Enhancement of functional maturation of human ipsc-derived neurons and regeneration with dynamics within supramolecular ikvav matrices

Abstract

To provide design of synthetic materials capable of more efficiently mimicking the structure and function of biological matrices.SOLUTION: Provided herein are peptide amphiphiles (PAs) comprising a bioactive peptide, nanofibers displaying the bioactive PAs, and methods of use thereof. The disclosed peptide amphiphiles comprise a hydrophobic tail, a structural peptide segment, a charged peptide segment, and a bioactive IKVAV peptide. The disclosed PAs may be used in cell culture methods and in methods of treating central nervous system injury.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Patent Application No. 62 / 796,425, filed January 24, 2019, which is incorporated by reference in its entirety.

[0002] [Technical field] Provided herein are peptide amphiphiles (PAs) containing bioactive peptides, nanofibers exhibiting bioactive PAs, and methods of their use. In some embodiments, provided herein are peptide amphiphiles for use in cell culture methods. In other embodiments, provided herein are peptide amphiphiles for use in methods of treating nervous system injury.

[0003] [background] The extracellular matrix (ECM) is a hierarchical milieu of water, matrix proteins, and other chemical factors that provides a microenvironment that orchestrates cellular behavior and plays an essential role in injury and disease as well as in the developing and adult central nervous system (CNS). The molecular complexity and dynamic organization of the ECM allows it to spatiotemporally bind to many specific transmembrane receptors, which in turn trigger multiple vital cellular responses through a process known as signal transduction. The rapid evolution of synthetic materials in recent years has made it possible to recapitulate aspects of the ECM with substantially improved in vitro cell culture platforms and tissue engineering approaches. Traditional synthetic substrates and matrices have proven to be of limited utility, in large part due to their inability to capture the dynamic properties of the ECM in different in vivo contexts such as development, maturation, disease progression, and maintenance of homeostasis. The design of synthetic materials that can more efficiently mimic the structure and function of biological matrices remains a challenging goal.

[0004] [overview] Provided herein are peptide amphiphiles (PAs) containing bioactive peptides, nanofibers exhibiting bioactive PAs, and methods of their use. In some embodiments, the peptide amphiphiles may be used in methods of generating neuronal cells. In other embodiments, the peptide amphiphiles may be used in methods of treating central nervous system injuries, such as spinal cord injuries.

[0005] In some embodiments, a peptide amphiphile is provided herein. The peptide amphiphile comprises a hydrophobic tail, a structural peptide segment, a charged peptide segment, and a bioactive peptide. Further described herein is a nanofiber comprising the peptide amphiphile described herein. The nanofiber may further comprise one or more filler peptide amphiphiles. The filler peptide amphiphile comprises a hydrophobic tail, a structural peptide segment, and a charged peptide segment. The filler peptide amphiphile does not comprise a bioactive moiety.

[0006] In some embodiments, the hydrophobic tail is an alkyl chain having 8 to 24 carbon atoms (C 8-24 In some embodiments, the hydrophobic tail may comprise a 16 carbon alkyl chain (C 16 )

[0007] In some embodiments, the structural peptide segment comprises V2A2, A2G2, or VEVA2. In some embodiments, the structural peptide segment has a propensity to form beta-sheet-like structures or other stabilizing interactions with adjacent structural peptide segments (e.g., to promote self-assembly of adjacent nanofibers). In some embodiments, the structural peptide segments have an overall propensity for beta-sheet structure formation of 4 or less.

[0008] In some embodiments, the charged peptide segment comprises an acidic, basic, or zwitterionic peptide segment. In some embodiments, the charged peptide segment comprises 2-4 glutamic acid (E) residues. For example, the charged peptide segment can comprise EE, EEE, or EEEE.

[0009] In some embodiments, the bioactive peptide comprises IKVAV. The bioactive peptide may be attached to the charged peptide segment by a linker. For example, the linker may be a single glycine (G) residue.

[0010] In some embodiments, the peptide amphiphile is IKVAV(G)E4V2A2-C 8-24 In another embodiment, the peptide amphiphile comprises IKVAV(G)E4A2G2-C 8-24 In some other embodiments, the peptide amphiphile comprises IKVAV(G)E4VEVA2-C 8-24 Contains:

[0011] In some embodiments, the filler peptide amphiphile is E4V2A2-C 8-24 In some embodiments, the filler peptide amphiphile comprises E4A2G2-C 8-24 In some other embodiments, the filler peptide amphiphile comprises E4VEVA2-C 8-24 Contains:

[0012] In some embodiments, the disclosed peptide amphiphiles or peptide amphiphile nanofibers can be used in cell culture methods. For example, the disclosed peptide amphiphiles or peptide amphiphile nanofibers can be used in neuronal cell culture methods. In some embodiments, the disclosed peptide amphiphiles or peptide amphiphile nanofibers can be used to culture motor neurons.

[0013] In some embodiments, methods of treating central nervous system (CNS) injury are provided herein, comprising administering to a subject suffering from CNS injury a pharmaceutical composition comprising a peptide amphiphile disclosed herein. In some embodiments, the CNS injury is a spinal cord injury. In some embodiments, the pharmaceutical composition is administered parenterally.

[0014] [Brief description of the drawings] Figure 1A–L. Characterization of supramolecular nanofibers formed by peptide amphiphiles (PAs). (A) Schematic molecular structure of IKVAV PA, which contains (B) V2A2, (C) A2G2, and (D) VEVA2. (E–G) Cryo-TEM micrographs of (E) V2A2, (F) A2G2, and (G) VEVA2 in KCl and NaCl aqueous solutions ([PA] = 0.01 wt%, [KCl] = 3 mM, [NaCl] = 150 mM). (H–J) SEM micrographs of (H) V2A2, (I) A2G2, and (J) VEVA2. (K) FT-IR spectra (1500–1800 cm) of film samples of V2A2 (red), A2G2 (blue), and VEVA2 (orange). -1 (L) WAXS profiles of V2A2 (red), A2G2 (blue), and VEVA2 (orange) in KCl and NaCl aqueous solutions ([PA] = 5.3 mM, [KCl] = 3 mM, [NaCl] = 150 mM).

[0015] Figure 2A-I. Dynamics within supramolecular IKVAV PA induce distinct effects on human motor neuron (MN) signals. (A) Fluorescence depolarization profiles (λ) at 25 °C of DPH (2.8 µM) embedded in IKVAV PA containing V2A2 (red), A2G2 (blue), and VEVA2 (orange) in KCl and NaCl aqueous solutions. ex = 336 nm, λ em= 450 nm) ([PA] = 100 μM, [KCl] = 3 mM, [NaCl] = 150 mM). (B) Self-assembled structure and coarse-grained view of a single IKVAV PA fiber containing V2A2 (red), A2G2 (blue), and VEVA2 (orange) PA after 10 μs. The micrograph contains a partial periodic image through the fiber formation axis in the simulation box (in blue). Water and ions have been omitted for clarity. (C) Kinetic analysis of the IKVAV PA containing V2A2 (red), A2G2 (blue), and VEVA2 (orange) structure formed in B. (D) Fluorescence emission spectra (λ 2.8 μM) at 25 °C of DPH (2.8 μM) embedded in IKVAV PA containing V2A2 (red), A2G2 (blue), and VEVA2 (orange) in KCl and NaCl aqueous solutions. ex = 336 nm) ([PA] = 100 μM, [KCl] = 3 mM, [NaCl] = 150 mM). (E) Water content analysis of IKVAV PA containing V2A2 (red), A2G2 (blue), and VEVA2 (orange). (F) Representative three-dimensional SIM micrograph (left) and shaded reconstruction (right) of MN neurites (TUJ-1, green) containing b-1 integrin (pink, ITGB1) cultured on A2G2 and laminin. (G) Intensity analysis of ITGB1 in MNs cultured on A2G2 (blue) and laminin (black) matrices at 72 h. (H) Western blot analysis of ITGB1 and downstream kinases (ILK, pFAK, FAK) in MNs cultured on supramolecular IKVAV matrices containing V2A2 (red), A2G2 (blue), and VEVA2 (orange) at 72 h and (I) normalized protein levels. Scale bar: 10 mm. Data are the average of at least four independent differentiations. All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0016] Figure 3A-O. Effect of intermolecular cohesion of A2G2-containing IKVAV PA on human motor neuron (MN) maturation. (A) Schematic of human MN differentiation and its culture on supramolecular matrix. (B) Western blot and (C) normalized protein expression levels (after 60 days) related to integrin activation (ITGB1), signaling pathways (ILK) and motor neuron maturation (ChAT) in MNs cultured on supramolecular IKVAV matrix containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black). All values ​​were normalized to actin. (D) Schematic of integrins and signaling pathways mediating cell behavior upon dynamic presentation of A2G2-containing IKVAV. All values ​​were normalized to actin or total FAK expression. (E) Volcano plots depicting proteomic changes of MNs cultured on A2G2 vs laminin. Mean log2 (fold change) vs P-value (-log 10 ) are shown. Proteins up- and downregulated by 2-fold change and FDR<0.05 are labeled by green and red dots, respectively. (F) Subset of most significant GO terms enriched in up- and downregulated groups of proteins identified in MNs cultured in A2G2 vs. laminin coatings. (G) Cell viability assessed by LDH concentration released into cell medium (day 60). (H) ChAT in MNs cultured on IKVAV PA containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) matrices. +Quantification of cells (after 60 days). (I,J) Confocal microscopy images of human MNs cultured on (I) A2G2 and (J) laminin matrices. (K) Analysis of total neurite processes of human MNs cultured on IKVAV PA containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) matrix (after 60 days). (L) Sholl analysis of dendritic branching of MNs cultured in vitro for 60 days on V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (gray). (M) Representative representative confocal micrograph of human MNs cultured on IKVAV PA containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (white) matrix (after 60 days). Cells were stained with the motor neuron marker ChAT (green), microtubule-associated protein-2 (MAP-2, red) and nuclei (DAPI). (N) DAPI channel micrograph of the image shown in M. (O) Histogram analysis of cell distribution on the different matrices referenced in M ​​and N. Scale bar: (M, N) 100 μm. Data are the average of at least three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0017] Figure 4A-P. Impact of molecular dynamics within supramolecular assemblies on functional maturation and regeneration. (A,B) Representative confocal micrographs (day 50) of synaptic vesicles (postsynaptic PS95 and presynaptic SYN-1) distributed along human MN neurites cultured on (A) A2G2 and (B) laminin matrices. (C) Intensity analysis of PSD95 and SYN-1 on MNs cultured on A2G2 and laminin coatings. (D) Western blots and (E) normalized protein levels of presynaptic and postsynaptic markers (SYN-1 and PS95, respectively) in human MNs cultured on A2G2 (blue) and laminin (black). All values ​​were normalized to actin. (F) Brightfield images of human MNs cultured for 40 days on MEA plates coated with A2G2 supramolecular peptide amphiphiles. Plots representing (G) the difference in spike counts and (H) the number of bursts per electrode and (I) synchrony index in MN cultures in various IKVAV PA, laminin or glial coatings. (J) Two-photon microscopy images (days 48-49) of human MNs cultured on A2G2 and laminin matrix and filled with Texas Red dextran through the patch electrode. (K) Percentage of neurons grown on A2G2 and laminin matrix that can repeatedly fire action potentials. (L) Representative examples of action potentials from MNs grown on A2G2 and laminin matrix. MNs grown on A2G2 were significantly larger and had faster rise and fall velocities. (M) Schematic of a dorsoventral contusion injury in a mouse spinal cord. (N) Basso Mouse Scale (BMS) scores for animals treated with saline solution (control), V2A2 (red) and A2G2 (blue). (O) Western blot and (P) normalized protein levels of beta-1 integrin receptor (ITGB1), various ECM proteins (fibronectin and laminin), astroglial GFAP and neuronal GAP43 markers in spinal cords treated with saline solution (control), V2A2 (red) and A2G2 (blue). All values ​​were normalized to GAPDH. In vivo data are the average of at least eight animals.All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0018] Figure 5A-F. MARTINI bead type and charge for (a) V2A2, (b) A2G2, (c) VEVA2 and (d) VE(-)VA2. (e) Charge amount of PA. (f) Raw radial distribution function for each scaffold bead of V2A2 (insert), showing in blue the area integrated to give the water contacts for the first solvation sphere.

[0019] Figure 6A-F. Liquid chromatography-mass spectrometry of IKVAV PA. (ac) Purified (a)C 16 V2A2E4GIKVAV, (b)C 16 A2G2E4GIKVAV(c)C 16 Analytical high-performance liquid chromatography (analytical HPLC) traces of VEVA2E4GIKVAV. (d-f)(d)C 16- V2A2E 4- GIKVAV, (e)C 16 A2G2E4GIKVAV and (f)C 16 Electrospray ionization mass spectrometry (ESI-MS) of VEVA2E4GIKVAV.

[0020] Figure 7A-I. Cryo-TEM and SEM images of IKVAV PA. (a-c) Schematic molecular structure, (a, d, g) C 16 V2A2E4GIKVAV, (b, e, h)C 16 A2G2E4GIKVAV(c,f,i)C 16 (d–f) Cryo-TEM and (g–i) SEM micrographs of VEVA2E4GIKVAV.

[0021] Figure 8A-D. TEM images of DPH-embedded IKVAV PA. (a) Schematic molecular structure of 1,6-diphenyl-1,3,5-hexatriene (DPH). (b-d) DPH-embedded PA stained with uranyl acetate. (b) C 16- V2A2E4GIKVAV, (c)C 16 A2G2E4GIKVAV and (d)C16 TEM micrograph of VEVA2E4GIKVAV ([PA]=100 μM, [DPH]=2.8 μM in KCl and NaCl aqueous solutions ([KCl]=3 mM, [NaCl]=150 mM).

[0022] Figure 9A-F. TEM images of TMA-DPH-embedded IKVAV PA. (a) Schematic molecular structure of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH). (b-d) TMA-DPH-embedded IKVAV PA stained with uranyl acetate. (b) C 16 V2A2E4GIKVAV(V2A2), (c)C 16 A2G2E4GIKVAV(A2G2) and (d)C 16 TEM micrographs of VEVA2GIKVAV (VEVA2) in KCl and NaCl aqueous solutions ([KCl] = 3 mM, [NaCl] = 150 mM), [PA] = 100 μM, [TMA-DPH] = 2.8 μM. (e) Fluorescence depolarization profiles (λ ) at 25 °C of TMA-DPH (2.8 μM) embedded in V2A2, A2G2, and VEVA2 in KCl and NaCl aqueous solutions. ex = 336 nm, λ em = 450 nm) ([PA] = 100 μM, [KCl] = 3 mM, [NaCl] = 150 mM). (f) Fluorescence emission spectra (λ ) at 25 °C of TMA-DPH (2.8 μM) embedded in V2A2, A2G2, and VEVA2 in KCl and NaCl aqueous solutions. ex =336nm) ([PA]=100μM, [KCl]=3mM, [NaCl]=150mM).

[0023] Figure 10. DPH location to hydrocarbon tails within lipid bilayers.

[0024] Figure 11A-F. Simulation results for IKVAV PA nanofibers. (a-c) Self-assembled structures and coarse-grained representations of single fibers of (a) V2A2, (b) A2G2, and (c) VEVA2IKVAV PA after 10 μs. The fibers are labeled C to indicate the patch core. 16All but the tail is shown transparent. The micrograph contains a partial periodic image through the fiber forming axis. The simulation box is shown in blue. Water and ions have been omitted for clarity. (d-f) Graphs showing the root mean square deviation (RMSD) versus time for (d) V2A2, (e) A2G2, and (f) VEVA2. The RMSD plots are the average of five independent simulations.

