:0.00001, the cell adhesion-inducing layer may be too thin compared to the polymer microparticles, resulting in a reduced adhesion between the cells and the microcarriers during cell culture. If the ratio exceeds 1:0.1, the cell adhesion-inducing layer may be too thick compared to the polymer microparticles, resulting in a change in physical properties such as the hydrophilicity of the polymer microparticles, thereby reducing dispersibility.

[0127] The microcarriers may have a cell adhesion, calculated by the following formula, of 2000% or more, or 2500% or more, or 3000% or more, or 4000% or less, or 2000% to 4000%, or 2500% to 4000%, or 3000% to 4000%. [Formula] Cell attachment = (number of cells after incubating microcarriers in cell culture medium at 37℃ for 7 days / number of cells initially contained in the cell culture medium) x 100

[0128] The cell culture medium, cells, and culture conditions are not particularly limited, and various previously known cell culture mediums, cells, and culture conditions can be applied without limitation. The method for measuring the cell count is also not particularly limited, and various previously known cell count measuring methods can be applied without limitation.

[0129] The microcarriers have a cell adhesion calculated by the above formula of 2000% or more, or 2500% or more, or 3000% or more, or 4000% or less, or 2000% to 4000%, or 2500% to 4000%, or 3000% to 4000%, thereby improving the cell attachment force of the microcarriers, thereby increasing the surface efficiency through three-dimensional expansion culture, thereby enabling mass expansion of adherent cells.

[0130] On the other hand, when the cell adhesion calculated by the above formula does not fall within the above-mentioned range, the cell attachment force on the surface of the microcarrier is weak, making it difficult to perform three-dimensional expansion culture sufficiently.

[0131] Meanwhile, the microcarrier may further include a primer polymer layer formed on the surface of the polymer microparticle.

[0132] That is, the surface of the polymeric microparticle may further include a mixed layer of one primer polymer layer and one cell adhesion-inducing layer. In the mixed layer of one primer polymer layer and one cell adhesion-inducing layer, the order of lamination is not particularly limited, and any structure in which a cell adhesion-inducing layer is laminated on a primer polymer layer, or a structure in which a primer polymer layer is laminated on a cell adhesion-inducing layer, may be applied.

[0133] Meanwhile, the primer polymer layer acts as an adhesive layer capable of introducing functional polymers onto the surface of the polymer microparticles, thereby enabling a polymer layer for cell attachment to be effectively introduced onto the microcarrier surface and stably maintained even during culture. The primer polymer layer may include, but is not limited to, any one or more selected from the group consisting of L-dihydroxyphenylalanine (L-DOPA), dopamine, polydopamine, norepinephrine, epinephrine, epigallocatechin, and derivatives thereof as a catechol derivative capable of inducing aqueous phase adhesion.

[0134] The microcarrier may be a cell culture microcarrier.

[0135] 2.Cell complex According to another embodiment of the present invention, there may be provided a cell complex comprising the microcarrier of the above embodiment and a cell attached on the surface of the microcarrier. The content relating to the microcarrier may include all of the content described above in the above embodiment.

[0136] In the cell complex of the other embodiment, the cells are stably attached to the microcarrier to form a complex, thereby increasing the cell survival rate and achieving a high transplantation rate in vivo.

[0137] The cells are adherent animal cells, examples of which are not limited to, 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.

[0138] The density of the cells is 1.02 g / cm 3 More than 1.1g / cm 3 It may be less than.

[0139] The cells may be attached onto the surface of the microcarriers, i.e., the cells may be in direct contact with the surface of the microcarriers, or the cells may be in contact with the surface of another layer that is in contact with the surface of the microcarriers.

[0140] 3.Medical Compositions According to another embodiment of the present invention, a medical composition may be provided that includes the microcarrier of the one embodiment or the cell complex of the other embodiment. The content related to the microcarrier may include all of the content described above in the one embodiment. The content related to the cell complex may include all of the content described above in the other embodiment.

[0141] That is, when the pharma- ceutical active substance of the embodiment is applied for medical purposes, a medical composition can be provided.

