KR100493461B1 - Natural polymers bonded adhesion molecular, their preparation and their use - Google Patents

Abstract

The present invention relates to a natural polymer attached with an adhesive molecule, a method for producing the same, and a use thereof. More particularly, the present invention relates to a natural polymer substituted with a carboxyl group having an acetyl group bonded to an acetyl group and an adhesive molecule to a hydroxyl group of a natural polysaccharide polymer. The present invention relates to a method and its use, which is excellent in biocompatibility, excellent in biodegradability, inexpensive compared to synthetic polymers, and specifically combined with cells and tissues, so that extracellular matrix, artificial organs, tissue reconstruction material, and catheter It can be used in coating materials or drug delivery materials.

Description

NATURAL POLYMERS WITH Adhesion Molecules, Method of Manufacturing the Same, and Uses thereof {NATURAL POLYMERS BONDED ADHESION MOLECULAR, THEIR PREPARATION AND THEIR USE}

The present invention relates to a natural polymer attached with an adhesive molecule, a method for producing the same, and a use thereof. More particularly, the present invention relates to a natural polymer substituted with a carboxyl group having an acetyl group bonded to an acetyl group and an adhesive molecule to a hydroxyl group of a natural polysaccharide polymer. It relates to a method and its use.

Multinational pharmaceutical companies and research institutes in the US, Europe and Japan are interested in the discovery of biocompatible natural polymers that are expected to be highly biocompatible in recent years in order to prevent various harmful effects from the use of synthetic polymers. However, except for tablets or coatings for oral administration of drugs, these materials are rarely commercialized.

Currently, natural polymers are mainly used in anti-adhesion membranes, injectables, transdermal absorbents, and cosmetics industries. Specifically, anti-adhesion means the adhesion of each organ which is the most fatal side effect after surgery, and the anti-adhesion anti-adhesion market for anti-adhesion is expected to exceed about US $ 100 million in 2003 in the US alone. Interceed Co. of the United States has developed and marketed a TC-7 membrane (US Pat. No. 5,629,294) using oxidized cellulose. Genzyme Co. has developed a hyaluronic acid (HA). Seprafilm (US Pat. No. 5,017,229 (1991)), which combines carboxymethylcellulose (CMC) with carboxymethylcellulose, is commercially available. In particular, Seprafilm is expected to be effective as an anti-adhesion barrier that can separate the physically wounded tissue from the tissue, but the clinical evaluation of various malignant factors in the abdominal cavity has not yet been efficiently performed. As such, marketability is not increasing significantly.

Development of injection materials using natural polymers can be divided into the case of producing microspheres of about 10 to 100 μm and using them for local injection and the case of making them to 1 μm or less and using them as intravenous nanoparticles. In the case of Spear, Professor SS Davis of the University of Nottingham, UK, succeeded in delivering a vaccine by attaching PEG to dextran by surface modification. M. Linaudo (M.) of Joseph Fourier University, France The use of starch has been reported by the Rinaudo professor team, as well as the use of casein, a natural protein, by the team of Professor EP Goldberg of the University of Florida. Intravenous nanoparticles have been reported by Sunamoto's team at the Japanese University of Hardness, Japan, to combine hydrophobic cholesterol with hydrophobic cholesterol to produce nanoparticles of less than 100 μm and then attempt to deliver insulin using them. The intravenous nanoparticles are not yet marketed, but are expected to form a huge market worldwide as the availability of gene carriers, specific long-term targeting carriers, etc. increases rapidly.

