Self-assembling peptide amphiphiles displaying transforming growth factor beta 1 (TGF-β1) mimetic epitopes

Abstract

Provided herein are self-assembling peptide amphiphiles (PAs) containing bioactive transforming growth factor beta 1 (TGF-β1) mimetic epitopes, high aspect ratio nanostructures of PAs exhibiting TGF-β1 mimetic epitopes, and methods for enhancing cartilage regeneration / repair and / or treatment of osteoarthritis and other musculoskeletal injuries and diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 227,097, filed July 29, 2021, which is incorporated by reference in its entirety.

[0002] Sequence Listing The text of the computer readable sequence listing filed herewith, entitled "39648-601_SEQUENCE_LISTING", created on July 28, 2022, having a file size of 10,544 bytes, is hereby incorporated by reference in its entirety.

[0003] Provided herein are self-assembling peptide amphiphiles (PAs) containing bioactive transforming growth factor beta 1 (TGF-β1) mimetic epitopes, high aspect ratio nanostructures of PAs exhibiting TGF-β1 mimetic epitopes, and methods for enhancing cartilage regeneration / repair and / or treatment of osteoarthritis and other musculoskeletal injuries and diseases. [Background technology]

[0004] Cartilage degeneration is one of the most widespread and incurable musculoskeletal disorders. Articular cartilage is essential for joint movement and is constantly subjected to high loads and repetitive impacts, which over time can lead to degeneration, including osteoarthritis (OA). 1 OA causes painful disability and comorbidity for over 32 million adults in the United States, a number that is predicted to increase with the aging population. 2 Cartilage regeneration faces several unique biological and mechanical challenges that are poorly addressed by existing therapies and results in $72 billion in direct medical costs nationwide. 2 Currently, there are no approved disease-modifying OA drugs, and current treatments are palliative rather than regenerative, aiming to slow disease progression or postpone inevitable total joint replacement. 3、4Microfracture remains the gold standard for clinical cartilage repair. 5 Recently, new biomaterials and growth factor strategies have emerged as promising engineering approaches. However, these strategies often face problems of non-biodegradability, low mechanical strength, and off-target immune effects. 6、7 To promote cartilage regeneration, biomaterial scaffolds must withstand the mechanical loads of the joint environment while promoting the biomolecular signaling necessary for chondrogenic differentiation and maintenance. Summary of the Invention

[0005] Provided herein are self-assembling peptide amphiphiles (PAs) containing bioactive transforming growth factor beta 1 (TGF-β1) mimetic epitopes, high aspect ratio nanostructures of PAs exhibiting TGF-β1 mimetic epitopes, and methods for enhancing cartilage regeneration / repair and / or treatment of osteoarthritis and other musculoskeletal injuries and diseases.

[0006] In some embodiments, provided herein is a composition comprising a bioactive peptide amphiphile (PA), the bioactive peptide amphiphile (PA) comprising (i) a hydrophobic non-peptide segment, (ii) a β-sheet forming peptide segment, (iii) an acidic peptide segment, and (iv) a TGF-β1 mimetic epitope peptide.

[0007] In some embodiments, the TGF-β1 mimetic epitope peptide comprises an amino acid sequence having 3 or less (e.g., 3, 2, 1, 0) substitutions to CESPLKRQC (SEQ ID NO:1). In some embodiments, the TGF-β1 mimetic epitope peptide has at least 50% sequence similarity (e.g., 50%, 60%, 70%, 80%, 90%, 100%, or ranges therebetween) to CESPLKRQC (SEQ ID NO:1).

[0008] In some embodiments, the hydrophobic non-peptide segment of the bioactive peptide amphiphile comprises an acyl chain. In some embodiments, the acyl chain is a C6-C 20 (For example, C6, C8, C 10 , C 12 , C 14 , C 16 , C 18 , C 20 ).

[0009] In some embodiments, the beta-sheet forming peptide segments of the bioactive peptide amphiphiles contain a combination of 2-6 V and A residues. In some embodiments, the beta-sheet forming peptide segments of the bioactive peptide amphiphiles and charged peptide amphiphiles are selected from VVVAAA (SEQ ID NO:3), AAAVVV (SEQ ID NO:4), AAVV (SEQ ID NO:5), VVAA (SEQ ID NO:6), AA, VV, VA, or AV.

[0010] In some embodiments, the acidic peptide segment of the bioactive peptide amphiphile comprises a combination of one to four Glu (E) and / or Asp (D) residues, hi some embodiments, the acidic peptide segment is selected from E, EE, EEE, D, DD, DDD, ED, DE, EDE, DED, EDD, and DEE.

[0011] In some embodiments, the bioactive PA comprises a backbone PA selected from C16-AAEE (SEQ ID NO: 7), C16-AEAE (SEQ ID NO: 8), and C16-VVVAAAEEE (SEQ ID NO: 9). In some embodiments, the TGF-β1 mimetic epitope peptide is CESPLKRQC (SEQ ID NO: 1) cyclized via a disulfide. In some embodiments, the TGF-β1 mimetic epitope peptide is linked to the backbone PA by a lysine linker.

[0012] In some embodiments, the compositions (e.g., nanostructures) herein further comprise a diluted PA comprising (i) a hydrophobic non-peptide segment, (ii) a β-sheet forming peptide segment, and (iii) a charged peptide segment.

[0013] In some embodiments, the hydrophobic non-peptide segment of the diluted peptide amphiphile comprises an acyl chain. In some embodiments, the acyl chain is a C6 to C 20 (For example, C6, C8, C 10 , C 12 , C 14 , C 16 , C 18 , C 20 ).

[0014] In some embodiments, the beta-sheet forming peptide segments of the diluted peptide amphiphiles contain a combination of 2-6 V and A residues. In some embodiments, the beta-sheet forming peptide segments of the bioactive peptide amphiphiles and charged peptide amphiphiles are selected from VVVAAA (SEQ ID NO:3), AAAVVV (SEQ ID NO:4), AAVV (SEQ ID NO:5), VVAA (SEQ ID NO:6), AA, VV, VA, or AV.

[0015] In some embodiments, the acidic peptide segment of the diluted peptide amphiphile comprises a combination of one to four Glu (E) and / or Asp (D) residues, hi some embodiments, the acidic peptide segment is selected from E, EE, EEE, D, DD, DDD, ED, DE, EDE, DED, EDD, and DEE.

[0016] In some embodiments, the diluent PA comprises a backbone PA selected from C16-AAEE (SEQ ID NO: 7), C16-AEAE (SEQ ID NO: 8), and C16-VVVAAAEEE (SEQ ID NO: 9).

[0017] In some embodiments, compositions (e.g., nanostructures) herein comprise 5%-95% (mol) of bioactive peptide amphiphile and 5%-95% (mol) of dilute peptide amphiphile. For example, 5-95% can include 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or ranges therebetween (e.g., 25-75%).

[0018] In some embodiments, provided herein is a method of promoting cartilage repair or regeneration comprising administering to a subject suffering from cartilage defect or damage a PA nanostructure composition herein.

[0019] In some embodiments, provided herein is a method of treating osteoarthritis or a musculoskeletal injury or disease, comprising administering a PA nanostructure composition to a subject suffering from osteoarthritis or a musculoskeletal injury or disease.

[0020] In some embodiments, provided herein is a method for preventing osteoarthritis or musculoskeletal injury or disease, comprising administering a PA nanostructure composition to a subject at high risk for osteoarthritis or musculoskeletal injury or disease. [Brief description of the drawings]

