KR100993006B1 - Chitosan coated alginate sponge and method for preparing thereof - Google Patents

Abstract

The present invention relates to an alginic acid sponge coated with chitosan, and a method for preparing the same, and more particularly, to crosslinking, neutralizing, and neutralizing an alginic acid sponge intermediate prepared by molding, freeze-drying an aqueous alginic acid solution, and coating and neutralizing chitosan for physical and tissue engineering. It relates to an alginic acid sponge having a and a method for producing the same.

Chitosan, alginic acid sponge, crosslinking, neutralization

Description

Chitosan coated alginate sponge and method for preparing honey}

The present invention relates to an alginic acid sponge coated with chitosan and a method for preparing the same, and more particularly, to cross-linking an alginic acid sponge intermediate prepared by molding and freeze-drying an aqueous alginic acid solution, and to coating and neutralizing chitosan, which is suitable for medical and tissue engineering. It relates to an alginic acid sponge having a and a method for producing the same.

Alginic acid is a polysaccharide of marine plants that is equivalent to cellulose of land plants. It is a linear copolymer composed of α- (1 → 4) -L-gluronic acid and β- (1 → 4) -D-mannuronic acid.

In general, alginic acid is a major component present between the cell membrane and the cell membrane of brown algae, which is the most productive algae among marine organisms, and has been commercially obtained from laminaria and giant kelp, and is widely used as a water-soluble sodium alginate salt. Water-soluble alginic acid salts are thickeners, stabilizers, emulsifiers, microcapsule materials, etc. in food, pharmaceutical, and textile industries due to their viscosity, biodegradability, nontoxicity, and easy gel formation by polyvalent metal ions (eg Ca ²⁺ ). It is widely used.

In addition, a variety of products using alginic acid have been sold in the fields of medical wound coating materials, topical hemostatic agents, etc., by utilizing the properties of alginic acid, such as biodegradability, hygroscopicity, hemostatic action, and biocompatibility.

The alginate wound coating material is the most widely used non-woven fabric made of calcium alginate fiber prepared by spinning an aqueous sodium alginate solution in a coagulation bath consisting of calcium chloride aqueous solution, for example Kaltostat (Convatec, USA), Sorbsan (Bertek, UK) , Nu-DERM (Johnson & Johnson, USA), and Tegagen (3M, USA). However, the alginate wound coating material in the form of the nonwoven fabric has a problem in that short fibers separated from the nonwoven fabric remain after being attached to the skin damage site and are difficult to be transformed into various forms, so that a specific molding is required in the field of tissue culture. There is a problem that it is not suitable to use.

Chitosan has a structure linked to (1,4) -2-amino-2-deoxy-β-o-glucan by deacetylation of chitin. It is mainly found in shells of arthropods such as crabs and shrimps. Its annual biosynthesis is 1010-1011 tons, the second most abundant organic matter after cellulose on earth.

Previous attempts have been made to use chitin as a material for the treatment and tissue engineering of wounds due to its inherent antimicrobial and biocompatibility, but its use has been limited due to the limitation of solvents that can dissolve chitin. However, chitosan is easily soluble in weak acids, so it is actively applied to hematology, immunology, wound healing, drug delivery, and molding materials.

To date, there have been chitosan wound coating materials in the form of fibers, films, and nonwoven fabrics, but each form has various problems such as the dropping of short fibers in the nonwoven fabrics and the lack of the ability to absorb wound secretions in the fiber and film forms. Due to its difficulty, it is not suitable for use in the field of tissue culture in which specific molding is required.

Meanwhile, after the reaction of alginic acid with chitosan in Korean Patent Publication No. 1999-49107, water is added to adjust the pH of the mixed solution to 2 to 3 to form an electrolyte complex, which is dissolved after washing, washed and lyophilized to form an electrolyte composite sponge. Although a method for preparing the present invention is disclosed, moldability and flexibility are reduced because of molding into a precipitate, which is an electrolyte composite, and there is a problem in that the manufacturing process is complicated because the manufacturing process requires centrifugation and washing. In addition, Korean Patent Laid-Open Publication No. 2002-75539 discloses a wound coating material prepared by mixing a water-soluble chitosan oligomer and alginic acid in an aqueous solution, but has a problem in that the shape is uneven and it is difficult to control the size and mechanical properties of the pores.

