KR20200046811A - Composition for coating cells comprising tyrosinase and method for coating cells - Google Patents

Abstract

The present invention relates to a composition for coating cells, which comprises phenolic amine and tyrosinase, to a cell coating method using the same, and to living cells comprising a polymer coating layer. Cell surfaces can be quickly coated with phenolic polymers in mild conditions without substantially impairing the viability of chemically unstable cells.

Description

Composition for cell coating containing tyrosinase and cell coating method using same {Composition for coating cells comprising tyrosinase and method for coating cells}

The present invention relates to a composition for cell coating comprising a phenolic amine and tyrosinase, a method for coating a cell using the same, and living cells comprising a polymer coating layer.

Various cells in nature have survived in the harsh environment around cells by strengthening the cell walls to preserve the genetic information of the cells. The cell walls of these single cells have also contributed to the protection of each species, but have become more important as applications such as cell-based reactors, cell delivery systems, diagnostic devices and biological sensors have been developed.

The application conditions of these application fields are often harsh environments, and in order for devices including cells to have reliability in the application fields, additional protection to protect the cells so that the cells can preserve viability in the harsh environment is required. Means are needed.

To protect cells in harsh environments, artificial spores can be introduced through mimicking of spore structures found in nature. For the implementation of such a spore structure, an alternating lamination method of a polymer electrolyte having different charges through a LbL method (Layer-by-Layer) or a coating method of an inorganic material may be proposed. Through the artificial spore structure, cell resistance to external stressors can be enhanced, and long-term stability and performance of a cell-based device including living cells can be improved. However, the artificial spore structure by the LbL method or the coating of the inorganic material may improve the stability of the cell, but as a dense layer is formed on the cell, it acts as a barrier for the diffusion of the material, so that the performance of the cell may decrease in various applications. There is a problem.

Meanwhile, a method of coating a polymer through hydrophilic monomer polymerization on the surface of a cell may be proposed. However, the polymerization of the monomer may cause damage to the cell by applying chemical stress as the cell is exposed to the active radical, and the viability of the living cell may be reduced by the added monomer compound and side reaction products, so it is directly applied to the chemical polymerization method There are limitations that are difficult to do. In addition, due to the nature of the chemical reaction, there is a problem in that it does not have a selectivity on a specific surface, so it is difficult to conformally coat the entire cell surface and to freely control the thickness of the coating layer.

Korean Registered Patent Publication No. 11766302 discloses a technique for coating cells through tannic acid and iron salts in order to avoid chemical stress on the cells. However, because the iron salt induces the oxidation reaction of tannic acid and chemical stress is applied to the cells, in the case of mammalian cells sensitive to reaction conditions (eg, Jurkat cells), there is a problem of poor viability.

Therefore, it is necessary to develop a new cell coating method that can conformally coat the entire cell surface without reducing the viability of cells under mild reaction conditions.

Korean Registered Patent Publication No.1766302

An object of the present invention is to provide a composition capable of coating a cell by polymerizing a hydrophilic monomer on a cell surface under mild reaction conditions.

Another object of the present invention is to provide a cell coating method capable of forming a uniform conformal polymer coating layer on a chemically unstable cell surface.

Another object of the present invention is to provide a living cell comprising a water-insoluble hydrophilic phenolic polymer coating layer in which the viability of the cell is substantially preserved.

Another object of the present invention is to provide a core-shell particle comprising a lipid structure core and a water-insoluble hydrophilic phenolic polymer coating layer.

In order to solve the problems as described above, the composition for cell coating according to the present invention includes a phenolic amine and a tyrosinase.

The phenolic amine of the cell coating composition according to an aspect of the present invention includes a compound represented by the following formula (1).

[Formula 1]

In Chemical Formula 1,

R ¹ is any one selected from hydrogen, hydroxy, carboxylic acid and salts thereof;

R ² are each independently (C1-C10) alkyl, (C1-C10) alkenyl, (C3-C20) cycloalkyl, (C3-C20) heterocycloalkyl, (C6-C20) aryl, (C3-C20) Heteroaryl, nitro, cyano, -C (= O) R ¹¹ and -C (= O) OR ¹² , or any combination of two or more selected from the group consisting of;

R ¹¹ and R ¹² are each independently hydrogen, (C1-C10) alkyl, (C3-C20) cycloalkyl, (C3-C20) heterocycloalkyl, (C6-C20) aryl and (C3-C20) heteroaryl Any one or a combination of two or more selected from the group consisting of;

L is a divalent linking group;

m is an integer from 1 to 3;

When m is 2 or more, adjacent substituents may be connected to each other to form a ring;

The alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl of R ² , R ¹¹ and R ¹² are each independently hydroxy, carboxylic acid, (C1-C10) alkoxy, (C1-C10) alkyl It may be substituted with any one or more substituents selected from the group consisting of carbonyl, halogen, amine, cyano, nitro and salts thereof.

R ¹ of the phenolic amine of the cell coating composition according to an aspect of the present invention is hydrogen or hydroxy;

The L is (C1-C10) alkylene or (C1-C10) alkenylene, -CH ₂ -of the alkylene or alkenylene is -N (R ¹³ )-, -C (= O) NH-,- C (= O) O- and -O- may be substituted by any one selected from the group consisting of, wherein R ¹³ is selected from the group consisting of hydrogen, (C1-C10) alkyl and amino (C1-C10) alkyl ,

The alkylene and alkenylene of L are further one or more substituents selected from the group consisting of halogen, hydroxy, amine, carboxylic acid, (C1-C10) alkoxy, (C1-C10) alkylcarbonyl and salts thereof. Can be substituted.

The phenol-based amine of the composition for cell coating according to an aspect of the present invention may more specifically include a compound represented by a compound represented by the following Chemical Formula 2 or Chemical Formula 3.

[Formula 2]

[Formula 3]

In the above formulas 2 and 3, R ³ and R ⁴ are each independently hydrogen, hydroxy or carboxylic acid.

The polymer formed on the cell according to an aspect of the present invention may be a water-insoluble hydrophilic phenol-based polymer.

In addition, the present invention provides a method for coating a cell, comprising the steps of mixing the cell coating composition and cells as described above; And forming a coating layer on the cells.

The coating layer of the cell coating method according to an aspect of the present invention has a thickness of 1 nm to 100 nm, and the surface roughness (R _RMS ) may be 10 nm or less.

The cells of the method for coating cells according to an aspect of the present invention may include any one or more selected from T cells, B cells, and natural killer (NK) cells.

In addition, the present invention is a cell; And a water-insoluble hydrophilic phenolic polymer coating layer on the cell.

The water-insoluble hydrophilic phenolic polymer of living cells according to an aspect of the present invention may be derived from an enzymatic oxidation reaction of any one or more compounds selected from the group consisting of phenolic amino acids, phenolic dipeptides and phenolic tripeptides. have.

In addition, the present invention is selected from a cell-based sensor (bio-catalyst) and cell-on-a-chip (cell-on-a-chip) comprising a living cell on which a water-insoluble hydrophilic phenolic polymer coating layer on a cell is formed. It provides a device including any one.

In addition, the present invention provides a method for preparing core-shell particles, the method comprising preparing a lipid structure dispersion; Mixing the coating composition comprising a phenolic amine and a tyrosinase in the dispersion; And forming a coating layer on the lipid structure.

The lipid structure of the method for preparing core-shell particles according to an aspect of the present invention may be a liposome or a nanoemulsion.

The present invention also provides core-shell particles, wherein the core-shell particles include a lipid structure core and a water-insoluble hydrophilic phenolic polymer coating layer on the lipid structure.

The core-shell particle according to an aspect of the present invention includes a hollow in the lipid structure core, and the lipid structure and the water-insoluble hydrophilic phenol-based polymer relate to hollow core-shell particles that are physically bound.

