KR101841111B1 - Hyaluronic acid derivatives and composition for cell-surface engineering using the same - Google Patents

Abstract

The present invention relates to a hyaluronic acid derivative for liver target-directed delivery through intravenous injection of cells and a method for producing the hyaluronic acid derivative. More particularly, the present invention relates to a method for producing a hyaluronic acid derivative having the ability to modify the surface of a cell and at the same time having biocompatibility, biodegradability, and liver-target-directed delivery characteristics, and a liver-targeted cell delivery system of the hyaluronic acid derivative .

Description

HYALURONIC ACID DERIVATIVES AND COMPOSITION FOR CELL-SURFACE ENGINEERING USING THE SAME BACKGROUND OF THE INVENTION 1. Field of the Invention [0001]

TECHNICAL FIELD The present invention relates to a hyaluronic acid derivative, a method for producing the same, and a cell-reforming composition and a liver-targeted cell transporter for treating diseases comprising hyaluronic acid derivatives using the same.

The cell delivery system is a system for delivering the cell itself and the drug of the genetically modified cell carrier through various injection routes such as arterial injection, intravenous injection, subcutaneous injection, local injection, and intraperitoneal injection. Do.

Stem cells, immune cells, etc. can be used as a cell therapy. In particular, mesenchymal stem cells have relatively few immune responses, have immunoregulatory ability, and are capable of differentiating into various cells. Therefore, they are widely used for cell carrier, disease treatment and tissue regeneration for drug delivery.

At this time, cell transfer to a desired site is one of important factors to be considered in various cell delivery systems including mesenchymal stem cells. Currently, cells are delivered to the desired site in a variety of ways, but compared to intravenous injection, local injections and arterial injections can cause risks and side effects such as bleeding and tissue damage. Thus, intravenous delivery of cells to the desired site is a simpler and more ideal method.

Currently, studies are under way to transfer immune cells or stem cells into the liver via intravenous and hepatic / hepatic arterial injections to treat liver diseases such as liver cancer or liver cirrhosis. However, in order for the cells to have a cancer-targeting orientation, the cells must be cultured for a certain period of time in vitro, and when cells are injected through intravenous injection, a large number of cells remain in the lungs, In addition, portal vein / hepatic artery injection should be accompanied by open surgery. Therefore, it is necessary to develop a liver-mediated delivery system of cells that can be applied to the treatment of all liver diseases efficiently through simple intravenous injection.

An object of the present invention is to provide a hyaluronic acid derivative or a salt thereof containing a unit represented by the following formulas (1) to (3); Or a hyaluronic acid derivative containing the unit represented by the following general formulas (1) and (4), or a salt thereof.

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

In the above formulas,

P is a water soluble protein;

X is -COR ¹ , -CONR ² H-R ¹ or -CONH-R ² -NH-R ¹ ;

Wherein R ¹ is selected from the group consisting of a vinyl group, a thiol group, a poly-L-lysine, a lipid, an acrylate group, a maleimide group or N-hydroxysuccinimide (NHS) ;

R ² is an alkyl group having 1 to 6 carbon atoms;

Wherein the sum of m, n and 1 is an integer of 50 to 10,000, the sum of n and 1 is an integer of 5 to 5,000 and 1 is an integer of 1 to 100; s is an integer of 1 to 2000;

It is still another object of the present invention to provide a process for producing the hyaluronic acid derivative or a salt thereof.

It is a further object of the present invention to provide a composition for cell surface modification comprising the hyaluronic acid derivative or a salt thereof.

It is still another object of the present invention to provide a cell modified with a hyaluronic acid derivative or a salt thereof or a pharmaceutical composition comprising the same as an active ingredient.

It is still another object of the present invention to provide a method for treating liver disease by administering a pharmaceutical composition comprising the hyaluronic acid derivative or its salt as a surface active agent.

It is still another object of the present invention to provide a method for targeting liver cells to an active substance using the hyaluronic acid derivative, which comprises the step of administering the hyaluronic acid derivative to a subject in need thereof.

As a result of the studies conducted by the present inventors, hyaluronic acid, which is a biopolymer excellent in biodegradability and biocompatibility, has liver-target transferring characteristics and is thus used as a cell surface modifying agent for hepatic transfer of cells using hyaluronic acid having excellent liver- It is possible to transfer the cells to the liver, not to the lungs. By using such characteristics, it has been developed a cell-reforming composition for treating diseases including hyaluronic acid derivatives and a method for producing the same, and it has been confirmed that the composition can be effectively applied to the treatment of liver disease by intravenous injection of a cell therapy agent. .

Cells whose surface has been modified with a hyaluronic acid derivative according to the present invention can be targeted to the liver through intravenous injection. More specifically, it can be safely applied to human body by using a hyaluronic acid derivative having biocompatibility, biodegradability and liver-target transferring properties and capable of efficiently modifying the cell surface, Which can be applied to a drug delivery system and tissue engineering of a cell carrier, which can be applied to a delivery system, and an intravenous injection of hepatocyte-derived cells, which is surface-modified with the hyaluronic acid derivative produced thereby, ≪ / RTI >

An example of the present invention is a hyaluronic acid derivative comprising a unit represented by the following formulas (1) to (3) or a salt thereof; Or a hyaluronic acid derivative comprising a unit represented by the following general formulas (1) and (4), or a salt thereof:

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

In the above formulas,

P is a water soluble protein;

X is -COR ¹ , -CONR ² H-R ¹ or -CONH-R ² -NH-R ¹ ;

Wherein R ¹ is selected from the group consisting of a vinyl group, a thiol group, a poly-L-lysine, a lipid, an acrylate group, a maleimide group or N-hydroxysuccinimide (NHS) ;

R ² is an alkyl group having 1 to 6 carbon atoms;

Wherein the sum of m, n and 1 is an integer of 50 to 10,000, the sum of n and 1 is an integer of 5 to 5,000 and 1 is an integer of 1 to 100; s is an integer of 1 to 2000;

Another example of the present invention is a cell surface modification composition comprising a hyaluronic acid derivative represented by the following formula (5) or a salt thereof:

[Chemical Formula 5]

P is a water soluble protein; The sum of m, n and 1 is an integer of 50 to 10,000, the sum of n and 1 is an integer of 5 to 5,000, and 1 is an integer of 1 to 100.

Preferably, the water-soluble protein may be wheat germ agglutinin (WGA) protein. A specific example of the WGA may be a protein having the amino acid sequence of SEQ ID NO: 1.

The water-soluble protein bound to the hyaluronic acid derivative is characterized by maintaining the secondary structure of the water-soluble protein before binding even in the state of being bound to hyaluronic acid.

The water-soluble protein is conjugated to hyaluronic acid to facilitate interaction with N-acetyl-glucosamine and / or sialic acid, one of the glycoproteins present on the cell surface, Specifically, the amine group of the N-terminal of the water-soluble protein and the aldehyde of the hyaluronic acid derivative may be chemically bonded.

Particularly, it is possible to prevent the reaction with another amino acid such as lysine having other amine groups in the protein by bonding under the specific pH condition at the time of bonding, and thus maximize the activity of the protein bound to hyaluronic acid, It is possible to maximize the reforming.

Another example of the present invention is a cell surface modification composition comprising a hyaluronic acid derivative represented by the following formula (6) or a salt thereof:

 [Chemical Formula 6]

X is -COR ¹ , -CONR ² HR ¹ or -CONH-R ² -NH-R ¹ ; Wherein R ¹ is selected from the group consisting of a vinyl group, a thiol group, a poly-L-lysine, a lipid, an acrylate group, a maleimide group or N-hydroxysuccinimide (NHS) ; R ² is an alkyl group having 1 to 6 carbon atoms; Wherein the sum of m and s is an integer of 50 to 10,000 and s is an integer of 1 to 2000.

Preferably, in Formula 6, X is -COR ¹ , R ¹ is lipid, and the total sum of m and s may be an integer of 50 to 10,000.

Preferably, in Formula 6, X is -COR ¹ , R ¹ is a thiol group, and the total sum of m and s may be an integer of 50 to 10,000.

Preferably, in Formula 6, X is -CONH-R ² -NH-R ¹ , R ¹ is maleimide, R ² is hexyl, and the total sum of m and s is 50 to 100, Lt; / RTI >

When X of the hyaluronic acid derivative is a lipid, a new lipid is inserted into a cell membrane composed of a lipid bilayer. When X is NHS, acrylate, thiol, or maleimide, To covalently bind to the amine or thiol groups of the protein.