Natural polymers have very good mucosal adhesion properties as well as biological stability and can be very advantageously used for drug delivery system through nasal and oral mucosa. For a long time, a scientific approach to external preparations has been attempted to develop relatively good products that have improved percutaneous permeability. In particular, since the 1970s, various percutaneous permeability measurement models and devices have been developed to improve the percutaneous permeability. GD Searle's scopolamine patch is used to improve the short duration of skin remaining, the shortcoming of typical external preparations, and to achieve systemic treatment effects to improve the patient's convenience and maximize the therapeutic effect. Various hormones and analgesics in pharmaceutical companies such as Alza, Boehringer Ingelheim, Galaxo, Lederle, Novartis, Janssen and Schwarz. It has been developing various patches such as antihypertensives and nicotine preparations. Sales increased from $ 1.9 billion in 1995 to $ 4 billion in 1998 and are expected to generate $ 5.3 billion in 2000 (Source: Technology Catalyst International). This is the highest growth rate when compared to the route of administration of the drug.

In addition, natural polymers are used in the cosmetics industry.In addition to the simple level of skin protection and skin care, natural polymers are researched and developed with a lot of manpower and money to develop functional cosmetics for the purpose of physiological activities such as anti-aging, wrinkle removal, and antioxidant. There is a situation. Many multinational companies have already found success in applying products containing functionalities such as propolis and curdlan.

As described above, although the utility of natural polymers is very high, the biocompatibility, biostability, and biodegradability of these derivatives through themselves and chemical modifications are not completely disclosed, and thus are subject to many industrial restrictions.

Thus, the present inventors prepared a natural polymer substituted with a carboxyl group having an acetyl group bonded to an acetyl group and an adhesion molecule to a hydroxyl group of a natural polysaccharide polymer, and the natural polymer has excellent biocompatibility, excellent biodegradability, and The present invention was completed by finding that it can specifically bind.

It is an object of the present invention to provide a natural polymer having an adhesive molecule attached thereto, a method for producing the same, and a use thereof.

In order to achieve the above object,

The present invention provides a natural polymer substituted with a carboxyl group having an acetyl group in which an acetyl group and an adhesive molecule are bonded to a hydroxy group of a natural polysaccharide polymer.

In addition, the present invention provides a method for producing the natural polymer.

The present invention also provides a use of the natural polymer.

Hereinafter, the present invention will be described in detail.

The present invention includes a natural polymer substituted with a carboxyl group having an acetyl group in which an acetyl group and an adhesion molecule are bonded to a hydroxyl group of a natural polysaccharide polymer.

The natural polysaccharide polymer uses pullulan (pullulan), curdlan (chidlan), chitosan (chitosan) or chondroitin sulfate (chondroitin sulfate), the natural polysaccharide polymer is excellent in biocompatibility, excellent biodegradability, degradation products In view of this stable sugar, it can be used as a biomaterial. In addition, the price is cheaper than the synthetic polymer, there is an advantage that exists in abundance in Korea.

The carboxyl group which has the amide group which the adhesive molecule substituted by the hydroxy group of the said polysaccharide couple | bonded is represented by following General formula (1).

(Wherein R is an adhesive molecule)

The R is an adhesion molecule, specifically a monomer represented by the following formula (2), the weight average molecular weight of 12,000 heparin or GRGDS represented by SEQ ID NO: 1, consisting of YIGSR represented by SEQ ID NO: 2 and IKVAV represented by SEQ ID NO: 3 It is selected from the group of peptides.

The substitution degree of the carboxyl group which has the amide group which the said adhesive molecule couple | bonded is 10 to 30%, Preferably it is 15 to 20%. When the substitution degree is less than 10%, the adhesion molecule and the binding force is weak, if more than 30% there are too many carboxyl groups that do not bond with the adhesion molecule adversely affects the interaction with the cells.

The natural polysaccharide polymer is an amphiphilic polymer having amphiphilic (amphiphilic), a portion of the hydroxy group of the natural polysaccharide polymer is substituted with an acetyl group, and some of the hydroxyl group and the carboxyl group of the insoluble monomer is composed of an ester bond. The degree of substitution of the acetyl group is 20 to 40%, preferably 23 to 30%, more preferably 25%. If the degree of substitution of the acetyl group is less than 20%, the hydrophilicity is too large, and if it is 40% or more, the hydrophilicity is too low to have amphiphilicity.

In addition, the present invention includes a method for producing the natural polysaccharide polymer.

