KR20040018927A - Microbeads of natural polysaccharide and hyaluronic acid and processes for preparing the same - Google Patents

Abstract

PURPOSE: A microbead of natural polysaccharide and hyaluronic acid and its preparation method are provided, to improve biocompatibility, swelling in an aqueous solution and physical stability. CONSTITUTION: The microbead comprises a natural polysaccharide selected from alginic acid and chitosan; and hyaluronic acid or its salt. Preferably the microbead is an alginic acid-hyaluronic acid microbead, a chitosan-hyaluronic acid microbead, or a chitosan-coated alginic acid-hyaluronic acid microbead. Preferably the hyaluronic acid or its salt has a molecular weight of 100,000-3,000,000; the chitosan has a molecular weight of 300-10,000,000; and the bead has an average diameter of 30-1,000 micrometers. The method comprises the steps of mixing an alginic acid aqueous solution and a hyaluronic acid aqueous solution; and spraying the mixture solution to a salt blend aqueous solution of a divalent or trivalent cationic salt and sodium chloride. Preferably the concentration of the cationic salt is 0.01-15 M and that of sodium chloride is 0.01-45 M.

Description

Microbeads of natural polysaccharides and hyaluronic acid and preparation method thereof {MICROBEADS OF NATURAL POLYSACCHARIDE AND HYALURONIC ACID AND PROCESSES FOR PREPARING THE SAME}

The present invention relates to a microbead consisting of natural polysaccharides selected from alginic acid or chitosan and hyaluronic acid, and a method for preparing the same. More specifically, the present invention relates to a biobead having excellent biocompatibility and superior swelling and physical stability. , Alginic acid-hyaluronic acid microbeads, chitosan-hyaluronic acid microbeads, alginic acid-hyaluronic acid microbeads coated with chitosan, and the like, and a method of preparing the same. Microbeads according to the present invention having such characteristics can be used in various applications as biocompatible materials, such as wrinkle treatments, cosmetic aids, drug carriers.

Hyaluronic acid is a biopolymer material in which a repeating unit consisting of N-acetyl-D-glucosamine and D-glucuronic acid is linearly connected, and is present in eyeglasses, synovial fluids of joints, and chicken clams. Hyaluronic acid and its derivatives are described in the applicant's Korean Patent No. 354299 (name of the invention: a cross-linked amide derivative of hyaluronic acid and a method for preparing the same), the contents of which are incorporated herein by reference. .

Hyaluronic acid derivatives have been developed for various purposes such as anti-adhesion films or gels, anti-wrinkle agents, molding aids, arthritis agents, drug carriers, and in particular, researches on the use of anti-wrinkle agents and cosmetic aids as commercial uses (F. Manna, M. Dentini, P. Desider, O. De Pita, E. Mortilla, B. Maras, Journal of European Academy of Dematology and Venereology, 13 (1999) 183-192).

The microparticles produced by hyaluronic acid have biocompatibility, excellent physical stability and biodegradability. For example, US Pat. Nos. 6,066,340 and 6,039,970 are synthesized by combining ethyl alcohol or benzyl alcohol with a carboxyl group of hyaluronic acid. One hyaluronic acid derivative is disclosed. This material is insoluble in water and soluble in organic solvents such as dimethyl sulfoxide. Therefore, there are known examples in which solid microparticles are prepared by an emulsion solvent extraction method using such hydrophobic hyaluronic acid derivatives. However, conventional microparticles prepared using hyaluronic acid and the like have a strong toxic organic solvent, have a size of several tens of micrometers or less, and have problems with low swelling and physical properties.

On the other hand, alginic acid is a polysaccharide richly contained on the surface of brown algae and is a compound obtained by random combination of (1-4) -bound mannuronic acid and gluronic acid without branch chains. Alginic acid beads can be easily prepared at room temperature without an organic solvent, and its preparation can be achieved under mild and inert conditions in which cells can live and is biodegradable in vivo.

U.S. Patent No. 5,459,054 has shown that the collection of cells or transgenic cells using alginic acid with high content of gluronic acid has the effect of reducing immunity in transplantation. U. S. Patent No. 5,472, 648 also discloses a method for producing micro-sized microbeads through controlled oscillating nozzles and for making alginic acid microbeads with a constant degree of cure. However, such alginic acid microbeads have a problem that they are not excellent in viscoelasticity and high in swelling.

Therefore, an object of the present invention is to provide a novel biocompatible microbead that can solve the problems of the prior art.

That is, an object of the present invention is to provide a microbead that can be useful in various environments in the body by having excellent biocompatibility, excellent swelling ability with respect to aqueous solutions such as water, saline and excellent physical stability.

It is another object of the present invention to provide various manufacturing methods which can efficiently produce such microbeads by a simple method.

The present inventors have conducted extensive research and numerous experiments in order to achieve this purpose, and the microbeads formed by physical and / or chemical bonding of natural polysaccharides and hyaluronic acid selected from alginic acid and chitosan are biocompatible, swellable, and physically stable. It discovered that the back is excellent and came to complete this invention.

1 is an IR analysis of microbeads having a molecular weight of 500,000 of sodium hyaluronate among Examples 2, 4 and 8 of the present invention;

2 is a photograph taken with a scanning electron microscope of the microbead prepared in Example 8 of the present invention;

3 is a photograph taken with an optical microscope in a state in which the microbead prepared in Example 8 of the present invention is dispersed in distilled water;

4a and 4b are photographs taken with an optical microscope, respectively, of the microbeads prepared in Example 11 of the present invention in a dry state and in a physiological saline solution;

5A and 5B are photographs taken with an optical microscope of the microbeads prepared in Example 12 of the present invention in a state of being dispersed in physiological saline;

6 is a photograph taken with an optical microscope of the microbead prepared in Example 14 of the present invention in a state dispersed in physiological saline;

7 is a graph showing the viscoelasticity of chitosan-hyaluronic acid microbeads prepared in Example 17 of the present invention in comparison with conventional hyaluronic acid;

8 is a photograph taken with an optical microscope of the microbeads prepared in Example 18 of the present invention in a state dispersed in physiological saline;

9 is a graph showing the viscoelasticity of the chitosan-hyaluronic acid microbead prepared in Example 19 of the present invention compared with the conventional hyaluronic acid.

Accordingly, the present invention provides a highly swellable biocompatible microbead composed of natural polysaccharides selected from alginic acid and chitosan and hyaluronic acid.

The microbeads according to the present invention can be broadly classified into three types according to the types and inclusion forms of natural polysaccharides: alginic acid-hyaluronic acid microbeads (a), chitosan-hyaluronic acid microbeads (b), and chitosan coated. Alginic acid-hyaluronic acid microbeads (c). Among the three types of microbeads, the binding form of chitosan and hyaluronic acid (and / or alginic acid) can be subdivided into those which are physical and chemical.

Since chitosan has an amine group that can accept hydrogen ions and has a positive charge under acidic or neutral conditions, negative charges and electrostatic attraction of carboxyl groups of hyaluronic acid can occur to form microbeads. "Physical bond". On the other hand, such physical bonding alone does not have a relatively good physical stability and viscoelasticity, in particular in the aqueous solution can be dispersed in a state that does not maintain a certain form, there are some limitations to use in the body for a specific use. Thus, inducing amide bonds between the amine groups of the chitosan and the carboxyl groups of the hyaluronic acid can produce microbeads with better physical stability and viscoelasticity, which can be referred to as "chemical bonds". Alginic acids with carboxyl groups, like hyaluronic acid, can also form chemical bonds with chitosan. Thus, when the microbead of the present invention contains chitosan, i.e., in the chitosan-hyaluronic acid microbead (b) and the alginic acid-hyaluronic acid microbead (c) coated with chitosan, the chitosan is hyaluronic acid and / or It is more preferred to form chemical bonds with alginic acid.

Among the three types of microbeads, chitosan-hyaluronic acid microbeads (b) and alginic acid-hyaluronic acid microbeads (c) coated with chitosan, which have relatively high swelling, physical stability and viscoelasticity, are more preferable. Do.

The microbeads of the present invention may further contain other components within the scope of not impairing the effects of the present invention.

In the following, the content of the present invention will be described in more detail. For reference, unless stated otherwise, various ranges defined herein, for example, molecular weight range, concentration range, temperature range, reaction time range, and the like are understood as conditions for carrying out the present invention more efficiently.

One. Microbead Components

As hyaluronic acid (sometimes abbreviated as "HA" in the drawings, etc.) used in the present invention, both hyaluronic acid itself and salts thereof can be used. The hyaluronic acid salt includes both inorganic salts such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronic acid, zinc hyaluronate, cobalt hyaluronic acid, and organic salts such as tetrabutylammonium hyaluronic acid. In some cases, a combination of two or more thereof may be used.

The molecular weight of the hyaluronic acid and salts thereof is preferably 100,000 to 3,000,000 in order to form uniform and stable microbeads.

Alginic acid itself or a salt thereof may be used as the alginic acid (sometimes abbreviated as "AA" in the drawings and the like) used in the present invention. Representative examples of the alginate salt include sodium alginate, potassium alginate, calcium alginate, magnesium alginate and the like. Alginic acid is composed of a combination of mannuronic acid and guluronic acid, and it is present that the gluron acid is 50% or less and 50% or more, all of which can be used in the present invention. Since the alginic acid-hyaluronic acid microbeads among the microbeads according to the present invention are formed by gluronic acid, microbeads of slightly different properties may be formed depending on the content thereof.

