JP5855651B2 - Self-assembling peptides incorporating modifications and methods of use thereof - Google Patents

Description

BACKGROUND OF THE INVENTION The extracellular matrix (ECM) is composed of various types of macromolecules, including both proteins and polysaccharides, which form a three-dimensional environment in which cells exist in the body and between cells. Constitutes a space-filling material; The ECM can also be organized into a sheet-like layer known as the basement plate or basement membrane. ECM consists primarily of locally secreted molecules that organize into a scaffold that stabilizes and supports the physical structure of cell layers and tissues. However, rather than just an inert substrate for cell attachment, the ECM constitutes an environment rich in biological information. Various biomolecules related to ECM and ECM (e.g., secreted locally or transported elsewhere to specific sites) have a significant impact on many aspects of cell behavior and phenotype. It is accepted to control processes such as exertion, migration and proliferation, influence cell development and differentiation, and affect cell shape and function. The structure of the ECM is then affected by the cells in the ECM. These cells not only secrete many ECM constructs, but also assist in matrix patterning. Thus, it is clear that cellular ECM interactions are extremely important. There is a need for synthetic compositions and materials for tissue engineering purposes that allow the creation of a cellular environment that mimics important aspects of the natural cellular environment without the disadvantages associated with products from natural sources. It is left. For applications involving implantation in the body, the specific need for such compositions and substances that do not elicit or minimally elicit an immune or inflammatory response, and the specific needs of degradable compositions and substances in the body There remains a need. There also remains a need in the art for compositions and materials that affect cell properties and functions in desirable ways.

It has been previously reported that a class of custom- assembled peptide scaffolds have a wide range of uses such as three-dimensional (3-D) cell culture, drug delivery, regenerative medicine and tissue engineering [1a]-[5a ]. The class of self- assembling peptide materials can spontaneously assemble into well-ordered nanofibers and scaffolds of about 10 nm fiber diameter with 5-200 nm pores and a moisture content greater than 90% [6a]. These peptide skeletons have a 3-D nanofiber structure similar to natural extracellular matrix such as collagen. Furthermore, the skeleton is biodegradable by various proteases in the body that have excellent biocompatibility with tissue [7a]. In addition, these scaffolds can be modified and functionalized by direct elongation of peptides using known biologically functional peptide motifs to promote specific cellular responses. Functional RADA16, a family of these peptide skeletons, has been studied for bone, cartilage, nerve regeneration and promotion of angiogenesis [8a]-[11a].

  In the treatment of periodontal disease, induced tissue regeneration [12a]-[14a], bone graft [15a], [16a], enamel matrix derivatives [17a]-[19a] and the use of growth factors [20a]-[ Many surgical techniques have been developed to regenerate periodontal tissue such as 25a]. However, there are concerns regarding the use of these animal-derived biomaterials, such as the risk of transferring infectious agents from animals to humans and the difficulty of handling animal-derived biomaterials. Accordingly, there remains a need in the art for improved methods for periodontal tissue regeneration.

The present invention relates to novel class of self-assembling peptide (self-assembling peptides), the composition, methods for their preparation and methods of use thereof. The invention also encompasses a method for tissue regeneration, a method for increasing production of extracellular matrix protein, and a therapeutic method comprising administering a self- assembling peptide.

In one aspect, the invention relates to a self- assembling peptide comprising the sequence VEVK (SEQ ID NO: 1), wherein the peptide can self- assemble . In a further aspect, the invention encompasses a self- assembling peptide having the sequence VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3). The invention also includes a self- assembling peptide comprising the sequence VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3).

In another aspect, the invention relates to a self- assembling peptide, wherein the self- assembling peptide is:
a. a first amino acid domain that mediates self- assembly , wherein the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1), which can self- assemble in an isolated form ;and
b. Contains a second amino acid domain that does not mediate self- assembly in isolated form.

  In some embodiments, the second amino acid domain includes a biologically active motif. In certain further aspects, the first amino acid domain is VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3). In a further aspect, the biologically active motif is a laminin cell adhesion motif. In yet a further aspect, the second amino acid domain is PDGSR (SEQ ID NO: 4), YIGSR (SEQ ID NO: 5), IKVAV (SEQ ID NO: 6), LRE (SEQ ID NO: 7), RNIAEIIKDI (SEQ ID NO: 8). ), RYVVLPR (SEQ ID NO: 9), LGTIPG (SEQ ID NO: 10), PVGLIG (SEQ ID NO: 11) and GPVGLIG (SEQ ID NO: 12). In certain embodiments, the second amino acid domain comprises RGD peptides such as PRGDS (SEQ ID NO: 13), YRGDS (SEQ ID NO: 14) and PRGDSGYRGDS (SEQ ID NO: 15). In certain further embodiments, the second amino acid domain comprises a matrix metalloproteinase cleavable substrate. An exemplary matrix metalloproteinase cleavable substrate comprises the sequence PVGLIG (SEQ ID NO: 16).

In yet another aspect of the invention, the invention relates to a self- assembling peptide, wherein the self- assembling peptide is:
a. a first amino acid domain that mediates self- assembly , wherein the domain comprises alternating hydrophobic and hydrophilic amino acids that are complementary and structurally compatible; Self- assembles into a macroscopic structure when present in an unmodified form; and
b. a second amino acid domain that does not mediate self- assembly in an isolated form, wherein the second amino acid domain comprises a biologically active motif, and the second amino acid domain comprises PDGSR (sequence Number: 4), YIGSR (SEQ ID NO: 5), LRE (SEQ ID NO: 7), RNIAEIIKDI (SEQ ID NO: 8), RYVVLPR (SEQ ID NO: 9), LGTIPG (SEQ ID NO: 10) and GPVGLIG (SEQ ID NO: Comprising a sequence selected from the group consisting of 12),
including.

  In certain aspects, the first amino acid domain of the peptide comprises a plurality of RADA (SEQ ID NO: 17) peptide subunits, such as AcN-RADARADARADARADA-CONH2 (SEQ ID NO: 18). In yet another aspect, the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1), eg, VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3).

In yet another aspect, the invention regenerates dental tissue in a patient, comprising administering an effective amount of a self- assembling peptide to the dental tissue of a patient in need of dental tissue regeneration. Wherein the self- assembling peptide is:
a. a first amino acid domain that mediates self- assembly , wherein the domain comprises alternating hydrophobic and hydrophilic amino acids that are complementary and structurally compatible; Self- assemble into a macroscopic structure when present in a modified form; and
b. a second amino acid domain that does not mediate self- assembly in an isolated form, wherein the second amino acid domain comprises a biologically active motif;
including.

In some aspects, the tooth tissue is periodontal ligament tissue and the self- assembling peptide is administered to the peridontium. In a further aspect, the biologically active motif is a laminin cell adhesion motif, RGD peptide or matrix metalloprotease cleavable substrate. In a further embodiment, the second amino acid domain is PDGSR (SEQ ID NO: 4), YIGSR (SEQ ID NO: 5), IKVAV (SEQ ID NO: 6), LRE (SEQ ID NO: 7), RNIAEIIKDI (SEQ ID NO: 8). , RYVVLPR (SEQ ID NO: 9), LGTIPG (SEQ ID NO: 10), PVGLIG (SEQ ID NO: 19), GPVGLIG (SEQ ID NO: 12), PRGDS (SEQ ID NO: 13), YRGDS (SEQ ID NO: 14), PRGDSGYRGDS A sequence selected from the group consisting of (SEQ ID NO: 15) and PVGLIG (SEQ ID NO: 19). In yet another aspect, the first amino acid domain comprises a plurality of RADA (SEQ ID NO: 17) peptide subunits, such as AcN-RADARADARADARADA-CONH2 (SEQ ID NO: 18). In a further aspect, the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1), such as VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3).

The present invention also treats periodontal disease in a patient, comprising administering an effective amount of a self- assembling peptide to the periodontal ligament of a patient in need of treatment of periodontal disease and / or regeneration of periodontal ligament tissue. And / or methods of regenerating periodontal ligament tissue, wherein the self- assembling peptide comprises:
a. a first amino acid domain that mediates self- assembly , wherein the domain comprises alternating hydrophobic and hydrophilic amino acids that are complementary and structurally compatible; Self- assemble into a macroscopic structure when present in a modified form; and
b. a second amino acid domain that does not mediate self- assembly in an isolated form, wherein the second amino acid domain comprises a biologically active motif;
including.

In another aspect, the present invention is a method of increasing extracellular matrix protein production in a tissue, comprising administering an effective amount of a self- assembling peptide to said patient tissue, wherein said self- assembling Peptides are:
a. a first amino acid domain that mediates self- assembly , wherein said domain comprises alternating hydrophobic and hydrophilic amino acids that are complementary and structurally compatible, said first domain Self- assembles into a macroscopic structure when present in an unmodified form; and
b. a second amino acid domain that does not mediate self- assembly in an isolated form, wherein the second amino acid domain comprises a biologically active motif;
including.

  In a further embodiment, the extracellular matrix is collagen.

The present invention further includes a method for regenerating damaged tissue in a patient comprising the step of administering an effective amount of a self- assembling peptide to a patient in need of regeneration of the damaged tissue, wherein Self- assembling peptides are:
a first amino acid domain that mediates self- assembly , wherein the domain comprises alternating hydrophobic and hydrophilic amino acids that are complementary and structurally compatible; the first domain; Self- assembles into a macroscopic structure when present in an unmodified form; and
b. a second amino acid domain that does not mediate self- assembly in an isolated form, wherein the second amino acid domain comprises a biologically active motif;
including.

  In certain aspects, the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1). Exemplary sequences comprising VEVK (SEQ ID NO: 1) include, for example, VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3). In a further embodiment, the patient has arthritis, neurological condition, muscle wasting condition, neuroendocrine disorder, muscle degeneration, muscle tendon defect, trauma, tissue necrosis, heart disorder, surgical resection Suffering from ischemic injury due to growth abnormalities, osteoporosis, fractures or peripheral vascular disease. In a further aspect, the damaged tissue is selected from the group consisting of skeletal tissue, bone tissue, tendon tissue, connective tissue or dental tissue.

In a further aspect, the present invention provides:
a. a first amino acid domain that mediates self- assembly , wherein the domain comprises alternating hydrophobic and hydrophilic amino acids that are complementary and structurally compatible; Self- assemble into a macroscopic structure when present in a modified form; and
b. a second amino acid domain that does not mediate self- assembly in an isolated form, wherein the second amino acid domain comprises a biologically active motif ;
And a skeleton for periodontal tissue regeneration.

  In some aspects, the biologically active motif is a laminin cell adhesion motif, RGD peptide or matrix metalloprotease cleavable substrate. In a further aspect, the second amino acid domain is PDGSR (SEQ ID NO: 4), YIGSR (SEQ ID NO: 5), IKVAV (SEQ ID NO: 6), LRE (SEQ ID NO: 7), RNIAEIIKDI (SEQ ID NO: 8). , RYVVLPR (SEQ ID NO: 9), LGTIPG (SEQ ID NO: 10), PVGLIG (SEQ ID NO: 19), GPVGLIG (SEQ ID NO: 12), PRGDS (SEQ ID NO: 13), YRGDS (SEQ ID NO: 14), PRGDSGYRGDS A sequence selected from the group consisting of (SEQ ID NO: 15) and PVGLIG (SEQ ID NO: 19). In yet another aspect, the first amino acid domain comprises a plurality of RADA (SEQ ID NO: 17) peptide subunits, such as AcN-RADARADARADARADA-CONH2 (SEQ ID NO: 18). In a further aspect, the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1) peptide subunit, eg, VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3).