[0021] [Figure 1] 1A-D: Chemical structures of PA and peptide molecules: (a) skeletal PA-a (SEQ ID NO: 7) (top) and skeletal PA-b (SEQ ID NO: 8) (bottom), where R=H, (b) TGF-β1 mimetic PA, (c) linear lnTGF-β1 PA, and (d) TGF-β1 mimetic peptide (SEQ ID NO: 1). [Diagram 2]2A-J: (a-f) Cryo-TEM micrographs of (a) scaffold PA-a, (b) TGF PA-a, (c) linear lnTGF PA-a, (d) scaffold PA-b, (e) TGF PA-b, and (f) linear lnTGF PA-b nanostructures. The scaffold PA nanostructures are assembled at 100 mol%, and the epitope nanostructures are co-assembled with diluted scaffold PA at 10 mol%. (g-j) Structural analysis of scaffold PA, TGF PA, and lnTGF PA nanostructures on each scaffold PA-a (blue shade) and scaffold PA-b (red shade) based systems. (g) SAXS patterns, (h) WAXS patterns, (i) CD spectra, and (j) FTIR spectra. All PA solutions were dissolved in 12 mM total PA, 30 mM NaCl, and water at pH 6.8. (***, p<0.001). [Diagram 3] 3A-C: Analysis of supramolecular dynamics of TGF-β1 mimetic PA assemblies, where TGF PA molecules are co-assembled with dilute scaffold PA molecules at 10 mol%. (a) Fluorescence anisotropy of TGF PA-a and TGF PA-b assemblies with TAMRA-labeled TGF-β1 mimetic epitopes. (b) 1H NMR spectrum peak intensity as a function of time and regression line for TGF PA-a and TGF PA-b assemblies. (c) 1H spin lattice relaxation rate of protons in TGF PA-a and TGF PA-b assemblies. [Figure 4]4A-F. Smad2 activation and downstream TGF-β1 pathway analysis in human chondrocytes treated with TGF PA. (a) Western blots of Sox9, phosphorylated Smad2 (p-Smad2), Smad2, and actin in chondrocytes treated with TGF PA-a and TGF PA-b at various co-assembly ratios and concentrations for 4 h in vitro. TGF-β1 protein (rhTGF-β1) and starvation medium (Strv) were used as positive and negative controls, respectively. (b) Quantitative densitometric analysis of western blot data from (a) showing the fraction of Smad2 that is phosphorylated (*, vs. rhTGF-β1; #, vs. 10% TGF PA-a 50 μM). (c) Western blot of p-Smad2 and Smad2 in chondrocytes treated with Strv, rhTGF-β1, TGF mimetic peptide, and different PA conditions (skeletal PA, TGF mimetic PA, and linear lnTGF PA) for 4 hours in vitro. (d) Quantitative densitometric analysis of Western blot data from (c) showing the fraction of Smad2 that is phosphorylated (*, vs. rhTGF-β1; #, vs. TGF PA-a). (e) Western blot of p-Smad2 and Smad2 in chondrocytes treated with Strv, rhTGF-β1, TGF PA-a, and TGF PA-b without (-) and with (+) the addition of pan-TGF-β neutralizing antibody 1D11, respectively. (f) Quantitative densitometric analysis of Western blot data from (e) showing the fraction of Smad2 that is phosphorylated (*, vs. rhTGF-β1-1D11; #, TGF PA-a β1-1D11). (#, p<0.05; ** / ##, p<0.01; *** / ###, p<0.001). [Diagram 5]5A-E ECM protein synthesis in human chondrocytes treated with TGF PA. (a) Western blots of collagen II, cartilage oligomeric matrix protein (COMP), Sox9, and aggrecan in chondrocytes treated with Ctrl, rhTGF-β1, TGF mimetic peptide, and different PA conditions (skeletal PA, TGF mimetic PA, and linear TGF PA) for 3 days in vitro. (b) Quantitative densitometric analysis of western blot data from (a) showing the fraction of each ECM protein normalized to actin (*, vs. rhTGF-β1; #, TGF PA-a). (c-d) Fluorescence micrographs of human chondrocytes treated with Ctrl, rhTGF-β1, TGF mimetic peptide, and different PA conditions (skeletal PA, TGF mimetic PA, and linear TGF PA) for 3 days in vitro. Cells were stained for collagen II (red) in (c), aggrecan (red), F-actin (green), and DAPI (nuclei, blue) in (d). (e) Fluorescence intensity quantification of collagen II and aggrecan from ICC micrographs of (c-d) (*, vs. rhTGF-β1; #, TGF PA-a). (* / #, p<0.05; **, p<0.01; *** / ###, p<0.001). [Figure 6] 6A-G: Mechanical characterization of PA hydrogels and chondrogenic behavior of human chondrocytes encapsulated in the hydrogels. (a-c) SEM micrographs of (a) scaffold PA-a only, (b) TGF PA-a, and (c) lnTGF PA-a PA gels. (d) Storage and loss modulus as a function of strain for scaffold PA-a only, TGF PA-a, and lnTGF PA-a PA gels. Flow strain and elastic modulus are indicated by vertical and horizontal lines, respectively. (e-g) Human chondrocytes encapsulated in PA hydrogels. 3D z-stack reconstructions of cells encapsulated in (e) scaffold PA-a gel, (f) TGF PA-a gel, and (g) lnTGF PA-a gel after 3 days in vitro. Cells were stained for F-actin (green) and nuclei (red), with the overlay areas between F-actin and nuclei appearing as yellow. [Figure 7]Cryo-TEM micrographs of TGF PA-a (top) and TGF PA-b (bottom) coassembled with diluted scaffolds PA-a and PA-b at 10, 25, 50, 75, and 100 mol %, respectively. [Figure 8] Cryo-TEM micrographs of linear lnTGF PA-a (top) and lnTGF PA-b (bottom) coassembled with diluted scaffolds PA-a and PA-b at 10, 25, and 50 mol %, respectively. [Figure 9] (b) Cryo-TEM micrograph of the TGF-mimetic peptide at 100 mol %. (b) Dynamic light scattering of micellar TGF-mimetic peptide aggregates at 100 mol %. [Figure 10] Dynamic light scattering of micellar TGF-mimetic peptide aggregates at 100 mol %. [Figure 11] 11A–B Structural analysis of TGF PA-a (top) and TGF PA-b (bottom) coassembled with diluted scaffolds PA-a and PA-b at 10, 25, 50, 75, and 100 mol%, respectively. (a) SAXS patterns, and (b) WAXS patterns of the TGF PA coassemblies. [Figure 12] 12A–B Structural analysis of linear lnTGF PA-a (top) and lnTGF PA-b (bottom) coassembled with diluted scaffolds PA-a and PA-b at 10, 25, and 50 mol %, respectively. (a) SAXS patterns, and (b) WAXS patterns of lnTGF PA coassemblies and peptide micelle structures. [Figure 13] 13A–B Secondary structure analysis of TGF PA-a (top) and TGF PA-b (bottom) coassembled with dilute scaffold PA-a and scaffold PA-b at 10, 25, 50, 75, and 100 mol %, respectively. (a) Circular dichroism and (b) FTIR spectra of the TGF PA coassemblies. [Figure 14] 14A–B Structural analysis of linear lnTGF PA-a (top) and lnTGF PA-b (bottom) coassembled with dilute scaffold PA-a and scaffold PA-b at 10, 25, and 50 mol %, respectively. (a) Circular dichroism and (b) FTIR spectra of lnTGF PA coassembled and peptide micelle structures. [Figure 15]TEM micrographs of TGF PA nanostructures in which 0.2 mol % of TGF PA molecules are conjugated to TAMRA dye and TGF PA is coassembled with diluted scaffold PA at 10 mol %. [Figure 16] 16A-B are 1H NMR spectra of protons in (a) TGF PA-a and (b) TGF PA-b. The methylene protons of the epitope ridine residues are shown in red, and the methyl protons of the terminal carbons of the alkyl chains are shown in blue. [Figure 17] 17A-B are proton positions used in T2-NMR for (a) TGF PA-a and (b) TGF PA-b. The methylene protons of the epitope ridine residues are shown in red, and the methyl protons of the terminal carbons of the alkyl chains are shown in blue. [Figure 18] 18A-B Viability assays testing the biocompatibility of TGF PA-a and TGF PA-b at various co-assembly ratios and concentrations. TGF-β1 protein (rhTGF-β1) and growth medium were used as controls. (a) Fluorescence micrographs of cells treated in vitro for 24 hours and subsequently stained for calcein AM (green, live) and propidium iodide (red, dead). (b) Quantification of cell survival based on cells / mm2 (#, vs. medium control). (*, p<0.05; ##, p<0.01). [Figure 19] 19A-B Viability assays testing the biocompatibility of TGF PA-a and TGF PA-b at various co-assembly ratios and concentrations. TGF-β1 protein (rhTGF-β1) and growth medium were used as controls. (a) Fluorescence micrographs of cells treated in vitro for 3 days and subsequently stained for calcein AM (green, live) and propidium iodide (red, dead). (b) Quantification of cell survival based on cells / mm2 (#, vs. medium control; ^, vs. 10% TGF PA-a 10 μM). (* / # / ^, p<0.05; ** / ## / ^^, p<0.01; *** / ### / ^^^, p<0.001). [Figure 20]20A-B Viability assays testing the biocompatibility of different PA conditions (TGF mimetic peptide, scaffold, TGF mimetic PA 10 mol% co-assembly, and linear lnTGF PA 10 mol% co-assembly) at various concentrations. TGF-β1 protein (rhTGF-β1) and growth medium were used as controls. (a) Fluorescence micrographs of cells treated in vitro for 24 hours and subsequently stained for calcein AM (green, live) and propidium iodide (red, dead). (b) Quantification of cell viability based on cells / mm2. (*, p<0.05; **, p<0.01; ***, p<0.001) [Figure 21] 21A-B Viability assays testing the biocompatibility of different PA conditions (TGF mimetic peptide, scaffold, TGF mimetic PA 10 mol% co-assembly, and linear lnTGF PA 10 mol% co-assembly) at various concentrations. TGF-β1 protein (rhTGF-β1) and growth medium were used as controls. (a) Fluorescence micrographs of cells treated in vitro for 3 days and subsequently stained for calcein AM (green, live) and propidium iodide (red, dead). (b) Quantification of cell survival based on cells / mm2. (*, p<0.05; **, p<0.01; ***, p<0.001) [Figure 22] Quantitative densitometric analysis of Western blot data from Figure 4a showing Sox9 expression normalized to actin (^, ​​vs. 10% TGF PA-b 10 μM; °, vs. 25% TGF PA-b 50 μM). (° / ^, p<0.05; ^^, p<0.01). [Diagram 23] LDH release from gel-encapsulated cells after 3 days in vitro. [Figure 24] 24A–F Rheological analysis of scaffold PA-a (a, d), TGF PA-a (b, e), and lnTGF PA-a gels (c, f). (a–c) Frequency sweeps showing the storage modulus (G') and loss modulus (G") for angular frequencies ranging from 0 to 100 rad / s. (d–f) Strain sweeps showing the storage modulus (G') and loss modulus (G'') for shear strains ranging from 0 to 100%. [Diagram 25]25A–B Mechanical properties of hybrid TGF PA gels. (a) Storage (G') and loss (G") moduli of TGF PA-a slurries with varying concentrations of crosslinked HA particles. (b) Strain required to fracture TGF PA-a hydrogels defined by the crossover point when G' = G" (**, p<0.01; ***, p<0.001). [Figure 26] 26A-C Implantation of TGF-mimetic PA slurry in a rabbit osteochondral defect model. After implantation into the medial condyle, slurry retention was tracked using dye-labeled PA molecules (a) 1 day, (b) 2 days, and (c) 7 days after surgery. Fluorescence imaging (middle row) and H&E staining (bottom row) of histological sections of the condyle showed good implant integration and biodegradation as new cartilage was formed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] definition Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before describing the materials and methods of the present invention, it should be understood that the present invention is not limited to the specific molecules, compositions, methodologies, or protocols described herein, as they may vary according to routine experimentation and optimization. It should also be understood that the terminology used herein is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. However, in case of conflict, the present specification including definitions shall prevail. Thus, in the context of the embodiments described herein, the following definitions shall apply.

[0024] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "peptide amphiphile" is a reference to one or more peptide amphiphiles and equivalents thereof known to those skilled in the art, and so forth.

[0025] As used herein, the term "comprise" and linguistic variations thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., without excluding the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term "consisting of" and linguistic variations thereof indicate the presence of the recited feature(s), element(s), method step(s), etc., and excludes any unrecited feature(s), element(s), method step(s), etc., except for impurities normally associated therewith. The phrase "consisting essentially of" indicates the recited feature(s), element(s), method step(s), etc., and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open term "comprising." Such embodiments encompass the multiple closed "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described using such language.

[0026] The term "amino acid" refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomeric forms, unless otherwise indicated, if their structures permit such stereoisomeric forms.

[0027] Naturally occurring amino acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0028] Unnatural amino acids include azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, naphthylalanine ("naph"), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tertiary butylglycine ("tBuG"), 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, and the like. Examples of suitable hydroxyl groups include, but are not limited to, lysine, homoproline ("hPro" or "homoP"), hydroxylysine, allo-hydroxylysine, 3-hydroxyproline ("3Hyp"), 4-hydroxyproline ("4Hyp"), isodesmosine, allo-isoleucine, N-methylalanine ("MeAla" or "Nime"), N-alkylglycines ("NAG") including N-methylglycine, N-methylisoleucine, and N-alkylpentylglycines ("NAPG") including N-methylpentylglycine. N-methylvaline, naphthylalanine, norvaline ("Norval"), norleucine ("Norleu"), octylglycine ("OctG"), ornithine ("Orn"), pentylglycine ("pG" or "PGly"), pipecolic acid, thioproline ("ThioP" or "tPro"), homolysine ("hLys"), and homoarginine ("hArg").

[0029] The term "amino acid analog" refers to a natural or unnatural amino acid in which one or more of the C-terminal carboxy group, the N-terminal amino group, and the side chain bioactive group are chemically blocked, reversibly or irreversibly modified, or otherwise changed to another bioactive group. For example, aspartic acid-(beta-methyl ester) is an amino acid analog of aspartic acid, N-ethylglycine is an amino acid analog of glycine, or alanine carboxamide is an amino acid analog of alanine. Other amino acid analogs include methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine ​​sulfoxide, and S-(carboxymethyl)-cysteine ​​sulfone.

[0030] As used herein, the term "peptide" refers to an oligomer to a short polymer of amino acids linked together by peptide bonds. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), a peptide is about 50 amino acids or less in length. A peptide can contain natural amino acids, unnatural amino acids, amino acid analogs, and / or modified amino acids. A peptide can be a subsequence of a naturally occurring protein or a non-natural (artificial) sequence.

[0031] As used herein, the term "artificial" refers to compositions and systems that are designed or prepared by man and do not occur in nature, for example, artificial peptides, peptoids, or nucleic acids that contain non-naturally occurring sequences (e.g., peptides that do not have 100% identity to a naturally occurring protein or fragment thereof).

[0032] As used herein, a "conservative" amino acid substitution refers to the replacement of an amino acid in a peptide or polypeptide with another amino acid that has similar chemical properties, such as size or charge. For purposes of this disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another: 1) Alanine (A) and Glycine (G), 2) Aspartic acid (D) and glutamic acid (E), 3) Asparagine (N) and Glutamine (Q), 4) arginine (R) and lysine (K), 5) isoleucine (I), leucine (L), methionine (M), and valine (V); 6) phenylalanine (F), tyrosine (Y), and tryptophan (W); 7) serine (S) and threonine (T), and 8) Cysteine ​​(C) and methionine (M).

[0033] Naturally occurring residues may be divided into classes based on common side chain properties, e.g., polar positive (or basic) (histidine (H), lysine (K), and arginine (R)), polar negative (or acidic) (aspartic acid (D), glutamic acid (E)), polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)), nonpolar fatty acids (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)), nonpolar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)), proline and glycine, and cysteine. As used herein, a "semi-conservative" amino acid substitution refers to the replacement of an amino acid in a peptide or polypeptide with another amino acid within the same class.

[0034] In some embodiments, unless otherwise specified, conservative or semi-conservative amino acid substitutions may also include non-naturally occurring amino acid residues that have similar chemical properties to the natural residues. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. The embodiments herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs.

[0035] Non-conservative substitutions may involve exchanging a member of one class for a member of another class.

[0036] As used herein, the term "sequence identity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequential composition of monomer subunits. The term "sequence similarity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) differ only by conservative and / or semi-conservative amino acid substitutions. "Percent sequence identity" (or "percent sequence similarity") is calculated by (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions that contain identical (or similar) monomers (e.g., the same amino acid occurs in both sequences, similar amino acids occur in both sequences) to obtain the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to obtain the percent sequence identity or percent sequence similarity. For example, if peptide A and peptide B are both 20 amino acids long and have identical amino acids at all positions except one, peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical characteristics (e.g., both were acidic), peptide A and peptide B will have 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to those in a portion of peptide C, peptides C and D will have 70% sequence identity, but peptide D will have 93.3% sequence identity with the optimal comparison window of peptide C. For purposes of calculating "percent sequence identity" (or "percent sequence similarity") herein, any gap in the aligned sequences is treated as a mismatch at that position.

[0037] Any polypeptide described herein as having a particular percent sequence identity or similarity (e.g., at least 70%) with a reference sequence ID number may also be expressed as having a maximum number of substitutions (or terminal deletions) relative to that reference sequence. For example, a sequence having at least Y% sequence identity (e.g., 90%) with SEQ ID NO: Z (e.g., 100 amino acids) may have a maximum of X substitutions (e.g., 10) with SEQ ID NO: Z, and thus may also be expressed as "having no more than X (e.g., 10) substitutions with SEQ ID NO: Z."