Chitosan (sometimes abbreviated as "Ch" in the drawings, etc.) used in the present invention is a concept including an oligomer thereof, and the chitosan and its oligomer are preferably a substance soluble in an aqueous solution of pH 2-8. The molecular weight of chitosan in the chitosan-hyaluronic acid microbead (b) is not particularly limited, and, for example, all of the oligomers of 1,600 or less (for example, oligomers of 300 to 1,600) and polymers of 1,600 to 10,000,000 may be used. have.

2. Microbead Manufacturing Methods

The microbeads of the present invention may be prepared by different manufacturing methods, as described below, depending on the type of components constituting the microbeads and their inclusion forms.

As described above, since the hyaluronic acid in the present specification includes both hyaluronic acid itself and salts thereof, the aqueous hyaluronic acid solution herein is an aqueous solution of hyaluronic acid, an aqueous solution of hyaluronic acid salt, and a mixture of hyaluronic acid and hyaluronic acid salt. Includes all aqueous solutions. In addition, since the alginic acid in the present specification is a concept including both alginic acid itself and salts thereof, the aqueous alginic acid solution here includes both aqueous solutions of alginic acid, aqueous solutions of alginic acid salts, and mixed aqueous solutions of alginic acid and alginic acid salts. Similarly, since chitosan in the present specification is a concept including both chitosan and oligomers thereof, the aqueous chitosan solution includes both an aqueous solution of chitosan, an aqueous solution of chitosan oligomer, and a mixed aqueous solution of chitosan and chitosan oligomer.

(One) Method for preparing alginic acid-hyaluronic acid microbead (a)

The alginic acid-hyaluronic acid microbead (a) is produced by spraying a mixed solution of an aqueous alginic acid solution and an aqueous hyaluronic acid solution onto an aqueous mixed salt solution of divalent or trivalent cation salt and sodium chloride. When the alginic acid / hyaluronic acid mixed solution is sprayed onto the mixed aqueous solution of divalent or trivalent cation salt / sodium chloride, solid microbeads are precipitated by ion exchange reaction of divalent or trivalent cation salt. The ratio (weight ratio) of alginic acid to hyaluronic acid in the mixed solution is preferably from 1:10 to 10: 1 in order to easily prepare solid microbeads. The concentration of alginic acid and hyaluronic acid in the mixed solution is preferably 0.1 to 10% by weight for easy spraying.

In some cases, the mixed solution may be prepared by adding hyaluronic acid to an aqueous alginic acid solution or adding alginic acid to an aqueous hyaluronic acid solution.

The divalent cation salt in the mixed salt aqueous solution may include calcium chloride, magnesium chloride, strontium chloride, barium chloride, and the like, and may be used in one or two or more thereof. In some cases, trivalent cation salts such as aluminum chloride may optionally be used. In the mixed salt aqueous solution, the concentration of the divalent or trivalent cation salt is preferably 0.01 to 15 M in order to obtain solid microbeads.

The reaction of alginic acid to form ionic bonds with divalent or trivalent cation salts proceeds very quickly. Thus, when only divalent or trivalent cation salts are used, these cation salts do not penetrate into the interior of the beads and form crosslinks by ionic bonds only on the surface of the beads by instantaneous reactions, thus obtaining a uniform crosslinking state. Can't. However, when sodium chloride is used together, the reaction of sodium chloride-alginic acid is competing for such divalent or trivalent cation salt-alginic acid reactions, so that these reaction rates can be controlled and thereby uniform throughout the beads. One crosslinking state can be obtained. In order to obtain such a uniform crosslinked state, the concentration of sodium chloride in the mixed salt aqueous solution is preferably 0.01 to 45 M.

The method of spraying the mixed solution of alginic acid / hyaluronic acid to the aqueous solution of divalent or trivalent cation salt / sodium chloride mixed salt is not particularly limited and may be carried out, for example, via a nebulizer.

It is preferable that the temperature at the time of manufacture of the said microbead is 0-40 degreeC, and room temperature (20-25 degreeC) is more preferable. In addition, the pH of the microbeads is preferably from 2 to 8. In addition, the duration for the production of microbeads in the reaction solution is not particularly limited, and is preferably 1 to 4 hours.

Recovery of the prepared microbeads can be carried out by various methods.

For example, the nitrogen gas may be dried for 2 to 24 hours to remove the water contained in the microbeads. After the water is removed, the microbeads are in a solid solid phase.

Alternatively, after slowly adding acetone, acetone aqueous solution (preferably 95% or more), C ₂ to C ₆ alcohols such as ₂ -propanol, and the like, the microbeads are formed into a solid solid after a predetermined time. It can also be recovered by centrifugation. The time for holding the reaction solution after adding acetone or the like is preferably about 2 to 24 hours.

It may be separated and / or purified by other conventional methods known in the art, and such representative examples include distillation (including distillation under atmospheric pressure and distillation under reduced pressure), recrystallization, column chromatography, ion exchange chromatography, gel chromatography. Chromatography, affinity chromatography, thin layer chromatography, phase separation, solvent extraction, dialysis, washing and the like. Purification can be carried out after each reaction or after a series of reactions.

The prepared microbeads may be washed with water, alcohols such as ethanol, ethers such as diethyl ether, acetone, and may be washed two or more times to obtain high purity microbeads.

(2) Method for preparing chitosan-hyaluronic acid microbead (b)

The chitosan-hyaluronic acid microbead (b), as described above, has an amide bond (chemically) between the amine group of the chitosan and the carboxyl group of the hyaluronic acid, so as to have a more suitable physical stability, viscoelasticity and high swellability for use as a biocompatible material. Connection). Such chitosan-hyaluronic acid microbeads (b) can be prepared by the following methods.

An exemplary first method of preparing chitosan-hyaluronic acid microbead (b) (1) is

(A) preparing a hyaluronic acid aqueous solution and a chitosan aqueous solution each containing a carboxyl group activator;

(B) dropping the two kinds of aqueous solutions into a solution containing an emulsifier to form microbeads; And

(C) separating and purifying the microbeads;

It is configured to include.

In some cases, between step (B) and step (C), acetone, acetone aqueous solution (preferably acetone 90% or more) in a solution containing microbeads in step (B), C ₂ to C It may further comprise the step of making the microbeads somewhat firm by adding ₆ alcohol and the like. This may be because the added acetone and the like swell out the water on the swelled chitosan-hyaluronic acid microbeads, and the slightly hardened microbeads do not stick to each other in the recovery process.

In some cases, after step (C), the method may further comprise the step of dispersing the microbead obtained in step (C) in acetone or acetone aqueous solution, and then adding a carboxyl group activator to induce an additional amidation reaction. have. This additional amidation reaction can form a greater number of amide bonds between the residual carboxyl groups of the hyaluronic acid and the residual amine groups of the chitosan, thereby forming microbeads with better physical stability and viscoelasticity.

An exemplary second preparation method (2) of chitosan-hyaluronic acid microbeads (b) is

(A1) preparing an aqueous hyaluronic acid solution and an aqueous chitosan solution, respectively;

(B1) dropping the two kinds of aqueous solutions into a solution containing an emulsifier to form microbeads;

(C1) separating and purifying the microbeads; And

(D1) dispersing the microbeads in acetone or an acetone aqueous solution, and then adding an carboxyl activator to induce an amidation reaction;

It is configured to include.

In the preparation method (2), the ionic bond (physical bond) between the carboxyl group of hyaluronic acid and the amine group of chitosan in the microbead of step (B1) is converted into an amide bond (chemical bond) in step (D1).

In some cases, between the step (B1) and step (C1), the microbeads are somewhat added by adding acetone, acetone aqueous solution, C ₂ to C ₆ alcohol, etc. to the solution containing the microbeads in step (B1). It may further comprise the step of making it robust.

Based on the above, various applications and modifications may be made, and they should be construed as being included in the scope of the present invention.

More specific conditions of the preparation method of the chitosan-hyaluronic acid microbead (b) are described in detail below, and the above two exemplary methods 1 and 2 will be described without any distinction.

The concentration of the aqueous chitosan solution and the concentration of the aqueous hyaluronic acid solution is preferably 0.00001% to 10% by weight, respectively. The mixing ratio of the chitosan aqueous solution and the hyaluronic acid aqueous solution is preferably 0.00001: 1 to 100: 1 for the smooth performance of the amidation reaction in the amine group of chitosan: carboxyl group of hyaluronic acid.

The emulsifier in the above production methods is not particularly limited, but preferred examples include lipids such as lecithin, phosphatidylcoline, phosphatidylethanolamine, phosphatidyl-serine, and phosphatidylinositol. And ester derivatives of fatty acids such as glyceryl stearate, sorbitan palmitate, sorbitan stearate, and the like, and derivatives thereof. Among them, sorbitan-based emulsifiers are more preferable, and examples thereof include polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40), Polyoxyethylene sorbitan monostearate (Tween 60), polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monolaurate (Span 20), sorbitan mono Stearate (Sorbitan monostearate: Span 60), sorbitan monooleate (Span 80) and the like. Among the examples of the various emulsifiers, lecithin, Tween 20, Tween 80, Span 80 and the like are particularly preferable.

The amount of the emulsifier added is preferably 0.05% by volume to 10% by volume with respect to the solution.

Examples of the solution containing an emulsifier include mineral oil (eg, mineral light white oil), methanol, a mixture thereof, a mixture of glycerol and 1-propanol, a mixture of glycerol and 2-propanol, a mixture of ethylene glycol and 1-propanol, A mixed liquid of ethylene glycol and 2-propanol, a mixed liquid of ethylene glycol and ethanol, a mixed liquid of ethylene glycol and methanol, a mixed liquid of ethylene glycol and acetone, and the like can be used.

