KR102597505B1 - A method of preparing polymeric micro particles, polymeric micro particles, medical composition, cosmetic composition, medical articles and cosmetic articles using the same - Google Patents

Abstract

According to the present invention, a method for producing polymer microparticles comprising additional crosslinking by an organic crosslinking agent after crosslinking by metal ions, polymer microparticles, medical compositions containing the same, cosmetic compositions, medical supplies, and beauty products can be provided. .

Description

Method for producing polymer microparticles, polymer microparticles, medical compositions containing the same, cosmetic compositions, medical supplies and beauty supplies THE SAME}

The present invention relates to a method for producing polymer microparticles capable of achieving excellent mechanical strength and stability, polymer microparticles, medical compositions containing the same, cosmetic compositions, medical supplies, and cosmetic products.

As the field of biopharmaceuticals and regenerative medicine expands, the demand for cell mass culture technology that can efficiently produce cells, tissues, microorganisms, etc. is increasing.

Adherent cells are cultured using microcarriers in a 3D bioreactor. Cells, culture medium, and microcarriers are placed in a bioreactor, and the culture medium is stirred to bring the cells and microcarriers into contact, allowing the cells to attach to the surface of the microcarrier and be cultured. The microcarrier used in this case is suitable for mass culture of cells because it provides a high surface area ratio (surface area/volume) on which cells can attach and proliferate. However, when expanding and culturing adherent cells using a microcarrier, a process of recovering cells through a cell detachment process is essential after the end of the culture. The cell detachment process induces cell detachment by using proteolytic enzymes or changing temperature. However, if this detachment process is added, there is a problem that manufacturing costs increase, making it less economical, and cell damage may occur. .

The development of new materials and processes to solve this problem is steadily progressing. In particular, in the case of cell therapy products that inject cells in vivo, efforts are being made to omit the separation and purification process by securing the biocompatibility of the microcarrier that cultivates the cells. there is. In this case, particles that are strong enough to withstand the stress exerted by the fluid around the carrier during the culture process and after injection into the body are needed.

In addition, in the case of transdermal drug delivery technology that delivers a microcarrier containing a drug or bioactive substance by mounting it on a microneedle, the microcarrier must use a polymer suitable for application to the living body and the stratum corneum of the skin. It must have sufficient strength to prevent particle deformation during the process of passing through the layer. Micro-carriers that stably penetrate the transdermis can deliver the loaded drug locally or systematically and act on the necessary lesion.

Hyaluronic acid, which is mainly used as a biocompatible material, is a biopolymer composed of N-acetyl-D-glucosamine and D-glucuronic acid, and the repeating units are linearly connected. It is abundant in the vitreous humor of the eye, synovial fluid of joints, and chicken comb. Hyaluronic acid is commonly used as a bioinjectable material due to its excellent biocompatibility and viscoelasticity, but its use by itself is limited as it is easily decomposed in vivo or under acid, alkali, etc. conditions. In addition, when applied to a microcarrier, hyaluronic acid exhibits a negative charge in the biological pH range, which causes a problem in that cell adhesion is significantly reduced.

In addition, gelatin is a polymer obtained by hydrolyzing collagen, a biological connective tissue, and is used as a scaffold for cell culture. Cells can be collected or cultured, but their strength is weak and sensitive to temperature, so there are efforts to improve their strength by introducing functional groups through chemical methods.

Accordingly, there is a need to develop microcarriers or polymer microparticles that are biocompatible and have excellent properties and stability, such as physical strength and stability against heat and enzymes.

The present invention is intended to provide a method for manufacturing polymer microparticles that can achieve excellent mechanical strength and stability.

In addition, the present invention is to provide polymer microparticles manufactured by the above manufacturing method.

Additionally, the present invention is to provide a medical composition containing the above polymer microparticles.

Additionally, the present invention is to provide a cosmetic composition containing the above polymer microparticles.

Additionally, the present invention is to provide a medical product containing the above medical composition.

Additionally, the present invention is intended to provide a beauty product containing the above-mentioned beauty composition.

In this specification, reacting a mixture containing a biocompatible polymer and a metal ion to form polymer crosslinked particles; and further crosslinking the polymer crosslinked particles in a polar solvent containing an organic crosslinker containing one or more reactive functional groups.

Additionally, in the present specification, a core comprising a first biocompatible polymer, a metal ion, and an organic crosslinking agent containing one or more reactive functional groups; and a shell surrounding all or part of the core and containing a second biocompatible polymer, a metal ion, and an organic cross-linking agent containing one or more reactive functional groups. Polymer microparticles having a core-shell structure comprising a provided.

The present specification also provides a medical composition comprising the polymer microparticles and a pharmaceutically active substance contained in the polymer microparticles.

Also provided herein is a cosmetic composition comprising the above polymer microparticles and a cosmetically effective substance contained in the polymer microparticles.

Also provided in this specification are medical supplies containing the above-mentioned medical composition.

Also provided herein is a beauty product comprising the above cosmetic composition.

Hereinafter, the manufacturing method of polymer microparticles, polymer microparticles, medical compositions, cosmetic compositions, medical supplies, and cosmetic products containing the same according to specific embodiments of the invention will be described in more detail.

Unless explicitly stated herein, terminology is intended to refer only to specific embodiments and is not intended to limit the invention.

As used herein, singular forms include plural forms unless phrases clearly indicate the contrary.

As used herein, the meaning of 'comprise' is to specify a specific characteristic, area, integer, step, operation, element and/or component, and to specify another specific property, area, integer, step, operation, element, component and/or group. It does not exclude the existence or addition of .

Additionally, 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 numbers. For example, within the scope of the present invention, a first component may also be referred to as a second component, and similarly, the second component may be referred to as a first component.

In this specification, (co)polymer refers to both polymers or copolymers. The polymer refers to a homopolymer composed of a single repeating unit, and the copolymer refers to a complex polymer containing two or more types of repeating units.

Since the present invention can be subject to various changes and can take various forms, specific embodiments will be illustrated and described in detail below. However, this is not intended to limit the present invention to a specific disclosed form, and should be understood to include all changes, equivalents, and substitutes included in the spirit and technical scope above.

As used herein, microparticles mean that the cross-section of the particle is circular or oval, and the ratio of the minor axis to the major axis (degree of sphericity) of the particle is in the range of 0.7 to 1.0. The length of the minor axis and major axis of the particle can be derived by taking an optical photograph of the particle and calculating the average value of 30 to 100 random particles in the optical photograph.

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

The Dn can be measured using a 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 measuring device (Horiba LA-960) to determine the particle size distribution by measuring the difference in diffraction patterns according to particle size when the particles pass through the laser beam. Calculate D10, D50, and D90 can be measured by calculating the particle diameters at points that are 10%, 50%, and 90% of the cumulative distribution of particle numbers according to diameter in the measuring device. More specifically, in this specification, diameter may mean D50.

In this specification, emulsion refers to a mixed phase in which one or more immiscible liquids of an oil phase or an aqueous phase are dispersed in a fine particle state (dispersoid) in another liquid (dispersion medium). Emulsions can be generally divided into macroemulsions, microemulsions, and nanoemulsions, depending on the particle size of the dispersed phase.