In a further aspect, the present invention provides:
a. a first amino acid domain that mediates self- assembly , wherein the domain comprises alternating hydrophobic and hydrophilic amino acids that are complementary and structurally compatible; Self- assemble into a macroscopic structure when present in a modified form; and
b. a second amino acid domain that does not mediate self- assembly in an isolated form, wherein the second amino acid domain comprises a biologically active motif;
A method for treating periodontal disease, comprising administering a skeleton for periodontal tissue regeneration to a patient in need of treatment for periodontal disease.

  In a further aspect, the method further comprises administering a solid biomaterial.

The present invention also includes a syringe having two compartments, wherein the first compartment comprises a peptide solution, the second compartment comprises a gelation fluid, the peptide solution comprising a self- assembling peptide Wherein the peptide is:
a. a first amino acid domain that mediates self- assembly , wherein the domain comprises alternating hydrophobic and hydrophilic amino acids that are complementary and structurally compatible; Self- assemble into a macroscopic structure when present in a modified form; and
b. a second amino acid domain that does not mediate self- assembly in an isolated form, wherein the second amino acid domain comprises a biologically active motif;
including.

The invention also encompasses a macroscopic scaffold comprising a plurality of self- assembling peptides comprising the sequence VEVK (SEQ ID NO: 1).

The invention further encompasses a macroscopic scaffold comprising a plurality of self- assembling peptides, wherein the peptides are:
a. a first amino acid domain that mediates self- assembly , wherein the domain comprises alternating hydrophobic and hydrophilic amino acids that are complementary and structurally compatible; Self- assemble into a macroscopic structure when present in a modified form; and
b. a second amino acid domain that does not mediate self- assembly in an isolated form, wherein the second amino acid domain comprises a biologically active motif;
And the scaffold further comprises living cells that bind to the surface of the scaffold or are encapsulated within the scaffold.

  In certain embodiments, the invention is a macroscopic skeleton, wherein the cells on the surface of the skeleton or cells encapsulated within the skeleton are periodontal ligament fibroblasts. In yet another embodiment, the invention is a macroscopic skeleton, wherein the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1), and optionally the cells are osteoblasts, cement buds. Cells, bone marrow cells, fibroblasts, periodontal ligament fibroblasts, mesenchymal cells, mesenchymal stem cells, adipose-derived cells, adipose-derived stem cells, periodontal ligament stem cells, pulp stem cells, stem cells derived from deciduous deciduous teeth and embryonic stem cells Selected from the group consisting of In a further embodiment, the cell is a periodontal ligament fibroblast.

The foregoing and other objects, features and advantages of the present invention will become apparent from the following more specific description of preferred embodiments of the invention, as illustrated in the accompanying drawings. In the drawings, like reference numerals refer to like parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.
FIG. 1 is a diagram showing teeth, gingiva, periodontal ligament, cementum and alveolar bone. Figure 2 is a bar graph showing the number of cells on each functionalized peptide scaffold using different mixing ratios after 2 weeks of cell culture (PRG (1: 9) is a mixture of PRG: RADA16 = 1: 9) (# Indicates t-test with p <0.01); PRG and RADA16 are described in detail in the Examples section). FIG. 3A is a photograph showing the morphology of cells on each functional peptide backbone after 2 weeks of cell culture. FIG. 3B is a photograph showing the morphology of cells on each functional peptide backbone after 2 weeks of cell culture. FIG. 4 is a confocal microscope image showing the migration of cells into the peptide backbone after 2 weeks of cell culture. FIG. 5 is a fluorescent immunostaining image of type I (green) collagen production and type III (red) collagen production on the peptide backbone after 6 weeks of cell culture. FIG. 6 shows a comparative study between periodontal ligament fibroblasts and gingival fibroblasts in a peptide backbone PDS (PDS is described in detail in the Examples section). FIG. 7 shows a comparative study between periodontal ligament fibroblasts and gingival fibroblasts in the peptide backbones vPDS_9, vYIG_9 and vIKV_9 (described in detail in the Examples section). FIG. 8 is a confocal microscope image showing that the functionalized peptide vPVG promotes cell migration. FIG. 9 is a bar graph showing the number of cells on each functionalized peptide backbone shown after 3 weeks of cell culture. (A; VEVK9, B; vPRG_9, C; vPVG_vPRG_9, D; vYIG_9, E; vPVG_YIG_9, F; vIKV_9, G; vPVG_vIKV_9, H; VEVK12, I; vPRG_12, J; vPVG_vPRG_12_L; VIKV_12, N; vPVG_vIKV_12). 10A and 10B are line graphs showing cell proliferation in the functionalized peptide backbone vPVG_vPRG_9. After 1 day, 7 days, 11 days and 21 days of culture, (A) cell number and (B) penetration depth were measured. FIG. 11A shows a molecular model of the functionalized peptide shown. FIGS. 11B and C show AFM images of VEVK12 (b) and vPVG_vPRG_9 (c).

DETAILED DESCRIPTION OF THE INVENTION A description of preferred embodiments of the invention follows.

  The word “a” or “an” is meant to include one or more unless specified otherwise. For example, the term “a peptide” encompasses one or more peptides, unless otherwise indicated.

The term “self- assembly ” is the process by which molecules or peptides form regularly shaped structures or aggregates in response to environmental conditions such as when added to an aqueous medium.

The term “self- assembling peptide” refers to a peptide comprising a self- assembling motif. Self- assembling peptides are peptides that can self- assemble into structures including but not limited to macroscopic membranes, nanostructures, and the like. Various self- assembling peptides and methods for their preparation have so far been described, for example, in U.S. Pat. Nos. 5,670,483; 5,955,343; 6,368,877; 2005/0181973 and 2007/0203062, the contents of each are incorporated herein by reference.

The term “self- assembled motif” refers to a peptide sequence or motif that can self- assemble .

The term “ionic self- assembled peptide” refers to a self- assembled peptide comprising a sequence of alternating hydrophobic and hydrophilic amino acids, wherein the hydrophilic amino acid is a charged amino acid. In some embodiments, the ionic self- assembling peptide comprises a sequence of alternating hydrophobic and hydrophilic amino acids, wherein the hydrophilic amino acids are acidic or basic amino acids. Ionic self- assembling peptides are described, for example, in US Pat. No. 5,670,483.

  As used herein, the term “amino acid” includes naturally occurring amino acids or non-naturally occurring amino acids. Non-naturally occurring amino acids are also referred to herein as “unnatural amino acids”. Naturally occurring amino acids are also referred to herein as “natural amino acids”. Natural amino acids are their well-known single letter: A for alanine, C for cysteine, D for aspartic acid, E for glutamic acid, F for phenylalanine, G for glycine, H for histidine, I for isoleucine, K for lysine, and leucine L, M for methionine, N for asparagine, P for proline, Q for glutamine, R for arginine, S for serine, T for threonine, V for valine, W for tryptophan and Y for tyrosine.

  The term “physiological pH” is a pH of about 7.

  The term “macroscopic” means having a dimension that is sufficiently large to be visible under magnification of 10 times or less. The macroscopic structure can be two-dimensional or three-dimensional. The terms “macroscopic structure” and “macroscopic material” are used interchangeably herein.

  A “biologically active peptide motif” or “biologically active motif” or “biologically active domain” is defined when a cell is contacted with a peptide containing a biologically active motif. A peptide motif that induces a phenotypic response or change in an appropriate cell type. Biologically active motifs are described, for example, in US Pat. No. 7,713,923, the contents of which are expressly incorporated herein by reference. In some aspects, the biologically active motif is a motif found in naturally occurring proteins. Biologically active peptide motifs can exist in isolated form or can exist as part of a larger polypeptide or other molecule. The ability of a peptide to elicit a response can be determined, for example, by comparing the response in the absence of the peptide (e.g., by mutating or removing the peptide when normally present in a larger polypeptide than normal). ) Can be determined. In some embodiments of the invention, phenotypic responses or changes include but are not limited to cell spreading, binding, adhesion, proliferation, secretion of extracellular matrix (ECM) molecules, or certain differentiated cell types. Promotion of the expression of phenotypic characteristics of

  As used herein, “isolated” is 1) separated from at least some of the components that are naturally associated with or normally associated with it; 2) human hands Means prepared or purified by a method comprising: and / or 3) non-naturally occurring.

  According to the present invention, a “peptide”, “polypeptide” or “protein” comprises a sequence of at least two amino acids linked together by peptide bonds. The amino acid sequences and formulas described herein are written according to convention such that the sequence is read from left to right, where the left corresponds to the N-terminus and the right corresponds to the C-terminus.

Amino acid domain that does not self-assembly condition results in self-organization of the unmodified self-assembling peptide as described below (e.g., ion concentration, peptide concentration, pH, temperature) below, as an isolated peptide An amino acid domain that does not self- assemble when present (ie, not bound or linked to a self- assembling peptide). “Do not self- assemble ” means that the amino acid domains or peptides do not form nanofilaments or nanofibers, do not form macroscopic structures, or typically either β-sheets, nanofibers or macroscopic structures. It means not forming.

  “Treat” or “treatment” means administration of a composition, compound or agent of an aspect of the invention to prevent or delay the onset of disease symptoms, complications or biochemical signs, alleviate or ameliorate the symptoms And / or preventing or inhibiting further development of the disease, condition or disorder.

  A “therapeutically effective amount”, alone or in combination with one or more other active agents, must control, reduce, inhibit, ameliorate, prevent, or otherwise treat one or more symptoms of the disease or condition being treated. This is the amount that can work.

  An “effective amount”, alone or in combination with one or more other active agents, is an amount sufficient to achieve the indicated goal. For example, in the context of tissue regeneration, an “effective amount” of a drug means that the amount of the drug is sufficient to effect tissue regeneration.

Self- Assembled Peptides As noted above, the present invention provides a first amino acid domain that can self- assemble when present in an unmodified form and a second amino acid domain that does not self- assemble in an isolated or unmodified form. Relates to methods and compositions comprising self- assembling peptides comprising Self- assembling peptides are peptides that can self- assemble into structures including, but not limited to, macroscopic membranes, nanostructures, and the like. Exemplary peptides that can be used in the first amino acid domain of such self- assembling peptides and peptides described herein are, for example, U.S. Patent Nos. 5,670,483, 5,955,343, 6,368,877, 7,098,028 and No. 7,449,180 and US Patent Application Publication Nos. 2005/0181973, 2007/0203062, and 2009/0162437 A1, the contents of each of which are expressly incorporated herein by reference.

Many self- assembled self-complementary peptides are designed by changing the amino acid sequence and following a periodic pattern. In some embodiments, the self- assembling peptide adopts a regular secondary structure, such as a β sheet structure, in solution (eg, an aqueous solution). This is due to the fact that the peptide contains two different surfaces, one hydrophilic and the other hydrophilic, and forms complementary ionic bonds with regular repeats on the hydrophilic surface. Can do. The side chain of the peptide is divided into two faces, a polar face composed of charged ionic side chains and a nonpolar face composed of hydrophobic groups. The ionic side chains are self-complementary to each other in that positively charged amino acid residues and negatively charged amino acid residues can form complementary ion pairs. Complementary ionic side chains are classified into several moduli: moduli I, II, III, IV, etc. and mixed moduli. The Modulus I peptide is a peptide in which one ionic residue is alternated with one positively charged residue and one negatively charged residue (− + − + − + − +). Modulus II peptides are peptides in which ionic residues alternate between two positively charged residues and two negatively charged residues (-++-++). The Modulus IV peptide is a peptide in which ionic residues alternate between four positively charged residues and two negatively charged residues (---- ++++).

Peptides that self- assemble in isolated form are self- assembled when present in an isolated form (in addition to the first amino acid domain that self- assembles in isolated form ). in order to distinguish non least one comprising an additional amino acid domain) "modified" self-assembling peptide or "functionalized" self-assembling peptides may be referred to herein as unmodified self-assembling peptide. Modified self- assembled peptides and hydrogels formed therefrom are also described herein as “functionalized”.

