KR100616464B1 - Epidermal Growth Factor-chitosan complexes and preparation method thereof - Google Patents

Abstract

The present invention is prepared by chemically bonding an amino group present in the carboxy group and chitosan functional groups present in the acidic amino acid of the epidermal growth factor (EGF) using a water-soluble carbodiimide (EGF) The present invention relates to an EGF-chitosan complex in which chitosan is chemically entrapped and a method for preparing the same. In the present invention, the epidermal growth factor (EGF) -chitosan complex is not only obtained in high yield, but also has excellent activity half-life and stability, cell nontoxicity, and wound up. Excellent wound healing effect.

Description

Epidermal growth factor-chitosan complexes and preparation method             

1 is a graph of absorbance standard curves of concentrations of pure EGF at 405 nm measured by ELISA assay;

2 is a sample-specific absorbance histogram of the EGF complex at 405 nm measured by ELISA assay;

3 is a graph showing the efficiency of the inclusion reaction to form an EGF-chitosan complex;

4 is an absorbance histogram of a negative or positive control to show the formation of an EGF complex;

5 is a graph showing the cytotoxicity test results of the EGF-chitosan complex;

6 is a graph showing cell growth stimulating effects of EGF and EGF-chitosan complexes;

7 is a graph showing the results of measuring stability of proteolytic degradation by trypsin of EGF and EGF-chitosan complexes;

8a to 8c and 9 are photographs showing the wound healing effect at the cut site of the EGF and EGF-chitosan complex.

The present invention relates to epidermal growth factor (EGF) -chitosan complexes and a method for preparing the same. More particularly, the amino acid of epidermal growth factor using water-soluble carbodiimide The present invention relates to an epidermal growth factor-chitosan complex chemically conjugated to an epithelial growth factor and chitosan, which is prepared by chemically bonding a carboxy group, which is a functional group present in a middle acidic amino acid, and an amino group, which is present in chitosan, and a method for preparing the same. .

The growth factor acts to promote cell growth, and epidermal growth factor (EGF), one of the growth factors, is part of many polypeptides that transmit signals that stimulate cell activity. to be. Therefore, due to the movement of molecules such as peptides and steroids, one cell and neighboring cells can interact, and further control cell function as a whole. EGF is a single polypeptide consisting of 53 amino acids and has been clinically applied to wounds and burns, foot ulcers, corneal ulcers and skin grafts. However, these growth factors have a lot of difficulties in use due to the short active half-life.

Previously reported water-soluble CM-cellulose and amylases (Wykes, J. et al., 1971), and water-soluble dextran and lysozyme, β-glucosidase (Vegarud, G. et al., 1975) or β By forming covalent bonds between amylases, enzyme-sugar complexes that have been stabilized against physical external factors such as pH, temperature, heat, oxygen and the like have been developed. In addition, when the dextran-enzyme complex was injected into the vein blood vessels of rats as a therapeutic agent, the residual activity of the enzyme was measured and showed higher activity than the original enzyme, and the half-life of the enzyme in the dextran-enzyme complex was also delayed. It became.

Recently, to the EGF using CDAP (1-cyano-4- dimethylaminopyridinium tetrafluoroborate) crosslinking agent, dextran-to form a ³ H complex with this stabilizing the EGF to exactly reach the EGF receptor on the cell membrane of cancer cells and the EGF Techniques for extending active half-life have been reported (Annelie, A., et al., 1991). However, in the above technique, the use of a CDAP crosslinking agent poses a human toxicity problem. In addition, since the preparation of the complex is based on a cyanylation reaction, non-specific binding of EGF to the three hydroxyl groups of dextran makes it difficult to control the reaction.

On the other hand, chitosan is a cationic polysaccharide in which glucose amine and N-acetylglucosamine are 1,4-β-linked, and can be obtained by deacetylating chitin obtained from crabs or other sources. Chitin is slowly degraded in vivo, and chitin and its degradation products are safe natural products. In the field of pharmaceuticals, chitosan has been used as a carrier for delayed release of drugs (Hou et al., Chem Pharm Bull 1985; 33 (9): 3986-3992) and the chitin itself is woven and used as a wound healing dressing. Has been. However, no attempt was made to increase the stability and applicability of growth factors by incorporating chitosan into growth factors to produce complexes.

