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WO2012008967A1 - Peptides autoassemblés comprenant des modifications et leurs procédés d'utilisation - Google Patents

Peptides autoassemblés comprenant des modifications et leurs procédés d'utilisation Download PDF

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Publication number
WO2012008967A1
WO2012008967A1 PCT/US2010/042257 US2010042257W WO2012008967A1 WO 2012008967 A1 WO2012008967 A1 WO 2012008967A1 US 2010042257 W US2010042257 W US 2010042257W WO 2012008967 A1 WO2012008967 A1 WO 2012008967A1
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WIPO (PCT)
Prior art keywords
seq
self
amino acid
peptide
acid domain
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PCT/US2010/042257
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English (en)
Inventor
Yoshiyuki Kumada
Shuguang Zhang
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Massachusetts Institute Of Technology
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Priority to PCT/US2010/042257 priority Critical patent/WO2012008967A1/fr
Priority to JP2013520692A priority patent/JP5855651B2/ja
Publication of WO2012008967A1 publication Critical patent/WO2012008967A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3865—Dental/periodontal tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses

Definitions

  • the extracellular matrix is composed of a diverse set of macromolecules, including both proteins and polysaccharides, which form the three dimensional environment within which cells exist in the body and constitute the space filling material between cells.
  • the ECM can also be organized into a sheet-like layer known as the basal lamina or basement membrane.
  • the ECM consists primarily of molecules that are secreted locally and assemble into a scaffold that stabilizes and supports the physical structure of cell layers and tissues. However, rather than being merely an inert substrate for cell attachment, the ECM constitutes an environment that is rich in biological information.
  • the ECM and various biomolecules associated with it (e.g., secreted locally or transported to a particular site from elsewhere), exert a significant influence on many aspects of cell behavior and phenotype, regulating processes such as migration and proliferation, influencing cell development and differentiation, and affecting cell shape and function.
  • the structure of the ECM is, in turn, influenced by the cells within it. Not only do these cells secrete many ECM constituents, but they also help to pattern the matrix. Thus it is evident that cell ECM interactions are of vital importance.
  • synthetic compositions and materials for tissue engineering purposes that would allow the creation of a cellular environment that mimics important aspects of the native cellular environment without the disadvantages associated with products derived from natural sources. For applications involving implantation into the body, there remains a particular need for such compositions and materials that elicit no or minimal immune or inflammatory response and for
  • compositions and materials that are degradable within the body are degradable within the body.
  • compositions and materials that would influence cell properties and functions in desirable ways There has previously been reported that a class of designer self-assembling peptide scaffolds have wide application including for three-dimensional (3-D) cell culture, drug delivery, regenerative medicine and tissue engineering [la]-[5a].
  • the class of self- assembling peptide materials can undergo spontaneous assembly into well-ordered nanofibers and scaffolds, ⁇ 10nm in fiber diameter with pores between 5-200nm and over 90% water content [6a].
  • These peptide scaffolds have 3-D nano fiber structures similar to the natural extracellular matrix including collagen.
  • the scaffolds are biodegradable by a variety of proteases in a body with superior biocompatibility with tissue [7a]. Moreover, these scaffolds can be modified and functionalized by direct extension of peptides with known biologically functional peptide motifs to promote specific cellular responses.
  • functionalized RADA16 has been studied for bone, cartilage, neural regeneration and angiogenesis promotion [8a]-[l la].
  • the invention relates to a novel class of self-assembling peptides, compositions thereof, methods for the preparation thereof and methods of use thereof.
  • the invention also encompasses methods for tissue regeneration, increasing the production of extracellular matrix proteins, and methods of treatment comprising administering self-assembling peptides.
  • the invention is directed to a self-assembling peptide comprising the sequence VEVK (SEQ ID NO: 1), wherein the peptide is capable of self-assembly.
  • 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).
  • the invention is directed to a self-assembling peptide wherein the self-assembling peptide comprises:
  • a first amino acid domain that mediates self-assembly wherein the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1) and wherein the domain is capable of self-assembly in isolated form; and
  • the second amino acid domain comprises a biologically active motif.
  • the first amino acid domain is VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3).
  • the biologically active motif is a laminin cell adhesion motif.
  • the second amino acid domain comprises a sequence selected from the group consisting of 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).
  • the second amino acid domain comprises an RGD peptide, such as PRGDS (SEQ ID NO: 13), YRGDS (SEQ ID NO: 14) and PRGDSGYRGDS (SEQ ID NO: 15).
  • 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).
  • the invention is directed to a self- assembling peptide wherein the self-assembling peptide comprises:
  • the first amino acid domain of the peptide comprises multiple RADA (SEQ ID NO: 17) peptide subunits, for example, AcN-RAD ARAD ARAD ARAD A-C ONH2 (SEQ ID NO: 18).
  • the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1), for example, VEVKVEVKV (SEQ ID NO: 2) or
  • VEVKVEVKVEVK (SEQ ID NO: 3).
  • the invention is directed to a method for regenerating a dental tissue in a patient in need thereof comprising administering to the dental tissue of said patient an effective amount of a self-assembling peptide, wherein the self-assembling peptide comprises:
  • 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, wherein said domain self- assembles into a macroscopic structure when present in unmodified form; and b.
  • the dental tissue is periodontal ligament tissue and the self-assembling peptide is administered to the peridontium.
  • the biologically active motif is a laminin cell adhesion motif, an RGD peptide or a matrix metalloprotease cleavable substrate.
  • the second amino acid domain comprises a sequence selected from the group consisting of 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 SEQ ID NO: 15
  • PVGLIG SEQ ID NO: 19
  • the first amino acid domain comprises multiple RADA (SEQ ID NO: 17) peptide subunits, for example AcN- RAD ARAD ARAD ARAD A-C ONH2 (SEQ ID NO: 18).
  • the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1), for example, VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3).
  • the invention also encompasses a method of treating periodontal disease and/or regenerating periodontal ligament tissue in a patient in need thereof comprising administering to the periodontium of said patient an effective amount of a self-assembling peptide, 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 and wherein said domain self- assembles into a macroscopic structure when present in unmodified form; and b. A second amino acid domain that does not mediate self-assembly in isolated form, wherein the second amino acid domain comprises a biologically active motif.
  • the invention is a method of increasing extracellular matrix protein production in a tissue comprising administering to said tissue of said patient an effective amount of a self-assembling peptide, wherein the self-assembling peptide comprises:
  • 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 and wherein said first domain self- assembles into a macroscopic structure when present in unmodified form; and b.
  • the extracellular matrix protein is collagen
  • the present invention additionally includes a method for regenerating a damaged tissue in a patient in need thereof comprising administering to said patient an effective amount of a self-assembling peptide, wherein the self-assembling peptide comprises:
  • 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 and wherein said first domain self- assembles into a macroscopic structure when present in unmodified form; and b.
  • the first amino acid domain comprises the sequence VEVK (SEQ ID NO: l).
  • Exemplary sequences comprising VEVK (SEQ ID NO: 1) include, for example, VEVKVEVKV (SEQ ID NO: 2) or VEVKVEVKVEVK (SEQ ID NO: 3).
  • the patient is suffering from arthritides, a neurological condition, a muscle wasting condition, a neuroendocrine disorder, muscular degeneration, musculotendenous failure, trauma, tissue necrosis, cardiac disorder, surgical resection, growth abnormalities, osteoporosis, fractures, or ischemic damage due to peripheral vascular disease.
  • the damaged tissue is selected from the group consisting of skeletal, bone, tendon, connective or dental tissue tissues.
  • the invention is directed to a scaffold for periodontal tissue regeneration comprising:
  • 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 and wherein said domain self- assembles into a macroscopic structure when present in unmodified form; and b.
  • the biologically active motif is a laminin cell adhesion motif, an RGD peptide or a matrix metalloprotease cleavable substrate.
  • the second amino acid domain comprises a sequence selected from the group consisting of 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).
  • the first amino acid domain comprises multiple RAD A (SEQ ID NO: 17) peptide subunits, for example AcN-RAD ARAD ARAD ARAD A-C ONH2 (SEQ ID NO: 18).
  • the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1) peptide subunits, for example, VEVKVEVKV (SEQ ID NO: 2) or
  • VEVKVEVKVEVK (SEQ ID NO: 3).
  • the invention is directed to a method of treating periodontal disease comprising administering to a patient in need thereof a scaffold for periodontal tissue regeneration comprising:
  • 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 and wherein said domain self- assembles into a macroscopic structure when present in unmodified form; and b.
  • the method further comprises administering a solid biomaterial.
  • the invention also encompasses a syringe having two compartments, wherein the first compartment comprises a peptide solution and the second compartment comprises a gelation fluid, wherein the peptide solution is an aqueous solution comprising a self assembling peptide wherein the peptide comprises:
  • 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 and wherein said domain self- assembles into a macroscopic structure when present in unmodified form; and b.