In one embodiment, the first amino acid domain that can self- assemble has alternating hydrophobic and hydrophilic amino acids. In some aspects, the first amino acid domain that can self- assemble is at least 8 amino acids in length. The optimal length of the peptide varies depending on the amino acid composition.

Peptides that can form ionized pairs between their hydrophilic side chains are referred to as “complementary” peptides. Peptides that can maintain a certain distance during pairing are said to be “structurally compatible”. Peptides that meet the above criteria are expected to self- assemble into a macroscopic membrane in a homogeneous peptide solution, and as used herein “self-complementary peptides” or “peptides having alternating hydrophobic and hydrophilic amino acids” ". Such macroscopic membranes can also be formed in a mixture of different components of the peptide when the peptides are complementary to each other and structurally compatible (each of the peptides alone does not form a membrane). ). Examples of mixtures of different peptides are (Lys-Ala-Lys-Ala) ₄ and (Glu-Ala-Glu-Ala) ₄ or (Lys-Ala-Lys-Ala) ₄ and (Ala- Asp-Ala-Asp) ₄ mixture. Although a macroscopic film is not formed in water, it is formed in a solution containing a salt. The presence of monovalent metal cations induces film formation, whereas divalent cations mainly induce unstructured aggregates. These macroscopic membranes are stable in a variety of aqueous solutions including but not limited to water, phosphate buffered saline (PBS), serum and ethanol.

In certain embodiments of the present invention, groups or radicals such as acyl groups (RCO--, where R is an organic group), e.g. acetyl groups (CH3CO--), may be present in an extra positive group. Note that it is present at the N-terminus of the peptide to neutralize charge (eg, charge that does not arise from the side chain of the N-terminal amino acid). Similarly, groups such as amine groups (NH2) can be used to neutralize extra negative charges (e.g., charges that do not arise from the side chain of the C-terminal amino acid) and accordingly the C-terminus is amide (- CONH2). Without wishing to be bound by any theory, neutralization of charge on the N-terminal and C-terminal molecules may facilitate self- assembly . The selection of other suitable groups is well within the scope of those skilled in the art.

Self-complementary peptides self- assemble into various macroscopic structures when exposed to ionic concentrations sufficient to form a macroscopic porous matrix (eg, containing monovalent cations). These matrices can take a variety of physical forms including, but not limited to, ribbons, tape-like structures, two-dimensional and three-dimensional frameworks, and the like. In one embodiment, the matrix is comprised of interwoven filaments having a pore diameter of about 10 to about 20 nm and a diameter of about 50 to about 100 nm. Detailed methods for the preparation of macroscopic structures are described in US Pat. Nos. 5,670,483, 5,955,343 and 6,368,877, the contents of each of which are expressly incorporated herein by reference. Self- assembly of self-complementary peptides involves dissolving the peptide in a solution that is substantially free of monovalent cations or containing such ions at a slightly lower concentration (e.g., less than about 10 nM), and then the ionic solute Or can be initiated by changing the pH of the solution. Organization of self-complementary peptides into macroscopic structures can also be initiated by adding the peptides to a solution containing a concentration of monovalent ions sufficient to initiate self- assembly .

In one embodiment, a self- assembling peptide that is expected to form a macroscopic structure in a homogeneous mixture has the following formula:

(Where:
Φ, Ψ and Γ represent neutral amino acids, positively charged amino acids and negatively charged amino acids that determine the composition and structure, respectively.
i, j, k and t are integers, indicating variables;
n represents the number of repeating units that also determine the length of the oligopeptide)
It is represented by one of

As described above, in some embodiments, self-assembling peptides self-assemble to form a network of nanofibers, higher than 99% when dissolved in water in the range of 1-10 mg / ml water This produces a hydrogel content. The nanofiber network results in hydrogel formation and can produce macroscopic structures of a size that can be preferably observed with the naked eye and can be three-dimensional. The peptide that forms the macroscopic structure may comprise 8-200 amino acids, 8-64 amino acids, 8-36 amino acids or 8-16 amino acids (inclusive). The concentration of the peptide prior to self- assembly can range, for example, from about 0.01% (0.1 mg / ml) to about 99.99% (999.9 mg / ml) (inclusive). Also, the concentration of the peptide prior to self- assembly can be about 0.1% (1 mg / ml) to about 10% (100 mg / ml) (inclusive), especially for cell culture and / or therapeutic applications. . In certain embodiments of the invention, the concentration of peptide prior to self- assembly is from about 0.1% (1 mg / ml) to about 5% (50 mg / ml) (inclusive), or about 0.5% (5 mg / ml). ~ About 5% (50 mg / ml) (inclusive). In certain embodiments, the concentration of the peptide prior to self- assembly is about 5 mg / ml, about 10 mg / ml, about 15 mg / ml or about 20 mg / ml.

If desired, a peptide backbone having a predetermined shape or volume can be formed. To form a scaffold with the desired geometry or dimensions, the aqueous peptide solution can be placed in a pre-shaped template, and the peptide can be added to the scaffold by adding ions as described herein. Self- organization is induced. Alternatively, ions can be added to the peptide solution shortly before placing the solution in the template, provided that the solution is carefully placed in the template before substantial organization occurs. Features of the obtained material, geometric structure and size of the time, as well as macroscopic peptide scaffold required for organizing the concentration and amount of the applied peptide solution, which is used to induce the organization of skeletal ions It is governed by parameters such as concentration, pH, specific self- assembling peptide sequence and casting equipment dimensions. If the peptide structure or backbone is implanted in the body, the shape can be selected based on the intended implantation site. The scaffold can be present according to various aspects of the invention, for example as a thin layer that covers the bottom of a conventional tissue culture or floats in solution. The layer can be, for example, several microns thick, such as 10 microns, 10-50 microns, 50-100 microns, 100-200 microns, and the like. The layer can include, for example, a plurality of β sheet layers. Self- assembled nanoscale scaffolds with different degrees of stiffness or elasticity can be formed. Typically, the peptide backbone has a low modulus in the range of 1-10 kPa, for example as measured in a standard cone-plate rheometer.

Non-limiting specific examples of self-complementary peptides that can be used in the compositions and methods described herein are listed in Table 1 below:

  VEVK9 and VEVK12 are described in more detail below. Other examples of self-complementary peptides are described, for example, in U.S. Patent Nos. 5,670,483, 5,955,343, 6,368,877, and U.S. Patent Application Publication No. 20090162437A1, the contents of each of which are expressly incorporated herein. Is done.

Furthermore, the amino acids of self- assembling peptides are natural amino acids. In another embodiment, the amino acids of the self- assembled motif include unnatural amino acids. In yet another aspect, the peptide can comprise L-amino acids, D-amino acids, natural amino acids, unnatural amino acids, or combinations thereof. Numerous classes of unnatural amino acids including D-amino acids have been described (Luo et al. (2008), PLoS ONE 3 (5): e2364. Doi: 10.1371 / journal.pone.0002364, the contents of which are referenced) Incorporated herein by reference). An exemplary unnatural amino acid is hydroxyproline. When L-amino acids are present in the scaffold, degradation produces amino acids that can be reused by, for example, cells in culture or cells in host tissue. The peptide can be synthesized chemically or purified from natural or recombinant sources, and the amino and carboxy termini of the peptide can be protected or unprotected. The peptide backbone can be formed from one or more different molecular species of peptides that are complementary and structurally compatible with each other. Peptides containing mismatched pairs, such as a repulsive pair of two similarly charged residues from adjacent peptides, can also form a structure when disruption is suppressed by stabilizing the interaction between the peptides .

Peptides containing the amino acid sequence VEVK (SEQ ID NO: 1) As mentioned above, several self- assembling peptides have been described in the literature. These self- assembling peptides experience spontaneous organization into structures such as nanofibers and macroscopic scaffolds. Exemplary peptides are complementary and structurally compatible and are composed of alternating hydrophilic and hydrophobic amino acid repeat units, and charged residues may contain alternating positive and negative charges. An exemplary self- assembling peptide is RADA-16-I (Ac-RADARADARADARADAARA-CONH2 (SEQ ID NO: 18). Functionalized scaffolds containing RADA-16-I are useful in bone, cartilage and nerve regeneration [6b-10b] Functionalized RADA-16-1 can yield relatively long peptide sequences, and under some circumstances it may be desirable to produce a shorter peptide backbone obtain.

The formation of self- assembled peptide backbones and their properties are affected by many factors, including the level of hydrophobicity. Thus, in addition to ionic complementary interactions, the degree of hydrophobic residues can affect the mechanical properties of the scaffold and the rate of self- assembly . The higher hydrophobicity of the peptide corresponds to the shorter peptide length required for self- assembly and the simpler backbone properties. Natural amino acids that are hydrophobic include alanine, valine, isoleucine, leucine, tyrosine, phenylalanine and tryptophan.

In some embodiments, the present invention relates to a self- assembling peptide comprising the amino acid sequence VEVK (SEQ ID NO: 1). In certain embodiments, a self- assembling peptide comprising the sequence VEVK (SEQ ID NO: 1) is 7-16 amino acids, 8-14 amino acids or 9-12 amino acids long. Non-limiting examples of peptides comprising VEVK (SEQ ID NO: 1) are VEVKVEVKV (SEQ ID NO: 2) and VEVKVEVKVEVK (SEQ ID NO: 3). In a further embodiment, the amino acid comprising VEVK (SEQ ID NO: 1) comprises the sequence VEVKVEVKV (SEQ ID NO: 2) or the sequence VEVKVEVKVEVK (SEQ ID NO: 3).

In a further aspect, the present invention provides:
a. a first amino acid domain that mediates self- assembly , wherein the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1), the peptide may self- assemble in an isolated form ;and
b. a second amino acid domain that does not mediate self- assembly in an isolated form, wherein the second amino acid domain comprises a biologically active motif;
Is a self- assembling peptide.

  Exemplary biologically active motifs are, for example, US Patent Application Publication Nos. 20090162437A1, 20050181973 and 20070203062, and Gelain et al. (2006). PLoS ONE 1 (1): el 19. doi: 10.1371 It is described in scientific and patent literature such as /journal.pone.0000119, the contents of which are expressly incorporated herein by reference. Exemplary biologically active motifs are also described in more detail below.

In a further aspect, the present invention encompasses a macroscopic scaffold comprising a plurality of self-assembling peptides, wherein said peptide sequence VEVK (SEQ ID NO: 1) comprises a self-assembled domain containing, self-organization It can be of. In one aspect, the present invention relates to an aqueous composition comprising a plurality of peptides containing self-assembly motifs, wherein each self-assembly motif, the amino acid sequence VEVK (SEQ ID NO: 1) comprises the peptide Can self- organize . In another embodiment, the present invention is a self- assembled nanostructure, wherein the nanostructure comprises a peptide comprising the sequence VEVK (SEQ ID NO: 1), wherein the peptide can self- assemble . In yet another embodiment, the invention is a method of preparing a self- assembled nanostructure comprising the step of forming an aqueous mixture of peptides comprising the sequence VEVK (SEQ ID NO: 1), wherein the peptide It may self-assemble under conditions suitable for self-assembly of peptides. In a further embodiment, the present invention is a macroscopic material comprising a plurality of peptides, wherein each peptide comprises the sequence VEVK (SEQ ID NO: 1), which can self- assemble . In some aspects, the material is comprised of a beta sheet. In a further aspect, the invention adds the macroscopic membrane of the invention to a cell culture medium containing cells, thereby forming a membrane / culture mixture; and b) sufficient mixture for cell growth A method for in vitro cell culture comprising the step of maintaining under mild conditions. In yet another embodiment, the invention is a macroscopic scaffold comprising a plurality of self- assembling peptides, wherein the peptides comprise a self- assembling motif comprising the amino acid sequence VEVK (SEQ ID NO: 1) the resulting self-assembled, the peptides self-assemble into β sheet macroscopic scaffold, the macroscopic scaffold live cells wrapped, the cells are present in the macroscopic scaffold in a three-dimensional arrangement. In certain embodiments, the peptide comprising the sequence VEVK (SEQ ID NO: 1) is VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3). In certain further aspects, the peptide comprising the sequence VEVK (SEQ ID NO: 1) is:
a. a first amino acid domain that mediates self- assembly , wherein the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1), the peptide may self- assemble in an isolated form ;and
b. a second amino acid domain that does not mediate self- assembly in an isolated form, wherein the second amino acid domain comprises a biologically active motif;
including.