Thus, the present inventors have studied to solve the above problems, unlike the existing method, the alginate aqueous solution formed by molding, freeze-dried crosslinking the alginate sponge intermediate material, chitosan coating and neutralization lacking the existing moldability The present invention has been completed by solving the problem of mechanical property control impossible, process complexity, and developing alginate sponges having suitable properties for medical and tissue engineering.

Accordingly, the present invention has the advantages of chitosan, such as antibacterial, biocompatibility, and alginic acid, such as hygroscopicity, hemostatic action, and also has a chitosan-coated alginic acid sponge having easy properties for wound healing and tissue engineering and Its purpose is to provide a method for producing the same.

The present invention is characterized by an alginate sponge coated with chitosan having a maximum bending angle (flexibility) of 90 ° or more, an apparent density of 0.005 to 0.1 g / cm ³ , and a saline absorption rate (%) of 100 to 700%.

In addition, the production method of the alginic acid sponge is another feature.

The present invention is described in more detail below.

The present invention relates to an alginic acid sponge prepared by molding and freeze-drying an aqueous alginic acid solution, to an alginic acid sponge coated with a chitosan, and to neutralizing the alginic acid sponge, and to a method for preparing the alginic acid sponge coated with neutralized chitosan.

The manufacturing process of the alginic acid sponge coated with chitosan according to the present invention will be described in detail as follows.

a) Alginate sponge intermediate

This step is to prepare a sponge intermediate material by molding and freeze-dried aqueous alginic acid solution.

The aqueous alginic acid solution is prepared by dissolving alginic acid in pure water. The alginic acid used herein is an alginic acid or an alginic acid alkali metal salt, and it is particularly preferable to use sodium alginate, potassium alginate, ammonium alginate, and the like having excellent water solubility. Moreover, it is preferable that the density | concentration of the alginic-acid aqueous solution is 0.1 to 30%. In this case, when the concentration is less than 0.1%, it is difficult to mold into a sponge, and if it exceeds 30%, it is difficult to dissolve and the flexibility of the manufactured sponge is inferior.

Molding of the aqueous alginic acid solution is carried out through a method such as injection into the molding die, coating, die casting, extrusion, and the like, preferably after degassing the aqueous alginic acid solution.

Alginate aqueous solution molded as described above is molded and lyophilized to prepare an alginate sponge intermediate.

b) preparation of alginic acid sponge coated with chitosan

This step is a step of crosslinking and chitosan coated the alginic acid sponge intermediate prepared in step a), followed by washing and drying to produce a chitosan coated alginic acid sponge.

The alginic acid sponge intermediate prepared above is immersed in a crosslinking solution and crosslinked, washed with water, and then immersed in an aqueous chitosan acid solution to coat chitosan. It is neutralized with alkaline solution, washed with water and dried.                     

At this time, it is possible to dissolve the crosslinking agent together in the chitosan acid aqueous solution to simultaneously proceed the crosslinking and chitosan coating process.

The chitosan used in the present invention preferably has a deacetylation degree of 50% or more and a molecular weight ranging from 10,000 to 1,000,000, more preferably a deacetylation degree of 85% or more and a molecular weight of 50,000 to 200,00. Use it. In this case, when using chitosan having a molecular weight of less than 10,000 may cause a phenomenon in the sponge, chitosan having a molecular weight of more than 1,000,000 is not preferable because there is a problem that the flexibility is very poor when forming a sponge.

The chitosan aqueous solution is preferably dissolved in chitosan 0.0001 to 20% (w / v) in 0.5 to 10% (v / v) acetic acid, hydrochloric acid, folic acid, citric acid, lactic acid and formic acid. In this case, if chitosan is dissolved at less than 0.0001% (w / v), the effect of chitosan coating cannot be obtained, and if it exceeds 20% (w / v), it is difficult to dissolve and the flexibility of the sponge is lowered.

The crosslinking agent used for the crosslinking of the alginic acid sponge intermediate may be a divalent metal salt or an organic crosslinking agent having a covalent binding ability. For example, as the divalent metal salt, calcium chloride, zinc chloride, or the like may be used, and an organic crosslinking agent having a covalent bond may be glutaraldehyde, dicyclohexyl carbodiimide, hexamethylene diisocyanate, or the like. The concentration of the crosslinking agent is not particularly limited and various concentrations may be used depending on the required performance and appearance of the sponge.