  • the invention also encompasses a macroscopic scaffold comprising a plurality self- assembling peptides wherein said peptides comprise the sequence VEVK (SEQ ID NO: 1).
  • the invention additionally encompasses a macroscopic scaffold comprising a plurality self-assembling peptides wherein said peptides comprise:
  • 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 and wherein said domain self- assembles into a macroscopic structure when present in unmodified form
  • a second amino acid domain that does not mediate self-assembly in isolated form wherein the second amino acid domain comprises a biologically active motif; wherein said scaffold further comprises living cells attached to its surface or encapsulated within the scaffold.
  • the invention is a macroscopic scaffold wherein the cells on the surface of the scaffold or encapsulated within the scaffold are periodontal ligament fibroblasts.
  • the invention is a macroscopic scaffold wherein the first amino acid domain comprises the sequence VEVK (SEQ ID NO: l) and optionally wherein the cells are selected from the group consisting of osteoblasts, cementoblasts, bone marrow cells, fibroblasts, periodontal ligament fibroblasts, mesenchymal cells, mesenchymal stem cells, adipose derived cells, adipose derived stem cells, periodontal ligament stem cells, dental pulp stem cells, stem cells from exfoliated deciduous teeth and embryonic stem cells.
  • the cells are periodontal ligament fibroblasts.
  • FIG. 1 is a drawing showing the tooth, gingival, periodontal ligament, cementum and alveolar bone.
  • FIGs. 3A and 3B are photographs showing cell morphology on each functionalized peptide scaffold after two weeks cell culture.
  • FIG. 4 shows confocal microscope images showing cell migration into peptide scaffolds after two weeks cell culture.
  • FIG. 5 are fluorescent immunostaining images type I (green) and type III (red) collagens production on peptide scaffolds after six weeks cell culture.
  • FIG. 6 shows a comparison study between periodontal ligament fibroblasts and gingival fibroblast on the peptide scaffold PDS (PDS is described in detail in the Examples section).
  • FIG. 7 shows a comparison study between periodontal ligament fibroblasts and gingival fibroblast on the peptide scaffolds vPDS_9, vYIG_9 and vIKV_9 (described in detail in the Examples section).
  • FIG. 8 is confocal microscope images showing that functionalized peptide vPVG promoted cell migration.
  • FIG. 9 is a bar graph showing cell numbers on each indicated functionalized peptide scaffold after three weeks cell culture.
  • FIGs. 10A and 10B are line graphs showing cell growth in functionalized peptide scaffold vPVG_vPRG_9.
  • FIG. 11 A shows molecular models of the indicated functionalized peptides.
  • FIG. 1 IB and C shows AFM images of VEVK12(b) and vPVG_vPRG_9(c).
  • self-assembly is a process of molecules or peptides forming regular shaped structures or aggregates in response to conditions in the environment, such as when added to an aqueous medium.
  • self-assembling peptide refers to a peptide comprising a self-assembling motif.
  • Self-assembling peptides are peptides that are capable of self-assembly into structures including, but not limited to, macroscopic membranes, nanostructures and the like.
  • Various self-assembling peptides and methods for preparation thereof have been described previously, for example in, U.S. Patent Nos. 5,670,483; 5,955,343; 6,368,877; 7,098,028; and 7,449,180 and U.S. Patent Application Publication Nos. 2002/0160471, 2005/0181973 and
  • self-assembling motif refers to a peptide sequence or motif capable of self-assembly.
  • ionic self-assembling peptide refers to a self-assembling peptide comprising an alternating sequence of hydrophobic amino acids and hydrophilic amino acids, wherein said hydrophilic amino acids are charged amino acids.
  • the ionic self-assembling peptide comprises an alternating sequence of hydrophobic amino acids and hydrophilic amino acids, wherein said hydrophilic amino acids are acidic or basic amino acids. Ionic, self-assembling peptides have been described for example in U.S. Pat. No. 5,670,483.
  • amino acid encompasses a naturally or non-naturally occurring amino acid.
  • Non-naturally occurring amino acids are also referred to herein as “non-natural amino acids.”
  • Naturally occurring amino acids are also referred to herein as "natural amino acids.”
  • Natural amino acids are represented by their well-known single-letter designations: 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, L for leucine, M for methionine, N for asparagines, 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.
  • physiologic pH is a pH of about 7.
  • macroscopic means having dimensions large enough so as to be visible under magnification of 10-fold or less.
  • a 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 a peptide motif that induces a phenotypic response or change in an appropriate cell type when the cell is contacted with the peptide comprising the biologically active motif.
  • Biologically active motifs have been described, for example, U.S. Pat. No. 7,713,923, the contents of which are expressly incorporated by reference herein.
  • a biologically active motif is a motif found in a naturally occurring protein.
  • the biologically active peptide motif can be present in isolated form or as part of a larger polypeptide or other molecule.
  • phenotypic responses or changes include, but are not limited to, enhancement of cell spreading, attachment, adhesion, proliferation, secretion of an extracellular matrix (ECM) molecule, or expression of a phenotype characteristic of a particular differentiated cell type.
  • ECM extracellular matrix
  • isolated means 1) separated from at least some of the components with which it is usually associated in nature or which naturally accompany it; 2) prepared or purified by a process that involves the hand of man; and/or 3) not occurring in nature.
  • a "peptide”, “polypeptide”, or “protein” comprises a sequence of at least two amino acids linked together by peptide bonds.
  • Amino acid sequences and formulae described herein are written according to convention such that the sequences are read from left to right wherein the left corresponds to the N-terminal end and the right corresponds to the C-terminal end.
  • amino acid domain that does not self-assemble is an amino acid domain that does not self-assemble when present as an isolated peptide (i. e., when not joined or linked to a self-assembling peptide) under conditions (e. g., ionic concentration, peptide concentration, pH, temperature) that would result in self-assembly of an unmodified self-assembling peptide as described below.
  • does not self-assemble is meant that the amino acid domain or peptide does not form nanofilaments or nanofibers, does not form a macroscopic structure, or typically, does not form either ⁇ - sheets, nanofibers, or a macroscopic structure.
  • Treating” or “treatment” includes the administration of the compositions, compounds or agents of aspects of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating or ameliorating the symptoms and/or or arresting or inhibiting further development of the disease, condition, or disorder.
  • a “therapeutically effective amount” is an amount which, alone or in combination with one or more other active agents, can control, decrease, inhibit, ameliorate, prevent or otherwise affect one or more symptoms of a disease or condition to be treated.
  • an “effective amount” is an amount which, alone or in combination with one or more other active agents is sufficient to achieve the indicated objective.
  • an "effective amount" of an agent in the context of regenerating tissue means that the amount of the agent is sufficient to result in tissue regeneration.
  • the present invention is directed to methods and compositions comprising self-assembling peptides comprising a first amino acid domain capable of self- assembly when present in unmodified form and a second amino acid domain that does not self-assemble in isolated or unmodified form.
  • Self-assembling peptides are peptides that are capable of self-assembly into structures include, but are not limited to, macroscopic membranes, nanostructures and the like.
  • Such self-assembling peptides and exemplary peptides that can be used in the first amino acid domain of a peptide described herein have been described, for example, in U.S. Patent Nos. 5,670,483, 5,955,343, 6,368,877, 7,098,028, and 7,449,180 and U.S. Patent Application Publication Nos. 2005/0181973, 2007/0203062 and 2009/0162437 Al, the contents of each of which are expressly incorporated by reference herein.
  • the self-assembling peptides assume regular secondary structures, for example, ⁇ - sheet structures, in solution (e.g., aqueous solution). This may be attributed to the fact that peptides contain two distinct surfaces, one hydrophilic and the other hydrophilic and form complementary ionic bonds with regular repeats on the hydrophilic surface.
  • the side-chains of the peptides partition into two faces, a polar face made up of charged ionic side chains and a nonpolar face made up of hydrophobic groups.
  • the ionic side chains are self- complementary to one another in that the positively charged and negatively charged amino acid residues can form complementary ionic pairs.
  • the complementary ionic sides have been classified into several moduli, i.e., modulus I, II, III, IV, etc., and mixed moduli.
  • Modulus I peptides are those wherein the ionic residues alternate with one positively and one negatively charged residue (-+-+-+-+).
  • Modulus II peptides are those wherein the ionic residues alternate with two positively and two negatively charged residues (—++—++).
  • Modulus IV peptides are those wherein the ionic residues alternate with four positively and two negatively charged residues (— ++ ++).
  • Peptides that self-assemble in isolated form may be referred to herein as unmodified self-assembling peptides to distinguish them from “modified” or “functionalized” self assembling peptides (which, in addition to a first amino acid domain that self-assembles in isolated form, further comprise one or more additional amino acid domains that do not self- assemble when present in isolated form).
  • Modified self-assembling peptides and hydrogels formed therefrom are also described herein as "functionalized.”