[0038] As used herein, the term "nanofiber" refers to an elongated or thread-like filament typically having a diameter of less than 100 nanometers (e.g., having a length dimension significantly greater than its width or diameter).

[0039] As used herein, the term "supramolecular" (e.g., "supramolecular complex", "supramolecular interaction", "supramolecular fiber", "supramolecular polymer", etc.) refers to non-covalent interactions between the resulting molecules (e.g., polymers, macromolecules, etc.) and the resulting multicomponent assemblies, complexes, systems, and / or fibers.

[0040] As used herein, the terms "self-assemble" and "self-assembly" refer to the formation of distinct, non-random, aggregate structures from component parts, which occur spontaneously through the random movement of the components (e.g., molecules) due solely to the inherent chemical or structural properties and attractive forces of those components.

[0041] As used herein, the term "peptide amphiphile" refers to a molecule that includes at least a non-peptide lipophilic (hydrophobic) segment, a structural peptide segment, and / or a charged peptide segment (often both), and optionally a biologically active segment (e.g., a linker segment, a biologically active segment, etc.). Peptide amphiphiles may exhibit a net charge at physiological pH, with either a net positive or negative charge, or may be zwitterionic (i.e., have both positive and negative charges). Certain peptide amphiphiles consist of or include (1) a hydrophobic, non-peptide segment (e.g., containing an acyl group of six or more carbons), (2) a structural peptide segment (e.g., beta-sheet forming), (3) a charged peptide segment, and (4) a biologically active segment (e.g., a linker segment).

[0042] As used herein and in the appended claims, the terms "lipophilic moiety" or "hydrophobic moiety" refer to a moiety (e.g., an acyl, ether, sulfonamide, or phosphodiester moiety) located at one end (e.g., C-terminus, N-terminus) of a peptide amphiphile, which may be referred to herein and elsewhere as a lipophilic or hydrophobic segment or component. The hydrophobic segment must be of sufficient length to provide amphiphilic behavior and to provide aggregate (or nanosphere or nanofiber) formation in water or another polar solvent system. Thus, in the context of the embodiments described herein, the hydrophobic component is preferably a compound of the formula: n-1 H 2n-1 They contain a single linear acyl chain of C(O)--, where n=2-25. In some embodiments, the linear acyl chain is a lipophilic group (saturated or unsaturated carbon), palmitic acid. However, other lipophilic groups may be used in place of the acyl chain, such as steroids, phospholipids, and fluorocarbons.

[0043] As used herein, the term "structural peptide" refers to a portion of a peptide amphiphile that is typically disposed between a hydrophobic segment and a charged peptide segment. Structural peptides are generally composed of 3-10 amino acid residues with non-polar, uncharged side chains (e.g., His (H), Val (V), Ile (I), Leu (L), Ala (A), Phe (F)) that are selected for their tendency to form hydrogen bonds or other stabilizing interactions (e.g., hydrophobic interactions, van der Waals interactions, etc.) with adjacent structural segments. In some embodiments, nanofibers of peptide amphiphiles with structural peptide segments exhibit linear or 2D structure when examined by microscopy and / or α-helical and / or β-sheet characteristics when examined by cyclic dichroism (CD).

[0044] As used herein, the term "beta (β) sheet-forming peptide segment" refers to a structural peptide segment that has a tendency to exhibit β-sheet-like properties (e.g., when analyzed by CD). In some embodiments, the amino acids in the beta (β) sheet-forming peptide segment are selected because they tend to form a beta sheet secondary structure. Examples of suitable amino acid residues selected from the 20 naturally occurring amino acids include Met (M), Val (V), Ile (I), Cys (C), Tyr (Y), Phe (F), Gln (Q), Leu (L), Thr (T), Ala (A), and Gly (G) (listed in order of tendency to form beta sheets). However, non-naturally occurring amino acids with similar beta sheet forming tendency may also be used. Peptide segments that can interact to form beta sheets and / or peptide segments that have a tendency to form beta sheets are understood (see, e.g., Mayo et al. Protein Science (1996), 5:1301-1315; incorporated herein by reference in its entirety).

[0045] As used herein, the term "charged peptide segment" refers to a portion of a peptide amphiphile that is rich in charged amino acid residues (e.g., greater than 50%, greater than 75%, etc.), or amino acid residues that carry a net positive or negative charge under physiological conditions. Charged peptide segments can be acidic (e.g., negatively charged), basic (e.g., positively charged), or zwitterionic (e.g., having both acidic and basic residues).

[0046] As used herein, the terms "carboxy-rich peptide segment," "acidic peptide segment," and "negatively charged peptide segment" refer to a peptide sequence of a peptide amphiphile that includes one or more amino acid residues having a side chain exhibiting a carboxylic acid side chain (e.g., Glu (E), Asp (D), or an unnatural amino acid). The carboxy-rich peptide segment may optionally contain one or more additional (e.g., non-acidic) amino acid residues. As will be apparent to one of skill in the art, unnatural amino acid residues, or peptidomimetics having acidic side chains, can be used. There can be from about 2 to about 7 amino acids, and / or about 3 or 4 amino acids in the segment.

[0047] As used herein, the terms "amino-rich peptide segment," "basic peptide segment," and "positively charged peptide segment" refer to a peptide sequence of a peptide amphiphile that includes one or more amino acid residues having a side chain exhibiting a positively charged acid side chain (e.g., Arg (R), Lys (K), His (H), or a non-natural amino acid, or a peptidomimetic). The basic peptide segment may optionally contain one or more additional (e.g., non-basic) amino acid residues. As will be apparent to one of skill in the art, non-natural amino acid residues having basic side chains can be used. There may be from about 2 to about 7 amino acids, and / or about 3 or 4 amino acids in the segment.

[0048] As used herein, the term "bioactive peptide" refers to an amino acid sequence that mediates the action of a sequence, molecule, or supramolecular complex associated with it. Structures (e.g., nanofibers) having peptide amphiphiles and bioactive peptides (e.g., TGF-β1 mimetic peptides, etc.) exhibit bioactive peptide functionality.

[0049] As used herein, the term "biocompatible" refers to materials and agents that are not toxic to cells or organisms. In some embodiments, a substance is considered to be "biocompatible" if its addition to cells in vitro results in about 10% or less cell death, usually less than 5%, more usually less than 1%.

[0050] As used herein, "biodegradable," as used herein to describe polymers, hydrogels, and / or wound dressings, means a composition that breaks down or is otherwise "broken down" under exposure to physiological conditions. In some embodiments, biodegradable materials break down by cellular mechanisms, enzymatic degradation, chemical processes, hydrolysis, etc. In some embodiments, the wound dressing or coating includes hydrolyzable ester bonds that provide the biodegradability.

[0051] As used herein, the phrase "physiological conditions" refers to the range of chemical (e.g., pH, ionic strength) and biochemical (e.g., enzyme concentrations) conditions likely to be encountered in the intracellular and extracellular fluids of a tissue. For most tissues, physiological pH ranges from about 7.0 to 7.4.

[0052] As used herein, the terms "treat", "treatment", and "treating" refer to reducing the amount or severity of a particular condition, disease state (e.g., osteoarthritis, cartilage damage, etc.), or symptom thereof, in a subject currently experiencing or suffering from the condition or disease state. These terms do not necessarily indicate a complete cure (e.g., complete disappearance of the condition, disease, or symptoms thereof). "Treatment" encompasses any administration or application of a therapeutic agent or technique to a disease (e.g., in a mammal, including a human), including inhibiting a disease, preventing its onset, relieving a disease, inducing regression, or restoring or repairing lost, deficient, or defective function, or stimulating an inefficient process.

[0053] As used herein, the terms "prevent", "prevention" and "preventing" refer to reducing the likelihood of a particular condition or disease state (e.g., osteoarthritis, cartilage deterioration, etc.) occurring in a subject not currently experiencing or suffering from that condition or disease state. The term does not necessarily indicate complete or absolute prevention. For example, "prevention of osteoarthritis" refers to reducing the likelihood of osteoarthritis occurring in a subject not currently experiencing or diagnosed with osteoarthritis. To "prevent osteoarthritis", a composition or method need only reduce the likelihood of osteoarthritis, not necessarily completely block any likelihood of it. "Prevention" encompasses any administration or application of a therapeutic agent or technique to reduce the likelihood of disease onset (e.g., in a mammal, including a human). Such likelihood may be assessed for a population or an individual.

[0054] As used herein, the terms "co-administration" and "co-administering" refer to the administration of at least two agents(s) or therapies (e.g., PA nanostructures exhibiting TGF-β1 mimetic peptides and one or more therapeutic agents) to a subject. In some embodiments, the co-administration of two or more agents or therapies is simultaneous. In other embodiments, a first agent / therapy is administered before a second agent / therapy. Those skilled in the art will appreciate that the formulations and / or routes of administration of the various agents or therapies used may vary. Appropriate dosages for co-administration can be readily determined by those skilled in the art. In some embodiments, when agents or therapies are co-administered, each agent or therapy is administered at a dosage that is less than the dosage appropriate for their administration alone. Thus, co-administration is particularly desirable in embodiments where the co-administration of agents or therapies reduces the required dosage of potentially dangerous (e.g., toxic) agent(s) and / or where the co-administration of two or more agents results in sensitization of the subject to the beneficial effects of one of the agents by the co-administration of the other agent. [Mode for carrying out the invention]

[0055] Provided herein are self-assembling peptide amphiphiles (PAs) containing bioactive transforming growth factor beta 1 (TGF-β1) mimetic epitopes, high aspect ratio nanostructures of PAs exhibiting TGF-β1 mimetic epitopes, and methods for enhancing cartilage regeneration / repair and / or treatment of osteoarthritis and other musculoskeletal injuries and diseases.

[0056] The technology involves peptide amphiphilic (PA) molecules and supramolecular PA nanostructures that mimic the chondrogenic activity of TGF-β1 for cartilage regeneration. Bioactive PA molecules conjugated to TGF-β1 mimetic epitopes can be co-assembled with dilute epitope-free molecules for self-assembly into high aspect ratio nanostructures that present bioactive TGF-β1 mimetic domains on their surface. These nanostructures enhance cell signaling and chondrogenic responses through the preferred presentation of epitopes on the PA nanostructures. These bioactive PA systems can enhance cartilage regeneration and repair without additional exogenous growth factors and can be used as acellular regenerative scaffolds to treat osteoarthritis as well as other musculoskeletal injuries and diseases.

[0057] In some embodiments, the peptide amphiphile molecules and compositions of the embodiments described herein are synthesized by preparative techniques well known to those of skill in the art, preferably standard solid-phase peptide synthesis, with the addition of fatty acids in place of standard amino acids at the N-terminus (or C-terminus) of the peptide to create lipophilic segments (however, in some embodiments, alignment of nanofibers is accomplished via techniques not previously disclosed or used in the art (e.g., extrusion through a mesh screen). Synthesis typically begins from the C-terminus where amino acids are added sequentially using either Rink amide resin (which after cleavage from the resin results in an --NH2 group at the C-terminus of the peptide) or Wang resin (which results in an --OH group at the C-terminus). Thus, some embodiments described herein encompass peptide amphiphiles having a C-terminal moiety that may be selected from the group consisting of --H, --OH, --COOH, --CONH2, and --NH2.

[0058] In some embodiments, the peptide amphiphile comprises a hydrophobic (non-peptide) segment linked to the peptide. In some embodiments, the peptide comprises a structural segment (e.g., hydrogen bond forming segments, beta sheet forming segments, etc.) and a charged segment (e.g., acidic segments, basic segments, zwitterionic segments, etc.). In some embodiments, the peptide further comprises a linker or spacer segment for added solubility, flexibility, distance between segments, etc. In some embodiments, the peptide amphiphile comprises a spacer segment (e.g., a peptide and / or non-peptide spacer) at the end of the peptide opposite the hydrophobic segment. In some embodiments, the spacer segment comprises peptide and / or non-peptide elements. In some embodiments, the spacer segment comprises one or more bioactive groups (e.g., an alkene, alkyne, azide, thiol, etc.). In some embodiments, the various segments may be connected by a linker segment (e.g., a peptide (e.g., GG) or a non-peptide (e.g., alkyl, OEG, PEG, etc.) linker).

[0059] The lipophilic or hydrophobic segment is typically incorporated at the N- or C-terminus of the peptide after the last amino acid coupling, and is composed of a fatty acid or other acid linked to the N- or C-terminal amino acid by an acyl bond. In aqueous solution, the PA molecule self-assembles (e.g., into cylindrical micelles (aka nanofibers)) with the lipophilic segment buried in its core and the bioactive peptide on its surface. The structural peptide undergoes intermolecular hydrogen bonding to form beta sheets oriented parallel to the long axis of the micelle.

