KR20130053479A - Application of biomimetic multi-block bio-polymers for tissue regeneration and tissue regeneration method thereof - Google Patents

Abstract

PURPOSE: An application of biomimetic multi-block bio-polymers for tissue regeneration and a tissue regeneration method thereof are provided to be effective in a wound treatment by at least one or more blocks among multiple blocks including peptide I of a cell adhesion function for tissue neogenesis. CONSTITUTION: As an application of biomimetic multi-block bio-polymers for tissue regeneration, blocks(110-160) of m(m is a fixed number of 1 to 100) connected in a row are repeated with n(n is the fixed number of 1 to 5,000). At least one block among blocks of m comprises peptide I of a cell adhesion function for tissue neogenesis. Each blocks more than one and not selected peptide I is one among peptide II having a structure of PGX (P is Lproline, G is ofglycine, and X is 19 L- or D- amino acid, except for L- or D-proline), peptide III having the structure of PGXX, peptide IV having the structure of PGXXX, peptide V having the structure of PGXGXX, and peptide VI of having a function with a mutual combination promotion. [Reference numerals] (110) Block 1; (120) Block 2; (130) Block 3; (140) Block 4; (150) Block 5; (160) Block 6

Description

Biomimetic multiblock biopolymer for tissue regeneration and tissue regeneration method using the same {Application of biomimetic multi-block bio-polymers for tissue regeneration and tissue regeneration Method

TECHNICAL FIELD The present invention relates to tissue regeneration, and more particularly, to a biomimetic multiblock biopolymer for tissue regeneration that supports more effective tissue regeneration using a multiblock biopolymer and a method for regenerating tissue using the same.

There has been a lot of information on the preparation of simple elastin biopolymers having a single repeating structure. For example, conventionally simple elastin biopolymers with a single repeating structure are polypeptides with simply repeated amino acid chains of Valine-Glycine-Valine-Alanine-Proline-Glycine, or tropoelastins with human cDNA genes, or L-proline. Four or five amino acids comprising were simply repeated polypeptides.

By the way, they exhibit a heat transfer phenomenon similar to that of the living elastin and exhibit the property of generating fibrous tissue, but there is a problem that the adhesion and interconnection of the cells are inferior to those of the living elastin. In addition, when expressing tropoelastin using a human cDNA gene, there was a problem that the genetic manipulation for producing elastin polypeptides suitable for use and purpose by containing an amino acid content of a desired component is not easy. In particular, biopolymers suitable for tissue regeneration have not been properly developed, and there has been a long-term demand for this.

Accordingly, an object of the present invention is to solve the problems of the prior art as described above, to provide a biomimetic multiblock biopolymer suitable for tissue regeneration and effective tissue regeneration method using the same.

The present invention for achieving the above object is repeated m blocks (m is an integer of 1 to 100) of n blocks (n is an integer of 1 to 5,000) are connected in a line, at least one block of the m blocks Is a peptide I of a cell adhesion function for tissue regeneration, wherein at least one block not selected as the peptide I is a peptide II having a structure of PGX (P is L-proline, G is ofglycine, and X is L -Or 19 L- or D-amino acids excluding D-proline), peptide III with the structure of PGXX, peptide IV with the structure of PGXXX, peptide V with the structure of PGXGXX, and mutual binding enhancement functions of multiblock biopolymers Disclosed is a configuration of a multiblock biopolymer for tissue regeneration, characterized in that one of the peptide VI.

The peptide I, which functions to recognize the cell surface antigen, may be composed of one or more of Arginine-Glycine-Aspartate (RGD) or Arginine-Glycine-Aspartate-Serine (RGDS), which are cell adhesion peptides.

And the peptide VI, which functions to promote cross-linking of the multiblock biopolymer, may include a binding site PAAAAKAAKZG or PAAAAKAAAKAG of human tropo elastin.

Meanwhile, the at least one block may be selected by selecting different peptides from the peptides II to VI.

And m may be composed of two to six.

The peptide I may be composed of ELP-RGD at a concentration of 100 uM RGD, for example, [VGRGD (VGVPG) 6] n (n = 10, 12, 15, 20).

The multiblock biopolymer for tissue regeneration may have a tissue regeneration function for treating wounds using peptide I which recognizes a cell surface antigen in the multiblock biopolymer and has binding ability.