  • the first amino acid domain which is capable of self-assembly has alternating hydrophobic and hydrophilic amino acids. In some aspects, the first amino acid domain which is capable of self-assembly is at least 8 amino acids in length. The optimal length of the peptides 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 which can maintain a constant distance upon pairing are referred to as “structurally compatible.”
  • Peptides meeting the criteria described above are expected to self-assemble into macroscopic membranes in homogenous peptide solutions and are referred to herein as “self-complementary peptides” or "peptides having alternating hydrophobic and hydrophilic amino acids.”
  • Such macroscopic membranes can also be formed of heterogenous mixtures of peptides (each of which alone would not form membranes) if the peptides are complementary and structurally compatible to each other.
  • heterogenous mixture of peptides is a mixture of (Lys-Ala-Lys-Ala) 4 and (Glu- Ala-Glu-Ala) 4 or a mixture of (Lys-Ala-Lys-Ala) 4 and (Ala-Asp-Ala-Asp) 4 .
  • Macroscopic membranes do not form in water but form in solution comprising salt. The presence of monovolent metal cations induces membrane formation but divalent cations primarily 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.
  • PBS phosphate buffered saline
  • RCO— an acyl group
  • CH3CO- - an acetyl group
  • the self-complementary peptides self-assemble into various macroscopic structures upon exposure to a sufficient concentration of ions (including, for example, monovalent cations) to form macroscopic porous matrices.
  • These matrices can assume various physical forms, including, but not limited to, ribbons, tape-like structures, two and three-dimensional scaffolds and the like.
  • the matrices are comprised of interwoven filaments about 10 to about 20 nm in diameter with a pore size of about 50 to about 100 nm in diameter.
  • Detailed methods for the preparation of macroscopic structure have been described in U.S. Patent Nos. 5,670,483, 5,955,343, and 6,368,877, the contents of each of which are expressly incorporated by reference herein.
  • Self-assembly of the self- complementary peptides can be initiated by dissolving the peptides in a solution that is substantially free of monovalent cations or contains only a low concentration of such ions (for example, less than about 10 nM) followed by the addition of an ionic solute to a peptide solution or by a changing the pH of the solution.
  • Assembly of the self-complementary peptides into macroscopic structures can also be initiated by the addition of the peptides to a solution comprising monovalent ions in a concentration sufficient to initiate self-assembly.
  • the self-assembling peptides that are expected to form macroscopic structures in homogenous mixtures are represented by one of the following formulae:
  • ⁇ , ⁇ , and ⁇ represent neutral, positively and negatively charged amino acids, respectively, which determine the composition and structure
  • i, j, k and t are integers and denote variable numbers
  • n represents the numbers of repeating units which also determines the length of oligopeptides.
  • self-assembling peptides self-assemble to form a network of nanofibers, resulting in hydrogels of water content higher than 99%, when dissolved water in a range of 1-10 mg/ml.
  • the nano fiber network can give rise to hydrogel formation, creating a macroscopic structure preferably of a size that can be observed with the naked eye and can be three-dimensional.
  • the peptides forming the macroscopic structure can contain between 8 and 200 amino acids, 8 to 64 amino acids, 8 to 36 amino acids, or 8 to 16 amino acids, inclusive.
  • the concentration of the peptides prior to self-assembly can range, for example, between about 0.01% (0.1 mg/ml) and about 99.99%) (999.9 mg/ml), inclusive.
  • the concentration of the peptides prior to self-assembly can be between about 0.1%) (1 mg/ml) and about 10%> (100 mg/ml), inclusive, particularly for cell culture and/or therapeutic applications.
  • the concentration of the peptides prior to self-assembly is between about 0.1%> (1 mg/ml) and about 5% (50 mg/ml), inclusive, or between about 0.5%> (5 mg/ml) and about 5% (50 mg/ml), inclusive.
  • the concentration of the peptides prior to self-assembly is about 5 mg/ml, about 10 mg/ml, about 15 mg/ml, or about 20 mg/ml.
  • peptide scaffolds can be formed with a predetermined shape or volume.
  • an aqueous peptide solution can be placed in a pre-shaped casting mold, and the peptides induced to self-assemble into a scaffold by the addition of an ion, as described herein.
  • the ion can be added to the peptide solution shortly before placing the solution into the mold, provided that care is taken to place the solution into the mold before substantial assembly occurs.
  • the resulting material characteristics, time required for assembly, and geometry and dimensions of the macroscopic peptide scaffold are governed by parameters including the concentration and amount of peptide solution that is applied, the concentration of ion used to induce assembly of the scaffold, the pH, the particular self-assembling peptide sequence, and the dimensions of the casting apparatus.
  • the shape can be selected based upon the intended implantation site.
  • the scaffold can exist as a thin layer, e.g., coating the bottom of a conventional tissue culture or floating in a solution, according to various embodiments of the invention.
  • the layer can, for example, be several microns thick, e.g., 10 microns, 10-50 microns, 50-100 microns, 100-200 microns, etc.
  • the layer can, for example, comprise multiple beta-sheets layers.
  • Self-assembled nanoscale scaffolds can be formed with varying degrees of stiffness or elasticity.
  • the peptide scaffolds typically have a low elastic modulus, e.g., in the range of 1-10 kPa as measured in a standard cone -plate rheometer.
  • VEVK9 and VEVK12 are described in more detail below.
  • Other examples of self- complementary peptides are described, for example, in U.S. Pats. 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.
  • the amino acids of the self-assembling peptides are natural amino acids.
  • the amino acids of the self-assembling motif comprise a non-natural amino acid.
  • the peptides may include L-amino acids, D-amino acids, natural amino acids, nonnatural amino acids, or a combination thereof. Numerous classes of non-natural 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 incorporated by reference herein).
  • An exemplary, non-natural amino acid is hyroxy-proline.
  • L- amino acids are present in the scaffold, degradation produces amino acids that may be reused, e.g., by cells in culture or by cells in a host tissue.
  • the peptides can be chemically synthesized or purified from natural or recombinant sources, and the amino- and carboxy- termini of the peptides may be protected or not protected.
  • the peptide scaffold can be formed from one or more distinct molecular species of peptides which are complementary and structurally compatible with each other. Peptides containing mismatched pairs, such as the repulsive pairing of two similarly charged residues from adjacent peptides, can also form structures if the disruptive force is dominated by stabilizing interactions between the peptides.
  • exemplary self-assembling peptides undergo spontaneous assembly into structures such as nano fibers and macroscopic scaffolds.
  • Exemplary peptides are complementary and structurally compatible and are composed of repeating units of alternating hydrophilic and hydrophobic amino acids, in which the charged residues can include alternating positive and negative charges.
  • An exemplary self-assembling peptide is RADA-16-I (Ac- RAD ARAD ARAD ARAD A-C ONH2 (SEQ ID NO: 18).
  • Functionalized scaffolds comprising RADA-16-I have been described as having utility in bone, cartilage and neural regeneration [6b- 10b]. Functionalized RADA-16-1 can result in a relatively long peptide sequence. It may, under some circumstances, be desirable to manufacture a shorter peptide scaffold.
  • self-assembling peptide scaffolds and properties thereof are influenced by many factors, including the level of hydrophobicity . Therefore, in addition to ionic complementary interactions, the extent of hydrophobic residues can influence the mechanical properties of the scaffolds and the rate of self-assembly. Higher hydrophobicity of a peptide corresponds to a shorter length of peptide required for self-assembly and easier scaffold properties.
  • Natural amino acids that are hydrophobic include alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, and tryptophan.
  • the present invention is directed to self-assembling peptides comprising the amino acid sequence VEVK (SEQ ID NO: 1).
  • the self-assembling peptide comprising the sequence VEVK (SEQ ID NO: 1) is 7 to 16 amino acid in length, 8 to 14 amino acids in length, or 9 to 12 amino acids in length.
  • Non- limiting examples of peptides comprising VEVK (SEQ ID NO: 1) are VEVKVEVKV (SEQ ID NO: 2) and VEVKVEVKVEVK (SEQ ID NO: 3).
  • the amino acid comprising VEVK (SEQ ID NO: l) comprises the sequence VEVKVEVKV (SEQ ID NO: 2) or the sequence VEVKVEVKVEVK (SEQ ID NO: 3).
  • the invention is a self-assembling peptide that comprises:
  • a first amino acid domain that mediates self-assembly wherein the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1) and wherein the peptide is capable of self-assembly in isolated form; and
  • the invention encompasses a macroscopic scaffold comprising a plurality self-assembling peptides wherein said peptides comprise a self-assembling domain comprising the sequence VEVK (SEQ ID NO: 1), wherein the peptide is capable of self- assembly.
  • the invention is directed to an aqueous composition comprising a plurality of peptides comprising a self-assembling motif, wherein each self-assembling motif comprises the amino acid sequence VEVK (SEQ ID NO: l) and wherein said peptides are capable of self-assembly.
