KR102010871B1 - Composition and Method for Cell Adhesion - Google Patents

Abstract

The present application discloses novel sequences of peptides and their use as bioadhesives. Peptides according to the present invention can be useful for cell cultures that require adhesion because they can adhere a bio-derived material such as cells or tissues to various objects or materials.

Description

Composition and Method for Cell Adhesion {Composition and Method for Cell Adhesion}

The present application is a technique for improving adhesion to various surfaces of cells, including protein based biological materials.

Bioadhesives that can be used for the adhesion of cells or tissues, or biological materials derived therefrom, are useful materials that can be widely used in various technical fields. For example, such materials may be used in the process of adhesion of tissues and tissues or tissues and cells in medicine, wound healing, regeneration or repair of tissues, or in culture of stem cells or normal cells, drug delivery carriers, It can be used for tissue filler, bioconjugation and the like.

Such bioadhesives require excellent adhesion and crosslinking ability as well as long-term functional maintenance, especially when used in vivo, and should have fewer 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. Bioadhesives using synthetic polymers have the disadvantage of poor adhesion in a solution environment.

US 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 is particularly limited in in vivo, and the adhesion and adhesion strength are not sufficient.

Korean Laid-Open Patent Publication No. 2006-005314 relates to a peptide that promotes cell attachment and spread, fragments thereof and derivatives thereof, wherein the integrin in the LG3 domain of the human laminin-5 α3 chain mediates integrin α3β1 binding and cell attachment and spread. -α3β1-dependent cell adhesion peptides and uses thereof are disclosed.

However, there is a need for the development of new bioadhesive materials with improved adhesion that can be applied in various environments based on various mechanisms while being less toxic than conventional ones.

The present invention is to provide a novel sequence of the adhesive polypeptide, the cell adhesion composition comprising the same, and its use, which can be applied in various environments with low toxicity.

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

[Formula 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,

X ² , X ³ and X ⁴ are the same as or different from each other as amino acids of any one of L, V, I, E, G and A, and one or more of X ² , X ³ or X ⁴ may be lacking. And

X ⁵ is an amino acid of K or R,

M is an integer of 1 to 5, when containing two or more, the amino acid sequence of each peptide is the same or different,

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

Said [X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ ] may be absent, 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 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,

One or more of X ⁸ and X ⁹ or X ¹⁰ and X ¹¹ may be absent,

N is 1 or 2,

X ¹² is F, K or R,

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

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

In another embodiment the peptide of [X ¹ -X ² -X ³ -X ⁴ -X ⁵ ] is any one 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 (SEQ ID NO: 70), QIVVK (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 (SEQ ID NO: 76), QLVIK (SEQ ID NO: 77), QLVAK (SEQ ID NO: 78), or QLVVR (SEQ ID NO: 79).

In another embodiment the peptide of [X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ ] is any one of: FRALPC (SEQ ID NO: 6), FREEPC (SEQ ID NO: 7), FRVVPC (SEQ ID NO: Number 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 of Formula IV 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).

In another embodiment X ¹ in Formula IV is polar Q, N, S, T, P or C, and X ² , X ³ and X ⁴ are each the same or different hydrophobicity of L, V, I, or A, X ⁵ is positively charged K or R, and in [X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ ], X ⁸ and X ⁹ or one of X ¹⁰ and X ¹¹ The above may be lacking: 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), or FRPC (SEQ ID NO: 14), or FR, wherein X ¹² is positively charged K or R, wherein m and n are 1 or 2, respectively, m or n In the case of 2, the amino acid sequence of each peptide is the same or different and the amino acid is a natural or unnatural amino acid of D- or L-type.

In one embodiment the 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 ] One of: QLVVK (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 (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 (SEQ ID NO: 76), QLVIK (SEQ ID NO: 77), QLVAK (SEQ ID NO: 78), or QLVVR ( SEQ ID NO: 79).

In one embodiment the peptide is represented by the 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 application provides a composition or use for cell adhesion comprising a peptide of amino acid sequence represented by any one of SEQ ID NO: 15 to 69.

In another aspect, the present invention also provides a composition for adhesion that enables adhesion of a cell comprising a peptide according to the present invention to a bio-derived material or a non-liver-derived material.

The adhesive or adhesive composition according to the present disclosure enables the adhesion of cells to organic or inorganic surfaces. Organics include cells, tissues, organs, and inorganics include metals such as precious 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, but is not limited thereto.

The peptides or compositions comprising them enable the adhesion of various materials, including biological and / or non-biological materials, to organic or inorganic surfaces, for example, non-biological materials can be metal, glass, plastic, metal or It includes a polymer synthetic resin, the biological material includes a cell, tissue, protein, lipid, sugar or nucleic acid or a combination thereof.

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

In another aspect the present disclosure also discloses a method of adhering two surfaces comprising at least one cell comprising a 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 culture vessel coated with a peptide according to the present invention or a cell culture medium or composition comprising the peptide according to the present invention.

In another aspect, the present disclosure provides a method of treating a peptide or a composition comprising the same according to the present invention with 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, and the remainder is an inorganic or organic material.