[0142] In this case, examples of medical effective substances are not limited, and effective substances suitable for the application of the polymer microparticles of the embodiment can be applied without limitation. That is, specific examples of the medical effective substances are not limited, and can include medical effective substances such as polyglycine, polyalanine, polyvaline, polyleucine, polyisoleucine, polyphenylalanine, polytryptophan, polyglutamic acid, polyproline, or polyaspartic acid, or elastin-derived polypeptides (repeated polymers of (valine-proline-glycine-valine-glycine)), and all substances with proven medical effects can be applied without limitation.

[0143] The amount of the medical effective substance added is not particularly limited, and the content can be used without restriction depending on the application and subject. For example, the medical effective substance can be included in a small amount, without restriction, of 0.0001 to 1,000 parts by weight based on 100 parts by weight of the polymeric microparticles, that is, in the polymeric microparticles.

[0144] 4. Beauty composition According to another embodiment of the present invention, a cosmetic composition may be provided that includes the microcarrier of the one embodiment or the cell complex of the other embodiment. The content related to the microcarrier may include all the content described above in the one embodiment. The content related to the cell complex may include all the content described above in the other embodiment.

[0145] That is, when the pharmaceutical active substance of one embodiment is applied for cosmetic purposes, a cosmetic composition may be provided.

[0146] In this case, examples of cosmetic effective substances are not particularly limited, and effective substances suitable for the intended use of the polymeric microparticles of the embodiment can be applied without limitation. That is, specific examples of the cosmetic effective substances are not limited, and include natural extracts, proteins, vitamins, enzymes, antioxidants, etc., and all substances with proven cosmetic effects can be applied without limitation.

[0147] The amount of the cosmetic active substance added is not particularly limited, and the content can be used without restriction depending on the application and subject. For example, the cosmetic active substance can be included in a small amount relative to the polymeric microparticles, that is, 0.0001 parts by weight to 1,000 parts by weight based on 100 parts by weight of the polymeric microparticles, without restriction on an excessive amount.

[0148] 5. Medical supplies According to another embodiment of the present invention, a medical product including the medical composition of the other embodiment may be provided. The medical composition may include all of the above-mentioned aspects.

[0149] Examples of the medical supplies are not limited to, but are suitable for cases where the medical supplies are inserted into the body or need to maintain their strength for a long period of time in order to realize the characteristics of the present invention, and examples of such medical supplies include internal implants, internally inserted drug delivery devices, transdermal patches, wound treatment agents, etc.

[0150] 6.Beauty supplies According to another embodiment of the present invention, a cosmetic product including the cosmetic composition of the other embodiment may be provided. The content related to the cosmetic composition may include all of the content described above in the other embodiment.

[0151] Examples of the beauty products are not particularly limited, but may include beauty creams, lotions, hair gels, packs, etc., in order to realize the characteristics of the present invention.

[0152] The structure of the cosmetic pack is not limited to a particular one, and may include, for example, a support and a cosmetically active substance delivery layer formed on the support and including the polymeric microparticles of the other embodiment. Examples of the support include woven fabric, nonwoven fabric, silicone, polyethylene terephthalate, polyethylene, polypropylene, polyurethane, metal mesh, polyester, etc. Effect of the Invention

[0153] According to the present invention, there can be provided a microcarrier that can achieve high crosslinking efficiency and production yield while also achieving stability, as well as confirming drug release characteristics without chemical modification of a protein or peptide pharmaceutical active substance, that can be immediately injected into the body after 3D culture without a cell detachment process, and that can provide a stable environment for adherent cells, thereby increasing cell survival rate and contributing to a high in vivo transplantation rate, as well as a cell complex, medical composition, cosmetic composition, medical product, and cosmetic product containing the same. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0154] The present invention will be described in more detail with reference to the following examples, however, the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0155] <Example: Production of polymer microparticles and microcarriers for cell culture> Example 1 (1) Production of polymer microparticles 0.15g of hyaluronate (weight average molecular weight: 500kDa) and 1g of gelatin (gel strength: 300g) were dissolved in distilled water at 1.0wt% and 12.5wt, respectively, to prepare a solution, and then the two solutions were mixed in a 1:1 volume ratio. Fluorescein isothiocyanate-polyproline (hydrodynamic radius: 2.5nm, reactive functional group content: 0.01mol / 10 3g) aqueous solution to a concentration of 600 μg / mL. The mixed solution (4 g) was discharged through the nozzle of a Buchi encapsulator and solidified in a 5 wt% FeCl3 (iron chloride) solution to produce particles. The solid particles were then washed five times with ethanol, and 0.5 ml (0.55 g) of 1,4-butanediol diglycidyl ether (BDDE) was added as a crosslinking agent to 25 g of the solid particle dispersion, and crosslinking reaction was carried out at room temperature for 4 days. The crosslinked particles were then washed with ethanol and collected using a mesh sieve with a mesh size of 45 μm.