Without a biologically active motif modified peptide dismisses the ability to form a macroscopic structure and self-organization, by incorporating additional domains that do not self-organizing, self-organization of such the above mentioned peptides It has been previously described that it is possible to modify the resulting peptides (eg, US Patent Application Publication No. 2009/0162437). Thus, in one embodiment, the self- assembling peptide comprises (a) a first amino acid domain that mediates self- assembly , wherein the domain is complementary and structurally compatible with a hydrophobic amino acid. Alternating hydrophilic amino acids, the domain self- assembles into a macroscopic structure when present in an unmodified form; and (b) a second amino acid domain that does not self- assemble in an isolated form. Including. Such self- assembling peptides are described, for example, in US Patent Application Publication Nos. 2005/0181973 and 2009 / 0162437A1, the contents of which are hereby incorporated by reference.

  In some embodiments of the invention, the second amino acid domain is a biologically active peptide motif. Biologically active motifs that can be used in accordance with the present invention include US Patent Application Publication No. 2005/0181973 and Gelain et al. (2006). PLoS ONE 1 (1): el 19. doi: 10.1371 / journal. pone.0000119, the contents of which are incorporated herein by reference. Biologically active motifs include, for example, short peptide sequences derived from proteins in the cell basement membrane that have been identified as being involved in several biological functions such as cell binding, proliferation, differentiation and migration. (Iwamoto et al. (1987). Science 238: 1132-34; Kleinman et al. (1989), PNAS 87: 2279-83; Koliakos et al. ((1989), J. Biol. Chem. 264, 2313) -2323) Various biologically active motifs have been identified in, for example, laminin, collagen IV, nidogen, proteoglycan and vascular endothelial cells, for example, the biologically active motif is PRGDSGYRGDS (SEQ ID NO: RGD peptides such as repetitive RGD sequences such as: 15) Another example of a biologically active motif is the sequence AASIKVAVSADR from the laminin alpha chain that promotes neurite outgrowth activity by PC12 cells Number: 49), human umbilical vein endothelial cells (HIVEC) Peptide CSRARKQAASIKVAVSADR (SEQ ID NO: 50) that induces degradation of the Matrigel matrix, and also sequences YIGSR (SEQ ID NO: 5), PDSGR (SEQ ID NO: 4) and RYVVLPR (SEQ ID NO: 4) located on the β1 chain of laminin SEQ ID NO: 9) promoted cell adhesion.The sequence KAFDITYVRLKF (SEQ ID NO: 51) from laminin γ1 chain also promoted HUVEC adhesion and tube formation, as well as neuronal cell adhesion and neurite outgrowth. The peptide sequence TAGSCLRKFS ™ (SEQ ID NO: 52) was found to bind specifically to heparin, eg, the RGD sequence found in nidogen acts as a cell binding site.

Non-limiting examples of biologically active motifs that can be used in accordance with the present invention are shown in Table 2 below:

  In some embodiments, the biologically active motif is an amino acid motif that is involved in tissue regeneration. In yet another embodiment, the biologically active motif is an amino acid motif involved in periodontal tissue regeneration. Non-limiting examples of biologically active motifs involved in periodontal tissue regeneration include, but are not limited to, laminin cell adhesion motifs, RGD peptides and matrix metalloproteinase degradable motifs.

  Laminin is a structural component that supports the cell and is a major component of the basement membrane that provides the cell with a beneficial microenvironment that regulates cell function. Laminin has been reported to have specific cell adhesion properties, to which periodontal ligament fibroblasts and / or osteoblasts can adhere (Palaiologou et al. (2001), J. Periodontal. 72: 798-807; Giannopoulou et al. (1996), J Dent Res 75: 895-902; Grzesik et al. (1998), J. Dent. Res. 77: 1606-1612). Non-limiting examples of laminin cell adhesion motifs include IKVAV (SEQ ID NO: 6), LGTIPG (SEQ ID NO: 10), RYVVLPR (SEQ ID NO: 9), PDSGR (SEQ ID NO: 4), YIGSR (SEQ ID NO: 5). ), LRE (SEQ ID NO: 7) and RNIAEIIKDI (SEQ ID NO: 8).

  The RGD peptide is a peptide containing an RGD (Arg-Gly-Asp) amino acid sequence. RGD is an important binding or recognition sequence for integrins, which are cell surface receptors that mediate cell-extracellular matrix (ECM) adhesion. RGD peptides are reviewed in, for example, Ruoslahti et al. (1996), Annual Review of Cell and Development Biology, 12: 697-715 and D'Souza et al., Trends in Biochemical Sciences 16: 246-50, respectively. Is expressly incorporated herein by reference. PRGDS (SEQ ID NO: 13) and YRGDS (SEQ ID NO: 14) are the most commonly occurring RGD motifs in natural proteins. Further exemplary RGD sequences are PRGDSGYRGD (SEQ ID NO: 15), GRGDSP (SEQ ID NO: 83). Repeating the RGD binding sequence increases the likelihood of binding to the cell. Binding is further affected by structural conformation because it increases the likelihood of effective conformation (Kantlehner et al. (2000)). A repetitive RGD sequence is a peptide sequence comprising at least two RGD sequences, eg PRGDSGYRGD (SEQ ID NO: 15) comprises two RGD sequences.

Matrix metalloproteinases (MMPs) belong to a family of proteases that degrade extracellular matrix components and play an important role in tissue regeneration. These proteins create a way for cells to expand, allow remodeling of the extracellular matrix, and release growth factors and other signals embedded from the extracellular matrix to stimulate cell differentiation and tissue growth. Incorporating MMP cleavable substrates into self- assembled peptide scaffolds is an attractive strategy for reshaping dynamic mechanisms for inducing cellular and tissue remodeling activity (Turk et al. (2001), Nat. Biotechnol. 19: 661-7). An exemplary MMP degradable motif is PVLIG (SEQ ID NO: 11).

In certain embodiments of the invention, the addition of non-self- assembled amino acid domains does not prevent the modified peptide from self- assembling , eg, forming nanofibers, macroscopic structures, or both. In certain further aspects, the modified peptide self- assembles to form a macroscopic structure composed of nanofibers. An unmodified self- assembling peptide can be modified in any of several ways described above that do not involve the addition of amino acids to the peptide, these modifications being referred to herein as modified and / or derivatized. It is understood that Modified self- assembling peptides of the present invention are different from naturally occurring molecules, for example, the peptides are not found in naturally occurring molecules, but one or more of the amino acid domains of the peptides of the present invention are May be present in naturally occurring molecules. As such, the peptide is considered “isolated” or “synthetic”, meaning that the entire sequence of the peptide does not occur naturally without human intervention.

A biologically active motif can be located proximal to the N-terminus or C-terminus of the first domain containing a peptide that self- assembles in isolated form (e.g., linked via a linking group) It is further understood. Biologically active motifs that can be the same or different can further be located at both the N-terminus and C-terminus of the first domain.

In certain embodiments of the present invention, conditions that self-assembly of the modified self-assembling peptide occurs are the same as the conditions corresponding unmodified self-assembling peptides to organize. In another aspect of the present invention, conditions that self-assembly of the modified self-assembling peptide occurs, corresponding unmodified self-assembling peptides differ from the conditions to organize. In this case, conditions for self-assembly of the modified self-assembling peptides are different (not compatible) unmodified self-assembling peptide is the same as the conditions for self-organization.

In one aspect, the second amino acid domain, the peptide to allow the organization of the first amino acid domain as to organize to form the nanofibers and / or macroscopic structure. In a preferred embodiment of the invention, the peptide forms a β sheet. In some embodiments of the invention, the amino acid domain has at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, or more such as 15 or 16 amino acids, 20 amino acids, etc. However, in general, it is desirable to limit the length of the second amino acid domain so that it does not interfere too much with self- assembly . For example, it may be preferable to limit the length of the second amino acid domain to 20 amino acids or less, 16 amino acids or less, 12 amino acids or less, 10 amino acids or less, 8 amino acids or less. It may be desirable to maintain a certain percentage of amino acids in the self-assembly portions and non-self-assembly moiety in the peptide. For example, in certain embodiments of the invention, it may be desirable for non-self- assembling domains to constitute 50% or less of the total number of amino acids in the peptide.

Under some circumstances, including a non-self- assembled domain between self- assembled parts rather than N-terminal or C-terminal is free from the structure in which the non-self- assembled parts are self- assembled in such cases The possibility of destroying self- organization may be higher. Furthermore, when included between two self- assembling moieties, the motif may have less chance of free interaction with molecules such as proteins present in the cell and / or culture environment. Thus, for various reasons, it may be preferable to add a non-self- assembled moiety to the N-terminus or C-terminus rather than inserting between two self- assembled moieties.

  In addition to amino acid domains whose sequences are derived from naturally occurring proteins such as those described above, growth factors, cytokines, chemokines, peptide hormones, peptide neurotransmitters, other biologically active peptides found in the body, etc. Amino acid domains derived from can also be used (see, for example, Goodman and Gilman, The Pharmacological Basis of Therapeutics, 10th edition, McGraw Hill, 2001 and Kandel et al, Principles of Neural Science, 4th edition, McGraw Hill, 2000) .

  Other biologically active motifs can be identified according to several criteria, including but not limited to those described by Yamada (1991), J. Biol. Chem, 266: 12809-12812. Yamada describes tests for biological relevance for the case of adhesion recognition sequences, but the criteria described can be more widely applied. A putative active peptide motif can also be active when conjugated to a substrate, but a synthetic peptide containing a sequence can be a carrier (e.g., IgG, even if it is inactive when adsorbed directly to a substrate such as glass or plastic) , Albumin, beads) can be identified as biologically active. Soluble forms of biologically active peptide motifs can competitively inhibit the function of intact proteins where the motifs are found in nature. Altering the peptide sequence can eliminate the function of the peptide. A biologically active peptide can bind to the same cellular receptor or naturally occurring biomolecule as the naturally occurring protein containing the peptide. A range of different peptide concentrations can be tested and various combinations can be used.

Two or more amino acid domains can be linked using methods known to those skilled in the art. Non-limiting examples include the use of linkers or bridges that can be one or more amino acids or different molecular entities. A linker domain consisting of one or more glycine (G) residues, eg 1, 2, 3, 4, 5 etc. glycine may be used. The use of glycine in the linker domain is advantageous because this amino acid is small and has a non-polar side chain, thus minimizing the possibility of substantial interaction with self- assembly . Another exemplary amino acid is alanine, or other amino acids having non-polar side chains may be used.