1A to 1D illustrate glass, zirconium (Zr), vinyl (Vinyl), polystyrene fiber (PS fiber), and PCL (non-biologically derived or non-biological material of the peptide (A) according to one embodiment of the present application). Polycaprolactone), titanium (Ti) and collagen (Col) results in showing the adhesion.
1A is a result of investigating the adhesion of the peptide to the glass through the detection of biotin bound to the glass after the peptide according to an embodiment of the present application to the glass disulfide bond, the signal of the biotin is It is adhered to the glass, and this result disappears upon decomposition of disulfide bonds by treatment with dithiothreitol (DTT), indicating that the bond is dependent on disulfide bonds.
FIG. 1B schematically illustrates the method used to perform the experiment of FIG. 1A, wherein the free-bonded peptide of the present disclosure is linked to Biotin, a labeling substance, through SH group, and the biotin may be detected as an antibody. This results in disappearance due to the treatment of DTT to separate biotin from the peptide.
FIG. 1C is the same as the method described in FIG. 1A but schematically illustrates an experiment to analyze adhesion to various non-biologically derived materials using fluorescein isothiocyanate (FITC) instead of biotin.
Figure 1d shows the results of the analysis, showing that the peptides of the present application has the adhesion to the surface of a variety of non-biologically derived material PBS (phosphate buffer saline), FITC-conjugate A, and FITC dye from the left to each material After adhesion, the fluorescent image is analyzed.
Figure 2a is a result of performing an adhesion test on the surface of the synthetic bone graft Bio-OSS ^® (Geistlich Pharma, Inc) of the FITC-labeled peptides according to an embodiment of the present application, the commercialized bone of the peptides according to the present application Excellent adhesion to the implant.
Figure 2b is a result of performing an adhesion test on the surface of the synthetic bone graft MBCP ^TM (Biomatlante, Biphasic calcuim phosphate) of the FITC-labeled peptide according to an embodiment of the present application, the commercialized bone graft material of the peptide according to the present application It shows excellent adhesion to. The fluorescence microscope magnification used was 200 times.
Figure 3a is a result of performing an adhesion test on the culture plate of MC3T3-E1, an anchorage-dependent cell of the peptide according to an embodiment of the present application, the cell containing the peptide according to the present invention was treated with a control group (PBS) ), Rough cell membrane boundary morphology, indicating increased adhesion of the cells to the culture dish by the peptides herein.
Figure 3b is a result of the adhesion of the peptide to the hydrophobic culture dish of the anchorage-dependent cells (MC3T3-E1) of the peptide according to an embodiment of the present application, PLL (poly-L-) which is a substance used for improving the conventional cell adhesion lysin) shows that the cells comprising the peptides according to the present invention have increased adhesion as compared to the control (treated with PBS and PLL) microscopically. Adhesion in the hydrophobic culture dish can compensate for the disadvantages of cell adhesion of various tissue-transfer materials (PCL, etc.) having hydrophobic properties, indicating a high industrial application value.
Figure 3c is the result of comparing the adhesion of the peptide to the hydrophobic culture plate of the anchorage-dependent cells (ST2) of the peptide according to an embodiment of the present invention, the cell containing the peptide according to the present application compared to the control group microscopic observation This indicates that the adhesion is increased.
Figure 3d is a result of thawing the anchorage-dependent cells (C2C12) of the peptide according to an embodiment of the present application, and comparing the adhesion ability to the culture dish with the medium control, the cells containing the peptide according to the present application under a microscope Results Compared to the control, the adhesion was increased. In particular, the cell adhesion ability immediately after thawing has a very important effect on the survival of the cell, and the results indicate that the peptide adhesive according to the present application may be usefully used to minimize the death of cells after thawing, and in the field of primary cell culture It can be usefully used at, indicating high industrial application value.
Figure 3e is a result of comparing the adhesion of the peptide to the hydrophilic culture dish of the mitomycin treated STO feeder cells according to an embodiment of the present invention with the negative control (Mock) and gelatin, the peptide according to the present application Comprising cells show increased adhesion as compared to the negative control and gelatin.
Figure 3f is a result of comparing the adhesion of various peptides (established cell line and primary cells) in the hydrophobic culture dish of the peptide according to an embodiment of the present application with a negative control (Mock) using a cell metabolism measurement . The results indicate that the peptides according to the present disclosure can increase their adhesion compared to negative controls in various kinds of cells.
Figures 4a to 4c is a result of analysis of the mechanism of cell adhesion of the peptide according to the present application.
4A shows that the cell adhesion by peptide A according to the present application does not require electrolyte on the surface of the cell (the result of EDTA treatment) or new protein synthesis (the result of CHX treatment), whereas the inhibition occurs initially by serum. This may be due to the primary binding of many types of GAG present in the serum to the peptide.
Figure 4b is a result of analyzing the association with GAG or collagen as a peptide cell adhesion promoting mechanism, the above figure is observed that the phenomenon appears by decomposing the potential target by the enzyme treatment as a result that is related to both collagen and GAG . Reduction of cell adhesion by collagenase treatment at least interacts with collagen. And hyaluronidase treatment reduced cell adhesion by hydrolyzing at least heparin sulphate, indicating interaction of heparin sulphate and peptide in GAG. The figure below shows the results of the analysis of the relationship with GAG, the results of the addition of heparin and C-sulfate significantly reduced the cell adhesion capacity promoted through the peptides according to the present invention in a concentration-dependent manner, these results are the result of It indicates the relationship with GAG. Changes in GAG concentrations have been reported to be associated with a variety of diseases, so the peptides according to the present application are novel, GAG binding / adhesive peptides that may be useful as new peptides that regulate GAGs in the blood or as drug carriers targeting GAGs. To indicate that it can.
Figure 4c is a result of the competitive treatment of the peptide according to the present invention and the soluble RGDS peptide in the non-peptide group according to the present invention when binding to soluble RGDS preferentially binds to the integrin (particulate molecule), the adhesion molecule to the bottom (substrate) Although significantly reduced the capacity, the peptide treatment group according to the present invention was shown to have no effect on the adhesion of cells to the treatment of RGDS, which means that the peptide according to the present invention is a cell separate from anchorage dependent cell adhesion through integrin. It has a mechanism of promoting adhesion.
Figure 5a is a schematic diagram showing a reporter system and an experimental procedure prepared for measuring the adhesion of the peptide to induced pluripotent stem cells (iPSC) according to the present application, the reporter system is induced pluripotent It includes a structure capable of measuring the activity of the Oct4 gene as an index of stem cell pluripotency (Stemness).
Figure 5b is a result of measuring the adhesion and maintenance of pluripotent mouse pluripotent stem cells using the construct according to Figure 5a by detecting the expression of Oct4 on day 2 and 3 of the cell culture, feeder cells and gelatin as a control in advance Coated petri dishes were used, and the peptides of the present application were added in a precoated or mixed manner to the cultures, both of which mediated the adhesion of iPSCs and also maintained the pluripotency.
Figure 5c is the same as Figure 5b, but as measured on the day 4 cell culture, the peptide according to the present invention was shown to mediate the adhesion of iPSC and also maintain the pluripotency.
Figure 6a is a result of observing the expression of the Alp (alkaline phosphotase) gene, another marker indicating the pluripotency, the peptides according to the present invention mediate adhesion of iPSC when precoated or mixed with the culture medium and also maintain pluripotency Appeared to.
6b is a result of observing the expression of the Nanog gene, which is another marker indicating pluripotency, through staining, and the peptide according to the present invention mediated adhesion of iPSC and maintained pluripotency even when precoated or mixed with a culture solution.
FIG. 6C is the same experiment as in FIG. 5B or the result of using the hESC, and shows that the hESC can grow well without any external environmental changes such as feeder cells or Matrigel.
Figure 7a is a schematic diagram showing a bioconjugation process using a peptide according to the present invention, using a linker DSS (Disuccinimidyl suberate) conjugated the peptide according to one embodiment of the present application to the bone morphogenic protein BMP2.
Figure 7b is a recombinant BMP treated group as a result of measuring the expression of Alp, a osteogenic differentiation gene 48 hours after culturing C2C12 cells after treatment of the peptide according to the present invention conjugated to BMP2 cell culture dish as shown in Figure 7a Alp activity was increased compared to (rhBMP2) or crosslinker treatment group (CL), indicating that cell adhesion and thereby bone differentiation were promoted by the peptide treatment herein.
8A to 8C show the results of testing the safety of the peptides according to the present invention using osteoblasts (MC3TC-E1).
Figure 8d is a result of testing the safety of the peptides according to the present invention is isolated from the monocytes (monocytes) of the mouse after the peptide stimulation is investigated whether the induction of inflammation occurs.
Figures 9a to 9c is a result of comparing the effect with the existing adhesive peptide.
Figure 9a is a spinal cord-derived neurons coated with a conventional peptide peptide laminin (laminin) and poly-L- ornithin (poly-L-ornithin) and coated with a peptide according to the present application according to an embodiment of the present application After incubation using the prepared culture plate is the result of analyzing CCK-8 at each time slot.
Figure 9b is a result of comparing and observed through an optical microscope after 4 days of culture.
Figure 9c is a result of verifying that the neuron-derived cells in the primary cultured cells coated with the peptide according to the present application according to an embodiment of the present application, the result of analysis by confocal microscopy after staining the neurolabel protein by immunofluorescence method to be. The above results indicate that the peptides according to the present invention have significantly better adhesion than the conventional ones.
10A to 10C show results of adhering progression of cells using hMSC to be promoted by a peptide according to the present invention, and are analyzed based on 6 hours after cell culture.
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 application, stress fiber formation has already been observed in a large number of cells.
10B is a graph quantifying the observation result.
Figure 10c is a result of measuring the geometric shape (cell aspect ratio: the ratio of the cell width / length, complete circle if 1, and represents the earliest state of the adhesion process) according to the cell adhesion according to the present application In the treated group of peptides, the maturation ability is shown to be significantly mature.
11A and 11B show the results of measuring cell adhesion promoting by peptides having various sequences according to the present application. Peptide names along the horizontal axis of Figure 11a is shown in SEQ ID NO: corresponding to Table 2. FIG. 11B is an experiment using a sequence generated by introducing various amino acid substitutions into the A peptide, which is one of the sequences of FIG. 11A, and the horizontal axis numbers indicate the sequence numbers of the peptides.
12 is a result showing the promotion of the established cell line epithelial cell adhesion capacity and maintenance of the peptide according to the present application.
Figure 13 is a result showing the primary epithelial cell adhesion promoting properties and the maintenance of properties of the peptides according to the present application.
In the figures, peptides A and A7-1 according to the present application have the same name.

In one aspect, the present application relates to a peptide represented by the following Chemical Formulas (I) to (IV), and a use thereof, which specifically allow a cell to attach or adhere to various materials or surfaces thereof, and specifically, a composition for cell adhesion comprising the same or It relates to a cell adhesion method.

Justice

Amino acid residues herein are denoted by single letter code and abbreviations are as follows: A, alanine; R, arginine; N, asparagine; D, aspartic acid; C, cysteine; E, glutamic acid; Q, glutamine; G, glycine; H, histidine; I, isoleucine; L, leucine; 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.

The term amino acid, as used herein, refers to 20 naturally occurring and non-natural amino acids, amino acids that are modified after translation in vivo, such as phosphoserine and phosphothreonine; And other rare amino acids such as 2-aminoadipic acid, hydroxylysine, nor-valine, nor-leucine; It includes modifications to improve stability in vivo, and includes both the enantiomers of the D- and L-forms.