[0025] Figure 12A-E. Simulation results of E-charged VE(-)VA2PA placed in the β-sheet region. (a, b) Self-assembled structure of the VE(-)VA2 fiber after 10 μs. (a) The β-sheet region is represented in orange, IKVAV in green, and the rest of the fiber in black. (b) The fiber is marked with C to indicate the patch core. 16 All but the tail is shown transparent. The micrograph includes a partial periodic image through the fiber forming axis. The simulation box is shown in blue. Water and ions have been omitted for clarity. (c) Water contact graph of VE(-)VA2 compared to V2A2, A2G2, and VEVA2. (d) Root mean square deviation (RMSD) versus time graph of the dynamism analysis of VE(-)VA2 compared to V2A2, A2G2, and VEVA2 in (a-b). The analysis in c-d is the result of five independent simulations.

[0026] Figure 13A-F. Liquid chromatography-mass spectrometry of scrambled VKIVA PA. (a-c) Purified (a) C 16- V2A2E 4- GVKIVA, (b)C 16 A2G2E4GVKIVA(c)C 16 Analytical high-performance liquid chromatography (analytical HPLC) traces of VEVA2E4GVKIVA. (d-f)(d)C 16- V2A2E 4- GIKVAV, (e)C 16 A2G2E4GVKIVA and (f)C 16 Electrospray ionization mass spectrometry (ESI-MS) of VEVA2E4GVKIVA.

[0027] Figure 14A-H. Simulation results of scrambled VKIVA-PA. (a-f) Self-assembled structures and coarse-grained representations of single fibers of (A,b) V2A2 (red), (c,d) A2G2 (blue), and (e,f) VEVA2 (orange) VKIVA after 10 μs. Color code is shown in the PA array. Micrographs include partial periodic images through the fiber forming axis. Simulation boxes are shown in blue. Water and ions have been omitted for clarity. (b,d,f) Fibers are shown in C to show their patchiness. 16 The entire structure is shown transparent except for the tail. The equilibrated structure of A2G2 is different from the typical fiber structure. (g) Water contact graphs of IKVAV and VKIVA-PA. (h) C of scrambled VKIVA and IKVAV PA. 16 Cluster analysis. 16 Quantifying the number of clusters has been used as a qualitative measure of disorder.

[0028] Figure 15A-F. Cryo-TEM micrographs of scrambled VKIVA-PA and scrambled IKVAV sequences (a, d) 16- V2A2E4GVKIVA, (b, e)C 16 A2G2E4GVKIVA(c,f)C 16 (a–c) Schematic molecular structure and (d–f) Cryo-TEM micrographs of VEVA2E4GVKIVA. Scale bar: 500 nm.

[0029] FIG. 16A-F. Liquid chromatography-mass spectrometry of skeletal PA. (a-c) Purified, (a) C 16 V2A2E4, (b)C 16 A2G2E4(c)C 16 Analytical high-performance liquid chromatography (analytical HPLC) traces of VEVA2E4. (d-f) (d) C 16 V2A2E4, (e)C 16 A2G2E4 and (f)C 16 Electrospray ionization mass spectrometry (ESI-MS) of VEVA2E4.

[0030] Figure 17A-F. Cryo-TEM micrographs of scaffold-PA. Scaffold arrangement (a, d) C 16 V2A2E4, (b, e)C 16 A2G2E4(c,f)C 16 (a–c) Schematic molecular structure and (d–f) Cryo-TEM micrographs of VEVA2E4.

[0031] FIG. 18A-K. Analysis of other laminin mimetic sequences. (a, c, e, g, i) C with bioactive epitopes (a, b) LGTIPG, (c, d) LRGDN, (e, f) PDGSR, (g, h) RGD, (i, j) YIGSR sequences after 10 μsec. 16 Cryo-TEM micrographs of A2G2E4G fibers and (b, d, f, h, j) self-assembled structure micrographs. The simulated micrographs contain partial periodic images through the fiber forming axis. The simulation boxes are shown in blue. Water and ions have been omitted for clarity. (k) Water contact analysis for LGTIPG, LRGDN, PDGSR, RGD, YIGSR sequences. IKVAV was used as a reference.

[0032] Figure 19A-I. Short-term analysis (72 h) of hiPSC-derived MNs on IKVAV PA matrices. (a) Schematic of the experimental paradigm. (b) Spectral illumination micrographs of MNs labeled with TUJ-1 (red) on A2G2IKVAV PA covalently linked to alexa-488 dye (green) after 72 h. (c) Number of cells / mm under different conditions at 72 h. 2 Quantification of. (d,e) Representative confocal micrographs of MNs cultured on IKVAV PA supramolecular fibers and laminin (white) coatings containing V2A2 (red), A2G2 (blue), and VEVA2 (orange). (d,e) Cells were consistently stained with neuronal (TUJ-1 and MAP-2, red) and motor neuron (ISL1 / 2 and ChAT, green) markers. Nuclear DNA was stained with DAPI, blue. (f-h) Representative confocal micrographs of MNs cultured on IKVAV PA supramolecular fibers and laminin (black) coatings containing V2A2 (red), A2G2 (blue), and VEVA2 (orange) at 72 h. (f) ISL1 / 2+ and (g) ChAT. + (h)TUJ-1 + Quantification of MN percentage. (i) Cell viability was assessed by LDH assay at 72 h. Scale bar: 50 μm. Data are the average of at least four independent differentiations. All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001), ns: not significant.

[0033] FIG. 20A-I. Short-term analysis of FOXA2 populations on IKVAV PA matrices. (a, b) Representative confocal micrographs of MNs cultured on (a) A2G2IKVAV PA and (b) laminin matrices at 72 hours. Cells were stained with the floor plate cell marker FOXA-2 (green), neuronal marker TUJ-1 (red) and nuclear marker DAPI (blue). (c) Western blot and (d) normalized levels of protein expression associated with the floor plate marker FOXA-2 and proliferation marker PH3 in MNs cultured on IKVAV PA supramolecular fibers containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) at 72 hours. (e, f) Representative confocal micrographs of MNs cultured on (e) A2G2IKVAV PA and (f) laminin matrices at 72 hours. Cells were stained with the floor plate marker FOXA-2 (green), neuronal marker TUJ-1 (white), proliferation marker KI67 (red) and nuclear marker DAPI (blue). (g-i) IKVAV PA supramolecular fiber containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) matrix at 72 h. (g) FOXA2 + , (h)KI67 + and (i) FOXA2 + / KI67 + Quantification of MN percentage. All values ​​were normalized to actin. Scale bar: 25 μm. Data are the average of at least three independent differentiations. All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0034] Figure 21A-G. ITGB1 expression in human MNs cultured on IKVAV PA. (a-d) Representative three-dimensional confocal micrographs of ITGB1 receptor expression in neurites of MNs (labeled with TUJ-1 in green) cultured on IKVAV PA supramolecular fibers containing V2A2, A2G2, VEVA2 and laminin coatings at 72 h. (f, g) Three-dimensional shadow reconstructions of confocal imaging conditions referenced in c and d. (e) Intensity analysis of ITGB1 in MNs cultured on V2A2 (red) and VEVA2 (orange) matrices at 72 h. Scale bar: 2 μm.

[0035] Figure 22A-F. Effect of αITGB1 blocking antibodies on attachment to IKVAV PA and viable MNs. (a) Schematic of hiPSC-derived MN differentiation and subsequent culture on supramolecular matrix in the presence of αβ-1 or β-4 ITG antibodies for 72 h. (b) Cell viability of ITGB1-treated MNs assessed by LDH levels released in the cell medium at 72 h. (c, d) Representative confocal micrographs of MNs treated with (c) β-1 integrin or (d) β-4 integrin blocking antibodies cultured on IKVAV PA supramolecular fibers containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) matrix at 72 h. Cells were stained with neuronal marker TUJ-1 (red), motor neuron marker ISL1 / 2 (green) and nuclei stained with DAPI (blue). (e, f) Number of cells / mm for conditions referred to in c and d, respectively. 2 Quantification of . Scale bar: 50 μm. Data are the average of three independent differentiations. All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001), ns: not significant.

[0036] Figure 23A-F. Effect of mechanical properties on laminin-associated signaling. (a) Schematic diagram of hiPSC-derived MN differentiation and its culture on different supramolecular matrix thicknesses. (b) Elastic modulus (gray and white graphs) and storage modulus (dark grey graphs) of thin coating and thick gel of supramolecular IKVAV matrix containing V2A2, A2G2 and VEVA2. (c,e) Western blot analysis and (d,f) normalized expression levels of ITGB1 receptor activation and signaling pathways (ILK, p-FAK, FAK) in human MNs cultured on (c,d) thick coating or (e,f) thin coating of supramolecular IKVAV matrix containing V2A2, A2G2, VEVA2 at 72 h. Laminin coating was used as a control. Data are the average of at least three independent experiments or differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0037] FIG. 24A-I. Profilometry of IKVAV PA and laminin coatings. (a-h) IKVAV containing (a,b) V2A2, (c,d) A2G2, (e,f) VEVA2 after 3 days (top images) and 60 days (bottom images) in vitro. Representative images of PA and (g, h) laminin coating. (i) Thickness analysis of IKVAV PA and laminin coating referenced in a-h. Data are the average of four independent experiments. All values ​​are presented as mean ± SD; ANOVA (***P<0.001).

[0038] Figure 25A-L. FM characterization of thin and thick IKVAV PA gels. (A-c) AFM topography images of thin coatings of IKVAV PA containing V2A2, (b) A2G2, and (c) VEVA2; (d-f) elastic modulus distributions of thin coatings from a-c; (g-i) optical microscopy images of thick gels of IKVAV PA indentation experiments containing V2A2, A2G2, and VEVA2; (j-l) elastic modulus distributions of thick gels for the conditions referenced in g-i.

[0039] Figure 26A-F. Rheological measurements of IKVAV PA gels. (a-c) Strain sweeps showing storage modulus G' and loss modulus G'' in the shear strain range of 0-100 for supramolecular IKVAV PA containing (a) V2A2, (b) A2G2, and (c) VEVA2. (d-f) Frequency sweeps showing storage modulus G' for angular frequency range of 0-100 rad / s for the conditions referenced in a-c. Data are the average of n=3 gels. All values ​​are presented as mean ± SD.

[0040] Figure 27A-C. Time course of human MNs cultured on IKVAV PA matrix. Representative confocal micrographs of MNs cultured on IKVAV PA supramolecular fiber and laminin (black) matrix containing V2A2, A2G2, VEVA2 at (a) 30 days, (b) 45 days, and (c) 60 days in vitro. Cells were stained with the neuronal marker MAP-2 (red), the motor neuron marker ChAT (green), and nuclei were stained with DAPI (blue). Scale bar: 100 μm.

[0041] FIG. 28A-F. Long-term culture of human MNs on IKVAV PA matrix. (a, c, e) Representative brightfield images and (b, d, f) confocal micrographs of MNs cultured on IKVAV PA supramolecular fiber and laminin matrix containing V2A2, A2G2, VEVA2 at (a, b) 30 days, (c, d) 45 days, and (e, f) 60 days in vitro. Cells were stained with the neuronal marker MAP-2 (red), the motor neuron marker ChAT (green), and nuclei were stained with DAPI (blue). Scale bar: 25 μm. Data are the average of six independent differentiations.

[0042] FIG. 29A-D. Characterization of human MNs on IKVAV PA at different time points. (a, c) Western blot analysis and (b, d) normalized protein levels of (a, b) ITGB1 receptor activation, motor neuron markers ChAT and ISL1 / 2 and (ILK-1), and (c, d) neuronal markers MAP-2 and TUJ-1 in human MNs cultured on supramolecular IKVAV matrix containing V2A2 (red), A2G2 (blue), VEVA2 (orange). Data are the average of at least three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001), ns: not significant.

[0043] Figure 30A-C. Differential ITGB1 expression in human MNs cultured on A2G2IKVAV PA and laminin. (a, b) Representative three-dimensional SIM micrographs of MN neurites (TUJ-1, green) and ITGB1 receptor expression (pink) cultured on (a) A2G2 and (b) laminin matrices at 60 days. (c) Intensity analysis of ITGB1 in MNs cultured on A2G2 (blue) and laminin matrices at 60 days in vitro. Scale bar: 10 μm.

[0044] Figure 31A-C. Assessment of MN maturation by gene ontology (GO) analysis of differentially expressed proteins in MNs cultured on A2G2IKVAV PA and laminin-coated surfaces. Tandem mass spectrometry revealed differentially expressed proteins between MNs plated on laminin-coated surfaces, which are associated with GO terms directly related to human MN maturation (Ho et al., 2016). (a) From the differentially expressed proteins in the A2G2 condition, we identified 21 of 58 (36%) GO terms related to human MN maturation. MN maturation-related terms were also identified from the list of upregulated (b) and downregulated (c) proteins in cultures plated on A2G2IKVAV PA versus laminin-coated surfaces. Bar graphs represent p-values ​​of MN maturation-related GO terms identified in the protein dataset. Pie charts represent the percentage of GO terms obtained in this study compared to previously described GO terms related to MN maturation ( Ho et al., 2016 ).

[0045] Figure 32A-C. Morphological analysis of human motor neurons (MNs) on IKVAV PA matrices. (a, b) Representative confocal micrographs of single human MNs cultured on IKVAV PA supramolecular fiber and laminin matrices containing V2A2, A2G2, VEVA2 at 60 days in vitro. Cells were stained with (a) the neuronal marker MAP-2 (red). (b, c) Analysis of (b) soma size and (c) number of primary neurites of MN cultures at 60 days. Scale bar: 25 μm. Data are the average of at least three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0046] Figure 33A-D. IKVAV PA coating after 60 days in vitro. (a, b) Representative structured illumination microscopy (SIM) micrographs of human MNs cultured on IKVAV PA containing V2A2, A2G2, and VEVA2. MNs were stained with TUJ-1 (red) and IKVAV PA was covalently linked with alexa-488 dye (green). (c, d) Scanning electron micrographs showing neurites of MNs cultured on IKVAV PA containing A2G2. Scale bars: (c) 5 μm, (d) 2 μm.

[0047] Figure 34A-C. Synthesis and liquid chromatography-mass spectrometry of IKVAV peptide for modification of glass coverslips. (a) Schematic molecular structure of IKVAV peptide. (b) Analytical high-performance liquid chromatography (analytical HPLC) trace of purified IKVAV peptide. (c) Electrospray ionization mass spectrometry (ESI-MS) of IKVAV peptide.

