KR20060002331A - A method for preparing chitosan/poly -glutamic acid polyelectrolyte complex - Google Patents

Abstract

The present invention relates to a method for producing a chitosan / poly gamma glutamic acid polymer electrolyte composite, and retains the excellent functions such as biocompatibility, antimicrobial, biodegradability of chitosan, structural and physical defects appearing when making a sponge or film with chitosan There is an excellent effect of providing a method for producing a chitosan / poly gamma glutamic acid polymer electrolyte composite exhibiting excellent properties such as high mechanical properties, structural uniformity, high cell affinity, high swelling, and the like.

In addition, the present invention chitosan / poly gamma glutamic acid polyelectrolyte complex, such as wound dressing, cell culture scaffold, artificial kidney hemodialysis membrane, artificial lung membrane, corneal graft material or contact lens material, tissue compatibility and blood compatibility material It can be applied to various fields.

Chitosan, Poly Gamma Glutamic Acid, Polymer Electrolyte Complex, Scaffold, Film

Description

Method for preparing chitosan / poly gamma glutamic acid polyelectrolyte complex {A method for preparing chitosan / poly γ-glutamic acid polyelectrolyte complex}

1 is a graph showing the degree of colloid (stable turbid dispersion) formation of the polymer electrolyte complex according to the mixing ratio of chitosan / poly gamma glutamic acid.

Figure 2 is a graph showing the degree of colloid (stable turbid dispersion) formation of chitosan / poly gamma glutamic acid polyelectrolyte complex with pH change.

Figure 3 shows a SEM photograph of the scaffold made of the present invention colloidal chitosan / poly gamma glutamic acid polyelectrolyte complex.

Figure 4 is a graph showing the pore size of the polymer electrolyte composite according to the mixing ratio of chitosan / poly gamma glutamic acid of the present invention.

5 is a graph showing the tensile strength of the film prepared with a polymer electrolyte composite according to the mixing ratio of chitosan / poly gamma glutamic acid of the present invention.

Figure 6 is a graph measuring the cell suitability of the scaffold prepared with the colloidal chitosan / poly gamma glutamic acid polyelectrolyte complex of the present invention, (a) shows the results of measuring cell adhesion, (b) shows the cell proliferation The measurement results are shown.

The present invention relates to a method for preparing chitosan / poly gamma glutamic acid polymer electrolyte composite. More specifically, the present invention retains excellent functions such as biocompatibility, antimicrobiality, and biodegradability of chitosan, and poly gamma, which is an anionic polymer, to overcome structural and physical defects that occur when sponges or films are made of chitosan. The present invention relates to a method for preparing a biomaterial having high properties such as high mechanical properties, cell affinity, swelling by introducing glutamic acid into chitosan and introducing a polymer electrolyte complex.

As chitosan is known to have excellent functions such as biocompatibility, antimicrobial activity, and biodegradability, research on this has been actively conducted. However, even though they have excellent functions, they have problems such as scaffolds and films having physical properties or structural defects. Thus, in order to solve these problems, blends of chitosan with other polymers ( Blends are being actively researched.

The method of blending chitosan and other polymers includes forming a polymer electrolyte complex with an anionic polymer and a chitosan having a cationic property. In the 1960s, Michaels et al. First applied a polymer electrolyte composite as a biomaterial. The polymer electrolyte composite is characterized by plasticization by water and electrolyte, so that it has flexibility when wetted, and exhibits high ion permeability selectivity, ion exchangeability, high water permeability, and high permeability to electrolyte and water-soluble low molecular materials. Using these properties, high molecular electrolyte complex membranes are highly regarded as hemodialysis membranes of artificial kidneys and membranes of artificial lungs, and are being actively researched as corneal graft materials or contact lens materials, tissue compatibility and blood compatibility materials. Conventionally, carboxylmethyl cellulose, dextran sulfate, alginic acid, polyvinyl alcohol sulfate as anionic polymers to form chitosan and a polymer electrolyte complex ), Carboxym ethyldextran, heparin, collagen, poly acrylic acid and the like have been used.