  • the invention is a self-assembled nanostructure, wherein the nanostructure comprises a peptide comprising the sequence VEVK (SEQ ID NO: 1) and wherein said peptides are capable of self-assembly.
  • the invention is a method of preparing a self-assembled nanostructure comprising forming an aqueous mixture of peptides comprising the sequence VEVK (SEQ ID NO: 1), wherein said peptides are capable of self-assembly, under conditions suitable for self-assembly of the peptides.
  • the invention is a macroscopic material comprising a plurality of peptides, wherein each peptide comprises the sequence VEVK (SEQ ID NO: l), wherein said peptides are capable of self-assembly.
  • the material is composed of of ⁇ -sheets.
  • the invention is a method for in vitro cell culture comprising adding a macroscopic membrane of the invention to a cell culture medium comprising cells, thereby forming a membrane/culture mixture; and b) maintaining the mixture under conditions sufficient for cell growth.
  • the invention is a macroscopic scaffold comprising a plurality self-assembling peptides, wherein said peptides comprise a self-assembling motif comprising the amino acid sequence VEVK (SEQ ID NO: 1) and wherein said peptides are capable of self-assembly wherein said peptides self-assemble into a ⁇ -sheet macroscopic scaffold; and wherein said macroscopic scaffold encapsulates living cells, said cells being present in said macroscopic scaffold in a three-dimensional arrangement.
  • the peptide comprising the sequence VEVK (SEQ ID NO: 1) is VEVKVEVKV (SEQ ID NO: 2) or
  • the peptides comprising the sequence VEVK (SEQ ID NO: 1) comprises: a. A first amino acid domain that mediates self-assembly, wherein the first amino acid domain comprises the sequence VEVK (SEQ ID NO: 1) and wherein the peptide is capable of self-assembly in isolated form; and
  • 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 and wherein said domain self-assembles into a macroscopic structure when present in unmodified form; and (b) a second amino acid domain that does not self-assemble in isolated form.
  • a first amino acid domain that mediates self-assembly
  • the domain comprises alternating hydrophobic and hydrophilic amino acids that are complementary and structurally compatible and wherein said domain self-assembles into a macroscopic structure when present in unmodified form
  • a second amino acid domain that does not self-assemble in isolated form.
  • the second amino acid domain is a
  • Bioly active motifs that can be used according to the present invention include those described in U.S. Pat. App. Pub. No. 2005/0181973 and in Gelain et al. (2006). PLoS ONE 1(1): el 19. doi: 10.1371/journal.pone.0000119, the contents of which are incorporated by reference herein. Biologically active motifs include, for example, short peptide sequences from proteins in the cellular basement membrane that have been identified as participating in several biological functions such as cell attachment, proliferation, differentiation and migration (Iwamoto et al. (1987). Science 238: 1132-34; Kleinman et al. (1989), PNAS 87: 2279-83; Koliakos et al.
  • the biologically active motif can be an RGD peptide, such as a repetitive RGD sequence such as PRGDSGYRGDS (SEQ ID NO: 15).
  • AASIKVAVSADR SEQ ID NO: 49
  • the sequence AASIKVAVSADR SEQ ID NO: 50
  • the sequences YIGSR SEQ ID NO: 5
  • PDSGR SEQ ID NO: 4
  • RYVVLPR SEQ ID NO: 9 located on the ⁇ chain of laminin promoted cell adhesion.
  • the sequence KAFDITYVRLKF (SEQ ID NO: 51) from the laminin ⁇ chain also promoted HUVEC adhesion and tube formation, as well as neuronal cell adhesion and neurite outgrowth.
  • the peptide sequence TAGSCLRKFSTM (SEQ ID NO: 52) from type IV collagen was found to specifically bind to heparin. Additionally, RGD sequences, found, for example, in nidogen, serve as cell attachment site.
  • Non- limiting examples of biologically active motifs that can be used according to the invention are shown in Table 2 below.
  • the biologically active motif is an amino acid motif that participates in tissue regeneration. In yet another embodiment, the biologically active motif is an amino acid motif that participates in periodontal tissue regeneration.
  • Non-limiting examples of biologically active motifs that participate in periodontal tissue regeneration include, but are not limited to, laminin cell adhesion motifs, an RGD peptide and matrix metalloproteinase degradable motifs.
  • Laminin is the main component of the basement membrane which is both a structural component supporting cells and provides cells with an instructive microenvironment that modulates their functions. It has been reported that laminin has specific cell adhesion properties, to which periodontal ligament fibroblasts and/or osteoblasts can adhere to (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 are 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).
  • RGD peptide is a peptide that comprises contains an RGD (Arg-Gly-Asp) amino acid sequence.
  • RGD is the key binding or recognition sequence for integrins, cell surface receptors that mediate adhesion between cells and the extracellular matrix (ECM).
  • ECM extracellular matrix
  • RGD peptides have been reviewed, for example, in 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, the contents of each of which are expressly incorporated by reference herein.
  • PRGDS SEQ ID NO: 13
  • YRGDS SEQ ID NO: 14
  • RGDSGYRGD SEQ ID NO: 15
  • GRGDSP SEQ ID NO: 83
  • MMPs Matrix metalloproteinases
  • the addition of the non self-assembling amino acid domain does not prevent the modified peptide from self-assembling, e.g., to form nanofibers, a macroscopic structure, or both.
  • the modified peptide self-assembles to form a macroscopic structure composed of nanofibers.
  • an unmodified self-assembling peptide can be altered in any of a number of ways described above that does not include addition of amino acids to the peptide and these alterations are referred to herein as alteration and/or derivitazation.
  • the modified self- assembling peptides of the invention are distinct from naturally occurring molecules, for example, they are not found in naturally occurring molecules, although one or more of the amino acid domains in an inventive peptide may occur in a naturally occurring molecule.
  • the biologically active motif can be placed (e.g., attached via a linking group) closer to the N-terminal end or to the C-terminal end of the first domain comprising a peptide that self-assembles in isolated form.
  • Biologically active motifs which can be the same or can be different, can additionally be placed at both the N-terminal and C-terminal ends of the first domain.
  • the conditions under which self-assembly of the modified self-assembling peptide occurs are the same as the conditions under which the corresponding unmodified self-assembling peptide assembles. In other embodiments of the invention, the conditions under which self-assembly of the modified self-assembling peptide occurs are different from the conditions under which the corresponding unmodified self- assembling peptide assembles. In this case, the conditions for self-assembly of the modified self-assembling peptide are the same as the conditions under which a different (non- corresponding) unmodified self-assembling peptide self-assembles.
  • the second amino acid domain permits assembly of the first amino acid domain so that the peptide assembles to form nanofibers, and/or a macroscopic structure.
  • the peptide forms beta-sheets.
  • an amino acid domain is 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, e. g., 15 or 16 amino acids, 20 amino acids, etc.
  • it will generally be desirable to limit the length of the second amino acid domain so as not to interfere too greatly with self-assembly.
  • the length of the second amino acid domain may be 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, etc. It may be desirable to maintain a certain ratio of amino acids in the self-assembling and non self-assembling portions of the peptide. For example, in certain embodiments of the invention it may be desirable for the non self-assembling domain to constitute 50% of less of the total number of amino acids in the peptide.
  • amino acid domains which sequences are derived from naturally occurring proteins such as those mentioned above, amino acid domains from growth factors, cytokines, chemokines, peptide hormones, peptide neurotransmitters, other biologically active peptides found in the body and the like can also be used (See, for example, Goodman and Gilman, The Pharmacological Basis of Therapeutics, 10th Ed. McGraw Hill, 2001 and Kandel et al, Principles of Neural Science, 4th ed., McGraw Hill, 2000).
  • a putative active peptide motif can be identified as biologically active when a synthetic peptide containing the sequence displays activity after conjugation to a carrier (e.g., IgG, albumin, beads), even if inactive when adsorbed directly on a substrate such as glass, plastic, etc., though they may also display activity when conjugated to a substrate.
  • a carrier e.g., IgG, albumin, beads
  • a soluble form of a biologically active peptide motif can competitively inhibit the function of an intact protein in which the motif is naturally found. Alteration of 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 a naturally occurring protein containing the peptide. A range of different peptide concentrations can be tested, and various combinations can be used.
  • the two or more amino acid domains can be joined using methods known to those of ordinary skill in the art.
  • Non-limiting examples include the use of a linker or bridge, which may be one or more amino acids or a different molecular entity.
  • a linker domain consisting of one or more glycine (G) residues, e.g., 1, 2, 3, 4, 5, etc. glycines, can be used.
  • G glycine
  • the use of glyicine in the linker domain is advantageous because this amino acid is small and has a nonpolar side chain, thus minimizing the likelihood of substantial interference with self- assembly.
  • Other exemplary amino acids are alanine or other amino acids having nonpolar side chains can also be used.