Positively charged amino acids, negatively charged amino acids, polar uncharged amino acids and nonpolar aliphatic amino acids herein are determined according to the chemical properties of the side chains and are known in the art and those skilled in the art will be able to select appropriate ones. For example, positively charged amino acids include K or R, negatively charged amino acids include D or E, polar uncharged amino acids include S, T, C, P, N or Q, and nonpolar aliphatic Amino acids may include G, A, L, V, M or I.

As used herein in reference to amino acids, in the natural and unnatural the former refers to compounds of the form found in cells, tissues or in vivo, the latter means that these compounds have been artificially modified for various purposes.

As used herein, the term peptide or polypeptide refers to a molecule in which monomers of amino acids are covalently linked, and are used interchangeably and are read from the N-terminus to the C-terminus unless otherwise indicated. It enhances stability in vivo or with native amino acids or their degradation products, synthetic peptides, recombinantly produced peptides, peptide mimetics (typically synthetic peptides), and peptide analogs such as peptides and semipeptoids It includes the modified for. 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 , backbone modifications, Side chain modifications. Methods for preparing peptide mimetic compounds are known in the art and may be referred to, for example, as described in Quantitative Drug Design, CA Ramsden Gd., Choplin Pergamon Press (1992).

As used herein, the term adhesion includes adhesion and adsorption, and includes reversible and irreversible adhesion, in other aspects by one or more chemical bonds including covalent bonds, ionic bonds, van der Waals bonds, hydrogen bonds, and the like. Adhesion. In another aspect it is meant that two materials or surfaces of the same or different materials are in tightly coupled state to achieve the desired effect. For example, one of two different materials is a cell or tissue, and the other material is an inorganic or inorganic surface, or an organic or organic surface comprising a cell or tissue.

As used herein, inorganic or inorganic surfaces are used interchangeably and include materials free of electron distribution due to their good electrical conductivity or inorganic in the conventional sense that do not contain carbon, silicon or nitrogen, etc., especially materials whose surfaces are hydrophobic. Examples thereof include metals such as precious metals including iron, copper, gold, silver and platinum, titanium or aluminum; Ceramics such as zirconia; Calcium apatite crystals such as hydroxyapatite; Polymer synthetic resins such as polyethylene; Glass; And combinations thereof, but is not limited thereto.

As used herein, organic or organic surface is used interchangeably and means a carbon-based compound, particularly a compound derived from an organism. Examples include, but are not limited to, cells, tissues, organs, or in vitro or in vivo cultures thereof.

The term surface according to the present application is interpreted in the widest range and is present in two-dimensional or more materials, and is not limited to a specific shape and specific size. It includes the surface which has. For example, it is present in particles or materials on the nanometer to micrometer level, as well as on the order of several millimeters to several meters in size.

Cell adhesion herein includes attachment or adsorption of cells or tissues consisting of cells to other cells or tissues, or other organic or inorganic materials (surfaces).

Adhesive Peptide

In one embodiment the present application relates to a peptide of formula (I)

[Formula I]

[X ¹ -X ² -X ³ -X ⁴ -X ⁵ ] m

In formula (I)

X ¹ is any amino acid or a polar uncharged amino acid,

X ² , X ³ and X ⁴ are the same as or different from each other as amino acids of any one of L, V, I, E, G and A, and one or more of X ² , X ³ or X ⁴ may be lacking. And

X ⁵ is a positively charged amino acid,

M is an integer of 1 to 5, and when two or more are included, the amino acid sequence of each peptide is the same or different, and the amino acid is a natural or unnatural amino acid of D- or L-type or a derivative thereof.

The compound of formula (I) according to the present invention is not only involved in binding between cells or between cells and other surfaces as the first region, but can also vary the number of peptides according to the specific purpose according to the present application. The first region is a key region that can affect the secondary structure of the peptide, thereby maintaining the molecular properties of other functional regions described later.

In the peptide according to the present application, the first region may include one or more units composed of five amino acids, and when two or more units are included, each unit may be different or the same. The number of units may be included in various numbers depending on various functionalizations for the production, storage, delivery of peptides or the efficacy and use of the peptides according to the present application 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 a dog.

X ¹ in Formula (I) is any amino acid and in one embodiment is a polar uncharged amino acid, or in other embodiments is 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 the above X ² , X ³ or X ⁴ May be absent, and the X ² -X ³ -X ⁴ sequence can be, for example, but not limited to, 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 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 (SEQ ID NO: 73), QLIVK (SEQ ID NO: 74), QLAVK (SEQ ID NO: 75), QLVLK (SEQ ID NO: 76), QLVIK (SEQ ID NO: 77), It is designated as QLVAK (SEQ ID NO: 78), or QLVVR (SEQ ID NO: 79), but is not limited thereto.

In another aspect, the disclosure is directed to a peptide of Formula (I-II), including but not limited to, compounds of Formula (II) below, or further comprising one or more compounds of Formula (II) below.

[Formula II]

[X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ ] n,

In Formula II

X ⁶ is 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 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,

One or more of X ⁸ and X ⁹ or X ¹⁰ and X ¹¹ may be absent,

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

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

According to the present application, the compound of formula II, as the second region, when present as a peptide single molecule with the first region, can easily make an alpha-helix structure, imparting hydrophilic properties to the peptide according to the present application.

At least one first region of formula (I) and at least one second region of formula (II) may be included, and arrangement thereof may vary. For example, at least one peptide of formula (I) and (II) is linked to each other to have a structure of the peptide of formula (I-II), formula (I)-(II)-(II), or The peptides are each linked one by one, and then linked again to include a structure such as Formula I-Formula II-Formula II, or Formula I-Formula II-Formula I, or Formula II-Formula I- may include a structure such as Formula II. In the above structure, the order of Formulas I and II may be reversed.

In one embodiment of the present invention the peptide of Formula II 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: Number 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) of the present application, or a peptide comprising both Formulas (I) and (II) may further include 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 positive or negatively charged.

In one embodiment the positively charged amino acid 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 included in various arrangements of the first region of Formula I and the second region of Formula II, and one or more of Formula I and Formula II may be included as mentioned above. For example, formula (III) is a peptide of formula (I-II) in which at least one peptide of formula (I) and (II) is linked to each other, a structure of formula (I)-(II)-(II), (I)-(II) Or a N or C terminus of a peptide having a structure such as II, or a structure of Formula I-Formula II-I, or Formula II-Formula I-II.

In one embodiment, in particular, is linked to the N-terminus of the peptide comprising Formulas I and II.

In another embodiment, the present application relates to a peptide of formula IV of structure III-I-II represented by formula IV below.

[Formula 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 _,

For the description of each residue included in the formula (IV) can refer to the bar described above.

In another embodiment, Formula IV is defined as follows.

In formula IV, at [X ¹ -X ² -X ³ -X ⁴ -X ⁵ ] m,

X ¹ is any amino acid,

X ² , X ³ and X ⁴ are the amino acids of any one of L, V, I, E, G and A, the same or different, respectively, one, ^two , of X ² , X ³ or X ⁴ , Or may lack all three residues,

X ⁵ is an amino acid of K or R,

M is an integer of 1 to 5, when containing two or more, the amino acid sequence of each peptide is the same or different,

[X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ ] n may be absent, or

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

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 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,

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

N is 1 or 2,

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

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

In another embodiment any amino acid of X ¹ in Formula IV can be polar S, T, C, P, N or Q, wherein X ² -X ³ -X ⁴ are AAA, EEE, LVG, LVA, It may be LVL, LVV, LLA, LLL, or LLV, wherein the peptides of [X ¹ -X ² -X ³ -X ⁴ -X ⁵ ] are 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 (SEQ ID NO: 73), QLIVK (SEQ ID NO: Number 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), wherein [X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ ] peptides are 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, in the peptide of Formula IV, X ¹ is polar Q, N, S, T, P or C, and X ² , X ³ and X ⁴ are the same or different hydrophobicity of L, V, I, or A, wherein X ⁵ is positively charged K or R, and in the above [X ⁶ -X ⁷ -X ⁸ -X ⁹ -X ¹⁰ -X ¹¹ ], X ⁸ and X ⁹ are together and / or the X ¹⁰ and X ¹¹ may be absent together, and the sequence may be 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), or FRPC (SEQ ID NO: 14), or FR, wherein X ¹² is positively charged K or R, wherein m and n are each When 1 or 2 and m or n is 2, the amino acid sequence of each peptide is the same or different and the amino acid is a natural or unnatural amino acid of D- or L-type. In this case, X ² -X ³ -X ⁴ may be LVV, AAA, LVA, LVL, LLA, LLL, or LLV. In this case, the above [X ¹ -X ² -X ³ -X ⁴ -X ⁵ ] is QLVVK (SEQ ID NO: 1), 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 (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). In one embodiment the peptide sequence satisfying the above conditions is SEQ ID NO: 17, 19, 20, 21, 24, 25, 26, 27, 30, 31, 32, 33, 37, 38, 39, 40. 41, 42, 43, 44, 46. It has the amino acid sequence of 47, 48, 50, 51, 52 or 54.