Hereinafter, the present invention will be described in more detail.

One. Method for producing polymer microparticles

According to one embodiment of the invention, forming polymer cross-linked particles by reacting a mixture containing a biocompatible polymer and a metal ion; and further crosslinking the polymer crosslinked particles in a polar solvent containing an organic crosslinker containing one or more reactive functional groups. A method for producing polymer microparticles may be provided, including.

Conventional polymer microparticles are crosslinked after forming a W/O emulsion using oil, which inevitably involves an oil washing process, which not only results in poor process efficiency, but also poses a technical problem in that it is difficult to remove residual oil.

Accordingly, as in the method for producing polymer microparticles of the above embodiment, the present inventors maximized process efficiency by conducting an additional crosslinking reaction using an organic crosslinking agent containing one or more reactive functional groups after crosslinking by metal ions. At the same time, it was confirmed through experiments that the mechanical strength and stability of the micro particles were significantly improved, and the invention was completed.

Specifically, the biocompatible polymer refers to a polymer that can be directly injected into the human body in order to deliver effective substances applied to the human body. Specifically, the biocompatible polymers include Hyaluronic acid (HA), Carboxymethyl cellulose (CMC), alginic acid, pectin, carrageenan, chondroitin (sulfate), dextran (sulfate), chitosan, Polylysine, collagen, gelatin, carboxymethyl chitin, fibrin, agarose, pullulan, polylactide, polyglycolide (PGA), polylactide-glycolide copolymer (PLGA), poly polyanhydride, polyorthoester, polyetherester, polycaprolactone, polyesteramide, poly(butyric acid), poly(valeric acid), Polyurethanes, polyacrylates, ethylene-vinylacetate polymers, acrylic substituted cellulose acetates, non-degradable polyurethanes, polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonate polyolefins ), polyethylene oxide, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polymethacrylate, hydroxypropylmethylcellulose (HPMC), ethylcellulose (EC), hydroxypropylcellulose (HPC), cyclodextrin and one or more polymers selected from the group consisting of cellulose and copolymers of monomers forming such polymers.

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

Polymer microparticles manufactured using only hyaluronic acid are easily decomposed in vivo or under acid, alkali, etc. conditions, which limits their use, and their cell adhesion is significantly reduced. The mechanical properties of polymer microparticles are significantly reduced.

Accordingly, by using a mixture of hyaluronic acid (HA) and gelatin as a biocompatible polymer, excellent cell adhesion and mechanical properties can be achieved at the same time.

In this specification, hyaluronic acid may mean including both hyaluronic acid itself and hyaluronic acid salts. Accordingly, the hyaluronic acid aqueous solution may be a concept that includes all of an aqueous solution of hyaluronic acid, an aqueous solution of a hyaluronic acid salt, and a mixed aqueous solution of hyaluronic acid and a hyaluronic acid salt. The hyaluronic acid salt may be inorganic salts such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, organic salts such as tetrabutylammonium hyaluronic acid, and mixtures thereof.

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 realize various physical properties and biocompatibility.

As used herein, gelatin may refer to a protein obtained by treating animal-derived collagen with acid or alkali and subsequent extraction.

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 realize various physical properties and biocompatibility.

In one embodiment of the invention, the mixture of hyaluronic acid (HA) and gelatin contains 50 parts by weight or more and 500 parts by weight or less, and 100 parts by weight, based on 100 parts by weight of hyaluronic acid (HA). It may contain more than 500 parts by weight or less, or more than 100 parts by weight and less than 300 parts by weight.

If less than 50 parts by weight of gelatin is included per 100 parts by weight of hyaluronic acid (HA), the cell adhesion of the produced polymer microparticles may be poor, and 100 parts by weight of hyaluronic acid (HA) may have poor cell adhesion. If gelatin is included in an amount exceeding 500 parts by weight, the mechanical properties of the produced polymer microparticles may deteriorate. In other words, the mixture of hyaluronic acid (HA) and gelatin contains 50 to 500 parts by weight of gelatin based on 100 parts by weight of hyaluronic acid (HA), thereby providing excellent cell adhesion and mechanical properties. Polymer microparticles capable of realizing physical properties can be manufactured.

In one embodiment of the invention, the metal ion is iron ion (Fe ³⁺ ), aluminum ion (Al ³⁺ ), copper ion (Cu ²⁺ ), iron ion (Fe ²⁺ ), magnesium ion (Mg ²⁺ ), It may include one selected from the group consisting of barium ions (Ba ²⁺ ), calcium ions (Ca ²⁺ ), etc. More specifically, the metal ion may be iron ion, aluminum ion, or a mixture thereof.

In one embodiment of the invention, forming polymer cross-linked particles by reacting a mixture containing the biocompatible polymer and metal ions includes forming an aqueous solution in which the biocompatible polymer is dissolved; Adding the compound containing the metal ion to a polar solvent to form a solution containing the metal ion; and forming a mixed solution by mixing an aqueous solution droplet in which the biocompatible polymer is dissolved and a solution containing the metal ion.

Specifically, in the step of forming an aqueous solution in which the biocompatible polymer is dissolved, the aqueous solution in which the biocompatible polymer is dissolved contains 0.01% by weight or more and 10% by weight or less, 0.01% by weight, based on the weight of the aqueous solution in which the entire biocompatible polymer is dissolved. It may include more than 5% by weight and less than 5% by weight, more than 1% by weight and less than 5% by weight, more than 1% by weight and less than 3% by weight, more than 2% by weight and less than 3% by weight, and more than 2% by weight and less than 2.5% by weight.

As in one embodiment of the invention, after the crosslinking reaction using metal ions, an additional crosslinking reaction is carried out using an organic crosslinking agent containing one or more reactive functional groups, forming a W/O emulsion as in the prior art, followed by a crosslinking reaction. Compared to the case where it is used, microparticles with excellent mechanical strength and stability can be manufactured with a small amount of biocompatible polymer, such as 0.01% by weight or more and 10% by weight or less, as described above.

The step of adding the compound containing the metal ion to a polar solvent to form a solution containing the metal ion is not greatly limited, but can be formed, for example, by dispersing the compound containing the metal ion in a polar solvent such as ethanol. there is.

In addition, in the step of forming a mixed solution by mixing the aqueous solution droplet in which the biocompatible polymer is dissolved and the solution containing the metal ion, the size of the particles can be appropriately controlled using an encapsulator (BUCHI, B-390) device. there is.

As the method includes forming a mixed solution by mixing an aqueous solution droplet in which the biocompatible polymer is dissolved and a solution containing the metal ion, the metal ion is chelated to the biocompatible polymer and the biocompatible polymer is cross-linked through the metal ion. A structure can be formed.

As in one embodiment of the above invention, the step of forming polymer cross-linked particles by reacting a mixture containing a biocompatible polymer and a metal ion is performed, and the cross-linking reaction is carried out using only an organic cross-linking agent containing one or more reactive functional groups. Compared to the case where micro particles can be manufactured in a polar solvent without using oil, the oil washing process can be omitted, resulting in improved process efficiency.