  • modified peptides described in the examples were made by solid phase synthesis of the extended peptide, resulting in a linear chain, variations in which the modifying motif is conjugated or cross- linked to a side chain are also encompassed within the present invention.
  • Methods for achieving such conjugation or cross-linking are well known in the art.
  • a peptide containing a cysteine residue or any amino acid modified to include a sulfur atom
  • the modified self-assembling peptide may be a single linear polymer of amino acids joined by peptide bonds (a structure that may be preferred), or may have a branched structure in which two polymers of amino acids (each being a polymer of amino acids joined by peptide bonds) are attached to one another either covalently or non-covalently (e.g., via a biotin-avidin interaction).
  • Non-limiting examples of cross-linking methods include, but are not limited to, the glutaraldehyde method which couples primarily through the V-amino group and W-amino group, maleimide-sulfhydryl coupling chemistries (e.g., the maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) method), and periodate oxidation methods.
  • glutaraldehyde method which couples primarily through the V-amino group and W-amino group
  • maleimide-sulfhydryl coupling chemistries e.g., the maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) method
  • MBS maleimidobenzoyl-N-hydroxysuccinimide ester
  • numerous cross- linking agents are known.
  • cross-linking agents include, e.g., carboiimides, N- Hydroxysuccinimidyl- 4-azidosalicylic acid (NHS-ASA), dimethyl pimelimidate dihydrochloride (DMP), dimethylsuberimidate (DMS), 3,3'-dithiobispropionimidate (DTBP), etc.
  • NHS-ASA N- Hydroxysuccinimidyl- 4-azidosalicylic acid
  • DMP dimethyl pimelimidate dihydrochloride
  • DMS dimethylsuberimidate
  • DTBP 3,3'-dithiobispropionimidate
  • Bifunctional crosslinking reagents contain two reactive groups, thereby providing a means of covalently linking two target groups.
  • the reactive groups in a chemical crosslinking reagent typically belong to the classes of functional groups including succinimidyl esters, maleimides, and iodoacetamides.
  • a number of common schemes for forming a heteroconjugate involve the indirect coupling of an amine group on one biomolecule to a thiol group on a second biomolecule, usually by a two- or three step reaction sequence.
  • the high reactivity of thiols and their relative rarity in most biomolecules make thiol groups good targets for controlled chemical crosslinking. If neither molecule contains a thiol group, then one or more can be introduced using one of several thiolation methods.
  • the thiol-containing biomolecule may then be reacted with an amine-containing biomolecule using a heterobifunctional crosslinking reagent, e.g., a reagent containing both a succinimidyl ester and either a maleimide or an iodoacetamide.
  • a heterobifunctional crosslinking reagent e.g., a reagent containing both a succinimidyl ester and either a maleimide or an iodoacetamide.
  • Amine-carboxylic acid and thiol- carboxylic acid crosslinking may also be used.
  • l-Ethyl-3-(3- dimethylaminopropyl) carbodiimide (ED AC) can react with biomolecules to form "zero-length" crosslinks, usually within a molecule or between subunits of a protein complex. In this chemistry, the crosslinking reagent is not incorporated into the final product.
  • Disulfide crosslinks of cystines in proteins can be reduced to cysteine residues by dithiothreitol (DTT), tris-(2-carboxyethyl) phosphine (TCEP), or tris-(2- cyanoethyl)phosphine.
  • DTT dithiothreitol
  • TCEP tris-(2-carboxyethyl) phosphine
  • TCEP tris-(2- cyanoethyl)phosphine
  • Amines can be indirectly thiolated by reaction with
  • succinimidyl acetylthioacetate followed by removal of the acetyl group with 50 mM hydroxylamine or hydrazine at near- neutral pH. Tryptophan residues in thiol-free proteins can be oxidized to mercaptotryptophan residues, which can then be modified by
  • self-assembling peptides (including both modified and unmodified self-assembling peptides) self-assemble to form macroscopic structures under a variety of conditions, for example, upon the addition of monovalent cations to an aqueous peptide solution or upon the introduction of a peptide solution to a solution containing monovalent cations.
  • the peptides Prior to self-assembly, the peptides can be dissolved in a solution that is substantially free of monovalent ions (e.g., cations) or contains only a low concentration of such ions, e.g., less than 10, 5, 1, 0.5, or 0.1 mM.
  • Self-assembly may be initiated or substantially accelerated by the addition of an ionic solute to a peptide solution or by a change in pH.
  • an ionic solute for example, NaCl at a concentration of between 5 mM and 5 M induces the assembly of the peptides to form macroscopic structures within a few minutes. Lower concentrations of NaCl can also induce assembly but at a slower rate.
  • Certain of the peptides can also self-assemble in the absence of significant concentrations of ions, in a process that may be dependent on pH. For example, certain of the peptides may remain in solution at a pH of approximately 3.0 but may self-assemble when the pH is raised.
  • self- assembly can be initiated by introducing the peptides into a solution comprising ions, e.g., standard phosphate buffered saline (PBS), tissue culture medium, or a physiological fluid such as blood, cerebrospinal fluid (CSF), etc.
  • PBS standard phosphate buffered saline
  • CSF cerebrospinal fluid
  • the peptides can thus self- assemble at a location in vivo.
  • Preferred ions include monovalent cations such as Li+, Na+, K+, and Cs+.
  • the concentration of the ion is at least 5, 10, 20, or 50 mM in order to induce or substantially accelerate self-assembly.
  • concentration of the ion is at least 5, 10, 20, or 50 mM in order to induce or substantially accelerate self-assembly.
  • the strength of the resulting structure is increased in the presence of ions relative to the strength in the absence of ions, or at a lower ionic concentration (although it is noted that a plateau may be reached at which an increase in ion concentration does not result in increased strength).
  • the invention is a nanostructure comprising a plurality of self-assembling peptides of the invention.
  • Exemplary nanostructures include nanofilaments, nanofibers and nanoscaffolds.
  • the invention is a macroscopic structure comprising a plurality of self-assembling peptides described herein.
  • the macroscopic structure comprises homogenous self-assembling peptides.
  • the macroscopic structure comprises heterogenous self-assembling peptides.
  • the term "homogenous self-assembling peptides" refers to a plurality of identical or same self-assembling peptides.
  • heterogenous self-assembling peptides refers to a plurality of different self-assembling peptides.
  • the macroscopic structure is a macroscopic membrane.
  • the macroscropic membranes comprise, for example, a plurality of interwoven nanofilaments which in turn comprise the inventive self-assembling peptides.
  • the initial concentration of the peptide is a factor in the size and thickness of the membrane formed. In general, the higher the peptide concentration, the higher the extent of membrane formation.
  • the macroscopic membrane takes the form of ⁇ -sheets.
  • the invention is a macroscopic scaffold comprising a plurality of self-assembling peptides described herein wherein the self-assembling peptides self-assemble into a ⁇ -sheet macroscopic scaffold.
  • the invention encompasses methods of using the inventive self- assembling peptides and self-assembled structures thereof as cell culture supports, as self- assembled monolayers imprinted onto solid supports, for the repair and replacement of various tissues, as a scaffold to encapsulate living cells, as part of a controlled-release drug delivery system and for promoting hemostasis.
  • the self-assembling peptide comprises the sequence VEVK (SEQ ID NO: 1).
  • Other uses of self-assembling peptides have been described, for example in, U.S. Pat. App. Pub. Nos. 2002/0072074; 2002/0160471; 2004/0087013; 2004/0242469; 2005/0287186 and 2007/0203062, the contents of each of which are incorporated by reference herein.
  • the materials, membranes and filaments are useful in biomaterial applications, such as medical products (e.g., sutures), artificial skin or internal linings, slow-diffusion drug delivery systems supports for in vitro cell growth or culture and supports for artificial tissue for in vivo use.
  • biomaterial applications such as medical products (e.g., sutures), artificial skin or internal linings, slow-diffusion drug delivery systems supports for in vitro cell growth or culture and supports for artificial tissue for in vivo use.
  • the structures can additionally be used in numerous applications in which permeable and water insoluble material are appropriate, such as separation matrices (e.g., dialysis membranes, chromatographic columns).
  • the macroscopic membranes described herein are useful as slow-diffusion drug delivery vehicles. Because the membranes are resistant to degradation by proteases and stomach acid (pH 1.5), drug delivery vehicles made of these membranes could be taken orally.
  • the small pore size of the membranes also makes them useful as filters, for example, to remove virus and other microscopic contaminants.
  • the pore size (interfilament distance) and diameter of the filaments in the membranes can be varied by varying the length and sequence of the peptides used to form the membranes.
  • the macroscopic membranes can also be modified to give them additional properties.
  • the membranes can be further strengthened by cross-linking the peptides after membrane formation by standard methods.
  • Collagen can be combined with the peptides to produce membranes more suitable for use as artificial skin; the collagen may be stabilized from proteolytic digestion within the membrane.