In another aspect the present application relates to a peptide having cell adhesion shown by any one of SEQ ID NOs: 15-69.

However, the peptide according to the present application is not limited to the above sequence, and includes biologically equivalents thereof. Biological equivalents are those having additional modifications to the amino acid sequences disclosed herein, but having substantially the same activity as the polypeptides according to the present application.

Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues. Such amino acid variations are made based on the relative similarity of 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 all positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have a similar shape. Thus, considering this, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.

In one embodiment, conservative amino acid substitutions have occurred in a polypeptide according to the present disclosure. Conservative amino acid substitutions mean substitutions that do not substantially affect or reduce the activity of a particular polypeptide. For example, conservative substitutions occur 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 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 / pnas.89.22.10915; Reference may be made to WO2009012175 A1 and the like.

TABLE 1

Thus, the present application includes the sequences represented by SEQ ID NOs: 1 to 79 and those in which conservative substitutions have occurred.

In addition, in introducing amino acid variations, the hydrophobic index of amino acids can be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+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 hydrophobicity indexes are particularly important for imparting the interactive biological function of proteins. It is known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobicity index, substitutions between amino acids which exhibit a hydrophobicity index difference of preferably within ± 2, more preferably within ± 1 and even more preferably within ± 0.5 are advantageous.

It is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity.

As disclosed, for example, in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+ 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); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

Amino acid substitutions that do not alter the activity of the peptides according to the invention as a whole are also 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 / Gly, Thr / Phe And substitutions between Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

Therefore, if the above-described variations having biologically equivalent activity are included, the amino acid sequence disclosed herein or the nucleic acid molecule encoding the same includes those having substantially the same identity as disclosed herein. Substantial identity is at least 61% homology, more preferably, when aligning the sequence disclosed herein to any other sequence as closely as possible and analyzing the aligned sequence using algorithms commonly used in the art. Preferably a sequence that exhibits 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. See, eg, 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. Mol . Biol. (1990) 215: 403-10) is accessible from NBCI and the like, and can be used with blast, blastp, blasm, blastx, tblastn and tblastx. It can be used in conjunction with a sequence analysis program. BLSAT is accessible at www.ncbi.nlm.nih.gov/BLAST/ and a method for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

In one embodiment, the above-described peptides according to the present application may increase the stability by replacing the N- or C-terminus, especially the C-terminus, with a group such as a chemical non-reactive group such as NH2.

In addition, the peptides according to the present application may be modified with various compounds, for example, may be modified in such a manner as to bind various functional substances and the like to the epsilon amino group.

In another aspect the present application also relates to a polynucleotide encoding a polypeptide according to the present application as described above, a recombinant vector comprising said polynucleotide and a cell into which said recombinant vector is introduced. A polypeptide is one in which one or more polynucleotides encoding a polypeptide according to the present disclosure may exist, 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. The method of cloning into such a vector and the method of delivering the vector to prokaryotic or eukaryotic cells can be used in the art. Those skilled in the art will be able to use suitable ones.

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

The peptides according to the invention can adhere cells to inorganic or organic or surfaces thereof, or to other surfaces derived from various living or non-living organisms, or surfaces thereof. This feature can be used to mediate adhesion between one material and the same or different materials. For example, when using the peptide according to the present application, cells cultured in vitro can be adhered to a culture dish, or mediate cell-to-cell adhesion, or are combined with a labeling material such as a fluorescent material, cells, tissues, etc. It can be used for the labeling of a bioactive substance, for example, a substance such as bone morphogenetic protein can be combined with the peptide of the present application, and the treatment effect can be enhanced when the bioactive substance is expected to be treated in tissue. .

Although not limited by this theory, the peptide according to the present invention is a heparan sulfate, heparin, controtin sulfate in the components constituting the proteoglycans present in a large amount in the Extra Cellular Matrix (ECM) of the surface of the cell It shows affinity for GAG (glycoaminoglycan) such as (chondroitin sulfate). In addition, the peptide according to the present invention does not increase the adhesion ability through interaction with the cell membrane protein, and has a cell adhesion promoting mechanism separate from anchorage dependent cell adhesion through integrins in which the RGD peptide acts. Compared to RGD, up to 100-fold less and significantly less use was possible in terms of treatment concentration. Proteoglycans, for example, are cartilage-forming tissue components that have a wide range of applications in tissue regeneration and include materials that are directly used in a variety of clinical procedures, including plastic surgery.

Thus, the cells to which the peptides of the present application or compositions or methods comprising the same can be applied include copper, plants and some, such as cells, tissues, and organs derived from the copper, plants.

The cells to which the peptides or compositions according to the invention can be applied are not particularly limited and can be applied to plants, insects and animal cells, for example animal cells, in particular human-derived cells such as skin, dermis, endothelial, epidermis, It includes cells from muscle, myocardium, nerve, bone, cartilage, brain, epithelium, heart, kidney, pancreas, spleen, mouth, cornea or hair, and includes cells, primary cells or established cell lines in vivo.

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

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

The surface to which the peptide or the composition according to the present application or the method using the same may be applied 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 application can be applied includes all materials derived from a living body or non-living material including plastic, glass, polymer synthetic resin, and metal.

Uses of the peptides or adhesive compositions according to the present application include, but are not limited to: (1) adhesion between substrates in water (water or saline water); (2) orthopedic treatments such as bone, ligament, tendon, meniscus and muscle treatments and artificial material implants; (3) ophthalmic conjugation, such as treatment of perforation, fissures, incisions, etc., corneal transplantation, artificial corneal insertion; (4) dental abutments such as correction devices, dentures, crown mounts, rocking teeth fixation, broken tooth care and filler fixation; (5) surgical treatments such as vascular bonding, tissue bonding, artificial material implantation, wound closure; (6) graft conjugation of plants, conjugation in plants such as wound healing; (7) cell or tissue culture comprising stem cells; (8) a variety of base materials for medical devices including artificial organs, dental, surgical or ophthalmic medical devices, such as implants, bone fixatives, bone cages, guidewires, catheters and stents, and (9) bioactive materials. Biomaterials may be used, for example, in bioconjugation with bioactive materials, drugs, labels, or target materials.

In one embodiment the peptides or compositions herein are used for dental, ophthalmic or orthopedic treatment for transplantation or reconstruction of cells or tissues for the treatment of a disease, in which case the surface to which the adhesive composition herein can be applied This includes, but is not limited to, PLGA, hydroxyapatite, zirconium, titanium, iron, stainless steel, titanium, platinum, gold, alloys.

In another embodiment the peptides or compositions herein are used for adhesion of cells to a support. The cell adhesion surface or support herein includes cell culture dishes, microbeads, substrates, tissue implants, and the like, and can be used, for example, for culturing tissues or cells, in particular for culturing stem cells. In one embodiment according to the present application, when using the peptide of the present application, it showed a significantly higher adhesion compared to the material used for conventional cell adhesion.

In another embodiment the peptides or compositions herein are used for bioconjugation. Bioconjugation refers to a chemical method / means for linking two biomolecules in a stable covalent bond, wherein the peptides according to the present invention are directly or in combination with low molecular linkers, proteins comprising various biomolecules such as enzymes, hormones, It can be linked to nucleic acids, lipids or carbohydrates. Such bioconjugation may be used as a research tool, for detection, monitoring, or the like of a biochemical material, or may be conjugated with a therapeutic material as a therapeutic agent to increase the efficiency of treatment or to target treatment. In one embodiment according to the present invention is conjugated to the osteogenic protein, increasing the therapeutic effect of the osteogenic protein.