The modified peptides described in the examples are those made by solid phase synthesis of elongated peptides that yield a linear chain, but variants in which the modified motif is conjugated or cross-linked to the side chain are also contemplated by the present invention. Is included. Methods for achieving such conjugates or crosslinks are well known in the art. For example, a peptide containing a cysteine residue (or any amino acid modified to contain a sulfur atom) can be linked to a second peptide containing a sulfur atom by formation of a disulfide bond. Thus, in general, a modified self- assembling peptide can be a single linear polymer of amino acids linked by peptide bonds (preferably a possible structure) or two polymers of amino acids, each of which A polymer of amino acids linked by peptide bonds) may have a branched structure that is either covalently or non-covalently linked to each other (eg, via biotin-avidin interaction). Non-limiting examples of cross-linking methods include, but are not limited to, glutaraldehyde methods that are linked primarily through V and W amino groups, maleimide-sulfhydryl coupling chemistry (e.g., maleimidobenzoyl-N-hydroxysuccinimide ester). (MBS method), and periodate oxidation method. Many cross-linking agents are also known. Exemplary cross-linkers include, for example, carboiimide, N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), dimethylpimelimidate dihydrochloride (DMP), dimethyl suberimidate (DMS). ), 3,3′-dithiobispropionimidate (DTBP) and the like. For more information on conjugation methods and crosslinkers, see generally the journal, Bioconjugate Chemistry, published by the American Chemical Society, Columbus Ohio, PO Box 3337, Columbus, Ohio, 43210. In addition, the “Cross-Linking”, Pierce Chemical Technical Library, which was available at the website www.piercenet.com, and the Pierce Catalog first published in 1994-95 and references cited in this document and Wong See SS, Chemistry of Protein Conjugation and Crosslinking, CRC Press Publishers, Boca Raton, 1991. Bifunctional cross-linking reagents contain two reactive groups, thereby providing a means for covalently linking two target groups. The reactive groups in chemical cross-linking reagents typically belong to a class of functional groups including succinimidyl esters, maleimides and iodoacetamides. Some common schemes for forming heteroconjugates are typically the conversion of an amino group on one biomolecule to a thiol group on a second biomolecule by a two-step or three-step reaction sequence. Includes indirect coupling. The high reactivity of thiols in most biomolecules and their relatively rare rarity make thiol groups a good target for controlled chemical crosslinking. If neither molecule contains a thiol group, one or more can be introduced using one of several thiolation methods. The thiol-containing biomolecule can then be reacted with the amine-containing biomolecule using a heterobifunctional cross-linking reagent, such as a reagent comprising both succinimidyl ester and either maleimide or iodoacetamide. Amine-carboxylic acid bridges and thiol-carboxylic acid bridges can also be used. For example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC) reacts with biomolecules, usually in the molecule or between the subunits of the protein complex. -length) "crosslinks. In this chemistry, no cross-linking reagent is incorporated into the final product.

  Several methods are available for introducing thiols into biomolecules, including reduction of endogenous disulfides and conversion of amine, aldehyde or carboxylic acid groups to thiol groups. Disulfide bridges of cysteine in proteins can be reduced to cysteine residues with dithiothreitol (DTT), tris- (2-carboxyethyl) phosphine (TCEP), or tris- (2-cyanoethyl) phosphine. The amine can be indirectly thiolated by reaction with succinimidyl 3- (2-pyridyldithio) propionate (SPDP) followed by reduction of the 3- (2-pyridyldithio) propynyl conjugate with DTT or TCEP. The amine can be indirectly thiolated by reaction with succinimidyl acetylthioacetate followed by removal of the acetyl group using 50 mM hydroxylamine or hydrazine near neutral pH. Tryptophan residues in thiol-free proteins can be oxidized to mercaptotryptophan residues and then modified with iodoacetamide or maleimide.

As methods described above for the use of self-organization and self-assembling peptides (including both modified and unmodified self-assembling peptides) self-assembling peptides, for example of monovalent cations in aqueous peptide solution Upon addition or upon introduction of the peptide solution into a solution containing a monovalent cation, it self- assembles under various conditions to form a macroscopic structure. Prior to self- assembly, the peptide is dissolved in a solution that is substantially free of monovalent ions (e.g., cations) or contains only such ions at low concentrations, e.g., less than 10, 5, 1, 0.5, or 0.1 mM. obtain. Self- assembly can be initiated or substantially accelerated by the addition of ionic solutes to the peptide solution or a change in pH. For example, NaCl at a concentration of 5 mM to 5 M induces peptide organization and forms a macroscopic structure within minutes. Lower concentrations of NaCl can also induce organization, but at a slower rate. Some peptides can also self- assemble in processes that can depend on pH in the absence of significant concentrations of ions. For example, some peptides are maintained in solution at a pH of about 3.0, but can self- assemble as the pH increases. Alternatively, the peptide is introduced into a solution containing ions, such as standard phosphate buffered saline (PBS), tissue culture medium, or a physiological fluid such as blood, cerebrospinal fluid (CSF), etc. By doing so, self- organization can be started. Thus, peptides can self- assemble in vivo locations. Preferred ions include monovalent cations such as Li +, Na +, K + and Cs +. Preferably, the concentration of ions is at least 5, 10, 20 or 50 mM to induce or substantially accelerate self- assembly . One skilled in the art can select a preferred ion concentration based on the particular peptide sequence and / or concentration and the desired rate of organization . In general, the strength of the resulting structure increases in the presence of ions compared to the strength in the absence of ions or at lower ion concentrations (but the increase in ion concentration does not result in an increase in strength. Note that a plateau may be reached).

In one aspect, the invention is a nanostructure comprising a plurality of self- assembling peptides of the invention. Exemplary nanostructures include nanofilaments, nanofibers, and nanoskeletons. In another aspect, the present invention is a macroscopic structure comprising a plurality of self- assembling peptides described herein. In one embodiment, the macroscopic structure comprises a uniform self- assembling peptide. In another embodiment, the macroscopic structure comprises a self- assembling peptide consisting of heterogeneous components. The term “homogeneous self- assembling peptide” refers to a plurality of identical or identical self- assembling peptides. The term “self- assembling peptide consisting of different components ” refers to a plurality of different self- assembling peptides. It will be appreciated that self- assembling peptides may differ when two or more peptides are different in a mixture. In some embodiments, the macroscopic structure is a macroscopic membrane. The macroscopic membrane includes, for example, a plurality of woven nanofilaments containing the self- assembling peptides of the present invention. The initial peptide concentration is a factor in the size and thickness of the film formed. In general, the higher the peptide concentration, the higher the degree of film formation. In a further embodiment, the macroscopic membrane takes the form of a β sheet. In one aspect, the present invention is a macroscopic scaffold comprising a plurality of self-assembling peptides described herein, wherein the self-assembling peptides self-assemble into β sheet macroscopic scaffold .

The formation of self- organized structures preferentially stains the β-sheet structure and, after staining with Congo Red, a dye commonly used to visualize abnormal protein deposits in the tissue, Can be observed. Further structural details can be observed under magnification. Scanning electron microscopy (SEM) makes it possible to observe the structural details of the nanostructures. The β-sheet secondary structure of the membrane can be further confirmed by circular dichroism (CD) spectroscopy (Zhang et al (1993), PNAS 90: 3334-8; Zhang et al (1995). Biomaterials 16: 1385 Zhang & Rich 1997, PNAS 94, 23-28, Yokoi et al, 2005. PNAS 102, 8414).

In one aspect, the present invention provides the self- assembled peptide of the present invention and the self- assembled structure as a cell culture support, as a self- assembled monolayer imprinted on a solid support. It includes methods used for repair and replacement of various tissues, as a framework for encapsulating living cells, as part of a controlled delivery drug delivery system, and for promoting hemostasis. In certain aspects, the self- assembling peptide comprises the sequence VEVK (SEQ ID NO: 1). Other uses of self- assembling peptides include, for example, US Patent Application Publication Nos. 2002/0072074; 2002/0160471; 2004/0087013; 2004/0242469; 2005/0287186 and 2007/0203062. Each of which is incorporated herein by reference.

In addition, the macroscopic structures, macroscopic membranes and nanostructures formed by the self- assembly of the peptides described herein are stable in serum, proteolytic digestion and at alkaline and acidic pH. Being resistant and non-cytotoxic, the substances, membranes and filaments support pharmaceutical products (e.g. sutures), artificial skin or internal lining, delayed diffusion drug delivery systems for in vitro cell growth or culture Useful for biomaterial applications such as supports for artificial tissues for body and in vivo use. The structure can be further used in a number of applications where permeable and water insoluble materials such as separation matrices (eg dialysis membranes, chromatography columns) are appropriate. Because of its permeability, the macroscopic membrane described herein is useful as a delayed diffusion drug delivery vehicle. Since the membranes are resistant to degradation by proteases and gastric acid (pH 1.5), drug delivery vehicles made with these membranes can be taken orally. The small pore size membrane also makes it useful as a filter, for example, to remove viruses and other minute contaminants. The pore size (interfilament distance) and filament diameter in the membrane can be varied by changing the length and sequence of the peptides used to form the membrane.

  The macroscopic membrane can also be modified to impart additional properties. For example, the membrane can be further strengthened by crosslinking the peptide after formation of the membrane by standard methods. Collagen and the peptide can be combined to make a membrane that is more suitable for use as an artificial skin, where the collagen can be stabilized against proteolytic digestion in the membrane. In addition, vesicles can be made by combining the phospholipid and the peptide.

Macroscopic structures can also be useful for culturing cells. Addition of growth factors such as fibroblast growth factor to the macroscopic structure of the peptide can further improve binding, cell proliferation and neurite outgrowth. Porous macrostructures can also be useful for encapsulating cells. The pore size of the membrane can be large enough to allow diffusion of cell products and nutrient molecules. Generally, the cells are much larger than the pores and are contained. In another aspect, the present invention is a macroscopic scaffold comprising a plurality of self-assembling peptides of the present invention, wherein the self-assembling peptides self-assemble into β sheet macroscopic scaffold,該巨view A typical skeleton envelops live cells, which are present in the macroscopic skeleton in a three-dimensional arrangement. Macroscopic scaffolds can be prepared by incubating self- assembling peptides and living cells in an aqueous solution under conditions suitable for self- assembly . The invention also includes a method of tissue regeneration, wherein the method comprises administering to a mammal a macroscopic scaffold comprising a self- assembling peptide of the invention, wherein the peptide is β-sheet macroscopic Self- organized into a skeleton, the macroscopic skeleton envelops living cells, and the cells are present in the macroscopic skeleton in a three-dimensional arrangement. The wrapped cells are present in a macroscopic skeleton in a three-dimensional arrangement. In some embodiments, the methods are used for the treatment or prevention of cartilage defects, connective tissue defects, neural tissue defects, epithelial lining defects, endothelium lining defects or arthritis. The macroscopic skeleton can be administered orally, transdermally, intramuscularly, intravenously, subcutaneously or in any other suitable manner. In another aspect, the present invention is the addition of macroscopic film formed by self-assembly in aqueous solution of the self-assembling peptides of (a) the present invention, the cell culture medium containing the cells, thereby A method for cell culture in vitro comprising the steps of forming a membrane / culture mixture; and (b) maintaining the mixture under conditions sufficient for cell growth.

Macroscopic membranes investigate the nature of biological protein structures with unusual properties such as resistance to extreme insolubility and proteolytic digestion, including but not limited to aggregates of β-amyloid protein and aggregated scrapie protein It can also be used as a model system for The present invention also includes a method for nerve regeneration using a structure containing a self- assembling peptide. For example, nerve regeneration can be facilitated and induced by implanting self- assembled nanostructures along the exact pathway to their target.

