KR20180029930A - Composition and Method for Cell Adhesion - Google Patents

Abstract

Disclosed in the present invention are peptide of novel sequences which serves as a bio-adhesive material, and uses thereof. The peptide according to the present invention can be usefully used for cell culture which requires adhesion, since the peptide can adhere a substance derived from a living body such as a cell or a tissue to various objects or substances.

Description

[Composition and Method for Cell Adhesion]

The present application is a technique for improving the adhesion to various surfaces of cells containing biological substances based on proteins.

Biodegradable adhesives that can be used to adhere cells or tissues, or biological materials derived therefrom, are useful materials that can be widely used in a variety of technical fields. For example, such materials may be used in the medical field to adhere tissues and tissues or tissues and cells, to treat wounds, to regenerate or repair tissues, or to cultivate stem cells or general cells in the biotechnology field, Tissue filler, bioconjugation, and the like.

These biodegradable adhesives need to have excellent adhesive ability and crosslinking ability, as well as long-term function maintenance especially in living body, and have few side effects.

Examples of bioadhesive materials include cyanoacrylate, fibrin, gelatin, albumin, or polyurethane-based adhesive materials. Cyanoacrylates are commonly used for topical wound closure, but their use is limited due to the release of toxic substances. Biodegradable adhesives using synthetic polymers have a weak adhesive property in solution environment.

U.S. Patent Publication Nos. 2002-0022588 and WO 99/66964 disclose gelatin or albumin in which albumin is crosslinked with carbodiimide. However, the carbodiimide used for crosslinking is also a problem of toxicity, and its use is limited especially in the presence of an insulator, and adhesion and strength of adhesion are also insufficient.

Korean Patent Laid-Open Publication No. 2006-005314 relates to peptides, fragments and derivatives thereof for promoting cell attachment and spread, and relates to integrin α3β1 binding and integrin (LG3) domain of the human laminin-5α3 chain mediating cell adhesion and spread -? 3? 1-dependent cell adhesion peptides and uses thereof.

However, there is a need to develop a new bioadhesive material with improved adhesiveness that can be applied in various environments based on various mechanisms while being less toxic than the conventional ones.

The present invention provides a new stand-up heat-resistant adhesive polypeptide capable of being applied in a variety of environments with low toxicity, a composition for cell adhesion comprising the same, and a use thereof.

In one embodiment, the present invention provides a peptide of the following formula (IV) or a composition for cell adhesion comprising the same or a use for cell adhesion of the peptide.

(IV)

[X ¹² ] _1-15 - [X ¹ -X ² -X ³ -X ⁴ -X ⁵ ] m- [X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ ] n _,

In the above [X ¹ - X ² - X ³ - X ⁴ - X ⁵ ] m,

X < ¹ > is any amino acid,

Wherein X ² , X ³ and X ⁴ are amino acids of any one of L, V, I, E, G and A, which are the same or different, and at least one of X ² , X ³ or X ⁴ may be absent In addition,

X < ⁵ > is an amino acid of K or R,

M is an integer of 1 to 5, and when two or more amino acids are included, the amino acid sequences of the respective peptides are the same or different,

In n, the ^{[X 11 X 6 - X 7} - X 8 - X 9 - - X 10]

Wherein ^{[X 6 - X 7 - X} 8 - X 9 - X 10 - X 11] is or may be lacking, or

X < ⁶ > is an amino acid of any one of F, Y and W,

X ⁷ is an amino acid of K or R,

X ⁸ is an amino acid of any one of A, M and I,

X ⁹ is any amino acid of L, M and G,

Wherein X ¹⁰ is any amino acid,

X ¹¹ is an amino acid of any one of C, S and T,

At least one of X ⁸ and X ⁹ or X ¹⁰ and X ¹¹ may be absent,

Wherein n is 1 or 2,

X ¹² is F, K or R,

The amino acid is a D- or L-type natural or unnatural amino acid or a derivative thereof.

In one embodiment, any of the amino acids of X ¹ may be S, T, C, P, N or Q, and X ² - X ³ - X ⁴ may be AAA, EEE, LVG, LVA, LVL, LVV, LLA, LLL, or LLV.

In another embodiment, the peptide of [X ¹ - X ² - X ³ - X ⁴ - X ⁵ ] is any one of the following: QLVVK (SEQ ID NO: 1), QEEEK (SEQ ID NO: 2), QAAAK QVVK (SEQ ID NO: 71), QAVVK (SEQ ID NO: 72), QLLVK (SEQ ID NO: 73), QLIVK (SEQ ID NO: QLAVK (SEQ ID NO: 75), QLVLK (SEQ ID NO: 76), QLVIK (SEQ ID NO: 77), QLVAK (SEQ ID NO: 78), or QLVVR (SEQ ID NO: 79).

In another embodiment the ^{[X 6 - X 7 - X} 8 - X 9 - X 10 - X 11] of the peptide is any of the following: FRALPC (SEQ ID NO: 6), FREEPC (SEQ ID NO: 7), FRVVPC (SEQ ID NO: (SEQ ID NO: 8), FEALPC (SEQ ID NO: 9), YRALPC (SEQ ID NO: 10), WRALPC (SEQ ID NO: 11), FRALP (SEQ ID NO: 12), FRAL (SEQ ID NO: 13), FRPC

In another embodiment, the peptide of formula (IV) is represented by one of the following sequences:

RQLVVK (SEQ ID NO: 15); FRALPCRQLVVK (SEQ ID NO: 16); RQLVVKFRALPC (SEQ ID NO: 17); RQLVVKFRALPCRQLVVKFRALPC (SEQ ID NO: 18); RQLVVKFRALP (SEQ ID NO: 19); RQLVVKFRAL (SEQ ID NO: 20); KQLVVKFRALPC (SEQ ID NO: 21); RQKFRALPC (SEQ ID NO: 22); RQEEEKFRALPC (SEQ ID NO: 23); RQAAAKFRALPC (SEQ ID NO: 24); RQLVVKFRPC (SEQ ID NO: 25); RQLVVKFREEPC (SEQ ID NO: 26); RQLVVKFRVVPC (SEQ ID NO: 27); RQEEEKFREEPC (SEQ ID NO: 28); EQLVVEFEALPC (SEQ ID NO: 29); RQLVVKYRALPC (SEQ ID NO: 30); RQLVVKWRALPC (SEQ ID NO: 31); RNLVVKFRALPC (SEQ ID NO: 32); RSLVVKFRALPC (SEQ ID NO: 33); RQLVVK- (FRALPC) 2 (SEQ ID NO: 37); (R) 2-QLVVKFRALPC (SEQ ID NO: 38); (R) 5-QLVVKFRALPC (SEQ ID NO: 39); (R) 10-QLVVKFRALPC (SEQ ID NO: 40); (R) 15-QLVVKFRALPC (SEQ ID NO: 41); RQVVVKFRALPC (SEQ ID NO: 42); RQIVVKFRALPC (SEQ ID NO: 43); RQAVVKFRALPC (SEQ ID NO: 44); RQEVVKFRALPC (SEQ ID NO: 45); RQLLVKFRALPC (SEQ ID NO: 46); RQLIVKFRALPC (SEQ ID NO: 47); RQLAVKFRALPC (SEQ ID NO: 48); RQLEVKFRALPC (SEQ ID NO: 49); RQLVLKFRALPC (SEQ ID NO: 50); RQLVIKFRALPC (SEQ ID NO: 51); RQLVAKFRALPC (SEQ ID NO: 52); RQLVEKFRALPC (SEQ ID NO: 53); RQLVVRFRALPC (SEQ ID NO: 54); RQLVVKFKALPC (SEQ ID NO: 55); RQLVVEFEALPC (SEQ ID NO: 56); RQLVVKFRLLPC (SEQ ID NO: 57); RQLVVKFRILPC (SEQ ID NO: 58); RQLVVKFRVLPC (SEQ ID NO: 59); RQLVVKFRELPC (SEQ ID NO: 60); RQLVVKFRAAPC (SEQ ID NO: 61); RQLVVKFRAIPC (SEQ ID NO: 62); RQLVVKFRAVPC (SEQ ID NO: 63); RQLVVKFRAEPC (SEQ ID NO: 64); RQEEEEFEEEPC (SEQ ID NO: 65); RQLVVKFRALXC (SEQ ID NO: 66); RQLVVKFRALPS (SEQ ID NO: 67); RQLVVKFRALPT (SEQ ID NO: 68); Or RQLVVKFRALPX (SEQ ID NO: 69).

In another embodiment, X ¹ of the formula IV is a polarity of Q, N, S, T, P or C and X ² , X ³ and X ⁴ are each the same or different hydrophobic L, V, A and X ⁵ is a positively charged K or R, wherein X ⁸ and X ^9, or one of X ¹⁰ and X ^11, in [X ⁶ - X ⁷ - X ⁸ - X ⁹ - X ¹⁰ - X ¹¹ ] FRALPC (SEQ ID NO: 6), FREEPC (SEQ ID NO: 7), FRVVPC (SEQ ID NO: 8), FEALPC (SEQ ID NO: 9), YRALPC (SEQ ID NO: 12), FRAL (SEQ ID NO: 13), or FRPC (SEQ ID NO: 14), or FR, wherein X ¹² is a positively charged K or R, m and n are each 1 or 2, m or n In this case, the amino acid sequences of the respective peptides are the same or different, and the amino acids are D- or L-type natural or unnatural amino acids.

In one embodiment, X ² - X ³ - X ⁴ is LVV, AAA, LVA, LVL, LLA, LLL, or LLV, or the peptide of [X ¹ - X ² - X ³ - X ⁴ - X ⁵ ] QVVK (SEQ ID NO: 1), QAAAK (SEQ ID NO: 3), NLVVK (SEQ ID NO: 4), or SLVVK (SEQ ID NO: 5), QVVVK (SEQ ID NO: 70), QIVVK QLVK (SEQ ID NO: 73), QLIVK (SEQ ID NO: 74), QLAVK (SEQ ID NO: 75), QLVLK (SEQ ID NO: 76), QLVIK SEQ ID NO: 79).

In one embodiment, the peptide is represented by the following sequence: RQLVVKFRALPC (SEQ ID NO: 17); RQLVVKFRALP (SEQ ID NO: 19); RQLVVKFRAL (SEQ ID NO: 20); KQLVVKFRALPC (SEQ ID NO: 21); RQAAAKFRALPC (SEQ ID NO: 24); RQLVVKFRPC (SEQ ID NO: 25); RQLVVKFREEPC (SEQ ID NO: 26); RQLVVKFRVVPC (SEQ ID NO: 27); RQLVVKYRALPC (SEQ ID NO: 30); RQLVVKWRALPC (SEQ ID NO: 31); RNLVVKFRALPC (SEQ ID NO: 32); RSLVVKFRALPC (SEQ ID NO: 33); RQLVVK- (FRALPC) ₂ (SEQ ID NO: 37); (R) ₂ -QLVVKFRALPC (SEQ ID NO: 38); (R) ₅ -QLVVKFRALPC (SEQ ID NO: 39); (R) ₁₀ -QLVVKFRALPC (SEQ ID NO: 40); (R) ₁₅ -QLVVKFRALPC (SEQ ID NO: 41); RQVVVKFRALPC (SEQ ID NO: 42); RQIVVKFRALPC (SEQ ID NO: 43); RQAVVKFRALPC (SEQ ID NO: 44); RQLLVKFRALPC (SEQ ID NO: 46); RQLIVKFRALPC (SEQ ID NO: 47); RQLAVKFRALPC (SEQ ID NO: 48); RQLVLKFRALPC (SEQ ID NO: 50); RQLVIKFRALPC (SEQ ID NO: 51); RQLVAKFRALPC (SEQ ID NO: 52); Or RQLVVRFRALPC (SEQ ID NO: 54).

In another aspect, the present invention provides a composition for cell adhesion or a use comprising a peptide having an amino acid sequence represented by any one of SEQ ID NOS: 15 to 69.

In another aspect, the present invention also provides a composition for adhering, which makes it possible to adhere a cell comprising a peptide according to the present invention to a bio-derived material or a non-bio-derived material.