However, when the polymer is blended with chitosan, synthetic polymers such as poly acrylic acid have a problem of low cell affinity and biodegradability, and a high cost of natural polymers such as heparin. .

The inventors conducted experiments to solve the problems of the prior art, poly-gamma glutamic acid (poly γ-glutamic acid) is a low cost because it is a polyacrylic acid that is secreted in large quantities in microorganisms of the bacillus strain, chitosan And polymer electrolyte complex formation was found to be excellent, biodegradable, and increased cell affinity. In addition, when producing sponges and films, it has excellent properties such as uniform pore size, high porosity and mechanical properties, thereby allowing for dressing of wounds and scaffolds for cell culture. The present invention has been found to be a biomaterial that can be used in various fields such as hemodialysis membranes of artificial kidneys, membranes of artificial lungs, corneal graft materials or contact lens materials, histological synthesis, and blood compatibility materials.

Accordingly, an object of the present invention is to provide a method for preparing a chitosan and a polymer electrolyte composite that improves the conventional disadvantages related to the decrease in cell affinity, biodegradability and high cost in the preparation of a conventional chitosan and a polymer electrolyte complex.

The object of the present invention is to mix chitosan and poly gamma glutamic acid by using chitosan obtained from the shell of crab, shrimp, etc. as a cationic polymer, and using poly gamma glutamic acid obtained by separation and purification in Bacillus strain culture as an anionic polymer. Mix so that the ratio is from 100: 1 to 100: 30 or 99: 1 to 70:30 w / v or v / v% and dissolve to 1 v / v% acetic acid at a pH of 4 to 5 to the final 1 w / v%. Colloidal chitosan / poly gamma glutamic acid polyelectrolyte was obtained to obtain a scaffold, a film using the same to achieve the mechanical properties and cell compatibility.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of invention is demonstrated concretely.

The present invention provides a method for preparing a colloidal chitosan / poly gamma glutamic acid polyelectrolyte complex by mixing chitosan as a cationic polymer and poly gamma glutamic acid as an anionic polymer at a constant pH; Preparing a scaffold using the colloidal polyelectrolyte complex; Preparing a film using the colloidal polymer electrolyte composite and investigating its mechanical properties; And using the scaffold prepared above is composed of the step of examining the cell suitability of the polymer electrolyte complex of the present invention.

The composition of the polymer electrolyte composite according to the present invention is characterized by blending chitosan and poly gamma glutamic acid at 1 to 30 w / v or v / v%.

The method for preparing chitosan / poly gamma glutamic acid polyelectrolyte composite of the present invention comprises the following steps:

Deacetylation of chitin through protein removal (weak alkaline treatment), calcium removal (dilute hydrochloric acid treatment), pigment removal from shells such as crabs and shrimps to obtain chitosan,

Isolate and purify poly gamma glutamic acid from Bacillus strain culture,

The mixing ratio of chitosan and poly gamma glutamic acid is 100: 1 to 100: 30 or 99: 1 to 70:30 w / v or v / v% using the chitosan as a cationic polymer and poly gamma glutamic acid as the anionic polymer. Mixing so as to dissolve in 1 v / v% acetic acid to a final 1 w / v% at pH 4 to 5 to obtain a colloidal chitosan / poly gamma glutamic acid polyelectrolyte complex.

In the preparation method of the chitosan / poly gamma glutamic acid polyelectrolyte composite of the present invention, the pH range of the solvent dissolving chitosan and poly gamma glutamic acid is 4 to 5, and if higher than this, chitosan is precipitated, and if lower than this, polygamma glutamic acid is precipitated. do.

In the preparation method of the chitosan / poly gamma glutamic acid polymer electrolyte composite, the mixing ratio of chitosan and poly gamma glutamic acid is 100: 1 to 100: 30 or 99: 1 to 70:30, where w / v or v / v% is suitable. If it is lower than this, the properties of the polymer electrolyte composite are significantly decreased, and if it is higher than this, precipitation occurs and it becomes difficult to control the material.

When preparing the polymer electrolyte composite obtained according to the chitosan / poly gamma glutamic acid polyelectrolyte composite prepared by an application such as a sponge or a film, a process of washing acetic acid with ethanol and dH ₂ O should be essential.