[0048] Figure 35A-E. Effect of immobilized IKVAV on MN attachment. (a) Schematic of immobilized IKVAV peptide on coverslip surface. (b) Bright field image. (c, d) Representative confocal micrographs of MNs cultured on APTES and IKVAV peptide coating. Cells were stained with neuronal markers (c) ISL1 / 2 (green) and TUJ-1 (red) and (d) ChAT (green) and MAP-2 (red). Nuclei were stained with DAPI (blue). (e) MNs cultured on APTES and immobilized peptide (IKVAV) 2 Quantification of cell numbers per well. IKVAV PA containing V2A2, A2G2, VEVA2 and laminin matrix were used as controls. Scale bar: 100 μm. Data are the average of three independent differentiations. All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0049] FIG. 36A-C. Human MN attachment on scrambled VKIVA PA. (a) Representative brightfield and (b) confocal images of MNs cultured on a scrambled VKIVA PA sequence containing V2A2, A2G2, and VEVA2. Cells were stained with ChAT, MAP-2, and DAPI in b. (c) MNs in mm at conditions referenced in a, b. 2 Quantification of cell number per culture. IKVAV PA containing V2A2, A2G2, VEVA2 and laminin matrix were used as controls. Scale bar: 100 μm. Data are the average of three independent differentiations. All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0050] Figure 37A-O. Effects of IKVAV PA on other human iPSC-derived motor neurons (11a cell line). (a, b) Representative micrographs of MN-11a cultured for 72 hours on V2A2, A2G2, VEVA2 IKVAV PA supramolecular fibers and laminin matrix. Cells were stained with neuronal and motor neuron markers, TUJ-1 (red) and ISL1 / 2 (green) in (a); MAP-2 (red) and ChAT (green), respectively. Nuclei were stained with DAPI (blue). (c) mm at 72 hours cultured on IKVAV PA supramolecular fibers and laminin coating containing V2A2, A2G2, VEVA2. 2 Quantification of cell number per well. (c-f) (c) TUJ-1 + , (d)ISL1 / 2 + (f) ChAT + Quantification of MN percentage. (g) Representative confocal micrograph of human MN11a cultured on IKVAV PA containing V2A2, A2G2, VEVA2 and laminin matrix at 60 days. Cells were stained with MN markers ChAT (green), microtubule associated protein-2 (MAP-2, red) and nuclei (DAPI). (h) DAPI channel micrograph of image shown in g. (i) Histogram analysis of cell distribution on the different matrices referenced in g and h. (j) mm 2Quantification of cell numbers per cell and (k) ChAT cells cultured on IKVAV PA supramolecular fibers containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) coatings at 60 days. + Quantification of MN percentage. (l-m) Scanning electron microscopy (SEM) micrographs of MN cultured on (l) IKVAV PA containing A2G2 and (m) laminin. (n) Western blots and (o) normalized protein levels of ITGB1 receptor activation, neuronal marker MAP-2 and motor neuron marker ChAT. All values ​​were normalized to actin. Data are the average of three independent differentiations: Scale bars (a,b) 50 μm, (g,h) 100 μm, (l,m) 5 μm. Data are the average of three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0051] Figure 38A-L. Effects of IKVAV PA on human iPSC-derived cortical neurons (CxNs). (a) Schematic of iPSC-derived CxN differentiation and its culture on supramolecular matrix. (b-d) Representative micrographs of CxNs cultured on V2A2 (red), A2G2 (blue), VEVA2 (orange) IKVAV PA supramolecular fibers and laminin (white) matrix at (b) 72 h and (c, d) 60 days. Cells were stained with neuronal markers TUJ-1 (red), MAP-2 (green) and NEUN (red), as well as the cortical marker SATB-2 (green). Nuclei were stained with DAPI (blue). (e) mm at 72 h. 2 Quantification of cell number per cell. (f) Cell viability assessed by LDH release in the cell medium at 60 days. (g-j) TUJ-1 (g) cultured on IKVAV PA supramolecular fibers containing V2A2, A2G2, VEVA2 and laminin (black) coatings at 60 days. + , (h)MAP2 + ,(i) NEUN + and (j) SATB-2 +Quantification of neuronal percentages. (k) Western blots and (l) normalized protein levels of ITGB1 receptor activation, neuronal markers MAP-2, TUJ-1 and NEUN, and presynaptic marker SYP. All values ​​were normalized to actin. Data are the average of three independent differentiations. Scale bars: (b) 100 μm, (c, d) 50 μm. Data are the average of three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0052] FIG. 39A-G. Expression of functional markers in human motor neurons (MNs) cultured on V2A2 and VEVA2IKVAV PA. (a, b) Representative confocal micrographs of synaptic vesicles (SYN-1, presynaptic, and PS95, postsynaptic) distributed along the neurites of MNs cultured on IKVAV and (b) laminin matrices containing A2G2 after 40 days in vitro. (c, d) Mean intensity analysis of (c) PSD95 and (d) SYN-1 in MNs cultured on V2A2 and VEVA2. (f) Western blot analysis and (g) normalized protein levels of synaptic markers PSD95, SYN-1, and SYP, as well as neuronal marker DCX, in human MNs cultured on supramolecular IKVAV matrices containing V2A2, A2G2, VEVA2 and laminin coatings. Scale bar: data are the average of at least three independent differentiations. All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0053] FIG. 40A-F. Electrophysiological studies of human MNs cultured on IKVAV PA. (a) Schematic of multi-electrode array plate (MEA plate) for recording neuronal cultures. (b, c) Schematic of neurons (green) plated on (b) IKVAV PA and (c) laminin-coated MEA plate. (d) Bright field image of MNs cultured on IKVAV PA containing V2A2, A2G2, VEVA2 and laminin-coated MEA plate. (e) Weighted average firing rate and (f) number of burst electrodes in the same conditions as referenced in d. Scale bar: 100 μm. Data are the average of two independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001).

[0054] Figure 41A-C. Spinal cord injury study in mice. (a) Schematic of spinal cord injury at T10 / 11 in mouse model. (b) Impact force graphs of animals treated with V2A2, A2G2, V2A2E2 and saline solution (control). (c) Basso Mouse scale (BMS) for the conditions described in (b).

[0055] [Definition] Although any method and material similar or equivalent to those described herein can be used in carrying out or testing 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 these descriptions, because the specific molecules, compositions, methodologies, or protocols described herein can be changed according to routine experimentation and optimization.It should also be understood that the terms used in this description are only for describing specific versions or embodiments, and are not intended to limit the scope of the embodiments described herein.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Therefore, in the context of the embodiments described herein, the following definitions apply.

[0057] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references 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.

[0058] As used herein, the term "comprise" and linguistic variations thereof refer to 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. In contrast, the term "consisting of" and linguistic variations thereof refer to the presence of the recited feature(s), element(s), method step(s), etc., excluding any unrecited feature(s), element(s), method step(s), etc., except for impurities normally associated therewith. The phrase "consisting essentially of" refers to the recited feature(s), element(s), method step(s), etc., as well as any additional feature(s), element(s), method step(s), etc. that do not substantially affect the basic nature of the composition, system, or method. Many embodiments herein are described using the open term "comprising." Such embodiments encompass the clauses "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described using such language.

[0059] The term "amino acid" refers to natural amino acids, unnatural amino acids, and amino acid analogs in all D- and L-stereoisomeric forms, unless otherwise indicated, where the structure allows for such forms.

[0060] 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).

[0061] 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, tetra-butylglycine ("tBuG"), 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline ("hPro" or "homoP"), hydroxylysine, allo-hydroxylysine, 3-hydroxyproline ("3Hyp"), 4-hydroxyproline ("4Hyp"), and the like. pGly"), isodesmosine, allo-isoleucine, N-methylalanine ("MeAla" or "Nime"), N-alkylglycines ("NAG") (including N-methylglycine), N-methylisoleucine, 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").

[0062] 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 reversibly or irreversibly chemically blocked or modified 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.

[0063] 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.), peptides are about 50 amino acids or less in length. Peptides can contain natural amino acids, unnatural amino acids, amino acid analogs, and / or modified amino acids. Peptides can be subsequences of naturally occurring proteins or unnatural (artificial) sequences.

[0064] As used herein, the term "artificial" refers to compositions and systems that are designed or prepared by humans 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 naturally occurring proteins or fragments thereof).

[0065] 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 (e.g., 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).

[0066] Naturally occurring residues can be grouped into classes based on common side chain properties, for example: positive polarity (or basic) (histidine (H), lysine (K), and arginine (R)); negative polarity (or acidic) (aspartic acid (D), glutamic acid (E)); neutral polarity (serine (S), threonine (T), asparagine (N), glutamine (Q)); nonpolar aliphatic (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.

[0067] 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 natural residues. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in living 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.

[0068] Non-conservative substitutions may involve replacing a member of one class with a member of another class.

[0069] As used herein, the term "sequence identity" refers to the degree to which two polymeric sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequential composition of monomeric subunits. The term "sequence similarity" refers to the degree to which two polymeric 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 window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a particular window, etc.), (2) determining the number of positions that contain identical (or similar) monomers (e.g., the same amino acid is present in both sequences, similar amino acids are present in both sequences) to obtain the number of matched positions, (3) dividing the number of matched positions by the total number of positions within the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a particular window), and (4) multiplying the result by 100 to obtain the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids long and have identical amino acids at all positions except position 1, then peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical properties (e.g., both are acidic), then peptide A and peptide B have 100% sequence similarity. As another example, if peptide C is 20 amino acids long, peptide D is 15 amino acids long, and 14 of the 15 amino acids of peptide D are identical to some amino acids of peptide C, then peptides C and D have 70% sequence identity. However, peptide D has 93.3% sequence identity over 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.

[0070] Any polypeptide described herein as having a particular percent sequence identity or similarity (e.g., at least 70%) with a reference sequence ID number can 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) can have up to X (e.g., 10) substitutions compared to SEQ ID NO: Z, and thus can also be expressed as "having no more than X (e.g., 10) substitutions compared to SEQ ID NO: Z."

[0071] As used herein, the term "nanofiber" refers to elongated or thread-like filaments (e.g., having a length dimension significantly greater than its width or diameter) typically having a diameter of less than 100 nanometers.

[0072] As used herein, the term "scaffold" refers to a material capable of supporting the growth and differentiation of cells. As used herein, the term "supramolecule" (e.g., "supramolecular complex," "supramolecular interaction," "supramolecular fiber," "supramolecular polymer," etc.) refers to non-covalent interactions between molecules (e.g., polymers, macromolecules, etc.) and the resulting multi-component assemblies, complexes, systems, and / or fibers that are formed.

[0073] As used herein, the terms "self-assemble" and "self-assembly" refer to the formation of discrete, non-random aggregate structures from constituent parts; said assembly occurs spontaneously through the random movement of the constituent parts (e.g., molecules) due solely to the inherent chemical or structural properties and attractive forces of those constituent parts.

[0074] 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 bioactive segment (e.g., a linker segment, a bioactive segment, etc.). Peptide amphiphiles can exhibit a net charge at physiological pH, i.e., either a net positive charge or a net negative charge, or can be zwitterionic (i.e., carrying 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, (3) a charged peptide segment, and (4) a bioactive segment (e.g., a linker segment).

[0075] As used herein and in the appended claims, the term "lipophilic moiety" or "hydrophobic moiety" refers to a moiety (e.g., an acyl moiety, an ether moiety, a sulfonamide moiety, or a phosphodiester moiety) located at one end (e.g., the C-terminus, the N-terminus) of a peptide amphiphile, which may also be referred to herein and elsewhere as a lipophilic or hydrophobic segment or component. The hydrophobic segment should be of sufficient length to provide amphiphilic behavior and 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 having 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 palmitic acid, a lipophilic group (saturated or unsaturated carbon). However, other lipophilic groups (e.g., steroids, phospholipids, and fluorocarbons) may be used in place of the acyl chain.

[0076] As used interchangeably herein, the term "structural peptide" or "structural peptide segment" refers to a portion of a peptide amphiphile that is typically located between a hydrophobic segment and a charged peptide segment. A structural peptide is generally composed of 3-10 amino acid residues (e.g., His (H), Val (V), Ile (I), Leu (L), Ala (A), Phe (F)) with non-polar, uncharged side chains that are selected for their propensity to form hydrogen bonds or other stabilizing interactions (e.g., hydrophobic interactions, van der Waals interactions, etc.) with adjacent structural peptide segments. In some embodiments, nanofibers of peptide amphiphiles having structural peptide segments exhibit linear or 2D structures when viewed by microscopy and / or exhibit alpha-helices and / or beta-sheets when viewed by circular dichroism (CD). In some embodiments, peptide amphiphile nanofibers having structural peptide segments with an overall propensity for β-sheet structure formation of 4 or less exhibit less ordered characteristics (e.g., less ordered secondary structure such as less rigid β-sheet structures). In some embodiments, peptide amphiphile nanofibers having structural peptide segments with an overall propensity for β-sheet structure formation of 4 or less (e.g., A2G2) exhibit a tendency to form random coil structures.

[0077] As used herein, the term "beta (β) sheet-forming peptide segment" refers to a structural peptide segment that has a tendency to exhibit beta sheet-like characteristics (e.g., when analyzed by CD). In some embodiments, amino acids in a beta (β) sheet-forming peptide segment are selected for their tendency to form beta sheet secondary structures. 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 have a tendency to form beta sheets are understood (see, e.g., Mayo et al. Protein Science (1996), 5:1301-1315, which is incorporated herein by reference in its entirety).

[0078] As used herein, the term "charged peptide segment" refers to a portion of a peptide amphiphile that is rich (e.g., >50%, >75%, etc.) in charged amino acid residues or amino acid residues that have 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).

[0079] 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 include 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 may be from about 2 to about 7 amino acids in the segment, and / or about 3 or 4 amino acids.

[0080] 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 side chains exhibiting a positively charged acid side chain (e.g., Arg (R), Lys (K), His (H), or unnatural amino acids or peptidomimetics). The basic peptide segment may optionally include one or more additional (e.g., non-basic) amino acid residues. As will be apparent to one of skill in the art, unnatural amino acid residues having basic side chains can be used. There may be from about 2 to about 7 amino acids in the segment, and / or about 3 or 4 amino acids.

[0081] As used herein, the term "bioactive peptide" refers to an amino acid sequence that mediates the action of an associated sequence, molecule, or supramolecular complex. Peptide amphiphiles and structures (e.g., nanofibers) bearing bioactive peptides (e.g., IKVAV peptides) exhibit bioactive peptide functionality.

[0082] 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 deemed "biocompatible" if its addition to cells in vitro results in no more than about 10%, usually less than 5%, and more usually less than 1% cell death.

[0083] As used herein, "biodegradable" used to describe polymers, hydrogels, and / or wound dressings, as used herein, refers to compositions that degrade or otherwise "break down" upon 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 wound coating includes hydrolyzable ester bonds that provide the biodegradability.

[0084] 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.

[0085] As used herein, the terms "treat", "treatment" and "treating" refer to reducing the amount or severity of a particular condition, pathology (e.g., CNS injury), or symptom thereof, in a subject currently experiencing or suffering from the condition or pathology. The term does not necessarily indicate a complete cure (e.g., complete elimination of the condition, disease, or symptom). "Treatment" includes any administration or application of a therapy or technique to a disease (e.g., in a mammal, including a human), and includes inhibiting the disease, arresting its onset, relieving the disease, regressing, or restoring or repairing lost, deficient, or defective function; or promoting an inefficient process.

[0086] As used herein, the terms "prevent", "prevention" and "preventing" refer to reducing the likelihood of a particular condition or pathology (e.g., CNS injury) occurring in a subject not currently experiencing or suffering from that condition or pathology. The terms do not necessarily indicate complete or absolute prevention. For example, "preventing CNS injury" refers to reducing the likelihood of CNS injury occurring in a subject not currently experiencing or diagnosed with CNS injury. To "prevent CNS injury", a composition or method need only reduce the likelihood of CNS injury, not necessarily block any possibility of it. "Prevention" encompasses any administration or application of a treatment or technique to reduce the likelihood of a disease developing (e.g., in a mammal, including a human). Such likelihood can be assessed for a population or an individual.