The hollow of the core-shell particles according to an aspect of the present invention may include any one or more selected from the group consisting of water, oil and bioactive materials.

The composition for cell coating according to the present invention does not exert any chemical stress on the cell and does not substantially reduce the viability of the cell in that the hydrophilic monomer is rapidly polymerized using enzymes under mild reaction conditions that do not contain active radicals. There are advantages.

The composition for cell coating according to the present invention does not independently generate a polymer in an aqueous solution and is polymerized only on the cell surface to form a nanometer-thick uniform coating layer, and an advantage of forming a conformal coating layer on the cell surface There is this.

The cell coating method according to the present invention has the advantage of being able to provide live cells in which water-insoluble hydrophilic phenol-based polymers are physically bound on the cell surface while substantially preserving the viability of the cells.

The core-shell particles according to the present invention have the advantage of being able to utilize a variety of carriers by forming a hollow in the core by including a lipid structure in the core and a water-insoluble hydrophilic phenolic polymer in the shell.

FIG. 1 illustrates the production of melanin-based polymers according to the enzymatic oxidation reaction of phenol-based amines using tyrosinase, FIG. 1 (a) is a melanin-based polymer production mechanism, and FIG. 1 (b) is a tyrosinase The color change of the tyrosine solution after addition, FIG. 1 (c) shows the change in the UV-visible spectrum of the tyrosine solution after the addition of tyrosinase, FIG. 1 (d) shows the XPS spectrum of the melamine-based polymer, and FIG. e) shows the change in the thickness of the melamine-based polymer over time.
Figure 2 shows the FT-IR spectrum on a gold substrate after 6 hours of enzymatic oxidation of phenolic amine using tyrosinase according to an embodiment.
Figure 3 shows the surface structure of the film formed on the gold substrate according to the embodiment (a) to (c) is a photograph showing a scanning electron microscope (SEM) of the surface, (d) to (f) Is an AFM image, and the left, middle, and right images correspond to images after 1 hour, 3 hours, and 6 hours, respectively.
Figure 4 shows the results of the enzymatic oxidation reaction of various phenolic amines using tyrosinase, Figure 4 (a) is an optical color change according to the enzymatic oxidation reaction of each phenolic amine, Figure 4 (b) shows the FT-IR spectrum on a gold substrate, and FIG. 4 (c) shows the polymer film thickness.
Figure 5 shows the result of forming a coating layer of Jurkat cells, Figure 5 (a) is a TEM image after 6 hours following the enzymatic oxidation reaction, Figure 5 (b) is an enzymatic oxidation reaction using poly tyrosine and poly Comparison of cell viability during dopamine coating, FIG. 5 (c) shows an enzymatic oxidation reaction using tyrosine and a CLSM image when coated with polydopamine.
Figure 6 shows the optical SEM image of Jurkat cells before and after the formation of the coating layer.
Figure 7 shows the yeast cells after formation of the coating layer, Figure 7 (a) is the viability of the yeast cells according to the enzymatic oxidation time, Figure 7 (b) is the yeast cells after 6 hours of the enzymatic oxidation reaction The CLSM image is shown.

Hereinafter, a composition for cell coating according to the present invention, a cell coating method, living cells including a coating layer, a method for manufacturing core-shell particles, and hollow core-shell particles according to the present invention will be described in detail with reference to the accompanying drawings. The drawings introduced below are provided as examples in order to sufficiently convey the spirit of the present invention to those skilled in the art. Therefore, the present invention is not limited to the drawings presented below, and may be embodied in other forms, and the drawings presented below may be exaggerated to clarify the spirit of the present invention.

Unless otherwise defined in the technical terms and scientific terms used in the specification of the present invention, those skilled in the art to which the present invention pertains have the meanings commonly understood, and the present invention in the following description and accompanying drawings Descriptions of well-known functions and configurations that may unnecessarily obscure the subject matter are omitted.

Also, the singular form used in the specification of the present invention may be intended to include the plural form unless otherwise specified in the context.

In addition, in the specification of the present invention, the units used without specific reference are based on weight, and for example, units of% or ratio mean weight percent or weight ratio.

In addition, in the specification of the present invention, hydrophilic and hydrophobic means water-like properties and water-dislike properties, but when hydrophilicity and hydrophobicity are used simultaneously, it means a relative concept. In a specific example, in the case of a hydroxyl group (-OH) and an alkyl group, the hydroxyl group is a hydrophilic group and the alkyl group means a hydrophobic group.

In addition, in the specification of the present invention, water-insoluble means a property in which solubility in water is very low, and a polymer is water-insoluble means that the coating layer or film structure of the polymer does not dissolve or dissolve in water and has structural stability as a film. it means.

Hereinafter, a composition for cell coating according to the present invention will be described in detail.

The composition for cell coating according to the present invention includes a phenolic amine and a tyrosinase. The composition for cell coating may be an aqueous phase in which phenolic amine and tyrosinase are dissolved in water, and the aqueous phase composition is referred to as an aqueous composition. The cell coating composition may be a one-liquid composition dissolved in one solution phase, but may also be a two-liquid composition in which phenolic amine and tyrosinase are dissolved in separate solutions. The two-component composition may be mixed to coat the cells and then mixed with the cells to coat the cells, but the cells are mixed with at least one solution of the two-component composition, and then mixed with the other solution to coat the cells. Can also be used.

The phenolic amine is not particularly limited as long as it does not impair the object of the present invention, but may have a molecular weight of 100 or more and 5000 or less, specifically 2000 or less, and more specifically 1000 or less. The number of hydroxy of the phenolic amine may be 1 or more, preferably 1 to 10, more preferably 1 to 4, and most preferably 1 to 2.

The amine of the phenolic amine may be any one selected from the group consisting of primary amines, secondary and tertiary amines, but may preferably be primary amines, and the number of amines is 1 or more, preferably 1 to 10. Dogs, more preferably 1 to 4, and most preferably 1 to 2.

The tyrosinase is an enzyme that induces a hydroxylation reaction at the ortho position of a monophenol and can induce a continuous oxidation reaction by mediating a reduction reaction of oxygen molecules present in the aqueous phase. Tyrosinase is present in various plant and animal tissues such as fruits, vegetables and mushrooms, and the tyrosinase can be used without limitation in the present invention. As a non-limiting example, when tyrosine is used as a substrate, oxidative hydroxylation of the phenolic residue of tyrosine is promoted to be converted into catechol, and film deposition is performed through a subsequent series of catalytic oxidation reactions. To play a decisive role.

In more detail, tyrosine is a non-catecholamine and is converted to 3,4-dihydroxyphenylalanine (DOPA) by tyrosinase, and the DOPA is subsequently oxidized to DOPAquinone and DOPAchrome. It is converted to eumelanin, a kind of phenolic polymer, by oxidation polymerization. Tyrosinase can use a phenol-based amine as a substrate, and has the advantage of being able to form a coating layer on cells quickly and effectively in a neutral pH region.

The oxidation reaction of tyrosine by tyrosinase and the production of eumelamine therefrom can be explained by the following scheme 1.

[Scheme 1]

As can be seen from the structural unit of the melamine above, the main chain of the polymer does not dissolve in water because it contains an aromatic ring, but can exhibit high hydrophilicity because it contains a hydroxy group in the structural unit. The hydrophilicity may exhibit a negative potential with a large absolute value when measuring the surface potential, and may exhibit an effect of stably dispersing in water and having high viability even when cells are coated with a polymer.

The phenolic amine and tyrosinase in the cell coating composition may be mixed at a rate of 0.01 mg / U to 10,000 mg / U, preferably 0.1 mg / U to 1,000 mg / U, more preferably 1 mg / U to 100 mg / U.

In the composition for cell coating according to an example of the present invention, the phenolic amine may include a compound represented by the following Chemical Formula 1.

