KR20070082785A - Self-assembly nano-composites comprising hydrophilic bioactive peptides and hydrophobic materials - Google Patents

Abstract

A self-assembly nano structure comprising hydrophilic bioactive peptides and hydrophobic materials is provided to promote adhesion, proliferation and differentiation of cells in the damaged tissue by adhering the self-assembly nano structure on the surface of a biological material and permeating the bioactive peptides into the living body while the peptides maintains structural stability, and maximize the tissue regeneration restoring efficiency. The self-assembly nano structure comprises a hydrophilic bioactive peptide selected from a cell adhesion-inducing peptide, a tissue growth factor-derived peptide or a cell permeation peptide and a hydrophobic material selected from a hydrophobic hydrocarbon, a hydrophobic polymer, a hydrophobic peptide and silane, and is prepared by applying the more than CMC(critical micelle concentration) of hydrophilic bioactive peptide and hydrophobic material to the water system, wherein the hydrophilic bioactive peptide and hydrophobic material are linked by an amide bonding; and the hydrophobic material is modified by a carboxyl group or an amine group for amide bonding.

Description

Self-Assembly Nano-Composites Comprising Hydrophilic Bioactive Peptides and Hydrophobic Materials

1A and 1C show hydrophilic bioactive peptides, and FIGS. 1B and 1D show 3D structures of a combination of a hydrophilic bioactive peptide and a hydrophobic material.

Figure 2 is a graph of the concentration of the self-assembly to determine whether the self-assembly of the hydrophilic peptide-hydrophobic material conjugate using a pyrene technique to find a rapidly falling concentration (CMC). At this time, when the conjugate is applied to the aqueous solution at a concentration of CMC or more, nanostructures are formed by self-assembly.

Figure 3a is to confirm the degree of activation of FAK and ERK using a Western blot method to determine what signal transduction system in the nanostructures according to the present invention, 3b is shown graphically.

Figure 4 confirms the cell adhesion of the nanostructures according to the invention by confocal microscopy.

5 confirms the osteoblast differentiation capacity of the nanostructures according to the present invention.

Figure 6 confirms the gene expression capacity of the nanostructures according to the present invention.

Figure 7 confirms the cellular penetration of the nanostructures according to the present invention by confocal microscopy.

The present invention relates to a self-assembled nanostructure comprising a hydrophilic bioactive peptide and a hydrophobic material, and more specifically, a cell adhesion inducing peptide to be directly applied to tissue regeneration treatment as well as a peptide fixing technique to be used for surface treatment of a biomaterial. The present invention relates to a nanostructure containing a tissue growth factor-derived peptide or a cell permeation peptide.

In the healing and repair of damaged tissues such as bone tissue, skin, and nerves, biomaterials such as biocompatible polymers are used to produce and apply them as medical devices having specific forms such as shielding membranes, artificial periosteums, implants, artificial skins, and artificial nerve conduits. Many studies have been made to help the regeneration and restoration of damaged tissues. In particular, research is being conducted to utilize active substances that play an important role in the proliferation and differentiation of cells in contact with medical devices in order to improve the tissue regeneration ability of medical devices including the shielding membrane and implants and to shorten the treatment period. Among these, extracellular matrix (eg, Fibronectin) or certain tissue growth factors (eg, Bone Morphogenetic Protein, BMP) have been reported to be excellent in repairing and regenerating tissue damage in vivo. In addition, excellent tissue regeneration in actual clinical trials has been confirmed in a number of results.

However, most of these extracellular substrates and growth factors are relatively expensive, and high molecular weight proteins having molecular weights of several tens of KDa are difficult to maintain certain tertiary structures at all times in order to maintain their activity. In fact, in the case of administering these proteins in vivo, there is a disadvantage in that the activity is unstable in the body, the activity is lost, especially in a few minutes to lose the body in order to obtain the desired therapeutic effect must be administered a large amount.

In order to overcome the above disadvantages, the polymer biomaterials contain tissue growth factors to enhance the retention effect at the local area and maintain the drug concentration for a certain period by controlled drug release to maximize the tissue regeneration effect. Attempts have been made, but in this case, a small amount after drug release, but non-specifically, the transition to surrounding blood vessels is accompanied by distribution to other tissues.

Meanwhile, an important technical field in tissue engineering techniques using biomaterials such as polymers or ceramics is the improvement and enhancement of the biointerface between the material surface and cells and tissues. To this end, the growth protein-derived material that enhances the differentiation and proliferation of cells as well as the functional protein that regulates cell adhesion should be labeled on the surface of the material. In order to label the physiologically active protein on the surface of the biomaterial, physical coating was conventionally performed, but in this case, the protein coated on the surface had no interaction with the material surface, so local retention was difficult for a certain period of time. Early interactions also showed irregular results, making it difficult to predict the correct therapeutic effect. In addition, studies have been reported to improve the localization of growth factors by chemically attaching growth factors to the surface of the biomaterial, but the amount of relatively high molecular weight growth factors is limited to the surface. In addition, if the active material is chemically bound to the surface, if the surface of the raw material of the medical device has an amine group or a carboxyl group (e.g. chitosan, alginic acid), an easy chemical bond is possible, but for example, an implant If the surface is titanium or hydrophobic polymers (eg, polylactic acid, polyurethane), it is difficult to raise the adhesion efficiency of peptides or other bioactive substances by chemical methods. In order to compensate for these disadvantages, the method of polymerizing the surface of the porous polymer support for tissue engineering using a low temperature plasma apparatus to polymerize the hydrophilic equivalent on the surface of the support is 'a method of preparing a biodegradable porous engineering support for tissue engineering with improved cell affinity.' (Korean Patent Publication No. 52909), but it requires a separate low temperature plasma discharge device to modify the surface.

However, in the present invention, in order to solve the above problems, nanostructures that can be attached stably by self-assembly have been used. Until recently, many attempts have been made to manufacture nanostructures, most of which use nanofibers using polymers. It was a method of manufacturing by spinning technique. For example, there was a case of providing a composite support of biomimetic nanofibers and microfibers to induce tissue regeneration and a method of manufacturing the same (Korea Patent Publication No. 10-2005-0040187), which induces tissue regeneration. In order to maintain the activity of the high molecular weight protein, a specific tertiary structure had to be preserved at all times. In addition, the existing research to study the regenerative effect in tissue regeneration treatment was measured by the histological method of staining after transplantation of animals, but by this, whether the effect is due to the applied material itself, the interaction with surrounding tissues and cells It was difficult to determine whether it was a regeneration effect by action.

Meanwhile, 'self-assembly' used in the present invention is a source technology of nanotechnology, which artificially manipulates the pushing or pulling force between molecules to spontaneously form a reproducible nanostructure. It is a field that is highly likely to be technically fused and developed in the fields of electronic materials and new materials including biochips, nanotubes, and nanostructures required for the manufacture of highly integrated semiconductors.