The adhesive or adhesive composition according to the present invention enables adhesion of cells to organic or inorganic surfaces. The organics include cells, tissues and organs, and the minerals are metals such as noble metals including iron, copper, gold, silver and platinum, titanium or aluminum; Ceramics such as zirconia; Calcium apatite crystals such as hydroxyapatite; Polymeric synthetic resins such as vinyl, polystyrene, polyethylene; Glass; And combinations thereof. ≪ / RTI >

The peptides according to the present invention or compositions comprising them enable the attachment of a variety of materials, including biological and / or non-biological materials, to organic or inorganic surfaces such as, for example, non-biological materials such as metals, Polymeric synthetic resin, wherein the biological material includes cells, tissues, proteins, lipids, sugars or nucleic acids, or combinations thereof.

In another aspect, the disclosure also provides a cell culture composition comprising a peptide according to the present invention.

In another aspect, the disclosure also discloses a method of adhering two surfaces comprising at least one cell using the peptide according to the present disclosure or a composition comprising the same.

In another aspect, the present invention also provides a cell culture method characterized by using a cell culture medium, or composition comprising the peptide according to the present invention, or a peptide according to the present invention in a culture container coated with the peptide according to the present invention for culturing.

In another aspect, the present invention provides a method of treating a subject comprising treating a peptide or composition comprising a peptide according to the present invention to a first substance and / or a second substance; And contacting the two materials, wherein at least one of the first material or the second material is a cell and the remainder is an inorganic or organic material.

Figures 1A-1D illustrate non-living or non-biological material of peptide (A) according to one embodiment of the present invention, including glass, zirconium (Zr), vinyl, polystyrene fiber (PS fiber), PCL Polycaprolactone), titanium (Ti) and collagen (Col).
FIG. 1A is a graph showing the adhesion of a peptide of the present invention to a glass through detection of biotin bound to a glass according to an embodiment of the present invention and then binding to the glass through a disulfide bond. As a result, This result shows that the disulfide bond disappeared upon the treatment of DTT (dithiothreitol), indicating that the bond is dependent on the disulfide bond.
FIG. 1B schematically illustrates the method used to carry out the experiment of FIG. 1A, wherein the glass-bound peptide of the present invention is linked to biotin, a labeling substance, via an SH group, biotin can be detected as an antibody, This results in the disappearance due to the treatment of DTT to separate biotin from the peptide.
FIG. 1C is a graphical representation of an experiment for analyzing the adhesion to various non-living body-derived materials using FITC (fluorescein isothiocyanate) instead of biotin, as in the method described in FIG. 1A.
Figure 1d shows the results of the analysis and shows that the peptides of the present invention have adhesive properties on the surface of various non-living body-derived substances. From the left side, PBS (phosphate buffer saline), FITC-conjugate A and FITC dye are added to each material After the adhesion, the fluorescence image was analyzed.
FIG. 2A shows the results of adhesion test on the surface of Bio-OSS ^® (Geistlich Pharma, Inc), a synthetic bone graft material of FITC-labeled peptide according to one embodiment of the present invention. As a result, And exhibits excellent adhesion to a graft material.
Figure 2b is a result of the adhesion test for the surface of the MBCP ^TM (Biomatlante, Biphasic calcuim phosphate) of synthetic bone grafting material of the peptide labeled with FITC according to one embodiment of the invention, commercially available bone grafting material of the peptides according to the present . ≪ / RTI > The magnification of the fluorescence microscope used is 200 times.
FIG. 3A shows the result of the adhesion test of MC3T3-E1, anchorage-dependent cell of the peptide according to an embodiment of the present invention, to a culture dish. As a result, cells containing the peptide according to the present invention were observed with a microscope ), Indicating a rough cell membrane boundary morphology, indicating that the adhesion of the cells to the culture dish was increased by the peptide of the present invention.
FIG. 3B is a graph showing the adhesiveness of anchorage-dependent cells (MC3T3-E1) to a hydrophobic culture dish according to one embodiment of the present invention. As a result, PLL (poly-L- lysin). As a result, the cells containing the peptides according to the present invention showed increased adhesiveness compared to the control group (treated with PBS and PLL) by microscopic observation. The adhesiveness in such a hydrophobic culture dish indicates that various tissues having hydrophobic properties can compensate for the disadvantages of cell adhesion of food ingredients (PCL, etc.) and thus have high industrial application value.
FIG. 3c shows the results of comparing the adhesion of peptides to anhydrophobic culture dish of anchorage-dependent cells (ST2) according to one embodiment of the present invention in comparison with the medium control group. The cells containing the peptides according to the present invention were compared with the control group , Indicating that the adhesiveness is increased.
FIG. 3D shows the result of comparing the adhesion of the peptide to the culture dish after thawing the anchorage-dependent cells (C2C12) of the peptide according to one embodiment of the present invention and comparing it with the medium control group. The cells containing the peptide according to the present invention were observed under a microscope As compared with the control group, adhesion was increased. In particular, the adhesion ability of cells after thawing has a very important effect on the survival of cells. The above results can be usefully used to minimize the cell death after thawing of the peptide adhesive according to the present invention. In the culture field of primary cells And thus the industrial application value is high.
Figure 3e is a comparison of the adhesion of mitomycin-treated STO feeder cells to hydrophilic culture dishes of peptides according to one embodiment of the present invention with negative control (Mock) and gelatin, Included cells show increased adhesion as compared to negative control and gelatin by microscopic observation.
Figure 3f is the result of comparing the adhesion of various cells (established cell lines and primary cells) shown in this figure to the hydrophobic culture dish of the peptides according to one embodiment of the present invention with a negative control (Mock) using cell metabolism measurement . The results indicate that peptides according to the present invention can increase their adhesiveness compared to negative controls in various types of cells.
4A to 4C are results of analysis of the mechanism of cell adhesion of the peptide according to the present invention.
Figure 4a shows that cell adhesion by peptide A according to the present invention does not require cell surface electrolytes (EDTA treatment results) or new protein synthesis (results of CHX treatment), whereas results showing initial inhibition by serum , Suggesting that a large number of GAG present in the serum binds primarily to the peptide.
FIG. 4B shows the result of analyzing the association of GAG or collagen with the peptide cell adhesion promoting mechanism. As a result that the above figure is related to both collagen and GAG, the phenomenon is observed by decomposing a provisional target by enzyme treatment . The decrease in cell adhesion by collagenase treatment is at least the interaction with collagen. And the treatment with hyaluronidase at least reduced the cell adhesion by hydrolyzing heparin sulfate, indicating the interaction of heparin sulfate and peptide in GAG. As a result of the analysis of the relationship between GAG and heparin, it was found that the addition of heparin and C-sulfate significantly decreased the cell adhesion ability promoted by the peptide according to the present invention in a concentration-dependent manner. It shows the relation with GAG. The GAG concentration change has been reported to be related to various diseases. Thus, the peptide according to the present invention is a novel peptide having GAG binding / adhesive property and is useful as a drug delivery system for targeting a novel peptide or GAG that regulates GAG in the blood .
FIG. 4c shows that the peptides according to the present invention and the soluble RGDS peptides are competitively treated. In the non-peptide-treated group according to the present invention, when soluble RGDS is treated, they bind preferentially to the integrin molecule, But the peptide treatment group according to the present invention showed no effect on the cell adhesion even after treatment with RGDS. This shows that the peptide according to the present invention is a cell different from anchorage-dependent cell adhesion through integrins Adhesion promoting mechanism.
FIG. 5A is a graphical representation of a reporter system and an experimental procedure using the same to measure the adhesive performance of the peptide to induced pluripotent stem cells (iPSCs) according to the present invention. It is an indicator of the stameness of stem cells. It contains a structure capable of measuring the activity of the Oct4 gene.
FIG. 5B is a graph showing the results of measurement of Oct4 expression on the 2nd and 3rd day of cell culture using the construct according to FIG. 5A as a result of measuring the adhesion and maintenance of the differentiation potential of mouse induced pluripotent stem cells. Coated peptides were used and the peptides of the present invention were added in a precoated or mixed manner, both of which mediated the adhesion of iPSC and maintained the ability to differentiate.
FIG. 5C is the same as FIG. 5B. As a result of measurement on the fourth day of cell culture, the peptide according to the present invention mediated adhesion of iPSC and maintained the ability to differentiate.
FIG. 6a shows the results of observation of the expression of Alp (alkaline phosphotase) gene, which is another marker showing the ability to differentiate into pluripotency, in the case where the peptide according to the present invention is previously coated or mixed with culture medium, mediates adhesion of iPSC, .
FIG. 6B shows the results of observation of the expression of Nanog gene, which is a different marker for differentiating ability, through dyeing. It has been shown that the peptide according to the present invention mediates adhesion of iPSC and maintains the ability to differentiate in all cases when it is precoated or mixed with culture medium.
FIG. 6C shows the same experiment as in FIG. 5B or an experiment using hESC, indicating that the hESC can grow well without external environmental changes such as feeder cells or Matrigel.
FIG. 7A is a schematic representation of a bio-conjugation process using a peptide according to the present invention. DSS (Disuccinimidyl suberate) was conjugated to a bone forming protein BMP2 using a linker according to one embodiment of the present invention.
FIG. 7B is a graph showing the expression of the gene for Alpinism, Alp, after 48 hours of culturing C2C12 cells after treating the peptide according to the present invention conjugated to BMP2, as shown in FIG. 7A, (rhBMP2) or the cross-linking agent treatment group (CL), indicating that the peptide treatment of the present invention promoted cell adhesion and thereby osteogenesis.
8A to 8C are the results of testing the safety of the peptide according to the present invention using osteoblast (MC3TC-E1).
FIG. 8D is a result of testing the safety of peptides according to the present invention, and it is a result of investigating whether induction of inflammation occurs after stimulation of peptides after monocyte separation of mouse.
FIGS. 9A to 9C show the results of comparing effects with existing adhesive peptides.
FIG. 9A is a photograph showing the spinal cord-derived neurons coated with a peptide according to the present invention according to one embodiment of the present invention and a culture plate coated with laminin and poly-L-ornithin as a conventional peptide The results of CCK-8 analysis at each time point after incubation with the culture plate of the present invention.
FIG. 9B shows the result of comparison observation through an optical microscope after 4 days of culture.
FIG. 9c is a graph showing the results of a neuronal marker protein as a result of verifying that a neural cell coated with a peptide according to an embodiment of the present invention is a neuron-derived cell, and then analyzing the nerve marker protein through a confocal microscope after staining with immunofluorescence to be. The results show that the peptides according to the present invention have significantly superior adhesive properties than the conventional peptides.
FIGS. 10A to 10C are results of assaying 6 hours after cell culture as a result of confirming that adhesion progression of cells using hMSC is promoted by peptides according to the present invention.
Figure 10a shows that the negative control group still has a large number of actin-ring formation, which is the initial stage of adhesion, but in the case of the peptide treatment group according to the present invention, stress fiber formation has already been observed in a large number of cells.
FIG. 10B is a graph obtained by quantifying the observation result.
FIG. 10C is a graph showing the geometrical shape (cell aspect ratio: ratio of cell length / length, complete circle in 1 case, and initial state of adhesion process) according to cell adhesion, The peptides showed higher mature adhesiveness in the treated group.
11A and 11B are the results of measuring the cell adhesion promoting activity by the peptide having various sequences according to the present invention. The peptide names on the horizontal axis in Fig. 11A are represented by SEQ ID NOs. 11B is an experiment using a sequence generated by introducing various amino acid substitutions into A peptide which is one of the sequences of FIG. 11A, and the abscissa numbers indicate the sequence numbers of the peptides.
Figure 12 shows the results of promoting established cell line epithelial cell adhesiveness and retention of peptides according to the present invention.
FIG. 13 shows the results of showing promotion of primary epithelial cell adhesion ability and retention of characteristics of peptides according to the present invention.
Peptide A and A7-1 according to the present invention are the same names in the drawings.

In one embodiment, the present invention relates to peptides represented by the following general formulas (I) to (IV), which enable attachment or adherence of cells to various substances or surfaces thereof, and their use. Specifically, Cell adhesion method.

Justice

The amino acid residues herein are represented by a single letter code and the abbreviations are: A, alanine; R, arginine; N, asparagine; D, aspartic acid; C, cysteine; E, glutamic acid; Q, glutamine; G, glycine; H, histidine; I, isoleucine; L, lysine; K, lysine; M, methionine; F, phenylalanine; P, proline; S, serine; T, threonine; W, tryptophan; Y, tyrosine; V, valine; B, asparagine, aspartic acid; Z, glutamine, glutamic acid; X, any amino acid.