  • combining phospholipids with the peptides may produce vesicles.
  • the macroscopic structures can also be useful for culturing cells.
  • growth factors such as fibroblast growth factor
  • the porous macrostructure can also be useful for encapsulating cells.
  • the pore size of the membrane can be large enough to allow the diffusion of cell products and nutrients.
  • the cells are, generally, much larger than the pores and are, thus, contained.
  • the invention is a macroscopic scaffold comprising a plurality of self-assembling peptides of the invention, wherein the self-assembling peptides self-assemble into a ⁇ -sheet macroscopic scaffold and wherein said macroscopic scaffold encapsulates living cells and wherein said cells are present in said macroscopic scaffold in a three-dimensional arrangement.
  • the macroscopic scaffold can be prepared by incubating the self-assembling peptides and living cells in an aqueous solution under conditions suitable for self-assembly.
  • the invention also encompasses a method of regenerating a tissue, the method comprising administering to a mammal a macroscopic scaffold comprising the inventive self-assembling peptides are self-assembled in a ⁇ -sheet macroscopic scaffold, wherein said macroscopic scaffold encapsulates living cells, said cells being present in said macroscopic scaffold in a three-dimensional arrangement.
  • the encapsulated cells are present in the macroscopic scaffold in a three-dimensional
  • the method is used to treat or prevent a cartilage defect, connective tissue defect, nervous tissue defect, epidermal lining defect, endothelial lining defect, or arthritis.
  • the macroscopic scaffold can be administered orally, percutaneously, intramuscularly, intravenously, subcutaneously, or by any other appropriate mode.
  • the invention is a method for in vitro cell culture comprising: (a) adding a macroscopic membrane which is formed by self-assembly of the inventive self-assembling peptides in an aqueous solution to a cell culture medium comprising cells, thereby forming a membrane/culture mixture; and (b) maintaining the mixture under conditions sufficient for cell growth.
  • the macroscopic membranes can also be used as a model system for investigating the properties of biological proteins structures with such unusual properties as extreme insolubility and resistance to proteolytic digestion including, but not limited to aggregates of ⁇ -amyloid protein and aggregated scrapie protein.
  • the invention also encompasses method for the regeneration of nerves with the structures comprising self assembling peptides. For example, nerve regeneration can be promoted and directed by transplanting the self- assembled nanostructures along the correct path to their targets.
  • Peptide hydrogels described herein can be used in culturing cells and tissues. Such methods are described in detail in U.S. Patent Application Publication No. 2009/0162437A1.
  • cells and tissues can be cultured on the surface of a hydrogel structure.
  • cells can also be encapsulated within the hydrogel.
  • peptides and living cells can be incubated in an aqueous solution having an iso-osmotic solute at an appropriate concentration to support cell viability, under conditions that in which the peptides are not substantially self-assembled.
  • the solution contains a monovalent cation concentration of less than 10, 5, 1, or 0.1 mM or is substantially free of monovalent cations.
  • the solution can also contain less than less than 10, 5, 1, or 0.1 mM or be substantially free of other ionic species, e.g., other cations or anions.
  • Sufficient ion e.g., monovalent cation
  • ion e.g., monovalent cation
  • the solution can be contained in a pre-shaped mold dimensioned to establish a desired volume or shape of the macroscopic structure.
  • Self- assembly can also be effected by a change in pH (e. g., a rise from a low pH to a higher pH).
  • Cells and agents such as bioactive molecules (e.g., differentiation-inducing agents, proliferation agents), therapeutic compounds, can also be introduced into the peptide solution prior to self-assembly.
  • the self-assembly process then forms a structure that encapsulates the cells or molecules.
  • To achieve even distribution of the cells or molecules within the structure it can be desirable to thoroughly mix the solution prior to initiation of self-assembly. It can be desirable to maintain the cells or agents in a solution that contains substantially no ions or only low concentration of ions in order to avoid initiation or acceleration of self-assembly immediately upon combining the cells or agents with the peptide solution.
  • the cells are preferably maintained in an iso-osmotic solute such as sucrose prior to combination with the peptide solution.
  • the peptides themselves can be dissolved in an isoosmotic solution to which cells (e.g., a cell pellet) or agents are added.
  • the resulting composition may be mixed to achieve a more uniform distribution of cells and/or agents, following which 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 can be cultured on the surface of a peptide hydrogel structure in a similar manner to that in which they are cultured on a conventional substrate such as a tissue culture dish or slide, or a tissue culture dish or slide that is coated with a biologically derived material such as collagen, Matrigel, etc.
  • a biologically derived material such as collagen, Matrigel, etc.
  • cells can be cultured at any desired degree of confluence. If encapsulated, the cells are preferably present in the macroscopic structure in a three-dimensional arrangement.
  • Conditions for culturing should preferably be close to physiological conditions.
  • the pH of the culture medium should preferably be close to physiological pH, preferably between pH 6-8, for example about pH 7 to 7.8, in particular pH 7.4.
  • Physiological temperatures range between about 30° C. to 40° C.
  • Cells can be cultured on or within the peptide structure for any appropriate time, depending upon the cell number and density desired, the proliferation rate of the cells, and the time required for the desired cellular reprogramming to occur. These parameters will vary depending upon the particular cells and purposes for which the invention is to be used. One of ordinary skill in the art will be able to vary these parameters and to observe the effects of doing so, in order to determine the optimal time for maintaining cells in culture on or within the structure. In certain embodiments of the invention the cell are cultured for approximately 3 days, 7 days, 14 days, 21 days, 28 days, 56 days, or 90 days.
  • At least 40, 50, 60, 70, 80, 90, or 95% of the cells are viable 1, 2, 4, 6, or more weeks after formation of the macroscopic scaffold.
  • at least 50%, at least 60%, at least 70%, at least 80% or 90% of the cells are viable one day or one week after formation of the macroscopic scaffold.
  • any cell type can be cultured and/or encapsulated in accordance with the present invention including, but not limited to, vascular endothelial cells and precursors thereof, bone marrow cells, periosteal cells, perichondrial cells, fibroblasts, skeletal myoblasts or myocytes, neuronal cells, hippocampal cells, epidermal cells, non-vascular endothelial cells or smooth muscle cells, keratinocytes, basal cells, spinous cells, granular cells, embryonic stem cells, lung cells, immune system cells, ovarian cells, pancreatic cells, cervical cells, liver cells, foreskin cells or periodontal ligament fibroblast cells.
  • the cells can comprise embryonic, fetal, or adult stem cells, e.g., stem cells that are able to or can be induced to differentiate into any of the preceding cell types.
  • the cells are periodontal ligament fibroblasts.
  • the self-assembling peptides described above can also be used, for wound healing, tissue regeneration, periodontal tissue regeneration and/or in increasing matrix
  • Peptide hydrogel structures optionally comprising cells growing on the surface thereof or encapsulated within may be implanted into the body using any suitable method.
  • Non-limiting methods include surgical procedures and/or by injection.
  • Routes of administration including, but not limited to, oral,
  • percutaneous, intramuscular, intravenous, subcutaneous or parental routes may be utilized.
  • a person having skill in the art will readily be able to select an appropriate delivery technique.
  • the self-assembling peptides are administered using a syringe.
  • the peptides in solution are unassembled or minimally assembled (e.g., where the solution has not formed a gel) are administered to the patient and assembly occurs after administration.
  • the peptides have self-assembled in vitro and are introduced into the body as an assembled matrix. Such methods are described in detail in U.S. Patent Application
  • the invention is directed to a syringe having two compartments wherein the first compartment comprises a solution comprising a self-assembling peptide and a second compartment comprising a gelation fluid.
  • a gelation fluid is a fluid that when combined with the peptide solution results in the formation of a hydrogel.
  • Exemplary gelation fluid include, for example, fluids comprising monovalent cations as described in detail above.
  • the invention is a method of regenerating a damaged tissue in a patient in need thereof comprising administering a self-assembling peptide described herein.
  • tissue such that regeneration of said tissue would be therapeutically useful.
  • Such conditions include, but are not limited to, arthritides, various neurological conditions, neuroendocrine disorders, muscular degeneration, muculotendenous failure, age-related degeneration, trauma, necrosis, cardiac disorder and surgical resection.
  • the damaged tissue can for example be skeletal tissue, bone, tendon, connective or dental tissues.
  • the invention is a method of treating a periodontal disease and/or regenerating dental tissue.
  • the dental tissue is periodontal ligament tissue.
  • Exemplary periodontal diseases are periodontitis, gingivitis, periimplantitis and peri-implant mucositis.
  • Periodontitis gums recede from the teeth and form pockets that become infected.
  • Bacterial toxins and the immune system fighting the infection actually begin damaging the bone and connective tissue that hold teeth in place.
  • Periimplantitis is a complication after surgical implantation of an alloplastic material into the jawbone and affects the tissues around an osseointegrated implant in function, resulting in loss of supporting bone.