In other words, as the metal ion is chelated in the biocompatible polymer, the biocompatible polymer forms a crosslinked structure mediated by the metal ion. Compared to the case where the crosslinking reaction is carried out using only an organic crosslinker containing one or more reactive functional groups, oil is used. Since micro particles can be manufactured in a polar solvent without oil washing process, the effect of improving process efficiency can be realized by omitting the oil washing process.

In addition, the compound containing the metal ion may be included in an amount of 200 parts by weight or more and 1000 parts by weight, 300 parts by weight or more and 1000 parts by weight, or 500 parts by weight or more and 1000 parts by weight or less, based on 100 parts by weight of the biocompatible polymer. .

In the method for producing polymer microparticles according to an embodiment of the invention, as described above, an additional crosslinking reaction is performed after the crosslinking reaction by metal ions, and 200 parts by weight or more 1000 parts by weight based on 100 parts by weight of the biocompatible polymer Even by adding a small amount of a mixture containing metal ions as follows, polymer microparticles that achieve sufficient mechanical strength and sphericity can be manufactured.

If the content of the compound containing the metal ion is added in excess of 1000 parts by weight based on 100 parts by weight of the biocompatible polymer, a technical problem may occur in which the remaining amount of metal ion remains in the crosslinked particles.

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

As described above, after crosslinking by metal ions, an additional crosslinking reaction is performed using a crosslinking agent with 1 to 30 carbon atoms containing one or more reactive functional groups, thereby maximizing process efficiency and improving the mechanical strength and stability of the microparticles. This can be significantly improved.

The type of the reactive functional group is not greatly limited, but examples include a hydroxy group, an epoxy group, a carboxyl group, an amino group, a (meth)acrylate group, a nitrile group, a thiol group, an aldehyde group, or a vinyl group.

Specifically, the organic crosslinking agent containing one or more reactive functional groups may include one or more formyl groups or epoxy groups, or two or more formsyl groups. The formyl group or epoxy group may be a crosslinkable functional group that reacts with the biocompatible polymer described above to form crosslinked particles.

In one embodiment of the invention, the organic crosslinking agent containing one or more reactive functional groups is not greatly limited. Specifically, the cross-linking agent is glutaraldehyde, butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), and hexanediol diglycidyl ether (BDDE). 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl Ether (polytetramethylene glycol diglycidyl ether), neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol Polyglycidyl ether (glycerol polyglycidyl ether), tri-methylpropane polyglycidyl ether, bisepoxypropoxyethylene (1,2-(bis(2,3-epoxypropoxy)ethylene), pentae It may include one selected from the group consisting of pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, divinylsulfone, and epichlorohydrin. .

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

Meanwhile, in the step of additionally crosslinking the polymer crosslinking particles in a polar solvent containing an organic crosslinking agent containing one or more reactive functional groups, the organic crosslinking agent containing one or more reactive functional groups is added to 100 parts by weight of the biocompatible polymer. It may be included in an amount of 150 to 1000 parts by weight, 200 to 1000 parts by weight, 300 to 800 parts by weight, and 400 to 500 parts by weight.

In the method for producing polymer microparticles according to an embodiment of the invention, as an additional crosslinking reaction is performed after the crosslinking reaction as described above, the reactive functional group is added in an amount of 150 parts by weight to 1000 parts by weight based on 100 parts by weight of the biocompatible polymer. Even by adding a small amount of an organic crosslinking agent containing one or more, polymer microparticles that achieve sufficient mechanical strength and sphericity can be manufactured.

If the content of the organic cross-linking agent containing one or more reactive functional groups is added in excess of 1000 parts by weight based on 100 parts by weight of the biocompatible polymer, a technical problem may occur in which the remaining amount of unreacted cross-linking agent remains in the cross-linked particles.

Meanwhile, in the step of additionally crosslinking the polymer crosslinking particles on a polar solvent containing an organic crosslinking agent containing one or more reactive functional groups, the polar solvent is not greatly limited, but may include, for example, ethanol, N,N-dimethylformamide, , N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylcaprolactam, 2-pyrrolidone, N-ethylpyrrolidone, N-vinylpyrrolidone, dimethyl sulfoxide, tetra Methylurea, pyridine, dimethylsulfone, hexamethylsulfoxide, 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, 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.

In addition, in the step of further crosslinking the polymer crosslinking particles on a polar solvent containing an organic crosslinking agent containing one or more reactive functional groups, the polar solvent containing the organic crosslinking agent containing one or more reactive functional groups may be an alkaline mixed solvent. there is.

That is, the additional crosslinking reaction of the present invention can proceed on an alkaline mixed solvent in which a polar solvent is mixed with an aqueous alkaline solution. Examples of the alkaline aqueous solution are not greatly limited, but may be, for example, an aqueous sodium hydroxide solution.

Since the polar solvent containing the organic crosslinking agent containing one or more reactive functional groups is an alkaline mixed solvent, the efficiency of the crosslinking reaction can be increased by creating an environment favorable to the nucleophilic substitution reaction (S _N reaction).

2. Polymer microparticles

According to one embodiment of the invention, a core including a first biocompatible polymer, a metal ion, and an organic crosslinking agent including one or more reactive functional groups; and a shell surrounding all or part of the core and containing a second biocompatible polymer, a metal ion, and an organic cross-linking agent containing one or more reactive functional groups. Polymer microparticles having a core-shell structure comprising a can be provided.

The present inventors conducted research on polymer microparticles, crosslinked them with metal ions as described above, and then proceeded with additional crosslinking reactions to maximize process efficiency and improve the mechanical strength and cell adhesion of the microparticles. This significant improvement was confirmed through experiments and the invention was completed.

In one embodiment of the invention, the core includes a polymer matrix in which the first biocompatible polymer is cross-linked via an organic cross-linker containing a metal ion and one or more reactive functional groups, and the shell includes a polymer matrix in which the second biocompatible polymer is a metal. It may include a polymer matrix crosslinked through an organic crosslinker containing one or more ions and reactive functional groups.

Specifically, the polymer matrix includes a first cross-linked region where biocompatible polymers are cross-linked via metal ions; and a second cross-linking region in which the biocompatible polymer is cross-linked via an organic cross-linking agent containing one or more reactive functional groups.

The first crosslinking region refers to a crosslinking region formed by a crosslinking reaction between a biocompatible polymer and a metal ion, and the second crosslinking region includes one or more reactive functional groups instead of a crosslinking reaction between the biocompatible polymer and the metal ion. It may refer to a crosslinked region formed through a crosslinking reaction with an organic crosslinking agent and a crosslinked region formed through an additional crosslinking reaction of an organic crosslinking agent containing one or more functional groups reactive with the first crosslinking region. That is, the polymer microparticles of the above embodiment can be manufactured through a crosslinking reaction using an organic crosslinker containing a metal ion and one or more reactive functional groups.