As used herein, the term " amino acid " refers to 20 natural and unnatural amino acids, amino acids that are modified in vivo after translation, such as phosphocerine and phosphothreonine; And other rare amino acids such as 2-amino adipic acid, hydroxy lysine, nor-valine, nor-leucine; Including those modified to improve in vivo stability, and encompass both D- and L-forms which are enantiomers.

The positively charged amino acids, negatively charged amino acids, polarity uncharged amino acids and nonpolar aliphatic amino acids of the present invention are determined according to the chemical nature of the side chain and are well known in the art and can be selected by those skilled in the art. For example, the positively charged amino acid comprises K or R, the negatively charged amino acid comprises D or E, the polar uncharged amino acid comprises S, T, C, P, N or Q, The amino acid may comprise G, A, L, V, M or I.

The terms used herein in connection with amino acids refers to compounds in the form found in cells, tissues or in vivo, both natural and non-natural, the latter of which means that artificial modifications have been made to these compounds for various purposes.

As used herein, the term peptide or polypeptide refers to a molecule in which amino acid units are covalently linked and is used interchangeably and is read from the N-terminus to the C-terminus unless otherwise stated. It may be used in combination with native amino acids or their degradation products, synthetic peptides, recombinantly produced peptides, peptide mimetics (typically synthetic peptides), and peptide analogs such as peptoids and semipeptides or with improved in vivo stability Lt; / RTI > Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications such as CH ₂ -NH, CH ₂ -S, CH ₂ -S═O, CH ₂ -CH ₂ , Side chain modifications. Methods for preparing peptide mimetic compounds are well known in the art and can be found in, for example, Quantitative Drug Design, CA Ramsden Gd., Choplin Pergamon Press (1992).

As used herein, the term adhesive is intended to include attachment and adsorption, including reversible, irreversible adhesion, and in another aspect, by one or more chemical bonds including covalent bonds, ionic bonds, van der Waals bonds, Adhesion. In yet another aspect, it is meant that the surfaces of two materials or materials that are the same or different from each other are closely linked to achieve the desired effect. For example, one of the two different materials is a cell or tissue, and the other is an organic or inorganic surface, including an inorganic or inorganic surface, or a cell or tissue.

The inorganic or inorganic surfaces herein are used interchangeably and include materials which are free of distribution of electrons due to their excellent electrical conductivity or inorganic substances in the conventional sense that do not contain carbon, And examples thereof include noble metals including metals such as iron, copper, gold, silver and platinum; titanium or aluminum; Ceramics such as zirconia; Calcium apatite crystals such as hydroxyapatite; Polymeric synthetic resins such as polyethylene; Glass; And combinations thereof. ≪ / RTI >

Herein, the organic or organic surface is used interchangeably and refers to a carbon-based compound, in particular, a compound derived from an organism. But are not limited to, for example, cells, tissues, organs, or their in vitro or in vivo cultures.

The term surface according to the present application is interpreted in the widest range and is not limited to a specific shape or a specific size existing in a material of two or more dimensions and is not limited to a surface existing in a specific unit or molecule level material, And a surface having the same. For example, at levels ranging from several millimeters to several meters in size, as well as being present in particles or materials of the order of nanometers to micrometers.

Cell adhesion in the present application includes attaching or adsorbing a cell or a tissue composed of cells to another cell or tissue, or other organic or inorganic substance (surface).

Adhesiveness Peptides

In one aspect, the present invention is directed to a peptide of formula I:

(I)

[X ¹ - X ² - X ³ - X ⁴ - X ⁵ ] m

In the above formula (I)

Wherein X < ^{1 >} is any amino acid or polar uncharged amino acid,

Wherein X ² , X ³ and X ⁴ are amino acids of any one of L, V, I, E, G and A, which are the same or different, and at least one of X ² , X ³ or X ⁴ may be absent In addition,

Wherein X ⁵ is a positively charged amino acid,

M is an integer of 1 to 5, and when two or more amino acids are included, the amino acid sequences of the respective peptides are the same or different, and the amino acids are D- or L-type natural or unnatural amino acids or derivatives thereof.

The compounds of formula (I) according to the present invention are not only involved in intercellular or intercellular binding between cells and other surfaces as a first region, but can also be used in a variety of ways depending on the specific purpose according to the invention. The first region is a key region that can affect the secondary structure of the peptide and thus maintains the molecular characteristics of other functional regions described later.

In the peptide according to the present invention, the first region may include one or more monomers composed of five amino acids, and when two or more monomers are included, the monomers may be different or the same. The number of monomers may be varied depending on the functionalization of the peptides for the manufacture, storage, delivery or for the efficacy and utility of the peptides according to the present invention as described below. For example 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 Includes dogs.

In Formula I, X ¹ is any amino acid, and in one embodiment is a polar, uncharged amino acid, or in another embodiment, S, T, C, P, N or Q.

X ² , X ³ and X ⁴ in formula (I) are each one of L, V, I, E, G and A and each residue may be the same or different or at least one of X ² , X ³ or X ⁴ And X ² -X ³ -X ⁴ sequences may be, but are not limited to, for example, AAA, EEE, LVG, LVA, LVL, LVV, LLA, LLL, or LLV. In another embodiment, formula I may be X ¹ -LVV-X ⁵ , X ¹ -AAA-X ^5, or X ¹ -EEE-X ⁵ .

In one embodiment, the positively charged amino acid is K or R.

In one embodiment, the sequence of Formula I is selected from the group consisting of QLVVK (SEQ ID NO: 1), QEEEK (SEQ ID NO: 2), QAAAK (SEQ ID NO: 3), NLVVK (SEQ ID NO: 4), SLVVK (SEQ ID NO: 5), QVVVK QVVK (SEQ ID NO: 71), QAVVK (SEQ ID NO: 72), QLLVK (SEQ ID NO: 73), QLIVK (SEQ ID NO: 74), QLAVK (SEQ ID NO: 75), QLVLK QLVAK (SEQ ID NO: 78), or QLVVR (SEQ ID NO: 79), but is not limited thereto.

In another aspect, the present invention relates to a peptide of formula I-II, which is not limited to this sequence, further comprising a compound of formula (II) or a compound of formula (I)

≪ RTI ID = 0.0 &

[X ⁶ - X ⁷ - X ⁸ - X ⁹ - X ¹⁰ - X ¹¹ ] n,

In the above formula (II)

X ⁶ is one amino acid of F, Y and W,

X ⁷ is an amino acid of K or R,

X ⁸ is an amino acid of any one of A, M and I,

X ⁹ is an amino acid of any one of L, M and G,

X ¹⁰ is any amino acid,

X ¹¹ is an amino acid of any one of C, S and T,

At least one of X ⁸ and X ⁹ or X ¹⁰ and X ¹¹ may be absent,

Wherein n is 1 or 2, and the amino acid is a D- or L-type natural or unnatural amino acid or a derivative thereof.

The compound of formula (II) may be linked to the amino terminal (N-terminal) or carboxy terminal (C-terminal), or both the N-terminal and C-terminal of the compound of formula (I).

According to the present invention, the compound of the formula (II) is a second region, which when present together with the first region as a single peptide molecule can easily form an alpha -helical structure and confer hydrophilic properties on the peptide according to the present invention.

The first region of formula (I) and the second region of formula (II) may each contain one or more, and the arrangement thereof may vary. For example, peptides of the formulas I and II, respectively, may be linked together to form a peptide of the formula I-II, a peptide of the formula I-I-II or II, (I), (II), (III), (III), (IV), (V) or I < / RTI > In this structure, the order of the formulas (I) and (II) can be changed.

In one embodiment, the peptide of Formula II is selected from the group consisting of FRALPC (SEQ ID NO: 6), FREEPC (SEQ ID NO: 7), FRVVPC (SEQ ID NO: 8), FEALPC (SEQ ID NO: 9), YRALPC No. 11), FRALP (SEQ ID NO: 12), FRAL (SEQ ID NO: 13), or FRPC (SEQ ID NO: 14) or FR.

In another aspect, the peptide of Formula I, or a peptide comprising both Formulas I and II, may further comprise a third region represented by X ¹² _1-15 of Formula III at its N or C terminus, X ¹² is any amino acid that is positively or negatively charged.

In one embodiment, the amino acid positively charged in Formula III is K or R, or F, K or R.

In another embodiment, the negatively charged amino acid in Formula III is D or E.

Formula (III) may be comprised in various arrangements of the first region of Formula (I) and the second region of Formula (II), and each of Formula (I) and Formula (II) may include one or more each as mentioned above. For example, formula (III) is a peptide of formula (I-II) in which one or more peptides of formula (I) and (II) are linked, a peptide of formula (I) II, or to the N or C terminus of a peptide having the structure of formula (I) - (II) - (I) or (II) - (I)

In one embodiment, it is particularly linked to the N-terminus of a peptide comprising formula (I) and (II).

In another embodiment, the present invention is directed to a peptide of formula (IV): wherein R < 1 >

(IV)

[X ¹² ] _1-15 - [X ¹ - X ² - X ³ - X ⁴ - X ⁵ ] m - [X ⁶ - X ⁷ - X ⁸ - X ⁹ - X ¹⁰ - X ¹¹ ] n _,

The description of each residue included in the above formula (IV) can be referred to the above.

In another embodiment, formula IV is defined as:

In the formula (IV), in [X ¹ - X ² - X ³ - X ⁴ - X ⁵ ] m,

X < ¹ > is any amino acid,

One of said X ^2, X ³ and X ⁴ is L, V, I, E, G and with any of the amino acids of the A, and the same or different, and wherein X ^2, X ³ or X ^4, two, Or all three residues may be absent,

X < ⁵ > is an amino acid of K or R,

M is an integer of 1 to 5, and when two or more amino acids are included, the amino acid sequences of the respective peptides are the same or different,

Wherein ^{[X 6 - X 7 - X} 8 - X 9 - X 10 - X 11] n is or can be devoid, or

In n, the ^{[X 11 X 6 - X 7} - X 8 - X 9 - - X 10]

X < ⁶ > is an amino acid of any one of F, Y and W,

X ⁷ is an amino acid of K or R,

X ⁸ is an amino acid of any one of A, M and I,

X ⁹ is any amino acid of L, M and G,

Wherein X ¹⁰ is any amino acid,

X ¹¹ is an amino acid of any one of C, S and T,

Wherein X ⁸ and X ⁹ together and / or X ¹⁰ and X ¹¹ are Can be lacking together,

Wherein n is 1 or 2,

X ¹² is a positively charged K or R, and the amino acid is a D- or L-type natural or unnatural amino acid or a derivative thereof.

In one embodiment, the polypeptide of formula (IV) may be linked to two or more molecules, for example two molecules linked in the N-C direction.

In another embodiment, any of the amino acids of X ¹ in Formula IV may be of S, T, C, P, N or Q of polarity and X ² - X ³ - X ⁴ may be AAA, EEE, LVG, LVA, LVL, may be LVV, LLA, LLL, or LLV, the ^{[X 1 - X 2 - X} 3 - X 4 - X 5] the peptide of the QLVVK (SEQ ID NO: 1), QEEEK (SEQ ID NO: 2), QAAAK ( SEQ ID NO: 3), NLVVK (SEQ ID NO: 4), SLVVK (SEQ ID NO: 5), QVVVK (SEQ ID NO: 70), QIVVK (SEQ ID NO: 71), QAVVK (SEQ ID NO: 72), QLLVK No. 74), QLAVK (SEQ ID NO: 75), QLVLK (SEQ ID NO: 76), QLVIK (may be SEQ ID NO: 77), QLVAK (SEQ ID NO: 78), or QLVVR (SEQ ID NO: 79), wherein [X ⁶ - X ^{7 - X 8 - X 9 - X} 10 - peptide of X ^11] is FRALPC (SEQ ID NO: 6), FREEPC (SEQ ID NO: 7), FRVVPC (SEQ ID NO: 8), FEALPC (SEQ ID NO: 9), YRALPC (SEQ ID NO: 10) , WRALPC (SEQ ID NO: 11), FRALP (SEQ ID NO: 12), FRAL (SEQ ID NO: 13), FRPC (SEQ ID NO: 14) or FR.