This self-assembled nanostructure is based on the fact that several molecules having both hydrophilic and hydrophobic moieties in one molecule are gathered in water and assembled into a specific form, and a significant amount of peptides is significantly higher than other surface treatments. Because it is contained in the unit structure it can significantly increase the interaction with the initial cell. In addition, the self-assembled nanostructures containing the cellular recognition and intracellular differentiation protein recognition peptides are introduced into the cell and diagnosed, thereby simultaneously exerting the effect of diagnosing and treating the cell differentiation by the material applied. Discovery of physiologically active peptides and functional peptides in regenerative treatment and the development of constructs through them can significantly improve the diagnostic and therapeutic efficiency by maximizing the interaction between biomaterials and cells.

Although there are more advantages in immunogenicity and stability than other tissue growth factors in the discovery of peptides, the development of related peptides has not been actively studied until now, and moreover, it is possible to bind to other substances while maintaining the structural stability of peptides. The technique has not been reported yet.

Accordingly, the present inventors have solved the above problems of the prior art, and as a result of diligent efforts to develop a peptide-containing structure to be directly applied to tissue regeneration treatment as well as a peptide fixing technique to be used for surface treatment, the hydrophilic peptide and a hydrophobic substance The conjugate is applied to the water system above the critical micelle concentration to form a self-assembly nanostructure, wherein the self-assembled nanostructure is stably attached to the surface of the biomaterial and at the same time contained in the nanostructure. It was confirmed that the physiologically active peptide is efficiently permeated in vivo and exhibits an effective therapeutic effect on tissue regeneration, thereby completing the present 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.

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

In the present invention, the hydrophilic bioactive peptide may be characterized in that the cell adhesion inducing peptide, tissue growth factor-derived peptide or cell permeation peptide. Specifically, the cell adhesion inducing peptide is preferably a peptide having an amino acid sequence of RGD that is generally used, and more preferably to maintain structurally stable CGGRGDS (SEQ ID NO: 1) or the amino acid sequence of the RGD Peptides containing essentially any one of the designed CGGVACDCRGDCFC (SEQ ID NO: 2) can be used.

The tissue growth factor-derived peptides are those chemically synthesized by identifying those derived from the active region of tissue growth factors. Specifically, (a) amino acids of bone morphogenetic protein (BMP) -2, 4 and 6 Amino acid sequences at positions 2-18 in the sequence [BMP-2 (SEQ ID NO: 3), BMP-4 (SEQ ID NO: 4), and BMP-6 (SEQ ID NO: 5)]; Amino acid sequence at positions 16-34 (SEQ ID NO: 6), amino acid sequence at positions 47-71 (SEQ ID NO: 7), amino acid sequence at positions 73-92 (SEQ ID NO: 8), amino acid sequence at positions 88-105 (SEQ ID NO: 9), amino acid sequence at positions 283-302 (SEQ ID NO: 10), amino acid sequence at positions 335-353 (SEQ ID NO: 11), and amino acid sequence at positions 370-390 (SEQ ID NO: 12); Amino acid sequence at positions 74-93 (SEQ ID NO: 13), amino acid sequence at positions 293-313 (SEQ ID NO: 14), amino acid sequence at positions 360-379 (SEQ ID NO: 15), and amino acid sequence at positions 382-402; (SEQ ID NO: 16); Amino acid sequence at positions 91-110 (SEQ ID NO: 17), amino acid sequence at positions 397-418 (SEQ ID NO: 18), amino acid sequence at positions 472-490 (SEQ ID NO: 19), and amino acid sequence at positions 487-510; (SEQ ID NO: 20); And amino acid sequence SEQ ID NO: 21) at positions 98-117 of BMP-7, amino acid sequence at positions 320-340 (SEQ ID NO: 22), amino acid sequence at positions 390-409 (SEQ ID NO: 23), and amino acid sequence at positions 405-423 (SEQ ID NO: 24);

(b) amino acid sequence at position 62-69 (SEQ ID NO: 25), amino acid sequence at position 139-148 (SEQ ID NO: 26), amino acid sequence at position 259-277 (SEQ ID NO: 27), position 199-204 Amino acid sequence (SEQ ID NO: 28), amino acid sequence 15-1558 (SEQ ID NO: 29), amino acid sequence 275-291 (SEQ ID NO: 30), amino acid position 20-28 (SEQ ID NO: 31), position 65-90 Amino acid sequence (SEQ ID NO: 32), amino acids 150-170 (SEQ ID NO: 33) and amino acid sequences 280-290 (SEQ ID NO: 34);

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

(d) amino acid sequences at positions 100-120 (SEQ ID NO: 38) and amino acid sequences 121-140 (SEQ ID NO: 39) of platelet derived growth factors;

(e) the amino acid sequence at positions 23-31 (SEQ ID NO: 40) and the amino acid sequence at positions 97-105 of the acidic fibroblast growth factor (SEQ ID NO: 41);

(f) the amino acid sequence at positions 16-27 (SEQ ID NO: 42), the amino acid sequence at positions 37-42 (SEQ ID NO: 43), and the amino acid sequence at positions 78-84 of the basic fibroblast growth factor; No. 44) and amino acid sequences 107-112 (SEQ ID NO: 45);

(g) the amino acid sequence of positions 255-275 (SEQ ID NO: 46), amino acid sequence of positions 475-494 (SEQ ID NO: 47) and amino acid sequence of positions 551-573 (SEQ ID NO: 48) of the dentin sialoprotein;

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

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

(j) amino acid sequence at position 54-159 (SEQ ID NO: 65), amino acid sequence at position 160-268 (SEQ ID NO: 66), amino acid sequence at position 269-383 (SEQ ID NO: 67) of osteoblast specific cadherin (OB-cadherin); , Amino acid sequence at positions 384-486 (SEQ ID NO: 68) and amino acid sequence at positions 487-612 (SEQ ID NO: 69) is preferably used at least one selected from the group consisting of. More preferably, the peptide is easy to fix by adding a cysteine and a spacer such as CGG, CGGGGG, and CEEEEEEE at the N-terminus.

In addition, the cell penetrating peptide rich in arginine and having hydrophilicity may be characterized in that the peptide is essentially a peptide containing the amino acid sequence of SEQ ID NO: 70 (VSRRRRRRGGRRRR) or SEQ ID NO: 71 (YGRKKRRQRRR), but is not limited thereto. no. At this time, the peptide is more preferably added to the N-terminal cysteine and spacer such as CGG, CGGGGG, CEEEEEEE to facilitate the fixation.

The hydrophobic material may be any one or more selected from the group consisting of hydrocarbons, hydrophobic polymers (polycaprolactone, etc.), hydrophobic peptides, and silanes, but is not limited thereto.

In the present invention, the combination of the hydrophilic bioactive peptide and the hydrophobic material may be connected by an amide bond, wherein the hydrophobic material is modified with a carboxyl group or an amine group for the amide bond with the hydrophilic bioactive peptide. It can be characterized by.

The self-assembled nanostructure according to the present invention may be obtained by applying a conjugate of the hydrophilic bioactive peptide and a hydrophobic material to a water system above a critical micelle concentration (CMC).