Specifically, the step of reacting the natural polysaccharide polymer and acetic acid or acetic anhydride in the presence of a base to replace a part of the hydroxy group with an acetyl group (step 1);

Reacting the natural polysaccharide polymer substituted with the acetyl group with the insoluble monomer in the presence of a binder to generate an ester bond of a part of the hydroxy group and the carboxyl group of the insoluble monomer (step 2);

Reacting the obtained natural polysaccharide polymer with a carboxylating agent in the presence of a catalyst and a binder to replace a part of the hydroxy group with a carboxyl group (step 3); And

The obtained natural polysaccharide polymer and heparin and / or an adhesion molecule selected from peptide groups consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are reacted in the presence of a binder to replace some hydroxy groups with a carboxyl group having an amide group bonded to the adhesion molecule. It includes a method of producing a natural polysaccharide polymer consisting of a step (step 4).

Step 1 is acetylation to obtain the amphiphilic nature of the natural polysaccharide polymer, the base is preferably pyridine, the binder is N, N -dicyclohexyl carbodiimide ( N, N ' -dicyclohexyl carbodiimide) And / or dimethylaminopyridine. At this time, the reaction temperature is 50 to 60 ° C, and the reaction time is preferably 5 to 10 hours.

Step 2 is for insolubilizing the obtained natural polysaccharide polymer, and the insoluble monomer is preferably deoxycholic acid (hereinafter referred to as "DOCA"). The reaction mechanism of the above step is to generate an ester bond by reacting the hydroxy group of the polysaccharide with the carboxyl group of DOCA.

Step 3 is to carboxylate the natural polysaccharide polymer to conjugate the heparin and / or peptide having an amide group to the obtained natural polysaccharide polymer, wherein the carboxylating agent uses succinic anhydride, and the catalyst is preferably tree Ethylamine.

Step 4 is to replace an amide group having a carboxyl group bonded to a carboxyl group of a part of the hydroxy group of the obtained natural polysaccharide polymer.

In addition, the present invention includes an extracellular matrix, a tissue reconstruction material, a catheter coating material or a drug delivery material consisting of the natural polysaccharide polymer.

In order to examine the cell adhesion of the natural polysaccharide polymer of the present invention, the polysaccharide conjugated with the adhesion molecule GRGDS (SEQ ID NO: 1) was cultured PC12 cell line secreting dopamine affecting Parkinson's disease. As a result, as shown in FIG. 1 , the polysaccharides containing GRGDS increase rapidly with time, whereas the polysaccharides without GRGDS do not grow much over time. This is because GRGDS-recognized PC12 cells proliferate after adhesion, whereas anchorage-dependent PC12 cells do not adhere to polysaccharides to which ligands are not conjugated. The negatively charged cells are bound to adhere to the positively charged matrix. These nonspecific bindings are not only weak in interaction, but also inevitably broken in weak impact. However, receptor-mediated binding is not only strong, but also proliferates rapidly for signal transduction delivered by these receptors.

 In a microenvironment, cells become deficient in oxygen, glucose, nutrients, hormones and growth factors, or necrosis occurs due to changes in pH or metabolites. This necrosis is acute when cells are clustered or aggregated because of poor supply of nutrients and oxygen to the inside of the cluster. This is especially true when culturing cells in gels. In this study, this phenomenon can be observed when fixed-dependent cells are not adhered or proliferate well. Polysaccharides containing GRGDS and heparin will not rapidly necrosis over time, while polysaccharides without GRGDS and heparin may rapidly develop necrosis over time. This leads to overall necrosis in polysaccharides, whereas cells that recognize GRGDS and heparin continue to proliferate after adhesion but continue to grow in the core where no nutrients or oxygen are supplied. This is due to the poor viability of nonspecific binding cells.