[Formula 1]

In Chemical Formula 1,

R ¹ is any one selected from hydrogen, hydroxy, carboxylic acid and salts thereof;

R ² are each independently (C1-C10) alkyl, (C1-C10) alkenyl, (C3-C20) cycloalkyl, (C3-C20) heterocycloalkyl, (C6-C20) aryl, (C3-C20) Heteroaryl, nitro, cyano, -C (= O) R ¹¹ and -C (= O) OR ¹² , or any combination of two or more selected from the group consisting of;

R ¹¹ and R ¹² are each independently hydrogen, (C1-C10) alkyl, (C3-C20) cycloalkyl, (C3-C20) heterocycloalkyl, (C6-C20) aryl and (C3-C20) heteroaryl Any one or a combination of two or more selected from the group consisting of;

L is a divalent linking group;

m is an integer from 1 to 3;

When m is 2 or more, adjacent substituents may be connected to each other to form a ring;

The alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl of R ² , R ¹¹ and R ¹² are each independently hydroxy, carboxylic acid, (C1-C10) alkoxy, (C1-C10) alkyl It may be substituted with any one or more substituents selected from the group consisting of carbonyl, halogen, amine, cyano, nitro and salts thereof.

In Chemical Formula 1, L may be any divalent linking group, and may be attached to any position of the phenol ring, preferably to a para position. The alkyl, alkenyl and alkoxy include both straight and branched chains.

On the other hand, when two or more substituents are combined, it means that two or more substituents are covalently bonded to each other.For example, when (C1-C10) alkyl and (C6-C20) aryl are combined, *-(C1-C10) alkyl It means having a structure of-(C6-C20) aryl or *-(C6-C20) aryl- (C1-C10) alkyl.

The phenolic amine is a hydrophilic monomer containing at least one hydroxyl group in a molecule, and can be uniformly dissolved in an aqueous solution as a medium, and does not apply any chemical stress to the cell at the point of being rapidly polymerized by an enzyme, and the viability of the cell is substantially Does not decrease.

In the cell coating composition according to an embodiment of the present invention, the R ¹ of the compound represented by Formula ¹ is hydrogen or hydroxy; The L is (C1-C10) alkylene or (C1-C10) alkenylene, -CH ₂ -of the alkylene or alkenylene is -N (R ¹³ )-, -C (= O) NH-,- C (= O) O- and -O- may be substituted with any one selected from the group consisting of, wherein R ¹³ is selected from the group consisting of hydrogen, (C1-C10) alkyl and amino (C1-C10) alkyl Alkylene and alkenylene of L may be selected from the group consisting of halogen, hydroxy, amine, carboxylic acid, (C1-C10) alkoxy, (C1-C10) alkylcarbonyl and salts thereof It may be further substituted with any one or more substituents.

When L is substituted with any one or more substituents selected from the group consisting of halogen, hydroxy, amine, carboxylic acid, (C1-C10) alkoxy, (C1-C10) alkylcarbonyl and salts thereof, alkylene or alke It means that the hydrogen atom of nylene is substituted with the above substituent.

Specifically, L is (C1-C6) alkylene or (C1-C6) alkenylene, -CH ₂ -of the alkylene or alkenylene is -N (R ¹³ )-, -C (= O) NH -, -C (= O) O- and -O- may be substituted by any one selected from the group consisting of, wherein R ¹³ is selected from the group consisting of (C1-C6) alkyl and amino (C1-C6) alkyl It can be any one. In this case, the alkylene and alkenylene of L may be further substituted with one or more substituents selected from halogen, hydroxy, amine, carboxylic acid, (C1-C6) alkoxy, (C1-C6) alkylcarbonyl and salts thereof. have.

Each of R ² is independently (C1-C6) alkyl, (C1-C6) alkenyl, (C3-C10) cycloalkyl, (C3-C10) heterocycloalkyl, (C6-C10) aryl, (C3-C10) ) Heteroaryl, nitro, cyano, -C (= O) R ¹¹ and -C (= O) OR ¹² , any one or a combination of two or more selected from the group consisting of; The alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl of R ² , R ¹¹ and R ¹² are each independently hydroxy, carboxylic acid, (C1-C10) alkoxy, (C1-C10) alkyl It may be substituted with any one or more substituents selected from the group consisting of carbonyl, halogen, amine, cyano, nitro and salts thereof.

More specifically, L is (C1-C3) alkylene, and -CH ₂ -of the alkylene is -N (R ¹³ )-, -C (= O) NH-, -C (= O) O- And it may be replaced with any one selected from the group consisting of -O-, R ¹³ may be any one selected from the group consisting of (C1-C3) alkyl and amino (C1-C3) alkyl. At this time, the alkylene and alkenylene of L may be further substituted with one or more substituents selected from hydroxy, amine, carboxylic acid and salts thereof.

Each of R ² is independently (C1-C6) alkyl, (C1-C6) alkenyl, (C3-C10) cycloalkyl, (C3-C10) heterocycloalkyl, (C6-C10) aryl, (C3-C10) ) Heteroaryl is selected from the group consisting of any one or two or more combinations; The alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl of R ² are each independently hydroxy, carboxylic acid, (C1-C10) alkoxy, (C1-C10) alkylcarbonyl, halogen, amine And it may be substituted with any one or more substituents selected from the group consisting of salts.

In the composition for cell coating according to an example of the present invention, the phenol-based amine may be a compound represented by a compound represented by the following Chemical Formula 2 or Chemical Formula 3.

[Formula 2]

[Formula 3]

In the above formulas 2 and 3, R3 and R4 are each independently hydrogen, hydroxy or carboxylic acid.

Specifically, the phenolic amine may be one or more compounds selected from the following formula.

As illustrated in the above compound, the phenolic amine may include a structure in which a phenyl group and a primary amine are connected to each other through a divalent linking group interposed between ethylene groups, and the hydrogen atom of the ethylene group is in the group consisting of hydroxy and carboxy groups. It may be further substituted with any one selected. The phenolic amine may function as a substrate of tyrosinase by simultaneously containing a phenol group and an amine group, and may be oxidized by tyrosinase, and a phenolic polymer may be produced by subsequent oxidation and oxidation polymerization.

According to a specific example according to the present invention, tyrosinase is tyramine, dopamine, norepinephrine, 3,4-dihydroxyphenyalanine (DOPA), etc. A variety of phenolic amines can be used as substrates to induce oxidation reactions, and oxidation polymerization can be induced to produce phenolic polymers and to form films on cell surfaces. On the other hand, pyrocathecol, pyrogallol, 3,4-dihydroxybenzaldehyde, tannic acid, gallic acid, caffeic acid, etc. The phenolic compound of the oxidized polymerization did not form a film.

From the above results, the presence of the amine group (amine group, -NH ₂ ) of the phenolic amine plays an important role in the recognition of the substrate by tyrosinase, and in order to form a coating layer on the cell surface, the compound that is the substrate is It means that a phenol group and an amine group should be included in the phenol ring.

In the composition for cell coating according to an example of the present invention, the polymer formed on the cell may be a water-insoluble hydrophilic phenol-based polymer. As described above, as the phenolic amine is polymerized, the aromatic ring of the main chain structural unit has a low water solubility, so it is not soluble in water and can be stably coated on the cell surface due to its insoluble properties. If the main chain structural unit has a high hydrophobicity, the affinity may be inferior to the hydrophilic surface of the cell, and when the affinity is low, the reaction proceeds in the form of precipitation polymerization rather than being coated on the cell surface, so that polymer particles can be formed independently. However, the phenolic amine can exhibit high hydrophilicity when the structural unit contains a hydroxyl group during oxidation polymerization by tyrosinase, and can have high affinity by the interaction between the hydrophilic group of the phenolic polymer and the hydrophilic group on the cell surface. . The interaction between the hydrophilic groups can enable conformal coating by stably physically adsorbing the phenolic polymer produced by oxidation polymerization on the cell surface and uniformly growing on the cell surface.