Preferably, the maleimide-PEG and the maleimide-PEG-lipid are sequentially modified on the hyaluronic acid-thiol derivative by a time difference, and the resultant is subjected to a click reaction between the hyaluronic acid-thiol derivative and the maleimide The conjugated hyaluronic acid derivative further comprises a conjugate in which the maleimide, maleimide-PEG, or maleimide-PEG and lipid are chemically bonded to each other to maintain the cell modification surface thick to facilitate the rate at which the hyaluronic acid derivative enters the cell .

The hyaluronic acid is not limited in its constitution, but preferably has a molecular weight of 100,000 to 3,000,000 Da, more preferably 100,000 to 3,000,000 Da in order to optimize the use of the hyaluronic acid derivative or its salt for modifying the cell surface and transferring liver target- Hyaluronic acid having a molecular weight of 1,000,000 Da can be used.

The term "hyaluronic acid (HA)" used in the present invention refers to a polymer having a repeating unit represented by the following general formula (7), and includes all salt forms of hyaluronic acid It is used as a meaning.

(7)

In the above formula (7), m "may be an integer of 50 to 10,000.

Another example of the present invention is a method for producing a hyaluronic acid-aldehyde (HA-aldehyde) represented by the following formula (8) by reacting hyaluronic acid with an oxidizing agent under a dark condition to open a ring structure of hyaluronic acid Stage 1; And a second step of reacting the hyaluronic acid-aldehyde and the N-terminus of the water-soluble protein in an aqueous solution. The hyaluronic acid derivative or the salt thereof according to claim 1,

 [Chemical Formula 8]

[Chemical Formula 1]

(2)

(3)

In Formula 8, the sum of m and n is an integer of 50 to 10,000, and n is an integer of 5 to 5,000;

P is a water soluble protein;

The sum of m, n and 1 is an integer of 50 to 10,000, the sum of n and 1 is an integer of 5 to 5,000, and 1 is an integer of 1 to 100.

The hyaluronic acid derivatives or salts thereof can be applied to a method for producing a hyaluronic acid derivative or a salt thereof.

In the production method according to the present invention, the surface of a cell to be modified is reacted with a derivative moiety in a hyaluronic acid derivative, that is, a WGA protein, a thiol group, a lipid, NHS, an acrylate, a thiol or a maleimide to modify the cell surface. Particularly, when the cells are surface-modified with a hyaluronic acid derivative containing a carboxyl group and injected intravenously, the hyaluronic acid receptor for endocytosis (HARE), which is present in the CD44 or hepatic sinusoidal endothelial cell present in the liver, So that the target-oriented transfer characteristic can be maximized.

Preferably, the derivative may contain a water-soluble protein per molecule of the hyaluronic acid derivative in an amount of 1 to 10 molecules, more preferably 2 to 6 molecules.

In the hyaluronic acid derivative according to the present invention, the hyaluronic acid-aldehyde substitution ratio, which is defined as the molar ratio of the repeating units in which the alicyclic acid ring structure of all the hyaluronic acid repeating units is opened and the aldehyde group is formed, Or from 0 mol% to 100 mol% or less. Preferably, the substitution ratio of the hyaluronic acid-aldehyde after the first step is 10 to 50 mol%, more preferably 10 to 40 mol%.

The amount of water soluble protein such as WGA which can be bound by using the above range of substitution ratio is sufficient to allow efficient cell surface modification and sufficiently interact with the hyaluronic acid receptor present in the liver, There is an advantage that the effect can be obtained.

As described above, biocompatible, biodegradable polymer hyaluronic acid is bound to a water-soluble protein and can be safely applied to a human body. In addition, a ring structure is opened while leaving a carboxyl group binding to a hyaluronic acid receptor in a cell, To maximize hepatic tissue-specific transfer characteristics of hyaluronic acid, thereby being applicable to cell surface modifying agents as well as to development of a cell therapy agent for the treatment of liver diseases.

The oxidizing agent is not particularly limited as long as it can break the ring structure of hyaluronic acid, and sodium periodate (NaIO ₄ ) can be preferably used.

Preferably, the oxidizing agent may be added in an amount of 1 to 10 moles per molecule of hyaluronic acid, more preferably 1 to 5 moles per mole of hyaluronic acid. Preferably, the oxidizing agent can be reacted for 2 to 24 hours, preferably for 2 to 12 hours. When 1 molar equivalent of the oxidizing agent was added and the reaction was carried out for 4 hours, 10 mol% was substituted. When 5 molar equivalents were added and the reaction was carried out for 12 hours, a 40 mol% substituted derivative could be obtained.

After the reaction with the oxidizing agent, a step of dialysis with distilled water and purification may be further carried out.

Preferably, the second step may be carried out in a buffer solution. Preferably, the hyaluronic acid-aldehyde is dissolved in the buffer solution, and a water-soluble protein such as WGA is added to react the aldehyde group contained in the hyaluronic acid-aldehyde with the amine group at the N-terminal of the water-soluble protein . Since a water-soluble protein, for example, an amine group is present at the N-terminal end of WGA, the water-soluble protein used in the second step can be used as the water-soluble protein itself without any preparation. Since hyaluronic acid-aldehyde and water-soluble proteins are water-soluble, they can be dissolved in water and reacted in an aqueous solution state.

The buffer solution may be at a pH of from 5 to 7, more preferably at a pH of from 5 to 6.5, for example, the buffer solution is a sodium acetate buffer at a pH of from 5 to 6.5. By carrying out the conjugation process under the pH range having the above-mentioned range, it is possible to prevent the reaction between the hyaluronic acid-aldehyde and another amino acid such as an amino acid having another amine group in the protein, for example, lysine and the like, It is possible to increase the bioadhesion efficiency and the cell surface modification efficiency while maximizing the activity of the protein, particularly WGA.

Preferably, the water-soluble protein is reacted so that a molecular number of 1 to 10, preferably 2 to 6, per molecule of hyaluronic acid-aldehyde is bound.

In the second step, sodium cyanoborohydride (NaCNBH ₃ ) may be further added together with the water-soluble protein. The sodium cyanoborohydride serves to reduce the double bond formed after the reaction of the amine group of the N-terminal of the water-soluble protein with the aldehyde group of the hyaluronic acid derivative. The sodium cyanoborohydride may be added in an amount of 2 to 10 moles, preferably 2 to 5 moles, of the aldehyde group, depending on the substitution ratio of the hyaluronic acid-aldehyde, for 12 to 24 hours, preferably 12 to 18 hours .

On the other hand, since the aldehyde group has a very good characteristic of reactivity, it may further include a third step of blocking the remaining aldehyde groups after the second step without reacting with proteins using amines. The material used for the blocking is not particularly limited as long as it is capable of reacting with a carboxyl group or an aldehyde group. Amines, more preferably, the amines are linear or branched alkyl carbazates having 1 to 5 carbon atoms alkyl carbazate, or a lower alkanolamine having 1 to 5 carbon atoms. Such a blocking process can be represented by the following reaction formula (1).

[Reaction Scheme 1]

P in the above Reaction Scheme 1 may be a water-soluble protein, more preferably WGA. In the above Reaction Scheme 1, A is a blocking substance.

The blocking material A may be a straight or branched alkylcarbazate having 1 to 5 carbon atoms or a lower alkanolamine having 1 to 5 carbon atoms and preferably the alkylcarbazate is an ethyl carbazate or a Butylcarbazate, and the alkanolamine may be aminomethanol, aminomethanol, aminopropanol, aminobutanol, or aminopentanol.

Preferably 5 to 10 molar times, preferably 5 to 7 molar times, of the amine is added to the molar unit of the aldehyde group, and the reaction is carried out for 12 to 24 hours, preferably 12 to 18 hours. 100% blocking is possible by adding amines within the above range.

Preferably, said third step may comprise a reaction according to Scheme 2 below.

[Reaction Scheme 2]

P in the above Reaction Scheme 2 may be a water-soluble protein, more preferably a reaction formula of WGA.

More preferably, the method for preparing the hyaluronic acid derivative may include the reaction according to the reaction formula (3).

[Reaction Scheme 3]

On the other hand, when the alkylcarbazate is used as the amine, the reaction can be preferably carried out preferably under the condition of pH 5 to 7. To this end, the same acetate buffer as used in the first step is preferably used . When an alkanolamine is used, the reaction can be carried out preferably under the condition of pH 7 to 9. For this purpose, a phosphate buffer can be preferably used.