Specifically, the biocompatible polymer refers to a polymer that can be directly injected into the human body in order to deliver effective substances applied to the human body. Specifically, the biocompatible polymers include Hyaluronic acid (HA), Carboxymethyl cellulose (CMC), alginic acid, pectin, carrageenan, chondroitin (sulfate), dextran (sulfate), chitosan, Polylysine, collagen, gelatin, carboxymethyl chitin, fibrin, agarose, pullulan, polylactide, polyglycolide (PGA), polylactide-glycolide copolymer (PLGA), poly polyanhydride, polyorthoester, polyetherester, polycaprolactone, polyesteramide, poly(butyric acid), poly(valeric acid), Polyurethanes, polyacrylates, ethylene-vinylacetate polymers, acrylic substituted cellulose acetates, non-degradable polyurethanes, polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonate polyolefins ), polyethylene oxide, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polymethacrylate, hydroxypropylmethylcellulose (HPMC), ethylcellulose (EC), hydroxypropylcellulose (HPC), cyclodextrin and one or more polymers selected from the group consisting of cellulose and copolymers of monomers forming such polymers.

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

Polymer microparticles manufactured using only hyaluronic acid are easily decomposed in vivo or under acid, alkali, etc. conditions, which limits their use, and their cell adhesion is significantly reduced. The mechanical properties of polymer microparticles are significantly reduced.

Accordingly, by using a mixture of hyaluronic acid (HA) and gelatin as a biocompatible polymer, excellent cell adhesion and mechanical properties can be achieved at the same time.

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

In this specification, hyaluronic acid may mean including both hyaluronic acid itself and hyaluronic acid salts. Accordingly, the hyaluronic acid aqueous solution may be a concept that includes all of an aqueous solution of hyaluronic acid, an aqueous solution of a hyaluronic acid salt, and a mixed aqueous solution of hyaluronic acid and a hyaluronic acid salt. The hyaluronic acid salt may be inorganic salts such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, organic salts such as tetrabutylammonium hyaluronic acid, and mixtures thereof.

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 realize various physical properties and biocompatibility.

As used herein, gelatin may refer to a protein obtained by treating animal-derived collagen with acid or alkali and subsequent extraction.

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 realize various physical properties and biocompatibility.

Meanwhile, the polymer microparticles may have a core-shell structure. The core-shell structure is realized by the difference in reactivity between the biocompatible polymer and the organic crosslinker containing one or more metal ions and reactive functional groups, as the polymer matrix contained in the polymer microparticles contains two or more types of biocompatible polymers. It can be.

In the core-shell structure, the core contains a polymer matrix in which hyaluronic acid is cross-linked through an organic cross-linking agent containing at least one metal ion and a reactive functional group, relative to the total volume of the polymer matrix included in the core, exceeding 50% by volume, It may contain more than 60% by volume, more than 70% by volume, or more than 75% by volume. Additionally, it may be included in 100 vol% or less, less than 100 vol%, 95 vol% or less, or 90 vol% or less. In addition, more than 50 volume% but less than 100 volume%, more than 50 volume% but less than 100 volume%, more than 60 volume% but less than 100 volume%, more than 60 volume% but less than 95 volume%, more than 70 volume% but less than 95 volume%, 70 volume% It may contain more than 90% by volume or less, or more than 75% by volume and less than 90% by volume. That is, the core may contain an excess amount of hyaluronic acid relative to gelatin.

With respect to the total volume of the polymer matrix included in the core, hyaluronic acid contains more than 50% by volume of a polymer matrix cross-linked through an organic cross-linker containing a metal ion and one or more reactive functional groups, meaning that the corresponding region is of the total area. Distribution exceeding 50% can be confirmed visually or through measuring equipment.

Specifically, the calculation of the volume ratio of the polymer matrix in which hyaluronic acid is cross-linked through an organic cross-linker containing one or more metal ions and one or more reactive functional groups relative to the total volume of the polymer matrix included in the core is not greatly limited and can be used as a general measurement. It can be calculated using this method. For example, the characteristic peak (1650 cm ^-1 ) of gelatin in the manufactured polymer microparticles It can be confirmed by taking an IR photo of the hyaluronic acid characteristic peak (1080 cm ^-1 ) relativized for the hyaluronic acid.

In the core-shell structure, the shell contains more than 50% by volume of a polymer matrix in which gelatin is crosslinked through an organic crosslinking agent containing at least one metal ion and a reactive functional group, relative to the total volume of the polymer matrix included in the shell. It may contain more than 70% by volume, or more than 75% by volume. Additionally, it may be included in 100 vol% or less, less than 100 vol%, 95 vol% or less, or 90 vol% or less. In addition, more than 50 volume% but less than 100 volume%, more than 50 volume% but less than 100 volume%, more than 60 volume% but less than 100 volume%, more than 60 volume% but less than 95 volume%, more than 70 volume% but less than 95 volume%, 70 volume% It may contain more than 90% by volume or less, or more than 75% by volume and less than 90% by volume. That is, the shell may contain an excess amount of gelatin relative to hyaluronic acid.

With respect to the total volume of the polymer matrix contained in the shell, the gelatin contains more than 50% by volume of a polymer matrix crosslinked through an organic crosslinker containing a metal ion and one or more reactive functional groups, meaning that the corresponding area is 50% by volume of the total area. Distribution exceeding % can be confirmed visually or through measuring equipment.

The calculation of the volume ratio of the polymer matrix in which gelatin is cross-linked through an organic cross-linker containing one or more metal ions and one or more reactive functional groups relative to the total volume of the polymer matrix contained in the shell is not greatly limited and can be calculated by a conventional measurement method. You can. For example, the characteristic peak (1650 cm ^-1 ) of gelatin in the manufactured polymer microparticles You can check this by taking an IR photo.

A core containing more than 50% by volume of a polymer matrix in which hyaluronic acid is cross-linked through an organic cross-linking agent containing at least one metal ion and a reactive functional group, and gelatin is mediated through an organic cross-linking agent containing at least one metal ion and a reactive functional group. A core-shell structure including a shell containing more than 50% by volume of a cross-linked polymer matrix can be implemented depending on physical and chemical factors such as solubility, temperature reactivity, and ionic bonding. More specifically, in the polymer microparticle manufacturing process, gelatin, which has low solubility in polar solvents such as ethanol and high temperature reactivity, has reduced fluidity and is fixed to the particle surface to form a shell, thereby forming a relative As a result, more hyaluronic acid can be distributed inside the particles to form a core.

In particular, in the method for producing polymer microparticles of one embodiment, before the step of additionally crosslinking the polymer crosslinked particles in a polar solvent containing an organic crosslinker containing one or more reactive functional groups, a mixture containing a biocompatible polymer and a metal ion is reacted. By including the step of forming polymer crosslinked particles, the core-shell structure can become clearer due to the ionic bond between the metal ion and the carboxyl group contained in hyaluronic acid.

Meanwhile, the polymer microparticles may have an average diameter in distilled water of 1 ㎛ or more, 1 ㎛ or more and 450 ㎛ or less, 100 ㎛ or more and 450 ㎛ or less, or 200 ㎛ or more and 400 ㎛ or less, or 300 ㎛ or more and 400 ㎛ or less. When the average diameter of the microparticles satisfies the above-mentioned range, cell attachment and culture performance are excellent.

