KR101250923B1 - Coacervate formed from mussel adhesive proteins or mutants thereof and anionic polymer - Google Patents

Abstract

The present invention relates to a novel use of a coacervate formed of an anionic polymer in a mussel adhesive protein or a variant thereof. Specifically, a composition and a bioactive material for delivering a bioactive material using a coacervate formed by mixing a mussel adhesive protein and an anionic polymer It relates to a carrier. In the present invention, the coacervate formed by mixing the mussel adhesive protein and the anionic polymer has an activity capable of encapsulation of the bioactive material, and thus can be effectively used as an active ingredient of the composition for delivery of the bioactive material.

Description

Coacervate formed from mussel adhesive proteins or mutants according to mussel adhesive proteins or variants thereof

The present invention relates to a novel use of a coacervate formed of an anionic polymer in a mussel adhesive protein or a variant thereof. Specifically, a composition and a bioactive material for delivering a bioactive material using a coacervate formed by mixing a mussel adhesive protein and an anionic polymer It relates to a carrier.

Mussels, a marine organism, produce and secrete adhesive proteins, which allow mussels to attach themselves to wet solid surfaces, such as rocks in the ocean, and are not affected by waves or buoyancy effects. (JH Waite et al., 1983, Biological Review 58, 209-231; HJ Cha et al., 2008, Biotechnology Journal 3, 631-638).

Mussel adhesive proteins are known as strong natural adhesives compared to currently known chemical synthetic adhesives, and have the flexibility to bend, while exhibiting a tensile strength of about twice that of epoxy resins. Mussel adhesive proteins also have the ability to adhere to a variety of surfaces, including plastics, glass, metals, Teflon and biomaterials, and can adhere to wet surfaces in minutes. These properties have not yet been an issue in the field of chemical adhesives. In addition, adhesive proteins are known to not attack human cells or cause immune reactions, and thus have great potential for application in medical fields such as adhesion of living tissues and adhesion of broken teeth during surgery (J. Dove et al., 1986, Journal of American Dental Association 112). , 879). In particular, the mussel adhesive protein may be used in the field of cell surface adhesion technology, which is one of the very important techniques required for the field of cell culture and tissue engineering, that is, the cell culture surface for cell and tissue culture. It is an important technique for promoting the proliferation and differentiation of cells because it is an effective adhesive technique (M. Tirrell et al., 2002, Surf. Sci., 500, 61-83).

Meanwhile, coacervate is a type of colloidal material formed when anionic polymer electrolyte and cationic polymer electrolyte are mixed under specific conditions. When coacervate is formed, the absorbance of the solution increases, and the round sphere on the solution forms an external solution and Exist in isolation. In coacervate formation, the participant electrolyte separates from the solution, condenses and remains in a liquid phase, with changes in physical properties such as reduced surface tension and increased viscosity. Coacervates can also occur through the mixing of proteins with polyelectrolytes with opposite properties (C.G. de Kruif et al., 2004, Current Opinion in Colloid and Interface® Science 9, 340-349). Due to the low surface tension of coacervate, techniques have been reported for immobilizing functional substances such as drugs, enzymes, cells and food additives into microcapsules. Schmitt C. et al., 1998, Critical Review in Food Science and Nutrition 8, 689-753.

However, no studies have been made on the formation of coacervates from mussel adhesive proteins, and no studies have been made to use mussel adhesive proteins for the delivery of bioactive substances.

Accordingly, the present inventors completed the present invention by confirming that when a cationic mussel adhesive protein and an anionic polymer are mixed, coacetate is formed and can be usefully used to deliver a bioactive material.

Therefore, an object of the present invention is to provide a composition for delivery of a bioactive material comprising coacervate formed by mixing an anionic polymer in a mussel adhesive protein or a variant thereof.

The present invention also includes (a) a coacervate and a bioactive material formed by mixing an anionic polymer in a mussel adhesive protein or a variant thereof, and (b) the bioactive material is encapsulated inside the coacervate. It is an object to provide an active substance carrier.

In another aspect, the present invention comprises the steps of (a) mixing the coacervate and the bioactive material formed by mixing an anionic polymer in the mussel adhesive protein or a variant thereof, and (b) forming a coating around the bioactive material It is an object of the present invention to provide a method for producing a biologically active substance carrier.

In order to solve the above problems, the present invention provides a bioactive material delivery composition comprising a coacervate (coacervate) formed by mixing an anionic polymer in a mussel adhesive protein or a variant thereof.