[0156] (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 at room temperature for 2 hours under stirring. Excess coating material was washed with ethanol, and the particles were filtered through a 45 μm sieve and used as microcarriers for cell culture.

[0157] Example 2 Polymeric microparticles and microcarriers for cell culture were produced in the same manner as in Example 1, except that in (1) of Example 1, the content of 1,4-butanediol diglycidyl ether was added to 1 ml (1.1 g).

[0158] Example 3 Polymeric microparticles and microcarriers for cell culture were produced in the same manner as in Example 1, except that the nozzle size was changed to 200 μm in (1) of Example 1.

[0159] Example 4 Polymeric microparticles and microcarriers for cell culture were produced in the same manner as in Example 1, except that in (1) of Example 1, the nozzle size was changed to 200 μm and 1 ml (1.1 g) of 1,4-butanediol diglycidyl ether was added.

[0160] Example 5 Polymeric microparticles and microcarriers for cell culture were produced in the same manner as in Example 1, except that the nozzle size was changed to 300 μm in (1) of Example 1.

[0161] Example 6 Polymeric microparticles and microcarriers for cell culture were produced in the same manner as in Example 1, except that in (1) of Example 1, the nozzle size was changed to 300 μm and 1 ml (1.1 g) of 1,4-butanediol diglycidyl ether was added.

[0162] Example 7 The crosslinked particles produced in Example 1(1) were used as microcarriers for cell culture without being subjected to the dopamine coating in Example 1(2).

[0163] Example 8 A microcarrier for cell culture was produced in the same manner as in Example 7, except that 1 ml (1.1 g) of 1,4-butanediol diglycidyl ether was added.

[0164] Example 9 Microcarriers for cell culture were produced in the same manner as in Example 7, except that the nozzle size was changed to 200 μm.

[0165] Example 10 Microcarriers for cell culture were produced in the same manner as in Example 7, except that the nozzle size was changed to 200 μm and the amount of 1,4-butanediol diglycidyl ether added was 1 ml (1.1 g).

[0166] Example 11 Microcarriers for cell culture were produced in the same manner as in Example 7, except that the nozzle size was changed to 300 μm.

[0167] Example 12 Microcarriers for cell culture were produced in the same manner as in Example 7, except that the nozzle size was changed to 300 μm and the amount of 1,4-butanediol diglycidyl ether added was 1 ml (1.1 g).

[0168] <Comparative Example> Comparative Example 1: Production of polymeric microparticles 0.15g of hyaluronate (weight average molecular weight: 500kDa) and 1g of gelatin (gel strength: 300g) were dissolved in distilled water at 1.5wt and 10wt, respectively, to prepare solutions, and then the two solutions were mixed in a 1:1 volume ratio. Fluorescein isothiocyanate-polyproline (hydrodynamic radius: 1.7nm, reactive functional group content: 1.1mol / 10 3 g) aqueous solution to a concentration of 600 μg / mL. The mixed solution (4 g) was mixed with liquid paraffin solution (40 g) to prepare a mixed solution containing a water-in-oil (W / O) microemulsion. Then, 1.1 g (1 ml) of 1,4-butandiol diglycidyl ether (BDDE) was added as a crosslinking agent to 44 g of the mixed solution, and the crosslinking reaction was carried out at room temperature for 4 days, followed by washing with acetone, dichloromethane, and ethanol in that order to prepare crosslinked particles. At this time, the prepared crosslinked particles were collected using a mesh sieve with a mesh size of 45 μm.