The average diameter may mean the diameter at the 50 volume% point of the cumulative distribution of the number of particles according to diameter.

In the polymer microparticles of the above embodiment, based on the cross section having the longest diameter of the polymer microparticles, the thickness of the shell is 95% or less, 90% or less, 80% or less, and 75% or less of the longest diameter of the polymer microparticles. , may be 50% or less, 30% or less, 25% or less, or 20% or less. Additionally, based on the cross section having the longest diameter of the polymer microparticles, the thickness of the shell may be 0.01% or more, 1% or more, or 5% or more of the longest diameter of the polymer microparticles.

In addition, in the polymer microparticles of the above embodiment, based on the cross section having the longest diameter of the polymer microparticles, the thickness of the core is 5% or more, 10% or more, 20% or more, 25% or more of the longest diameter of the polymer microparticles. % or more, 50% or more, 70% or more, 75% or more, or 80% or more. Additionally, based on the cross section having the longest diameter of the polymer microparticles, the thickness of the core may be 99.99% or less, 99% or less, or 95% or less of the longest diameter of the polymer microparticles.

In addition, the polymer microparticles may have a sphericity degree of 0.9 or more and 1.0 or less, 0.93 or more and 1.0 or less, 0.94 or more and 0.99 or less, or 0.94 or more and 0.98 or less.

The degree of sphericity can be obtained by taking an optical photograph of polymer microparticles and calculating the average value of 30 to 100 random particles in the optical photograph.

In addition, the polymer microparticles have an average compressive strength of 0.1 mN or more, 0.1 mN or more and 100 mN or less, 0.3 mN or more and 100 mN or less when deformed to 25% of the average diameter for particles swollen with distilled water for more than 24 hours, It may be 0.35 mN or more and 100 mN or less, 0.35 mN or more and 30 mN or less, 0.35 mN or more and 10 mN or less, or 0.35 mN or more and 3 mN or less.

The average compressive strength may be the compressive strength of the n polymer microparticles when deformed to 25% of the average diameter divided by n.

For example, in this specification, the average compressive strength may be the compressive strength of 30 polymer microparticles when deformed to 25% of the average diameter divided by 30.

If the average compressive strength of the polymer microparticles is less than 0.1 mN, technical problems such as reduced stability may occur due to inferior mechanical strength of the polymer microparticles.

3. Medical composition

According to another embodiment of the present invention, a medical composition containing the polymer microparticles of the other embodiments and a pharmaceutically effective substance contained in the polymer microparticles may be provided. The content regarding the polymer microparticles may include all of the content described above in the other embodiments.

The pharmaceutically active substance may exist contained within the polymer microparticles.

Examples of the pharmaceutically effective substances are not greatly limited, and depending on the intended use of the polymer microparticles of one embodiment, any effective material suitable for the intended use may be applied without limitation. That is, specific examples of the pharmaceutically effective substances are not limited and include amphetaminil, arecolin, atrophine, bupranolol, buprenorphine, and capsaicin. ), carisoprodol, chlorpromazine, ciclopirox olamine, cocaine, desipramine, dyclonine, epinephrine , ethosuximide, floxetine, hydromorphine, imipramine, lidocaine, methamphetamine, melproic acid, methylphenidate ( Methylpenidate, morphine, oxibutynin, nadolol, nicotine, nitroglycerin, pindolol, prilocaine, procaine ), propanolol, rivastigmine, scopolamine, selegiline, tulobuterol, valproic acid, donepezil, etc. Drugs selected from the group consisting of EPO (Erythropoietin), Human Growth Hormone (hGH), Exenatide, GLP-1 (Glucagon-like peptide-1), insulin, CSF (Granulocyte colony-stimulating factor), There are peptide or protein-based drugs selected from the group consisting of estrogen, progesterone, parathyroid hormone (PTH), etc., and all pharmacological substances with proven pharmacological effects can be applied without limitation.

The amount of the pharmaceutically active substance added is also not greatly limited, and the content can be used without limitation depending on the application and target. For example, the active substance may be included in an amount of 0.0001 parts by weight or more and 1,000,000 parts by weight or less compared to 100 parts by weight of the polymer microparticles, and may be included in a small amount or excessive amount compared to the polymer microparticles.

4. Cosmetic composition

According to another embodiment of the present invention, a cosmetic composition containing the polymer microparticles of the other embodiments and a cosmetic active substance contained in the polymer microparticles may be provided. The content regarding the polymer microparticles may include all of the content described above in the other embodiments.

The cosmetically active substance may exist contained within the polymer microparticles.

Examples of the cosmetic active substances are not greatly limited, and depending on the application purpose of the polymer microparticles of the above embodiment, any effective substance suitable for the application may be applied without limitation. That is, specific examples of the cosmetically effective substances are not limited and include natural extracts, proteins, vitamins, enzymes, antioxidants, etc., and all substances with proven cosmetic effects are applicable without limitation.

The amount of the cosmetic active substance added is also not greatly limited, and the content can be used without limitation depending on the intended use and target. For example, the cosmetically active substance may be included in an amount of 0.0001 parts by weight or more and 1,000,000 parts by weight or less compared to 100 parts by weight of the polymer microparticles, and may be included in a small amount or excessive amount compared to the polymer microparticles.

5. Medical supplies

According to another embodiment of the present invention, a medical article containing the medical composition of the other embodiment may be provided. The content regarding the medical composition may include all of the content described above in the other embodiments.

Examples of the above medical products are not greatly limited, but in order to implement the characteristics of the present invention, they are suitable for cases where they are inserted into the body and used or where their strength must be maintained for a long period of time, for example, body implants, body insertable drug carriers, and transdermal patches. , wound treatments, etc.

6. Beauty supplies

According to another embodiment of the present invention, the present invention can provide a beauty product containing the cosmetic composition of the other embodiment. The content regarding the cosmetic composition may include all of the content described above in the other embodiments.

Examples of the above beauty products are not greatly limited, but in order to implement the characteristics of the present invention, examples include beauty creams, lotions, hair gels, packs, etc.

The structure of the beauty pack is not greatly limited, but may include, for example, a support and a cosmetically effective substance transfer layer formed on the support and including the polymer microparticles of the other embodiments. Examples of the support include woven fabric, non-woven fabric, silicone, polyethylene terephthalate, polyethylene, polypropylene, polyurethane, metal mesh, polyester, etc.

According to the present invention, a method for producing polymer microparticles capable of achieving excellent mechanical strength and cell adhesion, polymer microparticles, medical compositions containing the same, cosmetic compositions, medical supplies, and cosmetic products can be provided.

Figure 1 is an optical microscope (OM) photograph of the polymer microparticles of Example 1.
Figure 2 is an IR photograph of the gelatin characteristic peak (1650 cm ^-1 ) of the polymer microparticles of Example 1.
Figure 3 shows the characteristic peak (1650 cm ^-1 ) of gelatin of the polymer microparticles of Example 1. This is an IR photo of the hyaluronic acid characteristic peak (1080 cm ^-1 ) relativized to

The invention is explained in more detail in the following examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited by the following examples.

