WO2007094570A1

WO2007094570A1 - Self-assembly nano-composites comprising hydrophilic bioactive peptides and hydrophobic materials

Info

Publication number: WO2007094570A1
Application number: PCT/KR2007/000336
Authority: WO
Inventors: Yoon-Jeong Park; Jung Eun Choo
Original assignee: Seoul National University Industry Foundation
Current assignee: Seoul National University Industry Foundation
Priority date: 2006-02-17
Filing date: 2007-01-19
Publication date: 2007-08-23
Anticipated expiration: 2008-08-17
Also published as: KR20070082785A; KR100759495B1

Abstract

The present invention relates to self-assembled nanostructures, comprising hydrophobic bioactive peptides and hydrophobic materials, more specifically, to nanostructures, in which conjugates of hydrophobic bioactive peptides (e.g., cell adhesion-inducing peptides, tissue growth factor-derived peptides, cell- permeating peptides, etc.) and hydrophobic materials are self-assembled and which can not only be used for the surface treatment of biomaterials but also be applied directly for tissue regeneration therapy. The inventive nanostructures are formed through the process in which molecules, each having a hydrophilic moiety and a hydrophobic moiety, are self-assembled into a specific shape in an aqueous system. Accordingly, unit nanostructures can contain peptides in an amount much larger than that attainable by other surface treatment methods, and thus the nanostructures can considerably increase interactions with initial cells. Also, because the nanostructures can efficiently attach peptides, which can recognize cells and intracellular differentiation proteins, to the surface of biomaterials, the nanostructures can promote the adhesion, proliferation and differentiation of cells in injured tissue.

Description

SELF-ASSEMBLY NANO-COMPOSITES COMPRISING HYDROPHILIC BIOACTIVE PEPTIDES AND HYDROPHOBIC MATERIALS

TECHNICAL FIELD

The present invention relates to self-assembled nanostructures, comprising hydrophilic bioactive peptides and hydrophobic materials, and more particularly to nanostructures, in which conjugates of hydrophilic bioactive peptides (e.g., cell adhesion-inducing peptides, tissue growth factor-derived peptides and cell permeating peptides) and hydrophobic substances are self-assembled and which can be used for the surface treatment of biomaterials and can also be applied directly for tissue regeneration therapy.

BACKGROUND ART

In the regenerative healing of injured tissues, such as bone tissue, skin and nerve, many studies have been conducted to manufacture medical devices having specific shapes, such as membranes, artificial periosteum, implants, artificial skin and artificial nerve conduits, from biomaterials such as biocompatible polymers, and to apply the medical devices to facilitate the regeneration and repair of injured tissues. Particularly, studies have been conducted to use active substances playing an important role in the proliferation and differentiation of cells that are in contact with medical devices, including membranes and implants, in order to increase the tissue regeneration ability of the medical devices and reduce treatment period. Among them, extracellular matrixes (e.g., fibronectin) or specific tissue growth factors (e.g., bone morphogenetic protein (BMP)) have been reported to have an excellent ability of repairing and regenerating injured tissues in vivo, and have been demonstrated to show excellent tissue regeneration ability in actual clinical practice.

However, most of these extracellular matrixes and growth factors are high- molecular-weight proteins that are relatively expensive and have a molecular weight of a few tens of kDa, and a specific 3D structure thereof should always be kept in order to maintain the activity thereof. When these proteins are administered in vivo, they will be unstable in vivo, thus leading to a decrease in the activity thereof, and particularly, will be lost in vivo within a few minutes. For this reason, these proteins should be administered in high doses in order to obtain desired treatment effects.

To overcome such shortcomings, attempts have been made to increase effects at local sites by adding tissue growth factors and the like to polymer biomaterials and to maximize tissue regeneration effects by maintaining drug concentrations for a given time through controlled drug release. However, in this case, a small amount of released drugs are transferred non-specifically to the surrounding blood vessels and distributed into other tissues, thus causing side effects.

Meanwhile, important technical fields in tissue engineering techniques, which employ biomaterials such as polymers or ceramics, are the improvement and promotion of the biointerface between cells and tissues. For this purpose, functional proteins that regulate the adhesion of cells, and growth factor-derived substances that promote the differentiation and proliferation of cells, should be labeled on the surface of biomaterials.

In order to label the bioactive proteins on the surface of biomaterials, physical coating has been performed in the prior art. In this case, however, the proteins coated on the biomaterial surface do not have any interaction with the biomaterial surface, and thus are difficult to stay at local sites for a given time. Also, their initial interactions with cells are irregular, thus making it difficult to predict accurate treatment effects. Moreover, studies have been conducted to localize growth factors by chemically attaching the growth factors to the surface of biomaterials and then applying them, but the amount of the growth factors having a relatively high molecular weight, which are attached to the surface, is limited. Furthermore, in the case of chemically bonding bioactive materials to a surface, the chemical bonding can be easily achieved if the surface of the raw material of medical devices has amino groups or carboxyl groups (e.g., chitosan or alginic acid); but otherwise, for example, if the surface of implants is made of titanium or a hydrophobic polymer (e.g., polylactic acid or polyurethane), the efficiency of attaching peptides or other bioactive materials through chemical methods will be reduced.