Hereinafter, the self-assembled nanostructure including the hydrophilic bioactive peptide and the hydrophobic material according to the present invention will be described in detail.

First, in order to develop cell adhesion domains and peptides according to the present invention, amino acid sequences of active sites are isolated and extracted from antibodies that bind to indicator proteins such as physiologically active cytokines or intracellular differentiation, and their three-dimensional structure. After analyzing the isolated domains and the like to bind to the cells and to design a tissue growth factor-derived peptide from them. The active peptide synthesizes 5-15 amino acid sequences among the amino acid sequences of the entire tissue growth factor, and selects the most active amino acid sequences by testing cell adhesion, activity, and differentiation using them. The ends are chemically modified. The hydrophobic materials to be bound to these hydrophilic peptides are made of synthetic polymers such as polycaprolactone (molecular weight 2000), peptides containing alanine and glycine and hydrophobic amino acids, saturated hydrocarbon materials, or materials such as silane.

The peptide-hydrophobic conjugate prepared above was applied to an aqueous system and confirmed through Pyrene probe technique and electron microscopy. As a result, self-assembled nanostructures in the form of micelles and nanofibers were formed.

In the nanostructure according to the present invention, since each molecule having a hydrophilic portion and a hydrophobic portion is formed by self-assembly in a specific form in the water system, a significantly larger amount of peptide is contained in the unit structure than other surface treatments, and Can significantly increase interaction. In addition, by effectively attaching cell recognition and intracellular differentiation protein recognition peptides to the surface of the biomaterial can promote the attachment, proliferation and differentiation of cells in the damaged tissue. When the nanostructures according to the present invention are introduced into cells, intracellular differentiation, etc. can be imaged, diagnosed and measured to finally optimize tissue regeneration recovery efficiency.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example  1: Cell recognition and activity Peptide  synthesis

Using the solid phase synthesis method, peptides of SEQ ID NOs: 6, 17, 70, and 71 having cell recognition and activity were synthesized by peptide autosynthesizers, respectively. In other words, it was synthesized using a Rink resin (0.075 mmol / g, 100 to 200 mesh, 1% DVB crosslinking) combined with Fmoc- (9-Fluorenylmethoxycarbonyl) as a blocking group, and 50 mg of Rink resin in the synthesizer. After swelling the resin with DMF, 20% piperidine / DMF solution was used to remove the Fmoc-group. From the terminal C, 0.5M amino acid solution (solvent: DMF), 1.0M DIPEA (solvent: DMF & NMP), and 0.5M HBTU (solvent: DMF) were added at 5, 10, and 5 equivalents, respectively, for 1 hour under a nitrogen stream. I was. Each time the deprotection and coupling steps were completed, washing was performed twice with DMF and NMP. Fmoc-group was removed by deprotection even after the last amino acid was coupled (coupling).

Synthesis was confirmed using the ninhydrin test method. After the test was completed, the synthesized resin was dried with THF or DCM, and TFA cleavage cocktail was added at a rate of 20 ml per 1g of resin. Through the cocktail was dissolved resin and peptide dissolved. The filtered solution was removed using a rotary evaporator, and then cold ether was added or an excessive amount of cold ether was directly added to the TFA cocktail solution in which the peptide was dissolved to crystallize the peptide into a solid phase. Separated. At this time, the TFA cocktail was completely removed by washing and centrifuging with ether several times. The peptide thus obtained was dissolved in distilled water and lyophilized. The synthesized peptide was dissolved in distilled water, and then its synthesis was confirmed by measuring molecular weight using MALDI-TOF. Other peptides of SEQ ID NOs: 1-5, 7-16 and 18-69 can also be synthesized in the same manner as above.

YVPKPCCAPTKLNAISVLYF (OPD1): originated in BMP6 (SEQ ID NO: 17; FIG. 1A)

SDVGWNDWIVAPPGYHA (OPD2): sourced in BMP2 (SEQ ID NO: 6; FIG. 1C)

VSRRRRRRGGRRRR (LMWP): SEQ ID NO: 70

YGRKKRRQRRR (TAT): SEQ ID NO: 71

Example  2: active Peptide  Formula to stabilize the structure

Cysteine (CGG) was added to the N-terminus of the peptides prepared in Example 1 (OPD1, OPD2, LMWP, TAT) for structural stabilization (OPD1: CGGYVPKPCCAPTKLNAISVLYF, OPD2: CGGSDVGWNDWIVAPPGYHA). The synthesized active peptide was used to bind the hydrophobic material, it was confirmed by NMR, and the molecular weight was measured using MALDI-TOF or GPC.

Example  3: hydrophilic bioactivity Peptides and  Synthesis of Hydrophobic Materials

The hydrophilic physiologically active peptides OPD1 and OPD2 having cysteine (CGG) added to the N-terminus of the peptide prepared in Example 2 were bonded to the hydrophobic saturated hydrocarbon material having 16 carbon atoms using palmitic acid (alkyl OPD1 and alkyl OPD2). At this time, in order to facilitate the method of binding, palmitic acid, which is a substance having a carboxyl group at the end of the hydrophobic saturated hydrocarbon material, is used, and the amine group and the amide bond of the N terminal of the hydrophilic peptide are used. To be. The binding method was the same as the amino acid binding method of Example 1, but the reaction time was three times. The synthesized peptide having hydrophobic ends was dissolved in distilled water, and then its synthesis was confirmed by measuring molecular weight using MALDI-TOF.

1B and 1D show a 3D structure in which a hydrophobic substance is bound to a hydrophilic bioactive peptide.

Experimental Example  1: hydrophilic Peptide Nanostructure formation by self-assembly of hydrophobic conjugates and its Confirm

OPD2 with cysteine (CGG) added to the N-terminus prepared in Example 2 and the hydrophilic peptide-hydrophobic conjugate (alkyl OPD2) prepared in Example 3 were applied to the aqueous system, and these were self-assembled to micelles and nanofibers. It was confirmed using Pyrene to form a nanostructure of the form. The fluorescence of the solution was measured by diluting 10 times with a 1.0 × 10 ⁻⁶ M Pyrene solution (solvent-distilled water) from 1.0 mg / ml to 1.0 × 10 ⁻⁵ mg / ml. When a graph of fluorescence according to the concentration is drawn, the concentration of which the fluorescence changes rapidly represents the minimum concentration at which micelles are formed by self-assembly, which is shown in FIG. 2. The CMC (critical micelle concentration; concentration at which micelle formation begins) was found to be 0.1 mg / ml, indicating that OPD1 was slightly changed but alkyl OPD2 was notably changed, thus forming micelle at this concentration. . As a result, since the hydrophilic peptide-hydrophobic conjugate is nanostructured by self-assembly when applied to the aqueous system at a critical micelle concentration or higher, the peptide-hydrophobic conjugate was made to have a CMC concentration or higher in the following experiment.