The method of using the peptide and the composition according to the present application is based on the method of using a conventional bioadhesive composition, and a representative method is an application method. For example, specific usage, dosage amounts, and formulations of the peptides or compositions according to the present application may be referred to Cell-Tak ^® (BD Biosciences, USA) which is a commercially available product.

The composition according to the present application may be a solvent type, a water soluble type, or a solventless type, and may be used at 0.1 to 1000 ng / mm ² , particularly 1 to 100 ng / mm ² , based on the area of the surface to be applied, but is not limited thereto.

The composition according to the present application may be adjusted with the adhesion and the amount of application according to the treatment with a surfactant, an oxidizing agent, a crosslinking agent, or a filler, or by adjusting the concentration of the peptide.

Compositions comprising the peptides according to the invention allow the cultivation of the cells while maintaining all the characteristics of the culturing cells. The peptide according to the present application can be used as is in the conventional culture method of the cells to be applied. That is, the cells which need an appropriate number of cultures may be suspended in a conventionally available liquid medium or cell culture solution, and the peptide according to the present application may be added thereto. Subsequent exchange and subculture of the liquid medium can also be carried out in the same manner as the conventional method, and in this process, the peptide according to the present application may or may not be added.

Peptides according to the invention can be used in addition to the cell culture medium.

In addition, the peptide according to the present application can be used by fixing or coating on the solid surface of the culture vessel. As the culture vessel, any one conventionally used for cell culture such as a plate or a flask can be used. The culture vessel is sterilizable, and an inorganic material such as glass or an organic material such as polystyrene or polypropylene may be used.

As a method of fixing or coating the peptide according to the present invention on the solid surface of the culture vessel, a physical method such as adsorption or a chemical method such as a covalent bond may be applied. For example, a method by adsorption that is easy to operate is preferable.

In another aspect, the present invention also relates to a cell culture method using the peptide or the composition according to the present invention, characterized in that the use of the peptide or the composition according to the present invention in the culture of the cells.

In one embodiment of the method according to the invention, the peptide or the composition comprising the same according to the invention can be used fixed or coated on the solid surface of the culture vessel. The culture vessel that can be used in the present method is not particularly limited as long as it can culture cells, but is preferably used for cell culture. Cell culture containers include, for example, culture dishes, plates, flasks, chamber slides, tubes, roller bottles, spinner flasks, holographic fibers, microcarriers, beads, and the like. These culture vessels may be made of an inorganic material such as glass or an organic material such as polystyrene. The culture vessel may be a material such as polystyrene having high adsorption to proteins or peptides, or a treatment to increase adsorption, for example, hydrophilic treatment or hydrophobic treatment.

In another embodiment of the method according to the present invention, a method of fixing or coating the peptide according to the present application on the solid surface of the culture vessel may apply a physical method such as adsorption or a chemical method such as covalent bonding, but is easy to operate. One method by adsorption is preferred. In one embodiment, the peptide according to the present invention may be adsorbed by contacting a solution containing the peptide according to the present invention to a culture vessel surface such as a plate and removing the solvent after a certain time.

In another embodiment of the method according to the invention, the peptide according to the invention can be used in addition to a conventional cell culture medium. For example, an appropriate number of cells of interest may be suspended in a commonly available liquid medium or cell culture, and an appropriate amount of the peptide according to the present may be added thereto, or the peptide according to the present application may be added to the liquid medium before the cells are suspended. Can be added first. Subsequent exchange and subculture of the liquid medium can also be carried out in the same manner as in the conventional method, in which further peptides according to the present application may or may not be added.

The cell culture in the method according to the present invention may use conventional culture medium or culture conditions except for using the peptide according to the present application.

Conventional culture methods or culture conditions can be found in 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 (eg fetal calf serum, horse serum and human serum), for example, RPMI series, Eagles' 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., J. Exp. Med. 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. Med.Assoc. 199: 519 (1967)), F12 (Ham, Proc. Natl. Acad. Sci. USA 53: 288 (1965)), F10 (Ham, RG Exp. Cell Res. 29: 515 (1963)) , DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)), mixtures of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102: 255 (1980) ), Way-mouth's MB752 / 1 (Waymouth, CJ Natl. Cancer Inst. 22: 1003 (1959)), McCoy's 5A (McCoy, TA, et al., Proc. Soc.Exp. Biol. Med. 100: 115 ( 1959) and the MCDB series (Ham, RG et al., In Vitro 14:11 (19) 78)), but is not limited to such. The medium may include other components such as antibiotics or antifungal agents (eg, penicillin, streptomycin), glutamine, and the like.

The composition according to the present application may include only the peptide according to the present application alone, but may further include a known adhesive, an adhesive protein other than the adhesive protein of the present invention, and a material that may be included in a known cell adhesion composition. . In addition, the content of the component to be included can be appropriately adjusted within the range generally acceptable according to the kind or formulation of the component. When additional ingredients are included, the active ingredient peptide is included in an amount capable of maintaining adhesive activity, for example, it may be included in 0.01 to 80% by weight.

In another aspect the present disclosure also relates to a method of adhering two or more material surfaces using a peptide or composition disclosed herein.

In one embodiment the method according to the present invention comprises treating the peptide or composition according to the present invention with a first substance and / or a second substance; And contacting the two materials under conditions sufficient for adhesion to occur so that the two materials can adhere. The above sufficient conditions will vary depending on the kind of specific material to which adhesion is made by the peptide according to the present application, and those skilled in the art, based on the description of the present examples, the description of the present specification, and examples of appropriate conditions in consideration of the knowledge known in the art. For example, temperature, time, composition of the medium in which the adhesion takes place, and the like may be determined.

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

The first or second material according to the present application may be treated in whole or in only one for attachment. In one embodiment the adhesive or composition is treated on a mineral surface, such as a cell culture dish, and adding cells thereto causes the cells to adhere to the cell culture dish.

In another embodiment, the peptide or the composition according to the present invention may be added to a culture solution containing cells, which may be added to the cell culture dish. In another embodiment, inorganic surfaces such as bone grafts can be coated with an adhesive according to the present application and attached useful biological materials such as osteogenic proteins. Alternatively, a biological material useful for the adhesive according to the present invention may be conjugated and then attached to the inorganic surface.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example

Example  1.adhesive Peptide  Produce

Peptides used in the experiment were customized to be synthesized by the method of 9-fluorenylmethyloxycarbonyl (Fmoc) (Ruzen Sci, South Korea; Peptron, South Korea). In order to verify the reproducibility of the peptide effect, peptide synthesis was performed three times (ruzen) and two times (peptron) through two companies, and each compound was analyzed through independent experiments. Obtained.

Example  2. Adhesive Peptides and  Fluorescent material Conjugation  And various thereof Non-living Analysis of adhesion to sieve-derived surfaces

Peptide of Example 1 A (RQLVVKFRALPC; SEQ ID NO: 17) (corresponding to the 12mer (A) sequence of Table 1 below) is conjugated to the biotin (Thermo Scientific), and FITC (Sigma) to Cys through the SH group, respectively. Subsequently, it was treated with glass, PCL, Ti, Col, Zr, vinyl, and PS fibers (each 10 μM in PBS, washed once with PBS after 20 minutes of adsorption at room temperature), and adhesion was performed by LAS (Fuji, Japan). Detection was by FITC fluorescence image measurement used. In order to liberate the biotin covalently bound to the peptide, a reduction reaction was induced by treatment with DTT (100 mM in PBS, one wash after 20 minutes at room temperature). Only dye was used as a control.

The results are described in FIGS. 1A-1D, and the detection of the antibodies indicated that the biotin according to the present application was strongly adhered to the surface used in the experiment via the peptide according to the present application, in particular this effect disappeared by treatment of DTT (FIG. 1a), indicating that the biotin or FITC is attached to the glass through the peptides herein.

Example  3. Adhesiveness according to the present application Peptide  variety Nonliving  And Biologically derived  Adhesion Analysis for Materials

Basic experiments were performed in the same manner as in Example 2, and Bio-OSS ^® (Geistlich Pharma, Inc) and MBCP ^TM (Biomatlante), which are widely used as bone grafts, were used instead of glass. Treatment conditions were as follows: Bio-Oss particles were added to a 10 μM peptide-FITC conjugate for 10 minutes at room temperature, followed by 5 washes for 48 hours in PBS solution containing 0.05% tween-20. After treatment it was analyzed by confocal microscopy (Zeiss LSM-700 model with Zen 2011 software, x20).