The present invention also includes (a) a coacervate and a bioactive material formed by mixing an anionic polymer in a mussel adhesive protein or a variant thereof, and (b) the bioactive material is encapsulated inside the coacervate. It provides an active substance carrier.

In another aspect, the present invention comprises the steps of (a) mixing the coacervate and the bioactive material formed by mixing an anionic polymer in the mussel adhesive protein or a variant thereof, and (b) forming a coating around the bioactive material It provides a method for producing a biologically active substance carrier.

Hereinafter, the present invention will be described in more detail.

Mussel adhesive protein in the present invention is an adhesive protein derived from mussels, but preferably includes all mussel adhesive proteins described in WO2006 / 107183A1 or WO 2005/092920.

Preferably, the mussel adhesive protein is (a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4, (b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5, (c) the amino acid sequence of SEQ ID NO: 6 1 to 10 times It may be a polypeptide fused with a heterologous polypeptide selected from the group consisting of a polypeptide connected in succession (d) the polypeptide of (a), the polypeptide of (b) and the polypeptide of (c). In (c), the polypeptide is not limited thereto, but may be a polypeptide consisting of the amino acid sequence of SEQ ID NO. In addition, the polypeptide fused in (d) may be, but is not limited to, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

Mutants of the mussel adhesive protein in the present invention preferably include an additional sequence at the carboxyl or amino terminus of the mussel adhesive protein or some amino acids are substituted with other amino acids under the premise of maintaining the adhesion of the mussel adhesive protein. It may be. More preferably, a polypeptide consisting of 3 to 25 amino acids including RGD is linked to the carboxyl terminal or amino terminal of the mussel adhesive protein, or a polypeptide comprising 1 to 100% of the total number of tyrosine residues constituting the mussel adhesive protein, And 5 to 100% thereof may be substituted with 3,4-dihydroxyphenyl-L-alanine (DOPA).

3 to 25 amino acids including the RGD include, but are not limited to, RGD (Arg Gly Asp, SEQ ID NO: 8), RGDS (Arg Gly Asp Ser, SEQ ID NO: 9), RGDC (Arg Gly Asp Cys, SEQ ID NO: 10), RGDV (Arg Gly Asp Val, SEQ ID NO: 11), RGDSPASSKP (Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro, SEQ ID NO: 12), GRGDS (Gly Arg Gly Asp Ser, SEQ ID NO: 13), GRGDTP (Gly Arg Gly Asp Thr Pro, SEQ ID NO: 14), GRGDSP (Gly Arg Gly Asp Ser Pro, SEQ ID NO: 15), GRGDSPC (Gly Arg Gly Asp Ser Pro Cys, SEQ ID NO: 16), and YRGDS (Tyr Arg Gly Asp Ser, SEQ ID NO: 17 It may be one or more selected from the group consisting of).

The variant of the mussel adhesive protein linked to the polypeptide consisting of 3 to 25 amino acids including RGD at the carboxyl or amino terminus of the mussel adhesive protein is not limited thereto but is preferably a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 Can be.

The mussel adhesive protein in the present invention is not limited thereto, but may be preferably inserted into a conventional vector designed to express an external gene so that it can be mass-produced by genetic engineering method. The vector may be appropriately selected or newly produced according to the type and characteristics of the host cell for producing a protein. The method for transforming the vector into a host cell and the method for producing a recombinant protein from the transformant can be easily carried out by conventional methods. Methods such as selection, production, transformation, and expression of recombinant proteins described above can be easily carried out by those skilled in the art, and some modifications are also included in the present invention.

In the present invention, the anionic polymer may be used without limitation as long as it is a polymer material capable of forming coacetate by combining with the cationic mussel adhesive protein, preferably, a polymer lower than pI (Isoelectric point) of the cationic mussel adhesive protein, More preferably, the polymer may have a pI value of 2 to 6, even more preferably a polymer having a pI value of 2 to 4. Since coacervate is hardly formed when the pI value is above or below the pI value, it is preferable to use an anionic polymer within the pI range.

In the present invention, the anionic polymer is, for example, hyaluronic acid (hyaluronic acid), ferredoxin (ferredoxin), polystyrene sulfonic acid (poly styrene sulfonic acid), gum arabic, gelatin (gelatin), albumin (albumin), carbo Carbopol, high or low methoxyl pectin, sodium carboxymethyl guar gum, xanthan gum, whey protein, legamin legumin, carboxymethyl cellulose, alginate, carrageenan, sodium hexametaphosphate, sodium casinate, hemoglobin, heparin, and extracellular It may be one or more selected from the group consisting of polysaccharide B40 (exopolysaccharide B40), the average molecular weight of the anionic polymer is not limited thereto, but preferably a group consisting of 1kDa to 300kDa It may have a selected molecular weight and standing and more preferably from 10kDa to 100kD, more preferably from 17kDa to about 59kDa, and most preferably may have a molecular weight of 17kDa, 35kDa, or 59kD. This is because coacervate may not be formed when the molecular weight is above or below the molecular weight.