[0087] As used herein, the terms "co-administration" and "co-administering" refer to the administration of at least two agents or treatments (e.g., IKVAV PA nanofibers and one or more therapeutic agents) to a subject. In some embodiments, the co-administration of two or more agents or treatments occurs simultaneously. In other embodiments, a first agent / treatment is administered prior to a second agent / treatment. Those skilled in the art will appreciate that the formulations and / or routes of administration of the various agents or treatments used may vary. Appropriate doses for co-administration can be readily determined by those skilled in the art. In some embodiments, when agents or treatments are co-administered, each agent or treatment is administered at a lower dose than is appropriate for their administration alone. Thus, co-administration is particularly desirable in embodiments where the co-administration of agents or treatments reduces the required dose of a potentially harmful (e.g., toxic) agent and / or where the co-administration of two or more agents results in a sensitization effect of the subject to the beneficial effects of one of the agents via the co-administration of the other agent.

[0088] [Detailed Description] Provided herein are peptide amphiphiles (PAs) that contain bioactive peptides, nanofibers exhibiting bioactive PAs, and methods of their use.

[0089] In some embodiments, the peptide amphiphile molecules and compositions of the embodiments described herein are synthesized using preparative techniques well known to those of skill in the art, preferably by standard solid-phase peptide synthesis involving the addition of fatty acids to the N-terminus (or C-terminus) of the peptide in place of standard amino acids, resulting in the creation of lipophilic segments (although in some embodiments, alignment of the nanofibers is achieved via techniques not previously disclosed or used in the art, such as extrusion through a mesh screen). Synthesis typically begins at the C-terminus, to which 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.

[0090] In some embodiments, the peptide amphiphile comprises a hydrophobic segment (i.e., a hydrophobic tail) linked to the peptide. In some embodiments, the peptide comprises a structural peptide segment. In some embodiments, the structural peptide segment is a hydrogen bond forming segment or a beta sheet forming segment. In some embodiments, the structural peptide segment has a tendency to form random coil structures (e.g., an overall tendency for beta sheet structure formation of 4 or less). In some embodiments, the peptide comprises a charged segment (e.g., an acidic segment, a basic segment, a zwitterionic segment, etc.). In some embodiments, the peptide further comprises a linker or spacer segment to add solubility, flexibility, distance between segments, etc. In some embodiments, the peptide amphiphile comprises a spacer segment (e.g., a peptide and / or a non-peptide spacer) at the peptide end opposite the hydrophobic segment. In some embodiments, the spacer segment comprises a peptide and / or a non-peptide element. In some embodiments, the spacer segment comprises one or more bioactive groups (e.g., an alkene, an alkyne, an azide, a thiol, etc.). In some embodiments, the various segments may be linked by linker segments, such as peptide linkers (eg, GG) or non-peptide linkers (eg, alkyl, OEG, PEG, etc.).

[0091] 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 via an acyl bond. In aqueous solution, PA molecules self-assemble (e.g., into cylindrical micelles (aka nanofibers)), burying the lipophilic segments in their core and presenting the bioactive peptides on the surface. In some embodiments, the structural peptides undergo intermolecular hydrogen bonding to form beta sheets oriented parallel to the long axis of the micelle. In some embodiments, the structural peptides exhibit weak intermolecular hydrogen bonding, resulting in a less rigid beta sheet structure in the nanofiber.

[0092] 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 linked to a peptide segment (e.g., a peptide that comprises a segment having a preference for beta-strand structures or other supramolecular interactions) to produce 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 generally conical molecular shapes that affect the assembly of PAs (see, for example, JN Israelachvili Intermolecular and surface forces; 2nd ed.; Academic: London San Diego, 1992, the entirety of which is incorporated herein by reference). Other molecular shapes have similar effects on assembly and nanostructural architecture.

[0093] In some embodiments, to induce self-assembly of an aqueous solution of peptide amphiphiles, the pH of the solution can be changed (raised or lowered) or multivalent ions such as calcium or charged polymers or other macromolecules can be added to the solution.

[0094] 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) having 4 to 25 carbon atoms (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25), a fluorinated segment, a fluorinated alkyl tail, a heterocycle, an aromatic segment, a pi-conjugated segment, a cycloalkyl, an oligothiophene, or the like. In some embodiments, the hydrophobic segment comprises an acyl / ether chain (e.g., saturated) having 2 to 30 carbon atoms (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).

[0095] In some embodiments, the PA comprises one or more peptide segments. The peptide segments may comprise natural amino acids, modified amino acids, unnatural 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.

[0096] In some embodiments, the peptide amphiphile comprises a charged peptide segment, which can be acidic, basic, or zwitterionic.

[0097] 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 up to 7 residues in length and comprises at least 50% acidic residues. In some embodiments, the acidic peptide segment comprises (Xa) 1-7 and each Xa is independently D or E. In some embodiments, the acidic peptide segment comprises E 2-4 For example, in some embodiments, the acidic peptide segment comprises EE. In some embodiments, the acidic peptide segment comprises EEE. In other embodiments, the acidic peptide segment comprises EEEE.

[0098] 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 up to 7 residues in length and comprises at least 50% basic residues. In some embodiments, the acidic peptide segment comprises (Xb) 1-7 wherein each Xb is independently R, H, and / or K.

[0099] In some embodiments, the peptide amphiphile comprises a structural peptide segment. In some embodiments, the structural peptide segment is a beta-sheet forming segment. In some embodiments, the structural peptide segment exhibits weak hydrogen bonding and has a tendency to form random coil structures rather than rigid beta-sheet structures. In some embodiments, the structural peptide segment is rich in one or more of H, I, L, F, V, G, and A residues. In some embodiments, the structural peptide segment comprises an alanine and valine rich peptide segment (e.g., VVAA, VVVAAA, AAVV, AAAVVV, or other combinations of V and A residues). In some embodiments, the structural peptide segment comprises four or more consecutive A and / or V residues, or conservative or semi-conservative substitutions thereto. In some embodiments, the structural peptide segment comprises V2A2. In some embodiments, the structural peptide segment comprises an alanine and glycine rich peptide segment (e.g., AAGG, AAAGGG, or other combinations of A and G residues). In some embodiments, the structural peptide segment comprises A2G2.

[0100] In some embodiments, the structural peptide segment comprises 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 peptide segment comprises 2-16 amino acids in length and comprises four 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. In some embodiments, the structural peptide segment comprises VEVA2.

[0101] In some embodiments, the structural peptide segments have an overall propensity for forming beta-sheet structure of 4 or less (e.g., less than 4, less than 3.9, less than 3.8, less than 3.7, less than 3.6, less than 3.5, less than 3.4, less than 3.3, less than 3.2, less than 3.1, less than 3.0, less than 2.9, less than 2.8, less than 2.7, less than 2.6, less than 2.5, less than 2.4, less than 2.3, less than 2.2, less than 2.1, less than 2.0, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1 or less than 1).

[0102] The overall tendency for β-sheet structure formation can be calculated as the sum of the tendencies of each amino acid in the structural peptide segment to form a β-sheet structure. The tendency of each amino acid to form a β-sheet structure and the method of calculating it are described, for example, in Fujiwara, K., Toda, H. & Ikeguchi, M. Dependence of α-helical and β-sheet amino acid propensities on the The overall protein fold type is described in BMC Struct Biol 12,18(2012), the entire contents of which are incorporated herein by reference. Exemplary values ​​are shown in Table 1 below. For the purpose of calculating the overall tendency for the formation of a β-sheet structure of a structural peptide segment, the values ​​shown in the "Total Residues" column of Table 1 for each amino acid are added together. For example, for the A2G2 structural peptide segment, the overall tendency for the formation of a β-sheet structure is 0.75+0.75+0.67+0.67=2.84. A structural peptide segment may include any suitable number and combination of amino acids to achieve an overall tendency for the formation of a β-sheet structure of 4 or less.

[0103] [Table 1]

[0104] In some embodiments, a structural peptide segment having an overall tendency for beta-sheet structure formation of 4 or less indicates that the amino acids in the structural peptide segment have weaker interactions with adjacent molecules. For example, the structural peptide segment may exhibit weak hydrogen bonding ability. Thus, such structural peptide segments and peptide amphiphiles containing the same can generate more dynamic nanofiber structures. For example, the A2G2 structural peptide segment may exhibit a random coil structure rather than a rigid beta-sheet structure.

[0105] 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 peptide terminus opposite the hydrophobic segment. In some embodiments, the spacer or linker segment provides an attachment site for a bioactive group. In some embodiments, the spacer or linker segment provides a reactive group for functionalization of the PA (e.g., alkene, alkyne, azide, thiol, maleimide, etc.). 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 bioactive groups, substituents, branches, etc. In some embodiments, the linker segment is a single glycine (G) residue.

[0106] Suitable peptide amphiphiles for use in the materials herein, as well as methods of preparing PA and related materials, amino acid sequences for PA, and materials that find use with 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 ... No. 8,114,835; U.S. Patent No. 8,114,834; U.S. Patent No. 8,080,262; U.S. Patent No. 8,076,295; U.S. Patent No. 8,063,014; U.S. Patent No. 7,851,445; U.S. Patent No. 7,838,491; U.S. Patent No. 7,745,708; U.S. Patent No. 7,683,025; U.S. Patent No. 7,554,021; U.S. Patent No. 7,544,661; U.S. Patent No. 7,534,761; U.S. Patent No. 7,491,690; U.S. Patent No. 7,452,679; U.S. Patent No. 7,371,719; U.S. Patent No. 7,030,167; all of which are incorporated by reference herein in their entireties.

[0107] The properties of PA supramolecular structures (e.g., shape, rigidity, hydrophilicity, etc.) depend on the identity of the peptide amphiphile components (e.g., lipophilic segments, acidic segments, structural peptide segments, bioactive segments, etc.). For example, nanofibers, nanospheres, intermediate shapes, and other supramolecular structures are achieved by tuning the identity of the PA component moieties. In some embodiments, the properties of PA supramolecular nanostructures are altered by post-assembly manipulations (e.g., heating / cooling, stretching, etc.).

[0108] In some embodiments, the peptide amphiphile comprises: (a) a hydrophobic tail comprising an alkyl chain having 8 to 24 carbon atoms; (b) a structural peptide segment (e.g., comprising VVAA, AAGG, or VEVA); and (c) a charged segment (e.g., comprising EE, EEE, EEEE, etc.). In some embodiments, any PA within the scope described herein or within the skill of one in the art, comprising the components described herein, may find use herein.

[0109] In some embodiments, the peptide amphiphile comprises a bioactive moiety (e.g., an IKVAV peptide). 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.

[0110] In some embodiments, the bioactive moiety is a peptide identified in the extracellular matrix (ECM). For example, the bioactive moiety can be a peptide sequence found in collagen, elastin, fibronectin, or laminin. In some embodiments, the bioactive moiety is a peptide sequence found in laminin. For example, the bioactive moiety can be found in laminin-1, laminin-2, laminin-3, laminin-4, laminin-5, laminin-6, laminin-7, laminin-8, laminin-9, laminin-10, laminin-11, laminin-12, laminin-13, laminin-14, or laminin-15. In some embodiments, the bioactive moiety is a peptide sequence found in laminin-1. In certain embodiments, the bioactive moiety is IKVAV. In some embodiments, the bioactive moiety is a recombinant peptide. In some embodiments, the bioactive moiety is a peptide sequence that binds to a peptide or polypeptide of interest (e.g., an ECM protein).

[0111] In some embodiments, a peptide amphiphile comprises (e.g., from C-terminus to N-terminus or N-terminus to C-terminus): a bioactive peptide (e.g., an IKVAV peptide)-a charged segment (e.g., an E 2-4 etc.)-structural peptide segments (e.g., containing V2A2, A2G2, VEVA2, etc.)-hydrophobic tails (e.g., containing alkyl chains having 8 to 24 carbon atoms).

[0112] In some embodiments, the peptide amphiphile comprises (e.g., from C-terminus to N-terminus or N-terminus to C-terminus): a bioactive peptide (e.g., an IKVAV peptide), a flexible linker (e.g., comprising a G, etc.), a charged segment (e.g., an E 2-4 etc.)-structural peptide segments (e.g., containing V2A2, A2G2, VEVA2, etc.)-hydrophobic tails (e.g., containing alkyl chains having 8 to 24 carbon atoms).

[0113] In some embodiments, the PA further comprises an attachment segment or residue (e.g., K) for attaching a hydrophobic tail to the peptide portion of the PA. In some embodiments, the hydrophobic tail is attached to a lysine side chain.

[0114] 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 the PAs described herein. In some embodiments, the nanofibers include or consist of a PA exhibiting an IKVAV peptide. In some embodiments, the IKVAV peptide is exhibited on the surface of the nanofiber. In some embodiments, in addition to the PA exhibiting an IKVAV peptide, a filler PA is included in the nanofiber. In some embodiments, the filler PA is a peptide amphiphile as described herein (e.g., structural peptide segments, charged segments, hydrophobic segments, etc.), but lacks a bioactive moiety. In some embodiments, the filler peptide is a basic or acidic peptide lacking a bioactive moiety. In some embodiments, the filler PA and the IKVAV PA self-assemble into a nanofiber that includes both types of PA. In some embodiments, provided are nanostructures (e.g., nanofibers) assembled from peptide amphiphiles described herein.

[0115] In some embodiments, the filler peptides (e.g., basic peptides, acidic peptides, etc.) impart mechanical properties to the materials comprising the PA nanofibers described herein. In some embodiments, 0-75% (wt%) of bioactive IKVAV Nanofibers assembled from PA and 25-100% (wt%) of basic filler PA gel at basic pH conditions (e.g., pH 8.5-11). In some embodiments, nanofibers assembled from 75-100% (wt%) of bioactive IKVAV PA and 0-25% (wt%) of basic filler PA are liquid at basic pH conditions (e.g., pH 8.5-11). In some embodiments, nanofibers assembled from 0-20% (wt%) of bioactive IKVAV PA and 80-100% (wt%) of acidic filler PA gel at acidic pH conditions (e.g., pH 1-5). In some embodiments, nanofibers assembled from 20-80% (wt%) of bioactive IKVAV PA and 20-80% (wt%) of acidic filler PA gel at neutral pH conditions (e.g., pH 5-8.5). In some embodiments, nanofibers assembled from 80-100% (wt%) bioactive IKVAV PA and 0-20% (wt%) acidic filler PA are liquid at acidic pH conditions (eg, pH 1-5).

[0116] In some embodiments, the nanostructures are assembled from (1) a PA bearing a bioactive moiety (e.g., an IKVAV peptide) and (2) a filler PA (e.g., an acidic or basic PA that is unlabeled or does not exhibit a bioactive moiety). 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 VEGF PA. In some embodiments, the nanostructures (e.g., nanofibers) contain 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) contain 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 IKVAV PA to acidic and / or basic PA in the nanofiber determines the mechanical properties of the nanofiber material (e.g., liquid or gel) and under what conditions the material will assume different properties (e.g., gel when exposed to physiological conditions, liquefy when exposed to physiological conditions, etc.).

[0117] 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., without bioactive 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 that has highly charged glutamic acid residues at the ends of the molecule (e.g., the ends exposed to the surface). These negatively charged PAs allow gelation to occur between nanofibers via ionic crosslinking. In some embodiments, the filler PAs are non-bioactive PA molecules that have highly charged lysine residues at the ends of the molecules (e.g., the ends exposed to the surface). 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 ensuring the gelling ability of the nanofiber solution. In some embodiments, the solution is annealed to increase the viscosity and make the gelling mechanism stronger. These filler PAs can be, for example, those sequences (e.g., C) described in U.S. Pat. No. 8,772,228, which is incorporated by reference in its entirety herein. 16- VVVAAAEEE).

[0118] In some embodiments, the PA nanofibers described herein exhibit small cross-sectional diameters (e.g., <25 nm, <20 nm, <15 nm, about 10 nm, etc.). In some embodiments, the small cross-sectional area (~10 nm diameter) of the nanofibers allows the fibers to penetrate the brain parenchyma.