In an attempt to overcome this shortcoming, Korean Patent Registration No. 52909 discloses a method of polymerizing hydrophilic monomers on the surface of a support by modifying the surface of a porous polymer support for tissue engineering using a low-temperature plasma system. However, this method has a shortcoming in that a separate low-temperature plasma discharge system is required to modify the surface.

To solve the above described problems, the present inventors have paid attention to a nanostructure which can be stably attached by self-assembly. Till now, there have been various attempts to manufacture nanostructures, most of which were methods of manufacturing nanostructures by spinning of polymer-based nanofibers. For example, Korean Patent Publication No. 10-2005-0040187 discloses a bioniimic composite support of nanofiber/microfϊber for inducing tissue regeneration and a manufacturing method thereof, but the technology disclosed therein has a shortcoming in that a specific 3D structure should always be kept in order to maintain the activity of high-molecular- weight proteins for inducing tissue regeneration. Also, in existing studies on regeneration effects in tissue regeneration therapy, regeneration effects were measured using histological methods by staining after transplantation into animals, but it was difficult to determine whether the regeneration effects were induced by applied materials themselves or interactions with the surrounding tissues or cells.

As used herein, the term "self-assembly" refers to a fundamental technology of nanotechnology, which spontaneously forms reproducible nanostructures by artificially manipulating a repulsive or an attractive force between molecules. This self-assembly technology can be applied in the field of electronic material and new materials, including biochips for disease diagnosis and nanotubes, and in the manufacture of nanostructures required for the manufacture of highly integrated semiconductors, and is expected to be highly developed through combination with other technologies.

Such self-assembly nanostructures are based on the phenomenon in which a number of molecules, each having both a hydrophilic moiety and a hydrophobic moiety, are self-assembled into a specific shape in an aqueous system. These self-assembly structures can significantly increase interactions with initial cells, because unit nanostructures contain peptides in an amount significantly larger than that attainable by other surface treatment methods. Also, it is possible to simultaneously achieve cell differentiation potential, and diagnostic and therapeutic effects by applied materials by introducing nanoparticles directly into cells through self-assembly nanostructures containing cell-recognizing and intracellular differentiation protein-recognizing peptides to diagnose. In tissue regeneration therapy, the discovery of bioactive peptides and functional peptides and the development of structures using the same allow biomaterial-cell interactions to be fundamentally maximized, thus leading to a significant increase in diagnostic and therapeutic efficiencies.

Meanwhile, the use of peptides is more advantageous over the use of proteins such as tissue growth factors in terms of immunogenity, stability and the like, but the development of relevant peptides is not yet actively studied. Furthermore, a technique of binding peptides to other materials while maintaining the structural stability of the peptides is not yet reported.

Accordingly, the present inventors have made many efforts to solve the above- described problems occurring in the prior art and to develop a peptide-containing structure, which can be not only applied for the immobilization of peptides but also applied directly for tissue regeneration therapy. As a result, the present inventors have found that self-assembled nanostructures formed by applying conjugates of hydrophilic peptides and hydrophobic materials to an aqueous system at concentrations higher than the critical micelle concentration are stably attached to the surface of biomaterials and, at the same time, bioactive peptides contained in the nanostructures effectively permeate in vivo to show excellent tissue regeneration effects, thereby completing the present invention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a nanostructure in which a conjugate of a hydrophilic bioactive peptide and a hydrophobic material is self- assembled.

To achieve the above object, the present invention provides a nanostructure in which a conjugate of a hydrophilic bioactive peptide and a hydrophobic material is self-assembled.

In the present invention, the hydrophobic bioactive peptide is preferably a cell adhesion-inducing peptide, a tissue growth factor-derived peptide or a cell- permeating peptide. The hydrophobic material is preferably at least one selected from the group consisting of hydrocarbons, hydrophobic polymers, hydrophobic peptides and silanes

In the present invention, the hydrophilic bioactive peptide and the hydrophobic material are preferably linked by amide bond, and the hydrophobic material is modified with a carboxyl group or amine group for amide bond with the hydrophilic bioactive peptide.

The self-assembly nanostructures according to the present invention can be preferably obtained by applying hydrophilic bioactive peptide-hydrophobic material conjugates to an aqueous system at concentrations higher than the critical micell concentration (CMC).

Another features and embodiments of the present invention will be more clarified from the following detailed description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. l(a) and FIG. l(c) show the structures of hydrophilic bioactive peptides, and FIG. l(b) and FIG. l(d) show the 3D structures of conjugates of hydrophilic bioactive peptides and hydrophobic materials.

FIG. 2 is a graph showing the critical micelle concentration at which fluorescence intensity rapidly decreases, in which the micelle concentration is determined using a pyrene technique in order to examine whether the hydrophilic peptide- hydrophobic material conjugates are self-assembled.

FIG. 3(a) shows the results of Western blot analysis for the activation of FAK and ERK, conducted to examine which intracellular signaling system is activated by the nanostructures according to the present invention, and FIG. 3(b) is a graphic diagram showing the Western blot analysis results.