The present inventors conducted continuous research to develop epidermal growth factor with the maximum stability and applicability. As a result, the epithelium was formed by forming a complex of epithelial growth factor with chitosan, a biocompatible biodegradable material with self-sterilization. It was confirmed that the growth factor and the half-life of the cell growth factor in vivo can be increased, and completed the present invention.

Therefore, an object of the present invention can be prepared with high efficiency, the stability and the half-life in vivo and increased as well as having no cytotoxicity and intrinsic activity of epidermal growth factor-chitosan complex and its preparation method It is to provide.

The present invention provides an epithelial cell growth factor-chitosan complex in which an epithelial cell growth factor and chitosan are chemically conjugated, which are prepared by chemically combining a carboxyl group, which is a functional group present in an acidic amino acid, and an amino group present in chitosan. to provide.

The invention also

Iii) hydrolyzing chitosan to obtain a chitosan fraction with a molecular weight of 10 to 25 kDa,

Ii) reacting the chitosan obtained in i) with an epidermal growth factor in the presence of a water-soluble carbodiimide, for example, an EDC (1-ethyl- (dimethylaminopropyl) carbodiimide) condensation reagent,

Iii) separating the epidermal growth factor-chitosan complex obtained in ii)

Including, it provides a method for producing the complex.

Hereinafter, the present invention will be described in detail.

In the present invention, after modifying the molecular weight of chitosan to suit the purpose of application, by producing an epidermal growth factor-chitosan complex, the stability and applicability of the epidermal growth factor in vivo and maximally increased.

Looking at the manufacturing process of the EGF-chitosan complex of the present invention.

First, a low molecular weight chitosan fraction is prepared by performing a chitosan hydrolysis reaction. At this time, it is possible to increase the yield of the desired molecular weight fraction by adjusting the acid concentration, the reaction time, the reaction temperature. For example, 4% (w / w) chitosan acetic acid solution is neutralized titrated using aqueous NaOH solution and left at room temperature until no white precipitate is produced near pH 7.0. Thereafter, the precipitate was washed, dried, and purified, HCl was added, stirred in a cold bath, and reacted at 50 ° C. and 83 ° C. for 0.5 to 5 hours. Distilled water was added to the reaction solution to remove the precipitate, and methanol was added. The precipitate is again dissolved in distilled water and ultrafiltered to obtain a low molecular weight (about 10-25 kDa) chitosan fraction.

The low molecular weight chitosan fractions obtained are added with EDC in a 10-fold excess of the number of moles of EGF added. For example, 250 μl of 33.8 pmol to 6.76 nmol of EDC condensation reagent was added, followed by 500 μl of 1.69 pmol to 0.338 nmol of EGF, followed by shaking at 4 ° C. for about 2 minutes, followed by 800 pmol to 0.16 μmol of valence. 250 μl of decomposed chitosan is added, followed by reaction at room temperature for 4 hours. Thereafter, 100 µl of 0.13 M glycine solution is added to the solution, followed by further shaking for 90 minutes. After 90 minutes, dialysate at 4 ° C. for one day using dialysis membrane cut-off 12,000 to obtain the EGF-chitosan complex.

The yield of EGF-chitosan complexes prepared according to the method was 60.84-73.5%, which was much higher than the reported yield of EGF-dextran complexes using CDAP crosslinkers, 30-40%, but this yield was proportional to the amount of EGF. It did not appear to increase. The EGF-chitosan complex also showed no toxicity when tested for cytotoxicity in DME-medium (serum free). In addition, even after 3 days, the activity half-life was increased by showing higher cell growth sustained effect than pure EGF, and it was confirmed that the stability was superior to pure EGF. In addition, the treatment of the EGF-chitosan complex on the rat nodal site was observed over 14 days. As a result, pure EGF was observed on the 3 and 7 days at the site treated with the EGF-chitosan complex prepared in an amount of only 50% of the pure EGF amount. The same level of wound healing as the treated area was achieved. Therefore, the use of the EGF-chitosan complex as a wound healing agent is expected to yield at least four times higher economic benefits than the use of pure EGF.

Hereinafter, the present invention will be described in detail by way of examples, which are intended to help the understanding of the present invention, but are not intended to limit the scope of the present invention in any way.