As a specific example, the water-insoluble hydrophilic phenol-based polymer may be a heteroaromatic polymer containing a nitrogen atom, and more specifically, a melamine-based polymer. More specifically, it may be a colored melamine-based polymer having a color. The water-insoluble hydrophilic phenol-based polymer may have a weight average molecular weight of 1,000 to 5,000,000, specifically 5,000 to 1,000,000, and more specifically 10,000 to 500,000, but is not limited thereto.

In a specific example according to the present invention, the coated cell may be any one or more selected from the group consisting of mammalian immune cells, stem cells, eukaryotic cells, single cell green algae and lactic acid bacteria, or as floating cells and adhesive cells. It may be any one selected from the group consisting of.

Specific examples include T cells, B cells, natural killer (NK) cells, mesenchymal stem cells, hematopoietic stem cells, red blood cells, HeLa cells, and fibroblast lactobacillus caseis ( L.casei), L. acidophilus, L. bacillus, L. bulgaricus, B. longum, B. bifidum, B. bifidum, Actiregularis (Actiregularis), brewer's yeast (Saccharomyces cerevisiae), chlorella may be any one or more selected from, but this is only an example and is not limited thereto.

Hereinafter, a method for coating a cell according to the present invention will be described in detail.

The method of coating a cell according to the present invention comprises the steps of mixing a composition comprising phenolic amine and tyrosinase with cells; And forming a coating layer on the cells. The composition may be mixed with cells, and the aqueous phase of the phenolic amine may be 0.001 to 100 mg / ml, preferably 0.01 to 10 mg / ml, and more preferably 0.1 to 1 mg / ml. The concentration of tyrosinase may be 0.01 kU / ml to 5000 kU / ml, preferably 0.1 kU / ml to 500 kU / ml, more preferably 1 kU / ml to 50 kU / ml. In the above concentration range, it is possible to rapidly coat the cell surface while minimizing the chemical stress on the cell, and thus, the viability of the chemically unstable cell can be substantially preserved even after coating.

In particular, conditions such as temperature and pH of the cell coating method are not particularly limited in a range that does not impair the achievement of the object of the present invention. For example, the pH is 4 to 12, preferably 5 to 10, more preferably May be 7 to 8, the temperature may be 10 ℃ to 50 ℃, preferably 20 ℃ to 40 ℃, preferably in the range of room temperature. The time at which the cells are coated in contact with the composition may be 1 minute to 10 hours, preferably 10 minutes to 6 hours, but is not limited thereto. Coating conditions as described above can be appropriately selected within a range that does not affect the physical properties of the substrate or the viability of the cells to form a coating layer.

The aqueous solution may be a conventional buffered aqueous solution and phosphate buffered saline (PBS) or Tris may be exemplified, but this is only an example and is not limited thereto.

As a specific example, in the case of chemically unstable cells, when treated with a composition for cell coating under mild conditions with a neutral range of pH 7 to 8, it may be particularly preferable in that the viability of the cells can be substantially preserved. Accordingly, an example of chemically unstable cells may include any one or more immune cells selected from the group consisting of T cells, B cells, and NK cells, and the cells are preferably coated with high viability at a neutral pH range. You can. As a more specific example, in the case of human T lymphocytes or Jurkat cells, a coating layer may be formed with high viability under the above conditions, and the human T lymphocytes or Jurkat cells play an important role in the immune system, and through the enzymatic cell coating of the present invention This is particularly preferable because instability to external stress can be significantly improved.

In the cell coating method according to an example of the present invention, the thickness of the coating layer formed on the cell may have a thickness of 1 nm to 100 nm, and the surface roughness (R _RMS ) of the coating layer may be 10 nm or less. Preferably, the thickness of the coating layer may have a thickness of 10 nm to 80 nm, more preferably the thickness of the coating layer may have a thickness of 20 nm to 50 nm, the surface roughness (R _RMS ) of the coating layer is 1 nm or more and It may be 5 nm or less.

The phenolic amine is enzymatically oxidized by tyrosinase, and the phenolic polymer, which grows rapidly by subsequent oxidative polymerization, forms a coating layer uniformly on the cell surface. The thickness can be increased. In addition, the phenol-based polymer grown by the enzyme on the cell surface is stably physically adsorbed on the surface, and the conformal coating may be possible by uniformly growing the coating layer by continuous oxidation polymerization by tyrosinase. In the conventional chemical oxidation polymerization, a coating layer having irregularity and poor uniformity was obtained due to the adsorption in the form of nucleation locally on a part of the cell surface, but the cell coating method according to the present invention is significantly different from the existing characteristics. It has remarkable features, and this rapid thickness rise and conformal coating features are due to enzymatic oxidation.

On the other hand, when a cell is coated with a phenol-based polymer by chemically oxidative polymerization of a phenol-based amine without tyrosinase, the thickness of the coating layer does not increase rapidly. For example, oxidative polymerization of dopamine in Tris buffer at pH 8.5 produces polydopamine, and when coated on cells, it takes about 6 hours to form a 10 nm coating layer thickness. That is, it means that it takes a long time to form a sufficient thickness of the coating layer to protect the cells from the external environment, and accordingly, the cells are exposed to external stress continuously and can greatly affect the viability of the cells. In particular, in the case of higher cells, such as immune cells that are chemically unstable, when exposed to such external stress, the viability converges substantially to 0, and thus there is a problem that such chemical oxidation polymerization cannot be applied.

On the other hand, in order to alleviate this external stress, the pH can be adjusted to a neutral region of 8 or less, and chemically oxidative polymerization of the phenolic amine in the neutral region can coat the cells, but the phenolic amine does not undergo oxidation polymerization in the neutral region. There is a problem that can not be coated. A method of increasing the salt concentration may be used to induce oxidation polymerization in the neutral region, but it is also not preferable when the salt concentration is high because cells are damaged by high osmotic pressure.

However, the cell coating method according to the present invention may not exert external stress on chemically unstable cells because it undergoes oxidative polymerization in the neutral region, and the viability is substantial despite being chemically unstable cells by rapidly forming a coating layer on the cell surface. It represents a significant difference in that it can be preserved. In addition, the concentration of the salt may be the concentration of the salt and the isotonic solution of the living body, for example, may be less than or equal to 0.3M concentration, specifically 0.01M to 0.2M, more specifically 0.5 to 0.15M.

The viability of the live cells produced through the cell coating method according to the present invention may be 90% or more, and preferably 93% or more.

The present invention through the method of coating the cells as described above, the cells are also; And a water-insoluble hydrophilic phenolic polymer coating layer.

As the monomer forming the water-insoluble hydrophilic phenolic polymer coating layer, a phenolic amine as described above may be selected as a phenolic amine, but as a modified example according to the present invention, the phenolic amine is a phenolic amino acid, phenolic It may be any one or more compounds selected from the group consisting of dipeptides and phenolic tripeptides. A preferred example of the phenolic amino acid may be tyrosine (Y), and the phenolic dipeptide and phenolic tripeptide are formed by covalently bonding one or two amino acids selected from phenolic amino acids and 20 standard amino acids. May be

Examples of phenolic dipeptides include YF, YL, YI, YV, YA, YW, YC, YG, YP, YH, YK, and examples of phenolic tripeptides include YFY, YFF, YFI, YLA, YII, YVY, YAW, YCW, YWC, YGG, YIP, YPA, YHH, YKK, YKY, YVV, YHP, YHF, YAA, etc. may be exemplified, but this is not limited thereto. When a phenolic dipeptide or phenolic tripeptide is used as a substrate, tyrosine is used as a substrate by tyrosinase to form a main chain by oxidative polymerization, and an amino acid linked to tyrosine by a peptide bond has a branched chain of the polymer main chain. Can form a structural unit of the polymer.