By blocking the remaining aldehyde groups without participating in the reaction, it is possible to prevent the remaining aldehyde groups from reacting with other amine groups such as WGA bonded to the hyaluronic acid derivatives. In addition, it is possible to effectively prevent an unwanted reaction after the hyaluronic acid derivative according to one embodiment of the present invention enters the body, for example, to prevent the aldehyde group and various proteins present in the body from reacting non-specifically So that the efficiency of the desired reaction can be further improved.

As another example of the present invention, a hyaluronic acid derivative or a salt thereof containing a unit represented by the following formulas (1) to (3); Or a hyaluronic acid derivative comprising the unit represented by the following formulas (1) and (4) or salts thereof:

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

In the above formulas,

P is a water soluble protein;

X is -COR ¹ , -CONR ² H-R ¹ or -CONH-R ² -NH-R ¹ ;

Wherein R ¹ is selected from the group consisting of a vinyl group, a thiol group, a poly-L-lysine, a lipid, an acrylate group, a maleimide group or N-hydroxysuccinimide (NHS) ;

R ² is an alkyl group having 1 to 6 carbon atoms;

Wherein the sum of m, n and 1 is an integer of 50 to 10,000, the sum of n and 1 is an integer of 5 to 5,000 and 1 is an integer of 1 to 100; s is an integer of 1 to 2000;

As another example of the present invention, there is provided a liver target-directed cell delivery composition or liver target-directed cell delivery system comprising a hyaluronic acid derivative represented by the following formula (5) or (6)

[Chemical Formula 5]

 [Chemical Formula 6]

In Formula 5,

P is a water soluble protein;

Wherein the sum of m, n and 1 is an integer of 50 to 10,000, the sum of n and 1 is an integer of 5 to 5,000 and 1 is an integer of 1 to 100;

In Formula 6,

Wherein X is-CO R ¹ , -CON R ² H-R ¹ or -CONH- R ² -NH-R ¹ ;

Wherein R ¹ is selected from the group consisting of a vinyl group, a thiol group, a poly-L-lysine, a lipid, an acrylate group, a maleimide group or N-hydroxysuccinimide (NHS) ;

R ² is an alkyl group having 1 to 6 carbon atoms;

Wherein the sum of m and s is an integer of 50 to 10,000 and s is an integer of 1 to 2000.

The hyaluronic acid derivatives or salts thereof may be applied to a composition for transferring liver target cells or a composition for transferring liver target cells or a liver target-oriented cell delivery system comprising hyaluronic acid derivatives or salts thereof.

In addition, the present invention includes a cell modified with a hyaluronic acid derivative or a salt thereof, or a pharmaceutical composition containing the same as an active ingredient.

The hyaluronic acid derivative or a salt thereof may be applied to a cell modified with the hyaluronic acid derivative or a salt thereof or a pharmaceutical composition containing the same as an active ingredient.

The cell surface-modified with a hyaluronic acid derivative according to an embodiment of the present invention may be a cell that needs to be transferred to liver tissue, for example, a cell therapy agent for preventing or treating liver disease, or a stem cell for regeneration of liver tissue . The hyaluronic acid derivative or its salt, which is a biopolymer, is excellent in biodegradability and biocompatibility as well as liver target transferring property and can be used as a cell surface modifying agent to transfer the cell to the liver, not the lung.

The cell is not particularly limited, but may be a cell that can be used as a cell therapy agent or a tissue regeneration stem cell. Preferably, the cell is a mammalian cell, a mammalian cell-derived cell, or a genetically modified cell thereof. Stem cells, preferably mesenchymal stem cells or genetically modified cells thereof.

Accordingly, the cell surface-modified with the hyaluronic acid derivative or a salt thereof, or a pharmaceutical composition containing the same as an active ingredient, may have a prophylactic or therapeutic use for a disease depending on the use of the modified cell. Preferably, the disease is one or more selected from the group consisting of liver cancer, liver metastasis cancer, cirrhosis, cirrhosis, hepatitis, and liver fibrosis, but the present invention is not limited thereto and the specific disease may be determined according to a cell modified with a hyaluronic acid derivative or a salt thereof .

The pharmaceutical composition may be administered by one or more methods selected from the group consisting of arterial injection, intravenous injection, intramuscular injection, hepatic artery injection, subcutaneous injection, local injection, intraperitoneal injection, oral administration, intranasal administration and rectal administration, Preferably parenterally, more preferably intravenously. When using the intravenous administration method, it is possible to prevent side effects of hemorrhage and tissue damage due to the use of topical injection and arterial injection, and it can be administered simply because it can prevent open surgery due to the use of portal vein injection or hepatic arterial injection. In addition, when intravenous injection of a cell therapy agent for treatment of liver disease has been performed, a large number of cells remain in the lungs and cell transfer to the liver is not efficiently performed. However, the composition of the present invention can be effectively administered by intravenous injection, Can be transferred to the liver, which is simple to use, and has excellent marking and therapeutic effects.

The composition of the present invention may further comprise a filler, an excipient, a disintegrant, a binder, a lubricant, and the like. The composition of the present invention may also be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

The pharmaceutical composition of the present invention may be formulated to further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier refers to a carrier or diluent that does not significantly stimulate the organism and does not interfere with the biological activity and properties of the administered ingredients. The pharmaceutically acceptable carrier in the present invention may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more components of these components, If necessary, other conventional additives such as antioxidants, buffers and bacteriostats may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, or tablets such as aqueous solutions, suspensions, emulsions and the like.

The present invention also includes a method for preventing or treating a disease by administering a pharmaceutical composition comprising the hyaluronic acid derivative or a salt thereof as a surface active agent.

The hyaluronic acid derivative or its salt may be applied to the above-mentioned preventive or therapeutic method.

The disease may be a prophylactic or therapeutic use of a disease depending on a therapeutic use of a surface-modified cell, and preferably the disease may be a liver disease. For example, the liver disease may be at least one selected from the group consisting of liver cancer, liver metastasis cancer, liver cirrhosis, cirrhosis, hepatitis, and liver fibrosis, but the present invention is not limited thereto and specific diseases are determined depending on cells modified with hyaluronic acid derivatives or salts thereof .

Another example of the present invention includes a cell surface modification method using the hyaluronic acid derivative. The cell surface modification method comprises the steps of: (1) washing cells with a buffer solution and then treating trypsin; (2) removing the cells of step (1) and re-dispersing the cells in a buffer solution; (3) adding the hyaluronic acid derivative to the re-dispersed cells of step (2); (4) mixing the resultant of step (3), incubating, and washing with buffer solution.

The hyaluronic acid derivative or a salt thereof may be applied to a cell or cell surface modification method in which the surface is modified with the hyaluronic acid derivative or a salt thereof.

PBS may be used as the buffer solution used in steps (1), (2) and (4) of the cell surface modification method. Preferably, the buffer solution has a pH of 6 to 8, preferably a pH of 7 to 7.5, and a pH of 7.4 can be used. For example, the buffer solution used in the present invention may be a pH 7.4 phosphate or serum-free buffer.

The hyaluronic acid derivative of the step (3) may be added in a state dissolved in an aqueous solution.

The incubation reaction time in the step (4) may be 0 to 20 minutes, more preferably 5 to 10 minutes.

When WGA is bound to the hyaluronic acid derivative, the WGA concentration of the hyaluronic acid derivative may be 0 to 20 ug / mL, preferably 5 to 10 ug / mL based on the cell concentration of 1 × 10 6 / mL, It is possible to make the liver target efficiently with a low cytotoxicity.

The cell used for the cell surface modification using the hyaluronic acid derivative according to an embodiment of the present invention is not particularly limited but may be a cell that can be used as a cell therapy agent and is preferably a mammalian cell or a mammalian cell- Genetically modified cells, and may include, for example, red blood cells or stem cells, preferably mesenchymal stem cells.

The hyaluronic acid derivative can be safely applied to the human body and is also capable of reacting with a specific amino acid of a protein to maximize bioactivity of the protein. The hyaluronic acid derivative can improve the cell surface modification efficiency and enable liver- It can be applied as an effective cell carrier.

In addition to the liver-targeted delivery characteristics of hyaluronic acid derivatives capable of modifying the cell surface, mucoadhesive mucoadhesive may be used to deliver cells to the brain via nasal injection, It can stay longer in target organs longer than unmodified cells.