In another embodiment, the peptide satisfying the above condition may be an amino acid sequence of any one of SEQ ID NOS: 15 to 33, or 37 to 69.

In another embodiment, X ¹ in the peptide of formula (IV) is Q, N, S, T, P or C of polarity, X ² , X ³ and X ⁴ are each the same or different hydrophobic L, V, I, or A, and X ⁵ is a positively charged K or R, wherein X ⁸ and X ⁹ together and / or in the above [X ⁶ - X ⁷ - X ⁸ - X ⁹ - X ¹⁰ - X ¹¹ ] X ¹⁰ and are X ¹¹ may be lacking with, the sequence is FRALPC (SEQ ID NO: 6), FREEPC (SEQ ID NO: 7), FRVVPC (SEQ ID NO: 8), FEALPC (SEQ ID NO: 9), YRALPC (SEQ ID NO: 10), (SEQ ID NO: 11), FRALP (SEQ ID NO: 12), FRAL (SEQ ID NO: 13), or FRPC (SEQ ID NO: 14), or FR, wherein X ¹² is a positively charged K or R, 1 or 2, and m or n is 2, the amino acid sequences of the respective peptides are the same or different, and the amino acid is a D- or L-type natural or unnatural amino acid. In this case, X ² - X ³ - X ⁴ may be LVV, AAA, LVA, LVL, LLA, LLL, or LLV. In this case, [X ¹ -X ² -X ³ -X ⁴ -X ⁵ ] is preferably selected from QLVVK (SEQ ID NO: 1), QAAAK (SEQ ID NO: 3), NLVVK (SEQ ID NO: 4), SLVVK (SEQ ID NO: 70), QIVVK (SEQ ID NO: 71), QAVVK (SEQ ID NO: 72), QLLVK (SEQ ID NO: 73), QLIVK SEQ ID NO: 77), QLVAK (SEQ ID NO: 78), or QLVVR (SEQ ID NO: 79). In one embodiment, the peptide sequence that meets the above conditions is selected from the group consisting of SEQ ID NOS: 17, 19, 20, 21, 24, 25, 26, 27, 30, 31, 32, 33, 37, 38, 39, 40, 43, 44, 46, 47, 48, 50, 51, 52 or 54. [

In still another embodiment, the present invention relates to a peptide having cell adhesion ability represented by any one of SEQ ID NOS: 15 to 69.

However, the peptide according to the present invention is not limited to the above sequence, but includes biologically equivalents thereof. Biological equivalents are those which have undergone further modifications to the amino acid sequences disclosed herein but which have substantially the same activity as the polypeptides according to the present invention.

Such modifications include, for example, deletion, insertion and / or substitution of amino acid sequence residues. Such amino acid variations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are both positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have similar shapes. Thus, in view of this, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

In one embodiment, conservative amino acid substitutions have been made in the polypeptides according to the present invention. Conservative amino acid substitution refers to a substitution that does not substantially affect or diminish the activity of a particular polypeptide. For example, conservative substitution occurs at one or more amino acid residues.

Conservative amino acid substitutions are known in the art and are described, for example, in Table 1 based on Blosum (BLOcks Substitution Matrix) and are described in Creighton (1984) Proteins. W. H. Freeman and Company (Eds); And Henikoff, S .; Henikoff, J.G. (1992). "Amino Acid Substitution Matrices from Protein Blocks ". PNAS 89 (22): 10915-10919. doi: 10.1073 / pnS.89.22.10915; WO2009012175 A1 and the like can be referred to.

[Table 1]

Therefore, the present invention encompasses a sequence represented by SEQ ID NO: 1 to 79 and a sequence in which conservative substitution has occurred in the sequence.

Also, in introducing amino acid mutations, the hydrophobic index of amino acids can be considered. Each amino acid is assigned a hydrophobic index according to its hydrophobicity and charge: isoruicin (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

Such a hydrophobic index is particularly important in imparting an interactive biological function of the protein. It is known that substitution with an amino acid having a similar hydrophobicity index can retain similar biological activities. When the mutation is introduced by referring to the hydrophobic index, substitution among amino acids showing a hydrophobic index difference preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5 is advantageous.

It is also well known that substitution between amino acids with similar hydrophilicity values results in proteins with equivalent biological activity.

For example, as disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Aspartate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoru Isin (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

Amino acid substitutions that do not entirely alter the activity of the peptides according to the present invention are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). For example, the most commonly occurring substitutions are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / , Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

Therefore, considering the mutation having biological equivalent activity as described above, the amino acid sequence disclosed herein or the nucleic acid molecule encoding the same includes those having substantial identity with those disclosed herein. Substantial identity is determined by aligning the sequence disclosed herein with any other sequence as closely as possible and providing a sequence having at least 61% homology, and more preferably a sequence identity of at least 61%, when analyzed for sequences that have been aligned using algorithms commonly used in the art Means a sequence that exhibits 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are well known in the art. For example, Smith and Waterman, Adv . Appl . Math. (1981) 2: 482; Needleman and Wunsch, J. Mol . Bio. (1970) 48: 443; Pearson and Lipman, Methods in Mol . Biol . (1988) 24: 307-31; Higgins and Sharp, Gene (1988) 73: 237-44; Higgins and Sharp, CABIOS (1989) 5: 151-3; Corpet et al., Nuc . Acids Res. (1988) 16: 10881-90; Huang et al., Comp. Appl . BioSci . (1992) 8: 155-65 and Pearson et al., Meth . Mol . Biol . (1994) 24: 307-31. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. MoI . Biol. (1990) 215: 403-10) is accessible from NBCI and the like, and can be used as blast, blastp, blasm, blastx, tblastn and tblastx It can be used in conjunction with a sequence analysis program. BLSAT is available at www.ncbi.nlm.nih.gov/BLAST/, and the sequence homology comparison using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

In one embodiment, the peptides according to the present invention described above may be substituted with a group such as a chemically unreactive group such as NH2 or the like to increase the stability, for example, the N- or C-terminus, particularly the C-terminus.

The peptides according to the present invention can be modified into various compounds, for example, by modifying epsilon amino groups in such a manner that various functional materials and the like are bound to the peptides.

In another aspect, the disclosure also relates to a polynucleotide encoding a polypeptide according to the present invention, a recombinant vector comprising the polynucleotide, and a cell into which the recombinant vector is introduced. Polypeptides are those in which one or more polynucleotides encoding a polypeptide according to the present invention may be present or include a codon-optimized sequence, given that a particular amino acid is encoded by one or more codons. Furthermore, the vector enables the expression of the cloned gene in prokaryotic and / or eukaryotic cells. Methods for cloning into such a vector, and for transferring the vector to prokaryotic or eukaryotic cells, can be used, , One skilled in the art will be able to use the appropriate one.

In another aspect, the present invention relates to a use of a peptide defined by the above-mentioned chemical formula for cell adhesion, and relates to a cell adhesion composition and a cell adhesion method comprising the above-mentioned peptide.

The peptide according to the present invention can adhere cells to a variety of bio-derived or non-bio-derived substances or surfaces thereof, such as inorganic or organic substances or their surface or other aspect. With this feature, it is possible to mediate the adhesion between one substance and the same or different substances. For example, when a peptide according to the present invention is used, cells incubated with Invit can be adhered to a culture dish, or mediated through cell-cell adhesion, or combined with a labeling substance such as a fluorescent substance, And it is possible to increase the therapeutic effect when a substance such as a useful bioactive substance such as an osteogenic protein is bound to the peptide of the present invention and the bioactive substance is treated to a tissue expected to have an effect .

Although not limited to this theory, the peptides according to the present invention can be applied to components constituting proteoglycans, which are abundant in ECM (Extra Cellular Matrix) on the surface of cells, such as heparan sulfate, heparin, (chondroitin sulfate) and the like. In addition, the peptide according to the present invention does not increase the adhesiveness through interaction with the cell membrane protein, and has a cell adhesion promoting mechanism distinct from anchorage-dependent cell adhesion through the integrin acting on the RGD peptide. Compared with RGD, the treatment concentration was 100 times less and it was found to be much smaller. For example, proteoglycans are tissue components that form cartilage, and include materials that are very useful in tissue regeneration and that are directly utilized in various clinical procedures including molding.

Thus, the cells to which the peptides of the present invention or compositions or methods comprising them may be applied include those derived from animals, plants and parts of the body, such as cells, tissues, and organs.

Cells to which the peptide or composition according to the present invention can be applied are not particularly limited and can be applied to plants, insects and animal cells, for example animal cells, especially human-derived cells such as skin, dermis, endothelium, epidermis, Primary cells, or established cell lines, including cells derived from muscle, myocardium, nerve, bone, cartilage, brain, epithelium, heart, kidney, pancreas, spleen, oral cavity, cornea or hair.

In another aspect, the present invention relates to a composition for adhering a cell-derived bio-material or a non-bio-based material comprising the peptide disclosed in the present invention.

In another aspect, the present invention relates to a cell culture composition comprising a peptide according to the present invention.

The surface to which the peptide according to the present invention or the composition containing the same or the method using the same is applicable is not particularly limited and includes both hydrophilic or hydrophobic organic or inorganic surfaces. In one embodiment, the surface to which the composition of the present invention may be applied includes all of bio-derived material or non-bio-based material including plastic, glass, polymer synthetic resin, and metal.

The use of peptides or adhesive compositions according to the present application is not limited thereto, but may include (1) adhesion between substrates in water (water or saline water); (2) orthopedic treatments such as bones, ligaments, tendons, meniscus and muscle treatments and artificial material implants; (3) ophthalmic adhesions such as perforation, fissure, incision, corneal transplantation, and artificial corneal incision; (4) dental junctions such as correction devices, prosthetic dentures, crown placement, shaking teeth fixation, broken tooth treatment, and filler fixation; (5) surgical treatment such as vascular occlusion, cell tissue grafting, artificial material grafting, wound closure; (6) junctions in plants such as plant graft joining, wound healing; (7) cell or tissue culture including stem cells; (8) Medical devices such as artificial organs, dental, surgical or ophthalmic devices such as implants, bone removers, bone cages, guidewires, catheters and stents; and (9) For example, bioconjugation with a bioactive substance, a drug, a labeling substance, a target substance, or the like.

In one embodiment, the peptide or composition of the present disclosure is used for dental, ophthalmic or orthopedic treatment for implantation or reconstruction of a cell or tissue for the treatment of a disease, wherein the surface to which the present adhesive composition may be applied But are not limited to, PLGA, hydroxyapatite, zirconium, titanium, iron, stainless steel, titanium, platinum, gold, and alloys.

In yet another embodiment, the peptide or composition of the present disclosure is used to adhere cells to a support. The cell adhesion surface or support herein includes cell culture dishes, microbeads, basal materials, tissue implants and the like, and can be used, for example, in the culture of tissues or cells, particularly in the culture of stem cells. In one embodiment according to the present invention, when the peptide of the present invention was used, it showed much higher adhesive force than the substance used for conventional cell adhesion.

In another embodiment, the peptide or composition herein is used in bioconjugation. Bioconjugation refers to a chemical method / means for linking two biomolecules with stable covalent bonds. The peptide according to the present invention can be used directly or by using a low-molecular linker to bind various biomolecules such as enzymes, hormone- A nucleic acid, a lipid, or a carbohydrate. Such bioconjugation can be used as a research tool for detection and monitoring of biochemical substances, or can be conjugated with a therapeutic substance as a therapeutic agent to increase the efficiency of treatment or to be used for target therapy. In one embodiment according to the present application, it is conjugated to an osteogenic protein to increase the therapeutic effect of the osteogenic protein.

The method of using the peptide and the composition according to the present invention is based on a method of using a conventional bioadhesive composition, and a typical method is a coating method. For example, reference may be made to Cell-Tak ^® (BD Biosciences, USA), a commercially available product, for the specific usage, dosage and formulation of the peptide or composition according to the present invention.

The composition according to the present application may be in the form of a solvent, water-soluble or solventless, and may be used in an amount of 0.1 to 1000 ng / mm ² , especially 1 to 100 ng / mm ² , based on the area of the surface to which it is applied.