[0119] In some embodiments, the PA and nanofibers described herein may be incorporated into pharmaceutical compositions for use in methods of treating disease. For example, the PA and nanofibers described herein may be used in methods of treating or preventing nervous system damage in a subject. For example, the PA and nanofibers described herein may be used in methods for treatment of prevention of damage to the central nervous system (CNS), including the brain and spinal cord, or the peripheral nervous system (PNS), including the nerves and ganglia outside the brain and spinal cord. In some embodiments, the PA and nanofibers described herein may be used for treatment or prevention of damage to the CNS or PNS in a subject. In some embodiments, the damage is a spinal cord injury. The spinal cord injury may be cervical, lumbar, thoracic, sacral, or any combination thereof.

[0120] The injury can be a traumatic injury. Traumatic injury refers to injury caused by trauma (e.g., trauma caused by automobile accidents, falls, violence, sports injuries, surgical injuries, etc.). For example, the PAs and nanofibers described herein can be used to treat traumatic spinal cord injury. As another example, the PAs and nanofibers described herein can be used to treat traumatic brain injury (TBI). Alternatively, the injury can be a non-traumatic injury. For example, the injury can be a non-traumatic injury to the CNS (e.g., brain and / or spinal cord) or PNS caused, for example, by cancer, multiple sclerosis, inflammation, arthritis, spinal stenosis, tumors, blood loss, etc.

[0121] In some embodiments, a composition comprising the PA and / or nanofibers described herein is provided to a subject suspected of having a traumatic spinal cord injury. For example, the composition may be provided to a subject exhibiting one or more symptoms including loss of sensation and / or loss of motor control in one or more areas of the body (e.g., hands, arms, legs, feet, etc.), low blood pressure, inability to regulate blood pressure, inability to regulate body temperature, inability to sweat below the injury area, chronic pain, and / or swelling of the spinal cord. The composition may be provided to the subject to treat the injury. In some embodiments, treating the injury may prevent the worsening of one or more symptoms associated with the injury. In some embodiments, treating the injury may reduce and / or eliminate the severity of one or more symptoms associated with the injury.

[0122] A composition comprising a PA and / or nanofibers described herein may be provided to a subject at any suitable time following injury (e.g., traumatic spinal cord injury) to treat the injury. For example, the composition may be provided to a subject within 24 hours of injury (e.g., within 24 hours, within 12 hours, within 10 hours, within 9 hours, within 8 hours, within 7 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, or within 1 hour of injury). In some embodiments, the composition may be provided to a subject after a sustained period of more than 24 hours has elapsed following injury or diagnosis of injury.

[0123] The composition may be administered in any appropriate amount, depending on factors including the age of the subject, the weight of the subject, the severity of the injury, etc. The composition may be administered in combination with other appropriate treatments for the injury or preventative measures to prevent the severity of the injury from worsening.

[0124] In some embodiments, the PA and nanofiber compositions herein are formulated for delivery to a subject. Suitable routes of administration of the pharmaceutical compositions 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 intracerebroventricular administration. In some embodiments, the PA compositions are administered parenterally. In some embodiments, parenteral administration is by intrathecal, intracerebroventricular, or intraparenchymal administration. The PA compositions herein can be administered as the sole active agent or in combination with other pharmaceutical agents, such as other agents used in the treatment of nervous system injury in a subject.

[0125] In some embodiments, the PA and nanofibers (e.g., nano-IKVAV) described herein find use in cell culture methods. For example, further disclosed herein are scaffolds comprising peptide amphiphiles described herein. The scaffolds may comprise self-assembled peptide amphiphile nanofibers, at least a portion of the peptide amphiphiles comprising a hydrophobic tail, a structural peptide segment, a charged peptide segment, and an IKVAV bioactive peptide. The scaffolds may further comprise one or more filler peptide amphiphiles. The scaffolds described herein can support cell proliferation and differentiation. Thus, the scaffolds may be in a method for culturing cells. The method for culturing cells comprises contacting cells with the scaffold as described herein. In some embodiments, the scaffolds may be used as a coating for any desired cell culture tool (tissue culture plate, petri dish, glass slide, etc.).

[0126] Cells cultured on the scaffolds disclosed herein may exhibit improved properties compared to cells cultured in the absence of the disclosed scaffolds. For example, cells may exhibit improved differentiation, improved maturation, and / or improved long-term viability compared to cells cultured in the absence of the disclosed scaffolds.

[0127] In some embodiments, the scaffold can be used in a method of culturing neuronal cells. For example, the scaffold can be used in a method of culturing hiPSC-derived neuronal cells, such as hiPSC-derived motor neurons. For example, hiPSCs can be differentiated using any standard cocktail known to stimulate neural induction and promote ventral / caudal patterning. Following this initial differentiation into neurons, the neurons can be cultured on various IKVAV scaffolds. Culture on the disclosed IKVAV scaffolds can allow efficient generation and functional maturation of hiPSC-derived motor neurons.

[0128] [experiment] [material and method] [Material synthesis, preparation and characterization] PA synthesis and preparation: IKVAV PA molecules (C 16 V2A2E4GIKVAV, C 16 A2G2E4GIKVAV, C 16 VEVA2GIKVAV), VKIV scrambled PA molecule (C 16 V2A2E4GVKIVA, C 16 A2G2E4GVKIVA, C 16 VEVA2GVKIVA), and the backbone sequence (C 16 V2A2E4, C 16 A2G2E4, C 16VEVA2) was synthesized. PA was purified by reversed-phase high performance liquid chromatography (HPLC). HPLC was performed using a Phenomenex Kinetex column (C 18 Stationary phase, 5 μm, 100 Å pore size, 30.0 × 150 mm) was used. 5R The analysis was carried out on a Shimadzu Model Prominence Modular HPLC system equipped with a degassing unit, two LC-20AP solvent delivery units, an SPD-M20A diode array detector, and an FRC-10A fraction collector. HPLC was performed using a gradient of H2O / CH3CN containing 0.1% NH4OH (v / v) as eluent at a flow rate of 25.0 mL / min. The purity of the lyophilized PA was analyzed by liquid chromatography-mass spectrometry (LC-MS). LC-MS was performed using a Phenomenex Jupiter 4 μm Proteo 90 Å column (C 12 Stationary phase, 4 μm, 90 Å pore size, 1 × 150 mm) was used on an Agilent model 1200 Infinity Series binary LC gradient system. LC-MS was performed using a gradient of H2O / CH3CN containing 0.1% NH4OH (v / v) as eluent at a flow rate of 50 μL / min. Electrospray ionization mass (ESI-mass) analysis was performed on an Agilent model 6510 Quadrupole Time-of-Flight Electrospray ionization mass (ESI-mass) analysis was performed on an LC / MS spectrometer in positive scan mode. After lyophilization, the PA powder was reconstituted in 125 mM NaCl and 3 mM KCl solution and adjusted to pH 7.4 by adding 1 μL of 1 N NaOH in increments to ensure cytocompatibility and material concentration. A thermocycler (Eppendorf Mastercycler) was used to anneal the PA solution at 80 °C for 30 min and then slowly cool at 1 °C / min to reach a final temperature of 27 °C, ensuring uniform and controlled heating and cooling of all samples. To prepare PA-coated substrates, 24, 12, and 6-well polystyrene cell culture plates or / and 12- and 18-mm cover glass slips were incubated overnight at 37 °C in a poly- D-lysine (0.01 mg / mL). The next day, the plate was rinsed three times with MilliQ water and then coated with 30 μL of annealed PA (1 wt%) solution for 3 h. Before further use, excess PA solution was removed and the plate was gently rinsed with gelling solution (150 mM NaCl, 3 mM KCl, 25 mM CaCl2).

[0129] Immobilization of IKVAV peptide on glass surfaces: Borosilicate glass coverslips (12 mm diameter; Fisher Scientific) were modified with synthetic IKVAV peptide according to previously described techniques (Olbrich, KC et al Biomaterials 17, 759-764 (1996) and Kam, L. et al Biomaterials 23, 511-515 (2002)). Borosilicate glass coverslips were washed with 2% (v / v) micro-90 surfactant (Sigma Aldrich) at 60°C for 30 min, rinsed six times with distilled water, rinsed with ethanol, and then dried. Coverslips were plasma etched with O2 for 30 s (Harrick Plasma PDC-001-HP) and immediately incubated in a 2% (v / v) solution of (3-aminopropyl)triethoxysilane (Sigma Aldrich) in ethanol for 15 min. The coverslips were then rinsed twice with ethanol and twice with water, then dried in an oven. The IKVAV peptide (see FIG. 34) was then prepared at 50 nmol / mL in a solution of 1.25 mg / mL 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide (Arcos Organics) and 2% DMF (dimethylformamide (Sigma Aldrich)). The coverslips were incubated with this solution for 3.5 hours at 40° C. After incubation, the coverslips were washed sequentially with 100% acetic anhydride (Fisher Chemical), 2 M hydrochloric acid (Fisher Chemical), and 0.2 M sodium bicarbonate. After rinsing with excess water, the samples were sonicated in 4 M urea for 10 minutes, followed by washing with 1 M NaCl for 10 minutes, then rinsed with excess water and dried at 100° C. for 1 hour.

[0130] Transmission electron microscopy (TEM): 5 μL of sample solution (in H2O, [PA] = 0.01 wt%) was deposited onto a copper TEM grid with a carbon support film (Electron Microscopy Science) and held in place for 5 min using tweezers. The sample solution was removed by capillary action using filter paper and the grid was allowed to dry for 10 min. The sample was then immersed in 10 μL of an aqueous solution of uranyl acetate (2 wt%, Sigma). Aldrich) for 3 min and the solution was removed by capillary action through filter paper. Grids were allowed to dry for at least 2 h before imaging. TEM images were obtained using a Hitachi model HT-7700 electron microscope operated at 120 kV equipped with an Orius SC 1000A camera.

[0131] Cryogenic Transmission Electron Microscopy (cryo-TEM): Plunge-frozen for cryo-TEM samples were prepared using an FEI model Vitrobot Mark III. 6.5 μL of sample solution (in H2O, [PA] = 0.01 wt%) was deposited onto a plasma-cleaned copper TEM grid with a holey carbon support membrane (Electron Microscopy Science) and held in place using tweezers mounted on the Vitrobot. The specimen was blotted (blot offset: 0.5 mm, total blot: 1, wait time: 0 s, blot time: 5 s, drain time: 0 s) in a 100% humidity, 22 °C environment and plunged into a liquid ethane reservoir cooled with liquid nitrogen. The vitrified sample was stored in liquid nitrogen and then transferred to a Gatan cryo-TEM holder. Cryo-TEM images were obtained using a Hitachi model HT-7700 electron microscope equipped with an Orius SC 1000A camera and operating at an accelerating voltage of 120 kV.

[0132] Scanning Electron Microscopy (SEM): PA samples were fixed for 20 min in a mixture of paraformaldehyde (2.0%, Electron Microscopy Sciences), glutaraldehyde (2.5%, Electron Microscopy Sciences) in phosphate-buffered saline (1X, Gibco). The fixative was removed and water was exchanged for ethanol by incubating the samples in an ethanol solution gradient. The gradient consisted of increasing concentrations (30–100%) of 200-proof ethanol (Decon Laboratories, Inc.). Excess water was removed using a critical dry point (Tousimis Samdri-795). A 15-min purge cycle was used. The resulting dehydrated sample coverslip was mounted onto a stub using 12 mm carbon adhesive tape (Electron Microscopy Sciences) and coated with approximately 6 nm of osmium (Filgen, OPC-60A) to make the sample surface conductive for imaging. All images were taken using a Hitachi SU8030 SEM instrument at an accelerating voltage of 2 kV.

[0133] Atomic Force Microscopy (AFM): AFM imaging and force measurements were performed at room temperature on a Bioscope Resolve BioAFM / Nanoscope V system (Bruker, Santa Barbara) integrated on an Axio Oberver.D1m inverted optical microscope (Carl Zeiss, Inc.). A silicon nitride triangular probe (ScanAsyst Fluid, Bruker) with a nominal tip radius of about 20 nm and a spring constant of about 0.7 N / m was used for imaging and indentation of thin fibers. SiO2 beads with a diameter of 1 μm attached to a silicon nitride triangular cantilever (Novascan Technologies, Inc.) with a 30 nm gold coat were used for indentation tests on thick gels.

[0134] The deflection sensitivity of each probe was calibrated by repeated indentations on a clean glass slide in MilliQ water, and the cantilever spring constant was estimated by the thermal noise method. Before each measurement, the effective tip radius of the conical (sharp) probe was estimated as a function of indentation depth. Here, polycrystalline titanium tip characterization samples (RS-15M, Bruker) were used using the tip estimation function (NanoScope Analysis software, Bruker).

[0135] Indentation was performed in MilliQ water by contacting the AFM probe with the sample surface with a controlled loading force and recording the force-displacement curves during the loading-unloading cycle. During indentation, a maximum load of <10 nN was applied at each data point to avoid plastic deformation and keep the indentation within the elastic range. Measurements were performed by obtaining approximately 250 force curves per sample. Protein Unfolding and Nano-indentation Analysis Software (PUNIAS) was used to fit the force curves.

[0136] The elastic modulus of thick gels (several μm thick) was obtained by fitting the load-force curves to the Hertzian model:

[0137]

number

[0138] where F is the force, δ is the sample deformation, E is Young's modulus, ν is Poisson's ratio, and R is the radius of the indentation probe. To fit the force curves, the indentation depth was controlled to within 200 nm, i.e., 10% of the total film thickness, to minimize the effect of the hard substrate.

[0139] Force curves performed with a sharp probe on a thin fiber were analyzed by the DMT (Derjaguin, Muller and Toporov) model, which is based on the Hertzian model but includes a description of adhesion and is a standard model used in mechanical studies on small fibers [see BRNeugirg et al. Nanoscale 2016, 8, 8414-8426]. The force curve is described in the DMT model by the following equation:

[0140]

number

[0141] F0 is the adhesion force. The fitting of the force curves was performed by controlling the indentation depth to within 4-5 nm, i.e., 10% of the total film thickness, to minimize the effect of the hard substrate. All samples were considered incompressible, with a Poisson's ratio of 0.5.

[0142] Profilometry analysis of coated coverslips: Sample coverslips were prepared as previously described. The surfaces of the coverslips were imaged using a Zygo Nexview 3D Optical Profilometer. 16 V2A2E4GIKVAV(V2A2), C 16 A2G2E4GIKVAV(A2G2), C 16VEVA2GIKVAV (VEVA2), laminin-coated coverslips, and blank coverslips were tested in vitro for 72 hours and 60 days. A 10x objective with 2x zoom and 10 μm scan length was used to obtain images. Samples were allowed to dry for 10 minutes before imaging. A surgical blade was used to make a scratch on each coverslip, and at least 9 images were taken per sample condition. Surface thickness was analyzed using the area tool in Zygo's Mx software. The scratched surface was set as the reference plane, and the measured coating thickness was calculated as the average depth of the coating relative to the reference plane. For visual comparison, all images were normalized to a standardized scale with a minimum of -0.5 μm and a maximum of 2.4 μm.

[0143] Wide-angle X-ray scattering (WAXS): Measurements were performed at the 5ID of the Advanced Photon Source (APS) at Argonne National Laboratory using a fiber-coupled device (CCD) detector. The wavelength of the incident X-rays was 0.729 Å, and the incident energy was 13 keV. 150 μL of sample solution ([PA]=5.3 mM in aqueous solutions of NaCl and KCl ([NaCl]=150 mM and [KCl]=3 mM)) was introduced into a glass capillary of constant diameter and irradiated with X-rays for 3 seconds. During irradiation, the sample solution was continuously vibrated using a flow cell system with a flow rate of 10 μL / s.