The present invention relates to a hyaluronic acid derivative for single cell surface modification and a method for producing the hyaluronic acid derivative, and the cells coated with a hyaluronic acid derivative can be targeted to the liver through intravenous injection. Biocompatibility, biodegradability and liver-target transferring characteristics, and can be safely applied to the human body by using a hyaluronic acid derivative capable of efficiently modifying the cell surface, and is applicable to various liver transfusion systems through intravenous injection The present invention relates to a method for preparing a hyaluronic acid derivative for cell surface modification, which can be used for a drug delivery system and tissue engineering of a cell carrier, and an intravenous injection of a cell surface-treated with hyaluronic acid derivative can do.

The liver-targeted cell delivery system using the hyaluronic acid derivative of the present invention is a liver-targeted cell delivery system capable of conducting a reaction between a hyaluronic acid derivative and a cell in an aqueous solution by a simple method, Can be used as a cell therapy agent for treating one or more liver diseases selected from the group consisting of liver cancer, liver metastasis cancer, liver cirrhosis, cirrhosis, hepatitis and liver fibrosis. It can also be used for tissue regeneration, such as regenerating liver tissue by delivering stem cells to the liver. Therefore, the cell therapeutic agent using the hyaluronic acid derivative of the present invention can deliver the liver effectively during intravenous injection, and can exhibit an excellent therapeutic effect.

FIG. 1 schematically shows the liver target transferring system after surface modification of cells with hyaluronic acid derivatives.
FIG. 2 shows various hyaluronic acid derivatives that can be used in the present invention. FIG. 2 (a) shows lipid-bound hyaluronic acid derivatives, FIG. 2 (b) shows 1,6 hexane diamine-maleimide bonded hyaluronic acid derivatives, And Fig. 2D schematically illustrates the addition of maleimide-PEG, maleimide-PEG-lipid as a thiol-bonded hyaluronic acid derivative.
FIG. 3 is a schematic view illustrating a reaction process in a method for preparing a hyaluronic acid-aldehyde and a hyaluronic acid-protein derivative using the hyaluronic acid-aldehyde according to an embodiment of the present invention.
4 shows GPC results of WGA-conjugated hyaluronic acid derivatives prepared by the method according to an embodiment of the present invention.
5A is a result of a toxicity test on a stem cell (rMSC) of a WGA-conjugated hyaluronic acid derivative prepared by a method according to an embodiment of the present invention.
FIG. 5B shows IC ₅₀ values derived from the toxicity test results shown in FIG. 5A.
FIG. 6 is a result of a toxicity test on a stem cell (hMSC) of a WGA-conjugated hyaluronic acid derivative prepared by a method according to an embodiment of the present invention.
FIG. 7 shows a comparison of results of circular dichroism (CD) analysis of WGA and WGA-conjugated hyaluronic acid derivatives.
FIG. 8 schematically shows a method of modifying a cell surface using a WGA-conjugated hyaluronic acid derivative according to an embodiment of the present invention.
FIG. 9 shows changes in cell viability with time after modification of the cell surface using a WGA-conjugated hyaluronic acid derivative according to an embodiment of the present invention.
10 is a graph showing the zeta potential of a cell surface modified using a WGA-conjugated hyaluronic acid derivative according to an embodiment of the present invention.
FIGS. 11A and 11B show the result of modifying the cell surface using a WGA-conjugated hyaluronic acid derivative according to an embodiment of the present invention and confirming the cell surface through a confocal microscope and flow cytometry.
12A and 12B are results obtained by modifying the cellular surface of rMSC and hMSC using a WGA-conjugated hyaluronic acid derivative according to an embodiment of the present invention, and then observing cellular uptake with time using a confocal microscope .
13A and 13B show the effect of co-incubation of HA or WGA on the cell binding of WGA-conjugated hyaluronic acid derivative when the surface is modified with a WGA-conjugated hyaluronic acid derivative using a confocal microscope and a flow cytometer.
FIG. 14 shows the results of confirming the binding efficiency of WGA-conjugated hyaluronic acid derivative and HA to cells according to an embodiment of the present invention using a flow cytometer.
FIG. 15 shows the effect of the TRAIL protein on the secretory profile of the transgenic W19-conjugated hyaluronic acid derivative-modified transgenic cells according to the present invention over time according to the EILSA assay.
FIGS. 16A and 16B illustrate the transwell coculture assay and the in vitro drug effect of the surface modified genetically modified cell of cancer cells using the MTT assay. FIG.
17 is an optical microscope image showing whether red blood cells surface-modified with a WGA-conjugated hyaluronic acid derivative according to an embodiment of the present invention can bind to a macrophage Raw 264.7.
FIGS. 18A and 18B are ex vivo images obtained by intravenously injecting the surface-modified cells into an animal in order to confirm the cell targeting effect of the WGA-conjugated hyaluronic acid derivative using the IVIS imaging system.
Figure 19 is a graphical representation of the ex vivo images obtained in Figures 18a and 18b quantitatively.
FIG. 20 shows the result of confirming the distribution of stem cells by fluorescence microscope by sectioning lungs and liver taken from FIGS. 18A and 18B.
Figures 21a, 21b and 21c show the cells stained with WGA-conjugated hyaluronic acid derivatives conjugated with CFSE and Hilyte 647 through a confocal microscope to confirm the biodistribution of stem cells, This is an ex vivo image obtained using the IVIS imaging system.
Figure 22 is a graphical representation of the ex vivo images obtained in Figures 21b and 21c quantitatively.
FIG. 23 shows the results of confirming the distribution of stem cells by fluorescence microscope by sectioning the lungs and liver taken from FIGS. 21B and 21C.
Figure 24 is a photograph before incorporating a fluorescence microscope photograph of the liver in Figure 23;
FIG. 25 shows confirmation of coliboring of erythrocytes with a WGA-conjugated hyaluronic acid derivative conjugated with Cy5 and FITC through a confocal microscope.
26A and 26B are in vivo images obtained by intravenous injection of the co-labeled erythrocytes shown in FIG. 25 using the IVIS imaging system by time, and quantified and graphically shown.

Hereinafter, various embodiments of the present invention will be described. However, this is an example of the invention, and the scope of the invention is not limited thereby.

Example  One. WGA - Preparation and Characterization of Coupled Hyaluronic Acid Derivatives

1-1: hyaluronic acid- Aldehyde  Preparation of derivatives

Hyaluronic acid (HA) (MW = 100 kDa) was dissolved in water at a concentration of 10 mg / ml, and sodium periodate (NaIO ₄ ) was added at 1 mole per hyaluronic acid unit. And then dialyzed against distilled water for 3 days to obtain a hyaluronic acid-aldehyde derivative having a different substitution rate by freeze-drying for 3 days.

In order to analyze the replacement ratio of the hyaluronic acid-aldehyde derivative, the hyaluronic acid-aldehyde derivative thus prepared was dissolved in an acetate buffer of 100 mM and pH 5.2 at a concentration of 5 mg / ml, and each of the hyaluronic acid- Tert butyl carbazate (TBC) and sodium cyanoborohydride (NaCNBH3) were added to the reaction mixture for 24 hours to react with the hyaluronic acid-aldehyde derivative in which the TBC was bonded to the aldehyde group contained in the hyaluronic acid- Aldehyde-TBC was prepared and dialyzed with distilled water for 3 days using MWCO 7000 dialysis membrane. After 3 days of lyophilization, 1 H-NMR (DPX300, Bruker, Germany) was used to analyze the replacement rate of aldehyde.

In the 1 H-NMR spectrum of the hyaluronic acid-aldehyde-TBC derivative, in addition to the hyaluronic acid peak, three methyl (methyl) peaks of TBC representing nine hydrogens at d = 1.2 to 1.4 ppm were obtained. For quantitative analysis, the methyl resonance of the acetamido functional group of hyaluronic acid (d = 1.85-1.95 ppm) was defined as the internal standard of the acetamido moiety of hyaluronic acid.

The replacement ratio of the hyaluronic acid-aldehyde was calculated by comparing the peak area of d = 1.85 to 1.95 ppm and the peak area of d = 1.2 to 1.4 ppm in the result of 1 H-NMR analysis. As a result, a replacement ratio of about 10 mol% was derived, and it was confirmed that a hyaluronic acid-aldehyde derivative having a substitution ratio of about 10 mol% was produced.