Example 1: Preparation of EGF-chitosan complex using EDC condensation reagent

A 2% (w / w) chitosan acetic acid solution was prepared and neutralized titrated using an aqueous NaOH solution. It was left at room temperature until no more white precipitate formed near pH 7.0. After that, the precipitate was washed with distilled water, ethanol, ether and then purified by drying. The purified chitosan powder was dissolved in a 2% acetic acid aqueous solution at room temperature for about 12 hours to prepare a chitosan solution. Here Conc. HCl was added to run the reaction at 50 ° C. for about 30 minutes. After 30 minutes, one volume of distilled water of the hydrolyzate was added to induce precipitation of the unreacted material. 1 volume of methanol was added to the hydrolyzate from which the precipitate was removed to obtain a precipitate. The precipitate was again dissolved in distilled water and ultrafiltration was performed using YM10 (dialysis membrane cut-off 10 kDa) and YM3 (dialysis membrane cut-off 3 kDa) membranes. The molecular weight of the chitosan fraction finally obtained through the reaction was 23 kDa and 10 kDa.

The low molecular weight (1,0535 Da) chitosan obtained in the above process was added with EDC in 50 and 10 times excess of the number of moles of EGF added, respectively. That is, 250 [mu] l of 6.76 nmol / ml EDC solution was added to the reactor. 500 μl of a 2000 ng / ml EGF (Human recombinant, Sigma) solution was added thereto. After shaking for about 120 seconds, 250 μl of a 0.16 μmol / mL hydrolyzed chitosan (10,535 Da) solution was added, followed by reaction at room temperature for 4 hours. Thereafter, 100 µl of 0.13 M glycine solution was added to the solution, followed by further shaking for 90 minutes. After 90 min, dialysis membrane cut-off was used for 1 day dialysis at 4 ° C. to obtain an EGF-chitosan complex.

Example 2: Detection of EGF-chitosan Complexes by ELISA Method

The yield of the EGF-chitosan complex obtained in the same manner as in Example 1 was measured by ELISA method. Specifically, EGF-chitosan complexes and neat EGF were attached to 96-well plates and monoclonal antibodies against EGF (Sigma, anti-monoclonal Human EGF) were added thereto. Subsequently, anti-mouse IgG, which is a secondary antibody, was added, and then the substrate para-nitrophenyl phosphate (pNPP) was reacted with the secondary antibody to determine the amount of the EGF-chitosan complex according to the degree of color development.

The ELISA results for each concentration of pure EGF and the ELISA results of the EGF-chitosan complex are shown in FIGS. 1 and 2, respectively [EC: EGF-chitosan complex; The amount of pure EGF added during the preparation of the complex: 10 ng (EC10), 1,000 ng (EC1000), 2,000 ng (EC2000)], and the efficiency (%) of the inclusion reaction according to the concentration of EGF is shown in FIG. 3. In addition, in order to prove whether the EGF complex was actually formed, the ELISA results of the negative or positive control expected during the preparation of the EGF complex are shown in FIG. 4. As can be seen from FIGS. 2 and 3, the yield of the EGF-chitosan complex (denoted Pro5) was 60.84-73.5%, much higher than the reported yield of EGF-dextran complex using the CDAP crosslinker 30-40%. . In addition, as can be seen from Figure 4, the EGF complex prepared using the crosslinking agent was not lost during the dialysis process, but EGF of the EGF-chitosan mixture prepared without using the crosslinking agent was found to be lost during the dialysis process. Therefore, when the results of FIG. 4 are related to the results of FIGS. 2 and 3, it was concluded that the EGF complex was actually formed, and the results of FIGS. 2 and 3 were judged to be reliable. However, the color development did not increase in proportion to the amount of EGF.

Example 3: Cytotoxicity Test of EGF-chitosan Complex

To assess the cytotoxicity of the EGF-chitosan complex, 20 μl of the EGF-chitosan complex in the concentration range of 1-4 μg / ml was added to human dermal fibroblasts at 1 × 10 ⁴ cells / well concentration in DME medium (serum-free). Addition to 96-well plates containing dermal fibroblast) resulted in cytotoxicity by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay for 12 hours, 2 days and Measured over 3 days. The results are shown in FIG. As shown in FIG. 5, the EGF-chitosan complex was found to be cytotoxic.