In the present invention, the biologically active substance is a substance exhibiting a certain pharmacological activity when administered to a living body or applied to the skin surface, but is not limited thereto, and preferably may be at least one selected from the group consisting of drugs, enzymes, cells, and food additives. More preferably, anticancer agent, antibiotic, anti-inflammatory agent, hormone, hormonal antagonist, interleukin, interferon, growth factor, tumor necrosis factor, endotoxin, lymphokoxy, urokinase, streptokinase, tissue plasminogen activator, protease inhibitor, alkylphosphocholine, At least one selected from the group consisting of radioisotope labeling agents, surfactants, cardiovascular drugs, gastrointestinal drugs, and nervous system drugs. The anti-inflammatory agent is not limited to this, but may preferably be dexamethasone (dexamethasone).

Coacervate in the present invention refers to a type of colloid formed by combining the mussel adhesive protein or a variant thereof and an anionic polymer. That is, in the present invention, coacervate is formed by mixing an anionic polymer with the mussel adhesive protein or a variant thereof.

Coacetate in the present invention is not limited thereto, but may be preferably prepared in an aqueous solvent, more preferably methanol, ethanol, propanol, acetone, acetic acid aqueous solution, more preferably aqueous acetic acid solution, even more preferably 0.1% to It may be formed in 10% aqueous acetic acid solution, even more preferably 0.5-8% aqueous acetic acid solution.

When preparing the coacervate in the solvent, the appropriate pH is limited to this, but preferably may be pH 2.0 to pH 10.0, more preferably may be pH 2.0 to pH 6.0, more preferably pH 2.5 to pH 5.5 days Can be. If the pH is above or below the coacervate is not formed or the polymer may be modified. The amount of the mussel adhesive protein or a variant thereof and the anionic polymer added to the solvent is not limited thereto, but preferably 0.001 to 100% (w / v) relative to the total volume of the solvent, and more preferably 0.01 to 30%. (w / v).

Coacetate in the present invention is limited to this, but preferably may be formed by mixing the mussel adhesive protein or a variant thereof and an anionic polymer in a weight ratio of 1: 0.01 to 1:10, more preferably 1: 0.25 to 1: It may be formed by mixing at a weight ratio of 2.5, more preferably at a weight ratio of 1: 0.25 to 1: 2.33. This is because coacervate may not be effectively formed when the mixing ratio is above or below.

In the present invention, the appropriate temperature at the time of coacetate formation may be 4 to 100 ℃ and preferably 10 to 60 ℃, because if the above or less than the coacetate does not form or deformation of the polymer may occur.

Coacervate formed by mixing the mussel adhesive protein or a variant thereof and the anionic polymer as described above can effectively deliver the bioactive material.

In one embodiment of the present invention, a coacervate formed by mixing a mussel adhesive protein or a variant thereof with hyaluronic acid or heparin, and more preferably, a mussel adhesive protein or a variant of SEQ ID NO: 2 consisting of a polypeptide of SEQ ID NO: 1 or 3 In addition, heparin is mixed to form coacervate, and as an example of a bioactive substance, red pepper seed oil is further mixed. As a result, it can be seen that the coacervate forms a film around red pepper seed oil.

Therefore, the composition for delivering a bioactive material of the present invention is characterized in that it comprises a coacervate (coacervate) formed by mixing an anionic polymer in a mussel adhesive protein or a variant thereof.

The biologically active substance delivery composition of the present invention is not limited thereto, but may preferably be in the form of a pharmaceutical composition.

The composition of the present invention comprises 0.0001 to 50% by weight of the coacervate relative to the total weight of the composition. The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in addition to the active ingredients.

The composition of the present invention may be prepared by containing one or more pharmaceutically acceptable carriers in addition to the coacervates described above for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and one or more of these components. , A buffer solution, a bacteriostatic agent, and the like may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.

The composition comprising coacervate is intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal, nasal, mucosal, inhalation In vivo) and by oral route. Dosage varies depending on the subject's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of disease. The daily dosage is about 0.1 to 100 mg / kg, preferably 0.5 to 10 mg / kg, and more preferably administered once to several times a day.