[0169] Comparative Example 2: Production of polymeric microparticles In Comparative Example 1, the active substance was human growth hormone (hydrodynamic radius: 2.5 nm, reactive functional group content: 10 mol / 10) instead of fluorescein isothiocyanate-polyproline. 3 It was produced in the same manner as in Comparative Example 1, except that g) was used.

[0170] Comparative Example 3: Production of polymeric microparticles In Comparative Example 1, the active substance was replaced with bovine serum albumin (hydrodynamic radius: 3.8 nm, reactive functional group content: 20 mol / 10 3 It was produced in the same manner as in Comparative Example 1, except that g) was used.

[0171] Comparative Example 4: Production of polymeric microparticles In Comparative Example 1, the active substance was fluorescein isothiocyanate-polylysine (hydrodynamic radius: 1.9 nm, reactive functional group content: 50 mol / 10) instead of fluorescein isothiocyanate-polyproline. 3 It was produced in the same manner as in Comparative Example 1, except that g) was used.

[0172] Comparative Example 5: Production of polymeric microparticles In Example 1(1), the effective substance was changed to fluorescein isothiocyanate-polylysine (hydrodynamic radius: 1.9 nm, reactive functional group content: 50 mol / 10 3 The preparation was carried out in the same manner as in Example 1(1), except that the compound (g) was used.

[0173] Comparative Example 6: Production of microcarriers for cell culture The polymeric microparticles obtained in Example 1(1) were used as microcarriers for cell culture.

[0174] [Table 1]

[0175] <Experimental Example 1> The physical properties of the polymer microparticles prepared in the above Examples and Comparative Examples were measured by the following methods and are shown in Tables 2 and 3.

[0176] 1. Average diameter and swelling degree The average diameter of the polymeric microparticles of the above examples and comparative examples was measured, and the swelling degree was calculated from the average diameter. The swelling degree of the particles according to the present invention was calculated according to the following formula 1. [Formula 1] Swelling degree = {(average diameter in distilled water) 3 - (average diameter of dry particles) 3} / (average diameter of dry particles) 3

[0177] In this case, the closer the swelling value is to 0, the less swelling occurs, and the larger the value is, the more swelling occurs and the larger the particles become.

[0178] The average diameter of dry particles and the average diameter in distilled water were measured using an optical microscope (Olympus, BX53).

[0179] 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 them.

[0180] The sphericity according to the present invention was calculated as the average value of the ratio of the shortest diameter to the longest diameter (major diameter ratio) of 30 particles randomly selected in an optical photograph.

[0181] In this case, the closer the sphericity value is to 100, the closer the particle is to a sphere.

[0182] [Table 2]

[0183] As shown in Table 2, the polymer microparticles of the embodiment prepared by the preparation method of the present application, which includes a step of recovering the particles after the first crosslinking and then performing the second crosslinking, have a high sphericity of 80 or more, while at the same time, have an excellent degree of crosslinking, with a swelling index of 0.9 to 4.3.

[0184] 3. Release amount The polymeric microparticles of the Examples and Comparative Examples were dispersed at 5 mg / mL under each release condition shown in Table 3 below, and then cultured for 4 weeks at 37° C. The fluorescence intensity of the culture supernatant was measured at a wavelength of 495 nm using a Synergy HTX multi-mode plate reader (BioTek), and the fluorescein isothiocyanate-polypeptide content was calculated based on a calibration curve of the conjugate of the polypeptide and blocking compound (fluorescein isothiocyanate) used in the Examples and Comparative Examples.

[0185] [Table 3]

[0186] As shown in Table 3, the polymer microparticles of the examples prepared by the preparation method of the present application, which includes a step of recovering particles after the first crosslinking and then performing the second crosslinking, showed 100% release amount over a release period of 150 hours or more, demonstrating protein drug release characteristics without chemical modification of the pharmacologic active substance. On the other hand, the polymer microparticles of the comparative example, which were only subjected to the first crosslinking, showed a release amount of less than 20% over a release period of 150 hours or more, demonstrating very poor protein drug release characteristics due to chemical modification of the pharmacologic active substance.