An important function of PC12 cells, a type of nerve cell, is a dopamine-producing cell line that can resolve the dopamine deficiency that causes Parkinson's disease. Therefore, the method of culturing dopamine-releasing PC12 cells and transplanting them into patients with Parkinson's disease is already in clinical trials in Japan. As a result of culturing PC12 cells in GRGDS-attached natural polymer, more dopamine secretion was observed than native polymer without adhesive molecule (see Table 1 ). This process occurs when PC12 cells recognize GRGDS and adhere to the substrate to which the adhesion molecule is attached, and then produce dopamine in the cell through signal transduction in the cell membrane. Through the above results, it can be seen that the development of the extracellular matrix using the GRGDS-attached substrate is not so far as compared with the existing extracellular matrix, and the developed substrate is easy to process and easy to manage.

The natural polysaccharide polymer of the present invention having the above properties can be used for extracellular matrix, tissue reconstruction material, catheter coating material or drug delivery material.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention.

Example 1 Preparation of Natural Polymer with Adhesive Molecules

(Step 1) Hydrophobicization of Polysaccharides by Acetylation (hereinafter referred to as "PA")

As shown in Scheme 1 below, pullulan was chemically modified by acetylation to obtain amphiphilic properties.

After dissolving 2 g of furan in 50 ml of formamide, 6 ml of pyridine and 15 ml of acetic anhydride were added and reacted at 54 ° C. for 6 hours. The product obtained is obtained by precipitating the reaction in 200 ml of water and then recovering it and freezing and drying it again. Fulan acetate is confirmed by FT-IR and NMR.

(Step 2) pullulan derivative hydrophobized by insoluble monomer

In order to insolubilize the polysaccharide, an insoluble monomer, deoxycholic acid (hereinafter referred to as "DOCA"), was used to react an OH of the polysaccharide with COOH of the DOCA to generate an ester bond.

5 g of polysaccharide was added to 50 ml of dimethyl sulfoxide (DMSO) from which water was completely removed, and the temperature was raised to 60 ° C. to dissolve the polysaccharide, which was then allowed to stand at room temperature. Various concentrations of DOCA were added to the dissolved DMSO solution at room temperature, and then DCC (N, N'-dicyclohexyl carbodiimide) and DMAP (Dimethylaminopyridine) were added at the molar ratio. Dicyclohexylurea (DCU) produced during the reaction was removed by filtration, and the solution was dialyzed in distilled water and lyophilized to obtain a polysaccharide-DOCA conjugate.

(Step 3) Carboxylated PA Preparation (PA-COOH)

PA was reacted with succinic anhydride to carboxylate to conjugate heparin or peptide with amide groups. 5.0 g of PA was dissolved in 200 ml of 1,4-dioxane and then succinic anhydride, a carboxylating agent, was added to the flask. 2 g of dimethylaminopyridine (DMAP) in the same amount of triethylamine was used as catalyst. This reaction was continued for 24 hours at room temperature. During carboxylation, the reaction was placed in 300 ml of carbon tetrachloride to remove unreacted reactant. The solution was filtered and the filtrate was precipitated in cold diethyl ether.

(Step 4) Heparin or Peptide / Polysaccharide Conjugate Preparation (PA-CONH-GRGDS)

5 g of carboxylated polysaccharides were dissolved in 50 ml of dimethylsulfoxide (DMSO) completely dehydrated. Thereafter, various concentrations of heparin or peptide were added to the DMSO solution, and then N, N' -dicyclohexyl carbodiimide (DCC) and dimethylaminopyridine (DMAP) were added to the molar ratio for one day at room temperature. Reacted for a while. Dicyclohexylurea (DCU) produced during the reaction was removed by filtration, and the solution was dialyzed in distilled water and then lyophilized to prepare a heparin or peptide / polysaccharide conjugate. Heparin and peptide conjugated polysaccharides were analyzed by FT-IR and NMR.

Wherein R is a monomer And a peptide group consisting of heparin having a weight average molecular weight of 12,000 or GRGDS represented by SEQ ID NO: 1, YIGSR represented by SEQ ID NO: 2, and IKVAV represented by SEQ ID NO: 3).