Meanwhile, the bioactive material carrier of the present invention includes (a) a coacervate and a bioactive material formed by mixing an anionic polymer in a mussel adhesive protein or a variant thereof, and (b) the bioactive material is enclosed in the coacervate. It is characterized by.

The bioactive substance carrier is not limited thereto, but may preferably be a microcapsule.

The coacervate is not limited thereto, but as described above, the mussel adhesive protein or a variant thereof and an anionic polymer may be formed by mixing at a weight ratio of 1: 0.01 to 1:10 at pH 2.0 to pH 10.0.

The coacervate may form a coating around a bioactive material, for example, pepper seed oil, and may effectively encapsulate the bioactive material inside the coacervate, thereby forming a microcapsule which is a bioactive material carrier. Furthermore, there is a characteristic of maintaining the shape even after a long time formed as described above (see <Example 2>).

The size of the bioactive material carrier formed as described above is not limited thereto, and may be preferably 1 to 50 μm on a diameter basis.

Since the bioactive substance carrier of the present invention can be encapsulated regardless of whether the bioactive substance is hydrophobic or hydrophilic in the coacervate, the bioactive substance can be effectively delivered by encapsulating the hydrophobic or hydrophilic bioactive substance effectively. The bioactive material is not limited thereto, but may be preferably encapsulated in the form of dispersion or core in the coacervate. The encapsulation refers to encapsulation of the bioactive material by forming a film around the bioactive material.

The bioactive substance carrier of the present invention can be used as an active ingredient of a pharmaceutical composition, as described above.

Meanwhile, the method for preparing a bioactive material carrier of the present invention comprises the steps of: (a) mixing an anionic polymer and a bioactive material with a mussel adhesive protein or a variant thereof, and (b) the mussel adhesive protein or a variant thereof and an anionic polymer. The formed coacervate comprises forming a coating around the bioactive material.

In the step (a), the mixing means mixing an anionic polymer and a bioactive material with a mussel adhesive protein or a variant thereof at the same time, or more preferably, any one of the mussel adhesive protein or a variant thereof and an anionic polymer is dissolved. This refers to a case where the bioactive material is mixed with the solution, and then the mussel adhesive protein or a variant thereof and the other of the anionic polymer are further mixed to induce coacervate formation.

Specifically, the mixing ratio of the mussel adhesive protein or a variant thereof and the anionic polymer is the same as described above. In addition, mussel adhesive proteins or variants thereof and anionic polymers may be mixed with 0.0001 to 50% by weight in a solvent set to a suitable pH described above, but is not limited thereto. In addition, the bioactive material when mixed in the step (a) is not limited to this, but preferably 0.01 to 20% (v / v) by volume, more preferably 0.1 to 2% by volume in the solvent set to the appropriate pH described above It is preferable to mix in (v / v).

The type of solvent, proper pH, and suitable temperature for preparing the biologically active substance carrier are the same as those under which coacervate described above can be effectively formed.

Specifically, the titration solvent is not limited thereto, but may preferably be prepared in a water-soluble solvent, more preferably in aqueous phosphate solution or acetic acid solution, more preferably in acetic acid solution, even more preferably in an aqueous solution of 0.1% to 10% acetic acid. And even more preferably 0.5-8% acetic acid.

A proper pH is limited to this but preferably may be pH 2.0 to pH 10.0, more preferably may be pH 2.0 to pH 6.0, more preferably pH 2.5 to pH 5.5. If the pH is above or below the coacervate is not formed or the deformation of the polymer occurs because the bioactive material transporter can not be effectively formed.

In addition, the proper temperature may be 4 to 100 ℃ and preferably 10 to 60 ℃, because if the temperature is above or below the coacetate does not form or deformation of the polymer may occur.

In the present invention, the coacervate formed by mixing the mussel adhesive protein and the anionic polymer has an activity capable of encapsulation of the bioactive material, and thus can be effectively used as an active ingredient of the composition for delivery of the bioactive material.

