JP2013533271A5 - 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].

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 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 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. It relates methods, wherein the self-assembling peptides:
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.

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 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 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.

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.

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 US Pat. Nos. 5,670,483; 5,955,343; 6,368,877; 7,098,028 and 7,449,180 and US Patent Application Publication Nos. 2002/0160471, 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.

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.

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 ionic solutes in the peptide solution. 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 .

(式中：
Φ、ΨおよびΓは、それぞれ組成および構造を決定する中性アミノ酸、正荷電アミノ酸および負荷電アミノ酸を示し、
i、j、kおよびtは整数であり、変数を示し；
nは、オリゴペプチドの長さも決定する繰り返し単位の数を表す)
の1つで表される。 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:

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.

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.

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.

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.

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 may be initiated or substantially accelerated by the addition or change in pH of an ionic solute to the peptide solution. 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.

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 . Then self-assembly process forms a structure surrounding 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).

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 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.

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.

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.

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.

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.

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. .

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.

Claims (44)

a. A first amino acid domain that mediates self- assembly , wherein the first amino acid domain consists of the sequence VEVK VEVKV (SEQ ID NO: 2 ) or VEVKVEVKVEVK (SEQ ID NO: 3) , the domain being isolated Can self- assemble in different forms; 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 self- assembling peptide. Amino acid domain of the first is, V EVKVEVKV (SEQ ID NO: 2) Tona Ru claim 1, wherein the peptide. It said biologically active motif, laminin cell attachment motif of claim 1, wherein the peptide. It said biologically active motif is a motif which periodontal ligament fibroblasts can adhere claim 1, wherein the peptide. 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: comprising a sequence selected from the group consisting of 12), according to claim 1, wherein the peptide. Amino acid domain of said second, an RGD containing peptide, according to claim 1, wherein the peptide. 7. The peptide of claim 6 , wherein the second amino acid domain comprises the sequence PRGDS (SEQ ID NO: 13) or YRGDS (SEQ ID NO: 14). The peptide according to claim 6 , wherein the RGD peptide is PRGDSGYRGDS (SEQ ID NO: 15). The second amino acid domain comprises a matrix metalloproteinase cleavable substrate of claim 1, wherein the peptide. 10. The peptide of claim 9 , wherein the second amino acid domain comprises the sequence PVGLIG (SEQ ID NO: 11). 請 Motomeko 1-10 comprising Nde including a self-assembling peptides according to any one of A pharmaceutical composition for regenerating dental tissue in a patient. The pharmaceutical composition according to claim 11 , wherein the tooth tissue is periodontal ligament tissue. 請 Motomeko 1-10 comprising Nde including a self-assembling peptides according to any one of A pharmaceutical composition for treating periodontal disease in a patient. 請 Motomeko 1-10 comprising Nde either contains one term self-assembling peptides described, pharmaceutical compositions for increasing extracellular matrix protein production in the tissue. The increase is carried out in the absence of additional growth factors, claim 14 pharmaceutical composition. The pharmaceutical composition according to claim 14 , wherein the extracellular matrix protein is collagen. 請 Motomeko 1-10 comprising Nde including a self-assembling peptides according to any one of A pharmaceutical composition for regenerating damaged tissue in a patient. 18. The pharmaceutical composition of claim 17 , wherein the tissue is selected from the group consisting of skeletal, bone, tendon, connective tissue, and periodontal ligament tissue. a. The first amino acid domain that mediates self-assembly, wherein the first amino acid domain is SEQ VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3) Ru Tona; 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;
The made Nde including, scaffold for periodontal tissue regeneration. 20. A scaffold according to claim 19 , wherein the biologically active motif is a laminin cell adhesion motif. The laminin cell adhesion motif 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 (sequence) 21. The scaffold of claim 20 , comprising a sequence selected from the group consisting of: No. 9) and LGTIPG (SEQ ID NO: 10). 20. The backbone of claim 19 , wherein the second amino acid domain comprises an RGD peptide. 23. The backbone of claim 22 , wherein the second amino acid domain comprises the sequence PRGDS (SEQ ID NO: 13) or YRGDS (SEQ ID NO: 14). 24. The scaffold of claim 23 , wherein the RGD peptide is PRGDSGYRGDS (SEQ ID NO: 15). 21. The scaffold of claim 19 , wherein the second amino acid domain comprises a matrix metalloproteinase cleavable substrate. 26. The backbone of claim 25 , wherein the second amino acid domain comprises the sequence PVGLIG (SEQ ID NO: 19). 19. become Nde contains the backbone and solid biomaterials according pharmaceutical composition for treating periodontal disease in a patient. 28. The pharmaceutical composition according to claim 27 , wherein the biomaterial comprises calcium, phosphorus or a combination thereof. 28. The pharmaceutical composition of claim 27 , 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 being an aqueous solution comprising a self- assembling peptide. And the peptide is
a. The first amino acid domain that mediates self-assembly, wherein the first amino acid domain is SEQ VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3) Ru Tona; 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 a syringe. A macroscopic scaffold comprising a plurality of self- assembling peptides , the peptide comprising:
a. The first amino acid domain that mediates self-assembly, wherein the first amino acid domain is SEQ VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3) Ru Tona; 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;
Wherein the backbone further seen contains living cells that are enclosed in or bone in skeleton attached to a surface of the skeleton, said cell is a periodontal ligament fibroblasts or periodontal ligament stem cells, Macroscopic skeleton. A self-assembling peptide comprising the amino acid sequence VEVKGPVGLIGVEVK (SEQ ID NO: 82). The peptide according to claim 32, which consists of the amino acid sequence VEVKGPVGLIGVEVK (SEQ ID NO: 82). 33. A pharmaceutical composition for regenerating dental tissue in a patient comprising the self-assembling peptide of claim 32. 35. The pharmaceutical composition of claim 34, wherein the tooth tissue is periodontal ligament tissue. 35. A pharmaceutical composition for treating periodontal disease in a patient comprising the self-assembling peptide of claim 32. A skeleton for periodontal tissue regeneration, comprising the self-assembling peptide according to claim 32. The skeleton according to claim 37, comprising the amino acid sequence VEVKGPVGLIGVEVK (SEQ ID NO: 82). 38. A pharmaceutical composition for treating periodontal disease in a patient comprising the skeleton of claim 37 and a solid biomaterial. 40. The pharmaceutical composition of claim 39, wherein the biomaterial comprises calcium, phosphorus or a combination thereof. 40. The pharmaceutical composition of claim 39, wherein the biomaterial comprises pores having a diameter of about 100 microns to about 500 microns. 35. A syringe having two compartments, wherein the first compartment comprises a peptide solution, the second compartment comprises a gelation fluid, the peptide solution comprising self-assembly according to claim 32. 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). 45. The macroscopic scaffold of claim 43, further comprising a self-assembling peptide comprising the amino acid sequence VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3).

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