Chitosan / poly-gamma glutamic acid polyelectrolyte complex according to the present invention is cheaper than conventional biomaterials and has most properties that can be applied medically, so dressing of wound, scaffold for cell culture, hemodialysis membrane of artificial kidney, It can be applied in various fields such as membrane of artificial lung, corneal graft material or contact lens material, tissue compatibility and blood compatibility material.

The formation of the polymer electrolyte complex of the present invention was confirmed by dispersing the complex in an aqueous solution and measuring turbidity at 500 nm using a UV / VIS spectrophotometer (MECASYS, Opizen 2120UV). The microstructure of the scaffold made of the polymer electrolyte composite was observed through a HITACHI S-3500N SEM machine, and the mechanical property test and the cytocompatibility test were performed to investigate the applicability as a medical product.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

EXAMPLE

Example 1 Preparation of the Chitosan / Poly Gamma Glutamic Acid Polyelectrolyte Composite of the Invention

To prepare chitosan / poly gamma glutamic acid polyelectrolyte complex of the present invention, chitin is deacetylated through protein removal (weak alkaline treatment), calcium removal (dilute hydrochloric acid treatment), pigment removal process from shells of crab, shrimp, etc. to obtain chitosan, Poly gamma glutamic acid was isolated and purified from Bacillus strain culture. The mixture ratio of chitosan and poly gamma glutamic acid is 100: 1 to 100: 30 or 99: 1 to 70:30 w / v or v / v%, and the final mixture is mixed with 1 v / v% acetic acid at pH 4-5. Dissolved to 1 w / v%.

Experimental Example 1: The degree of colloid (stable turbid dispersion) formation of the polymer electrolyte composite according to the mixing ratio of chitosan / poly gamma glutamic acid

In order to determine the degree of colloidal (stable turbid dispersion) formation of the polymer electrolyte complex according to the mixing ratio of chitosan / poly gamma glutamic acid, while increasing the mixing ratio of chitosan and poly gamma glutamic acid according to Example 1, a UV / VIS spectrophotometer (MECASYS , Opizen 2120UV) was used to measure turbidity at 500 nm.

As shown in Figure 1, up to a mixing ratio of 100/30 to form a colloid (stable turbid dispersion) it was found that the precipitation occurs at a higher mixing ratio.

Experimental Example 2: The degree of colloid (stable turbid dispersion) formation of chitosan / poly gamma glutamic acid polyelectrolyte complex with pH change

To determine the degree of colloidal (stable turbid dispersion) formation of chitosan / poly gamma glutamic acid polyelectrolyte complex with pH change, using a UV / VIS spectrophotometer (MECASYS, Opizen 2120UV) while titrating from pH1 to pH10 with 10M NaOH solution Turbidity was measured at 500 nm.

As shown in FIG. 2, colloidal (stable turbid dispersion) was formed between pH 1.2 and 5.5 and showed the highest turbidity at pH 4.3. Precipitation occurred above pH 5.5.

Example 2 Scaffolds Prepared Using the Chitosan / Poly Gamma Glutamic Acid Polyelectrolyte Composites of the Invention

The colloidal (stable turbid dispersion) chitosan / poly gamma glutamic acid polymer electrolyte composite according to Example 1 was frozen at -20 ° C and lyophilized to prepare a scaffold, followed by 100% EtOH, 70% EtOH, 50% EtOH. After washing sufficiently, the mixture was finally washed with dH ₂ O, and lyophilized to prepare a dried scaffold. The microstructure was observed by taking a SEM photograph of the prepared scaffold. Figure 3 (a) is a scaffold made of chitosan, (b) is a scaffold made of a polyelectrolyte complex having a mixing ratio of chitosan / poly gamma glutamic acid 100/1, (c) is chitosan / poly gamma A scaffold made of a polymer electrolyte complex having a mixing ratio of glutamic acid of 100/10, and (d) shows a scaffold made of a polymer electrolyte composite having a mixing ratio of chitosan / poly gamma glutamic acid of 100/30.