The present invention also discloses a configuration of a tissue regeneration method for regenerating tissue using the aforementioned multiblock biopolymer.

As described above, according to the biomimetic multiblock biopolymer for tissue regeneration and the tissue regeneration method using the same according to the present invention, the present invention may provide more stable and improved expression of tissue regeneration by providing an excellent effect on wound healing. Can be.

Figure 1 schematically shows the wound shape of the experimental group applied to the ELP-RGD experimental method of the present invention.
As shown in Figure 2 is a photograph of the experimental group for measuring the relative area of each wound on the 1st day, 3rd 5th day 7th day after the skin wound induction.
Figure 3 is a graph showing the degree of wound shrinkage of the mouse according to the change in the concentration of ELP-RGD used in the experimental example of the present invention.
Figure 4 is a schematic diagram of a multiblock biopolymer according to an embodiment that can be used in the above experimental example.

Hereinafter, some embodiments of the present invention will be described in detail with reference to exemplary drawings. In adding reference numerals to the components of each drawing, it should be noted that the same reference numerals are assigned to the same components as much as possible even though they are shown in different drawings. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

In addition, in describing the component of this invention, terms, such as 1st, 2nd, A, B, (a), (b), can be used. These terms are intended to distinguish the constituent elements from other constituent elements, and the terms do not limit the nature, order or order of the constituent elements. When a component is described as being "connected", "coupled", or "connected" to another component, the component may be directly connected to or connected to the other component, It should be understood that an element may be "connected," "coupled," or "connected."

Hereinafter, an experimental example for tissue regeneration using the biomimetic multiblock biopolymer of the present invention will be described.

<Experimental Method>

First, the species and lineage of the experimental animals were selected from 12 C57BL / 6 mice with SPF male. The source of the mouse is Orient Bio. This is because the C57BL / 6 mice used in this test as the reason for the selection of the test system described above have abundant basic data as animal models of skin damage, which can be used for application of the test results. For acclimatization, the animals were purified for 1 week in the animal laboratory at Kyungpook National University. Purified and bred in the animal room at the Department of Pathology, College of Veterinary Medicine.

Breeding boxes and breeding densities were accommodated one by one in a polycarbonate breeding box for the entire test period. The breeding boxes and individuals were identified by identification card labeling by breeding list, and unidentified labeling was used for individual identification.

The feed was sterilized freely by irradiation (13.2 kGy) of experimental animal solid feed (PMI Nutritional International, Richmond, USA), and the water was freely ingested with filtered water.

In the experimental group and the configuration of the experiment, a total of 12 mice were used as shown in Table 1, and four skin wounds were formed on the dorsal region as shown in FIG. 1 per mouse. Two concentrations of ELP-RGD were treated for each mouse.

Group Surgery Treatment Number RGD 0uM Skin injury PBS n = 6 RGD 20uM RGD 20uM RGD 50uM RGD 50uM n = 6 RGD 100uM RGD 100uM

The wounds of the mice were uniformly formed using a skin punch of 4 mm in diameter, and treated with ELP-RGD by concentration immediately after hemostasis of the wound site. During the entire test period, the degree of shrinkage of the skin wound was visually observed and the area of the wound was measured.

The test substance used in this experiment is a protein having a ligand having an amino acid sequence of RGD (arginine-glycine-aspartic acid) with an ELP (elastin like polypeptide) as a template. The test substance will be described later in more detail with reference to the accompanying drawings.

<Observation item>

In order to observe the effect of the test substance on the skin wound shrinkage and regeneration, in order to observe the effect of the test substance on the process of shrinkage and regeneration of the skin wound, as shown in FIG. On the 5th day and 7th day, the photograph measured the relative area (short diameter ■ long diameter) of each wound. Statistical processing was performed on the average value of the relative area of the above-described wound and is shown in FIG.

The degree of wound shrinkage increased as the concentration of the test substance increased. This means that the test material plays an important role in wound contraction and is considered to be a tissue regeneration technology using specific protein structures different from existing polymers.

Statistical method

For statistical processing on the data derived from this study, the corresponding sample T-test was used to analyze the mean difference between two groups that were not independent of each other. This statistical analysis was performed using GraphPad InStat (version 3.05, Graph Pad Software Inc.).

3 is a graph showing the degree of wound shrinkage of the mouse according to the change in the concentration of ELP-RGD used in the experimental example of the present invention.