1-2: WGA - Preparation of conjugated hyaluronic acid derivatives

The hyaluronic acid-aldehyde derivative obtained in Example 1-1 was dissolved in an acetate buffer of 100 mM and pH 5.5 at a concentration of 5 mg / ml. Then, WGA having the amino acid sequence of SEQ ID NO: 1 was dissolved in one molecule of hyaluronic acid (100 kDa) ) Was added in an amount of 4 moles per molecule of hyaluronic acid-aldehyde chain, and the reaction rate was 95 mol% or more.

Sodium cyanoborohydride (NaCNBH ₃ ) was added in an amount of 5 moles based on 1 mole of the aldehyde according to the substitution ratio of the hyaluronic acid-aldehyde derivative analyzed according to the method of Example 1-1 and reacted for 24 hours to prepare WGA To prepare a hyaluronic acid derivative. Ethyl carbazate was added to the aldehyde in 5 moles of the aldehyde for blocking the remaining aldehyde without further reaction, followed by further reaction for 24 hours. In this example, amino-ethanol was used as the blocking agent. In this case, amino ethanol was added in 5-fold molar amount of the aldehyde, the pH was raised to 8 using NaOH, Further reaction. The reaction solution was dialyzed against a phosphate buffer of pH 7.4 and stored at -70 ° C.

1-3. GPC  By analysis WGA  Confirm binding

The formation of WGA-conjugated hyaluronic acid derivatives was confirmed by GPC (Gel Permeation Chromatography) analysis of the WGA-conjugated hyaluronic acid derivatives prepared in Example 1-2. GPC analysis of the WGA-conjugated hyaluronic acid derivative using HPLC (High Performance Liquid Chromatography) was carried out and the analysis conditions were as follows.

≪ GPC analysis condition >

Pump: Waters 1525 binary HPLC pump

Absorbance meter: Waters 2487 dual λ absorbance detector

Sampler: Waters 717 plus auto-sampler

Column: Waters Ultrahydrogel 1000 + Waters Ultrahydrogel 500

Mobile phase: PBS (pH 7.4)

Flow rate: 0.4 mL / min.

Measured wavelength: Dual detection at 210 nm and 280 nm.

The GPC analysis results are shown in Fig. 4 shows GPC results of WGA-conjugated hyaluronic acid derivatives prepared by the method according to an embodiment of the present invention.

As can be seen from FIG. 4, the retention time of the high molecular weight WGA-conjugated hyaluronic acid derivative peaked at 27 minutes compared with the peak of WGA at the retention time of 62 minutes. Thus, Respectively.

1-4: WGA - Quantitative analysis of conjugated hyaluronic acid derivatives

The WGA standard solution was prepared by diluting the 1 mg / ml WGA solution to 0.5, 1, 5, 10, 20, 30, and 50 μg / ml, respectively. The standard curve was obtained by Bradford assay. That is, WGA standard solution and Coomassie Protein Assay Reagent were mixed at a volume ratio of 1: 1, incubated at room temperature for 10 minutes, and absorbance was measured at 595 nm to obtain a standard curve.

The WGA-bound hyaluronic acid derivative prepared in Example 1-2 and WGA were diluted 100-fold and the absorbance was measured by the Bradford assay under the same conditions, and the content of WGA was determined by substituting the standard curve.

As a result, the ratio of the amount of WGA present in the WGA-conjugated hyaluronic acid derivative to the amount of WGA (feed) added in the synthesis of the WGA-conjugated hyaluronic acid derivative was 95% or more, Was found to be 95 mol% or more.

1-5: WGA - CD analysis of conjugated hyaluronic acid derivatives

Circular Dichroism (CD) was performed using WGA solution and WGA-conjugated hyaluronic acid derivative solution based on WGA concentration (0.25 mg / ml). The analysis conditions are as follows.

<CD analysis condition>

UV spectrophotometer: JASCO J-715

Measurement conditions: 180 to 250 nm, N ₂ atmosphere

A quartz cuvette: 2 mm path length

Raw data: 0.2 mm interval according to reaction time 1 second.

As shown in FIG. 7, the spectrum of WGA was in agreement with the peak of CD of WGA-conjugated hyaluronic acid derivative, and it was found that the secondary structure of WGA was retained in the state of being bonded to hyaluronic acid.

Example  2. WGA -Cell surface of conjugated hyaluronic acid derivative By reforming  Analysis of cell viability due to

2-1. WGA -Cell surface modification of conjugated hyaluronic acid derivatives

To confirm the cell surface modification of the WGA-conjugated hyaluronic acid derivative, FITC, a fluorescent substance, was attached to the hyaluronic acid derivative of Example 1-2 to modify the cell surface. Specifically, the WGA-conjugated hyaluronic acid derivative of Example 1-2 had a WGA concentration of 2 mg / ml, and FITC was added thereto in an amount of 5 times. A PD 10 column was used to remove FITC that was not conjugated to the WGA-conjugated hyaluronic acid derivative. The cell surface was modified using the thus prepared WGA-conjugated hyaluronic acid derivative-FITC.

To prepare cells modified with WGA-conjugated hyaluronic acid derivatives, cultured cells were washed with pH 7.4 phosphate buffer, treated with trypsin, removed from the culture dish and placed in a pH 7.4 phosphate buffer solution at a suitable cell concentration, &Lt; / RTI &gt; In this case, as a redispersion solution, a serum-free buffer solution other than a phosphate buffer solution is also possible. WGA-conjugated hyaluronic acid derivative solution was added to the microtube in which the cells were redispersed to such an extent as not to exhibit cytotoxicity, and the cell / WGA-conjugated hyaluronic acid derivative mixture was mixed well and incubated for 10 minutes on ice. -Linked hyaluronic acid derivative was allowed to react. Thereafter, the cells were washed with pH 7.4 phosphate buffer to remove WGA-bound hyaluronic acid derivatives not bound to the cell surface. Although FITC enters the cell, it has been confirmed that WGA-bound hyaluronic acid derivative binds to the cell surface due to WGA binding to the cell surface. On the other hand, it was confirmed that hyaluronic acid does not bind to the cell surface.

11 and 12, FIG. 11 shows the result of confirming the cell surface modification through a confocal microscope and flow cytometry, FIG. 12 shows a cellular uptake according to time under a confocal microscope As a result, it was confirmed that a WGA-conjugated hyaluronic acid derivative was present on the cell surface even after 1 hour.

10, when the cell surface was modified with a WGA-conjugated hyaluronic acid derivative, the charge density of the hyaluronic acid was much larger than that of the WGA-bound molecule, so that the zeta potential was lower than that of the unmodified control .

2-2: WGA - Cytotoxicity analysis of conjugated hyaluronic acid derivatives

Mesenchymal stem cells are seeded in 96 well plates at a concentration of 10 ⁴ cells / well and cultured for 24 hours. After 24 hours, 100 μl of the 1 mg / ml WGA solution and the WGA-bound hyaluronic acid derivative solution diluted to various concentrations based on the WGA concentration were added to a 96-well plate and incubated for 24 hours Respectively.

After 24 hours, the toxicity of WGA-conjugated hyaluronic acid derivatives to cells was assayed using MTT assay. The result of the toxicity test is shown in FIG. 5 and FIG. 6. FIG. 5A is a result of toxicity test of the WGA-conjugated hyaluronic acid derivative prepared by the method of the present invention to stem cells (rMSC) 5b shows IC ₅₀ values derived from the toxicity test results shown in FIG. 5A. FIG. 6 is a result of a toxicity test on a stem cell (hMSC) of a WGA-conjugated hyaluronic acid derivative prepared by a method according to an embodiment of the present invention.

As shown in FIGS. 5A and 6, the cytotoxicity of the WGA-conjugated hyaluronic acid derivative was found to be relatively inferior in cytotoxicity at a concentration of 10 μg / ml or less based on the WGA concentration, and was lower than that of WGA . As shown in FIG. 5B, it was confirmed that the IC ₅₀ value of the WGA-conjugated hyaluronic acid derivative prepared in Example 1 was about four times larger than that of WGA.

2-3: Cell surface By reforming  Analysis of cell viability due to

The cell surface was modified using the WGA-conjugated hyaluronic acid derivative of Example 1-2. The surface was modified on the basis of the appropriate WGA concentration determined in Example 2-1.