In the amidation reaction, the carboxyl group activator acts to activate the carboxyl group of hyaluronic acid so that the carboxyl group of the hyaluronic acid reacts with the amine group of the chitosan to form an amide bond. For example, it is separated into urea and is not bound to the reaction product. Preferably, in order to assist the action of the activator (e.g., increase the reactivity and reduce side reactions), an activation aid may be used together.

Preferred examples of the carboxyl group activator include carbodiimide-based compounds, and preferred examples thereof include 1-alkyl-3- (3-dimethylaminopropyl) carbodiimide (wherein alkyl is one to one carbon). 10 alkyl), 1-ethyl-3- (3- (trimethylammonio) propyl) carbodiimide (ETC), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide (CMC) Etc. can be mentioned. Especially, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) is especially preferable.

Preferred examples of the activity aid include N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2, 3-benzotriazine (HOOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxy-sulfosuccinimide (Sulfo-NHS), etc. are mentioned. Especially NHS is especially preferable.

The amount of the activator and the activity assistant may be determined by various factors such as their activity and the concentration of hyaluronic acid. For example, 0.00001 to 100 mg / ml of EDC and 0.00001 to 100 mg / ml of NHS are preferable. Can be used.

The temperature, pH, reaction time, recovery method of the prepared microbeads, etc. of the reaction solution for preparing the microbeads are not significantly different from those of the alginic acid-hyaluronic acid microbeads (a).

(3) Method for preparing alginic acid-hyaluronic acid microbead (c) coated with chitosan

Alginic acid-hyaluronic acid microbeads (c) coated with chitosan, specifically, microbeads (c) with beads of alginic acid-hyaluronic acid coated with chitosan can be prepared by the following methods.

The first exemplary method of preparing alginic acid-hyaluronic acid microbeads (c) coated with chitosan is

(A) preparing an aqueous solution of alginic acid and an aqueous solution of hyaluronic acid, respectively;

(B) spraying a mixed solution of an aqueous alginic acid solution and an aqueous hyaluronic acid solution to a divalent or trivalent cation salt and an aqueous sodium chloride solution to generate microbeads;

(C) coating the resulting microbeads in chitosan solution; And

(D) separating and purifying the microbeads from the reaction solution;

It is configured to include.

In the production method (1), an ionic bond (physical bond) is formed between the carboxyl group of hyaluronic acid and / or alginic acid and the amine group of chitosan in the microbead of step (C).

In the step (B), the mixed solution may be prepared by adding hyaluronic acid to the aqueous alginic acid solution or by adding alginic acid to the aqueous hyaluronic acid solution.

The mixing ratio of alginic acid and hyaluronic acid is the same as in the method for producing alginic acid-hyaluronic acid microbead (a).

Although the reaction amount of chitosan is preferably adjusted so that the ratio of the carboxyl group of alginic acid and / or hyaluronic acid and the amine group of chitosan is in the range of 1: 100-100: 1, it is not necessarily limited to the range.

In some cases, after step (D), the method may further include amidating the purified microbeads. As described above, since alginic acid and hyaluronic acid have a carboxyl group, and chitosan has an amine group, the carboxyl group of alginic acid, the carboxyl group of hyaluronic acid, or the amine group of alginic acid and hyaluronic acid as an amide bond which is a chemical bond By connecting, microbeads which are more excellent in swelling, physical stability and viscoelasticity can be produced. The amidation reaction can be carried out by, for example, dispersing the microbead obtained in step (D) in distilled water and reacting the carboxyl group activator with the addition of a carboxyl group activator and an activation aid. In some cases, the reaction may be performed by adding the microbead obtained in step (D) to distilled water in which the carboxyl group activator is dissolved or distilled water in which the carboxyl group activator and the activation reaction assistant are dissolved. The carboxyl group activator for the amidation reaction in the above production method, the kind of the activation aid and the amount thereof added are the same as in the production method of the chitosan-hyaluronic acid microbead (b).

In the further amidation reaction of the chitosan-hyaluronic acid microbead (b), acetone (strictly, some distilled water is contained in the bead or the solvent itself) is used. In the amidation reaction, distilled water is used as a solvent. In the preparation of chitosan-hyaluronic acid microbeads (b), acetone (a solvent capable of maintaining form and no form reaction) can be maintained since the microbeads cannot be dissolved and maintained in an aqueous solution such as distilled water until crosslinking is completed. It is necessary to use a solvent containing the same organic solvent and slightly distilled water (necessary for smooth crosslinking), but in the case of chitosan-hyaluronic acid microbeads (b), since it is not dissolved in an aqueous solution such as distilled water, it can be used as a solvent. .

A second exemplary method for preparing alginic acid-hyaluronic acid microbead (c) coated with chitosan is

(A1) preparing an aqueous alginic acid solution and an aqueous hyaluronic acid solution, respectively;

(B1) coating chitosan while spraying a mixed solution of an aqueous alginic acid solution and an aqueous hyaluronic acid solution onto an aqueous chitosan solution containing a divalent or trivalent cation salt and sodium chloride;

(C1) separating and purifying the microbeads from the reaction solution;

It is configured to include.

The production method (2) is different from the production method (1) in that chitosan is coated on its surface while alginic acid-hyaluronic acid microbeads are generated.

The mixing ratio of the alginic acid and the hyaluronic acid in the production method (2), the chitosan reaction amount and the like are the same as in the production method (1). Likewise, after step (C1), the method may further comprise performing amidation reaction of purified microbeads, and the carboxyl group activator, activation reaction aid, etc. for the amidation reaction may same.

3. Microbeads

The microbeads according to the present invention have various properties due to various reaction factors such as the type, content and form of the components constituting the microbeads, spraying conditions, and the presence or absence of chemical bonds (amide bonds).

The shape of the microbead according to the present invention is approximately spherical (see FIG. 2), and may have various particle diameters depending on reaction conditions, and thus the present invention is not particularly limited thereto.

The microbead according to the present invention is biocompatible and exhibits high swellability in aqueous solutions such as water and saline. Therefore, according to the properties of the microbeads, it can be used as a biocompatible material for a variety of uses, such as a post-adhesion anti-adhesion film or gel, wrinkle treatment, molding aid, arthritis treatment, drug delivery.

Particularly where high swelling and physical stability and viscoelasticity are required, the microbeads according to the invention, namely alginic acid-hyaluronic acid microbeads (a), chitosan-hyaluronic acid microbeads (b), and alginic acid coated with chitosan Of the hyaluronic acid microbeads (c), chitosan-hyaluronic acid microbeads (b) and alginic acid-microbeads (c) coated with chitosan are particularly preferred. Also in these microbeads (b and c), microbeads in which the amine group of chitosan form an amide bond with the carboxyl group of alginic acid and / or hyaluronic acid are more preferable.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto. For convenience of description, the contents of the alginic acid-hyaluronic acid microbead (a) and the alginic acid-hyaluronic acid microbead (c) coated with chitosan are described together in Examples 1 to 10 and Experimental Examples 1 to 3, and chitosan -Hyaluronic acid microbead (b) is described separately in Examples 11-20 and Experimental Examples 4-13.

Example

Example 1 Preparation of Alginic Acid-Haluronic Acid Microbeads-1

10 mg / ml sodium hyaluronate (molecular weight: 150,000, 250,000, 370,000, 500,000, manufacturer: LG Life Sciences Ltd.) 10 ml of each aqueous solution was prepared, and 100 mg of alginic acid was added thereto to prepare a mixture of sodium alginate and sodium hyaluronate. After the preparation, the mixture was stirred well. 10 ml of the mixed solution was sprayed through a nebulizer into 100 ml of a mixed aqueous solution of 200 mM calcium chloride and 150 mM sodium chloride. The sprayed particles were precipitated (140 mg, yield: 70%) by ion exchange in an aqueous calcium chloride-sodium chloride solution to change to solid microbeads. After 2 hours, the solid beads were centrifuged at 1,500 rpm, washed several times with distilled water and then dispersed in 20 ml of distilled water.

Example 2 Preparation of Alginic Acid-Haluronic Acid Microbeads-2

Microbeads were prepared in the same manner as in Example 1, except that 10 ml of an aqueous 20 mg / ml sodium hyaluronate (molecular weight: 150,000, 250,000, 370,000,500,000) solution was used (150 mg, yield: 50%). .

The IR analysis of the microbeads having a molecular weight of 500,000 sodium hyaluronate among the thus prepared microbeads is shown in FIG. 1.

Example 3 Preparation of Alginic Acid-Haluronic Acid Microbeads Coated with Chitosan-1

10 ml of 10 mg / ml sodium hyaluronate (molecular weight: 150,000, 250,000, 370,000, 500,000) aqueous solution was prepared, and 100 mg of alginic acid was added thereto to prepare a sodium hyaluronate-alginic acid mixed solution. Stirred. Further, chitosan (molecular weight: 1,600 or less, manufacturer: Eugene Bio) was dissolved in a mixed aqueous solution of 200 mM calcium chloride and 150 mM sodium chloride at a concentration of 5 mg / ml to prepare 100 ml of a reaction solution. The reaction solution was sprayed with a 10 mL sodium hyaluronate-alginic acid mixed solution through a nebulizer. The sprayed particles were ion exchanged in calcium chloride-sodium chloride solution to turn into solid beads (150 mg, yield: 75%) and its surface coated with chitosan. After 2 hours, the solid microbeads were centrifuged at 1,500 rpm, washed several times with distilled water, and then dispersed in 20 ml of distilled water.