Peptide hydrogel as described herein (e.g., SEQ VEVK (SEQ ID NO: 1) modified self-assembling peptides and / or unmodified self-assembling peptide comprising the) may be used in the culture of cells and tissues. Such a method is described in detail in US Patent Application Publication No. 2009 / 0162437A1. In one aspect, cells and tissues can be cultured on the surface of the hydrogel structure. In another embodiment, the cells can also be encapsulated in a hydrogel. To encapsulate the cells in the peptide structure, the peptide and living cells are placed in an aqueous solution having an isotonic solute at an appropriate concentration to support cell viability, under conditions that do not substantially self- assemble the peptide. Can be incubated. In certain embodiments of the invention, the solution contains monovalent cations at a concentration of less than 10, 5, 1 or 0.1 mM, or is substantially free of monovalent cations. The solution may also contain other ionic species less than 10, 5, 1 or 0.1 mM, such as other cations or anions, or may be substantially free of the other ionic species. Sufficient ions (eg, monovalent cations) are added to the solution to initiate self- assembly of the peptide into the macroscopic structure, thereby enclosing the cells by formation of the macroscopic structure. The wrapped cells are present in the macroscopic structure in a three-dimensional arrangement. The solution can be contained in a pre-shaped mold that is sized to establish the desired volume or shape of the macroscopic structure. Self- assembly can also be affected by changes in pH (eg, increasing from a low pH to a high pH).

Agents such as cells and bioactive molecules (eg differentiation inducers, proliferators), therapeutic compounds can also be introduced into the peptide solution prior to self- assembly . The self- assembly process then forms a structure that encloses the cell or molecule. In order to achieve a uniform distribution of cells or molecules in the structure, it may be desirable to thoroughly mix the solution before the onset of self- assembly . To avoid initiating or accelerating self- assembly, the cells or drug and peptide solution are combined and immediately maintained in a solution that is substantially free of ions or only contains a low concentration of ions. It may be desirable. In this case, preferably, the cells, prior to combining with the peptide solution, is maintained in isotonic solutes such as sucrose. The peptides themselves can be dissolved in cells (eg, cell pellets) or isotonic solutions to which drugs are added. The resulting composition can be mixed to achieve a more uniform cell and / or drug distribution, and then the composition is exposed to ions (e.g., ions are added to the composition, or The composition is mixed with a solution containing ions).

  Cells are cultured on tissue culture dishes or glass slides, or on conventional substrates such as tissue culture dishes or glass slides coated with biological materials such as collagen, Matrigel, etc. Can be cultured on the surface of the peptide hydrogel structure. In general, the cells can be cultured at any desired degree of confluence. When encased, the cells are preferably present in the macroscopic structure in a three-dimensional arrangement. The culture conditions should preferably be close to physiological conditions. For example, the pH of the culture medium should preferably be close to physiological pH, preferably pH 6-8, such as about pH 7-7.8, especially pH 7.4. Physiological temperatures range from about 30 ° C to 40 ° C.

  Cells are cultured on or within the peptide structure for any suitable time, depending on the desired cell number and density, the growth rate of the cells, and the time required for the desired cell reprogramming to occur. obtain. These parameters will vary depending on the particular cell and the purpose for which the invention is used. One skilled in the art can vary these parameters and observe the effect of doing so to determine the optimal time to maintain cells on or in culture in the structure. In certain embodiments of the invention, the cells are cultured for about 3 days, 7 days, 14 days, 21 days, 28 days, 56 days or 90 days.

  In some aspects, at least 40, 50, 60, 70, 80, 90, or 95% of the cells (either cultured or encapsulated on the surface), after formation of the macroscopic skeleton, Viable for 1, 2, 4, 6 or more weeks. In another embodiment, at least 50%, at least 60%, at least 70%, at least 80% or 90% of the cells are viable for 1 day or 1 week after formation of the macroscopic skeleton.

  Generally, vascular endothelial cells and their precursor cells, bone marrow cells, periosteum cells, perichondrial cells, fibroblasts, skeletal myoblasts or skeletal muscle cells, neurons, hippocampal cells, epithelial cells, non-vascular endothelial cells or smooth Muscle cells, keratinocytes, basal cells, spinous cells, granule cells, embryonic stem cells, lung cells, immune system cells, ovarian cells, pancreatic cells, cervical cells, liver cells, foreskin cells or periodontal ligament fibroblasts Any cell type, including but not limited to cells, can be cultured and / or enveloped according to the present invention. The cells can include embryonic, fetal, or adult stem cells, eg, stem cells that can be induced to differentiate into any of the aforementioned cell types. In certain aspects, the cell is a periodontal ligament fibroblast.

The self- assembling peptides described above can also be used to increase wound healing, tissue regeneration, periodontal tissue regeneration and / or matrix metalloprotease production in the target tissue. Peptide hydrogel structures optionally comprising cells that grow on or encapsulated within the surface of the peptide hydrogel structure can be implanted into the body using any suitable method. Non-limiting methods include surgical procedures and / or injection procedures. Any route of administration may be utilized including but not limited to oral, transdermal, intramuscular, intravenous, subcutaneous or parenteral routes. One skilled in the art can readily select an appropriate delivery technique.

Several different types of devices can be used to administer self- assembling peptides at the site of injury or at the site of tissue damage or regeneration (in other words, sites that require tissue regeneration). It will be appreciated that delivery can be accomplished using a syringe or catheter. In some aspects of the invention, the self- assembling peptide is administered using a syringe. In some embodiments, peptides in solution are not organized or minimally organized (eg, where the solution does not form a gel) and are administered to the patient, with organization occurring after administration. In a further aspect, the peptide is self- assembled in vitro and introduced into the body as an organized matrix. Such a method is described in detail in US Patent Application Publication No. 2009/0162437. In certain further embodiments, the invention relates to a syringe having two compartments, wherein the first compartment comprises a solution comprising a self- assembling peptide and the second compartment comprises a gelling solution. A gelling liquid is a liquid that, when combined with a peptide solution, results in the formation of a hydrogel. Exemplary gelling liquids include, for example, liquids containing monovalent cations described in detail above.

In certain aspects, the present invention is a method of regenerating damaged tissue in a patient in need of regeneration of the damaged tissue, comprising administering a self- assembling peptide described herein. As will be appreciated by those skilled in the art, tissue can be damaged in several conditions, so that tissue regeneration is therapeutically useful. Such conditions include, but are not limited to, arthritis, various neurological conditions, neuroendocrine disorders, muscle degeneration, muculotendenous failure, age-related degeneration, trauma, necrosis, cardiac disorders and surgical resection. It is done. The damaged tissue can be, for example, skeletal tissue, bone, tendon, connective tissue or dental tissue.

In some embodiments, the invention is a method of treating periodontal disease and / or regenerating tooth tissue. In certain embodiments, the tooth tissue is periodontal ligament tissue. Exemplary periodontal diseases are periodontitis, gingivitis, periimplantitis and peri-implant mucositis. In periodontitis, the gingiva recedes from the teeth and forms a pocket that is infected. The immune system fighting bacterial toxins and infections actually begins to injure the bones and connective tissues that hold the teeth in place. Peri-implantitis is a complication after surgical implantation of alloplastic material into the jaw bone, affecting the tissue surrounding the functional implant that is osseointegrated. Cause loss of support. In certain embodiments, a therapeutically effective amount of a self- assembling peptide is administered to the periodontal ligament. The periodontal ligament (peridontium) and the tissue that forms the periodontal ligament are illustrated in FIG. The periodontal ligament consists of four tissues: gingiva, periodontal ligament, cementum and alveolar bone. The gingiva is a pink keratinized mucosa that covers part of the tooth and part of the alveolar bone. Periodontal ligaments are a group of connective tissue fibers that connect teeth to alveolar bone. Cementum is a calcified structure that covers the lower part of the tooth. The alveolar bone is a set of protrusions from the jaw bones (maxilla and mandible) in which the teeth are embedded. The area where periodontal disease develops is the gingival crevice, which is a pocket between the tooth and the gingiva.

In a further aspect, the present invention is a skeleton for periodontal tissue regeneration comprising the self- assembling peptides described herein. As used herein in the context of tissue regeneration and / or periodontal tissue regeneration, the scaffold is a degradable hydrogel. In certain further aspects, the self- assembling peptide comprises a biologically active motif selected from the group consisting of a laminin cell adhesion motif, an RGD peptide, and a matrix metalloprotease cleavable substrate. In further embodiments, the self- assembling peptide comprises a laminin cell adhesion motif. The use of skeletons in periodontal tissue engineering is described, for example, by Ma et al. (2006) Scaffolding in tissue engineering. CRC Press Pp 438; Akman et al. (2010), J Biomed Mater Res 92 (3): 953-62; Hollister (2005), Nature Mat. 4: 518-524, the contents of which are expressly incorporated herein by reference. The skeleton, for example, to maintain tissue volume as a vehicle to deliver therapeutic and / or biologically active substances to the wound and to promote selective colonization and growth used. The goal of treating periodontal disease is the generation of periodontal ligaments. Traditional induced tissue regeneration for the treatment of periodontal disease mechanically blocks gingival tissue invasion (which is shown to be related to root resorption) and periodontal ligament fibroblasts [28a] which enables the combination of In some aspects of the invention, the skeleton for periodontal tissue regeneration biologically blocks gingival cell invasion and promotes periodontal ligament growth. For example, if the skeleton contains a laminin cell adhesion motif, periodontal ligament fibroblasts can adhere to the motif, but gingival cells are less capable of binding to the motif than periodontal ligament fibroblasts [ 27a-29a]. In one aspect, the present invention is a skeleton for tissue regeneration further comprising cells, wherein the cells are periodontal ligament fibroblasts. In yet another aspect, the scaffold includes a self- assembling peptide that includes a biologically active motif to which periodontal ligament fibroblasts can adhere.

  In a further aspect, the invention is a method of treating periodontal disease comprising administering a skeleton as described herein. The present invention further includes a method of treating periodontal disease comprising the steps of administering a scaffold of the present invention and further administering a solid biomaterial. In certain aspects, the solid biomaterial is osteoconductive. As will be appreciated by those skilled in the art, the solid biomaterial can be made of any suitable material, including but not limited to calcium and / or phosphorus. Exemplary materials include calcium triphosphate and hydroxylapatite. A further example of a biomaterial is made with beta tricalcium phosphate. Such a biomaterial is, for example, a GEM 21S® growth factor promoting matrix. The biomaterial can take, for example, powder form, mass form and / or granules. In a further aspect, the solid biomaterial includes pores having a diameter of about 100 to about 500 microns. The method of treating periodontal disease comprises administering a scaffold and solid biomaterial as described herein, and further comprising administering additional bioactive agents such as drugs (e.g., small molecules and peptides). Including. In some embodiments, the agent comprises a recombinant human protein and / or growth factor. In a further embodiment, the growth factor is a human recombinant growth factor protein. Exemplary recombinant human proteins include recombinant human platelet derived growth factor (rh-PDGF), recombinant human bone morphogenetic protein-7 (rh-BMP-7) and recombinant human basal fibroblast growth factor (rh- bFGF).

In a further aspect, the present invention relates to a method for increasing extracellular matrix protein production in a tissue comprising administering a self- assembling peptide described herein. A preferred extracellular matrix protein is collagen.

  The following examples further illustrate the present invention but should not be construed as limiting the scope of the invention in any way.

Exemplification Recently, several peptide molecules containing RGD and laminin cell adhesion motifs have been reported to promote the activity of periodontal ligament fibroblasts [27a]-[29a]. RGD (Arg-Gly-Asp) is well known to be a central binding sequence for cell binding, particularly working with integrins. Laminin is a major component of the basement membrane. The basement membrane is not only important as a structural component that supports the cell, but also provides the cell with a beneficial microenvironment that regulates cell function. Cell adhesion is the first stage of cell / material interactions and affects the ability of cells to grow, migrate and differentiate. Therefore, fully synthesized peptide scaffolds functionalized by RGD and laminin cell adhesion motifs show promise as simple, safe and inexpensive materials for periodontal treatment.

In the tests described below, we tested periodontal ligament fibroblast activity in vitro on two custom- assembled peptide scaffolds PRG and PDS. PRG is a peptide backbone RADA16 via direct binding to the 2 unit RGD binding sequence PRGDSGYRGDS (SEQ ID NO: 15). PDS is RADA16 via direct binding to the laminin adhesion motif PDSGR (SEQ ID NO: 4). These skeletons are responsible for cell adhesion, proliferation, migration and extracellular matrix protein production of periodontal ligament fibroblasts, especially the production of type I and type III collagen, which are the major extracellular matrix protein components of periodontal ligament. Significantly promotes [30a], [31a].