The surface-modified cells were seeded at a concentration of 10 ⁴ cells / well in a 96-well plate, and cell viability was tested using ez-cytoxysin for a defined time (0.5, 1, 1.5, 2, 3, Respectively. The results are shown in Fig. FIG. 9 shows changes in cell viability with time after modification of the cell surface using a WGA-conjugated hyaluronic acid derivative according to an embodiment of the present invention.

As shown in Fig. 9, it was confirmed that even when the cells were surface-modified with the WGA-conjugated hyaluronic acid derivative, there was no effect on cell viability.

2-4: WGA - Identification of cell surface modification mechanism of bound hyaluronic acid derivatives

5-Aminofluorescein or Hilyte 647 was conjugated to a WGA-conjugated hyaluronic acid derivative in substantially the same manner as in Example 2-1. The competitive binding test of WGA-conjugated hyaluronic acid derivatives was performed using this.

More specifically, when modifying the cell surface according to Example 2-1, 100 μl of a 100 kDa HA solution at a concentration of 10 mg / ml or 50 μl of a WGA solution at a concentration of 2 mg / ml, together with a WGA-conjugated hyaluronic acid derivative And surface modification was carried out. The concentration of WGA-conjugated hyaluronic acid derivatives in cells was confirmed by using a confocal microscope and a flow cytometer. Experimental results are shown in Figs. 13A and 13B. 13a and 13b show the effect of WGA-conjugated hyaluronic acid derivatives on cell binding when reacted with free HA or free WGA at surface modification with WGA-conjugated hyaluronic acid derivatives using a confocal microscope and a flow cytometer It is confirmed.

As shown in FIGS. 13A and 13B, the fluorescence intensity was the brightest in the surface modified cells without the competitive binding substance, and the fluorescence intensity was the weakest in the cells modified with the surface added with free WGA. In addition, the analysis of flow cytometry showed that the binding efficiency of WGA-conjugated hyaluronic acid derivatives to modified cells with free HA was higher by 97% than that of cells modified with free WGA (72.4%). That is, it was found that the WGA present in the WGA-conjugated hyaluronic acid derivative mainly binds to sialic acid present on the cell surface, thereby modifying the cell surface.

2-5: WGA - Confirmation of cell surface binding efficiency of conjugated hyaluronic acid derivatives

Hyaluronic acid and WGA-conjugated hyaluronic acid derivatives were used to determine the binding efficiency to the cell surface using flow cytometry. More specifically, Hilyte 647 amine was conjugated to the hyaluronic acid-aldehyde prepared in Example 1. WGA-conjugated hyaluronic acid derivatives having Hilyte 647 conjugated according to Example 1 were synthesized by adding WGA to half of this material. The cell surface was modified according to Example 2 using hyaluronic acid and WGA-conjugated hyaluronic acid derivatives (50, 25, 12.5, and 6.25 μg / ml) based on HA concentration, respectively, The cell surface binding efficiency was confirmed by an analyzer. FIG. 14 shows the results of confirming binding efficiency of WGA-conjugated hyaluronic acid derivative and HA to cells according to an embodiment of the present invention using a flow cytometer.

As can be seen from FIG. 14, it was confirmed that WGA-conjugated hyaluronic acid derivatives can modify the cell surface more efficiently than hyaluronic acid based on the same HA concentration.

division HA concentration (μg / ml) 50 25 12.5 6.25 HA 5.2% 2.1% 0.5% 0.5% HA-WGA 95.1% 93.6% 91.2% 89.6%

Example  3. WGA - Determination of the effect of conjugated hyaluronic acid derivatives on transgenic cells

ELISA assays were used to determine the effect on the TRAIL protein release profile by modifying the surface of genetically modified cells to secrete TRAIL protein into WGA-conjugated hyaluronic acid derivatives.

Transgenic cells (cells in which rat bone marrow-derived mesenchymal stem cells were genetically modified to secrete TRAIL protein, professor Seong Young Chul, Department of Bioscience and Biotechnology, POSTECH), and WGA-conjugated hyaluronic acid derivatives prepared in Example 1-2, The cells were cultured in a transwell cell insert to modify the surface of the genetically modified cell with the hyaluronic acid derivative. The cell culture medium was extracted at a predetermined time (culture time: 1, 4, 12, 24, 48, 72 hours) Respectively. This was analyzed using a TRAIL ELISA kit, and the results of the analysis are shown in FIG. FIG. 15 is a graph showing the effect of the TRAIL protein on the secretory profile of the transgenic WGA-conjugated hyaluronic acid derivative modified by the EILSA assay according to the present invention.

As can be seen in FIG. 15, transgenic cells surface-modified with WGA-conjugated hyaluronic acid derivatives showed TRAIL protein emission patterns similar to those of unmodified transgenic cells, and cell surface modification affected TRAIL protein release .

Example  4. WGA - surface with conjugated hyaluronic acid derivative Reformed  Of genetically modified cells in vitro  Check the effectiveness of treatment

A transwell co-culture assay was conducted to confirm the in vitro therapeutic effect on cancer cells using transgenic cells modified with WGA-conjugated hyaluronic acid derivatives.

 HepG2, a hepatoma cell, was seeded at a concentration of 1 × 105 cells / well in a 24-well plate. After 24 hours, the transgenic cells were seeded on the upper surface of the transwell at a concentration of 105 cells / well and incubated for 72 hours (FIG. 15A).

The genetically modified cells (cells obtained by transfection of a rat bone marrow-derived mesenchymal stem cell to secrete a TRAIL protein, Professor Sung Young Chul of the Department of Bioscience and Biotechnology, Pohang University of Science and Technology) were subjected to surface modification (HA / TRAIL- rMSC), control (TRAIL-rMSC) and rMSC were used. Because rMSC itself may have a therapeutic effect, it was used as a control for TRAIL-rMSC. After that, MTT assay for HepG2 was performed to perform a cytotoxicity test. FIG. 16 shows the result of confirming the in vitro drug activity of tumor-modified GMC cells against cancer cells using the MTT assay.

As can be seen from FIG. 16, it was found that the transgenic cells surface-modified with the WGA-conjugated hyaluronic acid derivative sufficiently exhibited the therapeutic effect on cancer cells.

Example  5 WGA - surface with conjugated hyaluronic acid derivative Reformed  Cell in vitro  Check binding

An optical microscope was used to confirm the in vitro binding of red cells modified with WGA-conjugated hyaluronic acid derivatives to macrophage Raw 264.7.

The surface of the erythrocytes extracted from the human was modified by the method of Example 2-1 to prepare a surface-modified red blood cell with a WGA-conjugated hyaluronic acid derivative. Raw 264.7 Macrophages were seeded in cell culture dishes and WGA-conjugated hyaluronic acid derivatives were added to the surface-modified red blood cells. Ten minutes later, the cells were washed with phosphate buffer solution to remove red blood cells not bound to macrophages. FIG. 16 shows the result of confirming whether or not red blood cells surface-modified with WGA-conjugated hyaluronic acid derivative can bind to macrophage Raw 264.7 through an optical microscope.

As can be seen in FIG. 17, it was confirmed that red blood cells surface-modified with WGA-conjugated hyaluronic acid derivatives can bind to macrophages due to hyaluronic acid, whereas non-surface modified red blood cells do not bind with macrophages.

Example  6. WGA - surface with conjugated hyaluronic acid derivative Reformed  Liver of stem cells Targeting (biodistribution)

In vivo body distribution of WGA-conjugated hyaluronic acid-derivatized stem cells was determined by IVIS imaging system and fluorescence microscope.

First, it was confirmed that hyaluronic acid was responsible for liver-targeted transfer of surface-modified stem cells with WGA-conjugated hyaluronic acid derivatives. WGA-FITC surface-modified stem cells and HA-WGA-FITC surface modified stem cells were added to Rat at 10 ⁶ cells were intravenously injected and sacrificed 4 hours later, and ex vivo imaging was performed using an IVIS imaging system (FIG. 18). Further, the extracted lungs and liver were sectioned, and stem cells distributed in lung and liver tissues were identified through a fluorescence microscope, and the results are shown in FIG.

FIGS. 18A and 18B are ex vivo images obtained by intravenous injection into an animal using a cell using an IVIS imaging system. FIG. Figure 19 is a graphical representation of the ex vivo images obtained in Figures 18a and 18b quantitatively. FIG. 20 shows the result of confirming the distribution of stem cells by fluorescence microscope by sectioning lungs and liver taken from FIGS. 18A and 18B.