The composition according to the present invention can be adjusted with the adhesive force and the application amount thereof by treating with a surfactant, an oxidizing agent, a cross-linking agent, or a filler, or by adjusting the concentration of the peptide.

A composition comprising a peptide according to the present invention enables culturing of cells while maintaining all the characteristics of the cells to be cultured. The peptides according to the present invention can be used as is in conventional culture methods for the cells to which they are applied. That is, a cell in which an appropriate number of cultures are required may be suspended in a liquid medium or a cell culture solution which can be commonly used, and the peptide according to the present invention may be added thereto. Subsequently, the liquid medium can be exchanged and subcultured in the same manner as in the conventional method. In this process, the peptide according to the present invention may or may not be added.

The peptide according to the present invention can be added to the cell culture medium and used.

The peptide according to the present invention can be used by fixing or coating on the solid phase surface of a culture container. As the culture container, any of those conventionally used for cell culture such as a plate or a flask can be used. The culture container may be sterilized, and an inorganic material such as glass or an organic material such as polystyrene or polypropylene may be used.

As a method for fixing or coating the peptide according to the present invention on the solid surface of a culture container, a chemical method such as a physical method such as adsorption or a covalent bond can be applied, and for example, a method based on adsorption which is easy to operate is preferable.

In another aspect, the present invention also relates to a method of cell culture using the peptide according to the present invention or a composition comprising the same, wherein the peptide according to the present invention or a composition comprising the peptide is used for culturing the cells.

In one embodiment of the method according to the present invention, the peptide according to the present invention or a composition comprising the peptide can be used by being fixed or coated on the solid phase surface of a culture container. The culture container which can be used in the present method is not particularly limited as long as it is capable of culturing cells, but it is preferable that the culture container is used for cell culture. Examples of the cell culture container include a culture dish, a plate, a flask, a chamber slide, a tube, a roller bottle, a spinner flask, a hollow fiber, a micro carrier, and a bead. These culture containers may be made of an inorganic material such as glass or an organic material such as polystyrene. The culture container may be made of a material such as polystyrene having a high adsorption property to a protein or a peptide, or a treatment for enhancing adsorption, for example, a hydrophilic treatment or a hydrophobic treatment.

In another embodiment of the method according to the present invention, a physical method such as adsorption or a chemical method such as covalent bonding can be applied as a method of fixing or coating the peptide according to the present invention on the solid surface of a culture container, A method by one adsorption is preferable. In one embodiment, the peptide according to the present invention can be adsorbed by contacting a solution comprising the peptide according to the invention to the surface of the culture vessel, such as a plate, and removing the solvent after a period of time.

In another embodiment of the method according to the present invention, the peptide according to the invention can be used in addition to conventional cell culture media. For example, by suspending an appropriate number of the desired cells in a conventionally available liquid medium or cell culture medium, and adding thereto an appropriate amount of the peptide according to the present invention, or the peptide according to the present invention, Lt; / RTI > Subsequent exchange and subculture of the liquid medium can be carried out in the same manner as in the conventional method. In this process, the peptide according to the present invention may be further added or not added.

In the method according to the present invention, cell culture can be carried out using conventional culture medium or culture conditions except for using the peptide according to the present invention.

Conventional culture methods and culture conditions can be found in the following references: R. Eibl et al., Cell and Tissue Reaction Engineering: Principles and Practice, Springer-Verlag Berlin Heidelberg 2008.

The medium used for the culture may or may not include serum (e.g., fetal bovine serum, horse serum and human serum), for example, RPMI series, Eagles ' s MEM (Eagle ' s minimum essential medium, Eagle, H. Science 130: 432 (1959)), α-MEM (Stanner, CP et al., Nat. New Biol. 230: 52 (1971)), Iscove's MEM (Iscove, N. et al. 147: 923 (1978)), 199 medium (Morgan et al., Proc. Soc. Exp. Bio. Med., 73: 1 (1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. 29: 515 (1963)), F10 (Ham, RG Exp. Cell Res. 29: 519 (1967)), F12 (Ham, Proc. Natl. Acad Sci. USA 53: 288 , A mixture of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 ), Way-mouth's MB752 / 1 (Waymouth, CJ Natl. Cancer Inst. 22: 1003 (1959)), McCoy's 5A (McCoy, TA, et al., Proc. Soc. Exp Biol. 1959) and the MCDB series (Ham, RG et al., In Vitro 14:11 (19 78)), but is not limited thereto. The medium may include other components such as antibiotics or antifungal agents (e.g., penicillin, streptomycin) and glutamine.

The composition according to the present invention may contain only the peptide according to the present invention alone, but may further include other known adhesives, adhesive proteins other than the adhesive proteins of the present invention, and substances that can be included in known cell adhesion compositions . The content of the additional component may be appropriately adjusted within a range normally permitted depending on the kind of the component and the formulation. When an additional component is included, the pepti, which is an active ingredient, is included in an amount capable of maintaining the adhesive activity, and may be included, for example, in an amount of 0.01 to 80% by weight.

In another aspect, the present disclosure also relates to a method of bonding two or more surfaces of matter using the peptide or composition herein.

In one embodiment, a method according to the present invention comprises the steps of: treating a first substance and / or a second substance with a peptide or composition according to the invention; And contacting the two materials so that the two materials can be adhered, under conditions sufficient for adhesion to occur. Such sufficient conditions will vary depending on the kind of the specific substance to be adhered to by the peptide according to the present invention, and those skilled in the art will be able to determine appropriate conditions For example, temperature, time, composition of the medium where adhesion occurs, and the like can be determined.

In one embodiment, the first material is a cell and the second material is an organic or inorganic material.

The first material or the second material according to the present invention may be treated for all or only one for attachment. In one embodiment, the adhesive or composition is treated on an inorganic surface, e.g., a cell culture dish, and cells are added to adhere to the cell culture dish.

In another embodiment, the peptide or composition according to the invention may be added to the culture medium containing the cells and added to the cell culture dish. In other embodiments, an inorganic surface, e. G., A bone graft, can be coated with an adhesive according to the present disclosure and a biomaterial such as an osteogenic protein can be attached thereto. Or it may be conjugated to a biological material useful in the adhesive according to the invention and then attached to the mineral surface.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Example  1. Adhesiveness Of peptide  Produce

The peptides used in the experiments were custom-made by 9-fluorenylmethyloxycarbonyl (Fmoc) method (Lucen Sci., Korea; Peptron, Korea). To verify the reproducibility of the peptide effect, peptides were synthesized three times (rugen) and two times (peptone), respectively, through two companies, and each compound was analyzed through independent experiments. .

Example  2. Adhesiveness Peptides and  Fluorescent Conjugation  And a variety thereof Gypsum Adhesion analysis on sieve-derived surface

The peptide of Example 1 was conjugated to Cys through an SH group with A (RQLVVKFRALPC; SEQ ID NO: 17) (corresponding to the 12-mer (A) sequence in Table 1 below) with Biotin (Thermo Scientific) and FITC (Sigma). Then, it was treated with glass, PCL, Ti, Col, Zr, vinyl, PS fiber (each 10 μM in PBS, 20 min at room temperature and washed once with PBS) Were detected by FITC fluorescence image measurement. The reduction reaction was induced by treatment with DTT (100 mM in PBS, 20 minutes at room temperature and then once washing) to liberate covalently bound biotin on the peptide. Only the dye was used as the control group.

The results are set forth in Figures 1A-1D, in which the detection of antibodies was strongly adhered to the surfaces used in the experiments via the peptides according to the invention, in particular the effect of which was that the DTT was disappeared by treatment 1a), indicating that biotin or FITC is attached to the glass through the parent peptide.

Example  3. Adhesion according to the present invention Of peptide  variety Non-living body  And Living organism origin  Adhesion Analysis to Substance

Basically the same experiment as in Example 2 was carried out and Bio-OSS ^® (Geistlich Pharma, Inc) and MBCP ^™ (Biomatlante), widely used as bone graft substitutes, were used instead of glass. The treatment conditions were as follows: Bio-Oss particles were added to a peptide-FITC conjugate at a concentration of 10 μM, reacted at room temperature for 10 minutes, then washed 5 times in PBS solution containing 0.05% tween-20 for 48 hours. And analyzed by confocal microscopy (Zeiss LSM-700 model with Zen 2011 software, x20).

In order to verify the adhesion of peptides to in vitro fertilization, tissue sections were obtained from various tissues and analyzed. To confirm the adhesion of the peptides, peptides according to the present invention, Cys- or FITC-conjugate, or Cys or FITC dyes were dissolved in PBS at a concentration of 10 μM, and then 10-20 μl of the aqueous solution was locally injected or injected with a syringe. The adhesion induction time in the tissue was fixed to 2 hours. After the sacrifice, tissue sections were washed three times for 30 minutes in PBS, and then tissue sections were prepared and analyzed by confocal microscope. The tissues were examined with cartilage, skin, hair, subcutaneous fat and eyeballs of mice. In case of cartilage, the collagenase was partially treated to expose elastase fiber structure and analyzed after strong washing.

The results are set forth in Figures 2A-2M, which show that the peptides according to the present invention can be adhered to a variety of non-living and bio-derived materials. Commercialization of Peptides According to the Present Application It shows excellent adhesiveness to bone graft materials, and in particular to tissues containing a large amount of proteoglycan components such as elastic tissues, connective tissues and tissues containing large amounts of collagen or hyaluronan, And that there is adhesion affinity to yeast, bacterial cell wall, and the like.

In addition, the peptide of the present invention is excellent in the adhesion of cartilage tissue (*) containing a large amount of GAG (glycoaminoglycan) such as heparan sulfate, heparin and chondroitin sulfate as main components of proteoglycan , Which is consistent with the experimental result of FIG. 4B to be described later. For reference, GAG contains four types of substances: hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin, heparan sulfates, and keratan sulfate. sulfate. These are all virtually comprised of amine-linked glucose or galactose, such as N-acetylglucosamine or N-acetylgalactosamine, since the peptides according to the present invention have high affinity , Indicating that the peptide according to the present invention can act as a target specifically.

Example  4. Adhesiveness Peptides  Adhesiveness improvement effect of various cells using

The materials used in this experiment were as follows: 10 mM peptide solution (stock solution, Stock solution) in PBS; Plastic hydrophobic cell culture dishes of 35 mm diameter (for bacterial culture, Corning); DMEM (Dulbecco Modified Eagle Medium, cell culture medium: Hyclone); Fetal bovine serum (Hyclone); And C2C12 (mouse myoblast, cells, ATCC, CRL-1772); MC3T3-E1 (mouse C57BL / 6 calvaria cell, ATCC, CRL-2593) and ST2 (bone marrow-derived stroma cell: EMBO J. 7: 1337? 1343, 1983); (ATCC, CRL-1573), SaOS2 (ATCC, HTB-85), HeLa (ATCC, CCL-2), ROS17 / 2.8, NIH3T3 (ATCC, CRL-1503) (Scientific, Cat. # 2630) hDPSC (Cells Tissues Organs 184: 105-16), RAW 264.7 (ATCC, TIB-71), hMSC (Lonza, PT-2501), hPDL Primary Bone Marrow Stromal Cell from mouse, Primary Mouse Calvaria (MC), Mouse Embryonic Fibroblast (MEF)

The experimental method is as follows.

The cell adhesion was observed by two methods of pre-coating the peptide and directly adding it to the cell culture without coating, and the results were consistent. When the coating method was employed, a certain concentration of peptide was added to a cell culture medium containing no PBS or monoclonal antibody, coated at room temperature for 30 minutes, the solution was removed, and the cells were added to measure the adhesion over various time periods. When the peptide was added to the culture medium, a certain level of the peptide was added to suspend the cells, the cells were suspended well and transferred to a culture dish. Cell adhesion measurements were performed as follows. Cell adhesion was assessed by F-actin for the general observation through optic microscope, actin filament structure and rough cell membrane boundary morphology using PBS and poly-L-lysin coated as a negative control when no peptide was added. The metabolism or DNA content of the cells was measured for staining and confocal microscopy analysis and for quantification of adhesion. The absorbance was measured using CCK-8 (Dojindo) and the amount of DNA was measured using the Picogreen assay kit (Life Technologies) according to the manufacturer's method. In order to quantify only the adhered cells, unadhered cells were discarded and washed twice with PBS, and only the cells attached to the surface were used for metabolism or DNA quantification.