As shown in FIG. 3, it can be seen that the higher the concentration of ELP-RGD, the higher the shrinkage effect of the wound from day1 to day5. However, from day6, the control group without RGD showed higher wound contraction effect than a certain concentration, for example, RGD 20uM and RGD 50uM. However, it can be seen that even in day7, ELP-RGD having a concentration of 100 uM of RGD provides a higher wound contraction effect than other concentrations or excluded controls. Accordingly, it can be seen that the use of ELP-RGD biopolymer having an appropriate RGD concentration, for example, RGD 100 uM concentration, for tissue regeneration, such as wound treatment, can obtain better tissue regeneration effect.

Figure 4 is a schematic diagram of a multiblock biopolymer according to an embodiment that can be used in the above-described experimental example.

Referring to FIG. 4, the multiblock biopolymer 100 according to an embodiment is a multiblock biopolymer based on a mimic peptide of human tropoelastin existing in nature.

The core of the elastin fibers of the vascular wall or skin, lungs, ligaments is derived from a single protein called human tropoelastin. The multiblock biopolymer 100 includes cell recognition functionality and cross-linking blocks while having a repeat structure that is more similar to the human tropoelastin structure.

Multiblock biopolymer 100 according to an embodiment of the present invention includes m blocks, for example, blocks 1 to 6 (110 to 160), and blocks 1 to 6 (110 to 160) n times (n is Integer from 1 to 5,000) may be repeated. Blocks 1 to 6 (110 to 160) may have a structure arranged in a row by selecting six different peptides from peptides I to VI. In particular, the biomimetic multiblock biopolymer 100 for tissue regeneration of the present invention may have a peptide I having a cell attachment function disposed at any one of m blocks.

Peptide I is a peptide that functions as a cell binding recognition function (1) Arginine-Glycine-Aspartate (RGD), (2) Arginine-Glycine-Aspartate-Serine (RGDS), (3) Cell and spore surface antigens of disease-causing microorganisms Recognition peptides for (4) one or more of peptides that recognize cell surface antigens that are specifically expressed in normal or abnormal higher cells (eg cancer cells). In particular, the peptide I for supporting the biomimetic multiblock biopolymer 100 for tissue regeneration may be composed of peptides including RGD or RGDS.

Peptide II may have the structure of PGX. P represents L-proline, G represents glycine, and X represents 19 L- or D-amino acids except L- or D-proline.

Peptide III may have the structure of PGXX, peptide IV may have the structure of PGXXX, and peptide V may have the structure of PGXGXX. P, G, and X are identical to peptide II.

Peptide VI has the structure of an elastin interaction site PAAAAKAAKZG or PAAAAKAAAKAG. Each of blocks 1 to 6 (110 to 160) may be selected independently from each other and may be different peptides selected from peptides I to VI, but is not limited thereto. That is, block 1 (110) is a peptide of one of peptides I to VI, block 2 (120) is a peptide not selected as block 1 (110) of peptides I to VI, and block 3 (130) is a peptide I to VI. The peptide is not selected as block 1 (110) or block 2 (120) of the VI, and block 4 (140) is selected as block 1 (110), block 2 (120) or block 3 (130) of the peptides I to VI. Unblocked peptide, block 5 (150) is a peptide not selected as block 1 (110), block 2 (120), block 3 (130) or block 4 (140) of the peptides I to VI, block 6 (160) ) Is a peptide not selected as Block 1 (110), Block 2 (120), Block 3 (130), Block 4 (140) or Block 5 (150) of the peptides I to VI. However, in the multiblock biopolymer 100 of the present invention, at least one block of the blocks may be composed of peptide 1 to support tissue regeneration function.

As described above, each of blocks 1 to 6 (110 to 160) may be one independently selected from peptides I to VI. For example, each of blocks 1 to 6 (110 to 160) may be identical to each other of I to VI. For example, block 1 110 and block 3 130 may be identical to each other. In this case, one of the blocks 1 to 6 (110 to 160) may be a peptide I including the cell adhesion peptide RGD or RGDS to enhance the adhesion of the cell and the biopolymer. One of the blocks 1-6 (110-160) may be a peptite VI comprising a binding site PAAAAKAAKZG or PAAAAKAAAKAG of human tropo elastin to enhance multiblock biopolymer 100 cross-linking.