Experimental Example 1 Degree of Substitution of Acetylated Flurane

The degree of substitution of the furan acetate prepared in Example 1 was measured using NMR, IR, GPC, and found to be 1.17 acetyl groups per molecule of glucose.

Experimental Example 2 Degree of Carboxylation of Fulan Acetate

The degree of substitution of the furan acetate prepared in Example 1 was measured using NMR, IR, GPC, and found to be 0.51 carboxyl groups per molecule of glucose.

Experimental Example 3 Measurement of Dopamine Secretion and Dopamine Secretion of PC12 Cell Line

PC12 cells, a pheochromocytoma cell line, were used to examine the interaction between the newly developed substrate and neurons. The PC12 cell line, a pheochromocytoma cell line, is reported to be able to solve the inhibition of dopamine secretion, which is a major cause of Parkinson's disease. The PC12 cell line was used to measure adhesion and secretion of dopamine in the substrate to which heparin and the peptide were conjugated. To this end, PC12 cells isolated from 0.05% tyrosine-0.02% EDTA were subdivided in a 96 well plate coated with peptides and heparin conjugated polysaccharides at a concentration of 2 × 10 ⁴ / ml. Incubated for 3 days in RPMI-10% FCS, and then incubated with fresh RPMI 1640 medium for 2-4 hours. The culture was harvested and stored frozen until dopamine measurement. In addition, in order to determine the dopamine secretion by long-term culture was incubated for 2 weeks under the above conditions. All experiments were performed three times.

(1) Cell adhesion experiment

Cell culture was performed using GRGDS conjugated polysaccharide as an adhesion molecule. Parkinson's disease, caused by the lack of dopamine that causes Parkinson's disease, can be treated with PC12 cell lines that secrete dopamine. First, PC12 cells were removed from the flask, and the sterile peptides and heparin conjugated polysaccharides were dispensed. At this time, the number of mixed PC12 cells was about 20,000. PC12 cells were incubated under 37% oxygen at 5% carbon and 95% oxygen. The results are shown in Fig.

As shown in FIG. 1 , it can be seen that the polysaccharides containing GRGDS increase rapidly with time, whereas the polysaccharides without GRGDS do not proliferate with time. This is because GRGDS-recognized PC12 cells proliferate after adhesion, whereas anchorage-dependent PC12 cells do not adhere to polysaccharides to which ligands are not conjugated. The negatively charged cells are bound to adhere to the positively charged matrix. These nonspecific bindings are not only weak in interaction, but also inevitably broken in weak impact. However, receptor-mediated binding is not only very strong, but also rapidly propagates by signal transduction delivered by these receptors.

(2) cell survival observation

In a microenvironment, cells become deficient in oxygen, glucose, nutrients, hormones and growth factors, or necrosis occurs due to changes in pH or metabolites. This necrosis is acute when cells are clustered or aggregated because of poor supply of nutrients and oxygen to the inside of the cluster. This is especially true when culturing cells in gels. In this study, this phenomenon can be observed when fixed-dependent cells are not adhered or proliferate well. Polysaccharides containing GRGDS and heparin will not rapidly necrosis over time, while polysaccharides without GRGDS and heparin may rapidly develop necrosis over time. This leads to overall necrosis in polysaccharides, whereas cells that recognize GRGDS and heparin continue to proliferate after adhesion but continue to grow in the core where no nutrients or oxygen are supplied. This is due to the poor viability of nonspecific binding cells.

(3) Dopamine Secretion Test

An important function of PC12 cells, a type of nerve cell, is a dopamine-producing cell line that can resolve the dopamine deficiency that causes Parkinson's disease. Therefore, the method of culturing dopamine-releasing PC12 cells and transplanting them into patients with Parkinson's disease is already in clinical trials in Japan. As a result of culturing PC12 cells in GRGDS-attached natural polymer, more dopamine secretion was observed than native polymer without adhesive molecule (see Table 1 ). This process occurs when PC12 cells recognize GRGDS and adhere to the substrate to which the adhesion molecule is attached, and then produce dopamine in the cell through signal transduction in the cell membrane. Through the above results, it can be seen that the development of the extracellular matrix using the GRGDS-attached substrate is not so far as compared with the existing extracellular matrix, and the developed substrate is easy to process and easy to manage.