Example 1: Preparation of polymer microparticles

Dissolve 200 mg of hyaluronic acid (weight average molecular weight: 500 kDa, manufacturer: SK Bioland) at 2 wt.% in 0.1N NaOH aqueous solution, and gelatin (gel strength: 300 g Bloom, manufacturer: Sigma, product name: G2500) in distilled water. ) Prepare 20 mL by mixing 10 mL of each solution of 250 mg dissolved at 2.5 wt.%, and then add this to 80 mL of ethanol solution to which 4 g of FeCl ₃ , a compound containing iron ions (Fe ³⁺ ), was added. The droplets formed using an encapsulator (BUCHI, B-390) were added, crosslinked at 4°C for 2 hours, and then washed with ethanol to prepare crosslinked particles.

After mixing 2.2 g of 1,4-Butandiol diglycidyl ether (BDDE) with an 80% ethanol solution containing 20% of 0.1N NaOH aqueous solution, the cross-linked particles were added, Polymer microparticles were prepared by crosslinking at room temperature for 3 days. The prepared particles were washed with ethanol and distilled water in that order, and then the prepared cross-linked particles were recovered using a mesh sieve with an opening size of 45 ㎛. The recovered cross-linked particles were filtered through a mesh sieve with an opening size of 500 ㎛, and the remaining cross-linked particles were analyzed.

An optical microscope (OM) photograph of the manufactured polymer microparticles is shown in Figure 1.

An IR photograph of the gelatin characteristic peak (1650 cm ^-1 ) of the prepared polymer microparticles is shown in Figure 2. Through the difference in intensity of the characteristic peak of gelatin (1650 cm ^-1 ), it was confirmed that gelatin was distributed in the shell of the manufactured polymer microparticles.

The characteristic peak of gelatin (1650 cm ^-1 ) of the manufactured polymer microparticles An IR photograph of the characteristic peak (1080 cm ^-1 ) of hyaluronic acid relativized to hyaluronic acid is shown in FIG. 3 . It was confirmed that hyaluronic acid was distributed in a high relative amount in the core of the manufactured polymer microparticles compared to gelatin.

Example 2: Preparation of polymer microparticles

Except that instead of the ethanol solution to which 4 g of FeCl ₃ , a compound containing iron ions (Fe ³⁺ ) was added, the ethanol solution to which 4 g of AlCl ₃ , a compound containing aluminum ions (Al ³⁺ ), was added was used. Polymer microparticles were prepared in the same manner as in Example 1.

Example 3: Preparation of polymer microparticles

The polymer was 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). Micro particles were prepared.

Comparative Example 1: Preparation of polymer microparticles

Dissolve hyaluronic acid (weight average molecular weight: 500 kDa, manufacturer: SK Bioland) and gelatin (gel strength: 300 g Bloom, manufacturer: Sigma, product name: G2500) in distilled water at 2 wt.% and 20 wt.%, respectively, and make 5 ml. After preparing each solution, the mixture of these two solutions was mixed with the liquid paraffin solution to prepare a mixed solution containing a microemulsion. Afterwards, 2.2 g of 1,4-Butandiol diglycidyl ether (BDDE) was added as a cross-linking agent to the mixed solution, and a cross-linking reaction was performed at room temperature for 5 days to prepare polymer microparticles. The prepared particles were washed with acetone, dichloromethane, and distilled water in that order, and then the prepared cross-linked particles were recovered using a mesh sieve with an opening size of 45 ㎛. The recovered cross-linked particles were filtered through a mesh sieve with an opening size of 500 ㎛, and the remaining cross-linked particles were analyzed.

Comparative Example 2: Preparation of polymer microparticles

Dissolve 2 wt.% hyaluronic acid (weight average molecular weight: 500 kDa, manufacturer: SK Bioland) in 0.1N NaOH aqueous solution, and gelatin (gel strength: 300 g Bloom, manufacturer: Sigma, product name: G2500) in distilled water. 20 mL was prepared by mixing 10 mL of each solution dissolved at 2.5 wt.%, and then added to 80 mL of an ethanol solution containing 4 g of FeCl ₃ , a compound containing iron ions (Fe ³⁺ ), with an encapsulator (BUCHI, B-390) The droplets formed using the device were added, crosslinked at 4°C for 2 hours, and then washed with ethanol and distilled water to prepare crosslinked particles. The prepared cross-linked particles were recovered using a mesh sieve with an opening size of 45 ㎛. The recovered cross-linked particles were filtered through a mesh sieve with an opening size of 500 ㎛, and the remaining cross-linked particles were analyzed.

Comparative Example 3: Preparation of polymer microparticles

200 mg of hyaluronic acid (weight average molecular weight: 500 kDa, manufacturer: SK Bioland) at a concentration of 2 wt.% in 0.1 N NaOH aqueous solution, and 250 mg of gelatin (gel strength: 300 g Bloom, manufacturer: Sigma, product name: G2500) Each was dissolved in distilled water at a concentration of 2.5 wt.% to prepare 10 ml each, and then the mixed solution of the two solutions was mixed with the liquid paraffin solution to prepare a mixed solution containing a microemulsion. Afterwards, 2.2 g of 1,4-Butandiol diglycidyl ether (BDDE) was added as a cross-linking agent to the mixed solution, and cross-linking was performed at room temperature for 5 days. After washing this in the order of acetone, dichloromethane, and distilled water, the prepared cross-linked particles were recovered using a mesh sieve with an opening size of 45 ㎛. The recovered cross-linked particles were filtered through a mesh sieve with an opening size of 500 ㎛, and the remaining cross-linked particles were analyzed.

Comparative Example 4: Preparation of polymer microparticles

Hyaluronate (weight average molecular weight: 500 kDa, manufacturer: SK Bioland) was dissolved in 0.1 N NaOH aqueous solution at a concentration of 2 wt.%, and gelatin (gel strength: 300 g Bloom, manufacturer: Sigma, product name: G2500) was dissolved in distilled water at a concentration of 2.5 wt.%. Each solution was dissolved in wt.% concentration to prepare 5 ml each, and then the mixture of the two solutions was mixed with the liquid paraffin solution to prepare a mixed solution containing a microemulsion. Afterwards, 2.2 g of 1,4-Butandiol diglycidyl ether (BDDE) was added as a cross-linking agent to the mixed solution, and cross-linking was performed at room temperature for 5 days. After washing this in the order of acetone, dichloromethane, and distilled water, the prepared cross-linked particles were recovered using a mesh sieve with an opening size of 45 ㎛. The recovered cross-linked particles were filtered through a mesh sieve with an opening size of 500 ㎛, and the remaining cross-linked particles were analyzed.