Example 4 Preparation of Alginic Acid-Haluronic Acid Microbeads Coated with Chitosan-2

Microbeads were prepared in the same manner as in Example 3, except that 10 ml of an aqueous 20 mg / ml sodium hyaluronate (molecular weight: 150,000, 250,000, 370,000, 500,000) solution was used (180 mg, yield: 60). %). IR analysis results for the microbeads having a molecular weight of 500,000 hyaluronic acid in the prepared microbeads are shown in FIG. 1.

Example 5 Preparation of Alginic Acid-Haluronic Acid Microbeads Coated with Chitosan-3

Microbeads (140 mg, yield: 70%) prepared in the same manner as in Example 1 were added to 20 ml of an aqueous solution of 5 mg / ml of chitosan (molecular weight: 1,600 or less), followed by stirring for 2 hours. The surface of the microbeads was coated with chitosan. The coated solid microbeads were centrifuged at 1500 rpm, washed several times and then dispersed in 20 ml of distilled water.

Example 6 Preparation of Alginic Acid-Haluronic Acid Microbeads Coated with Chitosan-4

Microbeads (150 mg, yield: 50%) prepared in the same manner as in Example 2 were added to 20 ml of 5 mg / ml aqueous solution of chitosan (molecular weight: 1,600 or less) and dispersed, followed by stirring for 2 hours. The surface of the microbeads was coated with chitosan. The coated solid microbeads were centrifuged at 1500 rpm, washed several times and then dispersed in 20 ml of distilled water.

Example 7 Preparation of Alginic Acid-Haluronic Acid Microbead Covalently Bonded with Chitosan-1

Microbeads (150 mg, yield: 75%) prepared in the same manner as in Example 3 were washed several times with distilled water, and then dispersed in 20 ml of distilled water. To this dispersion was added 50 mg of EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and 60 mg of NHS (N-hydroxysuccinimide), followed by amide with gentle stirring at room temperature. The reaction was carried out. After 4 hours, the solid microbeads were centrifuged at 1500 rpm, washed with distilled water, and then dispersed in 20 ml of distilled water.

Example 8 Preparation of Alginic Acid-Haluronic Acid Microbeads Covalently Bonded with Chitosan-2

Microbeads (180 mg, yield: 60%) prepared in the same manner as in Example 4 were washed several times with distilled water, and then dispersed in 20 ml of distilled water. 50 mg of EDC and 60 mg of NHS were added to such a dispersion solution, and an amidation reaction was carried out while gradually stirring at room temperature. After 4 hours, the solid microbeads were centrifuged at 1500 rpm, washed with distilled water, and then dispersed in 20 ml of distilled water. The IR analysis of the microbeads having a molecular weight of 500,000 sodium hyaluronate among the thus prepared microbeads is shown in FIG. 1.

Example 9 Preparation of Alginic Acid-Haluronic Acid Microbeads Covalently Bonded with Chitosan-3

Microbeads (140 mg, yield: 70%) prepared in the same manner as in Example 5 were washed several times with distilled water, and then dispersed in 20 ml of distilled water. 50 mg of EDC and 60 mg of NHS were added to such a dispersion solution, and an amidation reaction was carried out while gradually stirring at room temperature. After 4 hours, the solid microbeads were centrifuged at 1500 rpm, washed with distilled water, and then dispersed in 20 ml of distilled water.

Example 10 Preparation of Alginic Acid-Haluronic Acid Microbead Covalently Bonded with Chitosan-4

Microbeads (150 mg, yield: 50%) prepared in the same manner as in Example 6 were washed several times with distilled water, and then dispersed in 20 ml of distilled water. 50 mg of EDC and 60 mg of NHS were added to such a dispersion solution, and an amidation reaction was carried out while gradually stirring at room temperature. After 4 hours, the solid microbeads were centrifuged at 1500 rpm, washed with distilled water, and then dispersed in 20 ml of distilled water.

Experimental Example 1 IR Analysis of the Prepared Microbeads

Microbeads consisting only of hyaluronic acid and alginic acid (Example 2), alginic acid-hyaluronic acid microbeads coated with chitosan (Example 4), and alginic acid-hyaluronic acid microbeads bonded chitosan (Example 8) ), IR (infra-red) analysis was performed on microbeads having a molecular weight of 500,000 sodium hyaluronate. As a result, in the alginic acid-hyaluronic acid microbead (Example 4) coated with chitosan, a new peak of 1529.55 cm ^-1 was observed, unlike the alginic acid-hyaluronic acid microbead (Example 2), which is a free amine of chitosan. Group. In addition, the alginic acid-hyaluronic acid microbeads covalently bonded to chitosan (Example 8) showed a broad tendency around the peak around 1639.49 cm ⁻¹ , indicating that the amidation reaction of the amine group of the chitosan and the carboxyl groups of hyaluronic acid and alginic acid was performed. This is the result shown by the resulting amide bond.

Experimental Example 2 Properties of Hyaluronic Acid Microbeads

The shape and size (diameter) of the microbeads prepared in Examples 1 to 10 were observed through a scanning electron microscope and an optical microscope. Table 1 shows the size (diameter) of the microbeads according to the ratio of the components and the molecular weight of hyaluronic acid (HA).

HA (MW: 150,000) HA (MW: 250,000) HA (MW: 370,000) HA (MW: 500,000) Example 1 243.9 μm 307 μm 315 μm 350.7 μm Example 2 266.7 μm 303 μm 388 μm - Example 3 261.7 μm 318.8 μm 344.3 μm 345.8 μm Example 4 283.4 μm 350.6 μm 394.7 μm 426 μm Example 7 267.2 μm 345.2 μm 422.5 μm 418.3 μm Example 8 309.7 μm 343.1 μm 447.8 μm 444 μm

As can be seen in Table 1, as the molecular weight of hyaluronic acid increases, the viscosity increases and the size of the microbeads also tends to increase.

The photograph of the shape of the alginic acid-hyaluronic acid microbead covalently bonded with chitosan (Example 8) was taken by scanning electron microscope in FIG. 2, and the image of the microbeads in a swollen state when the dispersion was dispersed in distilled water was taken by optical microscope. Each of these is disclosed in FIG. 3. As can be seen in these photographs, the microbeads show a spherical shape before and after swelling.

Experimental Example 3 Measurement of Swelling Degree of Microbeads

Swelling degree was measured for some of the microbeads prepared in Examples 1, 4 and 8. The swelling degree is the weight (dry weight: W _dry ) measured after placing the microbeads in a petri dish in a vacuum oven at room temperature for 24 hours (dry weight: W _dry ), and after swelling in distilled water for 24 hours to remove water from the surface ( Swelling weight: W _wet ) was calculated using the following formula.

Swelling Degree = W _wet / W _dry

The results are shown in Table 2 below.

Dry weight (W _dry ) Swelling Weight (W _wet ) Swelling degree (W _wet / W _dry ) Example 1 (HA MW: 500,000) 2.1 mg 41.9 mg 19.95 Example 4 (HA MW: 250,000) 1.1 mg 47.1 mg 42.82 Example 8 (HA MW: 250,000) 1.3 mg 54.6 mg 42

As can be seen in Table 2 above, the alginic acid-hyaluronic acid microbeads covalently bonded with the alginic acid-hyaluronic acid microbeads (Example 4) and chitosan coated with chitosan than the alginic acid-hyaluronic acid microbeads (Example 1) It can be seen that Example 8) shows a swelling degree that is two times higher. This is believed to be due to the absorption of moisture by the free amine groups still remaining in the coated or crosslinked chitosan.

Example 11 Preparation of Chitosan-Haluronic Acid Microbeads-1 (using mineral oil + Tween 80)

5 ml of an aqueous 10 mg / ml sodium hyaluronate (molecular weight: 250,000, manufacturer: LG Life Sciences Ltd.) and 5 ml of an aqueous chitosan oligomer (molecular weight: 1,600 or less) of 5 mg / ml were prepared, respectively. To the aqueous sodium hyaluronate solution, 25 mg of EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) and 30 mg of NHS (N-hydroxysuccinimide) are added to 100 mg of hyaluronic acid. It was. The two aqueous solutions were simultaneously dropped in drops of 100 ml of mineral oil containing 4 ml, 5 ml and 6 ml of emulsifier Tween 80, respectively. The reaction was carried out with stirring for 2 hours, and then dried with nitrogen gas for 12 hours to remove water dispersed in the mineral oil. Microbeads precipitated in the mineral oil from which water was removed were recovered by centrifugation. The recovered microbeads were washed several times with ethanol, acetone and water and dried with nitrogen gas (weight of microbeads: 53.1 mg, 40 mg, 31 mg; yield: 70.8%, 53.3%, 41.3%).

Experimental Example 4 Properties of the Microbeads Prepared in Example 11

The shape and size (diameter) of the microbeads prepared in Example 11 were observed through an optical microscope, and the size (diameter) of the beads according to the amount of the emulsifier was classified into a dry state and a swelling state. As can be seen in Table 3, as the amount of emulsifier increases, the size of the beads tends to decrease.

Quantity of emulsifier (twin 80) 4 ml 5 ml 6 ml Bead diameter in dry state (㎛) 1500 250 25 Swelled Bead Diameter (㎛) 2000 600 100

The photographs of the chitosan-hyaluronic acid microbeads prepared using the 6 ml emulsifier (Twin 80) in Example 11 were observed using an optical microscope in a dried state and swelled by being dispersed in physiological saline. And 4b. It can be seen that it has a spherical shape as a whole in both the dry state (Fig. 4a) and the swelling state (Fig. 4b).