The formation of the self- assembled peptide backbone and its mechanical properties are known to be affected by several factors, one of which is the degree of hydrophobicity [11b-16b]. That is, in addition to ionic complementary interactions, the degree of the hydrophobic residues Ala, Val, Ile, Leu, Tyr, Phe, Trp (or single letter codes A, V, I, L, Y, F, W) Can significantly affect the mechanical properties of the skeleton and the rate of skeleton self- assembly . The higher hydrophobicity corresponds to the shorter length of peptide required for self- assembly, easier backbone formation and better mechanical properties. However, this property does not necessarily promote cell migration, so additional active motifs may be required to stimulate cell adhesion.

The adhesion between the cells and the matrix is very important for cell growth. Functionalization of the peptide backbone with the cell adhesion motif RGD (Arg-Gly-Asp) and laminin derived cell adhesion motifs has been shown to be effective in increasing cell proliferation, migration and differentiation [8b, 9b]. Recently, peptides sensitive to enzymatic cleavage of matrix metalloproteinases (MMPs) have been added to synthetic polymers and peptide-amphiphiles ^17-24 . MMPs belong to a family of proteases that degrade extracellular matrix components and thus play an important role in tissue regeneration. MMP creates space for cells to grow and migrate into the matrix, allowing extracellular matrix remodeling and tissue growth. In addition to cell adhesion motifs, incorporating MMP cleavable substrates into self- assembled peptide scaffolds is an attractive strategy that reshapes the dynamic mechanism for inducing remodeling activity in cells and tissues.

Design of two self- assembling peptides VEVK9 (Ac-VEVKVEVKV-CONH ₂ ) (SEQ ID NO: 2) and VEVK12 (Ac-VEVKVEVKVEVK-CONH ₂ ) (SEQ ID NO: 3), as well as a designed 2 unit RGD binding sequence ( Synthesizing an extension sequence with PRGDSGYRGDS (SEQ ID NO: 15)) or one of two laminin-derived cell adhesion motifs (YIGSR (SEQ ID NO: 5), IKVAV (SEQ ID NO: 6)) added to the C-terminus The functionalization of VEVK9 by is described below. Designed two-unit RGD sequences have been shown previously to be effective in osteoblast and endothelial cell growth [8b, 25b]. The self- assembling peptide VEVK9 has only 9 amino acid residues, and its functionalized peptide is shorter than RADA16-I, which has 16 amino acid residues, and VEVK9 is cell activity in a similar manner Has the potential to promote We also report the functionalization of VEVK9 using MMP-2 cleavable sequences. We inserted the MMP-2 cleavable sequence (PVGLIG ((SEQ ID NO: 19)) into the self- assembling peptide VEVK9, which was obtained by screening a combinatorial peptide library for the optimal MMP substrate. Selected [26b] Self- assembled peptide scaffold (RADA) ₃ PVGLIG (RADA) ₃ (SEQ ID NO: 84) functionalized with this sequence was shown to be degradable by MMP-2, Promising for use as an engineering skeleton [19b].

In summary, we tested the potential of functional motifs for periodontal tissue regeneration. We compared human primary periodontal ligament fibroblasts to compare the effects of these motifs on proliferation, migration and matrix protein production with the non-functionalized self- assembling peptide scaffolds RADA16, VEVK9, VEVK12 as controls. Cells (HPDLF) were used.

Example 1: Periodontal ligament fibroblast adhesion, migration and matrix protein production method on peptide backbone peptide backbone;
A 1% RADA16 solution was obtained as PuraMatrix (3DM Inc./BD Bioscience). VEVK9, VEVK12 and functionalized peptides were obtained from CPC Scientific (San Jose, CA) and dissolved in water at a final concentration of 1% (v / w). The functionalized peptide solution was then mixed with a 1% RADA16, VEVK9 or VEVK12 solution in a 1: 1 ratio. Each peptide solution was then loaded into cell culture plate inserts (BD Bioscience, Bedford, MA). The following medium was added to induce hydrogel formation.

Cell culture of periodontal ligament fibroblasts Isolated primary human periodontal ligament fibroblasts are commercially available from Lonza Inc. (HPDLF, Walkersville, MD) and cultured on normal cell culture flasks ( (SCGM, Walkersville, MD). Cells were plated at 2 × 10 4 cells on a gel in the insert. The culture medium was changed every 3 days. No further growth factors were used.

In subsequent experiments, cells on the gel were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% Triton X-100 for 5 minutes at room temperature. Fluorescent rhodamine phalloidin and SYTOX® green (Molecular Probes, Eugene, OR) were used to label F-actin and nuclei, respectively. Images were obtained using a fluorescence microscope (Axiovert 25, ZEISS) or a laser confocal scanning microscope (Olympus FV300).

Fluorescent immunostaining for visualization of type I and type III collagen.
After fixing the cells, a primary antibody against type I collagen (5% anti-type I collagen, Millpore, MA) was added and incubated at 37 ° C. for 40 minutes, then washed 6 times with PBS with 1% BSA. Secondary antibody (0.5% Alexa fluor 488 goat anti-rabbit IgG, Invitrogen) was added and incubated for an additional 40 minutes at 37 ° C. and then washed in the same manner. Thereafter, a primary antibody against type III collagen (0.5% anti-type III collagen, Millpore, MA) was added, incubated at 37 ° C. for 40 minutes, and then washed. Finally, a secondary antibody (0.5% Alexa fluor 594 goat anti-mouse IgG, Invitrogen) was added and incubated for an additional 40 minutes at 37 ° C. As a control, non-specific staining was performed by omitting the primary antibody.

Results and Discussion Cell Adhesion and Cell Proliferation The functionalized peptide scaffold promoted more cell adhesion and proliferation compared to RADA16, VEVK9 or VEVK12 (FIGS. 2, 3A and 3B). We also tested different mixing ratios. PRG (1: 9) means a mixing ratio of PRG: RADA16 = 1: 9. # Means p <0.01 t test.

Cell migration into the peptide backbone The functionalized peptide backbones PRG and PDS promoted more cell migration into the peptide backbone compared to RADA16 (Figure 4).

Matrix protein production Functionalized peptide scaffolds PRG and PDS promoted more type I and type III collagen compared to RADA16 (FIG. 5). It is well known that type I collagen and type III collagen are the major matrix proteins of the periodontal ligament.

Example 2: Selective cell regeneration of peptide backbone (comparison between periodontal ligament fibroblasts and gingival fibroblasts)
We tested the selective cell regenerative properties of the functional skeleton using two types of periodontal cells, periodontal ligament fibroblasts and gingival fibroblasts.

Methods Cell culture of gingival fibroblasts Human gingival fibroblasts are commercially available from ATCC (HGF-1) and were routinely grown in culture medium (DMEM + 10% FBS) on normal cell culture flasks. . Cells were plated at 2 × 10 4 cells on the gel in the insert. The culture medium was changed every 3 days.

Results and Discussion Functionalized peptide backbone PDS showed selective cell attachment and migration (Figure 6). Periodontal ligament fibroblasts spread on the surface of the skeleton migrated into the skeleton. In comparison, gingival fibroblasts did not adhere well to the skeleton and did not migrate into the skeleton at all. The peptide backbone RADA16 did not show migration in both fibroblasts (not shown). These results indicate that the skeleton provides space and favorable niche for the growth of periodontal ligament fibroblasts and has a good potential to reconstruct periodontal tissue, It suggests limiting the repopulation of space by cells. Functionalized peptide backbones vPDS_9, vYIG_9 and vIKV_9 also showed selective cell adhesion (FIG. 7).

Example 3: Migration of periodontal ligament fibroblasts into a peptide backbone functionalized with an MMP degradable motif.
We have implicitly tested the ability of peptide scaffolds functionalized with MMP degradable motifs by the effect of periodontal ligament fibroblast migration.

Methods Peptide backbone Functionalized peptides were obtained from CPC Scientific (San Jose, CA) and similarly dissolved in water at a final concentration of 1% (v / w). The functionalized peptide solution was then mixed with 1% VEVK9 or VEVK12 solution. Each peptide solution was loaded into a cell culture plate insert (BD Bioscience, Bedford, MA). The following media was added to induce hydrogel formation.

Results Peptide scaffolds vPVG_vPRG_9 and vPVG_vPRG_12 showed significant cell migration to the scaffold (FIG. 8). These results show that the MMP cleavage site PVGLIG (SEQ ID NO: 19) of the functionalized peptide sequence L is cleaved by MMP produced in periodontal ligament fibroblasts, which then move easily into the skeleton I suggest that I was able to.

Discussion In Examples 1-3 above, the inventors described the development of a self- assembling peptide scaffold that has similar properties to natural extracellular matrix proteins for periodontal tissue regeneration. For example, two sequence motifs were selected from the two unit RGD binding sequence PRGDSGYRGDS (SEQ ID NO: 15) and the laminin cell adhesion motif PDSGR (SEQ ID NO: 4). The two motifs appeared to be effective for 3-D proliferation of periodontal ligament fibroblasts and collagen production from the fibroblasts. The custom peptide backbone PRG contains an RGD cell binding motif for the integrin receptor. PRG has been reported to promote osteoblast activity for bone tissue regeneration and endothelial cell activity for angiogenesis [10a], [11a]. Another specialty peptide backbone PDS contains the laminin PDSGR (SEQ ID NO: 4) cell adhesion motif. Periodontal ligament fibroblasts have been reported to adhere to RGD motifs, fibronectin and laminin and express integrin subunits associated with their binding to extracellular matrix proteins [27a], [28a]. In this study, we showed that PRG and PDS promoted periodontal ligament fibroblast activity. This suggests that periodontal ligament fibroblasts really recognized the exposed adhesion motifs bound to their skeleton via integrin receptors for the RGD motif or laminin PDSGR cell adhesion motif. Periodontal ligament fibroblasts adhered to the surface of these peptides also recognized the adhesion motifs inside the skeleton in a similar manner and then migrated into these skeletons.

  Growth factors and mechanical signals are known to regulate the production of extracellular matrix proteins in fibroblasts [26a], [31a]-[38a]. Considering that no extra growth factors were added, our results show that biochemical and possibly mechanical signals from each different peptide backbone induced the production of extracellular matrix proteins. To suggest. Fibroblasts have previously been reported to translate mechanical signals through integrins into changes in extracellular matrix production [39a]-[41a]. These showed that extension of the matrix-integrin contact resulted in cytoskeletal-mediated signals by rearrangement of cytoskeletal components including actin filaments and recruitment of kinases such as focal adhesion kinase (FAK) and Src. Activation of FAK and Src further activates mitogen-activated protein kinase (MAPK) signaling pathway to promote gene transcription. Altered gene transcription results in translational and post-translational modifications that selectively synthesize and secrete extracellular matrix proteins. In our study, the interaction between fibroblast integrin-skeletal PRG and PDS cell adhesion motifs induces the intracellular signaling pathway described above, which then causes fibroblasts to undergo type I collagen and III Seems to synthesize and secrete type collagen. On the other hand, since the peptide backbone RADA16 does not have a cell adhesion motif, periodontal ligament fibroblasts appear to adhere to the peptide in a different manner. The interaction between charged residues of RADA16 and cell surface components plays a role in non-integrin-mediated cell binding to the peptide backbone [42a], and the cells are derived from or produced by serum in added culture media. [43a] is presumed to adhere to the peptide backbone via an adhesion protein that can be either Periodontal ligament fibroblasts appear to adhere to the peptide backbone RADA16 without interaction between the integrin-skeletal cell adhesion motif and produce no collagen as in the functionalized peptide backbones PRG and PDS . These results suggest that peptide backbone functionalization by cell adhesion motifs that interact with integrins may be useful for stimulating matrix protein production from fibroblasts.