The results are shown in Figures 3a-3f. As shown therein, the peptide according to the present invention can be useful for adhesion of various anchorage-dependent cells, for example, adhesion of primary cells, established cells and thawed cells, feeder cells, Which is superior to PLL.

Example  5. Adhesiveness Of peptide  Identification of adhesion mechanism

In the Examples, the following experiment was conducted to investigate the mechanism of peptide adhesion according to the present invention.

In this example, the following experiments were carried out to investigate the mechanism of adhesion of the peptide to peptides according to the present invention.

The cells used Engle-dependent cells (MC3T3-E1), and treated with the peptide according to the present invention (SEQ ID NO: 17) A peptide (10 μM). The adhesive ability of the peptides according to the present invention was determined by removing the unadhered cells with PBS and quantitating the DNA with the picogreen assay kit (Life Technologies) or the CCK-8 (Cell Counting Kit -8, Dojindo) was measured using the manufacturer's method.

(1) Whether protein is involved

First, adhesion performance was tested as described above after treatment with EDTA, cyclohexamide (CHX) (10 μM, 1 hour at 37 ° C.) or GAG, which is known to inhibit protein synthesis.

The results are shown in FIG. 4A. As shown in FIG. 4A, there was no difference in the adhesiveness even when EDTA or CHX was treated. That is, the cell adhesion by the peptide according to the present invention does not require the cell surface electrolyte (result of EDTA treatment) or new protein synthesis (result of CHX treatment), whereas the result shows that inhibition occurs early in the serum.

 This is probably due to the fact that many kinds of GAG present in the serum are primarily bound to the peptide. GAG (glycoaminoglycan) such as heparan sulfate, heparin, and chondroitin sulfate, which are major components of many types of proteoglycans present in the serum, As shown in Fig. Also, the peptide according to the present invention shows that it does not increase the adhesiveness through interaction with the cell membrane protein.

(2) Non-proteinaceous  Substance involvement

Next, we investigated the involvement of nonprotein substances and analyzed their association with proteoglycans, a carbohydrate lipid contained in the extracellular matrix (ECM) of the cell surface.

- In proteoglycans  About Of peptide  Molecular affinity

The major constituents of the proteoglycans are heparan sulfate, heparin, chondroitin sulfate, dermatan sulfate, and keratan sulafate, as well as glycosaminoglycans glycoaminoglycan (GAG) is a major component of the ECM, but maintains the structural integrity of the ECM structure to maintain the shape of the cell, as well as controlling cell adhesion and cell polarity do. This function of ECM not only induces adaptation of the cells to the environment, but also has a complex function of controlling various physiological roles because it is directly related to a complex series of metabolic processes.

The molecular affinity of the peptide of the present invention for proteoglycans is determined by (i) affinity chromatography, (ii) cell adhesion through competitive binding using purified GAG components, (iii) treatment of enzymes that specifically degrade ECM comprising GAG components And inhibition of cell adhesion promotion by peptides. In the case of enzyme treatment, hyaluronidase (Sigma) and collagenase (Sigma) were treated. Purified GAGs were obtained from hyaluronan (Sigma), chondroitin sulfate (Sigma), heparin (Sigma) and heparan sulfate (Sigma).

(i) Determination of interaction with peptide-heparin or peptide-N-acetylglucosamine (GlcNAc) using affinity chromatography

Heparin-agarose beads (Biovision) and GlcNAc-agarose beads (Sigma) were used for chromatography, respectively. The peptide (F-peptide) labeled with 10 ng of FITC in PBS-T solution was reacted with heparin-agarose beads and GlcNAc-agarose beads previously coated with BSA for 10 minutes at room temperature and washed three times in PBS-T solution After the cells were suspended in PBS solution, fluorescence was measured. In order to induce competitive binding, FITC labeled peptides (Cold) were added at 1: 1, 1:10 (10 times) and 1: 100 (100 times), respectively, and reacted with beads and fluorescence was measured. As a negative control, FITC dye alone was measured by reacting with each bead. Only the beads were suspended in PBS, transferred to a microplate, and the fluorescence value measured was used as a blank. The results shown in Figure 4b indicate that the peptide has a high affinity for GlcNAc, a component of heparin or GAG.

(ii) Cell adhesion performance through competitive binding using purified GAG components

The following is a result of inhibition of cell adhesion by adhesive peptides by competitive inhibition method by GAG treatment. As a result, addition of heparin, heparan sulfate, and chondroitin sulfate significantly inhibited cell adhesion of peptides. .

(iii) Inhibition of promotion of cell adhesion by peptides by treating enzymes that specifically degrade ECM containing GAG components: Fibril proteins such as collagen as well as proteoglycan constituting ECM include GAG. Hyaluronidase, which hydrolyzes collagen containing GAG and hydrolyzes hyaluronan, one of the GAGs, was investigated for its ability to promote cell adhesion by the peptide. This is also evident that the promotion of cell adhesion by peptides is correlated with GAG.

Experimental methods were as follows: (i) Competitive inhibition by purified GAG component treatment was performed by adding 5 mg / ml of each GAG to the cell suspension treated with trypsin, incubating at 37 ° C for 10 minutes, After transferring to a dish, the DNA of the adhered cells was quantified 30 minutes later and the degree of adhesion was measured. DNA quantification was performed using a picogreen assay kit (Life Technologies); (ii) Cell adhesion test by treatment with collagenase and hyaluronidase, the cells treated with trypsin were suspended in a medium containing no folate serum, followed by addition of each enzyme, followed by reaction at 37 ° C for 30 minutes. The enzyme was treated at 10,000 units / 10 ³ cell level. After the enzyme reaction, EDTA and folate serum were added to inactivate the enzyme, centrifuged, suspended in a fresh medium, and cultured in a peptide-coated culture dish for 2 hours . The cells were washed two times with PBS before the picogreen assay, and only the adhered cells were recovered to quantify the DNA.

The results are shown in Figure 4b. The results show that the peptides of the present invention have a high affinity for proteoglycans, and the tissue components forming cartilage have a wide application value in the field of tissue regeneration and adhesion to materials which are directly used in various clinical procedures including molding It shows the ability. Therefore, this indicates that the peptide of the present invention is a potential substance widely used for cartilage regeneration, drug delivery, and the like, with its excellent binding ability to the proteoglycan layer including cartilage.

(3) RGDS and  compare

The peptide peptide (10 μM) or the soluble RGDS peptide (0, 10, 100 and 1000 μM) according to the present invention was competitively treated with the cells for 10 minutes in a test tube with Engle-dependent cells (MC3T3-E1) After transferring to a culture dish, adhesion was induced again in a thermostat for 10 minutes. After 10 minutes, the culture medium was removed again and the cells were washed with PBS solution twice to remove the remaining unadsorbed cells. A new medium containing no peptide and CCK-8 (Dojindo) were mixed and incubated in a thermostat for 1 hour. The absorbance was measured. Absorbance was measured using a medium mixed with CCK-8 as a blank.

The results are shown in Figure 4c. As shown above, when the RGDS peptide alone was treated, the adhesion decreased in a concentration-dependent manner. On the other hand, when the peptide according to the present invention and the RGD peptide are competitively treated, the peptide-untreated group according to the present invention, when treated with the water-soluble RGD, binds to the integrin and significantly decreases the adhesiveness to the bottom (substrate) The treatment group showed no effect on cell adhesion even after treatment with RGD, indicating that the peptide according to the present invention has a cell adhesion promoting mechanism distinct from anchorage-dependent cell adhesion through RGD-mediated integrins will be.

These results indicate that the technology for cell adhesion or tissue regeneration using RGDS peptides has already been well established and that there is a difference in technology due to the different mechanism and commercialization techniques using RGDS are known to be inefficient. This is an excellent technique that can be achieved.

Example  6. Adhesiveness Peptides  Enhancement of embryonic stem cell adhesion using

Peptides were coated on the culture material at a concentration of 100 μM or nanostructures were produced to examine adhesion and pluripotency of mouse induced pluripotent stem cells and human embryonic stem cells as follows.

For human embryonic stem cells, hESC-media, mTeSR (purchased from hES-specific medium, Stem cell technology), Essential 8 (hES medium, purchased from Gibco BRL), and 10 ng / ml bFGF alone (culture condition to prevent the inhibition of cell adhesiveness by serum), and analyzed for compatibility with various existing culture techniques. In the case of the cells, the culture technique using a single cell was analyzed by culturing with a colony state and treating with 0.25% trypsin-EDTA. Matrigel (hESC) and gelatin (miPSC) were compared as a positive control for the feeder-free culture of ESC or iPSC. In addition, all embryonic stem cell cultures Were cultured in feeder cells used as a positive control.

The cells were transfected with a plasmid (Szabo et al., 2002, Mechanisms of Development Vol. 115: 157-160) having a structure capable of measuring the activity of the Oct4 gene as an indicator of the stameness of the embryonic stem cell described in FIG. ) Were analyzed by fluorescence microscopy (Fig. 5). In addition, expression of the other markers, Alp and Nanog, which express the differentiation ability, was stained using a specific antibody and observed with a fluorescence microscope, and the improvement of cell adhesiveness in hES cell culture was also examined (FIG. 6).

The results are shown in Figures 5 and 6. As shown in Figure 5, the peptides of the present invention were both precoated or added in a mixed manner to the culture medium, both of which mediated the adhesion of iPSC and maintained the ability to differentiate (Figure 5b) . In addition, in the case of the fourth day of cell culture, the peptide according to the present invention mediated the adhesion of iPSC and maintained the ability to differentiate (Fig. 5C). As shown in FIGS. 6A and 6B, peptides according to the present invention mediate the adhesion of iPSC and maintain their ability to differentiate in advance when they are pre-coated or cultured. Also, in experiments using hESCs, (Matrigel), and also maintains the ability to differentiate (Fig. 6c).

Example  7. Adhesion Peptides and  Protein Bio-conjugation

The materials used in this experiment were as follows:

The peptides synthesized in Example 1 were used at a concentration of 10 [mu] M (in PBS); 1.9 cm ² plastic cell culture dish (24-well plate, manufactured by Corning); DSS (Disuccinimidyl suberate, C ₁₆ H ₂₀ N ₂ O ₈ , Thermo Scientific Inc.); Recombinant human BMP2 (rhBMP2, BD bioscience); DMSO (dimethyl sulfoxide, solvent for dissolving DSS, Sigma-Aldrich); Tris-HCl, pH 7.0 (Stop solution); PBS (phosphate buffered saline, reaction solution);

The analysis concept is schematically shown in FIG. 7A. Referring to FIG. 7A, when a therapeutic protein such as BMP is connected to various peptides having desired bioactivity by using a cross-linking agent, the peptides of the present invention can be used to treat The concentration of the therapeutic protein can be increased, and the therapeutic effect can be maximized.

The experimental method is as follows. The peptide-rhBMP2 cross-linking reaction was performed according to the method of the cross-linker manufacturer used in principle, and in summary the procedure is as follows: (i) Peptide coating: 200 [mu] l of peptide solution (10 [mu] M of the peptide of Example 1 above in PBS solution) was dispensed and adsorbed at 4 [deg.] C for 18 hours; (ii) Formation of DSS-BMP2 complex: 20 molar excess of DSS for rhBMP2 was covalently bound for 1 h in 100 μl of the reaction solution. The rhBMP2 per 1.9 cm ² area was adjusted to be 300 ng. At this time, DSS, rhBMP2 and DSS-rhBMP2 were coated as the control, respectively. In this case, , Thus being used as a negative control in this case; (iii) Peptide-DSS-BMP2 complex formation: lOO [mu] l of DSS-BMP2 complex was carefully added to the pre-reacted peptide-coated dish for 24 hours at 4 ° C; (iv) quenching: 200 μl of 1 M Tris solution was added and allowed to stand at room temperature for 15 minutes to neutralize any remaining DSS after covalent bonding; (v) Washing: After washing 10 times with PBS solution, DMEM medium not containing fetal calf serum was added until cells were added, and then preliminarily stored at 37 ° C; (vi) Preparation of C2C12 suspension and osteoblast differentiation: 4x10 ⁵ C2C12 cells were suspended in DMEM medium containing 300 μl of 2% fetal calf serum, and cells were prepared. Peptide -DSS-BMP2 DMEM complexes were previously contained in the coated plate was removed, and inducing differentiation of a cell culture by addition of 300μl in an incubator of 5% CO _2/37 ℃ condition 48 hours cells; (vii) Cell differentiation assay: The ability of C2C12 cells to differentiate into osteoblasts was determined by the cytotoxicity of alkaline phosphotase (Alkaline phosphotase).