Example 12 Preparation of Chitosan-Haluronic Acid Microbeads-2 (Using Mineral Oil + Span 80)

5 ml of an aqueous 10 mg / ml sodium hyaluronate (molecular weight: 500,000) solution and 5 ml of a 5 mg / ml aqueous chitosan oligomer (molecular weight: 1600 mass or less) were prepared, respectively. To the aqueous sodium hyaluronate solution, 25 mg of EDC and 30 mg of NHS were added to 100 mg of hyaluronic acid. The two aqueous solutions were simultaneously dropped in drops of 100 ml of mineral oil containing 0.75 ml, 0.85 ml, 0.1 ml and 0.15 ml of emulsifier span 80, respectively. The reaction was carried out with stirring for 2 hours, and then dried with nitrogen gas for 12 hours to remove water dispersed in the mineral oil. Microbeads precipitated in the mineral oil from which water was removed were recovered by centrifugation. The recovered microbeads were washed several times with ethanol, acetone and water and dried with nitrogen gas (weight of microbeads: 91.2 mg, 89.5 mg, 79.5 mg, 70.3 mg; yield: 91.2%, 89.5%, 79.5 %, 70.3%).

Experimental Example 5 Properties of Microbeads Prepared in Example 12

The shape and size (diameter) of the microbeads prepared in Example 12 were observed through an optical microscope, and the size (diameter) of the beads according to the amount of the emulsifier was measured in a dry state and a swollen state, respectively. Indicated. As can be seen in Table 4, as the amount of emulsifier increases, the size of the beads tends to decrease.

Quantity of emulsifier (span 80) 0.75 ml 0.85 ml 0.1 ml 0.15 ml Dry Bead Diameter (㎛) No bead formation No bead formation 150 20 Swelled Bead Diameter (㎛) 250 50

5A and 5B show photographs taken using an optical microscope in a state in which microbeads prepared using 0.1 mL and 0.15 mL of emulsifier span 80 were dispersed and swelled in physiological saline, respectively. In the case of using 0.1 ml of Span 80 (FIG. 5A) and 0.15 ml of Span 80 (FIG. 5B), it can be seen that the overall shape is spherical.

Example 13 Preparation of Chitosan-Haluronic Acid Microbeads-3 (Using Mineral Oil + Span 80)

10 ml of an aqueous 5 mg / ml sodium hyaluronate (molecular weight: 1,500,000) solution and 5 ml of 0.5, 1 and 2 mg / ml chitosan (molecular weight: 110,000) aqueous solution were prepared, respectively. To the aqueous sodium hyaluronate solution, 25 mg of EDC and 30 mg of NHS were added to 100 mg of hyaluronic acid. The two aqueous solutions were simultaneously dropped in drops of 100 ml of mineral oil containing 0.1 ml, 0.15 ml and 0.2 ml of emulsifier span 80, respectively. The reaction was carried out with stirring for 2 hours, and then dried with nitrogen gas for 12 hours to remove water dispersed in the mineral oil. Microbeads precipitated in the mineral oil from which water was removed were recovered by centrifugation. The recovered microbeads were washed several times with ethanol, acetone and water and dried with nitrogen gas. Specific reaction conditions and the results are shown in Table 5 below.

Reaction Condition Span 80 HA (MW = 1,500,000) 0.5% (50 mg), 10 ml (EDC / NHS = 25/30 mg) Chitosan (CL113) 0.05% (2.5 mg), 5 ml HA (MW = 1,500,000) 0.5% (50 mg), 10 ml (EDC / NHS = 25/30 mg) Chitosan (CL113) 0.1% (5 mg), 5 ml HA (MW = 1,500,000) 0.5% (50 mg), 10 mL (EDC / NHS = 25/30 mg) Chitosan (CL113) 0.2% (10 mg), 5 mL 0.1 ml 9.2 mg (17.5%) 28.6 mg (52%) No bead formation 0.15 ml - 25.3 mg (46%) 39.8 mg (66.3%) 0.2 ml - 18.1 mg (32.9%) 22.4 mg (37.3%)

Experimental Example 6 Properties of the Microbeads Prepared in Example 13

The shape and size (diameter) of the microbeads prepared in Example 13 were observed through an optical microscope, and the size (diameter) of beads according to the amount of emulsifier and the amount of chitosan was measured in the swelling state, and is shown in Table 6 below. . As can be seen in Table 6, the size of the beads tends to decrease as the amount of emulsifier increases and the amount of chitosan decreases.

Reaction Condition Span 80 HA (MW = 1,500,000) 0.5% (50 mg), 10 ml (EDC / NHS = 25/30 mg) Chitosan (CL113) 0.05% (2.5 mg), 5 ml HA (MW = 1,500,000) 0.5% (50 mg), 10 ml (EDC / NHS = 25/30 mg) Chitosan (CL113) 0.1% (5 mg), 5 ml HA (MW = 1,500,000) 0.5% (50 mg), 10 mL (EDC / NHS = 25/30 mg) Chitosan (CL113) 0.2% (10 mg), 5 mL 0.1 ml 50 ~ 180㎛ 100 ~ 300㎛ No bead formation 0.15 ml - 50 ~ 120㎛ 90 ~ 150㎛ 0.2 ml - 30 ~ 100㎛ 25 ~ 80㎛

Example 14 Preparation of Chitosan-Haluronic Acid Microbeads-4 (Using Mineral Oil + Span 80)

5 ml of an aqueous 10 mg / ml sodium hyaluronate (molecular weight: 150,000, 500,000 and 1,148,000) solution, 10 ml of an aqueous 5 mg / ml sodium hyaluronate (molecular weight: 2,465,000) solution, and 0.2 mg / ml and 1 mg / ml chitosan (ML: 110,000) 5 ml of an aqueous solution was prepared, respectively. To the aqueous sodium hyaluronate solution, 10 mg of EDC and 12 mg of NHS were added to 100 mg of hyaluronic acid. The two aqueous solutions were simultaneously dropped in drops of 100 ml of mineral oil, each containing 0.1 ml of emulsifier Span 80. The reaction was carried out with stirring for 2 hours, and then dried with nitrogen gas for 12 hours to remove water dispersed in the mineral oil. Microbeads precipitated in the mineral oil from which water was removed were recovered by centrifugation. The recovered microbeads were washed several times with ethanol, acetone and water and dried with nitrogen gas. Specific reaction conditions and the results are shown in Table 7 below.

HA molecular weight chitosan 150,000 500,000 1,148,000 2,465,000 0.02% (1 mg) 0.2 mg (0.39%) 5.4 mg (10.59%) 5.7 mg (11.18%) 6.7 mg (13.14%) 0.1% (5 mg) 8.9 mg (16.18%) 19.6 mg (35.64%) 8.6 mg (15.64%) 25.1 mg (45.64%)

Experimental Example 7 Properties of Microbeads Prepared in Example 14

The shape and size (diameter) of the microbeads prepared in Example 14 were observed through an optical microscope, and the size (diameter) of the beads according to the molecular weight and concentration of the hyaluronic acid and the amount of chitosan was measured in the swollen state. It is shown in Table 8 below. As shown in Table 8, as the molecular weight of hyaluronic acid is high, the concentration is high, and the amount of chitosan increases, the size of the beads tends to increase.

HA molecular weight chitosan 150,000 (1%, 5 ml) 500,000 (1%, 5 ml) 1,148,000 (1%, 5 ml) 2,465,000 (0.5%, 10 ml) 0.02% (1 mg, 5 ml) 50 ~ 150 ㎛ 100 ~ 150 ㎛ 100 ~ 200 ㎛ 100 ~ 500 ㎛ 0.1% (5 mg, 5 ml) 50 ~ 200 ㎛ 50 ~ 300 ㎛ 500 ~ 1,000 ㎛ 50 ~ 300 ㎛

6 shows a photo taken with an optical microscope in a state in which a hyaluronic acid (molecular weight: 2,465,000) / chitosan (0.1%, 5 mg) microbeads prepared using 0.1 ml of an emulsifier span 80 is dispersed in physiological saline and swelled. It is. As shown in Figure 6, it can be seen that the overall has a spherical shape.

Example 15 Preparation of Chitosan-Haluronic Acid Microbeads-5 (Using Mineral Oil / Methanol Mixture + Tween 20)

10 ml of an aqueous 5 mg / ml sodium hyaluronate (molecular weight: 2,465,000) solution and 5 ml of a 0.5 mg / ml aqueous chitosan (molecular weight: 110,000) solution were prepared, respectively. To the aqueous sodium hyaluronate solution, 20 mg of EDC and 24 mg of NHS were added to 100 mg of hyaluronic acid. Simultaneously drop the two aqueous solutions into 100 ml of a mixture of mineral oil and methanol containing 5 ml of emulsifier Tween 20 (mineral oil: methanol = 7: 3, 6: 4, 5: 5 and 4: 6). Dropped. After the reaction was carried out with stirring for 1 hour, 100 ml of acetone was slowly added to harden the microbeads dispersed in the mixed solution. After 2 hours the microbeads were recovered by centrifugation. The recovered microbeads were washed several times with diethyl ether and acetone. After washing, to chitosan-hyaluronic acid microbeads (hereinafter sometimes abbreviated as "hyaluronic acid derivative" or "HA derivative") dispersed in 20 ml of acetone (HA derivative: 5 mg / ml), hyaluronic acid To 100 mg of the derivative, 10 mg of EDC and 12 mg of NHS were added, followed by a secondary reaction (amidation reaction) for 2 hours. The obtained microbeads were washed three times with acetone and then dried with nitrogen gas (weight of microbeads: 37.2 mg, 36.8 mg, 39.5 mg, 44.1 mg; yield: 70.87%, 70.1%, 75.24%, 84%). Hereinafter, after drying, it was prepared in physiological saline 2% and the rheological properties were measured using a rheometer, and diluted to confirm the properties of the swelling state.