As an alkaline solution used for neutralization, an aqueous sodium hydroxide solution (preferably 0.01-2.0 M aqueous sodium hydroxide), an aqueous ammonium hydroxide solution (preferably 0.05-2.0 M aqueous ammonium hydroxide), and an aqueous sodium hydroxide alcohol solution (preferably 0.01-) 2.0 M sodium hydroxide 30-100% aqueous alcohol solution, ammonium hydroxide aqueous alcohol solution (preferably 0.05-2.0 M aqueous ammonium hydroxide 30-100% alcohol) or reconstituted buffer solution (2.2 g NaHCO ₃ , 4.77 g HEPES (200 Mm) ) / 100 ml 0.05N NaOH) and the like can be used. More preferably, an aqueous 30-100% alcohol solution of 2.0 M sodium hydroxide or an aqueous 30-100% alcohol solution of 2.0 M ammonium hydroxide is used.

The sponge thus obtained is not only excellent in hygroscopicity, wound healing ability, antibacterial property, hemostatic property, but also excellent in moldability and flexible, and has a dense structure with uniform pores in the inner and outer layers of the sponge, making it suitable for use in medical or tissue engineering materials. Do.

In order to evaluate the flexibility of the chitosan coated alginic acid sponge, the maximum bending angle at which the structure of the sponge was not broken was adopted as an evaluation criterion. Sponge obtained by the present invention showed a maximum flexibility of 90 ° or more of the sponge showed excellent flexibility.

As an evaluation criterion for evaluating the denseness of the sponge structure, an apparent density relatively showing the average size of pores was adopted. The chitosan coated alginic acid sponges prepared in the present invention have an apparent density of 0.005 to 0.1 g / cm ³ .

If the maximum bending angle is less than 90 °, the sponge is not flexible enough to cover the curved wound effectively, and if the apparent density is less than 0.005g / cm ³ , the sponge's structure is sparse and dense and easily broken. If there is more than 0.1g / cm ³ , the structure is more dense than necessary, the surface is rough, there is a disadvantage that the flexibility and water absorption is low.

In addition, the alginic acid sponge coated with chitosan of the present invention has a saline absorption rate of 100 to 700%, and when manufactured to less than 100%, the absorbency of various exudates is lowered, so that the effect of wound treatment may not be sufficiently exhibited. If it is made higher than 700% by failing to create a wet environment of the wound site by the absorption of excess exudate, there is also a problem that the effect of wound healing is reduced.

As described above, the alginic acid sponge coated with chitosan according to the present invention has excellent physical properties for use in medical and tissue engineering.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1

Sodium alginate (Medium viscosity, SIGMA) was dissolved in pure water at a concentration of 1% by weight to prepare an aqueous sodium alginate solution. The aqueous solution was degassed by depressurizing for 4 hours, then poured into a plastic container and frozen at -40 ° C. It was freeze-dried for 60 hours to prepare an alginic acid sponge intermediate.                     

The alginic acid sponge intermediate was immersed in a 0.2 M aqueous calcium chloride solution for 20 minutes to be crosslinked and washed with water.

A chitosan acetic acid aqueous solution was prepared by dissolving chitosan in a 1% (v / v) acetic acid aqueous solution to a solution of 0.1% (w / v). The crosslinked alginic acid sponge was immersed in this chitosan acetic acid aqueous solution to coat chitosan on the surface, and this was immersed in a 2.0 M aqueous sodium hydroxide solution to neutralize and wash. It was frozen at -40 ° C and lyophilized for 30 hours to prepare an alginic acid sponge coated with chitosan.

Example 2

Alginate sponge intermediate was prepared in the same manner as in Example 1. Chitosan was dissolved in a 1% (v / v) acetic acid aqueous solution at a concentration of 0.2% (w / v), and then calcium chloride was added thereto to a concentration of 0.3 M. The alginic acid sponge intermediate prepared above was immersed in this solution for 30 minutes to perform crosslinking and chitosan coating.

Sodium hydroxide was dissolved in 2.0 M in ethanol and 1: 1 water solution. The sponge was immersed in a neutral state, washed with water, and then dried in a warm air dryer for 24 hours to prepare an alginic acid sponge coated with chitosan.

Example 3

Alginate sponge intermediate was prepared in the same manner as in Example 1.