[0144] Infrared (IR) spectroscopy: IR spectra of PA samples were recorded on a Bruker model Tensor37 spectrometer. 100 μL of sample solution ([PA] = 1 wt % in aqueous solution of NaCl and KCl ([NaCl] = 150 mM and [KCl] = 3 mM)) was lyophilized and the lyophilized powder was placed on an ATR sample stage equipped with a germanium crystal. IR spectra were scanned 32 times and then averaged.

[0145] DPH-embedded PA sample for fluorescence observation: A THF solution (2 μL) of 1,6-diphenyl-1,3,5-hexatriene (DPH; 2.8 mM) was added to 100 μL of an aqueous PA solution ([PA] = 2 mM, [KCl] = 3 mM, [NaCl] = 150 mM) and the mixture was incubated at 25 °C for 30 min. The mixture was then diluted with an aqueous solution of KCl and NaCl (1900 μL, [KCl] = 3 mM, [NaCl] = 150 mM) and incubated at 25 °C for 10 min to obtain a solution of DPH (2.8 μM)-embedded PA (100 μM). Fluorescence spectra were recorded on an ISS model PC1 spectrofluorometer using a quartz cell with a path length of 1 cm. Polarized fluorescence upon optical excitation was measured using 336 nm plane polarized light, and the fluorescence anisotropy r was calculated as follows:

[0146]

number

[0147] where I represents the fluorescence intensity, the subscripts V and H represent the vertical and horizontal orientations of the excitation and emission polarizers, respectively, and G represents the I HV / I HH and describes the relative sensitivity to vertical and horizontal polarization (J.R. Lakowicz, in Principles of Fluorescence Spectroscopy, Springer Science+Business Media, New York, ed. 3, 2006, pp. 353-382).

[0148] TMA-DPH embedded PA sample for fluorescence observation: To 100 μL of PA aqueous solution ([PA] = 2 mM, [KCl] = 3 mM, [NaCl] = 150 mM), an ethanolic solution of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH; 1.4 mM) (4 μL) was added and the mixture was incubated at 25 °C for 30 min. The mixture was again diluted with an aqueous solution of KCl and NaCl (1900 μL, [KCl] = 3 mM, [NaCl] = 150 mM) and incubated at 25 °C for 10 min to obtain a solution of TMA-DPH (2.8 μM) embedded PA (100 μM). Fluorescence spectra were recorded on an ISS model PC1 spectrofluorometer using a quartz cell with a path length of 1 cm.

[0149] Rheology: PA materials were prepared using the method described above. For all rheological observations, an MCR302 rheometer (Anton Paar) was used. The PA solution was placed on the sample stage (150 μL) and 150 mM CaCl2 solution (30 μL, final concentration 25 mM) was pipetted onto the underside of a 25 mm cone plate above the material. The instrument stage was set to 37 °C to simulate in vitro culture conditions. The plate was slowly lowered to the measurement position and a humidity collar was used to seal the sample plunger and prevent sample evaporation during each 45 min experimental run. During the first interval of each experiment, samples were equilibrated for 30 min at a constant angular frequency of 10 rad / s and 0.1% strain. After a plateau occurred, the storage and loss moduli (G' and G'') were recorded at the end of the interval. The angular frequency was incremented from 100 rad / s to 1 rad / s over 21 points. For all frequencies, G' and G'' were recorded. Finally, the % strain was incrementally increased from 0.1 to 100% over 31 points and G' and G'' were recorded.

[0150] [Simulation Procedure] The PA for the simulations was created in Avogadro and converted to the MARTINI force field CG representation using a modified version of martinize.py including the aliphatic tails and using coiled coil as the secondary structure choice. The last two E's (farthest from the aliphatic tail) and K's are charged, while the two first E's are treated as protonated, since they were found to be ideal for fiber formation in preliminary simulations. This difference in protonation state between assembled and free peptides has been reported previously. Thus, the final charge is (-2+1=)-1, except for the VE(-)VA2 control, which is -2 (Figure 12). The initial structure consists of 300 molecules randomly arranged and spaced a minimum of 3 Å apart, solvated with CG water, and fits into a cubic box 21.5 × 21.5 × 21.5 nm 3 Sufficient ions were added to neutralize the system in

[0151] This is 50 mM (C 16 V2A2E4GIKVAV(V2A2), C 16 A2G2E4GIKVAV(A2G2), C 16 These correspond to concentrations of 7.8, 7.4, and 8.3 wt% for VEVA2GIKVAV (VEVA2), respectively. This is within the range of concentrations commonly used to speed up self-assembly simulations and can be up to 10 times higher than in experimental systems. All visualizations were rendered using Visual molecular dynamics (VMD).

[0152] Coarse-grained molecular dynamics (CG-MD) simulations were performed in GROMACS 5.0.4, which was also used to analyze the simulations. For intermolecular interactions, a cutoff of 1.1 nm was used. Here, a reaction field with a relative permittivity of 15 was used for electrostatics, and a potential shift was used for Lennard-Jones interactions. All systems were minimized for 5000 steps or until the forces in the atoms converged to less than 2000 pN. Self-assembly simulations were performed using a 25 fs time step in the NPT ensemble. Temperature (303 K, τ T = 1ps) using the V rescaling algorithm and pressure (1 bar, τ P = 3 ps) we used Berendsen. The simulation was run for 100,000,000 steps, corresponding to an effective time of 10 μs.

[0153] For the first solvation sphere of each backbone and aliphatic tail bead, the integral of the radial distribution function was used to calculate the water contacts of the PA (Figure 5f), and a 4x factor was applied to convert the CG water to water molecules. Dynamism is measured as the variation of the root mean square deviation (RMSD) over the last 5 μs of the simulation (the fiber is equilibrated after 5 μs).

[0154] [Cell culture and analysis] Human induced pluripotent stem cell (iPSC) culture conditions: Induced pluripotent stem cell lines were derived by retroviral transduction of skin fibroblasts from healthy control individuals (11a: male, 36 years old; 18a: female, 51 years old) (Boulting et al, 2011 Nat Biotech). iPSCs were cultured on Matrigel (BD Biosciences) coated plates with mTeSR1 medium (Stem Cell Technologies) and passaged weekly using 1 mM EDTA or Accutase (Sigma). All cell cultures were maintained at 37°C and 5% CO2 and tested monthly for mycoplasma.

[0155] Motor neuron (MN) differentiation: iPSC cultures were dissociated at 70% confluency using Accutase and cultured for 10 min at 4°C with 10 μM ROCK inhibitor (Y-27632, DNSK International) in mTeSR1. 5 cells / cm 2 The following day (day 0), the medium was replaced with N2B27 medium (50% DMEM:F12, 50% Neurobasal supplemented with NEAA, Glutamax, N2 and B27; Gibco, Life Technologies) containing 10 μM SB431542 (DNSK International), 100 nM LDN-193189 (DNSK International), 1 μM retinoic acid (RA, Sigma) and 1 μM Smoothened agonist (SAG, DNSK International). Culture medium was changed daily until day 6, then switched to N2B27 medium supplemented with 1 μM RA, 1 μM SAG, 5 μM DAPT (DNSK International) and 4 μM SU5402 (DNSK International). Cells were fed daily until day 14, at which time MNs were dissociated using TrypLE Express (Gibco, Life Technologies) supplemented with DNase I (Worthington) and plated onto pre-coated poly-D-lysine / laminin or another IKVAV-coated surface. MNs were fed three times a week with neurobasal medium (NBM: NEAA, Glutamax, N2 and B27) supplemented with ascorbic acid (0.2 μg / ml; Sigma-Aldrich), BDNF, CNTF and GDNF (10 ng / mL, R&D systems), and 1% fetal bovine serum (FBS).

[0156] Cortical neuron differentiation: Cortical neuron differentiation from iPSCs was generated based on the protocol described in Zhang et al., 2015. iPSCs were dissociated using Accutase and, still in suspension with mTESR1 + 10 μM ROCK inhibitor, cells were transduced with three lentiviral different constructs: 1, expressing constitutively expressed rtTA; 2, co-expressing a puromycin resistance gene with Ngn2 in a tetracycline-inducible manner; 3, expressing GFP even in the presence of tetracycline. Cells were plated (9 × 10) on Matrigel-coated plates for 24 h. 5 cells / cm 2 The next day (day 1), the cells were cultured in knockout serum replacement medium (KOSR, Life Sciences, Inc.) supplemented with SB431542, LDN-193189, and XAV939 (DNSK International). Expression of the different constructs was induced by adding doxycycline (Sigma-Aldrich) in Life Technologies. On day 2, supplemented KOSR medium is diluted 1:1 in neural induction medium (NIM: DMEM / F12 (Life Technologies) + Glutamax (Life Technologies) + non-essential amino acids (Corning) + N2 (Life Technologies) + heparan sulfate (Sigma-Aldrich)). Doxycycline and puromycin are added and Ngn2-infected cells are sorted. The next day (day 3), cells are fed with NIM, doxycycline and puromycin. On day 4, cells are detached using Accutase and plated on another surface coated with NBM + doxycycline + BDNF. Medium was changed 3 times a week until analysis.

[0157] Cell viability assay: To assess cell viability, the CytoTox96® non-radioactive cytotoxicity assay (Promega) was used. It is a colorimetric assay that quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme released upon cell lysis. Cell media from cells cultured on different coated surfaces were collected at different culture time points and extracellular levels of LDH enzyme were measured by quantifying the conversion of tetrazolium salt to a red formazan product, read by absorbance at 490-492 nm. Analyses were performed in at least three independent differentiations, with a minimum of three technical replicates per condition. To perform β-integrin studies, motor neurons were treated with β1-integrin (1:1000) or β4-integrin (1:1000, Abcam, UK) antibodies for 72 h and LDH viability and cell attachment were analyzed for each condition.

[0158] Mass spectrometry:C 16 Motor neurons cultured for 60 days on A2G2E4GIKVAV (A2G2) and laminin coatings were digested with lysis buffer (0.5% SDS (Sigma Aldrich), 50 mM AmBic (Fisher), 50 mM NaCl (Sigma-Aldrich), and HALT protease inhibitor (ThermoFisher Scientific)) and briefly vortexed. Samples were sonicated for 30 seconds three times. To determine protein concentration in the treated samples, a BCA assay (ThermoFisher Scientific) was performed, using 100 μg protein / condition for analysis. Peptides were analyzed by LC-MS / MS. LC-MS / MS was performed using a Dionex UltiMate A 3000 Rapid Separation nanoLC and a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ mass spectrometer (ThermoFisher Scientific) were used. Peptides were separated with a 180-minute analytical gradient from 5% ACN / 0.1% FA to 40% ACN / 0.1% FA. The top 15 most abundant precursor ions in each MS1 scan were selected for fragmentation. Precursors were selected with an isolation width of 2 Da and fragmented by higher energy collision dissociation (HCD) at 30% normalized collision energy in a HCD cell.

[0159] All MS / MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.5.1) configured to search the SwissProt_2017_11 database (selected for Homo sapiens, unknown version, 20244 entries) for the digestive enzyme trypsin. Mascot was searched with fragment ions with a mass tolerance of 0.050 Da and parent ions with a tolerance of 10.0 PPM. Carbamidomethyl on cysteine ​​was identified in Mascot as the fixed modification. Deamidations on asparagine and glutamine, oxides on methionine, acetyl on the n-terminus, and TMT6plex on lysine and the n-terminus were identified in Mascot as variable modifications. Scaffold (version Scaffold_4.8.4, Proteome Software Inc., Portland, OR) was used to validate MS / MS-based peptide and protein identifications. They were identified as Peptide ions. Peptide identifications were accepted if they could be established with greater than 95.0% probability using scaffold delta mass correction by the Prophet algorithm (Keller, A et al Anal. Chem. 2002;74(20):5383-92). Protein identifications were accepted if they could be established with greater than 99.9% probability and contained at least two identified peptides. Protein probabilities were assigned using the Protein Prophet algorithm (Nesvizhskii, Al et al Anal. Chem. 2003;75(17):4646-58). Proteins that contained similar peptides and could not be distinguished based on MS / MS analysis alone were grouped to satisfy the principles of parsimony.

[0160] Western blot: For Western blot analysis, protein extracts were obtained from human iPSC-derived neuronal cultures after 14, 17, 30, 45 and 60 days, and total protein extracts were quantified using the BCA assay. 10–20 μg of protein from each sample was separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich) and incubated first with primary antibodies overnight at 4 °C, then with their corresponding secondary HRP-conjugated antibodies (1:3000; Invitrogen). Protein signals were detected by an ECL chemiluminescence system (Azure). As a control for protein loading, densitometric analysis normalized to actin was performed using ImageJ software (National Institutes of Health, USA). For quantification, samples obtained from three to six differentiations were analyzed.

[0161] Cellular immunofluorescence: For immunofluorescence, fixed human iPSC-derived neurons were incubated with primary antibodies overnight at 4 °C, then with their appropriate rabbit, mouse and goat anti-Alexa488, Alexa555 or Alexa647 secondary antibodies (1:500, Molecular Probes). DAPI (1:500, Molecular Probes) was used to stain nuclei. Finally, preparations were coverslipped with Immunoblot (Invitrogen) for imaging.

[0162] Antibodies used for Western blot and immunofluorescence: For Western blot and / or immunocytochemistry, the following primary antibodies were used: goat anti-choline acetyltransferase (ChAT, 1:1000, Millipore), mouse anti-ISLET1 / 2 (ISL1 / 2, 1:500, Hybridoma Bank, Iowa), mouse anti-FOXA-2 (1:1000, Santa Cruz), rabbit anti-microtubule-associated protein-2 (MAP-2, 1:1000, Biolegend), anti-mouse, rabbit and chicken β-tubulin III (TUJ-1, abcam and Biolegend), rabbit anti-postsynaptic density 95 (PSD95, 1:1000, Abcam), mouse anti-synapsin-1 (SYN-1, abcam), mouse anti-synaptophysin (SYP, 1:500, abcam), mouse anti-1-integrin (HUTS4, 1:500, abcam, Cell Signalling), rabbit anti-phosphorylated focal adhesion kinase (p-FAK, 1:1000, Cell Signalling), rabbit anti-focal adhesion kinase (FAK, 1:1000, Rabbit), anti-mouse actin (1:2000, Sigma Aldrich), rabbit anti-integrin-linked kinase (ILK, 1:1000, Cell Signalling), rabbit anti-laminin (1:1000, Sigma Aldrich), rabbit anti-Ki67 (1:500, Abcam), rabbit anti-GFAP (1:1000, Dako), rabbit anti-fibronectin (1:1000, Abcam), rabbit anti-GAP43 (1:500, Abcam), mouse anti-neuronal antigen N (NEUN, 1:1000, Millipore), and mouse anti-SATB-2 (1:250, Abcam).

[0163] Sholl analysis: Neuronal morphology analysis was performed using confocal micrographs of neurons immunolabeled with anti-MAP2 antibody by Fiji software (National Institutes of Health, USA). Maximal reconstructions were calibrated and adjusted for brightness and contrast, and subsequently semi-automatically traced using Simple Neurite Tracer (SNT). From the resulting neurite-traced images, the neuronal branching complexity was analyzed using the Sholl analysis Fiji plugin. This tool generates a series of concentric circles around the neuronal soma and calculates the number of neurites crossing the different circles. Graphical representation of these data shows the branching complexity along the neuronal arbor of one particular neuron. For data obtained from a minimum of three independent differentiations with normal distribution, one-way ANOVA followed by Bonferroni post-hoc test was performed for comparison of the four experimental groups.