Experimental Example 8 Properties Confirmation and Viscoelasticity Measurement of the Microbeads Prepared in Example 15

The shape and size (diameter) of the hyaluronic acid microbeads prepared in Example 15 were observed through an optical microscope, and the size (diameter) of the beads according to the mixing ratio of the mineral oil and methanol was measured in the swelling state. Shown in As shown in Table 9, as the ratio of methanol increases, the size of the beads tends to decrease, and relatively transparent beads are made.

Mineral oil: methanol 70: 30 60: 40 50: 50 40: 60 Microbead Diameter (μm) 500-800 500-800 300-500 300-500

The microbeads prepared in Example 15 were prepared at a concentration of 20 mg / ml, and then 1 ml of the microbeads was prepared, and the composite viscosity was measured at a frequency of 0.02 to 1 Hz using a rheometer to check rheological properties. (complex viscosity) was measured. Compound viscosity measured at a frequency of 0.02 Hz is shown in Table 10 below. Compared to 1,258,100 cP (0.02 Hz), which is a complex viscosity of hyaluronic acid (molecular weight: 2,465,000), it was confirmed that the complex viscosity of chitosan-hyaluronic acid microbeads was improved.

Mineral Oil: Methanol 70: 30 60: 40 50: 50 40: 60 Compound Viscosity (cP) 1,280,000 1,500,000 2,250,000 1,400,000

Example 16 Preparation of Chitosan-Haluronic Acid Microbeads-6 (using mineral oil / methanol mixture + Tween 20)

10 ml of an aqueous 5 mg / ml sodium hyaluronate (molecular weight: 2,465,000) solution and 5 ml of an aqueous 0.02 mg / ml chitosan (molecular weight: 110,000) solution were prepared, respectively. To the aqueous sodium hyaluronate solution, 20 mg of EDC and 24 mg of NHS were added to 100 mg of hyaluronic acid. The two aqueous solutions were simultaneously dropped in drops of 100 ml of a mixture of mineral oil and methanol containing 5 ml of emulsifier Tween 20 (mineral oil: methanol = 5: 5). After the reaction was stirred for 1 hour, 100 ml of acetone was slowly added to harden the beads dispersed in the mixed solution. After 2 hours the microbeads were recovered by centrifugation. The recovered microbeads were washed several times with diethyl ether, acetone and the like. After washing, 10, 15 and 20 mg of EDC and 12, 18 and 24 mg of NHS were added to the microbeads (HA derivative: 5 mg / ml) dispersed in 20 ml of acetone, based on 100 mg of the hyaluronic acid derivative. , The reaction was carried out for 2 hours. The obtained microbeads were washed three times with acetone and dried with nitrogen gas (weight of microbeads: 39.92 mg, 29.9 mg, 37.5 mg; yield: 79.44%, 59.68%, 74.85%). Hereinafter, after drying, it was prepared in physiological saline 2% and the rheological properties were measured using a rheometer, and diluted to confirm the properties of the swelling state.

Experimental Example 9 Viscoelasticity Measurement of the Microbeads Prepared in Example 16

The microbeads prepared in Example 16 were prepared at a concentration of 20 mg / ml, and then 1 ml of the microbeads was measured, and the composite viscosity was measured at a frequency of 0.02 to 1 Hz using a rheometer to check rheological properties. It was. Compound viscosity measured at a frequency of 0.02 Hz is shown in Table 11 below. Compared to 1,258,100 cP (0.02 Hz), a complex viscosity of hyaluronic acid (molecular weight: 2,465,000), the composite viscosity of chitosan-hyaluronic acid microbeads can be improved. Show a tendency.

EDC / NHS (mg) on secondary amidation EDC / NHS = 10/12 for 100 mg of HA derivative EDC / NHS = 15/18 for 100 mg of HA derivative EDC / NHS = 20/24 for 100 mg of HA derivative Compound Viscosity (cP) 2,590,000 1,470,000 1,290,000

Example 17 Preparation of Chitosan-Haluronic Acid Microbeads-7 (using mineral oil / methanol mixture + Tween 20)

50 ml of an aqueous 5 mg / ml sodium hyaluronate (molecular weight: 2,465,000) solution and 25 ml of an aqueous 0.02 mg / ml chitosan (molecular weight: 110,000) solution were prepared, respectively. The two aqueous solutions were simultaneously dropped in drops of 500 ml of a mixture of mineral oil and methanol (mineral oil: methanol = 5: 5) each containing 25 ml of emulsifier Tween 80. After the reaction was stirred for 1 hour, 500 ml of acetone was slowly added to harden the beads dispersed in the mixed solution. After 2 hours the microbeads were recovered by centrifugation. The recovered microbeads were washed several times with diethyl ether and acetone. After washing, beads (HA derivative: 5 mg / ml) dispersed in 20 ml of 90% acetone were treated with 1.49 mg, 3.39 mg, 5.24 mg, 6.23 mg, 12.32 mg, 22.17 mg, and 69 mg with respect to 100 mg of the hyaluronic acid derivative. And 70.35 mg EDC and 1.79 mg, 4.07 mg, 6.29 mg, 7.48 mg, 14.78 mg, 26.6 mg, 82.8 mg, and 84.42 mg NHS were added for 2 hours. The reacted microbeads were washed three times with acetone and then dried with nitrogen gas (weight of microbeads: 202.2 mg, 176.8. Mg, 173.95 mg, 192.86 mg, 182.97 mg, 228.46 mg, 230.81 mg, 241.98 mg; Yield: 80.72%, 70.59%, 69.44%, 76.99%, 73.04%, 91.2%, 92.14%, 96.6%). Hereinafter, after drying, it was prepared in physiological saline 2% and the rheological properties were measured using a rheometer, and diluted to confirm the properties of the swelling state.

Experimental Example 10 Viscoelasticity Measurement of the Microbeads Prepared in Example 17

The microbeads prepared in Example 17 were prepared at a concentration of 20 mg / ml, and then 1 ml of the microbeads was measured, and the composite viscosity was measured at a frequency of 0.02 to 1 Hz using a rheometer to check rheological properties. It was. Compound viscosity measured at 0.02 Hz frequency is shown in Table 12 below. Compared with 1,258,100 cP (0.02 Hz), a composite viscosity of hyaluronic acid (molecular weight: 2,465,000), the composite viscosity of chitosan-hyaluronic acid microbeads is improved. It is dispersed in physiological saline without swelling.

EDC / NHS (mg) EDC / NHS = 1.49 / 1.79 for 100 mg HA derivative EDC / NHS = 3.39 / 4.07 for 100 mg HA derivative EDC / NHS = 5.24 / 6.29 for 100 mg HA derivative EDC / NHS = 6.23 / 7.48 for 100 mg HA derivative Compound viscosity (cP) 1,640,000 3,110,000 4,830,000 6,620,000 EDC / NHS (mg) EDC / NHS = 12.32 / 14.78 for 100 mg HA derivative EDC / NHS = 22.17 / 26.6 for 100 mg HA derivative EDC / NHS = 69 / 82.8 for HA derivative 100 mg EDC / NHS = 70.35 / 84.42 for 100 mg of HA derivative Compound viscosity (cP) 2,680,000 Not measurable Not measurable Not measurable

The microbeads prepared in Example 17 were prepared at a concentration of 20 mg / ml, and then 1 ml of the microbeads was measured, and the composite viscosity was measured at a frequency of 0.02 to 1 Hz using a rheometer to check rheological properties. The results are shown in FIG.

Example 18 Preparation of Chitosan-Haluronic Acid Microbeads-8 (Using Methanol + Tween 80)

25 ml of an aqueous 10 mg / ml sodium hyaluronate (molecular weight: 2,465,000) solution and 12.5 ml of an aqueous 0.04 mg / ml chitosan (molecular weight: 110,000) solution were prepared, respectively. The two aqueous solutions were simultaneously dropped one drop into 250 ml methanol containing emulsifier Tween 80 in 2.5 ml, 7.5 ml, 10 ml and 12.5 ml, respectively. After the reaction was stirred for 1 hour, 250 ml of acetone was slowly added to harden the beads dispersed in the mixed solution. After 2 hours, the microbeads were recovered by centrifugation. The recovered microbeads were washed several times with diethyl ether and acetone. After washing, beads (HA derivative: 5 mg / mL) dispersed in 20 mL of 90% acetone were 27.67 mg, 37.34 mg (Twin 80 2.5 mL), 7.99 mg, 9.85 mg (100 mg for hyaluronic acid derivative). 7.5 ml of Tween 80), 10.27 mg, 10.7 mg (Tween 80 10 ml), 6 mg, 7.34 mg (Tween 80 12.5 ml) with EDC, 33.2 mg, 44.81 mg (Twin 80 2.5 ml), 9.59 mg, 11.82 mg (7.5 ml of twin 80), 12.32 mg, 12.84 mg (twin 80 10 ml), 7.2 mg, 8.81 mg (twin 80 12.5 ml) of NHS were added and the reaction was carried out for 2 hours. After washing three times with acetone, and dried with nitrogen gas. The reaction conditions and the results are shown in Table 13 below. Hereinafter, after drying, it was prepared in physiological saline 2% and the rheological properties were measured using a rheometer, and diluted to confirm the properties of the swelling state.