A chitosan acetic acid aqueous solution was prepared by dissolving chitosan in a 2% (v / v) acetic acid aqueous solution to give a solution of 0.5% (w / v). The crosslinked alginic acid sponge was immersed in the chitosan acetic acid aqueous solution to coat chitosan on the surface, and this was immersed in a 2.0 M aqueous ammonium hydroxide solution to neutralize and wash. It was frozen at -40 ° C and lyophilized for 30 hours to prepare an alginic acid sponge coated with chitosan.

Example 4

Alginate sponge intermediate was prepared in the same manner as in Example 1. Chitosan was dissolved in a 2% (v / v) acetic acid aqueous solution at a concentration of 0.5% (w / v), and then calcium chloride was added thereto to a concentration of 0.3 M. The alginic acid sponge intermediate prepared above was immersed in this solution for 30 minutes to perform crosslinking and chitosan coating.

Ammonium hydroxide was dissolved in 2.0M aqueous solution of ethanol and 1: 1 water. The sponge was immersed in a neutral state, washed with water, and then dried in a warm air dryer for 24 hours to prepare an alginic acid sponge coated with chitosan.

Test Example

The flexibility, average size of pores and water absorption of the alginic acid sponge coated with chitosan prepared in Examples 1 to 4 were measured, and the results are shown in Table 1 below.

1) Flexibility (°): The maximum bending angle at which the sponge structure is not broken was measured and evaluated.

2) Average pore size (g / cm 3): apparent density was measured and evaluated by comparative comparison.

3) Absorption rate (%): The sponge sample was dried in a dryer at 60 ° C. for 24 hours to measure its weight (A), and then immersed in 0.9% aqueous sodium chloride solution at 25 ° C. for 48 hours, and centrifuged in a centrifuge. After centrifugation at 160 G for 5 minutes, the weight (B) was measured, and the absorption rate was measured according to the following equation (1).

division Example 1 Example 2 Example 3 Example 4 Flexibility (°) 152 143 150 148 Average size of pores (g / cm ³ ) 0.0391 0.0311 0.0350 0.0368 Absorption rate (%) 310 299 302 315

As shown in Table 1, the alginic acid sponges prepared in Examples 1 to 4 showed excellent physical properties for use in medical and tissue engineering because of excellent flexibility, average pore size (structural density) and absorption rate. .

As described above, according to the present invention, alginic acid sponges coated with chitosan having physical properties usable for medical and tissue engineering can be manufactured by a simple process, and the sponges thus prepared have excellent flexibility, structural compactness, absorbency, and processability. It is suitable for use in medical and tissue engineering applications.

Claims (9)

a) molding an aqueous alginic acid solution and freeze drying to prepare an alginic acid sponge intermediate; b) crosslinking and rinsing the alginic acid sponge intermediate in an aqueous solution of a crosslinking agent; c) coating the chitosan by immersing the crosslinked alginic acid sponge in an aqueous chitosan acid solution; And d) neutralizing the coated sponge in an alkaline solution, followed by washing and drying The method of producing an alginic acid sponge coated with chitosan having a maximum bending angle (flexibility) of 90 ° or more, an apparent density of 0.005 to 0.1 g / cm ³ , and a saline absorption rate (%) of 100 to 700%. The method of claim 1, wherein the steps b) and c) are performed simultaneously. The method of claim 1, wherein the alginic acid of step a) is an alginic acid or an alginic acid alkali metal salt. The method of claim 1, wherein the crosslinking agent of step b) is a divalent metal salt or an organic crosslinking agent having a covalent bond. The method of claim 1, wherein the chitosan of step c) has a deacetylation degree of 50% or more and a molecular weight ranging from 10,000 to 1,000,000. The method of claim 1, wherein the acid of step c) is 0.5-10% acetic acid, hydrochloric acid, folic acid, citric acid, lactic acid or formic acid. The method of claim 1, wherein the alkaline solution of step d) is sodium hydroxide solution, ammonium hydroxide solution, sodium hydroxide solution, ammonium hydroxide solution or buffer solution (2.2g NaHCO ₃ , 4.77g HEPES (200Mm) / 100ml 0.05 N NaOH) method for producing an alginic acid sponge coated with chitosan. Alginate sponge, characterized in that produced by the manufacturing method of claim 1. delete