Rather than using the phenolic amine as a single amino acid material, the hydrophilicity and hydrophobicity of the polymer formed by oxidation polymerization can be controlled by including a dipeptide or tripeptide linked by a peptide bond, and the branch chain connected to the main chain can be controlled. Various modifications of the polymer may be possible by introducing residues of various amino acids. In one modification according to the present invention, after forming a water-insoluble hydrophilic phenolic polymer coating layer by tyrosinase, functional functionality can be imparted to living cells by introducing functional substituents to the residues of amino acids forming branch chains. As a non-limiting example, a specific sequence capable of targeting a specific cell may be covalently attached to a residue of an amino acid forming a branch chain, and the specific sequence may be located on the outer wall of the hydrophilic phenolic polymer coating layer. Viable cells modified with a specific sequence can be effectively targeted to a local site, and in another example, sensitivity to a specific substance by covalently binding a functional group capable of selectively binding to a specific substance to a residue of an amino acid forming a branch chain Can implement an improved biological device.

In addition, the present invention can be applied to various biological devices, including living cells as described above. For example, it may be a biological device including any one selected from a cell-based sensor, a biocatalyst, and a cell-on-a-chip.

 Hereinafter, a method of manufacturing core-shell particles according to the present invention will be described in detail.

The method for preparing core-shell particles according to the present invention includes preparing a lipid structure dispersion; Mixing the coating composition comprising a phenolic amine and a tyrosinase in the dispersion; And forming a coating layer on the lipid structure.

The lipid structure dispersion may mean that the lipid structure particles are dispersed in water or an aqueous solution, and may have the form of a transparent phase dispersion, a semi-transparent phase dispersion or an opaque dispersion, and lipid structure particles having various particle diameter ranges may be included without limitation. have.

The lipid structure particles may have an average particle diameter of 10 nm to 1,000 mm, specifically 20 nm to 100 mm, 30 nm to 10 mm, and more specifically 50 nm to 1 mm. May, but is not limited to this.

The lipid structure may be derived from phospholipids, and specific examples of phospholipids include glycerophospholipids, sphingophospholipids, phosphosphingolipid, phosphatidic acid (PA), phosphatidyl inositol, phosphatidylinositol, and phosphatidylinositol. Phosphatidylcholine (PC), Phosphoinositides, Phosphatidylserine (PS) and Phosphatidylethanolamine (PE), Lecithin and derivatives thereof. However, it is not limited to this.

The concentration of the phospholipid in the lipid structure dispersion may be 0.001% to 30% by weight, specifically 0.01% to 20% by weight, and more specifically 0.1% to 10% by weight, but is not limited thereto.

The concentration of the aqueous phase of the phenolic amine may be 0.001 to 100 mg / ml, preferably 0.01 to 10 mg / ml, more preferably 0.1 to 1 mg / ml, and the concentration of tyrosinase is 0.01 kU / ml. To 5000 kU / ml, preferably 0.1 kU / ml to 500 kU / ml, more preferably 1 kU / ml to 50 kU / ml, but is not limited thereto.

Conditions such as temperature and pH in the step of forming the coating layer are not particularly limited in the range that does not impair the achievement of the object of the present invention, for example, pH is 4 to 12, preferably 5 to 10, more preferably It may be 7 to 8, the temperature may be 10 ℃ to 50 ℃, preferably 20 ℃ to 40 ℃, preferably in the range of room temperature.

The time for forming the coating layer by mixing the lipid structure dispersion and the coating composition may be 1 minute to 10 hours, preferably 10 minutes to 6 hours, but is not limited thereto.

The lipid structure may include a lipid monolayer or a lipid bilayer, and when including a lipid monolayer, the inner phase corresponding to the core may be an oil phase, and a lipid monolayer is located on the surface of the oil phase. Meanwhile, when the lipid structure includes a lipid bilayer, the inner phase corresponding to the core may be an aqueous phase, and a lipid bilayer is positioned on the surface of the aqueous phase.

Specifically, the lipid structure may be a liposome or nanoemulsion. The liposomes and nanoemulsions are bio-friendly, and have the advantage of being able to capture water-soluble or fat-soluble bioactive substances therein and be used as a carrier.

The water-soluble or fat-soluble physiologically active substance may be vitamins, polyphenols, peptides, proteins, extracts, water-soluble synthetic compounds, fat-soluble compounds, poorly soluble compounds, fat-soluble proteins, etc. without limitation. When the lipid structure is a liposome, all of the bioactive substances as described above may be included without limitation, and in the case of a nanoemulsion, a fat-soluble bioactive substance may be preferably included.

The bioactive material may be included in the preparation of the lipid structure dispersion. Specifically, in the step of preparing a liposome or nanoemulsion, the physiologically active substance is mixed to prepare a lipid structure in which a bioactive substance is previously collected, and then a coating layer by phenolic amine and tyrosinase is formed. Since it is known to mix the bioactive substances in the production step of liposomes or nanoemulsions, detailed descriptions thereof will be omitted.

The liposome is a lipid construct comprising a lipid bilayer and is known in the art as a delivery vehicle for bioactive substances. Examples of liposomes include small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs), multivesicular vesicles (MVVs), and large multivesicular vesicles (MVVs) Various types of liposomes such as large multivesicular vesicle (LMVV) and oligolamellar vesicle (OLV) may be included.

The amount of aqueous medium forming the aqueous phase per amount of lipid in LMVV is greater than MLV, and compared to the MLV system having a similar size distribution, the aqueous phase has a greater amount of water-soluble bioactive material as it contains the lipid structure of the LMVV. It is preferable in that it may include.

On the other hand, in the case of MLV and MVV, it can be preferable as a carrier of a fat-soluble bioactive substance in that it can provide a space for a lipid bilayer to contain a large amount of a fat-soluble bioactive substance.

The liposome contains a water-soluble physiologically active substance including an aqueous phase in an inner phase corresponding to the core, and a lipid-soluble bioactive substance in a lipid bilayer, so that both the water-soluble and fat-soluble bioactive substances can be simultaneously contained in one lipid structure. Is advantageous in

The method for preparing the liposome may be a known method such as a thin film hydration method or an extrusion method, without limitation.

The nanoemulsion is an oil-in-water emulsion, and the inner phase corresponding to the core includes an oil phase and has a structure in which a lipid monolayer is located on the surface of the oil phase. The inner wound can provide a space for collecting fat-soluble bioactive substances, and is separated from the trauma phase by a lipid monolayer to stably collect and deliver the fat-soluble bioactive substances.

The nanoemulsion has high usability in the food, cosmetic, and medical fields due to the above properties, and also has an advantage in that it can control various viscoelastic rheological properties by controlling the composition and microstructure constituting the nanoemulsion. In particular, nanoemulsions can have high dispersibility by a simple process using relatively simple raw materials (water, oil, surfactant, phospholipids), and the nano-level small emulsion particles provide the effect of reducing gravity in the brown exercise process. It can have a high stability that does not cause creaming or precipitation during storage.

The content of oil in the lipid structure dispersion containing nanoemulsion particles may be 0.01% to 40% by weight of the total weight of the lipid structure, specifically 0.1% to 30% by weight, more specifically 1% to 20% by weight %, But this is only an example and is not limited thereto. The nano-emulsion, a known method such as agitation method, high pressure emulsification method, ultrasonic method can be used without limitation, detailed description is omitted.

The present invention provides a core-shell particle comprising a lipid structure core prepared from a method for preparing core-shell particles as described above and a water-insoluble hydrophilic phenolic polymer coating layer on the lipid structure.

The core-shell particles include a hollow in the lipid structure core, and the lipid structure and the water-insoluble hydrophilic phenolic polymer are physically bound. A phenolic polymer film may be formed only on the surface of the lipid structure particles by mixing the coating composition containing the phenolic amine and tyrosinase on the lipid structure. Since tyrosinase enzymatically oxidizes a phenolic amine to form a phenolic polymer film, it does not involve a chemical reaction with a phospholipid molecule on the surface of the lipid structure particle, so the phenolic polymer is physically bound to the phospholipid molecule. More specifically, the phenolic polymer film means a water-insoluble hydrophilic phenolic polymer.