  • a therapeutically effective amount of a self- assembling peptide is administered to the periodontium.
  • the peridontium and the tissues that make it up are illustrated in FIG. 1.
  • the periodontium consists of four tissues, gingival, periodontal ligament, cementum and alveolar bone.
  • the gingiva is a pink-colored keratinized mucus membrane that covers parts of the teeth and part of the alveolar bone.
  • the periodontal ligament is a group of connective tissue fibers that attach the tooth to alveolar bone.
  • the cementum is a calcified structure that covers the lower parts of the teeth.
  • the alveolar bone is a set of ridges from the jaw bones (maxillary and mandible) in which the teeth are embedded.
  • the area where periodontal disease is initiated is the gingival sulcus, a pocket between the teeth and the gums.
  • the invention is a scaffold for periodontal tissue regeneration comprising a self-assembling peptide described herein.
  • a scaffold is a degradable hydrogel.
  • 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.
  • the self- assembling peptide comprises a lamin cell adhesion motif.
  • Scaffolds are utilized, for example, to maintain tissue volume, as vehicles for delivering therapeutic and/or biologically active materials to the wound and for promoting selective colonization and proliferation.
  • a goal in the treatment of periodontal disease is the generation of periodontal ligament.
  • Conventional guided tissue regeneration for the treatment of periodontal disease mechanically blocks gingival tissue invasion (which has been shown to be associated with root resorption) while allowing the attachment of periodontal ligament fibroblasts [28a].
  • the scaffold for periodontal tissue regeneration biologically blocks gingival cell invasion and promotes periodontal ligament growth.
  • the scaffold includes a laminin cell adhesion motif, periodontal ligament fibroblasts are able to adhere to the motif whereas gingival cells have less ability to adhere to the motif than periodontal ligament fibroblasts [27a-29a].
  • the invention is a scaffold for tissue regeneration further comprising cells wherein the cells are periodontal ligament fibroblasts.
  • the scaffold comprises a self-assembling peptide comprising a biologically active motif that periodontal ligament fibroblasts are capable of adhering to.
  • the invention is a method of treating periodontal disease comprising administering a scaffold described herein.
  • the invention further encompasses a method of treating periodontal disease comprising administering a scaffold of the invention and further administering a solid biomaterial.
  • the solid biomaterial is osteoconductive.
  • the solid biomaterial can be fabricated of any appropriate material including, but not limited to, calcium and/or phosphorous. Exemplary materials comprise calcium triphosphate and hydroxylapatite.
  • An additional example of a biomaterial is made from ⁇ -tricalcium phosphate. Such a biomaterial is, for example, GEM 2 IS® Growth-Factor Enhanced Matrix.
  • the biomaterial can, for example, take the form of a powder, block forms and/or granules.
  • the solid biomaterial comprises pores having a diameter from about 100 to about 500 microns.
  • the method of treating periodontal disease comprises administering a scaffold described herein and a solid biomaterial and further comprising administration of an additional bioactive agent, such as a pharmacologic agent (including, for example, small molecules and peptides).
  • a pharmacologic agent includes, for example, small molecules and peptides.
  • the pharmacologic agent comprises a recombinant human protein and/or a growth factor.
  • the growth factor is a human recombinant growth factor protein.
  • Exemplary recombinant human proteins are recombinant human platelet-derived growth factor (rh-PDGF), recombinant human bone morphogenetic protein-7 (rh-BMP-7) and recombinant human basic fibroblast growth factor (rh-bFGF).
  • rh-PDGF platelet-derived growth factor
  • rh-BMP-7 recombinant human bone morphogenetic protein-7
  • rh-bFGF recombinant human basic fibroblast growth factor
  • the invention is directed to a method of increasing
  • extracellular matrix protein production in a tissue comprising administering a self-assembling peptide described herein.
  • a preferred extracellular matrix protein is collagen.
  • RGD Arg-Gly-Asp
  • Laminin is a main component of basement membrane. The basement membrane is not only important as a structural component supporting cells, but also gives to the cells an instructive microenvironment that modulates their function.
  • Cell adhesion is a first phase of cell/material interaction and influences the cell's capacity to proliferate, migrate and differentiation. Therefore the fully-synthesis peptide scaffolds functionalized by RGD and laminin cell adhesion motifs show promise as a simple, safe and inexpensive material for periodontal therapy.
  • PRG is peptide scaffold RADA16 through direct coupling to a 2-unit RGD binding sequence
  • PRGDSGYRGDS SEQ ID NO: 15
  • PDS is RADA16 through direct coupling to a laminin cell adhesion motif PDSGR (SEQ ID NO: 4).
  • PDSGR laminin cell adhesion motif
  • these scaffolds significantly promote periodontal ligament fibroblasts cell attachment, proliferation, migration and extracellular matrix protein production, especially type I and type III collagens, which are major extracellular matrix protein components of periodontal ligament [30a], [31a].
  • MMPs matrix metalloproteinases
  • VEVK9 Ac- VEVKVEVKV-CONH 2
  • VEVK12 Ac-VEVKVEVKVEVK- CONH 2
  • the functionalization of VEVK9 by synthesizing extended sequences with a designed 2-unit RGD binding sequence (PRGDSGYRGDS (SEQ ID NO: 15)) or one of two cell adhesion motifs derived from laminin (YIGSR (SEQ ID NO: 5), IKVAV (SEQ ID NO: 6)) added at the C-terminus.
  • the designed 2-unit RGD sequence has previously been shown to be effective in osteoblasts and endothelial cell growth [8b, 25b].
  • the self-assembling peptide VEVK9 has only 9 amino acid residues and its functionalized peptides are shorter than those of RADA16-I, which has 16-amino acid residues, and VEVK9 has the potential for promoting cell activities in a similar way.
  • the self-assembling peptide scaffold functionalized with this sequence has been shown to be degradable by MMP-2, showing promise for use as a tissue engineering scaffold [19b].
  • RADA16, VEVK9, VEVK12 we tested the potentiality of the functional motifs for periodontal tissue regeneration.
  • HPDLF human primary periodontal ligament fibroblasts
  • Example 1 Periodontal ligament fibroblasts attachment, migration and matrix protein production on peptide scaffolds
  • 1% RADA16 solution was obtained as PuraMatrix (3DM Inc./BD Bioscience).
  • VEVK9, VEVK12 and the functionalized peptides were obtained from CPC Scientific (San Jose, CA) and dissolved in water at final concentration of 1% (v/w). The functionalized peptide solutions were then mixed with 1% RADA16, VEVK9 or VEVK12 solution at a ratio of 1 : 1. Each peptide solution was then loaded in the cell culture plate insert (BD Bioscience, Bedford, MA). The medium described below was added to induce hydrogel formation.
  • Primary isolated human periodontal ligament fibroblasts were commercially obtained from Lonza Inc. (HPDLF, WalkersviUe, MD) and routinely grown in the culture medium (SCGM, WalkersviUe, MD) on regular cell culture flask. The cells were plated at 2 x 104 cells on the gel in the inserts. The culture medium was changed every three days. Additional growth factors were not used.
  • the cells on the gel were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 5 min at room temperature.
  • Fluorescent Rhodamin phalloidin and SYTOX® Green were used for labeling F-actin and nuclei, respectively. Images were taken using a fluorescence microscope (Axiovert 25, ZEISS) or laser confocal scanning microscope (Olympus FV300). Fluorescent immunostaining for type I and type III coUagens visualization.
  • the primary antibody for type I collagen (5% Anti-collagen type I, Millpore, MA) was added and incubated at 37° for 40 min, then washing six times with PBS with 1% BSA.
  • the second antibody (0.5% Alexa fluor 488 goat anti-rabbit IgG, Invitrogen) was added and incubated at 37° for another 40 min, then washing as well.
  • the primary antibody for type III collagen (0.5% Anti-collagen type III, Millpore, MA) was added and incubated at 37° for 40 min, then washing.
  • the second antibody (0.5%> Alexa fluor 594 goat ant-mouse IgG, Invitrogen) was added and incubated at 37° for another 40 min. Nonspecific staining as a control was performed by omitting primary antibodies.
  • the functionalized peptide scaffolds promoted greater cell attachment and
  • the functionalized peptide scaffolds PRG and PDS promoted greater cell migration into peptide scaffolds compared to RADA16 (FIG. 4).
  • the functionalized peptide scaffolds PRG and PDS promoted greater type I and type III coUagens compared to RADA16 (FIG. 5).
  • Type I and type III coUagens are well known as major matrix proteins of periodontal ligament.
  • Example 2 Selective cell reproduction of peptide scaffolds (Comparison between periodontal ligament and gingival fibroblasts)
  • Human gingival fibroblasts were commercially obtained from ATCC (HGF-1) and routinely grown in the culture medium (DMEM + 10%FBS) on regular cell culture flask. The cells were plated at 2 x 104 cells on the gel in the inserts. The culture medium was changed every three days.