[0164] Multi-electrode array (MEA) recordings: For electrophysiology studies, 12- and 24-well MEA plates were prepared using either polyethyleneamine (PEI) and laminin according to the Axion Biosystems protocol, or with PEI and various IKVAV PAs (C 16 V2A2E4GIKVAV(V2A2), C 16 A2G2E4GIKVAV(A2G2), C 16Human iPSC-derived motor neurons (18a) were seeded directly onto the coating at a density of 50,000 cells / well and cultured until day 40. As a control, primary mouse cortical astrocytes were seeded 2 days before culturing motor neurons. Spontaneous network and synchronized activity were recorded using an Axion Biosystems Maestro 768 channel amplifier and Axion Integrated Studios (AxIS) v2.4 software. The amplifier recorded simultaneously from all channels using a gain of 1200 and a sampling rate of 12.5 kHz / channel. After passing Butterworth bandpass filters (300–5000 Hz) online spike detection (threshold = 6 × root mean square of the noise on each channel) was performed with an Axon-adaptive spike detector. All recordings were performed at 37 °C with appropriate 5% CO2 / 95% O2. Spontaneous network activity was recorded for 5 min, three times per week, starting on day 20. Active electrodes were defined as having >5 spikes / min, and only wells with more than 10 active electrodes during the baseline recording period were used for analysis. Synchronized activity was defined as spike and burst activity occurring within 100 ms of each other in ≥25% of the electrodes in a well. Mean firing rate (Hz), network burst duration (s), and number of spikes per network burst were used as measures of neuronal activity, as they indicate maturation of neuronal functional properties. All data reflect sufficiently broad averages, where the reported value of n represents the number of wells per condition.

[0165] Electrophysiology: Whole-cell patch clamp was performed using 2-4 MΩ glass electrodes pulled from glass capillaries (product no. TW150F-4, World Precision Instruments, Sarasota, FL, USA) using a Flaming-Brown P-97 (Sutter Instrument Company, Novato, CA, USA). Electrodes were positioned using a Sutter Instrument MP-285 motorized micromanipulator (Sutter Instrument Company). Whole-cell patch clamp measurements were performed at room temperature using a Multiclamp 700B amplifier (Molecular Devices, Burlingame, CA, USA) and Winfluor software (University of Strathclyde, Glasgow, Scotland). Briefly, slices were perfused with modified Ringer's solution containing (in mM): 111 NaCl, 3.09 KCl, 25.0 NaHCO3, 1.10 KH2PO4, 1.26 MgSO4, 2.52 CaCl2, and 11.1 glucose. The solution was oxygenated with 95% O2 and 5% CO2, and the perfusion rate was 1.5–2.0 ml / min. Patch electrodes contained (in mM) 138 K-gluconate, 10 HEPES, 5 ATP-Mg, 0.3 GTP-Li and Texas Red dextran (75 μM, 3000 MW, from Invitrogen, Life Technologies, Grand Island, NY, USA). In voltage clamp mode, fast and slow transient capacitances were compensated as well as whole-cell capacitance using automatic capacitance compensation on the multiclamp. In current clamp, neurons were subjected to depolarizing current ramps to test I-on (current level at onset of firing), I-off (current level at cessation of firing) and frequency-current relationships. Between stimuli, neurons were held near -80 mV using hyperpolarizing currents. The neurons selected were large in size (input resistance <1000 MΩ) and had a resting membrane potential below -35 mV. The first action potential elicited by a depolarizing current ramp was used to measure all parameters, including I-ON (current at the onset of firing). The threshold voltage was defined as the voltage at which the slope exceeded 10 V / s.Action potential magnitude was measured using the overshoot (through 0 mV) minus threshold voltage. Action potential duration is measured at half the height of the action potential. Rise and fall rates are defined as the peak and valley of the first derivative of the action potential profile.

[0166] Cell Imaging / Analysis: Fluorescent preparations were viewed and photomicrographs taken using a Nikon A1R confocal laser scanning microscope equipped with a GaAsP detector, a Nikon Spectral Illumination Microcopy (N-SIM) or a Nikon Ti2-Widelfield.

[0167] For protein expression studies, images were acquired with a Nikon A1R confocal laser scanning microscope equipped with a GaAsP detector or a Nikon Spectral Illumination Microcopy (N-SIM). Using a ×100 oil immersion objective (NA=1.4), 3–8 images were taken at 0.125 μm intervals in the z direction with a resolution of 1024 × 1024 pixels. Acquisition parameters were kept identical for all scans. Two-dimensional average projection reconstruction of images, fluorescence analysis and quantification were performed using Fiji software (National Institutes of Health, USA).

[0168] For cell distribution, images were acquired with a Nikon A1R confocal laser scanning microscope equipped with a GaAsP detector, using a ×20 objective at 1024 × 1024 pixel resolution. Cell positions were analyzed with Elements software and plotted using the Matlab Knnsearch plug-in.

[0169] [Animal testing and analysis] Animals: All animal housing and procedures were performed in accordance with Public Health Service policies for human treatment and the use of laboratory animals, and all procedures were approved by the Northwestern University Institutional Animal Care and Use Committee.

[0170] Astrocyte culture: Glial cells were derived from the cerebral cortex of newborn mice (P0) as previously described. 2 × 10 cells at passage 1 were cultured on MEA plates with poly-D-lysine and laminin coating in Neurobasal containing 3% normal human serum (NHS), 1% penicillin-streptomycin (pen-strep, Thermofisher) and 2 mm l -glutamine. 5 cells / cm 2 IP-derived motor neurons were cultured in the presence of astrocytes for 40 days. Both the cellular composition and biochemical characterization of the control and reference glial conditions have been previously described.

[0171] Mouse spinal cord injury and animals: Experiments were performed using adult female CD-1 mice (body weight 25-30 g, 8 weeks old). Under aseptic conditions and general anesthesia, a laminectomy was performed to expose the spinal cord at the T11 level.

[0172] Severe injury was performed using an Infinite Horizontal Spinal Cord Impact System (IH-0400 Precision Systems and Instrumentation LLC, USA) with an impact force of 80 kdyn and a dwell time of 60 s. Impact-induced spinal cord displacement was measured for each animal. After injury, the skin was sutured using 9 mm wound clips (BD Biosciences) and the animals were allowed to recover on a heating pad to maintain body temperature. Buprenorphine anesthetic (0.05 mg / kg, subcutaneous, in 1 ml of sterile saline) was administered daily for 3 days after injury. To reduce the risk of infection, varitryl antibiotic (2.5 mg / kg, subcutaneous, in 1 ml of sterile saline) was administered daily for 3 days after injury. Bladders were manually expressed daily. Neurobehavioral analysis was performed using the Basso Mouse scale (BMS) for mice as published elsewhere.

[0173] Peptide amphiphile injections: 24 h after SCI, borosilicate glass capillary micropipettes (Sutter Instruments, Novato, CA) (outer diameter 100 μm) coated with Sigmacote (Sigma, St. Louis, MO) to reduce surface tension were used to inject peptide amphiphiles in saline solution (n = 16), 1 wt% C 16 A2G2E4GIKVAV PA (n=16), or C 16 Mice were given the V2A2E4GIKVAV PA (n=10) solution. A capillary was loaded onto a Hamilton syringe using a female Luer adapter (World Precision Instruments, Sarasota, FL) controlled by a Micro4 microsyringe pump controller (World Precision Instruments). Under isofluorane anesthesia, the autoclip was removed and the injury site was exposed. At 24 hours post-injury, the laminectomy is still intact and the bruising caused by the injury is evident. Using a stereotaxic Kopf apparatus, a micropipette was placed just dorsal to the injury. The micropipette was lowered to a depth of 750 μm measured from the dorsal aspect of the spine and 2.5 μl of the diluted amphiphile solution was injected at 1 μl / min. The micropipette was withdrawn at 250 μm intervals leaving a trail of PA (ventral to dorsal) in the spine. At the end of the injection, the pipette was left in place for an additional minute, after which the pipette was withdrawn and the wound closed. As control conditions, isotonic saline solution (0.9% NaCl, RICCA Chemical) and skeletal C were used. 16 V2A2E2 was used. For all experiments, the experimenters remained blinded to the identity of the animals.

[0174] Immunohistochemistry: Mice were perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.3). Mouse spines were then fixed for 8–12 h, cryoprotected, and cryopreserved. 40 μm thick sections were collected in cryoprotectant solution and stored at -30°C until further use. To characterize the phenotype of proteins inside the scaffolds, the following primary antibodies were used: mouse anti-NeuN (neuronal marker, 1:500; Millipore), rabbit anti-glial fibrillary acidic protein (GFAP, mature reactive glial cell marker, 1:1000–8000; Dako), goat anti-Iba1 (microglia and macrophage marker, 1:200; Abcam), mouse anti-Tuj-1 (neuronal marker, 1:10,000; Biolegend), rabbit anti-MAP2 ... The antibodies used were: rabbit anti-doublecortin (DCX, a neuronal marker, 1:1000; Abcam), rat anti-CD31 / PECAM (endothelial marker, 1:200; Millipore), rabbit anti-laminin (extracellular matrix and vascular marker, 1:500; Sigma-Aldrich), chicken anti-neurofilament (NF, a dendritic marker, 1:1000, Abcam), and goat anti-5HT (1:1000, a dendritic marker, Abcam).

[0175] [Statistical analysis] Statistical tests and parameters including definition and exact value of n are described in the corresponding figure legends. Results were considered significant if p<0.05. All data are presented as mean ± SD unless otherwise stated. Statistical evaluation was performed using paired Student's t-test, one-way analysis of variance (ANOVA), repeated measures ANOVA, or Friedman test, depending on the experimental design. When the number of measurements was limited, their nonparametric equivalents were used: Wilcoxon rank sum test, and Kruskal-Wallis one-way analysis of variance. Tukey's range test and Bonferroni method were applied to test post-hoc differences where appropriate. All statistical analyses were performed with GraphPad Prism 7 software.

[0176] [result] Supramolecular polymers are unique because their constituent monomers assemble via non-covalent interactions in a dynamic manner that exhibits fascinating features. Moreover, supramolecular materials can be tailored with various biological signals on their surfaces to induce a wide range of biological responses such as cell adhesion, migration, division and differentiation. Although the constituent supramolecular monomers are often designed to maximize the stability of the assembled structures, little attention has been paid to the dynamic features of the supramolecular scaffolds. As numerous important biological events are controlled by non-covalent interactions between biomolecules, the dynamic features of ECM-mimicking supramolecular materials can affect their biological functions. Here, supramolecular materials are demonstrated as a suitable platform to decipher the critical nature of dynamic ECM signals in the CNS. To investigate this effect, the influence of intermolecular cohesive forces between molecules on the resulting bioactivity was evaluated both in vitro and in vivo.

[0177] A series of peptide amphiphiles (PAs) composed of four main segments were developed: (1) hydrophobic tails that induce nanophase separation in aqueous environments, (2) hydrophobic amino acids that form intermolecular hydrogen bonds (H-bonds) to stabilize high aspect ratio nanofiber structures, (3) hydrophilic amino acids that promote the dissolution of the molecules in aqueous solvents, and (4) a bioactive peptide sequence that is designed to interact with cells and is exposed in the outermost layer of the assembly. For this study, PAs were functionalized with isoleucine-lysine-valine-alanine-valine (IKVAV), a bioactive pentapeptide sequence found in laminin-1.

[0178] To investigate the effect of intermolecular cohesion within supramolecular assemblies on cellular behavior, we designed three IKVAV-containing PA molecules with different H-bonding propensities (Fig. 1A). PAs containing a valine-valine-alanine-alanine (V2A2) H-bond-forming region, four glutamic acid (E4) and glycine residues (G) and exhibiting the hydrophobic epitope IKVAV on their surface had a smaller tendency to enter into bundles and thus increase the bioactivity of the epitope (Fig. 1B). To develop a series of IKVAV PAs with the same charge and the same bioactive units but different intermolecular cohesion, we newly designed alanine-alanine-glycine-glycine (A2G2) and valine-glutamic acid-valine-alanine-alanine (VEVA2)-containing PAs (Fig. 1C,D). A2G2 was expected to have weaker intermolecular interactions than V2A2 due to its reduced hydrophobicity. IKVAV PAs, V2A2, A2G2, and VEVA2, were synthesized by solid-phase peptide synthesis and were clearly characterized by liquid chromatography-mass spectrometry (Figure 6). Using cryo-TEM and scanning electron microscopy (SEM), we confirmed that these three PAs formed nanofibers in an aqueous environment (Figure 1E-J, Figure 2). Infrared (IR) spectroscopy of the samples was performed to evaluate the hydrogen bonding (H-bond) properties of the PA molecules within the nanofibers (Figure 1K). As shown in Figure 1K, V2A2 and VEVA2 were found to have a hydrogen bond at 1628 cm -1 At 1634 cm, A2G2 showed an amide I vibration band, indicating the presence of a peptide secondary structure rich in β-sheet structure. In contrast, A2G2 showed a vibration band at 1634 cm -1A2G2 showed a broad amide I band with a peak at 0.05, indicating that the peptide is mainly in a random coil conformation. Furthermore, liquid-phase wide-angle X-ray scattering (WAXS) was used to quantify the β-sheet content. As shown in Figure 1L, V2A2 and VEVA2 showed a Bragg peak with a d-spacing of 4.72 Å, which corresponds to the distance between the H-bonded β-sheet peptide strands. Furthermore, the Bragg peak observed for VEVA2 was stronger than that for VEVA2. This indicates that the VEVA2 ensemble contains a higher population of H-bonds than V2A2. However, A2G2 did not show a clear peak, as did infrared spectroscopy analysis, which indicates the presence of a random coil conformation. These evidences confirmed that the various β-sheet-forming domains of the PA molecule result in various H-bonding propensities.

[0179] Next, we used the fluorescence depolarization technique to investigate the dynamics of PA molecules within the assemblies (Figure 2A). Here, we utilized DPH (DPH: 1,6-diphenyl-1,3,5-hexatriene) embedded in PA nanofibers to measure the microviscosity of the inner hydrophobic core of another IKVAV PA. Notably, the incorporation of DPH into PA nanofibers does not degrade their nanostructure, as confirmed by their cryo-TEM micrographs (Figure 8). V2A2 and VEVA2 showed anisotropy as high as 0.4, close to that observed for saturated phospholipid bilayer assemblies, while interestingly, A2G2 showed a significantly lower anisotropy value (0.21). Furthermore, we performed fluorescence depolarization experiments using TMA-DPH embedded PA nanofibers (TMA-DPH: (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate)) (Figure 9). DPH is known to be buried deep inside the hydrophobic tails of amphiphilic molecules, whereas TMA-DPH is known to be anchored at the hydrophilic-hydrophobic interface of amphiphilic assemblies (Figure 10). Similar to what was observed for DPH-embedded PAs, A2G2 showed significantly lower anisotropy than V2A2 and VEVA2 (Figure 9e). These results indicate that the molecular motion of A2G2 within the assemblies is significantly more dynamic compared to V2A2 and VEVA2. As discussed in the above section, V2A2 and VEVA2 form intermolecular H-bonds and adopt an ordered β-sheet secondary structure within the assemblies, whereas A2G2 forms a disordered structure. These results indicate the importance of H-bond propensity in determining the dynamics of molecular motion. The assemblies of the three PAs were further studied using coarse-grained molecular dynamics simulations (CG-MD) using the MARTINI force field, which has been well adapted to the study of peptide-based self-assembled materials. The simulations reproduced the experimentally observed fiber formation for the three PA sequences (Figure 2B). Although the three PA nanofibers appear similar, the arrangement of the aliphatic tails indicates that the PA fibers in A2G2 are not arranged on a well-ordered core-shell arrangement ( Figure 11 a–c ).Dynamism analysis of the self-assembled equilibrated fibers showed a significant increase for A2G2 (Figure 2C, Figures 11d-f), which corroborates the results obtained by anisotropy analysis. Moreover, the fluorescence intensity observed for DPH-embedded A2G2 was significantly lower compared to that of V2A2 and VEVA2 (Figure 2D). PA water mapping in CG-MD simulations also showed high water contact with the aliphatic tails of A2G2 (Figure 2E). This behavior is probably due to the loss of ordered arrangement of the core-shell, indicating that the inner hydrophobic core of A2G2 nanofibers is much more hydrated compared to V2A2 and VEVA2. All these results suggest that the dynamics of PA molecules within the assembly, their degree of hydration and intermolecular order are closely related.