As a result, WGA-FITC-surface-modified stem cells showed similar fluorescence intensities in the liver and lungs, whereas WGA-conjugated hyaluronic acid derivative-modified stem cells clearly showed higher intensity in the liver than in the lungs And Fig. 19). The results of examining the lung and liver tissues sectioned by fluorescence microscopy also showed the above trend (Fig. 20). It was confirmed that hyaluronic acid produced liver-target ability by modifying the surface of stem cells with WGA-conjugated hyaluronic acid derivative.

Second, the in vivo body distribution of surface-modified stem cells and WGA-conjugated hyaluronic acid derivatives was confirmed. A WGA-conjugated hyaluronic acid derivative having Hilyte 647 conjugated with Hilyte 647 NHS ester was prepared by the method for producing WGA-conjugated hyaluronic acid derivative conjugated with FITC of Example 2-1. Stem cells (purchased from Neuromics Co., Ltd.) were first stained with CFSE (Sigma). Half of the stained cells were surface-modified with Hilyte 647 conjugated WGA-conjugated hyaluronic acid derivatives (Fig. 21A). The cells were intravenously injected with 106 cells per rat in Rat, and sacrificed 4 hours later, and ex vivo imaging was performed using IVIS imaging system (Fig. 21b, c). Further, the extracted lungs and liver were sectioned, and stem cells distributed in the lung and liver tissue were identified through a fluorescence microscope, and the results are shown in FIGS. 23 and 24. FIG. Cells modified with hyaluronic acid alone without WGA were excluded in vivo as they were judged to have reduced cell surface binding ability in vitro (eg, flow cytometry).

FIG. 21A shows the cells stained simultaneously with WGA-conjugated hyaluronic acid derivatives conjugated with CFSE and Hilyte 647 through a confocal microscope to confirm the biodistribution of stem cells. FIG. 21B and FIG. This is an ex vivo image obtained using the IVIS imaging system. Figure 22 is a graphical representation of the ex vivo images obtained in Figures 21b and 21c quantitatively. FIG. 23 shows the results of confirming the distribution of stem cells by fluorescence microscope by sectioning the lungs and liver taken from FIGS. 21B and 21C. FIG. 24 is a photograph before merge of a fluorescence microscope photograph of the liver in FIG. 23; FIG.

As a result, stem cells not surface-modified are mainly present in the lungs and WGA-binding hyaluronic acid derivatives are surface-modified in the liver (FIGS. 21 and 22). As a result of examining the liver tissue sectioned by fluorescence microscopy, And the fluorescence signal of Hilyte 647 conjugated to the WGA-conjugated hyaluronic acid derivative was observed at the same position (FIGS. 23 and 24). Through this, it was confirmed that liver target function was generated by modifying the surface of stem cells with WGA-conjugated hyaluronic acid derivative.

Example  7. WGA - surface with conjugated hyaluronic acid derivative Reformed  Of red blood cells Liver targeting

We used bovine red blood cells to identify biodistribution in BALB / c mice similar to stem cells.

To do this, the surface of red blood cells was stained with Cy5 (lumiprobe), and further surface modification was carried out using WGA-conjugated hyaluronic acid derivatives conjugated with FITC (sigma). This was confirmed by using a confocal microscope (Fig. 25). In vivo imaging was performed using the IVIS imaging system at predetermined time intervals (0.5, 2, 18, 48 hours) The results are shown in Fig. The hyaluronic acid aldehyde derivative showed high HA (high) and low HA (low).

As a result, it was confirmed by a confocal microscope that WGA-conjugated hyaluronic acid derivatives successfully bonded to Cy5 and FITC on the surface of erythrocytes can be colabelled (FIG. 25). As can be seen from FIG. 26, it was confirmed that the WGA-conjugated hyaluronic acid derivative surface-modified red blood cells having the best liver target-directing ability was most abundant and long-lived in the liver.

Example  8. Preparation of lipid-bound hyaluronic acid derivative (HA-lipid)

A HA solution having a molecular weight of about 100 kDa was prepared to give a concentration of 5 mg / ml during the reaction. To this was added NH ₂ -PEG-lipid 5 times the equivalent of HA and completely dissolved by stirring. EDC was added thereto four times as much as the HA equivalent and completely dissolved by stirring. Sulfo-NHS was added in the same amount as EDC and dissolved by stirring. The resulting mixed aqueous solution was reacted for 6 hours while maintaining the pH of 6 by adding 1 N HCl. After 6 hours, 1N NaOH was added to stop the reaction and the pH was raised to 7.4. The reaction product was placed in a dialysis tube (MW 7000 Da) and dialyzed against 100 mM NaCl aqueous solution for 60 hours. After that, dialysis was repeated for 25% ethanol and distilled water, respectively. The aqueous solution with impurities removed by dialysis was lyophilized for 3 days to obtain a pure HA-lipid derivative. The results of conjugating COOH and NH ₂ -PEG-lipid present in HA using the EDC / sulfo-NHS chemistry are shown in FIG.

Example  9. Maleimide -Linked hyaluronic acid derivative (HA- maleimide )

A HA solution having a molecular weight of about 100 kDa was prepared to give a concentration of 5 mg / ml during the reaction. Hexamethylendiamine, a kind of diamine, was added thereto in an amount equivalent to HA equivalent to 10 times, and was completely dissolved by stirring. EDC was added thereto four times as much as the HA equivalent and completely dissolved by stirring. Sulfo-NHS was added in the same amount as EDC and dissolved by stirring. The resulting mixed aqueous solution was reacted for 2 hours while adding 1 N HCl to maintain a pH of 6. After 2 hours, 1N NaOH was added to stop the reaction and the pH was raised to 7.4. The reaction product was placed in a dialysis tube (MW 7000 Da) and dialyzed against 100 mM NaCl aqueous solution for 60 hours. After that, dialysis was repeated for 25% ethanol and distilled water, respectively. The aqueous solution with impurities removed by dialysis was lyophilized for 3 days to obtain a pure HA-HMDA derivative as a precursor of HA-maleimide.

HA-HMDA was added with 3-maleimidopropionic acid 10 times the equivalent of HA and completely dissolved by stirring. EDC was added thereto four times as much as the HA equivalent and completely dissolved by stirring. Sulfo-NHS was added in the same amount as EDC and dissolved by stirring. The resulting mixed aqueous solution was reacted for 2 hours while adding 1 N HCl to maintain a pH of 6. After 2 hours, 1N NaOH was added to stop the reaction and the pH was raised to 7.4. The reaction product was placed in a dialysis tube (MW 7000 Da) and dialyzed against 100 mM NaCl aqueous solution for 60 hours. After that, dialysis was repeated for 25% ethanol and distilled water, respectively. The aqueous solution from which the impurities were removed through dialysis was freeze-dried for 3 days to synthesize a maleimide-introduced hyaluronic acid derivative.

Using the EDC / sulfo-NHS chemistry as described above, an amine group-induced HA conjugate was prepared by conjugating COOH and amine groups of hexamethylendiamine present in HA, and 3-maleimidopropionic acid was added to this conjugate using EDC / sulfo-NHS chemistry The result of derivation of the maleimide group is shown in Fig. 2B.