Twin 80 2.5 ml 7.5 ml 10 ml 12.5 ml EDC / NHS (mg) 27.67 / 33.2 37.34 / 44.81 7.99 / 9.59 9.85 / 11.82 10.27 / 12.32 10.7 / 12.84 6 / 7.2 7.34 / 8.81 yield 50.6 mg (20.2%) 48.2 mg (19.24%) 135.19 mg (53.97%) 137 mg (54.69%) 95.39 mg (38.08%) 104.61 mg (41.76%) 168.59 mg (67.3%) 184 mg (73.45%)

Experimental Example 11 Properties Confirmation and Viscoelasticity Measurement of the Microbeads Prepared in Example 18

The shape and size (diameter) of the microbeads prepared in Example 18 were observed through an optical microscope, and the size (diameter) of the beads according to the amount of the emulsifier Tween 80 was measured in a swelled state in physiological saline. It is shown in Table 14 below. As shown in Table 14, as the amount of the emulsifier increases, the size of the beads tends to decrease.

Twin 80 2.5 ml 7.5 ml 10 ml 12.5 ml Bead diameter (μm) Bead not formed 200-400 200-300 100-200

The microbeads prepared in Example 18 were prepared at a concentration of 20 mg / ml, and then 1 ml of the microbeads was measured, and the composite viscosity was measured at a frequency of 0.02 to 1 Hz using a rheometer to check rheological properties. . Compound viscosity measured at a frequency of 0.02 Hz is shown in Table 15 below. Compared to 1,258,100 cP (0.02 Hz), a composite viscosity of hyaluronic acid (molecular weight: 2,465,000), the composite viscosity of chitosan-hyaluronic acid microbeads can be confirmed to be improved. In physiological saline it is dispersed form without swelling.

EDC / NHS Compound viscosity (cP) 6 / 7.2 4,930,000 7.34 / 8.81 7,230,000 7.99 / 9.59 8,420,000 9.85 / 11.82 3,860,000 10.27 / 12.32 6,100,000 10.7 / 12.84 Not measurable 27.67 / 33.2 Not measurable 37.34 / 44.81 Not measurable

Example 19 Preparation of Chitosan-Haluronic Acid Microbeads-9 (Using Methanol + Tween 80)

40 ml of an aqueous 10 mg / ml sodium hyaluronate (molecular weight: 2,465,000) solution and 5 ml of an aqueous 0.12 mg / ml chitosan (molecular weight: 110,000) solution were prepared, respectively. The two aqueous solutions were simultaneously dropped in drops of 300 ml of methanol each containing 12 ml of emulsifier Tween 80. After the reaction was stirred for 1 hour, 300 ml of acetone was slowly added to harden the microbeads dispersed in the mixed solution. After 2, 4, 15 and 24 hours, respectively, the microbeads were recovered by centrifugation. The recovered microbeads were washed several times with diethyl ether and acetone. After washing, the microbeads (HA derivative: 6.67 mg / ml) dispersed in 15 ml of 90% acetone, 5.34 mg (acetone treatment time: 2 hours), 6.78 mg, 7.32 mg, for 100 mg of hyaluronic acid derivative, 8.3 mg, 8.8 mg, 9.58 mg (acetone treatment time: 4 hours), 4.967 mg (acetone treatment time: 15 hours), 6.48 mg (acetone treatment time: 24 hours) and 6.41 mg (acetone treatment time: 2 hours) ), 8.14 mg, 8.784 mg, 9.96 mg, 10.56 mg, 11.50 mg (acetone treatment time: 4 hours), 5.96 mg (acetone treatment time: 15 hours), 7.78 mg (acetone treatment time: 24 hours) The reaction was carried out for 2 hours. The obtained microbeads were washed three times with acetone and then dried with nitrogen gas. The reaction conditions and the results are shown in Table 16 below. Hereinafter, after drying, it was prepared in physiological saline 2% and the rheological properties were measured using a rheometer, and diluted to confirm the properties of the swelling state.

Acetone Treatment Time EDC / NHS (mg) yield 2 hours 5.34 / 6.41 237.32 mg (59.24%) 4 hours 6.78 / 8.14 291.6 mg (72.79%) 7.32 / 8.784 283.91 mg (70.87%) 8.3 / 9.96 283.46 mg (70.76%) 8.8 / 10.56 261.51 mg (65.28%) 9.58 / 11.50 276.33 mg (68.98%) 15 hours 4.967 / 5.96 317.11 mg (79.16%) 24 hours 6.48 / 7.78 326 mg (81.38%)

8 shows a photomicrograph of a form of chitosan-hyaluronic acid microbeads (acetone treatment time: 2 hours) in a state of being dispersed and swelled in physiological saline. As shown in Figure 8, it can be seen that the overall has a spherical shape.

Experimental Example 12 Viscoelasticity Measurement of the Microbeads Prepared in Example 19

The microbeads prepared in Example 19 were prepared at a concentration of 20 mg / ml, and then 1 ml of the microbeads was measured, and the composite viscosity was measured at a frequency of 0.02 to 1 Hz using a rheometer to check rheological properties. It was. Compound viscosity measured at a frequency of 0.02 Hz is shown in Table 17 below. Compared to 1,258,100 cP (0.02 Hz), which is a complex viscosity of hyaluronic acid (molecular weight: 2,465,000), it can be seen that the complex viscosity of chitosan-hyaluronic acid microbeads is improved.

Acetone Treatment Time EDC / NHS (mg) Compound viscosity (cP) 2 hours 5.34 / 6.41 9,940,000 4 hours 6.78 / 8.14 9,160,000 7.32 / 8.784 9,350,000 8.3 / 9.96 9,030,000 8.8 / 10.56 9,990,000 9.58 / 11.50 7,260,000 15 hours 4.967 / 5.96 8,400,000 24 hours 6.48 / 7.78 8,460,000

The microbeads prepared in Example 19 were prepared at a concentration of 20 mg / ml, and then 1 ml of the microbeads was measured, and the composite viscosity measured at a frequency of 0.02 to 1 Hz was measured using a rheometer to check rheological properties. 9 is shown.

Example 20 Preparation of Chitosan-Haluronic Acid Microbeads-10 (Using Methanol + Tween 80)

40 ml of an aqueous 10 mg / ml sodium hyaluronate (molecular weight: 2,465,000) solution and 5 ml of an aqueous 0.12 mg / ml chitosan (molecular weight: 110,000) solution were prepared, respectively. The two aqueous solutions were simultaneously dropped in drops of 300 ml of methanol each containing 12 ml of emulsifier Tween 80. After the reaction was stirred for 1 hour, 300 ml of 2-propanol was slowly added to harden the beads dispersed in the mixed solution. After 2 hours the microbeads were recovered by centrifugation. The recovered microbeads were washed several times with diethyl ether and acetone. After washing, beads (HA derivative: 6.67 mg / ml) dispersed in 15 ml of 90% acetone were added with 6.04 mg and 8.51 mg EDC and 7.25 mg and 10.21 mg NHS for 100 mg of the hyaluronic acid derivative. The reaction was carried out for 2 hours. After washing three times with acetone, it was dried with nitrogen gas (weight of microbead: 342.51 mg, 342.91 mg; yield: 85.5%, 85.6%). Hereinafter, after drying, it was prepared in physiological saline 2% and the rheological properties were measured using a rheometer, and diluted to confirm the properties of the swelling state.

Example 21 Preparation of Chitosan-Haluronic Acid Microbeads-11 (Using Methanol + Tween 80)

40 ml of an aqueous 10 mg / ml sodium hyaluronate (molecular weight: 2,465,000) solution and 5 ml of an aqueous 0.12 mg / ml chitosan (molecular weight: 110,000) solution were prepared, respectively. The two aqueous solutions were simultaneously dropped in drops of 300 ml of methanol each containing 12 ml of emulsifier Tween 80. After the reaction was stirred for 1 hour, 300 ml of acetone was slowly added to harden the microbeads dispersed in the mixed solution. After 4 hours the microbeads were recovered by centrifugation. The recovered microbeads were washed several times with diethyl ether and acetone. After washing, it was dispersed in 90% acetone to form solid beads. It was washed with acetone several times again and dried with nitrogen gas (weight of microbeads: 267.68 mg, 282.98 mg; yield: 66.82%, 70.64%). 50 mg of each dried chitosan-hyaluronic acid microbeads were dispersed in 7.5 ml of 90% acetone (HA derivative: 6.67 mg / ml), 1.75 mg, 2.25 mg, 2.75 mg, 3.25 mg, 3.75 mg, 4.25 mg, The reaction was carried out for 2 hours by adding 4.75 mg and 5.25 mg EDC and 2.1 mg, 2.7 mg, 3.3 mg, 3.9 mg, 4.5 mg, 5.1 mg, 5.7 mg and 6.3 mg NHS. After washing three times with acetone and dried with nitrogen gas (microbead weight: 44.75 mg, 43.67 mg, 47.09 mg, 47.5 mg, 45.93 mg, 46.16 mg, 40.08 mg, 48.99 mg; yield: 89.5%, 87.33% , 94.17%, 95%, 91.85%, 92.31%, 80.15%, 97.97%). Hereinafter, after drying, it was prepared in physiological saline 2% and the rheological properties were measured using a rheometer, and diluted to confirm the properties of the swelling state.

Experimental Example 13 Viscoelasticity Measurement of the Microbeads Prepared in Example 21

The microbeads prepared in Example 21 were prepared at a concentration of 20 mg / ml, and then 1 ml of the microbeads was measured, and the complex viscosity was measured at a frequency of 0.02 to 1 Hz using a rheometer to check rheological properties. It was. Compound viscosity measured at a frequency of 0.02 Hz is shown in Table 18 below. Compared with 1,258,100 cP (0.02 Hz), a complex viscosity of hyaluronic acid (molecular weight: 2,465,000), the complex viscosity of chitosan-hyaluronic acid microbeads improved, but viscoelasticity tended to decrease when the crosslinking ratio was too high, and physiological saline above It is dispersed in the form without swelling.