Experimental Example  2: hydrophilic Peptides and  Of nanostructures made of hydrophobic materials Intracellular  Signaling

Integrins on the cell surface to confirm which signaling systems in cells activated by the hydrophilic bioactive peptides OPD1 and OPD2 prepared in Example 2 and the hydrophilic peptide-hydrophobic conjugates alkyl OPD1 and alkyl OPD2 prepared in Example 3 The activation of Smad involved in Focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK) and BMP signaling through integrin was measured. After culturing HOS cells (Korea Cell Line Bank, KCLB No.21543) for 24 hours, the peptides and conjugates were dissolved in distilled water, treated with 1mg, and then cultured for 30 minutes. At this time, alkyl OPD1 and alkyl OPD2 are dissolved in distilled water to form self-assembled nanostructures. Subsequently, the medium was removed, washed with PBS pH 7.4 solution, 60 μl of cell lysis buffer, 100X protease inhibitor, 100X phosphatase inhibitor, Cells were scraped by adding 6 μl of 0.1 M PMSF. In order to measure the amount of protein in cells, BSA solution was used as a standard and bradford method. Protein solution of all samples was added with bromine phenol blue (5X dye) in a ratio of 1: 4 and then 1X dye. Each sample was diluted equally. Each sample was SDS-PAGE and developed on nitrocellulose membrane. After blocking for 1 hour with 5% skim milk / TBS-T solution, the primary antibody was reacted with 5% BSA / TBS-T solution in a ratio of 1: 1000 at room temperature for 4 hours. The secondary antibody was reacted in a 5% skim milk / TBS-T solution at a ratio of 1: 2000, and then reacted by adding the membrane in an ECL solution, exposing it to an X-ray film, and developing it. The thickness of the bands on the X-ray film was measured and their activation levels were compared.

Figure 3 shows the activation of the ERK and FAK in the order of no treatment (no treated), OPD1, alkyl OPD1, OPD2, alkyl OPD2, the hydrophilic bioactive peptide and hydrophilic peptide-hydrophobic conjugate activity compared to the untreated group The activity of the hydrophilic bioactive peptide and the hydrophilic peptide-hydrophobic conjugate was almost similar, although there was a slight difference.

Experimental Example  3: hydrophilic Peptides and  Cell adhesion test of nanostructures made of hydrophobic materials

To test the adhesion of HOS cells (Korea Cell Line Bank, KCLB No.21543) to the hydrophilic physiologically active peptides OPD1 and OPD2 prepared in Example 1 and the hydrophilic peptide-hydrophobic conjugates alkyl OPD1 and alkyl OPD2 prepared in Example 3 In order to coat each of the peptides and conjugates in a 4well chamber, cells were cultured for 1 hour to confirm their adhesion. At this time, the conjugate alkyl OPD1 and alkyl OPD2 formed a self-assembled nanostructure in the medium. After removing all of the medium, washed with PBS pH7.4 and stained the nucleus and cytoplasm was observed by the fluorescent microscope of the cell adhesion form (Fig. 4).

As a result, as shown in Figure 4, OPD1, OPD2, alkyl OPD1, alkyl OPD2 all have a good cell adhesion compared to NT, OPD2 was found to be slightly better than OPD1, having a hydrophobic material Alkyl OPD1 and alkyl OPD2 showed no difference in cell adhesion when compared to OPD1 or OPD2.

Experimental Example  4: Hydrophilic Peptide Of hydrophobic conjugates Osteoblast Eruption  Experiment

The hydrophilic physiologically active peptides OPD1 and OPD2 prepared in Example 1, and the hydrophilic peptide-hydrophobic conjugate alkyl OPD1 and alkyl OPD2 prepared in Example 3, were differentiated into osteoblasts of HOS cells (Korea Cell Line Bank, KCLB No.21543). In order to determine the effect, each of the peptides and the conjugate was added to the medium and cultured for 2 weeks, and then the degree of generation of calcein (calcein) was observed. At this time, the conjugate alkyl OPD1 and alkyl OPD2 formed self-assembled nanostructures in the medium. The cultured cells were fixed with 10% NBF solution, washed with PBS pH 7.4 solution, stained nuclei and cytoplasm with Naphthol / FRV / water 2: 1: 1 solution, washed with PBS pH 7.4 solution, and It was confirmed (FIG. 5).

As a result, as shown in Figure 5, osteoblast differentiation ability was better in the case of alkyl OPD1 than in the case of OPD1, it was shown that similar to alkyl OPD2 in the case of OPD2. They also showed better osteoblast differentiation than NT in all cases.

Experimental Example  5: Genes of Self-Assembled Nanostructures Expression ability  Experiment

Effects of the hydrophilic bioactive peptides OPD1 and OPD2 prepared in Example 1 and the hydrophilic peptide-hydrophobic conjugate alkyl OPD1 and alkyl OPD2 prepared in Example 2 on the gene expression of HOS cells (Korea Cell Line Bank, KCLB No.21543) To find out, the aqueous and non-treated culture dishes were coated with aqueous solution of physiologically active peptides containing peptides and hydrophobic substances, and then HOS cells were incubated for one week. After removing the medium and washing with PBS pH 7.4 solution, RNA was extracted from the cells. PCR was performed using the extracted RNA by treating primers of collagen type I, a characteristic protein expressed as the osteoblasts differentiated. In order to determine the expression results, after electrophoresis using 2% agarose gel was stained on EtBr and confirmed the expression of the protein using UV (Fig. 6). As a result, as shown in Figure 6, the expression capacity was found to be better than the NT in all cases, the remaining OPD1, alkyl OPD1, OPD2 and alkyl OPD2 was found to be similar.

Experimental Example  6: Intracellular  Cells of Nanostructures for Diagnostics Penetration

Hydrophilic peptide-hydrophobic substance conjugate was obtained and cultured in the same manner as in Examples 2 and 3 after labeling the FITC as a fluorescent substance to the peptide prepared in Example 1 (LMWP; SEQ ID NO: 70, TAT; SEQ ID NO: 71) The conjugated aqueous solution labeled with HOS cell culture was added. In this case, the binder is self-assembled in the aqueous solution to form a nanostructure. After 60 minutes, the cells were fixed and cell permeability was confirmed using confocal microscopy (FIG. 7).

As a result, as shown in FIG. 7, LMWP showed better cell permeability than TAT, and in the case of NT treated only with a fluorescent FITC solution, only nuclei were observed, so LMWP and TAT helped cell permeation. I could see that.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications and variations of the present invention can be readily used by those skilled in the art, and all such variations or modifications can be considered to be included within the scope of the present invention.

The present invention has the effect of providing a nanostructure in which a combination of a hydrophilic bioactive peptide and a hydrophobic material is self-assembly. In the nanostructure according to the present invention, since each molecule having a hydrophilic portion and a hydrophobic portion is formed by self-assembly in a specific form by gathering in an aqueous system, a significantly larger amount of peptide is contained in the unit structure than other surface treatments, and Can significantly increase the interaction of the cells, and by effectively attaching the cell recognition and intracellular differentiation protein recognition peptide to the surface of the biomaterial can promote the attachment, proliferation and differentiation of cells in the damaged tissue. In addition, when the nanostructure is introduced into a cell, intracellular differentiation, etc. may be imaged, diagnosed, and measured to finally optimize tissue regeneration recovery efficiency. In particular, in order to study the tissue regeneration effect, it was difficult to determine whether the existing regeneration effect by the interaction between the applied material itself, surrounding tissues, and cells, but when the nanostructure according to the present invention is introduced into the cell, diagnosis is possible. Finally, the cell differentiation diagnosis and treatment effect by the applied material can be exerted simultaneously.