FIG. 4 shows the results of confocal microscopy performed to examine the cell adhesion ability of the nanostructures according to the present invention.

FIG. 5 shows the osteoblast differentiation potential of the nanostructures according to the present invention.

FIG. 6 shows the gene expression level of the nanostructures according to the present invention.

FIG. 7 shows the results of confocal microscopy performed to examine the cell permeability of the nanostructures according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION, AND PPRFERRED EMBODIMENTS

In one aspect, the present invention relates to a nanostructure in which a conjugate of a hydrophilic bioactive peptide and a hydrophobic material is self- assembled.

The self-assembly nanostructures according to the present invention can be easily obtained by applying hydrophilic bioactive peptide-hydrophobic material conjugates to an aqueous system at concentrations higher than the critical micell concentration (CMC).

In the present invention, the hydrophobic bioactive peptide is preferably a cell adhesion-inducing peptide, a tissue growth factor-derived peptide or a cell- permeating peptide.

As the cell adhesion-inducing peptide, it is preferable to use a peptide having an amino acid sequence of RGD. More preferably, the cell adhesion-inducing peptide may be a peptide that essentially contains any one of CGGRGDS (SEQ ID NO: 1) and CGGVACDCRGDCFC (SEQ ID NO: 2) designed to structurally stably maintain the amino acid sequence of RGD.

As the tissue growth factor-derived peptide, it is possible to use a peptide that is identified from the active region of a tissue growth factor to chemically synthesize. Preferably, it is possible to use at least one selected from among the following peptides, and more preferably, cysteine or a spacer, such as CGG, CGGGGG or CEEEEEEE, is added to the N-terminal end of the selected peptide in order to make it easy to immobilize the peptide: (a) an amino acid sequence of positions 2-18 of bone morphogenetic protein (BMP)-2 (SEQ ID NO: 3), an amino acid sequence of positions 2-18 of BMP-4 (SEQ ID NO: 4), an amino acid sequence of positions 2-18 of bone morphogenetic protein (BMP)-6 (SEQ ID NO: 5), an amino acid sequence of positions 16-34 of BMP-2 (SEQ ID NO: 6), an amino acid sequence of positions 41-71 of BMP-2 (SEQ ID NO: 7); an amino acid sequence of positions 73-92 of BMP-2 (SEQ ID NO: 8), an amino acid sequence of positions 88-105 of BMP-2 (SEQ ID NO: 9), an amino acid sequence of positions 283-302 of BMP-2 (SEQ ID NO: 10), an amino acid sequence of positions 335-353 of BMP-2 (SEQ ID NO: 11) and an amino acid sequence of positions 370-390 of BMP-2 (SEQ ID NO: 12); an amino acid sequence of positions 74-93 of BMP-4 (SEQ ID NO: 13), an amino acid sequence of positions 293-313 of BMP-4 (SEQ ID NO: 14), an amino acid sequence of positions 360-379 of BMP-4 (SEQ ID NO: 15) and an amino acid sequence of positions 382-402 of BMP-4 (SEQ ID NO: 16); an amino acid sequence of positions 91-110 of BMP-6 (SEQ ID NO: 17), an amino acid sequence of positions 397-418 of BMP-6 (SEQ ID NO: 18), an amino acid sequence of positions 472-490 of BMP-6 (SEQ ID NO: 19) and an amino acid sequence of positions 487-510 of BMP-6 (SEQ ID NO: 20); and an amino acid sequence of positions 98-117 of BMP-7 (SEQ ID NO: 21), an amino acid sequence of positions 320-340 of BMP-7 (SEQ ID NO: 22), an amino acid sequence of positions 390-409 of BMP-7 (SEQ ID NO: 23) and an amino acid sequence of positions 405-423 of BMP-7 (SEQ ID NO: 24);

(b) an amino acid sequence of positions 62-69 (SEQ ID NO: 25), an amino acid sequence of positions 139-148 (SEQ ID NO: 26), an amino acid sequence of positions 259-277 (SEQ ID NO: 27), an amino acid sequence of positions 199- 204 (SEQ ID NO: 28), an amino acid sequence of positions 151-158 (SEQ ID NO: 29), an amino acid sequence of positions 275-291 (SEQ ID NO: 30), an amino acid sequence of positions 20-28 (SEQ ID NO: 31), an amino acid sequence of positions 65-90 (SEQ ID NO: 32), an amino acid sequence of positions 150-170 (SEQ ID NO: 33) and an amino acid sequence of positions 280-290 (SEQ ID NO: 34) of bone sialoprotein;

(c) an amino acid sequence of positions 242-250 (SEQ ID NO: 35), an amino acid sequence of positions 279-299 (SEQ ID NO: 36) and an amino acid sequence of positions 343-361 (SEQ ID NO: 37) of transforming growth factor;

(d) an amino acid sequence of positions 100-120 (SEQ ID NO: 38) and an amino acid sequence of positions 121-140 (SEQ ID NO: 39) of blood platelet derived growth factor;