The results are shown in Figure 7b. As shown above, Alp activity was increased as compared with the recombinant BMP-treated group (rhBMP2) or the cross-linking agent-treated group (CL), indicating that bone morphogenesis was promoted by the peptide treatment of the present invention.

Example  8. According to the present invention Of peptide  Stability analysis

The safety of the peptides according to the present invention was tested using osteoblasts (MC3TC-E1). (Fig. 8A), survival (Fig. 8B), and differentiating ability (Fig. 8C) after treatment of the peptide according to the present invention synthesized in Example 1 at a concentration of 0.1, MC3T3-E1 cell line or C2C12 cell line was used for the differentiation ability. The cell line was stimulated with osteogenic differentiation medium (6 days treatment) and BMP2 (rhBMP2, 10 ng / ml, 3 days treatment) , And the differentiation effect was carried out through cell staining. The results are shown in Figs. 8A to 8C, and do not affect the growth rate, survival rate, and ability to differentiate depending on the concentration.

The safety of the peptides according to the present invention was also tested using mouse monocytes. Mononuclear cells isolated from the bone marrow of mice were treated with the peptide according to the present invention and then examined for induction of inflammation. LPS was treated as a positive control and IL-1β (interleukin-1β), Tnf-α (Tumor necrosis factor-α), iNos (inducible nitric oxide synthase), Cox- 2) were measured by real time quantitative reverse transcription PCR method. As a result, it was confirmed that the peptides according to the present invention did not cause inflammation. The results are shown in Fig. 8d, and the peptides according to the present invention did not induce inflammation and were judged to be stable in vivo.

Example  9. Adhesiveness Peptides  Primary neuron Adhesive ability  Improvement and culture

In this example, the effects of adhesion peptides on neuronal cell adhesion and culture were compared. Peptides were coated on culture media at 100 μM level or nanostructures were prepared and neuron-derived cells were obtained from mouse embryonic brain tissue and spinal cord of white rats. The cells were analyzed by optical microscope and metabolism through CCK-8 was measured and analyzed. Laminin and poly-L-ornithin (Sigma) were mixed as a positive control at the manufacturer's concentration and method. To quantify cell adhesion, cells not adhered (each time) were washed with PBS And CCK-8 solution (Dojindo), which measures the metabolism of the cells, was added to the culture medium. After incubation for 1 hour, absorbance was measured at 450 nm.

The results are shown in FIG. FIG. 9A is a photograph showing the spinal cord-derived neurons coated with a peptide according to the present invention according to one embodiment of the present invention and a culture plate coated with laminin and poly-L-ornithin as a conventional peptide . The results showed that CCK-8 was analyzed at each time point after culturing using a culture dish prepared according to the present invention, and showed a large difference in adhesive ability when the peptide according to the present invention was coated (adsorbed). FIG. 9B shows the result of comparative observation through an optical microscope 4 days after the culture, and the same amount of cells was used, but the degree of adhesion was the most excellent in the peptide according to the present invention. FIG. 9c is a graph showing that the primary cultured cells coated with the peptide according to one embodiment of the present invention are nerve-derived cells, and the nerve cell-specific proteins (GFAP, MAP2 and Nestin) It is the result of analysis through confocal microscope after staining with immunofluorescence. The results show that the peptides according to the present invention have much better adhesion ability than the conventional ones.

Example  10. Human MSC  ( Mesenchymal  Stem Cell's Adhesive ability  Improvement effect

MSC cells (mesenchymal stem cells) were cultured for 6 hours after the peptide peptides according to the present invention were coated with sterilized coverslip glass at a level of 100 μM and the adhesion progression of the cells was measured using fluorescence staining reagent Cells were stained using Rhodamine Phalloidin dye (Life Technology Cat. # R415) and DAPI (Sigma D9542) dye staining nuclei (DNA), respectively. Cells were stained and analyzed by confocal microscopy. At this time, the cells were adhered to a coverslip which was not coated with the peptide peptide according to the present invention as a negative control. The results are shown in FIG. In FIG. 10A, the negative control group still formed actin-ring formation, which is the initial stage of adhesion. However, in the case of the peptide-treated group according to the present invention, stress fiber formation was observed in many cells after the step, FIG. 10B is a graph showing the results of such observation, FIG. 10C is a graph showing the geometric shape (cell aspect ratio: cell aspect ratio, (See Prager-Khoutorsky et al., 2011. Nature Cell Biology 13, 1457-1465), showing excellent mature adherence in the treated group of peptides according to the present invention.

Example  11. Various sequences Peptides  Used Adhesive ability  Experiment I

Peptides having the sequences and titles as shown in Table 2 below were synthesized in the same manner as in Example 1, and then their adhesiveness to heparin or N-acetylglucosamine (GAG) was measured using the same method as described in Example 4. [

[Table 2]

The results are shown in FIG. As shown therein, the peptides of the present invention can be used in a variety of ways, including those in which the first region, the second region and the third region are arranged in various combinations, or even when the sequence of each region is replaced with an amino acid as defined herein Indicating that the peptide according to the present invention can be effectively used for adhesion or adhesion of cells including stem cells.

Example  12. A variety of sequences Peptides  For components contained in ECM (Extra cellular matrix) of cell surface including stem cell used Adhesive ability  Experiment II

Amino acid substitution was performed in consideration of the chemical nature, size, charge characteristics, and experimental results of the substituent on the basis of the sequence of RQLVVKFRALPC (SEQ ID NO: 17) named A in Table 2, The peptide of the sequence was synthesized, and the adhesive ability was analyzed as in Example 11.

[Table 3]

The bolded portion in the sequence column in Table 3 is the substituted portion compared to the A sequence and the results are shown in Figure 11b, indicating that the peptides of the various sequences according to the present invention can be used effectively for cell adhesion or attachment .

Example  13. According to the present invention Of peptide  Attachment and maintenance of epithelial cells

Adhesion or adhesion to epithelial cell HaKaT using peptide A according to the present invention was examined. The results are shown in Fig. When the peptides according to the present invention were not treated, the HaKaT epithelial cells on the hydrophobic surface were not completely attached to the surface of the culture dish. As a result, the results of the clumping type, which is a bundle phenomenon between the cells, were shown. These results are the result of loss of cell characteristics or inherent polarity of epithelial cells due to failure of attachment. In contrast, in the case of peptide treatment according to the present invention, it was observed that the epithelial cells could maintain their morphology on the hydrophilic surface, which is a normal culturing condition. Epithelial cell intracellular junctions and monolayer formation were clearly observed. These results indicate that biological cell attachment of epithelial cells is well maintained due to the effect of the peptide.

On the other hand, the functional effects of the peptides on the primary culture of the pig-derived epithelial cells were examined. In general, when various epithelial cells including pig epithelial cells are cultured in vitro, a protein such as collagen is used as a substrate for cell adhesion. The reason for this is that when cultured in vitro, the epithelial cell characteristics are rapidly lost. Therefore, epithelial cell characteristics can be maintained by adding collagen matrix that is involved in adhesion and adhesion of epithelial cells in vivo. FIG. 13 shows the results of examining the effect of the collagen matrix and the peptide substrate on cell adhesion and morphological maintenance. In step P.3, the epithelial cells on the collagen matrix have already been modified in their morphological characteristics. In the epithelial cells, there is a clear junction between the cell and the epithelium. The morphological change of amorphous form observed in fibroblasts or mesenchymal stem cells was not observed. This can be seen as a result of loss of the inherent character of epithelial cells. In contrast, in the peptide treatment group according to the present invention, it can be observed that the characteristics are maintained even in cells of the same passage.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

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PRT <213> Artificial Sequence <220> <223> artificial <400> 17 Arg Gln Leu Val Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 18 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 18 Arg Gln Leu Val Val Lys Phe Arg Ala Leu Pro Cys Arg Gln Leu Val   1 5 10 15 Val Lys Phe Arg Ala Leu Pro Cys              20 <210> 19 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 19 Arg Gln Leu Val Val Lys Phe Arg Ala Leu Pro   1 5 10 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 20 Arg Gln Leu Val Val Lys Phe Arg Ala Leu   1 5 10 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 21 Lys Gln Leu Val Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 22 Arg Gln Lys Phe Arg Ala Leu Pro Cys   1 5 <210> 23 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 23 Arg Gln Glu Glu Glu Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 24 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 24 Arg Gl Ala Ala Ala Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 25 Arg Gln Leu Val Val Lys Phe Arg Pro Cys   1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 26 Arg Gln Leu Val Val Lys Phe Arg Glu Glu Pro Cys   1 5 10 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 27 Arg Gln Leu Val Val Lys Phe Arg Val Val Pro Cys   1 5 10 <210> 28 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 28 Arg Gln Glu Glu Glu Lys Phe Arg Glu Glu Pro Cys   1 5 10 <210> 29 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 29 Glu Gln Leu Val Val Glu Phe Glu Ala Leu Pro Cys   1 5 10 <210> 30 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 30 Arg Gln Leu Val Val Lys Tyr Arg Ala Leu Pro Cys   1 5 10 <210> 31 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 31 Arg Gln Leu Val Val Lys Trp Arg Ala Leu Pro Cys   1 5 10 <210> 32 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 32 Arg Asn Leu Val Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 33 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 33 Arg Ser Leu Val Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 34 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 34 Arg Gln Leu Val Val Gln Leu Val Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 15 <210> 35 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 35 Arg Gln Leu Val Val Gln Leu Val Val Gln Leu Val Val Lys Phe Arg   1 5 10 15 Ala Leu Pro Cys              20 <210> 36 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 36 Arg Gln Leu Val Val Gln Leu Val Val Gln Leu Val Val Gln Leu Val   1 5 10 15 Val Lys Phe Arg Ala Leu Pro Cys              20 <210> 37 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 37 Arg Gln Leu Val Val Lys Phe Arg Ala Leu Pro Cys Phe Arg Ala Leu   1 5 10 15 Pro Cys         <210> 38 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 38 Arg Arg Gln Leu Val Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 39 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 39 Arg Arg Arg Arg Gln Leu Val Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 15 <210> 40 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 40 Arg Arg Arg Arg Arg Arg Arg Arg Arg Gln Leu Val Val Lys Phe   1 5 10 15 Arg Ala Leu Pro Cys              20 <210> 41 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 41 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Gln   1 5 10 15 Leu Val Val Lys Phe Arg Ala Leu Pro Cys              20 25 <210> 42 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 42 Arg Gln Val Val Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 43 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 43 Arg Gln Ile Val Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 44 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 44 Arg Gl Ala Val Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 45 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 45 Arg Gln Glu Val Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 46 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 46 Arg Gln Leu Leu Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 47 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 47 Arg Gln Leu Ile Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 48 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 48 Arg Gln Leu Ala Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 49 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 49 Arg Gln Leu Glu Val Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 50 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 50 Arg Gln Leu Val Leu Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 51 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 51 Arg Gln Leu Val Ile Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 52 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 52 Arg Gln Leu Val Ala Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 53 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 53 Arg Gln Leu Val Glu Lys Phe Arg Ala Leu Pro Cys   1 5 10 <210> 54 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 54 Arg Gln Leu Val Val Arg Phe Arg Ala Leu Pro Cys   1 5 10 <210> 55 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 55 Arg Gln Leu Val Val Lys Phe Lys Ala Leu Pro Cys   1 5 10 <210> 56 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 56 Arg Gln Leu Val Val Glu Phe Glu Ala Leu Pro Cys   1 5 10 <210> 57 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 57 Arg Gln Leu Val Val Lys Phe Arg Leu Leu Pro Cys   1 5 10 <210> 58 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 58 Arg Gln Leu Val Val Lys Phe Arg Ile Leu Pro Cys   1 5 10 <210> 59 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 59 Arg Gln Leu Val Val Lys Phe Arg Val Leu Pro Cys   1 5 10 <210> 60 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 60 Arg Gln Leu Val Val Lys Phe Arg Glu Leu Pro Cys   1 5 10 <210> 61 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 61 Arg Gln Leu Val Val Lys Phe Arg Ala Ala Pro Cys   1 5 10 <210> 62 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 62 Arg Gln Leu Val Val Lys Phe Arg Ala Ile Pro Cys   1 5 10 <210> 63 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 63 Arg Gln Leu Val Val Lys Phe Arg Ala Val Pro Cys   1 5 10 <210> 64 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 64 Arg Gln Leu Val Val Lys Phe Arg Ala Glu Pro Cys   1 5 10 <210> 65 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 65 Arg Gln Glu Glu Glu Glu Phe Glu Glu Glu Pro Cys   1 5 10 <210> 66 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <220> <221> VARIANT <222> (11) <223> any amino acids <400> 66 Arg Gln Leu Val Val Lys Phe Arg Ala Leu Xaa Cys   1 5 10 <210> 67 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 67 Arg Gln Leu Val Val Lys Phe Arg Ala Leu Pro Ser   1 5 10 <210> 68 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 68 Arg Gln Leu Val Val Lys Phe Arg Ala Leu Pro Thr   1 5 10 <210> 69 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <220> <221> VARIANT <12> <223> any amino acids <400> 69 Arg Gln Leu Val Val Lys Phe Arg Ala Leu Pro Xaa   1 5 10 <210> 70 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 70 Gln Val Val Val Lys   1 5 <210> 71 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 71 Gln Ile Val Val Lys   1 5 <210> 72 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 72 Gln Ala Val Val Lys   1 5 <210> 73 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 73 Gln Leu Leu Val Lys   1 5 <210> 74 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 74 Gln Leu Ile Val Lys   1 5 <210> 75 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 75 Gln Leu Ala Val Lys   1 5 <210> 76 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 76 Gln Leu Val Leu Lys   1 5 <210> 77 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 77 Gln Leu Val Ile Lys   1 5 <210> 78 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 78 Gln Leu Val Ala Lys   1 5 <210> 79 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 79 Gln Leu Val Val Arg   1 5