[0060] In some embodiments, the compositions described herein comprise a PA building block that in turn comprises a hydrophobic segment and a peptide segment. In certain embodiments, a hydrophobic (e.g., a hydrocarbon and / or alkyl / alkenyl / alkynyl tail, or a steroid such as cholesterol) segment of sufficient length (e.g., 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, 20 carbons, 21 carbons, 22 carbons, 23 carbons, 24 carbons, 25 carbons, 26 carbons, 27 carbons, 28 carbons, 29 carbons, 30 carbons or more, or any range therebetween) is covalently attached to a peptide segment (e.g., a peptide comprising a segment that favors a beta-strand conformation or other supramolecular interactions) to provide a peptide amphiphilic molecule. In some embodiments, multiple such PAs self-assemble into nanostructures (e.g., nanofibers) in water (or aqueous solutions). In various embodiments, the relative lengths of the peptide segments and hydrophobic segments result in different PA molecular shapes and nanostructural architectures. For example, wider peptide segments and narrower hydrophobic segments result in a generally conical molecular shape that has an effect on the assembly of the PA (see, e.g., JN Israelachvili Intermolecular and surface forces; 2nd ed.; Academic: London San Diego, 1992; incorporated herein by reference in its entirety). Other molecular shapes have similar effects on the assembly and nanostructural architecture.

[0061] In some embodiments, to induce self-assembly of aqueous solutions of peptide amphiphiles, the pH of the solution may be altered (raised or lowered) or multivalent ions such as calcium, or charged polymers or other macromolecules may be added to the solution.

[0062] In some embodiments, the hydrophobic segment is a non-peptide segment (e.g., an alkyl / alkenyl / alkynyl group). In some embodiments, the hydrophobic segment comprises an alkyl chain (e.g., saturated) of 4 to 25 carbons (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25), fluorinated segments, fluorinated alkyl tails, heterocyclic rings, aromatic segments, pi-conjugated segments, cycloalkyls, oligothiophenes, etc. In some embodiments, the hydrophobic segment comprises an acyl / ether chain (e.g., saturated) of 2 to 30 carbons (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30).

[0063] In some embodiments, the PA comprises one or more peptide segments. The peptide segments may comprise natural amino acids, modified amino acids, non-natural amino acids, amino acid analogs, peptidomimetics, or combinations thereof. In some embodiments, the peptide segments comprise at least 50% sequence identity or similarity (e.g., conservative or semi-conservative) to one or more of the peptide sequences described herein.

[0064] In some embodiments, the peptide amphiphile comprises a charged peptide segment, which can be acidic, basic, or zwitterionic.

[0065] In some embodiments, the peptide amphiphile comprises an acidic peptide segment. For example, in some embodiments, the acidic peptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or more) acidic residues (D and / or E) in the sequence. In some embodiments, the acidic peptide segment comprises a length of up to 7 residues and comprises at least 50% acidic residues. In some embodiments, the acidic peptide segment comprises (Xa) 1-7and each Xa is independently D or E. In some embodiments, the acidic peptide segment comprises EE.

[0066] In some embodiments, the peptide amphiphile comprises a basic peptide segment. For example, in some embodiments, the acidic peptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or more) basic residues (R, H, and / or K) in the sequence. In some embodiments, the basic peptide segment comprises a length of up to 7 residues and comprises at least 50% basic residues. In some embodiments, the acidic peptide segment comprises a peptide segment represented by the formula (Xb) 1-7 wherein each Xb is independently R, H, and / or K.

[0067] In some embodiments, the peptide amphiphile comprises structural and / or beta sheet forming segments. In some embodiments, the structural segments are rich in H, I, L, F, V, and A residues. In some embodiments, the structural and / or beta sheet forming segments comprise alanine and valine rich peptide segments (e.g., AAVV, AAAVVV (SEQ ID NO: 4), or other combinations of V and A residues). In some embodiments, the structural and / or beta sheet peptides comprise four or more consecutive A and / or V residues, or conservative or semi-conservative substitutions thereto. In some embodiments, the structural and / or beta sheet forming peptide segments comprise four or more consecutive non-polar aliphatic residues (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)). In some embodiments, the structural and / or beta sheet forming peptide segments are 2-16 amino acids in length and contain 4 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or any range therebetween) non-polar aliphatic residues.

[0068] In some embodiments, the peptide amphiphile comprises a non-peptide spacer or linker segment. In some embodiments, the non-peptide spacer or linker segment is located at the terminus of the peptide opposite the hydrophobic segment. In some embodiments, the spacer or linker segment provides a site for attachment of a biologically active group. In some embodiments, the spacer or linker segment provides a reactive group (e.g., alkene, alkyne, azide, thiol, maleimide, etc.) for functionalization of the PA. In some embodiments, the spacer or linker is a substantially linear chain of CH2, O, (CH2)2O, O(CH2)2, NH, and C=O groups (e.g., CH2(O(CH2)2)2NH, CH2(O(CH2)2)2NHCO(CH2)2CCH, etc.). In some embodiments, the spacer or linker further comprises additional biologically active groups, substituents, branches, etc.

[0069] Peptide amphiphiles suitable for use in the materials herein, as well as methods for preparing PA and related materials, amino acid sequences for use in PA, and materials that can be used in PA, are described in the following patents: U.S. Pat. No. 9,044,514; U.S. Pat. No. 9,040,626; U.S. Pat. No. 9,011,914; U.S. Pat. No. 8,772,228; U.S. Pat. No. 8,748,569; U.S. Pat. No. 8,580,923; U.S. Pat. No. 8,546,338; U.S. Pat. No. 8,512,693; U.S. Pat. No. 8,450,271; U.S. Pat. No. 8,236,800; U.S. Pat. No. 8,138,140; U.S. Pat. No. 8,124,583; Nos. 8,114,835, 8,114,834, 8,080,262, 8,076,295, 8,063,014, 7,851,445, 7,838,491, 7,745,708, 7,683,025, 7,554,021, 7,544,661, 7,534,761, 7,491,690, 7,452,679, 7,371,719, and 7,030,167, all of which are incorporated herein by reference in their entireties.

[0070] The characteristics of the PA supramolecular structure (e.g., shape, rigidity, hydrophilicity, etc.) depend on the identity of the constituent parts of the peptide amphiphile (e.g., lipophilic segments, acidic segments, structural segments, bioactive segments, etc.). For example, nanofibers, nanospheres, intermediate shapes, and other supramolecular structures are achieved by tuning the identity of the PA constituent moieties. In some embodiments, the characteristics of the PA supramolecular nanostructure are altered by post-assembly manipulations (e.g., heating / cooling, stretching, etc.).

[0071] In some embodiments, the peptide amphiphile comprises (a) a hydrophobic tail comprising an alkyl chain of 8-24 carbons, (b) a structural segment (e.g., comprising VVAA), and (c) a charged segment (e.g., comprising KK, EE, etc.). In some embodiments, any PA within the scope described herein or within the skill of one of ordinary skill in the art, including the components described herein, may be used herein.

[0072] In some embodiments, the peptide amphiphile comprises a bioactive moiety (e.g., a TGF-β1 mimetic epitope). In certain embodiments, the bioactive moiety is the most C-terminal or N-terminal segment of the PA. In some embodiments, the bioactive moiety is attached to the end of a charged segment. In some embodiments, the bioactive moiety is exposed on the surface of the assembled PA structure (e.g., nanofiber). The bioactive moiety is typically, but not limited to, a peptide (e.g., a TGF-β1 mimetic epitope, etc.). In some embodiments, the bioactive moiety is a peptide sequence that binds to a peptide or polypeptide of interest, e.g., a growth factor. In some embodiments, a TGF-β1 mimetic epitope is provided as a PA bioactive moiety. In some embodiments, such a TGF-β1 mimetic epitope has at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or ranges therebetween) sequence identity with SEQ ID NO: 1 (CESPLKRQC). In some embodiments, the TGF-β1 mimetic epitope peptide is cyclized. In some embodiments, the TGF-β1 mimetic epitope is SEQ ID NO:1. In some embodiments, nanofibers are provided that include a bioactive PA that exhibits one or more of a peptide having at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or ranges therebetween) sequence identity to one of SEQ ID NO:1. In some embodiments, the bioactive peptide comprises a conservative or semi-conservative substitution to one of SEQ ID NO:1.

[0073] In some embodiments, the peptide amphiphile comprises (a) a hydrophobic tail comprising an alkyl chain of 8-24 carbons, (b) a structural segment (e.g., including VVAA (SEQ ID NO: 6), AAVV (SEQ ID NO: 5), VA, AV, AA, etc.), (c) a charged segment (e.g., including KK, EE, EK, KE, EEE, etc.), and a bioactive peptide (e.g., a TGF-β1 mimetic epitope). In some embodiments, the PA further comprises a linking segment or residue (e.g., K) for attaching the hydrophobic tail to a peptide portion of the PA. In some embodiments, the hydrophobic tail is attached to a lysine side chain. In some embodiments, the PA further comprises a linking segment or residue (e.g., K) for attaching a bioactive peptide to the structural segment.

[0074] In some embodiments, the peptide amphiphile comprises (e.g., C-terminus to N-terminus or N-terminus to C-terminus): a bioactive peptide (e.g., a TGF-β1 mimetic epitope)-a charged segment (e.g., including KK, EE, EK, KE, EEE, etc.)-a structural segment (e.g., including VVAA (SEQ ID NO: 6), AAVV (SEQ ID NO: 5), VA, AV, AA, etc.)-a hydrophobic tail (e.g., including an alkyl chain of 8 to 24 carbons).

[0075] In some embodiments, the peptide amphiphile comprises (e.g., C-terminus to N-terminus or N-terminus to C-terminus) a bioactive peptide (e.g., a TGF-β1 mimetic epitope)-charged segment (e.g., including KK, EE, EK, KE, EEE, etc.)-structural segment (e.g., including VVAA (SEQ ID NO: 6), AAVV (SEQ ID NO: 5), VA, AV, AA, etc.)-binding segment or peptide (e.g., K)-hydrophobic tail (e.g., including an alkyl chain of 8 to 24 carbons).

[0076] In some embodiments, the peptide amphiphile comprises (e.g., C-terminus to N-terminus or N-terminus to C-terminus) a bioactive peptide (e.g., a TGF-β1 mimetic epitope)-binding segment or a peptide (e.g., K)-charged segment (e.g., including KK, EE, EK, KE, EEE, etc.)-structural segment (e.g., including VVAA (SEQ ID NO: 6), AAVV (SEQ ID NO: 5), VA, AV, AA, etc.)-hydrophobic tail (e.g., including an alkyl chain of 8 to 24 carbons).

[0077] In some embodiments, the peptide amphiphile comprises (e.g., C-terminus to N-terminus or N-terminus to C-terminus) a bioactive peptide (e.g., a TGF-β1 mimetic epitope)-EEEAAAVVV (SEQ ID NO: 10)-hydrophobic tail (e.g., comprising an alkyl chain of 8 to 24 carbons).

[0078] In some embodiments, the peptide amphiphile comprises (e.g., C-terminus to N-terminus or N-terminus to C-terminus) a bioactive peptide (e.g., a TGF-β1 mimetic epitope)-EEAA (SEQ ID NO: 11)-hydrophobic tail (e.g., comprising an alkyl chain of 8 to 24 carbons).

[0079] In some embodiments, provided herein are nanofibers and nanostructures assembled from peptide amphiphiles described herein. In some embodiments, the nanofibers are prepared by self-assembly of PAs described herein. In some embodiments, the nanofibers comprise or consist of PAs exhibiting TGF-β1 mimetic epitopes. In some embodiments, the TGF-β1 mimetic epitopes are exhibited on the surface of the nanofibers. In some embodiments, in addition to the PAs exhibiting TGF-β1 mimetic epitopes, a filler PA is included in the nanofibers. In some embodiments, the filler PA is a peptide amphiphile described herein (e.g., structural segments, charged segments, hydrophobic segments, etc.), but lacks a biologically active moiety. In some embodiments, the filler peptide is a basic or acidic peptide lacking a biologically active moiety (e.g., V3A3K3, V3A3E3, etc.). In some embodiments, the filler PA and the TGF-β1 mimetic epitope PA self-assemble into nanofibers that include both types of PA. In some embodiments, nanostructures (eg, nanofibers) organized from the peptide amphiphiles described herein are provided.