(e) an amino acid sequence of positions 23-31 (SEQ ID NO: 40) and an amino acid sequence of positions 97-105 (SEQ ID NO: 41) of acidic fibroblast growth factor; (f) an amino acid sequence of positions 16-27 (SEQ ID NO: 42), an amino acid sequence of positions 37-42 (SEQ ID NO: 43), an amino acid sequence of positions 78-84 (SEQ ID NO: 44) and an amino acid sequence of positions 107- 112 (SEQ ID NO: 45) of basic fibroblast growth factor; (g) an amino acid sequence of positions 255-275 (SEQ ID NO: 46), an amino acid sequence of positions 475-494 (SEQ ID NO: 47) and an amino acid sequence of positions 551-573 (SEQ ID NO: 48) of dentin sialoprotein;

(h) an amino acid sequence of positions 63-83 (SEQ ID NO: 49), an amino acid sequence of positions 84-103 (SEQ ID NO: 50), an amino acid sequence of positions 104-116 (SEQ ID NO: 51) and an amino acid sequence of positions 121-140 (SEQ ID NO: 52) of heparin binding EGF-like growth factor;

(i) an amino acid sequence of positions 326-350 (SEQ ID NO: 53), an amino acid sequence of positions 351-371 (SEQ ID NO: 54), an amino acid sequence of positions 372-400 (SEQ ID NO: 55), an amino acid sequence of positions 401-423 (SEQ ID NO: 56), an amino acid sequence of positions 434- 545 (SEQ ID NO: 57), an amino acid sequence of positions 546-651 (SEQ ID NO: 58), an amino acid sequence of positions 1375-1433 (SEQ ID NO: 59), an amino acid sequence of positions 1435-1471 (SEQ ID NO: 60), an amino acid sequence of positions 1475-1514 (SEQ ID NO: 61), an amino acid sequence of positions 1515-1719 (SEQ ID NO: 62), an amino acid sequence of positions 1764-1944 (SEQ ID NO: 63) and an amino acid sequence of positions 2096-2529 (SEQ ID NO: 64) of cadherin EGF LAG seven-pass G-type receptor 3; and

(J) an amino acid sequence of positions 54-159 (SEQ ID NO: 65), an amino acid sequence of positions 160-268 (SEQ ID NO: 66), an amino acid sequence of positions 269-383 (SEQ ID NO: 67), an amino acid sequence of positions 384-

486 (SEQ ID NO: 68) and an amino acid sequence of positions 487-612 (SEQ ID

NO: 69) of osteoblast specific cadherin(OB-cadherin).

As the cell-permeating peptide that is rich in arginine and hydrophilic, it is preferable to use a peptide essentially containing an amino acid sequence of SEQ ID NO: 70 (VSRRRRRRGGRRRR) or SEQ ID NO: 71 (YGRKKRRQRRR). More preferably, cysteine or a spacer, such as CGG or CGGGGG, CEEEEEEE, is added to the N-terminal end of this peptide in order to make it easy to immobilize the peptide. In the present invention, the hydrophobic material is at least one selected from the group consisting of hydrocarbons, hydrophobic polymers (e.g., polycaprolactone), hydrophobic peptides and silanes, but the scope of the present invention is not limited thereto.

In the present invention, the hydrophilic bioactive peptide and the hydrophobic material are preferably linked by amide bonds, and the hydrophobic material is modified with a carboxyl group or amine group for amide bond with the hydrophilic bioactive peptide.

To prepare cell adhesion domains and peptides according to the present invention, amino acid sequences were isolated and extracted from the active regions of antibodies binding to bioactive cytokine or marker proteins of intracellular differentiation, the three-dimensional structures thereof were analyzed, domains binding to cells were screened, and tissue growth factor- derived peptides were designed from the domains. Among the amino acid sequences of all tissue growth factors, 5-15 amino acid sequences were synthesized, and they were used to study cell adhesion and to test activity, differentiation potential and the like, thus selecting amino acid sequences having the highest activity. Also, the terminal ends of these peptides were chemically modified. These hydrophilic peptides were linked with hydrophobic materials, including synthetic polymers such as polycaprolactone (molecular weight: 2000), peptides, such as alanine, glycine and hydrophobic amino acids, saturated hydrocarbon materials, and silanes, thus preparing hydrophobic peptide-hydrophobic material conjugates.

The prepared peptide-hydrophobic material conjugates were applied to an aqueous system, and analyzed through the pyrene probe technique and an electron microscope. As a result, it was seen that self-assembled nanostructures in the form of micells and nanofibers were formed. Because the nanostructures according to the present invention are formed through the process in which molecules, each having a hydrophobic moiety and a hydrophilic moiety, are self-assembled into a specific form in an aqueous system, it is possible to enable a unit nanostructure to contain peptides in an amount much larger than that attainable by other surface treatment methods. Accordingly, the nanostructures according to the present invention can markedly increase interactions with initial cells and can efficiently attach cell-recognizing and intracellular differentiation protein-recognizing peptides to the surface of biomaterials, and thus they can promote the adhesion, proliferation and differentiation of cells in injured tissue. When the nanostructures according to the present invention are introduced into cells, it is possible to image, diagnose and measure intracellular differentiation and the like, thus optimizing tissue regeneration and repair efficiency.