Meanwhile, the multiblock biopolymer 100 according to an embodiment has been described as including blocks 1 to 6 (110 to 160), but is not limited thereto. The multiblock biopolymer 100 according to one embodiment mimics the cell binding recognition peptide Arginine-Glycine-Aspartate (RGD) tripeptide or Arginine-Glycine-Aspartate-Serine (RGDS) tetrapeptide and elastin that are present in multicellular outer wall proteins. Three, four, five, six amino tetrapeptides, pentapeptides, nucleopeptides, and elastin cross-linking sites, including L-proline of structure, may all be included simultaneously.

One example of a multiblock biopolymer that can be applied to the above-described experimental example may be TGPG [VGRGD (VGVPG) 6] nWPC. Wherein genes of [VGRGD (VGVPG) 6] n (n = 10, 12, 15, 20) can be prepared by recursive directional ligation (RDL) from plasmids containing VGRGD (VGVPG) 6 gene. The cell adhesion affinity of [VGRGD (VGVPG) 6] 20 has properties similar to natural fibronectin, and [VGRGD (VGVPG) 6] 20 adheres to fibronectin, leaving more than 85% of fibroblast, leaving [VGRGD (VGVPG) 6] shows excellent adhesion property of 20. Cell spreading and proliferation can be facilitated by [VGRGD (VGVPG) 6] 20.

The present invention can be used as a material for tissue regeneration, multiblock biopolymers based on mimetic peptides of human tropotin elastin existing in nature, genes designating these biopolymer structures, production and purification of biopolymers, and biopolymers. .

In the above examples and experimental examples, a multiblock biopolymer is described in which six blocks in a row are repeated n (n is an integer of 1 to 5,000) and the two blocks in a row are 20 repeats. Although described, the present invention is not limited thereto. For example, a multiblock biopolymer in which m blocks in a line are repeated n n (n is an integer of 1 to 5,000) may have the same or substantially the same structural and physicochemical properties as the above Examples and Experimental Examples. Can provide.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Accordingly, the embodiments disclosed herein are intended to be illustrative rather than limiting, and the spirit and scope of the present invention are not limited by these embodiments.

The scope of protection of the present invention should be construed according to the following claims, and all the techniques within the scope of equivalents should be construed as falling within the scope of the present invention.

100: multiblock biopolymer 110: block 1
120: block 2 130: block 3
140: block 4 150: block 5
160: block 6

Claims (9)

M blocks (m is an integer from 1 to 100) connected in line are repeated n (n is an integer from 1 to 5,000),
At least one of the m blocks comprises peptide I of a cell adhesion function for tissue regeneration,
Each of the at least one block not selected as peptide I is peptide II having a structure of PGX (P is L-proline, G is ofglycine, X is L- or D-amino acid except L- or D-proline) ), Peptide III having a structure of PGXX, peptide IV having a structure of PGXXX, peptide V having a structure of PGXGXX, and peptide VI having a function of promoting mutual binding of multiblock biopolymers. Mimic multiblock biopolymers. The method of claim 1,
Peptide I, which functions to recognize cell surface antigens, is one or more of cell adhesion peptides such as Arginine-Glycine-Aspartate (RGD) or Arginine-Glycine-Aspartate-Serine (RGDS). Block Biopolymers. The method of claim 1,
Peptide VI, which functions to promote cross-linking of the multiblock biopolymer, comprises a binding site PAAAAKAAKZG or PAAAAKAAAKAG of human tropo elastin. The method of claim 1,
The at least one block is a biomimetic multiblock biopolymer for tissue regeneration, characterized in that different peptides are selected from the peptides II to VI. The method of claim 1,
The m is a biomimetic multiblock biopolymer for tissue regeneration, characterized in that two to six. The method of claim 1,
The peptide I is
RGD A biomimetic multiblock biopolymer for tissue regeneration, comprising ELP-RGD at a concentration of 100 uM. The method of claim 1,
The peptide I is
[VGRGD (VGVPG) 6] n (n = 10, 12, 15, 20) A biomimetic multiblock biopolymer for tissue regeneration. 8. The method according to any one of claims 1 to 7,
A biomimetic multiblock biopolymer for tissue regeneration, characterized in that it has a tissue regeneration function for the treatment of wounds using peptide I which recognizes a cell surface antigen and binds to the multiblock biopolymer. A tissue regeneration method for regenerating tissue using the multiblock biopolymer according to claim 8.

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