VEVK9 and VEVK12 are simple repeating units of the amino acid VEVK (valine-glutamic acid-valine-lysine) that self- assemble into nanofiber structures. The self- assembling peptide VEVK9 is functionalized with either RGD, laminin cell adhesion motif or MMP cleavable motif to mimic the extracellular matrix and enhance function during cell maintenance and cell culture. Functionalized peptides vPRG, vYIG and vIKV were synthesized with the VEVK9 sequence and additional motifs added to the C-terminus using solid phase synthesis. One motif contains two repeats of the RGD sequence (PRGDSGYRGDS (SEQ ID NO: 15)) and the other motifs are laminin cell adhesion motifs (YIGSR (SEQ ID NO: 5), IKVAV (SEQ ID NO: 6)) It is. A glycine residue was used as a linker between the self- organizing motif VEVKVEVKV (SEQ ID NO: 2) and the functional motif to provide a space to maintain the flexibility of the functional peptide. The MMP-2 cleavable motif (PVGLIG (SEQ ID NO: 11)) was inserted between two VEVK units. Compared to RADA16-I and its functionalized peptides, these peptides are short sequences, up to 20 amino acids (in vPRG) and appear to be economical. The peptide was solubilized in water at a concentration of 10 mg / ml (1% w / v). Peptides readily self- assemble to form a soft hydrogel. Mixing with the self- assembling peptides VEVK9 or VEVK12 facilitated self- assembly and gelation. Tapping mode AFM allowed us to observe without damaging the peptide filaments, so tapping mode AFM was used to analyze nanofiber formation. Self-assembling peptides VEVK12 and VEVK9 mixed with functionalized peptide vPRG and vPVG formed a nanofiber in aqueous solution.

  In order to evaluate cell adhesion on each peptide scaffold, the same number of cells were seeded on each scaffold and cultured for 3 weeks. Fibroblasts showed good adhesion and elongation on peptide backbones functionalized with vPRG, vYIG and vIKV, especially on VEVK9 mixed with vYIG and vIKV. In contrast, on the non-functionalized peptide backbones VEVK9 and VEVK12, the cells are scattered and have a round morphology. All functionalized peptides tested here significantly promoted cell proliferation, especially in scaffolds made with VEVK9.

Periodontal ligament fibroblasts are known to bind RGD motifs, fibronectin and laminin and express integrin subunits associated with their binding to extracellular matrix proteins [28b, 29b]. Therefore, inclusion of these cell adhesion motifs in the peptide backbone would promote fibroblast adhesion, proliferation, and 3-D migration through interaction with fibroblast integrin receptors. . Interestingly, these results showed a difference in the effect of the functional motif between the self- assembling peptides VEVK9 and VEVK12. The functionalized peptide motif contained in the peptide backbone VEVK9 is more effective than that in VEVK12 for cell proliferation. Functional motifs physically incorporated into skeletal nanofibers have been reported to affect cell growth ^8b . In this study, the functionalized peptide appears to be well integrated into the self- assembling peptide VEVK9 and form nanofibers with functional motifs.

  We have previously shown that cells spontaneously migrate into the 3D scaffold using 3-D image collection and 3-D image reconstruction obtained by confocal microscopy [8b] . Reconstitution of periodontal ligament fibroblasts in peptide backbone VEVK9, VEVK9 mixed with functionalized peptide vPRG only, VEVK9 mixed with vPRG and vPVG, VEVK9 mixed with vYIG and vPVG, and VEVK9 mixed with vIKV and vPVG image. Migrating cells were clearly identifiable by confocal imaging, and differences in the characteristics of these skeletons were seen. Only a small number of fibroblasts adhered to the peptide backbone with VEVK9, which remained near the surface. In the peptide backbone mixed with VEVK9 and functionalized peptide, many cells were found on and inside the backbone. Furthermore, the inventors have shown that the fibroblasts that initially adhered to the surface of the skeleton on day 1 proliferated and spontaneously migrated into the skeleton. The images show a significant increase in fibroblast proliferation and migration due to the effects of functionalized peptides vPRG and vPVG. It appears that fibroblasts on the surface of the skeleton have migrated into the skeleton, increasing its range of activity.

Periodontal ligament fibroblasts are known to contribute significantly to remodeling of periodontal tissue by secreting MMP-2 for degradation and synthesizing extracellular matrix proteins for replacement [30b]. It is also known that MMP regulation occurs by integrin binding, such as α _v β ₃ , the major RGD binding integrin [31b]. These suggest that fibroblasts recognized the RGD adhesion motif exposed in the skeleton via integrin receptors and adhered to the skeleton. The interaction between the integrin and the RGD motif results in MMP-2 production by fibroblasts. As a result, fibroblasts migrated deeper into the scaffold and cleaved the MMP-cleavable sites incorporated into the scaffold. Interestingly, in the case of VEVK9 mixed with vPVG alone (no vPRG), fibroblasts curled up on the surface of the skeleton and did not proliferate or migrate into the skeleton as in VEVK9. Thus, cell adhesion motifs that interact with integrins appear to play an important role as stimulators for proteolytic cell migration into an enzymatically degradable scaffold [21b]. In the case of scaffolds functionalized with laminin cell adhesion motifs vYIG and vIKV, fibroblasts appear to migrate into the scaffold in a similar manner.

Various functionalized peptide scaffolds have been developed and have shown great potential for tissue engineering and regenerative medicine [7b, 8b, 25b]. Most of these are functionalized with cell adhesion motifs derived from extracellular matrix proteins. The motif contributed to the initial interaction between cells and matrix, and cell adhesion that promoted cell proliferation involved cell migration. However, since these functionalized peptide backbones are not enzymatically degradable, their ability to promote cell migration is limited. In a non-degradable scaffold, cells appear to somehow navigate the space between the scaffold's nanofibers. Since amoeba-like cell migration depends on the mechanical properties of the peptide scaffold, strategies using cell adhesion motifs are limited to scaffolds with relatively large pores and soft nanofibers through which cells can pass. In contrast, a peptide backbone that can be enzymatically degraded can significantly increase cell migration. The peptide backbone functionalized with vPVG is degradable by enzymes and appears to promote proteolytic cell migration. The MMP cleavable motifs tested here can be useful for the functionalization of most self- assembling peptides without considering mechanical properties. Also, when combined with cell adhesion motifs in various proportions, it is possible to design a peptide backbone that is appropriate for a particular application. For example, these peptide backbones may be useful as replacements for naturally occurring extracellular matrix derived materials such as fibrin or collagen that require difficult purification procedures and have the risk of immunogenicity and disease transmission. .

  We have now shown that a complete customized peptide backbone significantly promotes periodontal ligament proliferation and migration. This is an important finding that these simple motifs can have dramatic effects on fibroblast activity. Creating a peptide backbone is much easier and cheaper than using complex and expensive soluble factors that exhibit similar cellular behavior. These peptide scaffolds have potential value as scaffold materials for tissue engineering and regenerative medicine, eg periodontal tissue reconstruction.

  The periodontal ligament supporting the dental organ consists of four tissues: gingiva, periodontal ligament, cementum and alveolar bone. The various compositions of the periodontal ligament reshape the periodontal wound healing and the cellular and extracellular components of the new periodontal ligament, cementum and alveolar bone due to the interaction of hard and soft connective tissue It is a complex process that involves selective reassembly of the root surface by the cells it obtains [44a]. The traditional method for periodontal tissue reconstruction is guided tissue regeneration, which eliminates or limits the reassembly of periodontal defects by epithelial cells and gingival connective cells, and periodontal ligament fibroblasts It can be induced by providing space and favorable gaps to maximize the selective migration, proliferation and differentiation of cement blasts and osteoblasts. Given the clinical use of these skeletons for periodontal tissue reconstruction, the skeletons need to provide selective cellular reassembly. It has been previously reported that the peptide backbone PRG can control osteoblast activity by changing the concentration of a custom peptide containing 2 units of RGD [10a]. It has also been reported that laminin, to which periodontal ligament fibroblasts and osteoblasts adhere better than gingival fibroblasts, has specific cell adhesion properties [27a]-[29a]. These suggest that these peptide backbones PRG and PDS with laminin cell adhesion motif may be useful as periodontal tissue fillers with selective cell reassembly properties.

For successful clinical use in periodontal tissue reconstruction, functionalized peptide scaffolds are required to allow selective cell reassembly and promote angiogenesis. It has been previously reported that peptide backbones functionalized with RGD can control osteoblast activity by varying the concentration of the functionalized peptide ⁸ . Laminin has also been reported to have specific cell adhesion properties that favor adhesion by periodontal ligament fibroblasts and osteoblasts rather than adhesion by gingival fibroblasts [28b, 29b]. Due to angiogenesis, the above-described peptide backbone functionalized with RGD has also been shown to promote endothelial cell growth [25b]. Furthermore, the functionalized peptide vPVG tested in this example appears to be effective for proteolytic migration of endothelial cells. Various functionalizations of self- assembling peptides allow optimization of skeletal materials for many applications, including periodontal tissue reconstruction.

In the above studies, specially-functionalized peptide scaffolds PRG and PDS have been shown to significantly enhance periodontal ligament fibroblast proliferation and migration in cell culture and production of extracellular matrix proteins type I and type III collagen. Has been. In addition, the inventors have developed and evaluated the self- assembling peptides VEVK9, VEVK12 and custom self- assembling peptide scaffolds functionalized with cell adhesion motifs and MMP cleavable sites. These functionalized peptide scaffolds have been shown to significantly enhance periodontal ligament fibroblast proliferation and migration in 3D cell culture, independent of scaffold stiffness. Thus, the special scaffolds described herein are widely useful in tissue remodeling and regenerative medicine.

  While the invention has been particularly shown and described with reference to preferred embodiments thereof, various changes in form and detail may be made without departing from the scope of the invention as encompassed by the appended claims. However, it will be understood by those skilled in the art that the present invention can be made.

Claims (13)

  A self-assembling peptide comprising the amino acid sequence VEVKGPVGLIGVEVK (SEQ ID NO: 82). Amino acid sequence VEVKGPVGLIGVEVK (SEQ ID NO: 82) consisting of, according to claim 1, wherein the peptide. A pharmaceutical composition for regenerating dental tissue in a patient, comprising the self-assembling peptide according to claim 1 . The pharmaceutical composition according to claim 3 , wherein the tooth tissue is periodontal ligament tissue. A pharmaceutical composition for treating periodontal disease in a patient, comprising the self-assembling peptide according to claim 1 . A skeleton for periodontal tissue regeneration comprising the self-assembling peptide according to claim 1 . The skeleton according to claim 6 , comprising the amino acid sequence VEVKGPVGLIGVEVK (SEQ ID NO: 82). A pharmaceutical composition for treating periodontal disease in a patient, comprising the skeleton of claim 6 and a solid biomaterial. The pharmaceutical composition according to claim 8 , wherein the biomaterial comprises calcium, phosphorus or a combination thereof. 9. The pharmaceutical composition of claim 8 , wherein the biomaterial comprises pores having a diameter of about 100 microns to about 500 microns. A syringe having two compartments, wherein the first compartment comprises a peptide solution, the second compartment comprises a gelation fluid, the peptide solution comprising the self-assembly of claim 1 . A syringe, which is an aqueous solution containing a peptide.   A macroscopic scaffold comprising a plurality of self-assembling peptides, wherein the peptide comprises the amino acid sequence VEVKGPVGLIGEVEVK (SEQ ID NO: 82). 13. The macroskeleton of claim 12 , further comprising a self-assembling peptide comprising the amino acid sequence VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3).