Examples

Hereinafter, the present invention will be described in further detail by examples. It will however be obvious to a person skilled in the art that these examples are provided for illustrative purpose only and are not construed to limit the scope of the present invention.

Especially, the following examples illustrated only peptides of SEQ ID NOs: 6, 17, 70 and 71 as hydrophilic bioactive peptides, it will be obvious to one skilled in the art that effects similar to those of the examples can be obtained, even when cell adhesion-inducing peptides of SEQ ID NOs: 1, 2 and the like, and tissue growth factor-derived peptides of SEQ ID NOs: 3-5, 7-16, 18-69 and the like, are synthesized and cysteine or the like is added to the terminal ends thereof.

Example 1: Synthesis of cell-recognizing and cell-activating peptides Peptides of SEQ ID NOs: 6, 17, 70 and 71, each having the ability to recognize and activate cells, were synthesized using a solid phase synthesis method with an automatic peptide synthesizer (see Table 1). Specifically, for the synthesis of the peptides, Rink resin (0.075 mmol/g, 100-200 mesh, 1% DVB crosslinking) having Fmoc-(9-Fluorenylmethoxycarbonyl) as a blocking group linked thereto was used. 50 mg of the Rink resin was placed in a synthesizer and swollen with DMF, and then the Fmoc-group was removed using a 20% piperidine/DMF solution. The resulting resin was allowed to react with each of 5 equivalents of a 0.5M amino acid solution (solvent: DMF), 10 equivalents of a l.OM DIPEA solution (solvent: DMF&NMP) and 5 equivalents of a 0.5M HBTU solution (solvent: DMF) under a nitrogen stream for 1 hour. After completion of each of deprotection and coupling steps, the resin was washed with two times with DMF and NMP. Even after coupling of the last amino acid, the resin was deprotected to remove Fmoc-group.

Table 1

Determination that the peptides have been synthesized was performed using a ninhydrin test method. The tested and synthesized resin was dried with THF or

DCM, and then a TFA cleavage cocktail was added to the resin in an amount of

20 ml per g of the resin. Then, the mixture solution was shaken for 3 hours and filtered to remove a cocktail containing the resin and peptide dissolved therein.

The filtered solution was removed using a vacuum rotary evaporator. Then, cold ether was added, or an excess amount of cold ether was added directly to the

TFA cocktail solution containing the peptide dissolved therein, so as to crystallize the peptide in a solid phase. The crystallized peptide was isolated by centrifugation. At this time, the TFA cocktail was completely removed by washing several times with ether and a centrifugation process. The peptides thus obtained were dissolved in distilled water and freeze-dried. The synthesis of the peptides was confirmed by dissolving the peptides in distilled water and measuring the molecular weight of the peptides using MALDI-TOF.

In addition, peptides of SEQ ID NOs: 1-5, 7-16 and 18-69 can also be synthesized in the same manner as described above.

Example 2; Modification for stabilizing structure of active peptides

For structural stabilization, cysteine (CGG) was added to the terminal end of each of the peptides (OPDl, OPD2, LMWP, and TAT) prepared in Example 1 (OPDl : CGGYVPKPCCAPTKLNAISVLYF, OPD2: CGGSDVGWNDWIVAPPGYHA). The synthesized active peptides were confirmed through NMR and the like, and the molecular weights thereof were measured using MALDI-TOF or GPC. The synthesized peptides were used for linking with hydrophobic materials.

Example 3: Synthesis of hydrophilic bioactive peptide-hydrophobic material conjugates

To the hydrophilic bioactive peptides OPDl and OPD2 having cysteine (CGG) added to the N-terminal end thereof, which were synthesized in Example 2, a hydrophobic saturated hydrocarbon material having 16 carbon atoms was linked using palmitic acid (alkyl OPDl and alkyl OPD2) (see Reaction Scheme below). At this time, to make the linking easy, amide linkage between the terminal end of the hydrophobic saturated hydrocarbon material and the amine group of the N- terminal end of the hydrophilic peptide was induced using palmitic acid having a carboxyl group. The linkage was carried out in the same manner as in the amino acid coupling method described in Example 1, except that the reaction time was 3 hours. The synthesis of the peptides having a hydrophobic terminal end was confirmed by dissolving the peptides in distilled water and then measuring the molecular weights of the peptides using MALDI-TOF.

FIG. l(b) and FIG. l(d) show the 3D structure of the hydrophilic bioactive peptides linked with the hydrophobic material.

Example 4; Formation of nanostructiire by self-assembly of hydrophilic peptide-hydrophobic material conjugates and confirmation thereof

OPD2 having cysteine (CGG) added to the N-terminal end thereof, prepared in Example 2, and the hydrophilic peptide-hydrophobic material conjugate (alkyl

OPD2), prepared in Example 3, were applied to an aqueous system, so that they were induced to form nanostructures by self-assembly in the form of micelles and nanofibers. The formation of the nanostructures was confirmed using pyrene.