1 is a result of measuring the change in absorbance when mussel adhesive protein fp-151 and anionic polymer hyaluronic acid (17kDa) is mixed at various pHs and ratios.
FIG. 2 shows the results of coacervate formation when mussel adhesive protein fp-151 and hyaluronic acid (17kDa) were mixed at 60:40 in an acetic acid solution at pH 2.5.
Figure 3 is the result of measuring the change in absorbance when mussel adhesive protein fp-131 and hyaluronic acid (17kDa) is mixed at various pH and ratio.
Figure 4 shows the results of coacervate formation when the mussel adhesive protein fp-131 and hyaluronic acid (17kDa) is mixed at 60:40 in an acetic acid solution of pH 2.5.
5 is a result of measuring the change in absorbance when mussel adhesive protein fp-151-RGD and hyaluronic acid (17kDa) are mixed at various pHs and ratios.
FIG. 6 shows coacervate formation when mussel adhesive protein fp-151-RGD and hyaluronic acid (17kDa) were mixed at 60:40 in an acetic acid solution at pH 2.5.
7 is a result of measuring the change in absorbance when mussel adhesive protein fp-151 and hyaluronic acid (35kDa) are mixed at various pHs and ratios.
8 shows coacervate formation when mussel adhesive protein fp-151 and hyaluronic acid (35kDa) were mixed at 60:40 in an acetic acid solution at pH 2.5.
9 is a result of measuring the change in absorbance when mussel adhesive protein fp-151 and hyaluronic acid (59kDa) are mixed at various pHs and ratios.
10 shows coacervate formation when mussel adhesive protein fp-151 and hyaluronic acid (59kDa) were mixed at 60:40 in an acetic acid solution at pH 2.5.
FIG. 11 shows coacervate formation when mussel adhesive protein fp-131 and hyaluronic acid (35kDa) were mixed in an acetic acid solution of pH 3.8 at 80:20.
FIG. 12 shows coacervate formation when mussel adhesive protein fp-131 and hyaluronic acid (59kDa) were mixed in an acetic acid solution of pH 3.8 at 80:20.
FIG. 13 shows the results of measuring changes in absorbance when mussel adhesive protein fp-151 and heparin were mixed at various pHs and ratios.
FIG. 14 shows coacervate formation when mussel adhesive protein fp-131 and heparin were mixed at 80:20 in acetic acid solution of various pH.
Figure 15 shows the results of coacetate formation when mussel adhesive protein fp-131 and ferredoxin were mixed at 70:30 in an acetic acid solution at pH 4.5.
FIG. 16 shows coacervate formation when mussel adhesive protein fp-5 and hyaluronic acid (35kDa) are mixed at 80:20 in an acetic acid solution at pH 4.6.
17 is a result of measuring the change in absorbance when mussel adhesive protein fp-5 and hyaluronic acid (35kDa) are mixed at pH 4.6.
FIG. 18 shows the results of immobilizing red pepper seed oil in microcapsules using coacervate using mussel adhesive protein fp-151 and hyaluronic acid (35kDa) and coacervate using mussel adhesive protein fp-131 and hyaluronic acid (35kDa).
19 is a result of confirming whether the microcapsules formed in FIG. 18 are maintained for 8 days.
20 is a result of immobilization of red pepper seed oil in microcapsules using coacervate using mussel adhesive protein fp-151 and heparin.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Reference Example  1>

Preparation of Mussel Adhesive Proteins

<1-1> mussel adhesive protein fp Preparation of -151

The mussel adhesive protein fp-151 is a fp-1 consisting of six decapeptides so that a decapeptide (decapeptide) consisting of 10 amino acids repeated about 80 times among mussel adhesive proteins fp-1 present in nature can be expressed in Escherichia coli. Variants were synthesized and produced in Escherichia coli after inserting the Mgfp-5 gene (Genbank No. AAS00463 or AY521220) between two fp-1 variants (DS Hwang et. Al., Biomaterials 28, 3560-3568, 2007). ).

Specifically, in the amino acid sequence of fp-1 (Genbank No. Q27409 or S23760), a fp-1 variant of SEQ ID NO: 7 (hereinafter, referred to as 6xAKPSYPPTYK) was prepared, in which a peptide consisting of AKPSYPPTYK represented by SEQ ID NO: 6 was repeatedly linked six times. Fp-151 of SEQ ID NO: 1 was prepared by combining 6xAKPSYPPTYK at the N-terminus of Mgfp-5 and 6xAKPSYPPTYK at the C-terminus of Mgfp-5.

<1-2> mussel adhesive protein fp -151- RGD Manufacturing

Fp-151-RGD of SEQ ID NO: 2 was prepared by adding the GRGDSP sequence selected from the fibronectin RGD group to the C-terminus of fp-151 of <Example 1-1>.

<1-3> mussel adhesive protein fp Preparation of -131

Mussel adhesive protein fp-131 is a gene of the mussel adhesive protein Mgfp-3A (Genbank No. BAB16314 or AB049579) that exists naturally between two fp-1 variants in the same manner as in fp-151 of Example 1-1. ) And then produced by E. coli.