In addition, in order to verify the adhesion of the peptide in vivo compensation, tissue sections were obtained after injection into various tissues. PBS, Cys- or FITC-Conjugate To confirm the adhesion of the peptide, the peptide according to the present invention, or Cys or FITC dye was dissolved in PBS at a concentration of 10 μM, and then 10-20 μl of an aqueous solution was applied topically or injected into a syringe. The adhesion induction time in the tissue was fixed to 2 hours, the experiment was performed, and after tissue extraction after sacrifice, the tissue sections were washed three times in PBS for 30 minutes and analyzed by confocal microscopy. The tissues that were tested were performed mainly on cartilage, skin, hair, subcutaneous fat and eye of the mouse. In the case of cartilage, collagenase was partially treated to expose the elastech fiber structure and then analyzed after strong washing.

The results are described in FIGS. 2A-2M, indicating that the peptides according to the present disclosure can be attached to a variety of materials from non-living and living organisms. Excellent adhesion to the commercially available bone graft material according to the present invention, in particular, tissues containing a large amount of proteoglycan components, such as elastic tissue, connective tissue and tissue containing a large amount of collagen or hyaluronan, And adhesive affinity to yeast and bacterial cell walls.

In addition, the peptide of the present invention has excellent adhesion in cartilage tissue (*) tissue containing a large amount of glycoaminoglycan (GAG), such as heparan sulfate, heparin, and chondroitin sulfate, which are the main components of proteoglycans. It is consistent with the experimental results of FIG. 4B described later. GAG has four types of substances: hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin and heparan sulfates, and keratan sulfate. sulfate). These are all composed of N-acetylglucosamine or N-acetylgalactosamine, which are in fact glucose or galactose with bound amine groups, which means that the peptides according to the present invention have high affinity for such hexasaccharides. It indicates that it is possible to target specific action of the peptide according to the present invention.

Example  4. Adhesive Peptides  Adhesion Enhancement Effect of Various Cells

The materials used in this experiment were as follows: 10 mM peptide solution (stock solution) in PBS; 35 mm diameter plastic hydrophobic cell culture dish (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); STO (feeder cells: ATCC, CRL-1503), HEK-293 (ATCC, CRL-1573), SaOS2 (ATCC, HTB-85), HeLa (ATCC, CCL-2), ROS17 / 2.8, NIH3T3 (ATCC, CRL -1658), RAW264.7 (ATCC, TIB-71), hMSC (Lonza, PT-2501), hPDL (HPLF) (Sciencell, Cat. # 2630) hDPSC (Cells Tissues Organs 184: 105-16) Separation from tissue), Primary Bone Marrow Stromal Cell from mouse (mBMSC), Primary Mouse Calvaria cell (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 culturing by directly adding to the cell culture medium without coating, and the results were consistent. When the coating method was taken, a constant concentration of peptide was added to the cell culture medium containing no PBS or fetal bovine serum, followed by coating at room temperature for 30 minutes. After removing the solution, the cells were added to measure adhesion over various times. In addition, when the peptide is added to the culture medium, a certain level of peptide is added together when the cells are suspended, and then the suspension is transferred to the culture dish. Cell adhesion measurements were performed as follows. For cell adhesion, F-actin was observed for general observation through optical microscope, actin filament structure, and rough cell membrane boundary pattern, with PBS and poly-L-lysin coated as a negative control, when no peptide was added. Cell metabolism or DNA amount was measured for staining and confocal microscopy and quantification for adhesion. Metabolism was measured for absorbance using CCK-8 (Dojindo), and the amount of DNA was measured for fluorescence using the Picogreen assay kit (Life Technologies) according to the manufacturer's method. In order to quantify only adhered cells, non-bonded cells were discarded and washed twice with PBS, and only the cells adhered to the surface were used for quantification of metabolic amount or DNA.

The results are described in FIGS. 3A-3F. As shown here, the peptide according to the present invention can be usefully used for adhesion of anchorage-dependent cells of various origins, for example, adhesion of primary cells, established cells and thawed cells, and feeder cells, and has been previously used. Compared to the PLL, the effect was excellent.

Example  5. Adhesive Peptide  Identification of adhesion mechanism

In the Examples, the following experiment was performed to identify the mechanism of peptide adhesion according to the present application.

In this example, the following experiment was performed to investigate the mechanism of cell adhesion by the peptide according to the present application.

The cells used anchorage dependent cells (MC3T3-E1), which were treated with the peptide (SEQ ID NO: 17) A peptide (10 μM) according to the present application. Adhesion by the peptide according to the present invention is not adhered cells are removed with PBS, DNA quantification using a picogreen assay kit (picogreen assay kit (Life Technologies)) or CCK-8 (Cell Counting Kit) -8, Dojindo) was used to measure the cell count using the manufacturer's method.

(1) Whether protein is involved

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

The results are shown in Figure 4a, as shown there is no difference in adhesion even when treated with EDTA or CHX. In other words, the cell adhesion by the peptide according to the present application does not require the electrolyte on the cell surface (the result of EDTA treatment) or the new protein synthesis (the result of CHX treatment), whereas the serum initially shows inhibition.

 This may be due to the primary binding of many types of GAG present in the serum to the peptide. This is not limited to this theory, but GAG (glycoaminoglycan) such as heparan sulfate, heparin, and chondroitin sulfate, which are the main components of many kinds of proteoglycans present in serum, are primarily used in peptides. It is judged to be the cause of the combination. In addition, the peptide according to the present invention is not to increase the adhesion ability through interaction with the membrane protein.

(2) Nonproteinaceous  Substance involvement

Next, we investigated the involvement of nonprotein materials and analyzed the relationship between proteoglycan, a carbohydrate lipid contained in a large amount of extracellular matrix (ECM) on the cell surface.

- Proteoglycan  About Peptide  Molecular affinity

Glycoaminoglycans such as heparan sulfate, heparin, chondroitin sulfate, dermatan sulfate, keratan sulfate, and other major constituents of proteoglycans Glycoaminoglycan (GAG) is also a major component of ECM, but maintains the structural integrity of ECM to maintain cell morphology, as well as regulate cell adhesion and cell polarity. do. This function of ECM not only induces the adaptation of cells to the environment, but also has a complex function of directly regulating a complex series of metabolic processes and regulating various physiological roles.

The molecular affinity of the peptides for proteoglycans can be determined by treating (i) affinity chromatography, (ii) cell adhesion through competitive binding using purified GAG components, and (iii) enzymes that specifically degrade ECMs comprising GAG components. Inhibition of cell adhesion promotion by peptides was verified through the above three methods. In the case of enzyme treatment, hyaluronidase (hyaluronidase (Sigma)) and collagenase (collagenase (Sigma)) were treated. Purified GAG used hyaluronan (Sigma), chondroitin sulfate (Sigma), heparin (Sigma), heparan sulfate (Sigma), respectively.

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

(ii) Investigation of cell adhesion through competitive binding using purified GAG components

The following shows the inhibition of cell adhesion by the adhesive peptide by the competitive inhibition method by GAG treatment, and the cell adhesion promotion by the peptide was significantly inhibited by the addition of heparin, heparan sulfate, and chondroitin sulfate. .

(iii) Inhibition of Promoting Cell Adhesion by Peptides by Treating Enzymes That Specificly Decompose ECM Containing GAG Components: Fibril proteins such as collagen as well as proteoglycans that make up the ECM include GAG. The cell adhesion promoting ability by the peptide was examined by treating the collagen containing GAG or by treating the hyaluronidase which hydrolyzes the hyaluronan, one of the GAGs. This also indicates that the promotion of cell adhesion by peptides correlates with GAG.

The experimental method was as follows: (i) Competitive inhibition by treatment with purified GAG component was added to each cell suspension treated with trypsin to 5 mg / ml and reacted at 37 ° C. for 10 minutes and then coated with peptide. After transfer to the dish 30 minutes after the adhesion of the DNA to the adherent cells were quantified. DNA quantification was performed using a picogreen assay kit (Life Technologies); (ii) Cell adhesion investigation by collagenase and hyaluronidase treatment was suspended in trypsin-treated cells in a medium containing no fetal bovine serum and reacted at 37 degrees for 30 minutes after each enzyme was added. Enzyme was treated at 10,000 unit / 10 ³ cell level. After enzyme reaction, EDTA and fetal bovine serum were added to inactivate enzyme activity, followed by centrifugation, suspended in fresh medium, and then incubated in a peptide-coated culture dish for 2 hours. . The measurement of adherent cells was performed twice with PBS before performing the picogreen assay, and then only adhered cells were recovered to quantify DNA.