Example 4: Activity half-life and stability evaluation of EGF-chitosan complex

The cell growth sustaining effect of the EGF-chitosan complex was measured by the following method.

Fibroblasts were dispensed in 96 well plates at a concentration of 1.0 × 10 ⁴ cells / well. The cells were incubated in an optimal environment for at least 4 hours for attachment. After 4 hours, 20 μl of a solution containing 10 ng / ml, 1000 ng / ml and 2000 ng / ml of the EGF-chitosan complex, respectively, was mixed with 180 μl of DMEM medium and freshly added to 96 wells.

The results are shown in Figure 6 (E10: EGF 10 ng / ml; E1000: EGF 1000 ng / ml; E2000: EGF 2000 ng / ml; EC10: EGF-chitosan complex 10 ng / ml; EC1000: EGF-chitosan complex 1000 ng / ml EC2000: 2000 ng / ml EGF-chitosan complex). As shown in FIG. 6, it was confirmed that the EGF-chitosan complex at a concentration of 2000 ng / ml showed a continuous growth effect even after 24 hours. This is much higher than the sustained cell growth of 2000 ng / ml of pure EGF. From this, it can be seen that the EGF of the EGF-chitosan complex has an active half-life in vivo.

In addition, EGF-chitosan complex and pure EGF was trypsinized at 37 ℃, the results are shown in FIG. As shown in FIG. 7, pure EGF was rapidly reduced to 30% or less of initial activity, whereas EGF-chitosan complex (denoted EGF-LMC) was found to be maintained to 80% of initial activity. From this, the EGF-chitosan complex EGF was determined to have a higher protective effect on the protease than pure EGF.

Example 5 Measurement of Wound Healing Effect of EGF-chitosan Complex

Four cuts were applied to the dorsal region of the RD rats and the wounds were dressed by containing 100 ng of the EGF-chitosan complex. The results are shown in FIGS. 8 and 9 (FIG. 8A: negative control (no treatment); FIG. 8B: positive control (200 ng of EGF treatment); FIG. 8C: test group (100 ng of EGF-chitosan complex treatment; Figure 9: A: No treatment; B: Water soluble chitosan treatment; C: 200 ng of EGF treatment; D: 100 ng of EGF-chitosan complex treatment) As can be seen from Figures 8 and 9, with the EGF-chitosan complex Treatment resulted in more than 95% of the wound healing effect after 7 days, which is very similar to the wound healing effect obtained by treatment with 200 ng of pure EGF, therefore, the EGF-chitosan complex with EGF combined with hydrolyzed chitosan was pure. It was judged to have about 2 times higher wound healing effect than EGF.

Epithelial cell growth factor-chitosan complex according to the present invention is not only obtained in high yield, but also has excellent activity half-life and stability, cell non-toxicity, and enables more effective wound healing than pure EGF for wounds. Compared to the case, it brings a great economic benefit.

Claims (7)

The epidermal growth factor and chitosan prepared by chemically combining the carboxyl group, which is a functional group present in the acidic amino acid among the epidermal growth factor (EGF), and the amino group present in the chitosan having a molecular weight of 10 to 25 kDa Chemically entrapped epithelial growth factor-chitosan complex. delete The complex according to claim 1, wherein the carboxy group of EGF and the amino group of chitosan are chemically bonded by using a water-soluble carbodiimide. The complex of claim 3 wherein the water soluble carbodiimide is EDC (1-ethyl- (dimethylaminopropyl) carbodiimide). The complex according to claim 1, which is used after being contained in a wound dressing. Vi) hydrolyzing chitosan to produce a chitosan fraction with a molecular weight of 10 to 25 kDa, Ii) reacting the chitosan obtained in iii) with EGF in the presence of a water-soluble carbodiimide, Iii) separating the EGF-chitosan complex obtained in ii) Including, the method of producing a complex according to any one of claims 1 to 3. A method of extending the activity half-life of epidermal growth factor by chemically bonding a carboxyl group, which is a functional group present in an acidic amino acid, among amino acids of epithelial cell growth factor and an amino group present in chitosan having a molecular weight of 10 to 25 kDa.

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