[0080] In some embodiments, the nanostructures are assembled from (1) a PA having a bioactive moiety (e.g., a TGF-β1 mimetic epitope) and (2) a filler PA (e.g., an acidic or basic PA that is unlabeled or does not exhibit a bioactive moiety, etc.). In some embodiments, the nanostructures (e.g., nanofibers) comprise 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% (or any range therebetween) of the TGF-β1 mimetic epitope PA. In some embodiments, the nanostructures (e.g., nanofibers) comprise 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% (or any range therebetween) of an acidic filler PA. In some embodiments, the nanostructures (e.g., nanofibers) comprise 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% (or any range therebetween) of a basic filler PA. In some embodiments, the ratio of TGF-β1 mimetic epitope PA to acidic and / or basic PA in the nanofiber determines the mechanical characteristics of the nanofiber material (e.g., liquid or gel) and under what conditions the material adopts various characteristics (e.g., gels upon exposure to physiological conditions, liquefies upon exposure to physiological conditions, etc.).

[0081] The peptide amphiphile (PA) nanofiber solution may include any suitable combination of PAs. In some embodiments, at least 0.05 mg / mL of the solution (e.g., 0.10 mg / ml, 0.15 mg / ml, 0.20 mg / ml, 0.25 mg / ml, 0.30 mg / ml, 0.35 mg / ml, 0.40 mg / ml, 0.45 mg / ml, 0.50 mg / ml, 0.60 mg / ml, 0.70 mg / ml, 0.80 mg / ml, 0.90 mg / ml, 1.0 mg / ml or more, or ranges therebetween) is filler PA (e.g., does not include peptide epitopes or other nanofiber surface-displayed moieties). In some embodiments, at least 0.25 mg / mL of the solution is filler PA. In some embodiments, the filler PA is a non-bioactive PA molecule with highly charged glutamic acid residues at the ends of the molecule (e.g., the surface-displayed ends). These negatively charged PAs allow gelation to occur between nanofibers via ionic crosslinking. In some embodiments, the filler PAs are non-bioactive PA molecules with highly charged lysine residues at the ends of the molecules (e.g., the surface-exposed ends). These positively charged PAs allow gelation to occur under basic conditions. The filler PAs provide the ability to incorporate other bioactive PA molecules into the nanofiber matrix while still preserving the gelling ability of the nanofiber solution. In some embodiments, the solution is annealed for increased viscosity and stronger gel mechanics. These filler PAs are described, for example, in U.S. Pat. No. 8,772,228 (e.g., C 16 -VVVAAAEEE (SEQ ID NO:9) (incorporated herein by reference in its entirety).

[0082] In some embodiments, the PA nanofibers described herein exhibit a small cross-sectional diameter (e.g., less than 25 nm, less than 20 nm, less than 15 nm, about 10 nm, etc.). In some embodiments, the small cross-sectional area of ​​the nanofibers (diameter about 10 nm) allows the fibers to penetrate the brain parenchyma.

[0083] In some embodiments, the PAs and nanostructures described herein are used to treat or prevent osteoarthritis, cartilage damage / deterioration, and other musculoskeletal injuries and diseases.

[0084] In some embodiments, the TGF-β1 mimetic epitope PA nanostructure composition herein is formulated for delivery to a subject. Suitable routes of administration of the pharmaceutical composition described herein include, but are not limited to, topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, intratympanic, intravisceral, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intraventricular administration. In some embodiments, the TGF-β1 mimetic epitope PA nanostructure composition is administered parenterally. In some embodiments, the parenteral administration is by intrathecal, intraventricular, or intraparenchymal administration.

[0085] The TGF-β1 mimetic epitope PA nanostructure compositions herein may be administered as the sole active agent or in combination with other pharmaceutical agents, such as other drugs used to treat osteoarthritis, cartilage damage / deterioration, and other musculoskeletal injuries and diseases.

[0086] In some embodiments, the PA nanostructures herein are provided as composites with one or more additional components. In some embodiments, the composites herein include PA nanostructures and biocompatible polymers. In some embodiments, the biocompatible polymers are in the form of particles (e.g., microparticles (e.g., diameters greater than 1 μm but less than 1 mm), nanoparticles (e.g., diameters greater than 1 nm but less than 1 μm), etc.). In some embodiments, the composites are in the form of slurries, pastes, gels, etc.

[0087] Suitable biocompatible polymers for use in the materials herein include PLA, PLLA, PGA, PGLA, PCL, chitosan, polylactide, polyglycolide, epsilon-caprolactone, polyhydroxyvaleric acid, polyhydroxybutyric acid, other polyhydroxy acids, polytrimethylene carbonate, polyamines, vinyl polymers, polyacrylic acids and their derivatives containing esters, polyethylene glycols, polydioxanones, polycarbonates, polyacetals, polyorthoesters, polyamino acids, polyphosphoesters, polyethersulfone ... The polyether polyether ester may be selected from the group consisting of steramides, polyfumarates, polyanhydrides, polycyanoacrylates, poloxamers, polyurethanes, polyphosphazenes, aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides, poly(iminocarbonates), polyoxaesters, polyamide esters, amine group-containing polyoxaesters, polyacetals, polyalkanoates, gelatin, collagen, elastin, polysaccharides, alginates, chitin, hyaluronic acid, and combinations thereof.

[0088] In some embodiments, the particles are of any suitable size and shape. In some embodiments, the particles are microparticles and have an average diameter of 1 μm to 1 mm (e.g., 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 1 mm, or ranges therebetween). In some embodiments, the particles are nanoparticles and have an average diameter of 1 nm to 1 μm (e.g., 1 nm, 2 nm, 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 1 nm, or ranges therebetween). In some embodiments, the particles are produced using any suitable technique, such as freezing (e.g., under liquid N2), drying, freeze-drying, lyophilization, crushing, grinding, exposure to a solvent (e.g., ethanol), sieving, and combinations thereof.

[0089] In an exemplary embodiment, the biocompatible particles and peptide amphiphile solution are mixed at 5 wt% biocompatible particles and 1 wt% PA in neutral pH water. Other ranges (e.g., 1 wt% to 20 wt% (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or any range therebetween) of biocompatible particles; 0.1 wt% to 10 wt% (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any range therebetween) of PA) may be used. In certain embodiments, the polymer (e.g., HA) is used at 0 wt% to 20 wt% and the PA is used at 0.05 wt% to 3 wt%. EXAMPLES

[0090] experiment Example 1 In vitro characterization of TGF-β1 mimetic supramolecular nanostructures Materials and Methods Synthesis and purification of PA All PA molecules were synthesized using standard fluorenylmethoxycarbonyl (Fmoc) synthesis on Wang resin (EMD Biosciences). PAs were purified by reversed-phase high performance liquid chromatography (HPLC). Mass spectra of each fraction of PA after HPLC purification were verified using direct infusion Q-ToF MS on an Agilent model 6520. PA purity was confirmed by liquid chromatography-electrospray ionization mass spectrometry (LCMS) using a Phenomenex Gemini C18 column over a 5% to 95% water to acetonitrile gradient with 0.1% ammonium hydroxide.

[0091] The TGF-β1 mimetic PA molecule was synthesized using Fmoc-Lys(Mtt)-OH lysine residues on the resin with orthogonal protecting groups. After Fmoc deprotection, C 16 -AAEE (SEQ ID NO: 7) or C 16The PA backbone of either -AEAE (SEQ ID NO: 8) was synthesized from the lysine α-amino group from the C-terminus to the N-terminus. After Mtt deprotection, PEG4 was synthesized from the lysine side chain amine, followed by the synthesis of the TGF-β1 mimetic epitope, which was cyclized by disulfide bond on the resin. After synthesis, the peptides were purified by high performance liquid chromatography and liquid chromatography mass spectrometry to ensure the cyclization of the peptides.

[0092] The purified confirmed fractions of each PA were pooled and the solution was freeze-dried by lyophilization. The diluted skeletal PA was co-assembled with the respective TGF-β1 mimetic PA or linear epitope PA at different molar percentages by dissolving the lyophilized powder in sterile water. The PA solution was adjusted to pH 7 using a sterile filtered solution of 1 M NaOH, then subjected to an ultrasonic bath for 30 min. The solution was thermally annealed at 80°C for 30 min and then cooled to 25°C at a rate of 1°C per min. The solution was then adjusted to 30 mM NaCl using a sterile solution of 150 mM NaCl, then annealed at 50°C for 30 min and cooled.

[0093] PA gel preparation Gels were made using a coassembly of 10 mol% epitope PA and diluted PA using a 2 wt% PA solution prepared as described above. 12-well chamber slides (Ibidi) were coated with 0.01 mg / mL poly-D-lysine (Sigma-Aldrich) solution and incubated overnight at 37°C. The wells were washed three times with water and air-dried for at least 30 min. 120 μL of PA solution was pipetted evenly into the wells, and a gelling solution consisting of 110 mM NaCl, 3 mM KCl, and 25 mM CaCl2 was pipetted on top of the PA solution. The gels were incubated at 37°C for 30 min, and then the excess gelling solution was removed.

[0094] Cryo-transmission electron microscopy A 300 mesh copper grid with lacey carbon film (Electron Microscopy Sciences) was glow discharged for 30 seconds in a PELCO easiGlow system (Ted Pella, Inc.). PA solution was prepared as previously described and then diluted to 1 mM in water prior to imaging. 7 μL of PA solution was placed on the grid, blotted, and plunge frozen in liquid ethane using an FEI Vitrobot Mark IV (FEI) maintained at 95-100% humidity. Vitrified specimens were transferred to a Gatan 626 cryoholder (Gatan) while immersed in liquid nitrogen and imaged with a JEOL 1230 TEM operated at an accelerating voltage of 100 kV. Liquid nitrogen temperature was maintained throughout imaging, and micrographs were captured with a Gatan 832 CCD camera.

[0095] Conventional Transmission Electron Microscopy The PA solution was diluted to 0.6 mM in water and immediately placed on a copper TEM mesh grid (Electron Microscopy Sciences) for 30 s, blotted twice with water, stained with 2% uranyl acetate, and air-dried for 15 min. Imaging was performed on an FEI Sprit G2 transmission electron microscope.

[0096] Dynamic Light Scattering DLS measurements were performed on a Malvern Zetasizer Nano ZSP light scattering spectrometer. TGF-β1 mimetic peptide alone without PA was prepared at 1 wt% as previously described. The temperature was kept at 25°C during sample measurements. Before each measurement, the sample was equilibrated for 30 s. The duration of each measurement was 10 s, and the measurement angle was 173° backscattering. The attenuator was automatically determined by the instrument, as was the number of accumulations for each run. Each measurement run was repeated three times.

[0097] X-ray scattering Experiments were performed at the Advanced Photon Source of Argonne National Laboratory, Dupont-Northwestern-Dow Collaborative Access Team Synchrotron Research Center, beamline 5ID-D. A 4.8 mM PA solution was prepared in a quartz capillary cell and exposed to 17 keV monochromatic X-rays five times for 2–3 s. The scattering intensity was recorded with a CCD detector placed 245 cm behind the sample. The collected 2D scattering images were averaged by azimuth integration using the data reduction program FIT2D and plotted against the wave vector q = (4π) sin(θ / 2), where d = 2π / q. A sample with only 30 mM NaCl buffer was background subtracted to obtain the final intensity. SAXS patterns were plotted on a log-log scale and fitted to a core-shell cylinder model as necessary. WAXS patterns were plotted on a linear scale and the peaks and minima in the data were identified using the MATLAB peak finder function.

[0098] Circular dichroism CD spectra were recorded on a JASCO model J-815 spectropolarimeter using a parallel plate quartz chamber with an optical length of 0.5 mm. PA solutions were diluted to 0.1 and 0.5 mM in water immediately prior to measurement. Spectra were collected over the wavelength range of 190-300 nm with a step size of 1 nm, a scan speed of 100 nm / min, and standard sensitivity. A high tension voltage was recorded for each sample to ensure that measurements were not saturated. An accumulation of three measurements was used and a sample with only 30 mM NaCl buffer was background subtracted to obtain the final spectrum. All PA concentrations were analyzed together to ensure the absence of linear dichroism, and the spectrum of the 0.5 mM PA solution was used for the final analysis.

[0099] Fourier transform infrared spectroscopy PA solutions were prepared under anhydrous conditions using the method described above. PA was solubilized in D2O, pH adjusted with DOH, and diluted to 12 mM PA and 30 mM NaCl using 150 mM NaCl in D2O. The PA solution was diluted to 5 mM in D2O immediately prior to measurement and then placed between two CaF2 windows with a separation of 50 μm. Transmittance was measured with a Bruker Tensor 37 FTIR spectrometer. 1 cm -1 Spectra were recorded over 25 scans at a resolution of 100 s, averaged, and background subtracted for 30 mM NaCl and air in the D2O buffer only sample to obtain the final spectrum. Peaks in the spectra were identified using the MATLAB peak finder function, and secondary structures were identified through peak positions.