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

(3) With RGDS  compare

After competitive treatment with an anchorage dependent cell (MC3T3-E1) the peptide peptide (10 μM) according to the present invention or soluble RGDS peptide (0, 10, 100 and 1000 μM) together with the cells in a test tube for 10 minutes After transferring to the culture dish, adhesion was induced again in a thermostat for 10 minutes. After 10 minutes, the culture medium was removed again and washed twice with PBS solution to remove the remaining unbonded cells, and mixed with fresh medium containing no peptide and CCK-8 (Dojindo) for 1 hour in a thermostat and then 450nm Absorbance was measured at. Absorbance was measured by blanking the medium mixed with CCK-8.

The results are described in Figure 4c. As shown here, when treated with RGDS peptide alone, the adhesion was reduced 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 binds to integrin and significantly reduces the adhesion to the bottom (substrate) when the water-soluble RGD is treated. In the treatment group, treatment with RGD did not affect cell adhesion at all, indicating that the peptide according to the present invention had a cell adhesion promoting mechanism distinct from anchorage dependent cell adhesion through integrins in which RGD acts. will be.

These results indicate that cell adhesion or tissue regeneration using RGDS peptides has already been well established, which indicates that there is a technical differentiation, and that commercialization techniques using RGDS are known to be inefficient. It is an excellent technology that can be shown.

Example  6. Adhesive Peptides  Embryonic Stem Cell Adhesion Enhancement Effect

Peptide was coated on the culture material at 100μM level or nanostructures were prepared to investigate the adhesion and pluripotency of mouse induced pluripotent stem cells and human embryonic stem cells as follows.

For human embryonic stem cells, hESC-media (general culture composition), mTeSR (hES-only medium, purchased from Stem cell technology), Essential 8 (hES-only medium, purchased from Gibco BRL), and 10 ng for each The cells were cultured under the condition of serum exclusion culture containing only / ml bFGF (a condition for preventing the cell's adhesion ability from being inhibited by serum) and analyzed for mutual compatibility with various existing culture techniques. In addition, the cells were treated with a colony culture method and 0.25% trypsin-EDTA to analyze the culture method using a single cell. To verify the effect of ESC or iPSC culture on feeder-free, we compared matrigel (hESC) and gelatin (miPSC) as positive controls, as well as all embryonic stem cell cultures. Cultivation was also performed in a feeder cell used as a positive control group.

The cells were plasmids having a structure capable of measuring the activity of the Oct4 gene as an indicator of the stem cells of embryonic stem cells described in FIG. 5A (Szabo et al., 2002, Mechanisms of Development Vol. 115: 157-160 ) Was analyzed by fluorescence microscope (FIG. 5). In addition, the expression of Alp and Nanog genes, which are markers of pluripotency, were observed by fluorescence microscopy by staining with specific antibodies, and whether cell adhesion was improved in hES cell culture was also examined (FIG. 6).

The results are described in FIGS. 5 and 6. As shown in FIG. 5, the peptides of the present disclosure have been shown to mediate adhesion of iPSCs while maintaining predifferentiation in both cases, either precoated or added in a mixed manner to the culture (FIG. 5B). . In addition, in the case of day 4 cell culture, the peptide according to the present invention was shown to mediate the adhesion of iPSC and also maintain the pluripotency (FIG. 5C). In addition, as described in Figures 6a and 6b, the peptide according to the present application was shown to mediate the adhesion of the iPSC and to maintain the pluripotency even when pre-coated or mixed with the culture medium, even in experiments using hESC feeder cells or Matrigel (Matrigel) mediated the adhesion of cells without the external environmental changes and also appeared to maintain the pluripotency (Fig. 6c).

Example  7. Adhesive Peptides and  Protein Bioconjugation

The materials used in this experiment were as follows:

Peptides synthesized in Example 1 were used at a concentration of 10 μM (in PBS); Plastic cell culture dish with a area of 1.9 cm ² (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, pH7.0 (Stop solution); Phosphate buffered saline, reaction solution;

The analysis concept is shown schematically in FIG. 7A. Referring to Figure 7a, by using a cross-linking agent to connect various substances having the desired bioactivity to the peptide of the present application, for example, therapeutic proteins such as BMP, due to the adhesion of the peptide of the present application, The concentration of the therapeutic protein can be increased, and the therapeutic effect can be maximized.

The experimental method is as follows. Peptide-rhBMP2 cross-linking reaction was carried out according to the method of the cross-linker manufacturer used basically, and in summary, the process was as follows: (i) Peptide coating: cell culture dish 200 μl of peptide solution (10 μM of the peptide of Example 1 in PBS solution) were aliquoted and adsorbed at 4 ° C. for 18 hours; (ii) Formation of DSS-BMP2 complex: 20 molar excess of DSS against rhBMP2 was induced for 1 hour in 100 μl of reaction solution. RhBMP2 per area of 1.9cm ² was adjusted to 300ng level, where DSS, rhBMP2, and DSS-rhBMP2 were coated as a control, respectively. In this case, all of them were observed to be substantially lost in the washing process without adsorption to the surface material of the culture plate. Therefore it was used as a negative control in this case; (iii) Peptide-DSS-BMP2 complex formation: l00 μl of DSS-BMP2 complex was carefully added to a pre-reacted peptide coated dish (overlay) and reacted at 4 ° C. for 24 hours; (iv) Stop reaction: neutralize all remaining DSS after covalent bonding by adding 200 μl of 1M Tris solution and standing at room temperature for 15 minutes; (v) washing: washed 10 times with PBS solution, and then left at 37 ° C. after addition of DMEM medium without fetal calf serum before cell addition; (vi) C2C12 suspension preparation and osteoblast differentiation: 4 × 10 ⁵ C2C12 cells were suspended in DMEM medium containing 300 μl of 2% fetal bovine serum and then 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 measurement: Differentiation capacity of C2C12 cells into osteoblasts was measured by cytostaining activity of alkaline phosphatase.

The results are described in Figure 7b. As shown here, Alp activity was increased compared to recombinant BMP treated group (rhBMP2) or crosslinker treated group (CL), indicating that bone differentiation was promoted by the peptide treatment herein.

Example  8. according to the present application Peptide  Stability analysis

The safety of the peptides according to the invention was tested using osteoblasts (MC3TC-E1). Peptides according to the present invention synthesized in Example 1 After treating the peptides according to the present application to the osteoblasts at concentrations of 0.1 μM, 1 μM, 10 μM and 100 μM, proliferation (FIG. 8A), survival (FIG. 8B) and differentiation capacity (FIG. 8C) In the case of differentiation capacity, MC3T3-E1 cell line or C2C12 cell line was used, and the cell line was stimulated with bone constituent medium (6 days treatment) and BMP2 (rhBMP2, 10ng / ml, 3 days treatment). Differentiation was induced, and the differentiation effect was performed through cell staining. Results are shown in Figures 8a to 8c, it did not appear to affect the proliferation rate, survival rate, differentiation according to the concentration.

In addition, the safety of the peptides according to the present application was tested using monocytes of mice. Monocytes isolated from the bone marrow of mice were added after treatment with the peptides according to the present application and examined for inflammation induction. LPS was treated as a positive control, and after 24 hours, inflammation markers IL-1β (interleukin-1β), Tnf-α (Tumor necrosis factor-α), iNos (Inducible nitric oxide synthase), and Cox-2 (cyclooxygenase- 2) were each measured using a real-time quantitative reverse transcription PCR method. As a result, it was confirmed that the peptide according to the present application does not cause inflammation. Results are shown in Figure 8d, the peptide according to the present application did not cause inflammation, it was determined to be a stable material in vivo.