The hollow of the core-shell particles may include water or oil, and optionally, the hollow may include a hollow shape as a subsequent drying process is included.

The lipid structure may be a liposome or a nanoemulsion, and in the case of a liposome, the hollow may be empty or water may be included, and in the case of a nanoemulsion, the hollow may be empty or oil may be included. As the lipid spheroid contains liposomes or nanoemulsions, the hollow may include any one or more selected from the group consisting of water, oil, and bioactive substances.

Hereinafter, the contents of the present invention will be described in more detail through examples. The examples are only intended to illustrate the present invention in more detail, and the scope of the present invention is not limited by them.

[Reagents and ingredients]

Tyrosinase from mushroom (> 1000 U mg ^-1 , Sigma) purchased mushroom-derived products, Rosell Park Memorial Institute 1640 medium (RPMI 1640; including L-glutamine and 26 mM HEPES, Welgene), bovine Fetal bovine serum (FBS, Welgene), penicillin-streptomycin (P / S, 5,000 U / mL of penicillin and 5,000 mg / mL of streptomycin, Welgene) are from Welgene. It was purchased and used.

Yeast extract peptone dextrose (YPD) agar (Duchefa Biochemistry) and YPD medium (Duchefa Biochemistry) were purchased from Duchefa Biochemistry and used. As the Jurkat cell, the one named Jurkat clone E6-1 (No. 40152) from the Korean Cell Line Bank was used, and the yeast ( Saccharomyces cerevisiae ) was used from the baker's yeast.

[Evaluation method: Evaluation by analyzer]

Fourier transform infrared spectroscopy (FTIR) was measured using a FTIR meter (Thermo Nicolet ^TM nexus FT-IR spectrophotometer) in an atmosphere purged with nitrogen, and the FT-IR spectrum was about 4,000 for each sample in the background. Scanned once and equalized.

To measure the thickness of the coating layer film, an ellipsometric (elliptical analysis) thickness was obtained using an ellipsometer (Gaertner L116s ellipsometer, Gaertner Scientific Corporation). A refractive index of 1.46 was used for all samples, and five different points were measured for each sample, and the average value was recorded.

Field-emission scanning electron microscopy (FE-SEM) was used to analyze the surfaces of the cells and the coating layer. After sputter-coating with platinum, imaging was performed using an imaging device (FEI Inspect F50 microscope) at an acceleration voltage of 10 kV. Meanwhile, a transmission electron microscopy was imaged using a LEO 912AB microscope (Carl Zeiss).

UV-visible absorption spectra (UV-visible absorption spectra) was measured with a UV-2550 spectrophotometer (Shimadzu), Atomic force microscopy (AFM) was performed using INNOVA-LABRAM HR800 (Horiba Jobin & Bruker). Mode was performed to image the surface.

X-ray photoelectron spectroscopy (XPS) was analyzed using a multi-purpose X-ray photoelectron spectrometer (Sigma Probe, Thermo VG Scientific) with an Al-Kα (1486.6eV) X-ray source, and the passing energy was 50.0. eV (wide scan) and 20.0 eV (individual narrow scan).

The contact angle was measured using a Phoenix 300 goniometer (Surface Electro Optics Co.) equipped with a video camera, and the average was determined by measuring the static contact angle of 3 uL droplets at 4 different locations for each sample.

Surface potential (zeta potential) and hydraulic particle size were measured using a Zetasizer Nano ZS (Malvern) through dynamic light scattering (DLS) method, and particle size was calculated based on the Smoluchowski model.

[Evaluation method: measuring cell viability]

To measure the viability of Jurkat cells, LIVE / DEAD®viability / cytotoxicity kit (calcein acetoxymethyl (calcein AM): 1.6 μM, ethidium homodimer-1: 4 μM in PBS) was used.

Jurkat cells were incubated with the kit for 20 minutes, washed with PBS, and observed with a confocal laser-scanning microscopy (CLSM, LSM 700 Confocal Microscope, Carl Zeiss).

The viability of yeast cells was measured by FDA and PI. Specifically, 5 uL of an FDA solution contained in acetone at a concentration of 10 mg / mL and PI acceptance at a concentration of 1 mg / mL) were prepared. Each solution was added to 1 mL of yeast sample, incubated for 20 minutes, washed with PBS and observed with CLSM.

[Test Example 1] Catalytic activity of tyrosinase

The catalytic activity of tyrosinase in aqueous solution was tested using tyrosine as a substrate and tyrosinase as a catalyst.

Tyrosine was diluted (pH 7.4) in PBS solution to a concentration of 0.4 mg / mL, and completely dissolved by stirring at 50 ° C, and then cooled to room temperature, 25 ° C, to prepare a transparent solution. A change in color on the solution phase was observed over 10 minutes, 30 minutes, 1 hour, 3 hours, and 6 hours by adding a tyrosinase solution having a concentration of 10 kU / mL in PBS.

In the initial stage of adding the tyrosinase solution, a transparent color was exhibited, but as shown in FIG. 1 (b), a change in color was observed from 10 minutes and gradually changed to a dark brown color.

As a result of observing the UV-visible light spectrum, as shown in FIG. 1 (c), the presence of cyclic amine (480 nm) and quinone (310 nm) can be confirmed, confirming that eumelanin was formed therefrom. Did.

[Example 1] Film formation by tyrosine precursor and tyrosinase catalyst on gold substrate

The gold substrate was prepared by thermally depositing 5 nm titanium and 100 nm gold on a silicon wafer (99.9999%). Before use, the gold substrate was washed with sonication in ethanol and acetone solution for 15 minutes.

Tyrosine was diluted (pH 7.4) in PBS solution to a concentration of 0.4 mg / mL, and completely dissolved by stirring at 50 ° C, and then cooled to 25 ° C at room temperature. After immersing the washed gold substrate in a 2 mL tyrosine solution, 2 mL of a 10 kU / mL concentration tyrosinase solution in PBS was added.

After impregnating the gold substrate with the solution for a predetermined time, the gold substrate was washed with deionized water to prepare a gold substrate with a hydrophilic phenolic polymer coating layer.

As a result of performing XPS analysis to analyze the coating layer of the gold base on which the hydrophilic phenolic polymer coating layer was formed, it was confirmed that a film was formed on the gold. As shown in FIG. 1 (d), the high resolution C 1s peaks were resolved into three peaks: 284.3 eV (CC and CH), 285.6 eV (CO and CN) and 287.8 eV (C = O and C = N). It can be, the three peaks were represented as a representative peak of eumelanin.

In addition, as a result of performing FT-IR analysis, as shown in FIG. 2, hydroxyl (OH), carboxyl (C = O) and amine (CN) peaks were observed at 3,200, 1,600 and 1,350 cm ⁻¹ , respectively. It was confirmed that the eumelanin structure was successfully formed.

As a result of checking the thickness of the coating layer by time, as shown in FIG. 1 (e), the thickness of the coating layer increased in proportion to time, and after 6 hours of reaction under the condition of pH 7.4, a film of about 29 nm thickness was It was confirmed that it was formed.

[Comparative Example 1] Film formation when no tyrosinase catalyst was added to the gold substrate

Other processes were performed in the same manner as in Example 1, except that the tyrosinase solution was not added. As a result, it was confirmed that no film was formed on the gold substrate.

[Comparative Example 2] Forming a polydopamine film on a gold substrate

In Example 1, instead of coating with tyrosine and tyrosinase, for the coating of polydopamine, a 2 mg / mL dopamine solution was prepared in Tris buffer, and the pH was adjusted to 8.5 using 1M NaOH. As a result of observing the film thickness over a period of time, the film thickness increased linearly, and after 6 hours, it was confirmed that a film having a thickness of 10 nm was formed.