Dopamine Secretion Measurement in Various Substrates Sample Substrate with GRGDS / Substrate without GRGDS Conventional dish 1.0 PA-COOH 1.05 PA-COGRGDS 1.45 Collagen 1.47

As described above, a natural polymer substituted with a carboxyl group having an acetyl group and an amide group bonded with an adhesive molecule to a hydroxy group of a natural polysaccharide polymer has excellent biocompatibility and excellent biodegradability, and is inexpensive and cheaper than synthetic polymers. Specific binding to the tissue can be used as extracellular matrix, artificial organs, tissue reconstruction material, catheter coating material or drug delivery material.

1 is a graph showing the adhesion experiment of pc21 cells in the present invention.

  (a) is the control,

  (b) includes PA-COOH (pullan substituted with carboxyl group),

  (c) includes PA-COGRGDS (Flurane with bonded molecules),

  (d) contains collagen (Collagen).

<110> CHOUNG, Pill-Hoon <120> NATURAL POLYMERS BONDED ADHESION MOLECULAR, THEIR PREPARATION AND THEIR USE <130> 2p-02-18c <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide 1 <400> 1 Gly Arg Gly Asp Ser 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide 2 <400> 2 Tyr Ile Gly Ser Arg 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide 3 <400> 3 Ile Lys Val Ala Val 1 5

Claims (9)

The functional groups of the following A), B) and C) are independently represented in the hydroxyl group of one natural polysaccharide polymer selected from the group consisting of pullulan, curdlan, chitosan and chondroitin sulfate. Natural polymers, characterized in that substituted. A) acetyl group B) dioxycholic acid as an insoluble monomer comprising a carboxyl group C) represented by Formula 2 below, wherein one adhesive molecule selected from the group consisting of heparin having a weight average molecular weight of 12,000, a peptide of SEQ ID NO: 1, a peptide of SEQ ID NO: 2, and a peptide of SEQ ID NO: 3 is amide-bound; Carboxyl groups. Formula 1 (In the above formula, R-NH- is an adhesion molecule.) Formula 2 delete delete delete Reacting one natural polysaccharide polymer selected from the group consisting of furan, curdlan, chitosan and chondroitin sulfate with acetic acid or acetic anhydride in the presence of a base to replace a portion of the hydroxy group of the polysaccharide with an acetyl group (step 1); Reacting a natural polysaccharide polymer substituted with an acetyl group with dioxycholic acid, an insoluble monomer, in the presence of a binder to generate a ester bond with a carboxyl group of the dioxycholic acid with a portion of the hydroxy group of the polysaccharide (step 2); Reacting the obtained natural polysaccharide polymer with a carboxylating agent in the presence of a catalyst and a binder to replace a part of the hydroxy group of the polysaccharide with a carboxyl group (step 3); And The obtained natural polysaccharide polymer and heparin, a peptide of SEQ ID NO: 1, a peptide of SEQ ID NO: 2 and one adhesion molecule selected from the group consisting of a peptide of SEQ ID NO: 3 in the presence of a binder to react with the carboxyl group substituted in step 3 Method of producing a natural polymer consisting of forming an amide bond between the amine groups (step 4). The method of claim 5, wherein the base is pyridine in the first step, the binder is N, N -, characterized in that, dicyclohexylcarbodiimide (N, N '-dicyclohexyl carbodiimide) or dimethylaminopyridine (dimethylaminopyridine) Natural polymer manufacturing method. delete 6. The method according to claim 5, wherein the carboxylating agent of step 3 is succinic anhydride and the catalyst is triethylamine. Extracellular matrix, artificial organs, tissue reconstruction material, catheter coating material or drug delivery material consisting of the natural polymer of claim 1.