Comparative Example 5: Preparation of polymer microparticles

Dissolve hyaluronic acid (weight average molecular weight: 500 kDa, manufacturer: SK Bioland) and gelatin (gel strength: 300 g Bloom, manufacturer: Sigma, product name: G2500) in distilled water at 2 wt.% and 2.5 wt.%, respectively, and make 5 ml. After preparing each solution, the mixture of these two solutions was mixed with the liquid paraffin solution to prepare a mixed solution containing a microemulsion. Afterwards, 2.2 g of 1,4-Butandiol diglycidyl ether (BDDE) was added as a cross-linking agent to the mixed solution, and cross-linking was performed at room temperature for 5 days. After washing this in the order of acetone, dichloromethane, and distilled water, the prepared cross-linked particles were recovered using a mesh sieve with an opening size of 45 ㎛. The recovered cross-linked particles were filtered through a mesh sieve with an opening size of 500 ㎛, and the remaining cross-linked particles were analyzed.

Comparative Example 6: Preparation of polymer microparticles

Alginate (manufacturer: Sigma, product name: 180947) and cellulose (manufacturer: Sigma, product name: C5678) were each dissolved at 2.5 wt.% in distilled water to prepare 20 mL by mixing 10 mL of each solution, which was then mixed with calcium ions ( Add the droplets formed using an encapsulator (BUCHI, B-390) device to 80 mL of ethanol solution containing 4 g of CaCl ₂ , a compound containing Ca ²⁺ ), and cross-link at room temperature for 2 hours, followed by ethanol. Cross-linked particles were prepared by washing with .

After mixing 2.2 g of 1,4-Butandiol diglycidyl ether (BDDE) with an 80% ethanol solution containing 20% of 0.1N NaOH aqueous solution, the cross-linked particles were added, Polymer microparticles were prepared by crosslinking at room temperature for 3 days. The prepared particles were washed with ethanol and distilled water in that order, and then the prepared cross-linked particles were recovered using a mesh sieve with an opening size of 45 ㎛. The recovered cross-linked particles were filtered through a mesh sieve with an opening size of 500 ㎛, and the remaining cross-linked particles were analyzed.

Comparative Example 7: Preparation of polymer microparticles

200 mg of hyaluronic acid (weight average molecular weight: 500 kDa, manufacturer: SK Bioland) at a concentration of 2 wt.% in 0.1 N NaOH aqueous solution, and 250 mg of gelatin (gel strength: 300 g Bloom, manufacturer: Sigma, product name: G2500) 20 mL was prepared by mixing 10 mL of each solution dissolved in distilled water at a concentration of 2.5 wt.%, and then encapsulated in 80 mL of ethanol solution to which 4 g of FeCl ₃ , a compound containing iron ions (Fe ³⁺ ), was added. (BUCHI, B-390) The droplets formed using a device were added, cross-linked at 4° C. for 2 hours, and then washed with ethanol to prepare cross-linked particles.

The cross-linked particles were added to 80 mL of an ethanol solution containing 4 g of CaCl ₂ , a compound containing calcium ions (Ca ²⁺ ), and subjected to a cross-linking reaction at 4°C for 2 hours to prepare polymer microparticles. The prepared particles were washed with ethanol and distilled water in that order, and then the prepared cross-linked particles were recovered using a mesh sieve with an opening size of 45 ㎛. The recovered cross-linked particles were filtered through a mesh sieve with an opening size of 500 ㎛, and the remaining cross-linked particles were analyzed.

Experimental example: Measurement of physical properties of polymer microparticles

The average diameter, degree of sphericity, strength, cell culture suitability, and stability of the polymer microparticles prepared in the above Examples and Comparative Examples were evaluated in the following manner.

1. Average diameter

The average diameter of the polymer microparticles of the above examples and comparative examples in distilled water was measured using a laser particle size analyzer (Horiba, Partica LA-960).

2. Sphericity

Optical photos (Olympus, BX53) of the polymer microparticles of the above examples and comparative examples were taken, and the degree of sphericity was calculated from these.

The degree of sphericity according to the present invention was calculated as the average value of the ratio (long-aspect ratio) of the longest diameter to the shortest diameter of any 30 particles in the optical photograph.

At this time, the closer the sphericity value is to 1, the closer it is to a sphere.

3. Robbery

For the polymer microparticles of the above examples and comparative examples, the strength of the microparticles was measured using texture analyzer equipment. 30 micro particles swollen with distilled water for 24 hours were placed in a single layer in the area below the flat cylindrical probe of the equipment equipped with a 5 N load cell. The initial trigger force was set to 1 mN, and the particles were compressed at a speed of 1 mm/s. The force when deformed to 25% of the average particle diameter was defined as compressive force.

The average compressive strength was calculated by dividing the compressive force by 30, which is the number of micro particles to be measured.

4. Cell culture suitability

A 6-well plate was filled with cell culture medium, polymer microparticles and cells were added, and the cells were cultured using a plate-rocking method. At this time, the temperature of the culture medium was maintained at 37°C, and the culture was performed for 3 days to confirm the number of cells cultured in the polymer microparticles.

At this time, cell culture suitability was evaluated based on the following criteria.

Suitable: When the number of cultured cells is 100% or more compared to the number of cells input.

Inadequacy: When the number of cultured cells is less than 100% compared to the number of cells input, or when microparticles are decomposed during culture.

5. Stability

The stability of polymer microparticles in a phosphate-buffered saline solution during sterilization using a high-temperature and high-pressure autoclave and the stability of the particles during long-term culture were evaluated based on the following criteria.

Suitable: When the weight reduction rate of dried polymer micro particles before and after using an autoclave is 20% or less.

Inadequacy: When the weight loss rate of dried polymer micro particles before and after using an autoclave exceeds 20%.

division Average diameter (㎛) Sphericity Average compressive strength (mN) Cell culture compatibility stability Example 1 317±21 0.95±0.08 2.10 fitness fitness Example 2 346±26 0.96±0.13 1.37 fitness fitness Example 3 319±32 0.95±0.05 0.37 fitness fitness Comparative Example 1 385±13 0.98±0.09 0.23 fitness incongruity Comparative Example 2 193±21 0.96±0.03 0.10 incongruity incongruity Comparative Example 3 Particle manufacturing is not possible Comparative Example 4 Particle manufacturing is not possible Comparative Example 5 Particle manufacturing is not possible Comparative Example 6 301±48 0.94±0.11 0.29 incongruity fitness Comparative Example 7 194±17 0.95±0.04 0.12 incongruity incongruity

As shown in Table 1, the polymer microparticles of the examples are not only suitable for cell culture as the number of cultured cells is more than 100% compared to the number of cells introduced, but also the weight reduction rate of the dried polymer microparticles before and after autoclave treatment is 20%. As shown below, it was confirmed that it was suitable for sterilization and long-term culture. In addition, the polymer microparticles of the examples showed an average compressive strength of 0.37 mN or more, demonstrating excellent mechanical properties, and at the same time, it was confirmed that the proportion of particles showing a high crosslink density was high.

In other words, it was confirmed that the polymer microparticles of the examples were suitable for cell culture, sterilization, and long-term culture while demonstrating excellent crosslinking density and mechanical properties.

On the other hand, the polymer microparticles of Comparative Example 1 showed a weight reduction rate of more than 20% of the dried polymer microparticles before and after autoclave treatment, making them unsuitable for sterilization and long-term culture, as well as an average compressive strength of 0.23 mN, showing inferior mechanical properties. It was confirmed that the proportion of particles showing low crosslinking density was high.