Specifically, a 6.Ox 10^"6M pyrene solution (solvent: distilled water) was serially diluted 10-fold from 1.0 mg/ml to 1.0x l0^~5 mg/ml, and the fluorescence intensity of the solution was measured. When a graph of fluorescence intensity versus concentration is plotted, the concentration at which the fluorescence intensity rapidly changes shows the minimum concentration at which micelles are formed by self-assembly (see FIG. 2). As a result, as shown in FIG. 2, the concentration at which micelles began to form (critical micelle concentration) was 0.1 mg/ml. At said concentration, OPDl showed a slight change, and alkyl OPD2 showed a definite change. Thus, it could be seen that micelles were formed at said concentration. When the conjugates were applied to an aqueous system at concentrations higher than said critical micelle concentration (CMC), they formed nanostructures by self-assembly. Accordingly, the hydrophilic peptide-hydrophobic material conjugates were used at concentrations higher than the CMC concentration in Examples below, because, when the hydrophilic peptide-hydrophobic material conjugates were applied to an aqueous system at concentrations higher than the CMC concentration, they would form nanostructures.

Example 5: Intracellular signaling of nanostructure consisting of hydrophilic peptide and hydrophobic material

In order to examine which intracellular signaling system is activated by the hydrophilic bioactive peptides OPDl and OPD2, prepared in Example 2, and the hydrophilic peptide-hydrophobic material conjugates alkyl OPDl and alkyl OPD2, prepared in Example 3, the activation of Smad, which is involved in the signaling of focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK) and BMP through cell surface integrin, was measured.

HOS cells (Korean Cell Line Bank, KCLB No. 21543) were cultured and subjected to starvation for 24 hours. Then, the cells were treated with 1 mg of each of the peptides and conjugates dissolved in distilled water, followed by culture for 30 minutes. The activity of the cultured cells was analyzed by Western blot. Herein, alkyl OPDl and alkyl OPD2 formed self-assembled nanostructures when they were dissolved in distilled water. Thereafter, the media were removed, the cells were washed with PBS (pH 7.4), and then scrapped by adding 60 μi of cell lysis buffer, 6 μi of IOOX protease inhibitor, 6 id of IOOX phosphatase inhibitor and 6 μi of 0.1 M PMSF thereto. To measure the amounts of proteins in the cells, BSA solution was used as a standard, the Bradford method was used, the protein solutions of all the samples were added with bromophenol blue-containing 5X dye at a ratio of 1 :4, and then the samples were diluted with IX dye, such that they had the same concentration. Each of the samples was subjected to SDS-PAGE and developed on a nitrocellulose membrane. Each of the samples was blocked with 5% skim milk/TBS-T solution for 1 hour, and allowed to react with a primary antibody (a 1 :1000 dilution in 5% BSA/TBS-T) at room temperature for 4 hours. Then, each of the samples was allowed to react with a secondary antibody (a 1 :2000 dilution in 5% skim milk/TBS-T). Then, each of the membranes was allowed to react with ECL solution, and exposed to an X-ray film, followed by development. The thickness of bands shown in the X-ray films was measured, thus comparing the activation between the samples.

Specifically, the activation of ERK and FAK was analyzed in the order of a non- treated group, OPDl, alkyl OPDl, OPD2, and alkyl OPD2. As a result, as shown in FIG. 3, the activities of the hydrophilic bioactive peptides and the hydrophilic peptide-hydrophobic material conjugates were higher than that of the non-treated group, and the activity of the hydrophilic bioactive peptides and the activity of the hydrophilic peptide-hydrophobic material conjugates were almost similar to each other.

Example 6: Cell adhesion test of nanostructure consisting of hydrophilic peptide and hydrophobic material

In order to test the adhesion of HOS cells (Korean Cell Line Bank, KCLB No. 21543) to the hydrophilic bioactive peptides OPDl and OPD2, prepared in Example 1 , and to the hydrophilic peptide-hydrophobic material conjugates alkyl OPDl and alkyl OPD2, prepared in Example 3, each of the peptides and the conjugates was coated on a 4-well chamber, and then the cells were cultured in the chamber for 1 hour, and the adhesion thereof was determined. Herein, the conjugates alkyl OPDl and alkyl OPD2 formed self-assembled nanostructures in the media. After completely removing the media, the cells were washed with PBS (pH 7.4), and then the cell nucleus and cytoplasm were stained, and the adhesion patterns of the cells were observed with a fluorescent microscope (see FIG. 4).

As a result, as shown in FIG. 4, all of OPDl, OPD2, alkyl OPDl, alkyl OPD2 had good cell adhesion compared to the non-treated group (NT), and OPD2 had cell adhesion slightly higher than that of OPDl . Also, alkyl OPDl and alkyl OPD2, having the hydrophobic substance, were not different from OPDl or OPD2 with respect to cell adhesion.