[0100] Fluorescence anisotropy PA coassemblies of diluted PA, TGF-β1 mimetic PA, and fluorescent dye-conjugated TGF-β1 mimetic PA were prepared in a ratio of 90:8:2 mol%. Dye-conjugated PA was synthesized as previously described with a tetramethylrhodamine (TAMRA) molecule conjugated to the N-terminal amine of the TGF-β1 mimetic epitope. PA solutions were prepared as previously described and diluted to 100 μM PA, 100 μM CaCl2, and 30 mM NaCl immediately prior to measurement. Fluorescence measurements were performed on an ISS PC1 photon-counting steady-state fluorescence spectrometer equipped with a 300 W xenon arc lamp with the power set to 18 A. An excitation slit width of 1 mm (8 nm bandwidth) and an emission slit width of 0.5 mm (4 nm bandwidth) were used. Fluorescence anisotropy measurements were performed at λex = 554 nm and λem = 580 nm using the formula:

number

[0101] Transverse relaxation nuclear magnetic resonance spectroscopy NMR spectra were acquired at 600 MHz on a Brucker Neo system with a QCI-F cryoprobe. NMR spectra of PA were recorded at 25 °C using TFA-d, H2O / D2O in a 9 / 1 ratio (D2O containing 0.05 wt% 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt) as the solvent. Chemical shifts are reported in parts per million (ppm). The 90° pulse width was 15 μs and a typical spectrum required 32 scans. Spin-spin relaxation rates were measured using a Carr-Purcell-Gill-Meiboom pulse sequence with a delay time of 0.2 ms in a variable loop. Peak intensity data were fitted to the following form, exponential:

number

[0102] cell culture C28 / I2 human articular chondrocytes (Millipore) were maintained in DMEM high glucose medium (Gibco) supplemented with 10% fetal bovine serum (Denville Scientific), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco) using standard cell culture techniques. Cells were passaged using 0.25% trypsin (Gibco) and used for experiments at passages 3–8.

[0103] PA cell treatment in vitro PA solutions (skeletal PA, TGF PA, lnTGF PA, and peptide only) were prepared under sterile conditions at 1% by weight as described above. For cell treatments less than 24 hours, PA treatment solutions or native rhTGF-β1 protein (R&D Systems) were diluted in starvation medium consisting of DMEM high glucose medium (Gibco) supplemented with 0.5% fetal bovine serum (Denville Scientific), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco). For cell treatments longer than 24 hours, PA treatment solutions or native rhTGF-β1 protein (R&D Systems) were diluted in complete growth medium. PA solutions were prepared at various concentrations of 1, 10, 50, and 100 μM, where the concentration refers to the concentration of epitope PA.

[0104] For cell viability assays, cells were seeded in 48-well plates at a density of approximately 15,400 cells / well and cultured for 24 hours. Cells were then treated with PA in solution for 24 hours or 3 days in vitro.

[0105] For Western blot of 4-hour PA treatment, cells were seeded in 6-well plates at a density of 600,000 cells / well and cultured for 24 hours. Cells were then serum-starved for 20 hours in DMEM high glucose medium (Gibco) supplemented with 0.5% fetal bovine serum (Denville Scientific), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco). Cells were then treated with PA in solution for 4 hours in vitro. For Western blot of 3-day PA treatment, cells were seeded in 6-well plates at a density of 230,000 cells / well and cultured for 24 hours. Cells were then treated with PA in solution for 3 days in vitro.

[0106] For immunocytochemistry (ICC), 12 mm glass coverslips were coated with sterile filtered 0.01 mg / mL poly-D-lysine (Sigma-Aldrich) solution and incubated overnight at 37°C. Coverslips were washed three times with sterile water and air-dried for at least 30 min. Cells were seeded on the coverslips at a density of approximately 3,000 cells / coverslip and cultured for 24 h. Cells were then treated with PA in solution for 3 days in vitro. After treatment, cells were fixed in 4% paraformaldehyde for 15 min at room temperature.

[0107] Viability assay For 2D viability assays, cells were cultured and treated with PA solution in vitro as previously described. After 24 h or 3 days of culture, cells were washed with HBSS (Gibco) and the medium was replaced with HBSS containing 2 μM calcein AM (Invitrogen) and 100 ng / mL propidium iodide (Sigma-Aldrich) for 30 min at 37 °C. Cells were washed with HBSS and imaged.

[0108] For 3D viability assays, cells were encapsulated in PA gels and cultured in vitro as described below. After 3 days of culture, media fractions were collected to measure the presence of lactate dehydrogenase (LDH), a cytosolic enzyme that is only released upon cell lysis, using the CyQUANT™ LDH Cytotoxicity Assay (Invitrogen).

[0109] Western blot Proteins were extracted from cells using Halt protease and phosphatase inhibitor cocktail (Thermo Scientific) and a BCA assay (Thermo Scientific) was performed to determine the protein content of each sample. Cellular proteins were loaded and separated using 4-20% SDS-PAGE gels (Bio-Rad). They were then electrophoretically transferred from the gel to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 10% milk solution (Bio-Rad) for 30 min, followed by overnight incubation with primary antibodies at 4 °C. The following primary antibodies were used: rabbit anti-pSmad2 (1:1000, Cell Signaling), rabbit anti-Smad2 (1:1000, Cell Signaling), rabbit anti-Sox9 (1:500, Abcam), mouse anti-aggrecan (1:500, ThermoFisher), rabbit anti-COMP / cartilage oligomeric matrix protein (1:500, Abcam), mouse anti-collagen II (1:500, ThermoFisher), and mouse anti-actin (1:1000, Novus Bio). Membranes were then incubated with the corresponding secondary HRP-conjugated antibodies (1:1000, ThermoFisher). Protein signals were detected using Radiance Bioluminescent ECL substrate (Azure Biosystems). Densitometric analysis, normalized to total receptor content or actin as a control for protein loading, was performed using ImageJ software. For quantification, experimental triplicate samples were analyzed and two different experiments were performed.

[0110] immunocytochemistry Fixed samples were permeabilized and blocked in a solution of 0.1% (v / v) Triton X-100 and 1% normal horse serum (Invitrogen) for 2 h at room temperature. Samples were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used: rabbit anti-Sox9 (1:500, Abcam), mouse anti-aggrecan (1:500, ThermoFisher), mouse anti-collagen II (1:500, ThermoFisher), and mouse anti-nuclear (1:500, Sigma-Aldrich). The next day, samples were incubated with AlexaFluor488 secondary antibody, AlexaFluor555 secondary antibody, Phalloidin AlexaFluor488, and / or Phalloidin AlexaFluor633 (1:1000, Invitrogen) for 2–3 h at room temperature. For 2D cell culture, samples were then incubated with DAPI (1:1000, Invitrogen) for 10 min at room temperature. If not already mounted on a glass coverslip, samples were mounted with Immu-Mount (Thermo Scientific) and imaged on a Nikon A1R Spectromicroscope. Image analysis was performed using ImageJ software.

[0111] Cell encapsulation To count, chondrocytes were trypsinized, resuspended in growth medium, then pelleted and counted at 6 x 10 cells / well in growth medium. 7The cells were resuspended to 1000 cells / mL. The cell solution was mixed 1:2 v / v with sterile PA solution prepared at 3 wt% in 30 mM NaCl. For gels encapsulating rhTGF-β1 protein (R&D Systems), soluble protein was added to the PA solution after thermal annealing. 120 μL of the PA / cell solution was mixed gently and thoroughly with a pipette and then placed in a PDL-coated 12-well chamber slide (Ibidi). Sterile gelling solution, consisting of 110 mM NaCl, 3 mM KCl, and 25 mM CaCl2, was pipetted on top of the PA / cell solution and the gels were incubated at 37 °C for 30 min. After incubation, excess gelling solution was removed and 250 μL of growth medium was added to each well. The medium was removed after 3 days for LDH viability assay as described above. For ICCs, after 3 days of in vitro culture, gels were fixed with 4% paraformaldehyde for 30 min at room temperature.

[0112] statistical analysis Statistical analysis was performed using MATLAB 2021 software. Analysis of variance (ANOVA) tests of significance with Tukey's post-hoc analysis were used for all multigroup analyses, except for cell area analysis, where the data were non-Gaussian and the Kruskal-Wallis test was used instead. Error bars represent standard error.

[0113] result Design of TGF-β1 mimicking PA nanostructures During the development of the embodiments herein, experiments were carried out to develop supramolecular nanostructures that mimic TGF-β1 signaling in cartilage through the presentation of the cyclic peptide CESPLKRQC (SEQ ID NO: 1). A TGF-β1 mimetic PA molecule (TGF PA) was designed by conjugating this peptide to the C-terminus of two different PA molecules via a tetra(ethylene glycol) spacer (FIG. 1). A linear non-cyclized derivative of the mimetic PA (lnTGF PA) presenting the epitope SESPLKRQS (SEQ ID NO: 2) was also designed. PA assemblies containing 100 mol% of the epitope PA molecules were unable to form long fibrous structures, likely due to the steric requirements of the epitope (FIG. 7). Each scaffold PA alone was unable to form a robust high aspect ratio structure. 37 , which has been shown to promote better cell viability and biological activity. 38、39 Therefore, non-bioactive diluted scaffold PA molecules (scaffold PA-a and scaffold PA-b) were co-assembled with the respective PAs conjugated to cyclic epitopes (TGF PA-a and TGF PA-b) or non-cyclic controls (lnTGF PA-a and lnTGF PA-b), respectively, to form long fibrous nanostructures displaying the epitopes on their surfaces. To investigate whether the bioactivity of the epitope is enhanced by its presentation on the PA supramolecular nanostructures, the soluble cyclic TGF-β1 mimetic peptide was also evaluated alone.

[0114] Material characterization of TGF-β1 mimetic PA nanostructures Since the molar ratio of bioactivity and dilution PA has been shown to affect nanostructure morphology, epitope presentation, and bioactivity. 28、30、31Various co-assembly ratios of epitope PA and the corresponding diluted scaffold PA (10, 25, 50, 75, and 100 mol%) were characterized to determine the optimal ratio for self-assembly into long, uniform structures that promote cell viability and bioactivity. Cryo-transmission electron microscopy (cryo-TEM) revealed clear differences in nanostructure morphology between different scaffold systems and co-assembly ratios (Figures 2a-f, 7, and 8). Only the diluted scaffold PA formed twisted ribbon-like structures with high aspect ratios that changed upon co-assembly with epitope PA molecules (Figures 2a and 2d). At a molar ratio of 10%, both the TGF PA and lnTGF PA systems formed high aspect ratio structures. TGF PA-a formed wide, helical ribbon-like structures with widths of 50-60 nm, and TGF PA-b formed one-dimensional cylindrical fibers (Figures 2b and 2e). When the molar ratio of TGF PA was increased above 50 mol%, both systems formed short fibers or micelles, presumably due to steric repulsion from the cyclic epitope (Figure 7). Both linear lnTGF PA systems formed narrow twisted ribbons with widths of 20-30 nm at 10 mol% (Figures 2c, 2f, and 8). Dynamic light scattering revealed that only the TGF-β1 mimetic epitope formed micelles with hydrodynamic diameters of about 2 nm and aggregates of micelles as large as 200 nm (Figures 9 and 10).

[0115] To more quantitatively characterize these morphologies observed in cryo-TEM, the nanostructures were analyzed in situ using small-angle and wide-angle X-ray scattering (SAXS and WAXS) (Figures 2g-h, 11, 12). SAXS intensity profiles plotted on a log-log scale could be linearly fitted to the low-q Guinier region with slopes of approximately -2 for TGF PA-a and -1 for TGF PA-b, indicating high aspect ratio two-dimensional and one-dimensional structures, respectively (Figure 2g). 40WAXS revealed clear differences in the molecular packing schemes between the two scaffold systems (Fig. 2h). Multiple sharp peaks in the TGF PA-a WAXS pattern indicated highly ordered crystalline packing, in contrast to the less ordered internal organization of TGF PA-b. Next, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy were performed to investigate the secondary structure of the assemblies and the degree of hydrogen bonding between PA molecules (Figs. 2i-j, 13, and 14). Scaffold PA-a, Scaffold PA-b, and TGF PA-a assemblies showed strong β-sheet-like character with CD maxima at approximately 195 nm, but 41 Both lnTGF PA assemblies had little or no β-sheet structure (Figure 2i), indicating that the linear epitope more severely interfered with the hydrogen bonds required for β-sheet formation. Similarly, FTIR spectra of both TGF PA-a and TGF PA-b assemblies showed a peak at approximately 1625–1640 cm -1 The canonical β-sheet amide I band of 42 , which was absent in both the lnTGF PA spectra (Figure 2j). Skeletal PA-a also exhibited a peak at approximately 1600 cm -1 The cytoplasmic PA showed a second peak at 100 nm, which was absent in all other samples (FIG. 2j). Different combinations of backbone PA and epitope PA led to changes in the internal order and secondary structure of the assembled bodies.