In addition, the polymer microparticles of Comparative Example 2 were observed to shrink due to the dissolution of polymers that were not crosslinked during the distilled water washing step. In addition, the microparticles are decomposed during cell culture, making them unsuitable for cell culture as the number of cultured cells is less than 100% compared to the number of cells input, and the weight loss rate of the dried polymer microparticles before and after autoclave treatment is more than 20%, indicating that they must be sterilized. And it was confirmed that it was unsuitable for long-term culture. In addition, it was confirmed that the average compressive strength was 0.1 mN, indicating inferior mechanical properties.

In addition, unlike Comparative Example 1, the polymer microparticles of Comparative Examples 3 to 5 did not form polymer microparticles as the concentration of the aqueous solution in which the biocompatible polymer was dissolved was adjusted to the same level as Example 1, resulting in a low It was confirmed that the formation of polymer microparticles was possible even at biocompatible polymer concentrations.

The polymer microparticles of Comparative Example 6 showed an average compressive strength of 0.29 mN in distilled water due to chemical crosslinking by a crosslinking agent, but were unsuitable for cell culture due to the use of alginate and cellulose, which are biocompatible polymers and do not have cell adhesion. I was able to confirm. In addition, as the metal ion is cross-linked using calcium ions (Ca ²⁺ ), it may react reversibly with the calcium ions present in the cell culture medium, which may lower the particle strength during cell culture.

As the polymer microparticles of Comparative Example 7 were manufactured only through ionic crosslinking, it was confirmed that some of the microparticles were decomposed during sterilization and cell culture, making them unsuitable for cell culture.

Claims (25)

reacting a mixture containing a biocompatible polymer and a metal ion to form polymer cross-linked particles; and
A step of further crosslinking the polymer crosslinked particles in a polar solvent containing an organic crosslinker containing one or more reactive functional groups,
A method for producing polymer microparticles, wherein the biocompatible polymer includes a mixture of hyaluronic acid and gelatin.
According to paragraph 1,
The step of forming polymer crosslinked particles by reacting the mixture containing the biocompatible polymer and metal ions is performed in a polar solvent.
According to paragraph 1,
The step of forming polymer crosslinked particles by reacting a mixture containing the biocompatible polymer and metal ions,
Forming an aqueous solution in which the biocompatible polymer is dissolved;
Adding the compound containing the metal ion to a polar solvent to form a solution containing the metal ion; and
A method for producing polymer microparticles comprising: mixing an aqueous solution droplet in which the biocompatible polymer is dissolved and a solution containing the metal ion to form a mixed solution.
According to paragraph 3,
The aqueous solution in which the biocompatible polymer is dissolved is
A method for producing polymer microparticles, comprising the biocompatible polymer in an amount of 0.01% by weight or more and 10% by weight or less, based on the weight of the aqueous solution in which all biocompatible polymers are dissolved.
According to paragraph 3,
A method for producing polymer microparticles, wherein the compound containing the metal ion is contained in an amount of 200 parts by weight or more and 1000 parts by weight or less based on 100 parts by weight of the biocompatible polymer.
According to paragraph 1,
In the step of further crosslinking the polymer crosslinked particles in a polar solvent containing an organic crosslinker containing one or more reactive functional groups,
A method for producing polymer microparticles, wherein the organic crosslinking agent containing one or more reactive functional groups is contained in an amount of 150 parts by weight or more and 1000 parts by weight or less based on 100 parts by weight of the biocompatible polymer.
According to paragraph 1,
The polar solvent is ethanol, N,N-dimethylformamide, N,N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylcaprolactam, 2-pyrrolidone, N-ethylpyrrolidone , N-vinylpyrrolidone, dimethylsulfoxide, tetramethylurea, pyridine, dimethylsulfone, hexamethylsulfoxide, 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, 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, ethylene glycol monobutyl ether acetate. A method for producing polymer microparticles, which is one selected from the group.
According to paragraph 1,
The organic crosslinking agent containing one or more reactive functional groups is,
A method for producing polymer microparticles containing one or more formyl groups or epoxy groups.
According to paragraph 1,
The metal ion includes one selected from the group consisting of iron ions, aluminum ions, copper ions, iron ions, magnesium ions, barium ions, calcium ions, etc.
delete According to clause 10,
The mixture of hyaluronic acid and gelatin,
A method for producing polymer microparticles, comprising 50 parts by weight or more and 500 parts by weight or less of gelatin based on 100 parts by weight of hyaluronic acid.
A core containing a first biocompatible polymer, a metal ion, and an organic crosslinking agent containing one or more reactive functional groups; and
A shell surrounding all or part of the core and containing a second biocompatible polymer, a metal ion, and an organic cross-linking agent containing one or more reactive functional groups;
The first biocompatible polymer includes hyaluronic acid,
The second biocompatible polymer includes gelatin,
Polymer microparticles with a core-shell structure.
According to clause 12,
The core includes a polymer matrix in which a first biocompatible polymer is cross-linked through an organic cross-linker containing a metal ion and one or more reactive functional groups,
The shell is a polymer microparticle comprising a polymer matrix in which a second biocompatible polymer is cross-linked via an organic cross-linker containing a metal ion and one or more reactive functional groups.
delete According to clause 13,
The core, with respect to the entire volume of the polymer matrix contained in the core,
Polymer microparticles comprising more than 50% by volume of a polymer matrix in which hyaluronic acid is crosslinked via an organic crosslinker containing a metal ion and one or more reactive functional groups.
According to clause 13,
The shell, with respect to the entire volume of the polymer matrix contained in the shell,
Polymer microparticles comprising more than 50% by volume of a polymer matrix in which gelatin is crosslinked via an organic crosslinker containing a metal ion and one or more reactive functional groups.
According to clause 12,
The polymer microparticles have an average diameter of 1 ㎛ or more in distilled water.
According to clause 12,
Based on the cross section having the longest diameter of the polymer microparticles,
Polymer microparticles wherein the thickness of the shell is 95% or less of the longest diameter of the polymer microparticles.
According to clause 12,
The organic crosslinking agent containing one or more reactive functional groups is,
Polymer microparticles containing a crosslinking agent having 1 to 30 carbon atoms and containing one or more reactive functional groups.
According to clause 12,
The polymer micro particles are
Polymer microparticles with an average compressive strength of 0.1 mN or more when deformed to 25% of the average particle diameter.
According to clause 12,
Polymer microparticles having a sphericity degree of 0.9 or more and 1.0 or less, which is the ratio (major aspect ratio) of the longest diameter to the shortest diameter of any particle in an optical photograph.
A medical composition comprising the polymer microparticles of claim 12 and a pharmaceutically active substance contained in the polymer microparticles.
A cosmetic composition comprising the polymer microparticles of claim 12 and a cosmetically effective substance contained in the polymer microparticles.
A medical article comprising the medical composition of claim 22.
A cosmetic product comprising the cosmetic composition of claim 23.