<110> Seoul National University Industry Foundation <120> Self-Assembly Nano-Composites Comprising Hydrophilic Bioactive          Peptides and Hydrophobic Materials <130> P06-B019 <160> 71 <170> KopatentIn 1.71 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> RGD containing peptide <400> 1 Cys Gly Gly Arg Gly Asp Ser   1 5 <210> 2 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> RGD containing peptide <400> 2 Cys Gly Gly Val Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys   1 5 10 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 3 Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg His   1 5 10 15 Pro     <210> 4 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-4 <400> 4 Cys Ser Pro Lys His His Pro Gln Arg Ser Arg Lys Lys Asn   1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-6 <400> 5 Cys Ser Ser Arg Lys Lys Asn Lys Asn Cys Arg Arg His   1 5 10 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 6 Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr His   1 5 10 15 Ala     <210> 7 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 7 Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile   1 5 10 15 Val Gln Thr Leu Val Asn Ser Val Asn              20 25 <210> 8 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 8 Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser   1 5 10 15 Met Leu Tyr Leu              20 <210> 9 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 9 Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr   1 5 10 15 Gln asp         <210> 10 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide BMP-2 <400> 10 Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg   1 5 10 15 His Pro Leu Tyr              20 <210> 11 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-2 <400> 11 Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser   1 5 10 15 Met Leu Tyr Leu              20 <210> 12 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-7 <400> 12 Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr   1 5 10 15 Gln Asp Met Val Val              20 <210> 13 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-4 <400> 13 Ser Ser Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile   1 5 10 15 Ser Met Leu Tyr Leu              20 <210> 14 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprptein <400> 14 Pro Lys His His Ser Gln Arg Ala Arg Lys Lys Asn Lys Asn Cys Arg   1 5 10 15 Arg His Ser Leu Tyr              20 <210> 15 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-4 <400> 15 Ser Ser Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala Ile   1 5 10 15 Ser Met Leu Tyr Leu              20 <210> 16 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-4 <400> 16 Ser Met Leu Tyr Leu Asp Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr   1 5 10 15 Gln Glu Met Val Val              20 <210> 17 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-6 <400> 17 Tyr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser   1 5 10 15 Val Leu Tyr Phe              20 <210> 18 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-6 <400> 18 Val Ser Ser Ala Ser Asp Tyr Asn Ser Ser Glu Leu Lys Thr Ala Cys   1 5 10 15 Arg Lys His Glu Leu Tyr              20 <210> 19 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-6 <400> 19 Tyr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser   1 5 10 15 Val Leu Tyr Phe              20 <210> 20 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-6 <400> 20 Ser Val Leu Tyr Phe Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr   1 5 10 15 Arg Asn Met Val Val Arg Ala Cys              20 <210> 21 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Partial sequence of BMP-7 <400> 21 Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile Ser   1 5 10 15 Val Leu Tyr Phe              20 <210> 22 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Partial peptide of BMP-7 <400> 22 Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu   1 5 10 15 Tyr Val Ser Phe Arg              20 <210> 23 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-7 <400> 23 Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile Ser   1 5 10 15 Val Leu Tyr Phe              20 <210> 24 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BMP-7 <400> 24 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr   1 5 10 15 Arg Asn Met             <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 25 Glu Glu Glu Gly Glu Glu Glu Glu   1 5 <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 26 Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu   1 5 10 <210> 27 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 27 Tyr Glu Thr Tyr Asp Glu Asn Asn Gly Glu Pro Arg Gly Asp Thr Tyr   1 5 10 15 Arg Ile         <210> 28 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 28 Glu Glu Gly Glu Glu Glu   1 5 <210> 29 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 29 Glu Glu Glu Glu Glu Glu Glu Glu   1 5 <210> 30 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 30 Tyr Glu Ile Tyr Glu Ser Glu Asn Gly Glu Pro Arg Gly Asp Asn Tyr   1 5 10 15 Arg     <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 31 Lys Asn Leu His Arg Arg Val Lys Ile   1 5 <210> 32 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 32 Asp Ser Ser Glu Glu Asn Gly Asp Asp Ser Ser Glu Glu Glu Glu Glu   1 5 10 15 Glu Glu Glu Thr Ser Asn Glu Gly Glu Asn              20 25 <210> 33 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 33 Asp Glu Glu Glu Glu Glu Glu Glu Glu Gly Asn Glu Asn Glu Glu Ser   1 5 10 15 Glu Ala Glu Val Asp              20 <210> 34 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of bone sialoprotein <400> 34 Ser Glu Asn Gly Glu Pro Arg Gly Asp Asn Tyr   1 5 10 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of TGF <400> 35 Thr Gly Arg Arg Gly Asp Leu Ala Thr   1 5 <210> 36 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of TGF <400> 36 Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys   1 5 10 15 Cys Val Arg Gln Leu              20 <210> 37 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of TGF <400> 37 Leu Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val   1 5 10 15 Pro Gln Ala             <210> 38 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of PDGF <400> 38 Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser Pro Gly Gly   1 5 10 15 Ser Gln Glu Gln Arg              20 <210> 39 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of PDGF <400> 39 Ala Lys Thr Pro Gln Thr Arg Val Thr Ile Arg Thr Val Arg Val Arg   1 5 10 15 Arg Pro Pro Lys              20 <210> 40 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of AFGF <400> 40 Tyr Lys Lys Pro Lys Leu Leu Tyr Cys   1 5 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of AFGF <400> 41 Ile Ser Lys Lys His Ala Glu Lys   1 5 <210> 42 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BFGF <400> 42 Gly His Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys   1 5 10 <210> 43 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BFGF <400> 43 Pro Asp Gly Arg Val Asp   1 5 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BFGF <400> 44 Lys Glu Asp Gly Arg Leu Leu   1 5 <210> 45 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of BFGF <400> 45 Tyr Arg Ser Arg Lys Tyr   1 5 <210> 46 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of dentin sialoprotein <400> 46 Glu Asp Glu Gly Ser Gly Asp Asp Glu Asp Glu Glu Ala Gly Asn Gly   1 5 10 15 Lys Asp Ser Ser Asn              20 <210> 47 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of dentin sialoprotein <400> 47 Asp Asp Ala Asn Ser Glu Ser Asp Asn Asn Ser Ser Ser Arg Gly Asp   1 5 10 15 Ala Ser Tyr Asn              20 <210> 48 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of dentin sialoprotein <400> 48 Asp Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser   1 5 10 15 Ser Asp Ser Asp Ser Ser Asp              20 <210> 49 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of Heparin binding EGF like GF <400> 49 Asp Leu Gln Glu Ala Asp Leu Ala Leu Leu Arg Val Thr Leu Ser Ser   1 5 10 15 Lys Pro Gln Ala Leu Ala              20 <210> 50 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of Heparin binding EGF like GF <400> 50 Ala Thr Pro Asn Lys Glu Glu His Gly Lys Arg Lys Lys Lys Gly Lys   1 5 10 15 Gly Leu Gly Lys              20 <210> 51 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of Heparin binding EGF like GF <400> 51 Lys Arg Asp Pro Cys Leu Arg Lys Tyr Lys Asp Phe Cys   1 5 10 <210> 52 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of Heparin binding EGF like GF <400> 52 Cys Lys Tyr Val Lys Glu Leu Arg Ala Pro Ser Cys Ile Cys His Pro   1 5 10 15 Gly Tyr His Gly              20 <210> 53 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 53 Pro Gln Tyr Asn Tyr Gln Thr Leu Val Pro Glu Asn Glu Ala Ala Gly   1 5 10 15 Thr Ala Val Leu Arg Val Val Ala Gln              20 25 <210> 54 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 54 Asp Pro Asp Ala Gly Glu Ala Gly Arg Leu Val Tyr Ser Leu Ala Ala   1 5 10 15 Leu Met Asn Ser Arg              20 <210> 55 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 55 Ser Leu Glu Leu Phe Ser Ile Asp Pro Gln Ser Gly Leu Ile Arg Thr   1 5 10 15 Ala Ala Ala Leu Asp Arg Glu Ser Met Glu Arg His Tyr              20 25 <210> 56 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 56 Leu Arg Val Thr Ala Gln Asp His Gly Ser Pro Arg Leu Ser Ala Thr   1 5 10 15 Thr Met Val Ala Val Thr Val              20 <210> 57 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 57 Glu Gln Ala Gln Tyr Arg Glu Thr Leu Arg Glu Asn Val Glu Glu Gly   1 5 10 15 Tyr Pro Ile Leu Gln Leu Arg Ala Thr Asp Gly Asp Ala Pro Pro Asn              20 25 30 Ala Asn Leu Arg Tyr Arg Phe Val Gly Pro Pro Ala Ala Arg Ala Ala          35 40 45 Ala Ala Ala Ala Phe Glu Ile Asp Pro Arg Ser Gly Leu Ile Ser Thr      50 55 60 Ser Gly Arg Val Asp Arg Glu His Met Glu Ser Tyr Glu Leu Val Val  65 70 75 80 Glu Ala Ser Asp Gln Gly Gln Glu Pro Gly Pro Arg Ser Ala Thr Val                  85 90 95 Arg Val His Ile Thr Val Leu Asp Glu Asn Asp Asn Ala Pro Gln Phe             100 105 110 <210> 58 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 58 Ser Glu Lys Arg Tyr Val Ala Gln Val Arg Glu Asp Val Arg Pro His   1 5 10 15 Thr Val Val Leu Arg Val Thr Ala Thr Asp Arg Asp Lys Asp Ala Asn              20 25 30 Gly Leu Val His Tyr Asn Ile Ile Ser Gly Asn Ser Arg Gly His Phe          35 40 45 Ala Ile Asp Ser Leu Thr Gly Glu Ile Gln Val Val Ala Pro Leu Asp      50 55 60 Phe Glu Ala Glu Arg Glu Tyr Ala Leu Arg Ile Arg Ala Gln Asp Ala  65 70 75 80 Gly Arg Pro Pro Leu Ser Asn Asn Thr Gly Leu Ala Ser Ile Gln Val                  85 90 95 Val Asp Ile Asn Asp His Ile Pro Ile Phe             100 105 <210> 59 <211> 59 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 59 Asp Asp Asn Val Cys Leu Arg Glu Pro Cys Glu Asn Tyr Met Lys Cys   1 5 10 15 Val Ser Val Leu Arg Phe Asp Ser Ser Ala Pro Phe Leu Ala Ser Ala              20 25 30 Ser Thr Leu Phe Arg Pro Ile Gln Pro Ile Ala Gly Leu Arg Cys Arg          35 40 45 Cys Pro Pro Gly Phe Thr Gly Asp Phe Cys Glu      50 55 <210> 60 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 60 Glu Leu Asp Leu Cys Tyr Ser Asn Pro Cys Arg Asn Gly Gly Ala Cys   1 5 10 15 Ala Arg Arg Glu Gly Gly Tyr Thr Cys Val Cys Arg Pro Arg Phe Thr              20 25 30 Gly Glu Asp Cys Glu          35 <210> 61 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 61 Glu Ala Gly Arg Cys Val Pro Gly Val Cys Arg Asn Gly Gly Thr Cys   1 5 10 15 Thr Asp Ala Pro Asn Gly Gly Phe Arg Cys Gln Cys Pro Ala Gly Gly              20 25 30 Ala Phe Glu Gly Pro Arg Cys Glu          35 40 <210> 62 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 62 Val Ala Ala Arg Ser Phe Pro Pro Ser Ser Phe Val Met Phe Arg Gly   1 5 10 15 Leu Arg Gln Arg Phe His Leu Thr Leu Ser Leu Ser Phe Ala Thr Val              20 25 30 Gln