[0116] To further probe the intermolecular dynamics and mobility of individual PA molecules within the assemblers, fluorescence anisotropy (FA) was used to measure the rotational diffusion of TAMRA-labeled epitopes within TGF PA nanostructures (Figure 3a). TGF PA was co-assembled with dilute scaffold PA at 10 mol%, and 0.2 mol% of TGF PA molecules were conjugated with TAMRA on a cyclic epitope. It was confirmed that the TAMRA fluorophore did not interfere with self-assembly, and PA nanostructures with TAMRA-labeled epitopes formed the same morphology as unlabeled assemblers (Figure 15). FA revealed significantly lower anisotropy values ​​in TGF PA-a compared to TGF PA-b, indicating increased epitope mobility in terms of rotational diffusion (Figure 3a). This higher rotational diffusion may allow epitopes on TGF PA-a assemblers to change conformation or extend from the nanostructure and bind cellular receptors.

[0117] To better understand the internal dynamics of the assemblies and the mobile diffusion of individual PA molecules, transverse relaxation nuclear magnetic resonance (T2-NMR) spectroscopy was used to measure the spin-lattice relaxation (T2) rates of the methylene protons on the ε carbon (Hε) of the lysine residues of the CESPLKRQC (SEQ ID NO: 1) cyclic epitope (observed at 2.64 ppm) and the methyl protons on the terminal carbon of the alkyl tail at the peptide N-terminus (0.77 ppm) (Figures 3b-c, 16, and 17). In contrast to FA, which measures only the rotational diffusion of the labeled epitope, T2-NMR can also probe the mobile diffusion of PA molecules within the assemblies and infer intermolecular aggregation. The relaxation rates of the alkyl tail protons in TGF PA-a assemblies were much higher than those in TGF PA-b assemblies, almost 5 times higher (Figures 3b-c). This higher relaxation rate was consistent with lower mobile diffusion and stronger intermolecular aggregation, as explained by the attractive forces between PA molecules that stabilize the supramolecular structure. T2-NMR measurements from the alkyl tail protons confirmed our WAXS results, indicating that TGF PA-a assemblies had high internal order and stronger intermolecular aggregation compared to TGF PA-b. In contrast, the epitope protons in both TGF PA-a and TGF PA-b assemblies had similar low relaxation rates (Figure 3b-c). This indicated that TGF PA-a assemblies had high internal order, but the epitopes displayed on the nanostructured surface were flexible and mobile. This coexistence of internal crystallinity and epitope mobility revealed that the epitope PA molecules significantly influenced the supramolecular dynamics within the TGF PA-a nanostructure. In the TGF PA-a assemblies, the cyclic epitope PA molecules may have caused localized disruption in the crystal structure and provided greater mobility for the epitope. Similar T2-NMR measurements of alkyl tail protons in scaffold PA assemblies revealed increased supramolecular dynamics at the periphery of scaffold PA-a assemblies compared to scaffold PA-b (Table 1). These interplays of scaffold and epitope PA dynamics in TGF PA-a were unexpected because they demonstrated that even when the inner core of the assemblies is highly ordered and aggregated, the epitope and periphery can still exhibit dynamic mobility.The TGF PA-a nanostructures also have an increased surface area due to their wide belt-like morphology, which likely presents circular epitope molecules with a dynamic range of motion to interact more favorably with cells. [Table 1]

[0118] Cellular responses to TGF-β1 mimetic peptide amphiphile nanostructures Considering the differences in morphology, internal order, and supramolecular dynamics between TGF PA-a and TGF PA-b nanostructures, the effect of these differences on bioactivity was analyzed by treating human articular chondrocytes in vitro with PA solutions diluted in culture medium. Biocompatibility was measured by treating cells with each TGF PA solution at different co-assembler ratios and concentrations, and then viability was analyzed after 24 h and 3 days of culture (Figures 18 and 19). After 24 h, all PA treatments induced high viability (300 cells / mm3) at both 10 μM and 50 μM. 2 After 3 days, nearly all 10 μM PA treatments showed similarly high viability (>400 cells / mm 2 The 25 mol% and 50 mol% coassemblies varied in both biocompatibility and morphology, whereas the 10% coassembly, which formed a uniform fibrous structure, revealed consistent concentration- and time-dependent biocompatibility. The total PA concentration was varied in the 10 mol% epitope coassembly to determine the optimal concentration for biocompatibility. Cells were treated in solutions containing various concentrations of each TGF PA, lnTGF PA, scaffold PA, peptide alone, or native recombinant human TGF-β1 (rhTGF-β1) (Figures 20 and 21). After 24 hours of culture, all PA treatments except for 100 μM scaffold PA-b and lnTGF PA-b resulted in high viability (>300 cells / mm 2 After 3 days of culture, all PA treatments up to 10 μM showed high viability (>300 cells / mm 2Although we showed that 50 μM and 10 μM PA treatments were appropriate concentrations for treatment time points up to 24 h and 3 days, higher concentrations significantly reduced viability, likely due to excessive signaling or excess PA material that accumulated on top of the cells after 3 days. These results informed the experimental design that 50 μM and 10 μM PA treatments were appropriate concentrations for treatment time points up to 24 h and 3 days, respectively.

[0119] After 3 days, the scaffold PA-a system, TGF PA-a, and lnTGF PA-a exhibited greater biocompatibility than the corresponding scaffold PA-b system. This difference in biocompatibility was unexpected because all of the assemblies formed long, high aspect ratio structures, a physical attribute that is generally attributed to enhanced cell viability. 35、43 The molecules of each system differ only in the order of two adjacent amino acids in the non-bioactive PA backbone. However, WAXS and T2-NMR spectroscopy revealed less internal order and lower relaxation rates for TGF PA-b assemblies, suggesting increased mobile diffusion and weaker internal aggregation that may have led to disruption of the cell lipid membrane and cell death via dissociation of TGF PA-b molecules from the nanofibers. Conversely, the high internal order and relaxation rates of TGF PA-a assemblies indicated stronger internal aggregation that allowed the high aspect ratio nanostructures to remain intact, coexist and interact with cells.

[0120] TGF-β1-mimicking PA nanostructures activate intracellular signaling During the development of the embodiments herein, experiments were performed to evaluate the ability of PA materials to activate intracellular TGF-β1 signaling in chondrocytes. For TGF PA-a nanostructures, it was hypothesized that the wide belt-like morphology, crystalline internal order, and dynamic epitopes would contribute to enhanced bioactivity for chondrogenic signaling. Co-assemblies and concentration sweeps were performed by treating cells with different co-assembly ratios (10, 25, and 50 mol%) and concentrations (10 and 50 μM) of each TGF PA, rhTGF-β1 as a positive control, or starvation medium as a negative control. After 4 hours of treatment, Western blot analysis was performed to evaluate the levels of phosphorylated Smad2 (p-Smad2) and Sox9 (Figures 4a-b and 22). Smad2 phosphorylation is the first step of canonical TGF-β1 signaling in cartilage after receptor binding. Upon activation, phosphorylated Smad2 and Smad3 form a complex with Smad4, which translocates to the nucleus and initiates transcription (including transcription of chondrogenic genes) 11、12 10 mol% co-assembly and TGF-PA-a at 50 μM showed strong p-Smad2 activation. Similarly, high co-assembly ratios induced little cellular response, preventing the enhanced signaling previously seen with long fiber structures, possibly because short fibers and micelles displayed less favorable conformational or distribution epitopes. Sox9 is a nuclear transcription factor expressed during early mesenchymal stem cell condensation and was not expected to be significantly upregulated in mature chondrocytes. 10 There was no significant upregulation of Sox9, but all conditions showed baseline levels of expression, indicating a healthy chondrocyte phenotype (Figure 22).

[0121] Based on the biocompatibility and bioactivity results, experiments were performed at 10 mol% coassembly for all epitope PA assemblies, 50 μM for short-term (<24 h) treatments, and 10 μM for long-term (>24 h) treatments. Cells were treated with 50 μM of each TGF PA, lnTGF PA, scaffold PA, peptide alone, or rhTGF-β1 for 4 h. Western blot analysis showed no activation by scaffold PA, linear epitope PA, or peptide alone, confirming the hypothesis that both cyclization of the epitope and its presentation on the PA nanostructures conferred bioactivity (Figure 4c-d). TGF PA-a upregulated p-Smad2 more significantly than all other PA treatments, including its constituent isomer TGF PA-b. This further supported the hypothesis that the inner PA scaffold influences bioactive signaling via morphology and internal dynamics, enhancing cell receptor interaction with epitopes more preferably presented on TGF PA-a assemblies. Next, to study the nature of TGF-β1 activation by the mimetic PA nanostructures, cells were treated with TGF PA-a, TGF PA-b, or rhTGF-β1 with or without the addition of 1D11, a pan-TGF-β neutralizing antibody, each of which has been shown to neutralize all three isoforms of TGF-β in vitro and in vivo. 44~46 Western blot analysis revealed that 1D11 effectively neutralized TGF PA-a, TGF PA-b, and rhTGF-β1 in activating p-Smad2 (Fig. 4e-f). Comparing the activation of p-Smad2 with or without 1D11, the neutralizing effect of 1D11 on TGF PA-a was statistically significant, but its effect on TGF PA-b was not significant. This was most likely because TGF PA-a upregulated p-Smad2 more than TGF PA-b. These results confirmed that the mimetic TGF PA-a nanostructures activated intracellular TGF-β1 signaling in chondrocytes.

[0122] TGF-PA nanostructures enhance ECM synthesis in chondrocytes The main role of chondrocytes in articular cartilage is to maintain the ECM.1、8 The synthesis of cartilaginous proteins was analyzed after 10 μM PA treatment for 3 days in vitro. Western blot was used to measure the levels of collagen II (Col2a1), cartilage oligomeric matrix protein (COMP), and aggrecan (Acan), all essential components of articular cartilage. 10、12 , and the transcription factor Sox9 (Figure 5a-b) were analyzed. In general, TGF PA-a upregulated ECM synthesis more significantly than all other PA treatments. Treatment with skeletal PA, linear epitope PA, and peptide alone again showed low activation, further demonstrating that the presentation of cyclic epitopes on the PA nanostructures is important for bioactivity. Interestingly, cells treated with TGF PA-a had significantly higher levels of collagen II than cells treated with native rhTGF-β1 protein. Chondrocyte signaling is mainly mediated by integrin-collagen II binding. 47 , and mechanical activation of endogenous TGF-β1 sequestered in the ECM. 48 These results indicate that the morphology and supramolecular dynamics of TGF-PA-a nanostructures may physically present mimicking epitopes more favorably for receptor binding, as mediated by physical cell-ECM interactions such as cellular signaling.

[0123] To confirm these results from Western blot, immunocytochemistry (ICC) staining and fluorescence intensity image analysis were performed on cells treated under the same conditions as Western blot (Fig. 5c-d). Confocal microscopy imaging results were consistent with the Western blot results and revealed that the mimetic TGF PA-a induced significantly more collagen II production than native rhTGF-β1 (Fig. 5c). These results indicate that the TGF PA-a nanostructure may have presented epitopes with optimal orientation, density, and / or dynamic motion for receptor binding compared to the native protein in solution. In addition, the native TGF-β1 protein has a half-life of only 2-3 min in its active form in vivo. 49Thus, PA nanostructures may have delayed epitope degradation and improved activation kinetics relative to the native protein. Both rhTGF-β1 and TGF PA-a significantly increased aggrecan synthesis at similar levels. Cells treated with the mimetic peptide alone showed little upregulation of either protein, again indicating that the peptide alone is unable to mimic TGF-β1. Staining of F-actin also revealed that cells treated with rhTGF-β1 and TGF PA-a maintained a healthy chondrocyte phenotype and prevented hypertrophy, as evidenced by regular, round or oval morphology, and compact cell size (Figure 5c-d). These results indicated that TGF PA-a not only enhanced ECM synthesis but also maintained a mature chondrocyte phenotype without hypertrophy over a longer in vitro culture period.