Claims (13)

A composition for cell adhesion comprising a peptide of the following formula:
[X ¹² ] _1-15 - [X ¹ -X ² -X ³ -X ⁴ -X ⁵ ] m- [X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ ] n _,
In the above [X ¹ - X ² - X ³ - X ⁴ - X ⁵ ] m,
X &lt; ¹ &gt; is any amino acid,
Wherein X ² , X ³ and X ⁴ are amino acids of any one of L, V, I, E, G and A, which are the same or different, and at least one of X ² , X ³ or X ⁴ may be absent In addition,
X &lt; ⁵ &gt; is an amino acid of K or R,
M is an integer of 1 to 5, and when two or more amino acids are included, the amino acid sequences of the respective peptides are the same or different,
In n, the ^{[X 11 X 6 - X 7} - X 8 - X 9 - - X 10]
Wherein ^{[X 6 - X 7 - X} 8 - X 9 - X 10 - X 11] is or may be lacking, or
X &lt; ⁶ &gt; is an amino acid of any one of F, Y and W,
X ⁷ is an amino acid of K or R,
X ⁸ is an amino acid of any one of A, M and I,
X ⁹ is any amino acid of L, M and G,
Wherein X ¹⁰ is any amino acid,
X ¹¹ is an amino acid of any one of C, S and T,
At least one of X ⁸ and X ⁹ or X ¹⁰ and X ¹¹ may be absent,
Wherein n is 1 or 2,
X ¹² is F, K or R,
The amino acid is a D- or L-type natural or unnatural amino acid or a derivative thereof.
The method according to claim 1,
Wherein any of the amino acids of X ¹ is S, T, C, P, N or Q.
The method according to claim 1,
Wherein X ² - X ³ - X ⁴ is AAA, EEE, LVG, LVA, LVL, LVV, LLA, LLL, or the LLV, cell adhesion composition.
The method according to claim 1,
Wherein the peptide of [X ¹ - X ² - X ³ - X ⁴ - X ⁵ ] is any one of the following:
QVVK (SEQ ID NO: 1), QEEEK (SEQ ID NO: 2), QAAAK (SEQ ID NO: 3), NLVVK (SEQ ID NO: 4), SLVVK QLVK (SEQ ID NO: 73), QLIVK (SEQ ID NO: 74), QLAVK (SEQ ID NO: 75), QLVLK (SEQ ID NO: 76), QLVIK (SEQ ID NO: 79).
The method according to claim 1,
Wherein ^{[X 6 - X 7 - X} 8 - X 9 - X 10 - X 11] peptide composition for any of the cell adhesion of the following:
FRALPC (SEQ ID No. 6), FREEPC (SEQ ID No. 7), FRVVPC (SEQ ID No. 8), FEALPC (SEQ ID No. 9), YRALPC (SEQ ID No. 10), WRALPC (SEQ ID NO: 13), FRPC (SEQ ID NO: 14) or FR.
The cell adhesion composition according to any one of claims 1 to 5, wherein the peptide is represented by any one of the following sequences:
RQLVVK (SEQ ID NO: 15);
FRALPCRQLVVK (SEQ ID NO: 16);
RQLVVKFRALPC (SEQ ID NO: 17);
RQLVVKFRALPCRQLVVKFRALPC (SEQ ID NO: 18);
RQLVVKFRALP (SEQ ID NO: 19);
RQLVVKFRAL (SEQ ID NO: 20);
KQLVVKFRALPC (SEQ ID NO: 21);
RQKFRALPC (SEQ ID NO: 22);
RQEEEKFRALPC (SEQ ID NO: 23);
RQAAAKFRALPC (SEQ ID NO: 24);
RQLVVKFRPC (SEQ ID NO: 25);
RQLVVKFREEPC (SEQ ID NO: 26);
RQLVVKFRVVPC (SEQ ID NO: 27);
RQEEEKFREEPC (SEQ ID NO: 28);
EQLVVEFEALPC (SEQ ID NO: 29);
RQLVVKYRALPC (SEQ ID NO: 30);
RQLVVKWRALPC (SEQ ID NO: 31);
RNLVVKFRALPC (SEQ ID NO: 32);
RSLVVKFRALPC (SEQ ID NO: 33);
RQLVVK- (FRALPC) 2 (SEQ ID NO: 37);
(R) 2-QLVVKFRALPC (SEQ ID NO: 38);
(R) 5-QLVVKFRALPC (SEQ ID NO: 39);
(R) 10-QLVVKFRALPC (SEQ ID NO: 40);
(R) 15-QLVVKFRALPC (SEQ ID NO: 41).
RQVVVKFRALPC (SEQ ID NO: 42);
RQIVVKFRALPC (SEQ ID NO: 43);
RQAVVKFRALPC (SEQ ID NO: 44);
RQEVVKFRALPC (SEQ ID NO: 45);
RQLLVKFRALPC (SEQ ID NO: 46);
RQLIVKFRALPC (SEQ ID NO: 47);
RQLAVKFRALPC (SEQ ID NO: 48);
RQLEVKFRALPC (SEQ ID NO: 49);
RQLVLKFRALPC (SEQ ID NO: 50);
RQLVIKFRALPC (SEQ ID NO: 51);
RQLVAKFRALPC (SEQ ID NO: 52);
RQLVEKFRALPC (SEQ ID NO: 53);
RQLVVRFRALPC (SEQ ID NO: 54);
RQLVVKFKALPC (SEQ ID NO: 55);
RQLVVEFEALPC (SEQ ID NO: 56);
RQLVVKFRLLPC (SEQ ID NO: 57);
RQLVVKFRILPC (SEQ ID NO: 58);
RQLVVKFRVLPC (SEQ ID NO: 59);
RQLVVKFRELPC (SEQ ID NO: 60);
RQLVVKFRAAPC (SEQ ID NO: 61);
RQLVVKFRAIPC (SEQ ID NO: 62);
RQLVVKFRAVPC (SEQ ID NO: 63);
RQLVVKFRAEPC (SEQ ID NO: 64);
RQEEEEFEEEPC (SEQ ID NO: 65);
RQLVVKFRALXC (SEQ ID NO: 66);
RQLVVKFRALPS (SEQ ID NO: 67);
RQLVVKFRALPT (SEQ ID NO: 68); or
RQLVVKFRALPX (SEQ ID NO: 69).
The method according to claim 1,
Wherein X &lt; ¹ &gt; is Q, N, S, T, P or C of polarity,
X ² , X ³ and X ⁴ are each the same or different hydrophobic L, V, I, or A,
Wherein X &lt; ⁵ &gt; is a positively charged K or R,
Wherein ^{[X 6 - X 7 - X} 8 - X 9 - X 10 - X 11] In, X ⁸ and X ^9, or wherein X ¹⁰ and X one or more of the ¹¹ may be lacking, FRALPC (SEQ ID NO: 6), FRALP (SEQ ID NO: 7), FRVVPC (SEQ ID NO: 8), FEALPC (SEQ ID NO: 9), YRALPC (SEQ ID NO: 10), WRALPC FRPC (SEQ ID NO: 14), or FR,
Wherein X ¹² is a positively charged K or R,
Wherein m and n are each 1 or 2 and m or n is 2, the amino acid sequences of the respective peptides are the same or different, and the amino acid is a natural or unnatural amino acid of D- or L- Composition.
8. The method of claim 7,
Wherein X ² - X ³ - X ⁴ is LVV, AAA, LVA, LVL, LLA, LLL, or LLV.
8. The method of claim 7,
Wherein the peptide of [X ¹ - X ² - X ³ - X ⁴ - X ⁵ ] is any one of the following:
QVVK (SEQ ID NO: 1), QAAAK (SEQ ID NO: 3), NLVVK (SEQ ID NO: 4), or SLVVK (SEQ ID NO: 5), QVVVK (SEQ ID NO: 70), QIVVK QLLVK (SEQ ID NO: 73), QLIVK (SEQ ID NO: 74), QLAVK (SEQ ID NO: 75), QLVLK (SEQ ID NO: 76), QLVIK (SEQ ID NO: 77), QLVAK (SEQ ID NO: 78), or QLVVR (SEQ ID NO: 79).
10. The method of claim 9,
Wherein the peptide is represented by the following sequence:
RQLVVKFRALPC (SEQ ID NO: 17);
RQLVVKFRALP (SEQ ID NO: 19);
RQLVVKFRAL (SEQ ID NO: 20);
KQLVVKFRALPC (SEQ ID NO: 21);
RQAAAKFRALPC (SEQ ID NO: 24);
RQLVVKFRPC (SEQ ID NO: 25);
RQLVVKFREEPC (SEQ ID NO: 26);
RQLVVKFRVVPC (SEQ ID NO: 27);
RQLVVKYRALPC (SEQ ID NO: 30);
RQLVVKWRALPC (SEQ ID NO: 31);
RNLVVKFRALPC (SEQ ID NO: 32);
RSLVVKFRALPC (SEQ ID NO: 33);
RQLVVK- (FRALPC) ₂ (SEQ ID NO: 37);
(R) ₂ -QLVVKFRALPC (SEQ ID NO: 38);
(R) ₅ -QLVVKFRALPC (SEQ ID NO: 39);
(R) ₁₀ -QLVVKFRALPC (SEQ ID NO: 40);
(R) ₁₅ -QLVVKFRALPC (SEQ ID NO: 41);
RQVVVKFRALPC (SEQ ID NO: 42);
RQIVVKFRALPC (SEQ ID NO: 43);
RQAVVKFRALPC (SEQ ID NO: 44);
RQLLVKFRALPC (SEQ ID NO: 46);
RQLIVKFRALPC (SEQ ID NO: 47);
RQLAVKFRALPC (SEQ ID NO: 48);
RQLVLKFRALPC (SEQ ID NO: 50);
RQLVIKFRALPC (SEQ ID NO: 51);
RQLVAKFRALPC (SEQ ID NO: 52); or
RQLVVRFRALPC (SEQ ID NO: 54).
A composition for cell adhesion comprising a peptide having an amino acid sequence represented by any one of SEQ ID NOS: 15 to 69.
11. A method for adhering a cell to an inorganic or organic surface, the method comprising treating the cell with a composition according to any one of claims 1 to 11.
12. A method of treating a composition comprising: treating a composition according to any one of claims 1 to 11 to a first material and / or a second material; And contacting the two materials, wherein at least one of the first material or the second material is a cell.

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