Gln Ser Gly Leu Leu Phe Tyr Asn Gly Arg Leu Asn Glu Lys His          35 40 45 Asp Phe Leu Ala Leu Glu Leu Val Ala Gly Gln Val Arg Leu Thr Tyr      50 55 60 Ser Thr Gly Glu Ser Asn Thr Val Val Ser Pro Thr Val Pro Gly Gly  65 70 75 80 Leu Ser Asp Gly Gln Trp His Thr Val His Leu Arg Tyr Tyr Asn Lys                  85 90 95 Pro Arg Thr Asp Ala Leu Gly Gly Ala Gln Gly Pro Ser Lys Asp Lys             100 105 110 Val Ala Val Leu Ser Val Asp Asp Cys Asp Val Ala Val Ala Leu Gln         115 120 125 Phe Gly Ala Glu Ile Gly Asn Tyr Ser Cys Ala Ala Ala Gly Val Gln     130 135 140 Thr Ser Ser Lys Lys Ser Leu Asp Leu Thr Gly Pro Leu Leu Leu Gly 145 150 155 160 Gly Val Pro Asn Leu Pro Glu Asn Phe Pro Val Ser His Lys Asp Phe                 165 170 175 Ile Gly Cys Met Arg Asp Leu His Ile Asp Gly Arg Arg Val Asp Met             180 185 190 Ala Ala Phe Val Ala Asn Asn Gly Thr Met Ala Gly Cys         195 200 205 <210> 63 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 63 Pro His His Phe Arg Gly Asn Gly Thr Leu Ser Trp Asn Phe Gly Ser   1 5 10 15 Asp Met Ala Val Ser Val Pro Trp Tyr Leu Gly Leu Ala Phe Arg Thr              20 25 30 Arg Ala Thr Gln Gly Val Leu Met Gln Val Gln Ala Gly Pro His Ser          35 40 45 Thr Leu Leu Cys Gln Leu Asp Arg Gly Leu Leu Ser Val Thr Val Thr      50 55 60 Arg Gly Ser Gly Arg Ala Ser His Leu Leu Leu Asp Gln Val Thr Val  65 70 75 80 Ser Asp Gly Arg Trp His Asp Leu Arg Leu Glu Leu Gln Glu Glu Pro                  85 90 95 Gly Gly Arg Arg Gly His His Val Leu Met Val Ser Leu Asp Phe Ser             100 105 110 Leu Phe Gln Asp Thr Met Ala Val Gly Ser Glu Leu Gln Gly Leu Lys         115 120 125 Val Lys Gln Leu His Val Gly Gly Leu Pro Pro Gly Ser Ala Glu Glu     130 135 140 Ala Pro Gln Gly Leu Val Gly Cys Ile Gln Gly Val Trp Leu Gly Ser 145 150 155 160 Thr Pro Ser Gly Ser Pro Ala Leu Leu Pro Pro Ser His Arg Val Asn                 165 170 175 Ala Glu Pro Gly Cys             180 <210> 64 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of cadherin EGF LAG seven-pass G-type receptor 3 <400> 64 Cys Pro Cys Arg Pro Gly Ala Leu Gly Arg Gln Cys Asn Ser Cys Asp   1 5 10 15 Ser Pro Phe Ala Glu Val Thr Ala Ser Gly Cys Arg Val Leu Tyr Asp              20 25 30 Ala Cys Pro Lys          35 <210> 65 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of OB-cadherin <400> 65 Gly Trp Val Trp Asn Gln Phe Phe Val Ile Glu Glu Tyr Thr Gly Pro   1 5 10 15 Asp Pro Val Leu Val Gly Arg Leu His Ser Asp Ile Asp Ser Gly Asp              20 25 30 Gly Asn Ile Lys Tyr Ile Leu Ser Gly Glu Gly Ala Gly Thr Ile Phe          35 40 45 Val Ile Asp Asp Lys Ser Gly Asn Ile His Ala Thr Lys Thr Leu Asp      50 55 60 Arg Glu Glu Arg Ala Gln Tyr Thr Leu Met Ala Gln Ala Val Asp Arg  65 70 75 80 Asp Thr Asn Arg Pro Leu Glu Pro Pro Ser Glu Phe Ile Val Lys Val                  85 90 95 Gln Asp Ile Asn Asp Asn Pro Pro Glu Phe             100 105 <210> 66 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of OB-cadherin <400> 66 Leu His Glu Thr Tyr His Ala Asn Val Pro Glu Arg Ser Asn Val Gly   1 5 10 15 Thr Ser Val Ile Gln Val Thr Ala Ser Asp Ala Asp Asp Pro Thr Tyr              20 25 30 Gly Asn Ser Ala Lys Leu Val Tyr Ser Ile Leu Glu Gly Gln Pro Tyr          35 40 45 Phe Ser Val Glu Ala Gln Thr Gly Ile Ile Arg Thr Ala Leu Pro Asn      50 55 60 Met Asp Arg Glu Ala Lys Glu Glu Tyr His Val Val Ile Gln Ala Lys  65 70 75 80 Asp Met Gly Gly His Met Gly Gly Leu Ser Gly Thr Thr Lys Val Thr                  85 90 95 Ile Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Lys Phe             100 105 <210> 67 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of OB-cadherin <400> 67 Pro Gln Arg Leu Tyr Gln Met Ser Val Ser Glu Ala Ala Val Pro Gly   1 5 10 15 Glu Glu Val Gly Arg Val Lys Ala Lys Asp Pro Asp Ile Gly Glu Asn              20 25 30 Gly Leu Val Thr Tyr Asn Ile Val Asp Gly Asp Gly Met Glu Ser Phe          35 40 45 Glu Ile Thr Thr Asp Tyr Glu Thr Gln Glu Gly Val Ile Lys Leu Lys      50 55 60 Lys Pro Val Asp Phe Glu Thr Glu Arg Ala Tyr Ser Leu Lys Val Glu  65 70 75 80 Ala Ala Asn Val His Ile Asp Pro Lys Phe Ile Ser Asn Gly Pro Phe                  85 90 95 Lys Asp Thr Val Thr Val Lys Ile Ser Val Glu Asp Ala Asp Glu Pro             100 105 110 Pro Met Phe         115 <210> 68 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of OB-cadherin <400> 68 Pro Met Phe Leu Ala Pro Ser Tyr Ile His Glu Val Gln Glu Asn Ala   1 5 10 15 Ala Ala Gly Thr Val Val Gly Arg Val His Ala Lys Asp Pro Asp Ala              20 25 30 Ala Asn Ser Pro Ile Arg Tyr Ser Ile Asp Arg His Thr Asp Leu Asp          35 40 45 Arg Phe Phe Thr Ile Asn Pro Glu Asp Gly Phe Ile Lys Thr Thr Lys      50 55 60 Pro Leu Asp Arg Glu Glu Thr Ala Trp Leu Asn Ile Thr Val Phe Ala  65 70 75 80 Ala Glu Ile His Asn Arg His Gln Glu Ala Gln Val Pro Val Ala Ile                  85 90 95 Arg Val Leu Asp Val Asn Asp Asn Ala Pro             100 105 <210> 69 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> partial peptide of OB-cadherin <400> 69 Lys Phe Ala Ala Pro Tyr Glu Gly Phe Ile Cys Glu Ser Asp Gln Thr   1 5 10 15 Lys Pro Leu Ser Asn Gln Pro Ile Val Thr Ile Ser Ala Asp Asp Lys              20 25 30 Asp Asp Thr Ala Asn Gly Pro Arg Phe Ile Phe Ser Leu Pro Pro Glu          35 40 45 Ile Ile His Asn Pro Asn Phe Thr Val Arg Asp Asn Arg Asp Asn Thr      50 55 60 Ala Gly Val Tyr Ala Arg Arg Gly Gly Phe Ser Arg Gln Lys Gln Asp  65 70 75 80 Leu Tyr Leu Leu Pro Ile Val Ile Ser Asp Gly Gly Ile Pro Pro Met                  85 90 95 Ser Ser Thr Asn Thr Leu Thr Ile Lys Val Cys Gly Cys Asp Val Asn             100 105 110 Gly Ala Leu Leu Ser Cys Asn Ala Glu Ala Tyr Ile Leu Asn         115 120 125 <210> 70 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> oligo peptide <400> 70 Val Ser Arg Arg Arg Arg Arg Arg Gly Gly Arg Arg Arg Arg   1 5 10 <210> 71 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> oligo peptide <400> 71 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg   1 5 10