[0124] Three-dimensional TGF-PA scaffolds maintain chondrocyte phenotype Because TGF-β1 mimetic PA nanostructures effectively activated intracellular TGF-β1 signaling and ECM synthesis, and due to the important role of three-dimensional ECM organization in articular cartilage structure and function, experiments were conducted during the development of the embodiments herein to investigate their function as three-dimensional gel networks. Because TGF PA-a showed bioactivity, studies focused on hydrogels composed of scaffold PA-a, TGF PA-a, and lnTGF PA-a. PA hydrogel networks were prepared by exposing PA solutions to calcium ions. 26To confirm that the differences in bioactivity were not caused by differences in the mechanical properties of the hydrogels, scanning electron microscopy (SEM) and rheology were performed, demonstrating that the PA hydrogels each had similar morphology and viscoelastic behavior (Fig. 6a-d, Table 2). The three PA gels had similar flow strain and elastic modulus, indicating that they may behave similarly with shear strain in vivo. We then encapsulated chondrocytes within the PA gels and cultured them in vitro for 3 days to measure biocompatibility and observe chondrogenic behavior. The cells survived the encapsulation process and maintained high viability as measured by the levels of lactate dehydrogenase (LDH), a cytoplasmic enzyme that is only released upon cell lysis in the culture medium (Fig. 23). The morphology and distribution of the encapsulated cells were examined using F-actin ICC staining and confocal microscopy (Fig. 6e-g). Increased cell migration or clustering may be indicative of osteoarthritis, dedifferentiation, and reduced chondrogenic capacity, so we used a 3D CT image of the chondrocytes in the culture medium. 50~53 The phenotypic and migratory responses of chondrocytes within the PA gels were analyzed. Although there was no significant difference in viability, the cells in each PA gel behaved notably differently. Despite the same concentration of cells being encapsulated, there were far fewer cells visible within the majority of scaffold PA-a and lnTGF PA-a gels compared to the TGF PA-a gels (Figure 6e-g). In contrast, cells in the TGF PA-a gels remained within the gel and survived for 3 days in vitro, indicating that the TGF PA-a hydrogels provided the necessary bioactive signals for a sustainable microenvironment. Furthermore, the cells exhibited a compact, oval morphology and were well-dispersed throughout the TGF PA-a gels, similar to their chondrogenic behavior in native cartilage. These cell-PA interactions indicated that in addition to inducing a chondrogenic TGF-β1 response, our TGF-β1-mimetic PA nanostructures maintained healthy chondrocyte phenotype and behavior within a 3D ECM environment. [Table 2]

[0125] Example 2 Slurry formulation and in vivo data method PA / HA slurry formulation As described for the in vitro data, PA solutions were prepared at 2 wt% and then mixed with various amounts of cross-linked hyaluronic acid particles. The resulting slurries were physically agitated and then microcentrifuged for 15 min before being stored at 4°C for 24 h to allow complete hydration of the HA particles. The slurry material was loaded into 1 mL syringes with 19-gauge needles and kept on ice until use during surgery. Fluorescently labeled PA was produced by covalent functionalization of carboxytetramethylrhodamine (TAMRA) to the C-terminus of diluted scaffold PA-a. For fluorescently labeled PA experiments, the final co-assembly ratio was 10 mol% epitope PA, 88 mol% scaffold PA, and 2 mol% TAMRA PA.

[0126] Rheology Measurements were performed on an Anton Paar MCR302 rheometer equipped with a 25 mm parallel plate fixture. 120 μL of PA / HA hybrid slurry was placed on the bottom plate and 30 μL of 50 mM CaCl2 / 75 mM NaCl gelling solution was placed on the top fixture. The fixture was lowered to a gap of 0.5 mm for 10 min, during which an oscillatory strain of 0.1% was applied at an angular frequency of 10 rad / s. The gap height was varied to keep the normal force at 0 N. A strain sweep was then performed at 10 rad / s from 0.1% to 100% strain to measure the strain at which the gel broke.

[0127] Rabbit osteochondral defect model New Zealand white rabbits (2.8-3.2 kg) were used for this procedure. Prior to surgery, the rabbits were clipped across the surgical site and sterilized with chlorhexidine scrub. For all rabbits, a 3 cm curvilinear incision was made on the lateral aspect of the patellar tendon. The joint capsule and synovium were incised across the lateral aspect of the joint. According to our established protocol, circular osteochondral defects (2-3 mm diameter) approximately 3 mm deep were created bilaterally in the medial condyle of each rabbit. Approximately 30 μL of PA / HA slurry was expelled from the syringe and filled into the defect space. After filling the defects with PA / HA slurry, approximately 30 μL of sterile 50 mM CaCl2, 75 mM NaCl gelling solution was dripped onto the top of each defect. After irrigating the joints with saline, the synovium and joint capsule were closed in one layer using 4-0 absorbable sutures in a continuous pattern. Additionally, the muscular layer and subcutaneous tissue were closed with 4-0 absorbable sutures, and the skin was closed with 4-0 or 5-0 absorbable sutures in a subcuticular pattern. After euthanasia, the surgical joints were harvested and the gross appearance was recorded by digital photography. The operated medial condyle of each group was then cut into small bone blocks containing the defect and its associated underlying subchondral bone using a band saw. The bone blocks were fixed in 10% neutral buffered formalin and then decalcified using an EDTA / sucrose decalcification solution (20% EDTA in 5% sucrose). After decalcification, the blocks were cut in half through the center of the defect using a razor blade, and both parts were embedded in the same paraffin block. Serial 5-μm-thick sections were prepared and stained with H&E to evaluate the general morphology of the implantation site. Both stained (histology) and unstained (fluorescence) sections were imaged using a TissueGnostics microscope.

[0128] result To test the bioactivity of TGF-mimetic PA in vivo, a solution of TGF PA-a nanofibers was mixed with cross-linked hyaluronic acid (HA) particles to create a robust injectable slurry formulation that could be implanted into cartilage defects and could withstand shear forces within the joint. Various ratios of this hybrid material were tested using a 2 wt% PA solution mixed with different amounts of HA (2-6 wt%). The storage modulus and strain required to fracture the hybrid gel increased with increasing concentrations of HA (Figure 25). Compared to the PA gel alone, the hybrid PA / HA slurry had a 3-4 times higher strain to fracture, highlighting the increased ductility and resistance to shear of the hybrid material. Although the high concentration of HA improves the stiffness and toughness of the gel, it also results in increased volume expansion in saline, which can lead to displacement of the material after implantation. Therefore, to balance mechanical toughness with minimal swelling, a 2 wt% PA + 4 wt% HA slurry formulation was used for the in vivo studies.

[0129] To test the material retention in cartilage defects in vivo, hybrid slurries with fluorescently labeled PA were implanted into osteochondral defects in a rabbit condyle model (Figure 26). The PA / HA slurry (pink color) was clearly visible and localized in the defect after implantation. Joint explants 1 and 2 days after surgery showed good retention of the implant in the defect, as a robust clot was macroscopically observed and a clear fluorescent signal was detected at the implant site. Histological staining showed good integration of the implant with the surrounding bone and cartilage tissue, which is important for robust cartilage regeneration. After 7 days, the defect site showed macroscopically good tissue filling and early stages of cartilage regeneration in histological slices. No fluorescent signal was detected from the PA material, suggesting that the implant initiated the tissue regeneration process and then biodegradation as expected.

[0130] It should be understood that the foregoing detailed description and accompanying examples are merely illustrative and should not be construed as limiting the scope of the present disclosure, which is defined solely by the appended claims and equivalents.

[0131] Various changes and modifications to the embodiments of the present disclosure will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those related to the chemical structures, substituents, derivatives, intermediates, compounds, compositions, formulations, or methods of use of the present disclosure, may be made without departing from the spirit and scope thereof.

[0132] Any patents and publications referenced herein are hereby incorporated by reference in their entirety.

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Claims

1. A composition comprising a bioactive peptide amphiphilic substance (PA), wherein the bioactive peptide amphiphilic substance (PA) comprises (i) a hydrophobic non-peptide segment, (ii) a β-sheet forming peptide segment, (iii) an acidic peptide segment, (iv) a TGF-β1 mimetic epitope peptide, and the composition.

2. The TGF-β1 mimetic epitope peptide comprises an amino acid sequence having three or fewer substitutions relative to CESPLKRQC (SEQ ID NO: 1), or The TGF-β1 mimetic epitope peptide has 100% sequence similarity with CESPLKRQC (SEQ ID NO: 1), The composition according to claim 1.

3. The composition according to claim 1, wherein the hydrophobic non-peptide segment of the bioactive peptide amphiphilic substance comprises an acyl chain.

4. wherein the acyl chain is C ６ to C ２０ The composition according to claim 3, comprising.

5. The β-sheet forming peptide segment of the bioactive peptide amphiphilic substance comprises a combination of 2 to 6 V residues and A residues, and / or The acidic peptide segment of the bioactive peptide amphiphilic substance comprises a combination of 1 to 4 Glu (E) residues and / or Asp (D) residues, and the composition according to claim 1.

6. The β-sheet forming peptide segment of the bioactive peptide amphiphilic substance and the charged peptide amphiphilic substance is selected from VVVAAA (SEQ ID NO: 3), AAAVVV (SEQ ID NO: 4), AAVV (SEQ ID NO: 5), VVAA (SEQ ID NO: 6), AA, VV, VA, or AV, and / or The acidic peptide segment is selected from E, EE, EEE, D, DD, DDD, ED, DE, EDE, DED, EDD, and DEE, and the composition according to claim 5.

7. The composition according to claim 1, comprising the backbone PA of the bioactive peptide amphiphilic substance selected from C16-AAEE (SEQ ID NO: 7), C16-AEAE (SEQ ID NO: 8), and C16-VVVAAAE EE (SEQ ID NO: 9).

8. The composition according to claim 7, wherein the TGF-β1 mimetic epitope peptide is CESPLKRQC (SEQ ID NO: 1) cyclized via a disulfide bond.

9. The composition according to claim 7, wherein the TGF-β1 mimetic epitope peptide is linked to the backbone PA by a lysine linker.

10. (i) a hydrophobic non-peptide segment, (ii) a β-sheet forming peptide segment, (iii) The composition according to claim 1, further comprising a diluted PA, which comprises a charged peptide segment.

11. The composition according to claim 10, wherein the hydrophobic non-peptide segment of the diluted peptide amphiphile comprises an acyl chain.

12. wherein the acyl chain is C ６ -C ２０ The composition according to claim 11, comprising.

13. The β-sheet forming peptide segment of the diluted peptide amphiphile comprises a combination of 2 to 6 V residues and A residues, and / or The composition according to claim 10, wherein the acidic peptide segment of the diluted peptide amphiphile comprises a combination of 1 to 4 Glu (E) residues and / or Asp (D) residues.

14. The β-sheet forming peptide segment of the diluted peptide amphiphile is selected from VVVAAA (SEQ ID NO: 3), AAAVVV (SEQ ID NO: 4), AAVV (SEQ ID NO: 5), VVAA (SEQ ID NO: 6), AA, VV, VA, or AV, and / or The composition according to claim 13, wherein the acidic peptide segment of the diluted peptide amphiphile is selected from E, EE, EEE, D, DD, DDD, ED, DE, EDE, DED, EDD, and DEE.

15. The composition according to claim 10, comprising a backbone PA of the diluted peptide amphiphile selected from C16-AAEE (SEQ ID NO: 7), C16-AEAE (SEQ ID NO: 8), and C16-VVVAAAEEE (SEQ ID NO: 9).

16. The composition according to claim 10, comprising 5% to 95% (mol) of a bioactive peptide amphiphile and 5% to 95% (mol) of a diluted peptide amphiphile.

17. The composition according to claim 10, wherein the bioactive peptide amphiphile and the diluted peptide amphiphile co-assemble to form a helical ribbon-like structure with an average diameter of 50 to 60 nm.

18. A composite material comprising the composition according to any one of claims 1 to 17 and a biocompatible polymer.

19. The biocompatible polymer is hyaluronic acid, The composite according to claim 18, wherein the hyaluronic acid is present as hyaluronic acid particles. **Claim 20**: The composition according to any one of claims 1 to 17 for promoting cartilage repair or regeneration, treating osteoarthritis or musculoskeletal injury or disease, or preventing osteoarthritis or musculoskeletal injury or disease in a subject suffering from cartilage defect or injury, suffering from osteoarthritis or musculoskeletal injury or disease, or having a high risk of osteoarthritis or musculoskeletal injury or disease. **Claim 21**: The composite according to claim 18 for promoting cartilage repair or regeneration, treating osteoarthritis or musculoskeletal injury or disease, or preventing osteoarthritis or musculoskeletal injury or disease in a subject suffering from cartilage defect or injury, suffering from osteoarthritis or musculoskeletal injury or disease, or having a high risk of osteoarthritis or musculoskeletal injury or disease.

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