Example 7: Test of osteoblast differentiation potential of hvdrophilic peptide-hydrophobic material conjugates

In order to examine the effects of the hydrophilic bioactive peptides OPDl and OPD2, prepared in Example 1, and of the hydrophilic peptide-hydrophobic material conjugates alkyl OPDl and alkyl OPD2, prepared in Example 3, on the differentiation of HOS cells (Korean Cell Line Bank, KCLB No. 21543) into osteoblasts, each of the peptides and the conjugates was cultured in media for 2 weeks, and then the production of calcein in the media was observed. Herein, the conjugates alkyl OPDl and alkyl OPD2 formed self-assembled nanostructures in the media. The cultured cells were immobilized with a 10% NBF solution, and washed with PBS (pH 7.4). Then, the nucleus and cytoplasm of the cells were stained with a naphthol/FRV/water (2: 1 :1) solution, washed with PBS (pH 7.4) and then observed with a microscope (see FIG. 5).

As a result, as shown in FIG. 5, alkyl OPDl showed osteoblast differentiation potential higher than that of OPDl, and OPD2 showed osteoblast differentiation potential similar to that of alkyl OPD2. Also, the hydrophilic bioactive peptides and the conjugates all showed excellent osteoblast differentiation potential compared to that of the non-treated group (NT).

Example 8: Test of gene expression level of self-assembled nanostructures

In order to examine the effects of the hydrophilic bioactive peptides OPDl and OPD2, prepared in Example 1, and of the hydrophilic peptide-hydrophobic material conjugates alkyl OPDl and alkyl OPD2, prepared in Example 3, on the expression of HOS cells (Korean Cell Line Bank, KCLB No. 21543), a bioactive peptide aqueous solution having each of the peptides and the conjugates were coated on a culture dish and a non-treated culture dish, and then HOS cells were cultured on the dishes for 1 week. After the media were removed, the cells were washed with PBS (pH 7.4), and then RNA was extracted from the cells. The extracted RNA was subjected to PCR using a primer of collagen type I, a characteristic protein that is expressed with the differentiation of osetoblasts. To examine the results of expression of the protein, the PCR products were electrophoresed on 2% agarose gel and stained with EtBr, and the expression of the protein was examined using UV (see FIG. 6).

As a result, as shown in FIG. 6, all of OPDl, alkyl OPDl, OPD2 and alkyl OPD2 showed excellent expression level compared to that of NT, and were similar to each other with respect to expression level.

Example 9; Cell permeability of nanostructures for intracellular diagnosis

The peptides (LMWP: SEQ ID NO: 70; TAT: SEQ ID NO: 71) prepared in Example 1 were labeled with fluorescent material FITC, and hydrophilic peptide- hydrophobic material conjugates were prepared from the peptides in the same manner as in Examples 2 and 3. Then, the labeled conjugates in aqueous solution were added to HOS cell culture media. Herein, the conjugates were self-assembled in aqueous solution to form nanostructures. After 60 minutes, the cells were immobilized, and the cell permeability of the nanostructures was observed using confocal microscopy (see FIG. 7).

As a result, as shown in FIG. 7, LMWP showed excellent cell permeability compared to TAT, and in the case of NT treated with fluorescent material FITC solution alone, nuclei were observed. This suggests that LMWP and TAT facilitated cell permeation.

INDUSTRIAL APPLICABILITY

As described and proven above in detail, the present invention provides the nanostructures, in which the hydrophilic bioactive peptide-hydrophobic material conjugates are self-assembled. The nanostructures according to the present invention are formed through the process in which molecules, each having a hydrophilic moiety and a hydrophobic moiety, are self-assembled into a specific shape in an aqueous system. Accordingly, unit nanostructures can contain peptides in an amount much larger than that attainable by other surface treatment methods, and thus the nanostructures can considerably increase interactions with initial cells. Also, they can efficiently attach cell-recognizing and intracellular differentiation protein-recognizing peptides to the surface of biomaterials, and thus the nanostructures can promote the adhesion, proliferation and differentiation of cells in injured tissue. In particular, it was difficult in the prior art to determine whether tissue regeneration effects were induced by applied materials themselves or interactions with the surrounding tissues or cells, however, when the nanostructures according to the present invention are introduced into cells, that can be determined, and thus can simultaneously achieve cell differentiation potential, and diagnostic and therapeutic effects by applied materials.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMSWhat is Claimed is:

1. A nanostructure in which a conjugate of a hydrophilic bioactive peptide and a hydrophobic material is self-assembled.

2. The nanostructure according to claim 1, wherein the hydrophobic bioactive peptide is a cell adhesion-inducing peptide, a tissue growth factor- derived peptide or a cell-permeating peptide.

3. The nanostructure according to claim 2, wherein the cell adhesion-inducing peptide has an amino acid sequence of RGD.

4. The nanostructure according to claim 3, wherein the cell adhesion-inducing peptide essentially contains an amino acid sequence of CGGRGDS (SEQ ID NO: 1) or CGGVACDCRGDCFC (SEQ ID NO: 2).