(Per 100 mg of HA) EDC / NHS (mg) Compound viscosity (cP) 3.5 / 4.2 3,100,000 4.5 / 5.4 4,590,000 5.5 / 6.6 6,140,000 6.5 / 7.8 7,330,000 7.5 / 9 1,710,000 8.5 / 10.2 1,340,000 9.5 / 11.4 356,000 10.5 / 12.6 Not measurable

As described above, since the microbead according to the present invention is biocompatible and has high swelling property, the microbead may be used in various applications as a biocompatible material such as a wrinkle treatment agent, a molding aid, a drug carrier, and the like. In particular, chitosan-hyaluronic acid microbeads, in which the amine groups of chitosan form amide bonds with the carboxyl groups of hyaluronic acid and / or alginic acid, and alginic acid-hyaluronic acid microbeads coated with chitosan, have very high swelling properties and viscoelastic properties. Since it is excellent, it has a feature that can be usefully used under various environments in the human body.

Although the present invention has been described in detail with reference to specific examples, it will be apparent to those skilled in the art that various changes and modifications can be made within the scope and spirit of the present invention, and such modifications and variations belong to the appended claims. will be.

Claims (36)

A high swellable biocompatible microbead consisting of natural polysaccharides selected from alginic acid and chitosan and hyaluronic acid or salts thereof. The microbead according to claim 1, wherein the microbead is an alginic acid-hyaluronic acid microbead (a), a chitosan-hyaluronic acid microbead (b), or an alginic acid-hyaluronic acid microbead coated with chitosan (c). Bead. 3. The microbead according to claim 2, wherein the chitosan is connected with hyaluronic acid and / or alginic acid by an amide bond. 2. The microbead according to claim 1, wherein the microbead is chitosan-hyaluronic acid microbead (b) in which chitosan is connected by hyaluronic acid and an amide bond, or chitosan in which chitosan is connected by hyaluronic acid and / or alginic acid and an amide bond. Microbeads, characterized in that the alginic acid-hyaluronic acid microbead (c) coated with. The method according to claim 1, wherein the hyaluronic acid is hyaluronic acid itself, a salt thereof, or a combination thereof, the alginic acid is alginic acid itself, a salt thereof, or a combination thereof, and the chitosan is chitosan itself, an oligomer thereof, Or a combination thereof. The hyaluronic acid salt according to claim 5, wherein the hyaluronic acid salt is an inorganic salt such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, or tetrabutylammonium hyaluronic acid, or the like. Combination of two or more thereof, wherein the alginic acid salt is one or more selected from the group consisting of sodium alginate, potassium alginate, calcium alginate and magnesium alginate. 6. The microbead of claim 5, wherein the hyaluronic acid and its salt have a molecular weight of 100,000 to 3,000,000. The microbead according to claim 2, wherein the chitosan has a molecular weight of 300 to 10,000,000. The microbead according to claim 1, wherein the average particle diameter of the beads is 30 to 1000 mu m. A method for producing the alginic acid-hyaluronic acid microbead according to claim 1, prepared by spraying a mixed solution of an aqueous alginic acid solution and an aqueous hyaluronic acid solution onto an aqueous solution of a divalent or trivalent cation salt and sodium chloride. The microbead according to claim 10, wherein the ratio (weight ratio) of alginic acid to hyaluronic acid in the mixed solution is from 1: 10 to 10: 1, and the concentration of alginic acid and hyaluronic acid is from 0.1 to 10% by weight. The method according to claim 10, wherein the cationic salt in the mixed salt aqueous solution is at least one selected from the group consisting of calcium chloride, magnesium chloride, strontium chloride, barium chloride and aluminum chloride. The production method according to claim 10, wherein the concentration of the cationic salt is 0.01-15 M, and the concentration of sodium chloride is 0.01-45 M. (A) preparing a hyaluronic acid aqueous solution and a chitosan aqueous solution each containing a carboxyl group activator; (B) dropping the two kinds of aqueous solutions into a solution containing an emulsifier to form microbeads; And (C) separating and purifying the microbeads; A method for producing chitosan-hyaluronic acid microbeads according to claim 1, which comprises a. Acetone, an acetone aqueous solution (90% or more of acetone), and / or C ₂ to C are added to the solution containing microbeads in step (B) between the steps (B) and (C). _The method of claim ₆ , further comprising the step of making the microbeads somewhat firm by adding alcohol. 15. The method according to claim 14, further comprising, after the step (C), dispersing the microbead obtained in the step (C) in an acetone or an acetone aqueous solution, and then adding a carboxyl activator to induce an additional amidation reaction. Manufacturing method characterized in that. (A1) preparing an aqueous hyaluronic acid solution and an aqueous chitosan solution, respectively; (B1) adding the two kinds of aqueous solutions dropwise to a solution containing an emulsifier to form microbeads; (C1) separating and purifying the microbeads; And (D1) dispersing the microbeads in acetone or an acetone aqueous solution, and then adding an carboxyl activator to induce an amidation reaction; A method for producing chitosan-hyaluronic acid microbeads according to claim 1, which is composed of a compound. 18. The method of claim 17, wherein between step (B1) and step (C1), acetone, acetone aqueous solution, and / or C ₂ to C ₆ alcohol are added to the solution containing the microbeads in step (B1). And further comprising making the beads somewhat firm. 18. The method according to claim 14 or 17, wherein the concentration of the aqueous hyaluronic acid solution and the concentration of the chitosan aqueous solution is 0.00001 wt% to 10 wt%, respectively, and the mixing ratio of the hyaluronic acid aqueous solution and the chitosan aqueous solution for the amidation reaction is 1: 0.00001 to 18. 1: 100 (carboxyl group of hyaluronic acid: amine group of chitosan). The method of claim 14 or 17, wherein the emulsifier is a lipid such as lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, derivatives thereof, or glyceryl stearate, sorbitan palmitate, sorbitan stearate and the like. Method for producing an ester derivative of a fatty acid. 21. The method of claim 20, wherein the emulsifier is a sorbitan fatty acid ester derivative, polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40), polyoxyethylene sorbitan monostea To latex (Tween 60), polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monolaurate (Span 20), sorbitan monostearate (Span 60), and sorbitan monooleate (Span 80) One or two or more manufacturing method selected from the group consisting of. The method of claim 21, wherein the emulsifier is lecithin, Tween 20, Tween 80 or Span 80. 18. The method according to claim 14 or 17, wherein the amount of the emulsifier added is 0.05% by volume to 10% by volume with respect to the solution. The solution according to claim 14 or 17, wherein the solution containing an emulsifier is a mineral oil, methanol, a mixture thereof, a mixture of glycerol and 1-propanol, a mixture of glycerol and 2-propanol, a mixture of ethylene glycol and 1-propanol, A mixed liquid of ethylene glycol and 2-propanol, a mixed liquid of ethylene glycol and ethanol, a mixed liquid of ethylene glycol and methanol, or a mixed liquid of ethylene glycol and acetone. The method for producing microbeads according to claim 14, 16 or 17, wherein an activation reaction aid is used together with the carboxyl group activator. The method of claim 25, wherein the carboxyl activator is 1-alkyl-3- (3-dimethylaminopropyl) carbodiimide, wherein alkyl is alkyl having 1 to 10 carbon atoms, 1-ethyl-3- (3- (Trimethylammonio) propyl) carbodiimide (ETC), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide (CMC), and the like. -Hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysulfosuccinimide (Sulfo-NHS) and the like. The method according to claim 26, wherein the carboxyl activator is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and the activating aid is NHS. (A) preparing an aqueous solution of alginic acid and an aqueous solution of hyaluronic acid, respectively; (B) spraying a mixed solution of an aqueous alginic acid solution and an aqueous hyaluronic acid solution to a divalent or trivalent cation salt and an aqueous sodium chloride solution to generate microbeads; (C) coating the resulting microbeads in chitosan solution; And (D) separating and purifying the microbeads from the reaction solution; A method for producing an alginic acid-hyaluronic acid microbead coated with chitosan according to claim 1, comprising: 29. The process of claim 28, further comprising, after step (D), carrying out the amidation reaction of the purified microbeads. (A1) preparing an aqueous alginic acid solution and an aqueous hyaluronic acid solution, respectively; (B1) coating chitosan while spraying a mixed solution of an aqueous alginic acid solution and an aqueous hyaluronic acid solution onto an aqueous chitosan solution containing a divalent or trivalent cation salt and sodium chloride; And (C1) separating and purifying the microbeads from the reaction solution; A method for producing an alginic acid-hyaluronic acid microbead coated with chitosan according to claim 1, comprising: 31. The process according to claim 30, further comprising, after step (C1), carrying out the amidation reaction of the purified microbeads. The production method according to claim 28 or 30, wherein the reaction amount of chitosan is such that the ratio of the carboxyl group of alginic acid and / or hyaluronic acid and the amine group of chitosan is from 1: 100 to 100: 1. The amidation reaction according to claim 29 or 31, wherein the microbeads obtained in the previous step are dispersed in distilled water and reacted by adding a carboxyl group activator or a carboxyl group activator and an activation aid. Manufacturing method. 34. The method of claim 33, wherein the carboxyl activator is 1-alkyl-3- (3-dimethylaminopropyl) carbodiimide, wherein alkyl is alkyl having 1 to 10 carbon atoms, 1-ethyl-3- (3- (Trimethylammonio) propyl) carbodiimide (ETC), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide (CMC), and the like. -Hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysulfosuccinimide (Sulfo-NHS) and the like. 35. The method of claim 34, wherein the carboxyl activator is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and the activating aid is NHS. Use of the microbead according to claim 1 as a biocompatible material.