Specifically, in the amino acid sequence of fp-1 (Genbank No. Q27409 or S23760), a fp-1 variant of SEQ ID NO: 7 (hereinafter, referred to as 6xAKPSYPPTYK) prepared by repeating the peptide consisting of AKPSYPPTYK described in SEQ ID NO: 6 six times Fp-131 of SEQ ID NO: 3 was prepared by combining the 6xAKPSYPPTYK at the N-terminus of Mgfp-3 and 6xAKPSYPPTYK at the C-terminus of Mgfp-3.

<1-4> mussel adhesive protein fp Manufacture of -5

Mussel adhesive protein fp-5 is produced by Escherichia coli genes of naturally occurring mussel adhesive protein Mgfp-5 (Genbank No. AAS00463 or AY521220) (DS Hwang et. Al., Applied and environmental microbiology, 3352-3359, 2004).

< Example  1>

Mussel adhesive protein Of coacervate  formation

Coacervate is a type of colloid produced by mixing anionic electrolyte polymer and cationic electrolyte polymer at a specific ratio under specific pH conditions. Since the absorbance of the solution increases when the coacetate is formed, the absorbance is mainly measured to confirm the formation of coacetate (V. Ducel et. Al., Colloids and Surfaces a-Physicochemical and Engineering Aspects, 232, 239-247). , 2004). The following examples confirm whether coacetate is formed when the mussel adhesive protein prepared in the reference example and the negative electrolyte polymer are mixed.

<1-1> mussel adhesive protein fp -151 and hyaluronic acid (17 kDa ) Coacervate  formation

When mussel adhesion protein fp-151 prepared in <Reference Example 1-1> and hyaluronic acid, a negative electrolyte polymer, were mixed, it was confirmed whether coacetate was formed.

Specifically, the mussel adhesive protein fp − dissolved in 5% acetic acid (pH adjusted with sodium hydroxide) at a concentration of 0.05% (w / v) was dissolved in hyaluronic acid (Lifcore Biomedical; Minesota, USA) having a molecular weight of 17 kDa. 151 was mixed while increasing the proportion of the solute (mussel adhesive protein and hyaluronic acid) in increments of 10% (w / w). After the mixing, the absorbance at 600 nm wavelength was measured using a UV-spectrphotometer (Optizen 3220UVbio, Mecasys, Daejeon, Korea) and the results are shown in FIG. 1.

As shown in FIG. 1, it was found that coacervate was best formed when the weight ratio of fp-151 and hyaluronic acid (17kDa) at pH 2.5 was 58:42, 63:37 at pH 3.0, and 68:32 at pH 3.5. .

In addition, it was confirmed whether the increase in absorbance was due to the formation of coacervate through a micrograph and the results are shown in FIG. 2.

As shown in FIG. 2, it was confirmed that a circular body having a size of about 10 μm was formed, which was a coacervate formed by mussel adhesive protein fp-151 and hyaluronic acid (17kDa).

<1-2> mussel adhesive protein fp -131 and hyaluronic acid (17 kDa ) Coacervate  formation

Except for using the mussel adhesive protein fp-131 prepared in <Reference Example 1-3> was confirmed whether the coacervate is formed in the same manner as in <Example 1-1> and the results are shown in Figures 3 and 4 is described.

3 and 4, the weight ratio of fp-131 to hyaluronic acid (17kDa) was 41:59 at pH 2.5, 56:44 at pH 3.0, and coacervate was formed at 59:41 at pH 3.5. Could.

<1-3> mussel adhesive protein fp -151- RGD And hyaluronic acid (17 kDa ) Coacervate  formation

Except for using the mussel adhesive protein fp-151-RGD prepared in <Reference Example 1-2> was confirmed whether coacetate is formed in the same manner as in <Example 1-1> and the results are shown in Figure 5 And FIG. 6.

As shown in FIGS. 5 and 6, the (17kDa) weight ratio of fp-151-RGD and hyaluronic acid is best formed at 60:40 at pH 2.5, 70:30 at pH 3.0, and 73:27 at pH 3.5. Could confirm. In particular, when fp-151-RGD was used rather than fp-151, the absorbance was increased more.

<1-4> mussel adhesive protein fp -151 and hyaluronic acid (35 kDa ) Coacervate  formation

Except for using hyaluronic acid (35kDa) was confirmed whether coacervate is formed in the same manner as in <Example 1-1> and the results are shown in Figures 7 and 8.