5. The nanostructure according to claim 2, wherein the tissue growth factor- derived peptide is at least one selected from the group consisting of:

(a) an amino acid sequence of positions 2-18 of bone morphogenetic protein (BMP)-2 (SEQ ID NO: 3), an amino acid sequence of positions 2-18 of

BMP-4 (SEQ ID NO: 4), an amino acid sequence of positions 2-18 of bone morphogenetic protein (BMP)-6 (SEQ ID NO: 5), an amino acid sequence of positions 16-34 of BMP-2 (SEQ ID NO: 6), an amino acid sequence of positions

41-71 of BMP-2 (SEQ ID NO: 7); an amino acid sequence of positions 73-92 of

BMP-2 (SEQ ID NO: 8), an amino acid sequence of positions 88-105 of BMP-2

(SEQ ID NO: 9), an amino acid sequence of positions 283-302 of BMP-2 (SEQ

ID NO: 10), an amino acid sequence of positions 335-353 of BMP-2 (SEQ ID NO: 11) and an amino acid sequence of positions 370-390 of BMP-2 (SEQ ID NO: 12); an amino acid sequence of positions 74-93 of BMP-4 (SEQ ID NO: 13), an amino acid sequence of positions 293-313 of BMP-4 (SEQ ID NO: 14), an amino acid sequence of positions 360-379 of BMP-4 (SEQ ID NO: 15) and an amino acid sequence of positions 382-402 of BMP-4 (SEQ ID NO: 16); an amino acid sequence of positions 91-110 of BMP-6 (SEQ ID NO: 17), an amino acid sequence of positions 397-418 of BMP-6 (SEQ ID NO: 18), an amino acid sequence of positions 472-490 of BMP-6 (SEQ ID NO: 19) and an amino acid sequence of positions 487-510 of BMP-6 (SEQ ID NO: 20); and an amino acid sequence of positions 98-117 of BMP-7 (SEQ ID NO: 21), an amino acid sequence of positions 320-340 of BMP-7 (SEQ ID NO: 22), an amino acid sequence of positions 390-409 of BMP-7 (SEQ ID NO: 23) and an amino acid sequence of positions 405-423 of BMP-7 (SEQ ID NO: 24);

(c) an amino acid sequence of positions 242-250 (SEQ ID NO: 35), an amino acid sequence of positions 279-299 (SEQ ID NO: 36) and an amino acid sequence of positions 343-361 (SEQ ID NO: 37) of transforming growth factor; (d) an amino acid sequence of positions 100-120 (SEQ ID NO: 38) and an amino acid sequence of positions 121-140 (SEQ ID NO: 39) of blood platelet derived growth factor;

(i) an amino acid sequence of positions 326-350 (SEQ ID NO: 53), an amino acid sequence of positions 351-371 (SEQ ID NO: 54), an amino acid sequence of positions 372-400 (SEQ ID NO: 55), an amino acid sequence of positions 401-423 (SEQ ID NO: 56), an amino acid sequence of positions 434- 545 (SEQ ID NO: 57), an amino acid sequence of positions 546-651 (SEQ ID NO: 58), an amino acid sequence of positions 1375-1433 (SEQ ID NO: 59), an amino acid sequence of positions 1435-1471 (SEQ ID NO: 60), an amino acid sequence of positions 1475-1514 (SEQ ID NO: 61), an amino acid sequence of positions 1515-1719 (SEQ ID NO: 62), an amino acid sequence of positions 1764-1944 (SEQ ID NO: 63) and an amino acid sequence of positions 2096-2529 (SEQ ID NO: 64) of cadherin EGF LAG seven-pass G- type receptor 3; and

(J) an amino acid sequence of positions 54-159 (SEQ ID NO: 65), an amino acid sequence of positions 160-268 (SEQ ID NO: 66), an amino acid sequence of positions 269-383 (SEQ ID NO: 67), an amino acid sequence of positions 384- 486 (SEQ ID NO: 68) and an amino acid sequence of positions 487-612 (SEQ ID NO: 69) of osteoblast specific cadherin(OB-cadherin).

6. The nanostructure according to claim 5, wherein the tissue growth factor- derived peptide has an addition of cysteine at the N-terminal thereof.

7. The nanostructure according to claim 6, wherein said cysteine is added via spacer selected from the group consisting of: CGG, CGGGGG and CEEEEEEE.

8. The nanostructure according to claim 1, wherein the cell-permeating peptide essentially contains an amino acid sequence of SEQ ID NOs: 70 or 71.

9. The nanostructure according to claim 8, wherein the cell-permeating peptide has an addition of cysteine at the N-terminal thereof.

10. The nanostructure according to claim 9, wherein said cysteine is added via spacer selected from the group consisting of: CGG, CGGGGG and CEEEEEEE.

11. The nanostructure according to claim 1 , wherein the hydrophobic material is at least one selected from the group consisting of hydrocarbons, hydrophobic polymers hydrophobic peptides and silanes.

12. The nanostructure according to claim 1, wherein the hydrophilic bioactive peptide and the hydrophobic material are linked by amide bond.

13. The nanostructure according to claim 1, wherein the hydrophobic material is modified with a carboxyl group or amine group for amide bond with the hydrophilic bioactive peptide.

14. The nanostructure according to any one claim among claims 1~13, which is obtained by applying hydrophilic bioactive peptide-hydrophobic material conjugates to an aqueous system at concentrations higher than the critical micell concentration (CMC).