  • the functionalized peptide scaffold PDS showed selective cell attachment and migration (FIG. 6). Periodontal ligament fibroblasts spread on the surface of the scaffold and migrated into the scaffolds. In comparison, gingival fibroblasts didn't adhere to the scaffold well and migrate into the scaffold at all. The peptide scaffold RADA16 did not show migration in both fibroblasts (not shown). These results suggest that the scaffold may have good potential to provide space and favorable niche to grow periodontal ligament fibroblasts and reconstruct periodontal tissue, restricting the repopulation of the space by gingival fibroblasts.
  • the functionalized peptide scaffolds vPDS_9, vYIG_9 and vIKV_9 also showed selective cell attachments (FIG. 7).
  • the functionalized peptide was obtained from CPC Scientific (San Jose, CA) and dissolved in water at final concentration of 1% (v/w) as well. The functionalized peptide solutions were then mix with 1% VEVK9 or VEVK12 solution. The each peptide solution was loaded in the cell culture plate insert (BD Bioscience, Bedford, MA). The medium described below was added to induce hydrogel formation.
  • the peptide scaffolds vPVG vPRG 9, vPVG_vPRG_12 showed significant cell migration into the scaffolds (FIG. 8). These results suggest that MMP cleavage sites PVGLIG (SEQ ID NO: 19) of functionalized peptide sequence L were degraded by MMP periodontal ligament fibroblasts produced and then the fibroblasts could migrate into the scaffold easily.
  • PDSGR SEQ ID NO:4 cell attachment motif of laminin. It has been reported that periodontal ligament fibroblasts adhered to RGD motif, fibronectin and laminin, and expressed the integrin subunits related to the attachment to these
  • Activation of FAK and Src further activate mitogen activated protein kinase (MAPK) signaling pathway to promote gene transcription.
  • the altered gene transcription leads to translational and post-translational modification to selectively synthesize and secrete extracellular matrix proteins.
  • MAPK mitogen activated protein kinase
  • the interaction between integrin of the fibroblasts - cell adhesion motifs of the scaffolds PRG and PDS triggers an intracellular signaling pathway described above, then the fibroblasts synthesize and secrete type I and III collagens.
  • peptide scaffold RADA16 does not have cell adhesion motif, periodontal ligament fibroblasts seem to adhere to peptide by a different way.
  • VEVK9 and VEVK12 are simple repeating units of amino acids VEVK (Valine- Glutamate-Valine-Lysine) which self-assemble into a nanofiber structure.
  • the self- assembling peptide VEVK9 was functionalized either with RGD, laminin cell adhesion motifs or an MMP cleavable motif in order to mimic extracellular matrix to enhance cell maintenance and function in cell cultures.
  • the functionalized peptides vPRG, vYIG and vIKV were synthesized with the VEVK9 sequence plus an additional motif added to the C-terminus using solid phase synthesis.
  • One motif contains two repetitions of the RGD sequence (PRGDSGYRGDS (SEQ ID NO: 15)), and the others are cell adhesion motifs of laminin (YIGSR (SEQ ID NO: 5), IKVAV(SEQ ID NO: 6)). Glycine residues were used between the self-assembling motif VEVKVEVKV (SEQ ID NO: 2) and the functional motif as a spaced linker to keep the flexibility of the functional peptides.
  • the MMP-2 cleavable motif (PVGLIG (SEQ ID NO: 1 1) was inserted between two VEVK units.
  • these peptides are short sequences, maximum 20 amino acids (in vPRG), and seem to be cost-effective.
  • the peptides were solubilized in water at a concentration of lOmg/ml (1%, w/v). The peptides readily undergo self-assembly to form soft hydrogels. Mixing with self-assembling peptide VEVK9 or VEVK12 facilitated self-assembly and gelation. Tapping mode AFM was used to analyze the formation of nanofibers because this system allowed us to observe peptide filaments without damaging them . Self-assembling peptides VEVK12 and VEVK9 mixed with functionalized peptides vPRG and vPVG formed nanofibers in aqueous solutions.
  • periodontal ligament fibroblasts adhere to the RGD motif, fibronectin and laminin and express the integrin subunits related to the attachment to these extracellular matrix proteins [28b, 29b].
  • the inclusion of these cell adhesion motifs in the peptide scaffolds seems to promote the fibroblasts' adhesion, proliferation and 3-D migration through the interaction with integrin receptors of the fibroblasts.
  • VEVK9 VEVK9
  • peptide scaffolds with VEVK9 mixed with functionalized peptides a large number of cells appeared on surface and inside of the scaffolds.
  • the fibroblasts initially attached to the surface of the scaffold at day 1 proliferated as well as migrated into the scaffold spontaneously.
  • the images exhibit significant increases in fibroblast proliferation and migration due to the effects of the functionalized peptides vPRG and vPVG. It appears that the fibroblasts on the surface of the scaffold migrated into the scaffold to enlarge their sphere of activity.
  • periodontal ligament fibroblasts contribute greatly to the 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, for example by ⁇ ⁇ ⁇ 3 , which is the main RGD-binding integrin [31b]. They suggest that the fibroblasts recognized the exposed RGD adhesion motifs of the scaffold via integrin receptors to adhere to the scaffold. The interaction between the integrin and the RGD motifs yields MMP-2 production by the fibroblasts. As a result, the fibroblasts moved deeper into the scaffold, breaking the MMP cleavable sites
  • a variety of functionalized peptide scaffolds have been developed and shown a great potential for tissue engineering and regenerative medicine [7b, 8b, 25b]. Most of them are functionalized with cell adhesion motifs derived from extracellular matrix proteins. They are responsible for the first interaction between cell and matrix, cell adhesion to promote cell growth included cell migration. But since these functionalized peptide scaffolds are not enzymatically degradable, their ability to promote cell migration is limited. In the nondegradable scaffolds, cells seem to squeeze through the spaces between nanofibers of the scaffolds. As an ameboid cell migration depends on the mechanical properties of the peptide scaffold, the strategy of using a cell adhesion motif is limited to scaffolds with relatively large pores and soft nanofibers which cells can penetrate.
  • enzymatically degradable peptide scaffolds enable a significant increase in cell migration.
  • the peptide scaffolds functionalized with vPVG seem to be enzymatically degradable and promoted proteolytic cell migration.
  • the MMP cleavable motif tested here may be useful to functionalize most of self-assembling peptides without considering the mechanical properties.
  • these peptide scaffolds may be useful as alternatives for naturally occurring extracellular matrix derived materials such as fibrin or collagen, which require difficult purification procedures and carry the risks of immunogenicity and disease transmission.
  • the periodontium the supporting teeth apparatus, consists of four tissues, gingival, periodontal ligament, cementum and alveolar bone.
  • the diverse composition of the periodontium makes periodontal wound healing a complex process because of the interaction between hard and soft connective tissues, implying the selective repopulation of the root surface by cells capable of reforming the cellular and extracellular components of new periodontal ligament, cementum and alveolar bone [44a].
  • Guided tissue regeneration is a conventional method for periodontal tissue reconstruction, which could be driven by excluding or restricting the repopulation of periodontal defects by epithelial and gingival connective cells, providing space and favorable niche to maximize periodontal ligament fibroblasts, cementoblasts and osteoblasts to migrate selectively, proliferate and differentiate. Considering the clinical use of these scaffolds for
  • peptide scaffolds PRG could control osteoblasts activities by changing the concentration of the designer peptide containing two unit of RGD [10a]. It also has been reported that laminin has specific cell adhesion properties, which periodontal ligament fibroblasts and osteoblasts could adhere well, compared with gingival fibroblasts [27a]-[29a]. They suggest that these peptide scaffolds PRG and PDS with laminin cell adhesion motif might be useful as periodontal tissue filler with selective cell repopulation properties.
  • peptide scaffolds For successful clinical use of in periodontal tissue reconstruction, functionalized peptide scaffolds would be required to allow selective cell repopulations and promote angiogenesis. It has been previously reported that the peptide scaffold functionalized with RGD could control osteoblasts' activities by changing the concentration of the
  • Peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision Proc Natl Acad Sci U S A 103: 5054-5059.
  • Transforming growth factor beta is a bifunctional regulator replication and collagen synthesis in osteoblast-enriched cell cultures from fetal rat bone. J Biol Chem 262: 2869-2874.

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Abstract

L'invention concerne une nouvelle classe de peptides autoassemblés, leurs compositions, leurs procédés de préparation et leurs procédés d'utilisation. L'invention concerne également des procédés de régénération de tissus, d'augmentation de la production de protéines de matrice extracellulaire, et des procédés de traitement comprenant l'administration de peptides autoassemblés.
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JPWO2014065017A1 (ja) * 2012-10-26 2016-09-08 株式会社ジェイ・エム・エス 人工血管、および、人工血管の製造方法
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