As shown in FIGS. 7 and 8, the weight ratio of fp-151 and hyaluronic acid (35kDa) was 59:41 at pH 2.5, 70:30 at pH 3.0, and coacervate was formed at 77:27 at pH 3.5. Could. Particularly, when 35kDa hyaluronic acid was used rather than 17kDa hyaluronic acid, the absorbance increased more than that of coacervate.

<1-5> mussel adhesive protein fp -151 and hyaluronic acid (59 kDa ) Coacervate  formation

Except for using hyaluronic acid (59kDa) was confirmed whether coacervate is formed in the same manner as in <Example 1-1> and the results are shown in Figure 9 and 10.

9 and 10, the weight ratio of fp-151 and hyaluronic acid (59kDa) was 62:38 at pH 2.5, 71:29 at pH 3.0, and coacervate was formed at 75:25 at pH 3.5. Could. In particular, when the use of 59kDa hyaluronic acid than 17kDa hyaluronic acid increases the absorbance was found to be better formed than coacervate.

<1-6> mussel adhesive protein fp -131 and Ferredoxin  Used Coacervate  formation

Except for using fp-131 prepared in <Reference Example 1-3> as the mussel adhesive protein, when the weight ratio of fp-131 and peredoxin is 70:30 at pH 4.5, <Example 1-1> It was confirmed whether coacervate is formed in the same manner as in and the results are shown in FIG. 17.

As shown in FIG. 15, coacervate was formed when the mussel adhesive protein fp-131 and ferredoxin were used.

<1-7> mussel adhesive protein fp -131 and hyaluronic acid (35 kDa ) Coacervate  formation

Except for using the mussel adhesive protein fp-131 prepared in <Reference Example 1-3> when the weight ratio of fp-131 and hyaluronic acid (35kDa) is 80:20 at pH 3.8, <Example 1-5 It was confirmed whether coacervate was formed in the same manner as> and the results are shown in FIG. 11.

<1-8> mussel adhesive protein fp -131 and hyaluronic acid (59 kDa ) Coacervate  formation

Except for using the mussel adhesive protein fp-131 prepared in <Reference Example 1-3> when the weight ratio of the fp-131 and hyaluronic acid (59kDa) is 80:20 at pH 3.8, <Example 1-6 It was confirmed whether coacervate is formed in the same manner as> and the results are shown in FIG. 12.

<1-9> mussel adhesive protein fp -151 and heparin ( heparin ) Coacervate  formation

Absorbance measurement experiments of heparin (sigma) and fp-151 were performed at pH 4.0, pH 5.0, and pH 5.5, respectively. Heparin was dissolved in 100mM sodium acetate buffer having the pH at a concentration of 0.02% (w / v), and fp-151 dissolved in the same solution was mixed in 10% increments by 10% and the absorbance was specified. Are described in FIGS. 13 and 14.

As described in FIGS. 13 and 14, when the weight ratio of fp-151 and heparin was 90:10 at pH 5.0, the absorbance was the highest and it was confirmed that coacetate was formed in FIG. 14.

<1-10> mussel adhesive protein fp -5 and hyaluronic acid (35 kDa ) Coacervate  formation

Except for using the mussel adhesive protein fp-5 prepared in <Reference Example 1-4> was confirmed whether coacervate is formed in the same manner as in <Example 1-4> and the results are shown in Figure 16 and FIG. It described in 17.

As shown in FIG. 16 and FIG. 17, it was found that the absorbance was most increased when the weight ratio of fp-5 and hyaluronic acid (35kDa) was mixed at pH 4.6 at 80:20.

< Example  2>

Coacervate  Microcapsule forming activity

<2-1> mussel adhesive protein fp -151 and hyaluronic acid (35 kDa Formed by Coacervate  Microcapsule forming activity

10 g / L of fp-151 was dissolved in 100 mM acetate buffer adjusted to pH 3.8 with sodium hydroxide, and then stirred for 10 minutes to add 1% of red pepper seed oil to emulsify it. After the stirring, hyaluronic acid was additionally added to the emulsion of red pepper seed oil so that the mass ratio of hyaluronic acid to fp-151 was 8: 2 (fp-151: hyaluronic acid).

After the addition, whether or not microcapsules were formed was confirmed by fluorescence microscopy (Olympus) using fluorescence with pepper seed oil and the results are shown in FIG. 18. In addition, it is confirmed whether the microcapsules are maintained until 8 days have elapsed and the results are shown in FIG. 19.

As shown in FIG. 18 and FIG. 19, the coacervate formed by the mussel adhesive protein and hyaluronic acid has an activity capable of forming microcapsules, and in particular, the microcapsules formed as described above are maintained as they are even after 8 days. Can be.