The above results are in sharp contrast to those of Example 1, whereas in the case of a coating by an enzymatic oxidation reaction, a coating layer by a chemical oxidation reaction is formed in a harsh environment for cells compared to a point in which a coating layer is rapidly formed despite mild conditions. The thickness did not form quickly. From this, it can be confirmed that the coating layer formed by the enzymatic oxidation reaction is formed at a rate about 3 times higher than the coating by the chemical oxidation reaction.

As a result of performing the surface analysis of Example 1 and Comparative Example 2, as shown in Figure 3, the film of the coating layer of Example 1 was obtained a very uniform film compared to the polydopamine film of Comparative Example 2, a scanning electron microscope (SEM) hardly found aggregates. The surface roughness (R _RMS ) of the film of the coating layer of Example 1 was calculated through AFM analysis, and the results were measured for each 1 hour, 3 hour, and 6 hour reaction, and were 1.44 nm, 3.79 nm, and 8.16 nm, respectively. This is supported by the obtained. From the above results, the cell coating method according to the present invention means that it works efficiently to form a uniform film under mild conditions.

[Example 2] Film formation using tyramine (TY) precursor

In Example 1, a film was formed in the same manner as in other procedures, except that tyramine was used instead of tyrosine as a precursor.

[Example 3] Film formation using dopamine (DA) precursor

In Example 1, a film was formed in the same manner as in other procedures, except that dopamine (DA) was used instead of tyrosine as a precursor.

[Example 4] Film formation using norepinephrine (NE) precursor

In Example 1, a film was formed in the same manner as in other procedures, except that norepinephrine (NE) was used instead of tyrosine as a precursor.

[Example 5] Film formation using 3,4-dihydroxyphenylalanine precursor

In Example 1, a film was formed in the same manner as in the other procedures, except that 3,4-dihydroxyphenylalanine was used instead of tyrosine as a precursor.

[Comparative Example 3]

In Example 1, a film was formed in the same manner as in the other procedures, except that pyrocatechol having the following structure was used as the precursor.

[Comparative Example 4]

In Example 1, except for using pyrogallol (pyrogallol) as a precursor, the other procedures were the same to form a film.

[Comparative Example 5]

In Example 1, a film was formed in the same manner as in other procedures, except that gallic acid was used as the precursor.

[Comparative Example 6]

In Example 1, a film was formed in the same manner as in other procedures, except that caffeic acid was used as the precursor.

[Comparative Example 7]

In Example 1, a film was formed in the same manner as in other procedures, except that 3,4-dihydroxybenzaldehyde was used as the precursor.

[Comparative Example 8]

In Example 1, a film was formed in the same manner as in other procedures, except that tannin acid having the following structure was used as a precursor.

As a result of experiments of Examples 2 to 5, as shown in FIG. 4, tyrosinase can effectively induce an enzymatic oxidation reaction using tyrosine as well as various phenolic amines as a substrate, and a film on a substrate. It can be confirmed that it can form. Depending on the substrate, the thickness of the film is not different, but a film having a thickness of 10 nm to 20 nm was formed, and as a result of performing FTIR analysis of the resulting film, hydroxyl (OH) and carboxyl (C) as shown in FIG. 3. = O) and amine (CN) peaks were observed at 3,200, 1,600 and 1,350 cm ^-1 , respectively, confirming that the melanin structure was successfully generated.

On the other hand, when tyrosinase was not included, the film was hardly formed or had a thickness of 2 nm or less, so that the film was formed slowly and inefficiently. The above results suggest that tyrosinase plays an important role when forming a coating layer using a phenolic amine.

As a result of the experiments of Comparative Examples 3 to 8, no film was formed even when tyrosinase was used as a catalyst. These results mean that it is necessary to include at least one amino group (-NH2) in the precursor phenolic compound, and the number of hydroxyl groups or the presence of other substituents in the phenol ring is a film by enzymatic oxidation reaction and oxidation polymerization. This suggests that there is no correlation with formation.

[Example 6] Formation of phenolic polymer coating layer on Jurkat cells

Jurkat clone was suspended in a T75 cell culture flask containing 15 mL of RPMI 1640 medium containing 10% FBS and 1% P / S as a medium for cell culture, and humidified incubator at 37 ° C and 5% carbon dioxide environment Were cultured in.

After reaching 80% culture, cells were subcultured at a ratio of 1/15 in another T5 cell culture flask. After the passaged cells were separated by centrifugation, the cells were washed with RPMI 1640 medium, and the Jurkat cells were taken out of the cell culture medium with a pipette so that the cell weight was 0.1 mg. The extracted Jurkat cells were dispensed into 2 mL of RPMI 1640 medium in which tyrosine was dissolved at a concentration of 0.4 mg / mL, and then 2 mL of a tyrosinase solution at a concentration of 10 kU / mL was added. After 6 hours, Jurkat cells having a coating layer were collected and washed with RPMI 1640 medium to finally prepare Jurkat cells having a phenolic polymer coating layer.

As a result of observing the TEM and SEM images of the Jurkat cells on which the phenolic polymer coating layer was formed, it was confirmed that the phenolic polymer was uniformly conformal coated on the cells, as shown in FIG. 5 (a).

Meanwhile, the viability of the Jurkat cells on which the phenolic polymer coating layer was formed was measured. As a result, as shown in Figure 5 (b), after 6 hours, it was confirmed that the viability of Jurkat cells was 96.5%, and these results can also be confirmed from the CLSM image of Figure 5 (c).

FIG. 6 shows a scanning electron micrograph of Jurkat cells and Jurkat cells on which a phenolic polymer coating layer was formed. Jurkat cells have a somewhat rough cell surface, but a very smooth coating layer was formed as the phenolic polymer coating layer was uniformly coated. Can be confirmed. These results suggest that the phenolic polymer coating layer has low surface roughness even on the cell surface, as confirmed in Example 1.

[Example 7] Formation of phenolic polymer coating layer on yeast

A single colony of yeast was obtained from the YPD agar plate and cultured in YPD medium with gentle shaking at 33 ° C. for 30 hours. The cultured yeast was washed with PBS, and the yeast sample was dispensed into 2 mL of a tyrosine PBS solution at a concentration of 0.4 mg / mL, and then 2 mL of a tyrosinase solution at a concentration of 10 kU / mL was added. After 6 hours, the yeast was recovered and washed with PBS to prepare a yeast finally formed with a phenolic polymer coating layer.

As a result of measuring the viability of yeast in the same manner as in Example 6 above, it was confirmed that the viability of yeast after 94 hours was 94%, and this viability is equivalent to that of yeast before being coated. This means that it has no effect on viability. The results can also be confirmed from the CLSM image of FIG. 7B.

[Example 8] Formation of phenolic polymer coating layer on nanoemulsion cells

A nanoemulsion was prepared in which tocopherol acetate, a derivative of vitamin E, is a bioactive substance and is trapped inside particles. Saturated lecithin extracted from soybean and hydrogenated (phosphatidylcholine content: 70 to 0.15 g, 0.3 g of rapeseed sterol with 5 mol of polyethylene glycol added), 0.5 g of caprylic / capric triglyceride, 1 g of ethanol and 0.5 g of tocopherol acetate were heated and dissolved at 65 ° C., then heated to 70 ° C. and mixed with 8.5 g of distilled water dissolved to 5,000 rpm with a homogenizer (T 25 digital ULTRA-TURRAX®IKA®) The mixture was emulsified for 10 minutes, and pre-emulsion made through emulsification was added to ultrasonic waves for 10 minutes to prepare a translucent nanoemulsion, and then ethanol was evaporated under reduced pressure.