Example  9. Adhesive Peptides  Primary neurons used Adhesion  Enhancement and Cultivation

In this example, the effect on neuronal cell adhesion and culture was compared with the existing adhesive peptide. Peptides were coated on the culture material at 100 μM level or nanostructures were prepared to obtain neuron-derived cells from mouse embryonic brain tissue and spinal cord of rats, and the adhered cells were analyzed by light microscopy and metabolism through CCK-8. At this time, Laminin and poly-L-ornithin (Sigma) were mixed and coated according to the manufacturer's concentration and method as a positive control group.In order to quantify cell adhesion, unadhered cells were washed with PBS (for each hour). CCK-8 solution (Dojindo), which measures the metabolism of the culture medium and cells, was mixed and added, and then absorbed at 450 nm after incubation for 1 hour.

The results are described in FIG. Figure 9a is a spinal cord-derived neurons coated with a conventional peptide peptide laminin (laminin) and poly-L- ornithin (poly-L-ornithin) and coated with a peptide according to the present application according to an embodiment of the present application After culturing using the prepared culture plate was analyzed by CCK-8 for each time period, showed a significant difference in adhesion capacity under the conditions in which the peptide is coated (adsorption) according to the present application. Figure 9b is a result of comparing the observation through the optical microscope after 4 days, the same amount of cells were used, but the degree of adhesion was the most excellent in the peptide according to the present application. Figure 9c is a result of verifying by using a marker specific to neurons cells that are primary cultured cells coated with a peptide according to the present application according to an embodiment of the present application, neuron marker proteins (GFAP, MAP2 and Nestin) After staining with immunofluorescence, the result was analyzed by confocal microscopy. The results indicate that the peptides according to the present application have significantly better adhesion than the existing ones.

Example  10. Human MSC  ( Mesenchymal  Stem Cell) Adhesion  Enhancement effect

Peptides according to the present invention were coated with glass sterilized coverslip at 100 μM level, and then cultured with MSC cells (Mesenchymal Stem cells) for 6 hours. Cells were stained using Rhodamine Phalloidin stain (Life Technology Cat. # R415) and DAPI (Sigma D9542) stain, which stains the nucleus of cells (DNA), and analyzed by confocal microscopy. At this time, as a negative control, the cells were adhered to the coverslip which is not coated with the peptide according to the present application and compared. The results are described in FIG. In FIG. 10A, 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 many cells after the step. Figure 10b is a graph quantifying these observations, Figure 10c is a cell aspect ratio (cell aspect ratio: the ratio of the cell to vertical, according to the cell adhesion, 1 is a perfect circular and the initial state of the adhesion process Prager-Khoutorsky et al., 2011. See Nature Cell Biology 13, 1457-1465) as a result of showing a significantly mature adhesion in the treatment group of the peptides according to the present application.

Example  11. of various sequences Peptides  Used Adhesion  Experiment I

Peptides having the sequence and name as shown in Table 2 below were synthesized as in Example 1, and then adhesion to heparin or N-acetylglucosamine (GAG) was measured using the same method as described in Example 4.

TABLE 2

The results are described in FIG. As shown therein, the peptides of the present disclosure are directed to components contained in a cell even when the first region, the second region and the third region are arranged in various combinations or the sequence of each region is substituted with an amino acid as defined herein. Adhesion ability, which indicates that the peptide according to the present application can be effectively used for adhesion or attachment of cells including stem cells.

Example  12. Diverse Sequences Peptides  The components contained in the extra cellular matrix (ECM) on the cell surface including the stem cells used Adhesion  Experiment II

Based on the RQLVVKFRALPC (SEQ ID NO: 17) sequence designated as A sequence in Table 2, amino acid substitution was performed in consideration of the chemical properties, size, charge characteristics, and experimental results of Example 11, and the like, as shown in Table 3 below. Peptides of the sequence were synthesized and analyzed for adhesion as in Example 11.

TABLE 3

The bolded part in the sequence column of Table 3 is a substituted part compared to the A sequence, and the result is described in FIG. 11B, which indicates that peptides of various sequences according to the present invention can be effectively used for adhesion or attachment of cells. To indicate.

Example  13. In accordance with the present application Peptide  Epithelial cell adhesion and maintenance

Adhesion or adhesion to epithelial cell HaKaT using peptide A according to the present application was investigated. The results are shown in FIG. When the peptides according to the present invention were not treated, HaKaT epithelial cells at the hydrophobic surface did not completely adhere to the culture plate surface. The result was a clumping form, which is a phenomenon of cell aggregation. This result is due to the loss of cell characteristics and the intrinsic polarity of the epithelial cells due to the failure to adhere. On the contrary, in the case of peptide treatment according to the present application, it was observed that epithelial cells can maintain their morphology on the hydrophilic surface under normal culture conditions. Epithelial cell-specific cell junctions and monolayer formation were clearly observed. These results indicate that biological cell adhesion of epithelial cells is well maintained due to the effect of peptides.

On the other hand, the functional effect of the peptide on the primary culture (primary culture) of pig-derived epithelial cells was investigated. Generally, in vitro culture of epithelial cells of various individuals, including porcine epithelial cells, uses collagen-like proteins as substrates for cell attachment. The reason for this is that when cultured in vitro, epithelial cell characteristics are rapidly lost. Therefore, it is possible to maintain more epithelial cell characteristics by injecting collagen substrates involved in the dominance and adhesion of epithelial cells in vivo. Figure 13 is the result of examining the effect on the cell adhesion and morphology between the collagen substrate and the peptide substrate according to the present application. In step P.3, epithelial cells on the collagen matrix can be seen that their morphological characteristics have already been altered. In epithelial cells, a clear junction of cells, ie, cells, is observed and the characteristics of typical monolayers are well reflected. Amorphous cell junctions were not observed, but amorphous morphological changes in fibroblasts or mesenchymal stem cells were observed. This can be seen as a result of the loss of the uniqueness of epithelial cells. In contrast, the peptide treatment group according to the present application can be observed that the characteristics are maintained even in the cells of the same passage.

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

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artificial <400> 8 Phe Arg Val Val Pro Cys   1 5 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 9 Phe Glu Ala Leu Pro Cys   1 5 <210> 10 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 10 Tyr Arg Ala Leu Pro Cys   1 5 <210> 11 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 11 Trp Arg Ala Leu Pro Cys   1 5 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 12 Phe Arg Ala Leu Pro   1 5 <210> 13 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 13 Phe Arg Ala Leu   One <210> 14 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 14 Phe Arg Pro Cys   One <210> 15 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 15 Arg Gln Leu Val Val Lys   1 5 <210> 16 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> artificial <400> 16 Phe Arg Ala Leu Pro Cys Arg Gln Leu Val Val Lys   1 5 10 <210> 17 <211> 12 <212> 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 Gln 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 Arg Arn 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 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 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 Gln 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 <222> (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 cell adhesion composition comprising a peptide of amino acid sequence as set forth in SEQ ID NO: 6, 15 to 22, 24 to 27, 29 to 37, 42 to 64, 67 or 68. The method of claim 1,
The first amino acid residue of the amino acid sequence set forth in SEQ ID NOs: 15-20, 22, 24-27, 30-37, 42-64, 67 and 68 is substituted with lysine,
The first amino acid residue of the amino acid sequence set forth in SEQ ID NO: 21 is substituted with arginine, and
The first amino acid residue of the amino acid sequence disclosed in SEQ ID NO: 29 is substituted with aspartic acid, the composition for cell adhesion.
The method of claim 1,
The first amino acid residue of the amino acid sequence set forth in SEQ ID NOs: 15-22, 24-27, 30-37, 42-64, 67 and 68 is substituted with aspartic or glutamic acid residues, and
The first amino acid residue of the amino acid sequence disclosed in SEQ ID NO: 29 is substituted with lysine or arginine residues, the composition for cell adhesion.
The method of claim 1,
The peptide of the amino acid sequence set forth in SEQ ID NO: 15-20, 22, 24-27, 30-37, 42-64, 67 or 68 further comprises up to 14 arginine residues at its N-terminus, and
The peptide of the amino acid sequence set forth in SEQ ID NO: 21 further comprises up to 14 lysine residues at its N-terminus, composition for cell adhesion.
The method according to any one of claims 1 to 4,
N- or C- terminus of the polypeptide is modified to a non-reactive group, the composition for cell adhesion.
A method of adhering a cell to an inorganic or organic surface comprising the step of treating the cell according to any one of claims 1 to 4, wherein the cell does not comprise cells in the human body.
Treating the composition according to any one of claims 1 to 4 with a first material and / or a second material; And contacting the two materials, wherein at least one of the first or second material is a cell and the cells do not include cells in the human body.

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