Example  10. Thiol -Linked hyaluronic acid derivative (HA- SH )

First, cystamine was introduced into HA by using EDC / sulfo-NHS chemistry, and DTT or TCEP treatment was performed for 1 to 2 hours to break the S-S bond. 200 mg of hyaluronic acid (HA) (MW = 100 kDa) was dissolved in 20 ml of distilled water and then 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and sulfo-NHS were added to the HA Fold, and activated for 30 min. Cystamine dihydrochloride was then added at a 20-fold molar ratio to the carboxyl group of HA and the pH of the solution was adjusted to 4.8, followed by overnight incubation. After the reaction was completed, the solution was subjected to ethanol precipitation to remove impurities and the remaining precipitate was dissolved in 20 ml of distilled water to obtain tris (2-carboxyethyl) phosphine (TCEP) The pH of the solution was adjusted to 6 and the reaction was allowed to proceed for 4 hours. After the reaction was completed, impurities were filtered through ethanol precipitation, and the remaining precipitate was dissolved in 20 ml of distilled water and lyophilized for 3 days to obtain HA-SH. Confocal microscope results of the prepared HA-SH are shown in FIG. 2C.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Hyaluronic acid derivatives and composition for cell-surface          engineering using the same <130> DPP20154789 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 171 <212> PRT <213> WGA protein <400> 1 Gln Arg Cys Gly Glu Gln Gly Ser Asn Met Glu Cys Pro Asn Asn Leu   1 5 10 15 Cys Cys Ser Gln Tyr Gly Tyr Cys Gly Met Gly Gly Asp Tyr Cys Gly              20 25 30 Lys Gly Cys Gln Asp Gly Ala Cys Trp Thr Ser Lys Arg Cys Gly Ser          35 40 45 Gln Ala Gly Gly Ala Thr Cys Thr Asn Asn Gln Cys Cys Ser Gln Tyr      50 55 60 Gly Tyr Cys Gly Ply Gly Ala Gly Tyr Cys Gly Ala Gly Cys Gln Gly  65 70 75 80 Gly Pro Cys Arg Ala Asp Ile Lys Cys Gly Ser Gln Ala Gly Gly Lys                  85 90 95 Leu Cys Pro Asn Asn Leu Cys Cys Ser Gln Trp Gly Phe Cys Gly Leu             100 105 110 Gly Ser Glu Phe Cys Gly Gly Gly Cys Gln Ser Gly Ala Cys Ser Thr         115 120 125 Asp Lys Pro Cys Gly Lys Asp Ala Gly Gly Arg Val Cys Thr Asn Asn     130 135 140 Tyr Cys Cys Ser Lys Trp Gly Ser Cys Gly Ile Gly Pro Gly Tyr Cys 145 150 155 160 Gly Ala Gly Cys Gln Ser Gly Gly Cys Asp Gly                 165 170

Claims (28)

A hyaluronic acid derivative comprising a unit represented by the following formulas (1) to (3) or a salt thereof; or
A cell surface modification composition comprising a hyaluronic acid derivative or a salt thereof, comprising a unit represented by the following formulas (1) and (4):
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

In the above formulas,
Wherein P is a wheat germ agglutinin protein which is a water soluble protein;
X is -COR ¹ , -CONR ² H-R ¹ or -CONH-R ² -NH-R ¹ ;
Wherein R ¹ is selected from the group consisting of a vinyl group, a thiol group, a poly-L-lysine, a lipid, an acrylate group, a maleimide group or N-hydroxysuccinimide (NHS) ;
R ² is an alkyl group having 1 to 6 carbon atoms or alkylene;
Wherein the sum of m, n and 1 is an integer of 50 to 10,000, the sum of n and 1 is an integer of 5 to 5,000 and 1 is an integer of 1 to 100; s is an integer of 1 to 2000; delete The composition of claim 1, wherein X is -COR ¹ , R ¹ is a lipid or thiol group, and the sum of m and s is an integer from 50 to 10,000. The method of claim 1, wherein X is -CONH-R ² -NH-R ¹ , R ¹ is maleimide, R ² is hexyl, and the sum of m and s is an integer of 50 to 10,000 , Composition. The composition of claim 1, further comprising a conjugate wherein the composition is chemically conjugated with maleimide, maleimide-PEG, or maleimide-PEG and lipid. delete The composition of claim 1, wherein the composition modifies the cell surface to deliver the cells to liver tissue. The composition according to claim 1, wherein the cell is at least one selected from the group consisting of red blood cells, stem cells and genetically modified cells thereof. The cell surface modification composition according to claim 1, which comprises a hyaluronic acid derivative represented by the following formula (5) or a salt thereof:
[Chemical Formula 5]

In Formula 5,
P is a water soluble protein;
The sum of m, n and 1 is an integer of 50 to 10,000, the sum of n and 1 is an integer of 5 to 5,000, and 1 is an integer of 1 to 100. The cell surface modification composition according to claim 1, which comprises a hyaluronic acid derivative represented by the following formula (6) or a salt thereof:
[Chemical Formula 6]

In Formula 6,
X is -COR ¹ , -CONR ² H-R 1 or -CONH-R ² -NH-R ¹ ;
Wherein R ¹ is selected from the group consisting of a vinyl group, a thiol group, a poly-L-lysine, a lipid, an acrylate group, a maleimide group or N-hydroxysuccinimide (NHS) ;
R ² is an alkyl group having 1 to 6 carbon atoms;
Wherein the sum of m and s is an integer of 50 to 10,000 and s is an integer of 1 to 2000. A hyaluronic acid derivative comprising a unit represented by the following formulas (1) to (3) or a salt thereof; or
A pharmaceutical composition for the prevention or treatment of liver disease, comprising a cell surface-modified with a hyaluronic acid derivative or a salt thereof comprising the unit represented by the following formulas (1) and (4) as an active ingredient:
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

In the above formulas,
Wherein P is a wheat germ agglutinin protein which is a water soluble protein;
X is -COR ¹ , -CONR ² H-R ¹ or -CONH-R ² -NH-R ¹ ;
Wherein R ¹ is selected from the group consisting of a vinyl group, a thiol group, a poly-L-lysine, a lipid, an acrylate group, a maleimide group or N-hydroxysuccinimide (NHS) ;
R ² is an alkyl group having 1 to 6 carbon atoms or alkylene;
Wherein the sum of m, n and 1 is an integer of 50 to 10,000, the sum of n and 1 is an integer of 5 to 5,000 and 1 is an integer of 1 to 100; s is an integer of 1 to 2000; 12. The composition of claim 11, wherein the composition has liver-specific properties. 12. The composition of claim 11, wherein the cell is at least one selected from the group consisting of red blood cells, stem cells, and genetically modified cells thereof. 12. The composition according to claim 11, wherein the liver disease is at least one selected from the group consisting of liver cancer, liver metastatic cancer, liver cirrhosis, cirrhosis, hepatitis, and liver fibrosis. delete A first step of producing a hyaluronic acid-aldehyde (HA-aldehyde) by opening a ring structure of hyaluronic acid by reacting hyaluronic acid with an oxidizing agent under dark conditions; And
A second step of reacting the hyaluronic acid-aldehyde and the N-terminus of the water-soluble protein in an aqueous solution to prepare a hyaluronic acid derivative or a salt thereof, wherein the hyaluronic acid derivative comprises a unit represented by the following Chemical Formulas 1 to 3:
[Chemical Formula 1]

(2)

(3)

Wherein P is a wheat germ agglutinin protein which is a water soluble protein;
The sum of m, n and 1 is an integer of 50 to 10,000, the sum of n and 1 is an integer of 5 to 5,000, and 1 is an integer of 1 to 100. delete 17. The method according to claim 16, wherein the hyaluronic acid-aldehyde is a compound represented by the following structural formula:
[Chemical Formula 8]

In Formula 8,
The sum of m and n is an integer of 50 to 10,000, and n is an integer of 5 to 5,000. 17. The method according to claim 16, wherein the hyaluronic acid derivative is a protein having a molecular number of 1 to 10 molecules per molecule of the hyaluronic acid-aldehyde. 17. The method according to claim 16, wherein the substitution ratio of aldehyde groups after the first step is 10 to 50 mol%. 17. The method according to claim 16, wherein the oxidizing agent is added in an amount of 1 to 10 moles per unit of the hyaluronic acid derivative. 17. The method according to claim 16, wherein the oxidizing agent is sodium periodate. 17. The method of claim 16, wherein the second step is carried out in a buffer solution at a pH of from 5 to 7. 17. The process according to claim 16, wherein the second step further comprises the reaction of sodium cyanoborohydride. 17. The method according to claim 16, further comprising a third step of blocking remaining aldehyde groups not reacted with the protein represented by the following Reaction Scheme 1:
[Reaction Scheme 1]

In the above Reaction Scheme 1,
P is a water soluble protein;
Wherein the sum of m, n and 1 is an integer of 50 to 10,000, the sum of n and 1 is an integer of 5 to 5,000 and 1 is an integer of 1 to 100;
A is an amine blocking material. 26. The process according to claim 25, wherein A is a straight or branched alkylcarbazate having 1 to 5 carbon atoms, or a lower alkanolamine having 1 to 5 carbon atoms. The method of claim 26, wherein the alkylcarbazate is ethylcarbazate or tertbutylcarbazate, the alkanolamine is selected from the group consisting of aminomethanol, aminomethanol, aminopropanol, aminobutanol, , Or aminopentanol. 26. The method according to claim 25, wherein the third step is performed by adding amines to the molar unit of the aldehyde group in an amount of 5 to 10 moles and reacting for 12 to 24 hours.

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