Figure 4 shows the pore size of the polymer electrolyte composite according to the mixing ratio of chitosan / poly gamma glutamic acid.

As shown in Figure 3, it can be seen that the uniformity of the pores in the scaffold prepared using the polymer electrolyte complex than the chitosan scaffold is significantly increased, the pore size in Figure 4 is a mixture ratio of chitosan / poly gamma glutamic acid It can be observed that as it increases, it decreases.

Example 3: Investigation of mechanical properties using a film prepared using the present invention chitosan / poly gamma glutamic acid polymer electrolyte composite

A film was prepared using the colloidal (turbid dispersion) chitosan / poly gamma glutamic acid polymer electrolyte composite according to Example 1 and the mechanical properties of the film were investigated. The chitosan / poly gamma glutamic acid colloid (turbid dispersion) was naturally dried at room temperature, thoroughly washed with 100% EtOH, 70% EtOH, 50% EtOH, and finally with dH ₂ O, and then dried again to prepare a dried film. Tensile strength was measured.

As shown in FIG. 5, it was found that the tensile strength increased as the mixing ratio of chitosan / poly gamma glutamic acid increased.

Example 4 Cell Compatibility of Scaffolds Prepared with Inventive Chitosan / Poly Gamma Glutamic Acid Polyelectrolyte Complexes

In order to investigate the cellular suitability of the scaffold made of chitosan / poly gamma glutamic acid polyelectrolyte complex, NHDF (Normal Human Dermal Fibroblast) in scaffold made of chitosan / poly gamma glutamic acid polyelectrolyte complex and scaffold made of chitosan ) Was cultured and the cell number was measured by MTT analysis. Figure 6 (a) is the result of measuring the cell adhesion, (b) shows the result of measuring the cell proliferation (cell proliferation).

As shown in Figure 6, the cell adhesion test did not find any difference between the chitosan and the polymer electrolyte complex. In the cell proliferation test, the scaffolds prepared with the polyelectrolyte complex showed more cell proliferation compared to the chitosan scaffold, and the chitosan / polyglutamic acid mixing ratio was the highest at 100/10 and 100/20. In the above mixing ratio, cell proliferation was decreased.

As described through the above Examples and Experimental Examples, the present invention relates to a method for preparing chitosan / poly gamma glutamic acid polymer electrolyte composite, and retains excellent functions such as biocompatibility, antimicrobial, biodegradability of chitosan, A method for preparing a chitosan / poly gamma glutamic acid polymer electrolyte composite exhibiting excellent properties such as high mechanical properties, structural uniformity, high cell affinity, and high swelling by overcoming the structural and physical defects that appear when making a sponge or film. There is an outstanding effect.

In addition, the present invention chitosan / poly gamma glutamic acid polyelectrolyte complex, such as wound dressing, cell culture scaffold, artificial kidney hemodialysis membrane, artificial lung membrane, corneal graft material or contact lens material, tissue compatibility and blood compatibility material It can be applied to various fields and is a very useful invention in the biomaterials industry.

Claims (3)

Deacetylation of chitin through protein removal (weak alkaline treatment), calcium removal (dilute hydrochloric acid treatment), pigment removal from shells such as crabs and shrimps to obtain chitosan, Isolate and purify in Bacillus strain culture to obtain poly gamma glutamic acid, The mixing ratio of chitosan and poly gamma glutamic acid is 100: 1 to 100: 30 or 99: 1 to 70:30 w / v or v / v% using the chitosan as a cationic polymer and poly gamma glutamic acid as the anionic polymer. Method of producing a chitosan / poly gamma glutamic acid polyelectrolyte composite, characterized in that the mixture is dissolved so that the final 1 w / v% in 1 v / v% acetic acid at pH 4 to 5 to obtain a colloidal polyelectrolyte complex. A chitosan / poly gamma glutamic acid polyelectrolyte complex, which is prepared according to the method of claim 1. A scaffold comprising chitosan / poly gamma glutamic acid polyelectrolyte composite having a mixing ratio of chitosan and poly gamma glutamic acid of 100: 1 to 100: 30 or 99: 1 to 70:30 w / v or v / v%.

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