<2-2> mussel adhesive protein fp -131 and hyaluronic acid (35 kDa Formed by Coacervate  Microcapsule forming activity

Except for using fp-131 as the mussel adhesive protein, the same method as in <Example 2-1> was performed to determine whether the microcapsule forming activity of the coacervate formed by the mussel adhesive protein fp-131 and hyaluronic acid (35kDa) was determined. The results are shown in FIGS. 18 and 19.

As shown in FIG. 18 and FIG. 19, the coacervate formed by the mussel adhesive protein and hyaluronic acid has an activity capable of forming microcapsules, and in particular, the microcapsules formed as described above are maintained as they are even after 8 days. Can be.

<2-3> mussel adhesive protein fp Formed by -151 and heparin Coacervate  Microcapsule forming activity

Microcapsules of coacervate formed by mussel adhesive protein fp-151 and heparin in the same manner as in <Example 2-1>, except that heparin was used instead of hyaluronic acid and the mass ratio of fp-151 and heparin was 9: 1. It was confirmed whether there was a forming activity and the results are shown in FIG. 20.

<110> POSTECH Foundation <120> Coacervate formed from mussel adhesive proteins or mutants          about and anionic polymer <130> DPP20103801KR <160> 17 <170> Kopatentin 1.71 <210> 1 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> FP-151 <400> 1 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu      50 55 60 Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser  65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys                  85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly             100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr         115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr     130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys                 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro             180 185 190 Pro Thr Tyr Lys         195 <210> 2 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> FP-151-RGD <400> 2 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu      50 55 60 Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser  65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys                  85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly             100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr         115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr     130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys                 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro             180 185 190 Pro Thr Tyr Lys Gly Arg Gly Asp Ser Pro         195 200 <210> 3 <211> 172 <212> PRT <213> Artificial Sequence <220> <223> FP-131 <400> 3 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ala      50 55 60 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly  65 70 75 80 Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn                  85 90 95 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser Ala             100 105 110 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro         115 120 125 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro     130 135 140 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 145 150 155 160 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu                 165 170 <210> 4 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> FP-3 <400> 4 Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly   1 5 10 15 Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp              20 25 30 Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr          35 40 45 <210> 5 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> FP-5 <400> 5 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His   1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr              20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys          35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg      50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser  65 70 75 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> fragment sequence derived from FP-1 <400> 6 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys   1 5 10 <210> 7 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> FP-1 <400> 7 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro   1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys              20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr          35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys      50 55 60 <210> 8 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 1 <400> 8 Arg Gly Asp   One <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 2 <400> 9 Arg Gly Asp Ser   One <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 3 <400> 10 Arg Gly Asp Cys   One <210> 11 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 4 <400> 11 Arg Gly Asp Val   One <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 5 <400> 12 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro   1 5 10 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 6 <400> 13 Gly Arg Gly Asp Ser   1 5 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 7 <400> 14 Gly Arg Gly Asp Thr Pro   1 5 <210> 15 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 8 <400> 15 Gly Arg Gly Asp Ser Pro   1 5 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 9 <400> 16 Gly Arg Gly Asp Ser Pro Cys   1 5 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 10 <400> 17 Tyr Arg Gly Asp Ser   1 5

Claims (21)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete (a) mixing a bioactive material with a mussel adhesive protein consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3,
(b) adding heparin to the mixture of the mussel adhesive protein and the bioactive material, and
(C) a method of producing a bioactive material carrier comprising the step of coacetate formed by the mussel adhesive protein and heparin to form a coating around the bioactive material. The method of claim 16, wherein in step (a), the mussel adhesive protein and heparin are mixed at a weight ratio of 1: 0.01 to 1:10 at a pH of 2.0 to 10.0. The method of claim 16, wherein the bioactive substance carrier is a microcapsule. The method of claim 16, wherein the bioactive material is an anticancer agent, antibiotic, anti-inflammatory agent, hormone, hormonal antagonist, interleukin, interferon, heparin, enzyme, growth factor, tumor necrosis factor, endotoxin, lymphokoxy, urokinase, streptokinase, tissue plasmin A method for producing a bioactive substance carrier, which is at least one selected from the group consisting of a gen activator, a protease inhibitor, an alkylphosphocholine, a radioisotope labeling substance, a surfactant, a cardiovascular drug, a gastrointestinal drug, and a nervous system drug. The method of claim 16, wherein the average molecular weight of heparin is 1 kDa to 300 kDa. 21. A bioactive substance carrier prepared by the method of any one of claims 16-20.

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