After dispensing 1 g of the nanoemulsion into 2 mL of a tyrosine solution at a concentration of 0.4 mg / mL, 2 mL of a tyrosinase solution at a concentration of 10 kU / mL was added. After 6 hours, the nanoemulsion particles on which the coating layer was formed were recovered and washed three times with distilled water to finally prepare nanoemulsion particles on which the phenolic polymer coating layer was formed.

The tyrosine solution and the nanoemulsion particles not treated with tyrosinase were separated into emulsions during separation and purification, but were not obtained in the form of particles. It was confirmed that the shape was maintained and the phenolic polymer coating layer was stably formed on the surface of the nanoemulsion.

[Comparative Example 9] Formation of polydopamine (PD) coating layer on Jurkat cells

Jurkat cells were prepared in the same manner as in Example 6. In Example 6, instead of coating with tyrosine and tyrosinase, for a polydopamine coating, a 2 mg / mL concentration of dopamine solution was prepared with RPMI medium, and the pH was adjusted to 8.5 using 1M NaOH. Then, Jurkat cell samples were added to the dopamine solution. After 6 hours, Jurkat cells were recovered and washed with RPMI 1640 medium.

As a result of measuring the viability of Jurkat cells, as shown in FIG. 5 (b), the viable cells, Jurkat cells, were completely killed by polydopamine coating, resulting in a viability of 0, forming aggregates, This result can also be confirmed from the CLSM image of FIG. 5 (c).

When the results of Example 6 and Comparative Example 9 are put together, in the case of Jurkat cells prepared by the cell coating method of the present invention, the phenolic polymer is uniformly conformal coated on the cell surface and is almost equivalent to Jurkat cells on which the coating layer is not formed. It can be seen that it shows viability. That is, the cell coating method of the present invention is particularly useful for chemically unstable cells. In contrast, Jurkat cells coated by a chemical oxidation reaction showed a viability of 0, suggesting that the coating of a phenolic polymer by a chemical oxidation reaction is not applicable to chemically unstable cells.

[Comparative Example 10] Formation of a polydopamine (PD) coating layer on yeast

Yeast was prepared in the same manner as in Example 7. In Example 7, instead of coating with tyrosine and tyrosinase, for a polydopamine coating, a 2 mg / mL dopamine solution was prepared in PBS, and pH was adjusted to 8.5 using 1M NaOH. Thereafter, yeast was added to the dopamine solution. After 6 hours, the yeast was recovered and washed with PBS to prepare a yeast finally formed with a phenolic polymer coating layer.

As a result of measuring the viability of yeast in the same manner as in Example 7 above, it was confirmed that the survival rate of yeast was 54% after 6 hours.

From the results of Examples 6 to 7, and Comparative Examples 9 and 10, the enzymatic oxidation reaction using tyrosinase and the coating by oxidative polymerization hardly affect the viability of the cells, and have high cellular compatibility. can confirm. These results can increase the applicability of living cells by forming a coating layer stably and uniformly without affecting the viability even for chemically unstable cells through the cell coating method according to the present invention. It means that it can have high usability.

On the other hand, in order to measure the surface potential (zeta potential) of the particles, a phenolic polymer coating layer was formed in the same manner as in Example 7 on the negatively charged polystyrene particles having an average particle diameter of 4.97 mm instead of cells. As a result of measuring the surface of the polystyrene particles on which the phenolic polymer coating layer was formed, it was -22.6 mV before coating but -84.2 mV after coating.

The above results indicate that the main chain of the phenolic polymer contains an aromatic ring, and thus, although not soluble in water, it can exhibit high hydrophilicity because it contains one or more hydroxy groups in its structural unit. The high absolute value of the surface potential means high water dispersibility, and even if the cells are coated with a polymer, it can stably disperse in water and have an effect of having high viability.

Claims (16)

Cell coating composition comprising a phenol-based amine and tyrosinase. The composition for cell coating according to claim 1, wherein the phenolic amine comprises a compound represented by the following Chemical Formula 1.
[Formula 1]

In Chemical Formula 1,
R ¹ is any one selected from hydrogen, hydroxy, carboxylic acid and salts thereof;
R ² are each independently (C1-C10) alkyl, (C1-C10) alkenyl, (C3-C20) cycloalkyl, (C3-C20) heterocycloalkyl, (C6-C20) aryl, (C3-C20) Heteroaryl, nitro, cyano, -C (= O) R ¹¹ and -C (= O) OR ¹² , or any combination of two or more selected from the group consisting of;
R ¹¹ and R ¹² are each independently hydrogen, (C1-C10) alkyl, (C3-C20) cycloalkyl, (C3-C20) heterocycloalkyl, (C6-C20) aryl and (C3-C20) heteroaryl Any one or a combination of two or more selected from the group consisting of;
L is a divalent linking group;
m is an integer from 1 to 3;
When m is 2 or more, adjacent substituents may be connected to each other to form a ring;
The alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl of R ² , R ¹¹ and R ¹² are each independently hydroxy, carboxylic acid, (C1-C10) alkoxy, (C1-C10) alkyl It may be substituted with any one or more substituents selected from the group consisting of carbonyl, halogen, amine, cyano, nitro and salts thereof. According to claim 2,
R ¹ is hydrogen or hydroxy;
The L is (C1-C10) alkylene or (C1-C10) alkenylene, -CH ₂ -of the alkylene or alkenylene is -N (R ¹³ )-, -C (= O) NH-,- C (= O) O- and -O- may be substituted with any one selected from the group consisting of, wherein R ¹³ is selected from the group consisting of hydrogen, (C1-C10) alkyl and amino (C1-C10) alkyl ,
The alkylene and alkenylene of L are further one or more substituents selected from the group consisting of halogen, hydroxy, amine, carboxylic acid, (C1-C10) alkoxy, (C1-C10) alkylcarbonyl and salts thereof. Cell coating composition, which can be substituted. According to claim 2,
The phenolic amine is a composition for cell coating comprising a compound represented by a compound represented by the following formula 2 or formula 3.
[Formula 2]

[Formula 3]

In Chemical Formulas 2 and 3,
R ³ and R ⁴ are each independently hydrogen, hydroxy or carboxylic acid. The method of claim 1
The polymer formed on the cell is a water-insoluble hydrophilic phenolic polymer cell coating composition. Mixing the cell and the cell coating composition of any one of the cells selected from claim 1 to claim 5; And forming a coating layer on the cells. The method of claim 6,
The coating layer has a thickness of 1 nm to 100 nm, the surface roughness (R _RMS ) of less than 10nm coating method of cells. The method of claim 6,
The cell is a method of coating a cell comprising any one or more selected from T cells, B cells and natural killer (NK) cells. cell; And a live cell comprising a water-insoluble hydrophilic phenolic polymer coating layer on the cell. The method of claim 9,
The water-insoluble hydrophilic phenol-based polymer is derived from an enzymatic oxidation reaction of any one or more compounds selected from the group consisting of phenolic amino acids, phenolic dipeptides and phenolic tripeptides, live cells. A device comprising any one selected from cell-based sensors, biocatalysts and cell-on-a-chips comprising the living cells of claim 9. Preparing a lipid structure dispersion;
Mixing the coating composition comprising a phenolic amine and a tyrosinase in the dispersion; And
Method of manufacturing a core-shell particles comprising; forming a coating layer on the lipid structure. The method of claim 12,
The lipid structure is a method for producing core-shell particles that are liposomes or nanoemulsions. A core-shell particle comprising a lipid structure core and a water-insoluble hydrophilic phenolic polymer coating layer on the lipid structure. The method of claim 14,
The core-shell particles include hollows in the lipid structure core, and the lipid structure and the water-insoluble hydrophilic phenol-based polymer are physically bonded, hollow core-shell particles. The method of claim 14,
The hollow includes any one or more selected from the group consisting of water, oil and bioactive materials, hollow core-shell particles.

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