Claims (14)

Nanostructures are self-assembly of a combination of a hydrophilic bioactive peptide and a hydrophobic material. The nanostructure of claim 1, wherein the hydrophilic bioactive peptide is a cell adhesion inducing peptide, a tissue growth factor-derived peptide, or a cell permeation peptide. 3. The nanostructure of claim 2, wherein the cell adhesion inducing peptide has an amino acid sequence of RGD. The nanostructure of claim 3, wherein the cell adhesion inducing peptide essentially contains the amino acid sequence of SEQ ID NO: 1 (CGGRGDS) or SEQ ID NO: 2 (CGGVACDCRGDCFC). The nanostructure of claim 2, wherein the tissue growth factor-derived peptide is any one or more peptides selected from the group consisting of: (a) amino acid sequences of positions 2-18 of each of the amino acid sequences of bone morphogenetic protein (BMP) -2, 4 and 6 [BMP-2 (SEQ ID NO: 3), and BMP-4 (SEQ ID NO: Number 4) and for BMP-6 (SEQ ID NO: 5)]; Amino acid sequence at positions 16-34 (SEQ ID NO: 6), amino acid sequence at positions 47-71 (SEQ ID NO: 7), amino acid sequence at positions 73-92 (SEQ ID NO: 8), amino acid sequence at positions 88-105 (SEQ ID NO: 9), amino acid sequence at positions 283-302 (SEQ ID NO: 10), amino acid sequence at positions 335-353 (SEQ ID NO: 11), and amino acid sequence at positions 370-390 (SEQ ID NO: 12); Amino acid sequence at positions 74-93 (SEQ ID NO: 13), amino acid sequence at positions 293-313 (SEQ ID NO: 14), amino acid sequence at positions 360-379 (SEQ ID NO: 15), and amino acid sequence at positions 382-402; (SEQ ID NO: 16); Amino acid sequence at positions 91-110 (SEQ ID NO: 17), amino acid sequence at positions 397-418 (SEQ ID NO: 18), amino acid sequence at positions 472-490 (SEQ ID NO: 19), and amino acid sequence at positions 487-510; (SEQ ID NO: 20); And amino acid sequence SEQ ID NO: 21) at positions 98-117 of BMP-7, amino acid sequence at positions 320-340 (SEQ ID NO: 22), amino acid sequence at positions 390-409 (SEQ ID NO: 23), and amino acid sequence at positions 405-423 (SEQ ID NO: 24); (b) amino acid sequence at position 62-69 (SEQ ID NO: 25), amino acid sequence at position 139-148 (SEQ ID NO: 26), amino acid sequence at position 259-277 (SEQ ID NO: 27), position 199-204 Amino acid sequence (SEQ ID NO: 28), amino acid sequence at positions 151-158 (SEQ ID NO: 29), amino acid sequence at positions 275-291 (SEQ ID NO: 30), amino acid at position 20-28 (SEQ ID NO: 31), 65-90 Amino acid sequence of positions (SEQ ID NO: 32), amino acid position 150-170 (SEQ ID NO: 33) and amino acid sequence of position 280-290 (SEQ ID NO: 34); (c) the amino acid sequence of positions 242-250 (SEQ ID NO: 35), the amino acid sequence of positions 279-299 (SEQ ID NO: 36) and the amino acid sequence of positions 343-361 (SEQ ID NO: 37) of the transforming growth factor ; (d) amino acid sequences at positions 100-120 (SEQ ID NO: 38) and amino acid sequences 121-140 (SEQ ID NO: 39) of platelet derived growth factors; (e) the amino acid sequence at positions 23-31 (SEQ ID NO: 40) and the amino acid sequence at positions 97-105 of the acidic fibroblast growth factor (SEQ ID NO: 41); (f) the amino acid sequence at positions 16-27 (SEQ ID NO: 42), the amino acid sequence at positions 37-42 (SEQ ID NO: 43), and the amino acid sequence at positions 78-84 of the basic fibroblast growth factor; No. 44) and amino acid sequences 107-112 (SEQ ID NO: 45); (g) the amino acid sequence of positions 255-275 (SEQ ID NO: 46), amino acid sequence of positions 475-494 (SEQ ID NO: 47) and amino acid sequence of positions 551-573 (SEQ ID NO: 48) of the dentin sialoprotein; (h) amino acid sequence at position 63-83 (SEQ ID NO: 49), amino acid sequence position 84-103 (SEQ ID NO: 50), 104-116 position of heparin binding EGF-lke growth factor; Amino acid sequence of SEQ ID NO: 51 and amino acid sequence of position 121-140 (SEQ ID NO: 52); (i) the amino acid sequence of positions 326-350 (SEQ ID NO: 53), the amino acid sequence of positions 351-371 (SEQ ID NO: 54), and the amino acid sequence of positions 372-400 of the cadherin EGF LAG seven-pass G-type receptor 3 55), amino acid sequence of positions 401-423 (SEQ ID NO: 56), amino acid sequence of positions 434-545 (SEQ ID NO: 57), amino acid sequence of positions 546-651 (SEQ ID NO: 58), amino acid sequence of positions 1375-1433 (SEQ ID NO: 59), amino acid sequence at position 1435-1471 (SEQ ID NO: 60), amino acid sequence at position 1475-1514 (SEQ ID NO: 61), amino acid sequence at position 1515-1719 (SEQ ID NO: 62), position 1764-1944 Amino acid sequence (SEQ ID NO: 63) and amino acid sequence at positions 2096-2529 (SEQ ID NO: 64); And (j) amino acid sequence at position 54-159 (SEQ ID NO: 65), amino acid sequence at position 160-268 (SEQ ID NO: 66), amino acid sequence at position 269-383 (SEQ ID NO: 67) of osteoblast specific cadherin (OB-cadherin); , Amino acid sequence at positions 384-486 (SEQ ID NO: 68) and amino acid sequence at positions 487-612 (SEQ ID NO: 69). The nanostructure of claim 5, wherein the tissue growth factor-derived peptide has cysteine added to the N-terminus. The nanostructure of claim 6, wherein the cysteine is added in the form of a spacer selected from the group consisting of CGG, CGGGGG, and CEEEEEEE. The nanostructure of claim 1, wherein the cell permeation peptide essentially contains the amino acid sequence of SEQ ID NO: 70 (VSRRRRRRGGRRRR) or SEQ ID NO: 71 (YGRKKRRQRRR). The nanostructure of claim 8, wherein the cell penetrating peptide has cysteine added to the N-terminus. The nanostructure of claim 9, wherein the cysteine is added in the form of a spacer selected from the group consisting of CGG, CGGGGG, and CEEEEEEE. The nanostructure of claim 1, wherein the hydrophobic material is selected from the group consisting of hydrophobic hydrocarbons, hydrophobic polymers, hydrophobic peptides, and silanes. The nanostructure of claim 1, wherein the conjugate of the hydrophilic bioactive peptide and the hydrophobic material is linked by an amide bond. The nanostructure of claim 1, wherein the hydrophobic material is modified with a carboxyl group or an amine group for amide bond with the hydrophilic bioactive peptide. The nanostructure according to any one of claims 1 to 13, wherein the conjugate of the hydrophilic bioactive peptide and the hydrophobic material is obtained by applying to a water system at a concentration higher than the critical micelle concentration (CMC).

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