[0187] <Experimental Example 2> The physical properties of the microcarriers for cell culture prepared in Example 1 and Comparative Example 6 were measured by the following methods and are shown in Table 4.

[0188] 4. Coating layer thickness The thickness of the coating layer formed on the surface of the polymer microparticles in the cell culture microcarrier was measured through cross-sectional TEM images.

[0189] 5.Cell adhesion Mesenchymal stem cells (density: 1.05 g / cm) were placed in a 100 mL vertical wheel bioreactor (PBS). 3 The microcarriers for cell culture were filled with a culture medium containing 100% ethanol, and the microcarriers for cell culture were poured into the culture medium and stirred. After culturing at 37°C for 7 days, the number of cells cultured in the microcarriers for cell culture was confirmed. The cell adhesion of the microcarriers was evaluated by comparing the number of cells initially put in according to the following formula. Cell attachment = (number of cells after incubating microcarriers in cell culture medium at 37℃ for 7 days / number of cells initially contained in the cell culture medium) x 100

[0190] [Table 4]

[0191] As shown in Table 4, it was confirmed that the cell culture microcarrier of Example 1 had significantly improved cell adhesion compared to Comparative Example 6 by including a 0.1 μm cell adhesive material coating layer on the surface of the polymer microparticles.

Claims

1. a polymer matrix containing a biocompatible polymer; and a polymer matrix having a reactive functional group content of 1 mol / 10 ３ 1. A microcarrier comprising polymeric microparticles containing a polypeptide having a molecular weight of 1000 to 2000 g or less, and further comprising a cell attachment-inducing layer formed on the surface of the polymeric microparticles.

2. 2. The microcarrier of claim 1, wherein the hydrodynamic radius of the polypeptide is between 1 nm and 30 nm.

3. The microcarrier of claim 1 , wherein the reactive functional group is one or more selected from the group consisting of an amine group, a hydroxyl group, a thiol group, a phenylhydroxy group, and an imide group.

4. The content of the reactive functional group is 1 mol / 10 ３ A polypeptide having a length of 0.1 g or less is The microcarrier of claim 1, comprising a polypeptide having at least 90 mol % of reactive functional groups on its surface substituted with a blocking compound.

5. 10. The microcarrier of claim 1, wherein the polypeptide comprises a polypeptide having a functional group that is a conjugate of a blocking compound and a reactive functional group.

6. The microcarrier according to claim 1 , wherein the polymeric microparticles have an average diameter in distilled water of 1 μm or more and 1,000 μm or less.

7. The microcarrier according to claim 1 , wherein the polymeric microparticle has a swelling degree of 10 or less according to the following mathematical formula 1: [Formula 1] Swelling degree = {(average diameter in distilled water) ３ - (average diameter of dry particles) ３ } / (average diameter of dry particles) ３

8. The polymeric microparticles are 2. The microcarrier of claim 1, wherein after incubation for 150 hours or more, the amount of the pharmacologic active substance released is 20% or more and 100% or less under release conditions of pH 5.0 or more and 30°C to 40°C.

9. The polymer matrix has a first crosslinked region in which a biocompatible polymer is crosslinked via a first crosslinking agent; and The microcarrier according to claim 1 , further comprising a second crosslinked region in which the biocompatible polymer is crosslinked via a second crosslinking agent.

10. The microcarrier of claim 1 , wherein the biocompatible polymer comprises hyaluronic acid and gelatin.

11. The microcarrier of claim 1, wherein the cell attachment-inducing layer comprises one or more cell attachment substances selected from the group consisting of gelatin, collagen, fibronectin, chitosan, polydopamine, poly-L-lysine, vitronectin, peptides containing RGD, acrylic polymers containing RGD, lignin, cationic dextran, and derivatives thereof.

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

13. The microcarrier of claim 1 , wherein the microcarrier has an average diameter of between 1 μm and 1,000 μm.

14. The microcarrier according to claim 1, wherein the microcarrier has 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

15. The microcarrier of claim 1; and a cell attached on said microcarrier surface.

16. A medical composition comprising any one of the microcarriers of claim 1 or the cell complexes of claim 15.

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

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

19. A cosmetic product comprising the cosmetic composition according to claim 17.

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