[0180] V2A2, A2G2, and VEVA2 containing the scrambled sequence VKIVA were also synthesized by solid-phase peptide synthesis and characterized by liquid chromatography-mass spectrometry (Figure 13). Simulations showed that the scrambled sequence VKIVA bound to V2A2 and VEVA2 could form fibers (Figure 14), as corroborated by Cryo-TEM (Figure 15). However, A2G2 bound to VKIVA had high water content and low order. This may be related to the experimental deficiency of fiber formation by Cryo-TEM. The PA molecular backbone sequence also cannot form fibers (Figures 16 and 17). This suggested that the IKVAV sequence plays an important role in fiber formation. Finally, other laminin mimetic sequences (LGTIPG, LRGDN, PDGSR, RGD, YIGSR) could not drive fiber formation with the A2G2 β-sheet sequence. The water content in the aliphatic core was a good quantitative measure of this tendency (Figure 18).

[0181] To test the impact of molecular dynamics within supramolecular assemblies on cellular behavior, we next explored the use of human induced pluripotent stem cell (hiPSC)-derived neurons on three PA matrices and determined the efficiency with which each PA could generate functional human motor neurons. iPSCs are powerful tools for disease modeling and complex tissue regeneration due to their ability to differentiate into any single cell type in the body. The development of in vitro models utilizing hiPSC-derived neuronal cells has proven to provide novel insights into disease mechanisms and therapeutic strategies. Nevertheless, these models still exhibit significant technical limitations, such as inefficient maturation, abnormal aggregation and low long-term viability, and need to be improved to better resemble physiological conditions. Current strategies for culturing and maturing hiPSC-derived neurons on feeder-free culture systems rely on static two-dimensional (2D) matrices composed of recombinant proteins, such as laminin or fibronectin, identified in the ECM. Although classical systems such as plastic Petri dishes offer simplicity and convenience, it is now widely recognized that these materials fail to mimic key aspects of the in vivo environment, often resulting in non-physiological responses. Subjecting hiPSC-derived motor neurons (MNs) to a series of supramolecular scaffolds with different dynamic characteristics may provide an attractive model to study and clarify the influence of the strength of intermolecular cohesion on MN maturation, providing insight into ECM-cell interactions.

[0182] MNs were differentiated from hiPSCs by utilizing a small molecule cocktail. The small molecule cocktail first stimulates neuronal induction and then promotes ventral / caudal patterning. At day 14 of the differentiation protocol, representing the peak generation of postmitotic neurons (neuron efficiency of 80-90%), hiPSC-derived MNs were cultured on different IKVAV supramolecular scaffolds (Figure 19a, b). The response of iPSC-derived MN cultures on different coatings was evaluated by characterizing the cellular composition of the cultures 72 hours after plating. Neurons cultured on A2G2 expressed a higher percentage of β-III tubulin (TUJ-1) compared to conventional unpatterned PDL / laminin-coated coverslip conditions (laminin, 81%). + ) neurons (92%) (Fig. 19c). + ) and choline acetyltransferase (ChAT) + The efficiency of MN generation was also assessed by immunocytochemistry (ICC) for MN (Figure 19d-g). A higher percentage of ISL1 / 2 was detected in neurons grown on A2G2 (61%, 71%) compared to V2A2 (51%, 60%), VEVA2 (41%, 57%), and laminin (52%, 62%). + and ChAT + markers were visible (Fig. 19f, g). However, cell numbers in the laminin condition were significantly higher than those in the three IKVAV PAs (Fig. 19h). The higher number of neurons and reduced total cell numbers could be driven by differential cell attachment, cell survival and / or terminal differentiation of progenitor cells into different spinal cord cell lineages produced by the present differentiation protocol (Ziller et al., 2018). Although cell viability at day 17 did not show significant differences between the various conditions (Fig. 19i), cultures in laminin coatings showed a higher percentage of FOXA2+ floor plate cells (25%) compared to the IKVAV PA matrix (Fig. 20). Notably, FOXA + The percentage of cells expressing TUJ1 in different conditions +The differences were more pronounced between laminin and A2G2, and were complementary to the percentage of neurons in the presence of PA matrices. + A dramatic decrease in the number of cells was observed. As expected, the proliferation marker Ki67 was downregulated by FOXA2. + A2G2 was observed only in iPSC-derived iPSC-derived cells, indicating that the only proliferative cells present in the cultures were floor plate cells (FIG. 20). These results indicate that the IKVAV material, and in particular A2G2, improves neuronal purity in iPSC-derived cultures by reducing the attachment and / or proliferation of non-neuronal cells.

[0183] Since IKVAV is well known to bind to the transmembrane receptor β1-integrin, we next investigated the impact of the IKVAV peptide displayed on the three PA nanofibers. This was by analyzing the restoration of β1 integrin and the resulting intracellular signaling on hiPSC-derived MNs, a process known as "outside-in" signaling. Even though the three PAs tested had the same IKVAV epitope, A2G2 induced significantly higher levels of β1-integrin (ITGB1), as well as the downstream effectors integrin-linked kinase (ILK) and phosphorylated focal adhesion kinase (p-FAK), compared to V2A2, VEVA2, and laminin (Figures 2F-I, and Figure 21). FAK and ILK are important signaling scaffold proteins that cross many intracellular pathways in response to ECM stimuli by acting as signaling integrators. Thus, the epitope IKVAV displayed by the highly dynamic A2G2PA has better receptor accessibility compared to V2A2 and VEVA2. To determine whether cell binding and spreading is mediated solely by IKVAV-integrin activation, we used antibodies blocking laminin-interacting β-integrins (β1, β4) (Figure 22). Under β1-integrin antibodies, there was no cell attachment on the three IKVAV matrices, indicating that the bioactivity induced by the IKVAV surface depends on integrin engagement (Figure 22c, e). However, in the presence of β4-integrin antibodies, MNs showed similar attachment in all experimental conditions (Figure 22d, f). These results suggested that cell binding and spreading of MNs, especially on IKVAV matrices, is mediated by ITGB1. By changing the thickness of the PA matrix, the effect of mechanical properties on cell signal activation was evaluated (Figure 23a). The three IKVAV PA and laminin coatings used in the biological experiments showed a thickness of 200 nm by profilometry analysis (Figure 24).However, A2G2 fibers showed a modulus of 0.2 MPa, while V2A2 and VEVA2 showed higher moduli of 13 and 20 MPa, respectively (Figures 23 and 25). With increasing thickness of the three IKVAV PAs, the mechanical properties are significantly decreased, showing higher elastic and storage moduli for A2G2 (8 KPa, 5 KPa) compared to V2A2 (1 KPa, 1 KPa) and VEVA2 (4 KPa, 1 KPa) (Figures 23, 20, 21). Although these hydrogels have various stiffnesses depending on the thickness, no differential cell-receptor activation and signaling was observed for cells cultured on thin or thick gels (Figures 23c-f). These results suggest that the different mechanical properties of the materials do not affect the dynamic molecular motion of A2G2 and allow the same accessibility of IKVAV ligands to the receptors.

[0184] Since achieving functional maturation of hiPSC-derived neuronal cells in vitro remains a challenge in the art, we evaluated the time evolution of human MNs (Figures 3A, 27 and 23). MNs cultured in vitro for 30-60 days maintained higher expression of ITGB1, ILK and motor neuron marker ChAT compared to other conditions (Figures 3B,C, 27-30). We next verified whether differential cell signaling induced by IKVAV PA could improve the problems of reduced maturation levels and cell aggregation commonly observed in neuronal disease cell modeling where hiPSC-derived neurons are cultured for long periods without a feeder layer. Upon ligand binding, integrins are known to play a role in various neuronal processes, including survival, adhesion, differentiation, maturation, neuronal morphogenesis (Colognato & Tzvetanova, 2011; Gardiner, 2011; Kazanis & French-Constant, 2011; Myers, Santiago Medina & Gomez, 2011, reviewed in), and synaptic plasticity (Park & ​​Goda, 2016, reviewed in) (Figure 3D). This problem was addressed by utilizing a high-throughput and unbiased method, allowing us to investigate the proteomic changes that occurred in MNs cultured for 2 months on A2G2 and laminin coatings. Quantitative mass spectrometry of tandem mass tag-labeled samples identified 892 proteins with at least 99% confidence. Normalized intensity differences in 30.3% of identified proteins were observed between MNs cultured on A2G2 and laminin coatings (Figure 3E). To understand the cellular context of the downregulated (196) and upregulated (76) proteins, we performed gene ontology (GO) analysis. Consistent with the previously described changes in the laminin-ITGB1 signaling cascade, we observed that the most significant GO term associated with differentially expressed proteins in A2G2 cultures was the integrin pathway.Moreover, in the obtained dataset, cellular functions promoted by ITGB1 signaling (such as cell survival, proliferation, and cell-substrate transfer) were also enhanced (Figure 3F, Figure 3I). To validate these proteomic findings, a comprehensive analysis of cellular behavior was performed on all platforms. Cell viability and ChAT-positive cells were analyzed in various conditions (Figure 3G, H). Cells cultured on IKVAV matrices showed higher viability and maturation compared to laminin coating, especially on A2G2. Morphological characteristics of MNs cultured on laminin, as well as on IKVAV matrices including V2A2, A2G2, and VEVA2 (Figure 3I-L, Figure 27) were also analyzed. MNs on V2A2 and A2G2 showed larger soma size compared to MNs cultured on laminin coating. MNs cultured on A2G2 showed a significant increase in the number of processes and increased complexity of neuronal branching (Figure 3I-L, Figure 32). As current methods for culturing hiPSC-derived neuronal cells result in neuronal clustering, which precludes analysis of individual neurons and has been shown to induce cell death, cell-cell and cell-substrate interactions, we similarly investigated cell viability after 30-60 days in vitro on IKVAV matrices and laminin coatings (Figure 3M,N). Cell distribution analysis of confocal images showed that MNs cultured on IKVAV matrices, especially A2G2, exhibited highly homogenous distribution and network connections along the platform over time. MNs grown on laminin coatings showed heterogeneous neuronal growth and formed cell clusters along the entire platform. Correlating with these results, it was found that MNs cultured on laminin coatings exhibited a highly significant increase in cell death compared to cells cultured on the three IKVAV matrices (Figure 3G).

[0185] To corroborate MN interaction with supramolecular PA after 60 days in vitro, the presence of IKVAV and laminin coatings was analyzed by profilometry. Analyzing the platforms at short (72 h) and long (60 days) time points, the three supramolecular peptide amphiphile coatings showed little loss of thickness between 72 h and 60 days, whereas the laminin coating was significantly reduced after 60 days in vitro (Figure 24). Structured illumination microscopy (SIM) and SEM micrographs corroborated the presence of the coatings in the presence of cells (Figure 33). In agreement with the observations from the proteomic analysis, all these data indicate qualitative and quantitative differences in MN maturation between A2G2 and laminin coatings. Molecular Dynamics To confirm that IKVAV peptide presentation by supramolecular nanostructures plays a key role on cell behavior, IKVAV peptide was immobilized on solid surfaces to investigate the various molecular dynamics on IKVAV PA matrices containing V2A2, A2G2 and VEVA2 (Figures 34, 35). For this purpose, 3-aminopropyltriethoxysilane (APTES)-treated glass coverslips were functionalized with IKVAV peptide. On surfaces coated with immobilized IKVAV peptide, MN attachment was dramatically reduced and further analysis was not possible (Figure 35). Scrambled VKIVA PA containing V2A2, A2G2 and VEVA2 (Figure 36) was also tested. MNs cultured on coatings of the three scrambled sequences barely survived at 72 h. Finally, other types of motor neurons (MN-11A) and cortical neurons (Cx-18A) were tested with V2A2, A2G2 and VEVA2, which display the IKVAV sequence, and the cells attached, survived, proliferated and activated the ITGB1 pathway in a manner similar to that described above (Figures 37 and 38).

[0186] One of the main obstacles for stem cell-based models of neurological diseases is the difficulty in promoting functional maturation in hiPSC-derived neurons that can accurately reflect physiological conditions. To demonstrate that molecular dynamics within A2G2 supramolecular assemblies induce functional maturation in hiPSC-derived MNs, we monitored the electrophysiological state by analyzing the expression and electrophysiological properties of synaptic vesicles after 40–60 days in vitro. MNs cultured on A2G2 expressed higher levels of the postsynaptic marker PSD95, as well as the presynaptic markers SYN-1 and SYP-1, suggesting an increase in synaptic networks (Figure 4A–E). Cells on V2A2 and VEVA2 showed similar expression of synaptic markers compared to laminin (Figure 39). Next, the cellular network activity of MNs cultured on the different platforms was assessed using a multi-electrode array (MEA) approach (Figure 4F–I). Consistent with the reduced aggregation mentioned above, MNs cultured on A2G2 showed an increased number of active electrodes compared to V2A2, VEVA2 and laminin cultures. Furthermore, MNs grown in A2G2 showed higher spontaneous and synchronized activity compared to V2A2, VEVA2 and laminin coatings. Also, MNs on A2G2 were able to fire longer network bursts with significantly more spikes per network burst than those on VEVA2 and laminin (Figure 4F-I, Figure 4C). A widely used approach to promote both morphological and functional maturation of human neurons derived from hiPSCs is to co-culture neurons with astrocytes. Compared to the commonly used substrate laminin, astrocytes significantly enhanced the complexity of neuronal dendrites, the expression of ion channels and neurotransmitter receptors, as well as the frequency and amplitude of synaptic events. MNs cultured on astrocyte monolayers showed higher excitability than A2G2 material. These results demonstrate that the A2G2 supramolecular matrix enhances neuronal network activity at levels comparable to neurons cocultured with astrocytes.To further investigate the electrophysiological maturity of MNS on A2G2, we performed whole-cell patch clamp recordings on MNs grown in A2G2 and commercial laminin.Electrophysiological development of MNs is characterized by their ability to fire repeatedly and the generation of spikes of larger amplitude (Carrascal et al., 2005; Tadros et al., 2015). As neuronal development progresses, more voltage-sensitive ion channels are inserted into the cell membrane, generating faster and larger action potentials. At day 50, MNs grown on A2G2 showed characteristics of more mature MNs: more MNs were able to fire repeatedly (Fig. 4J,K; Table 2), and their action potentials were larger in amplitude and had faster rise and fall kinetics, as shown in Fig. 4L. Markers of electrophysiological development of human MNs grown on astrocytes are comparable to the development of human MNs grown on A2G2 matrices. Human embryonic stem cell-derived MNs grown on astrocytes (recorded at P48-50 for a previous study) had similar properties (including similar values ​​of I-on, voltage threshold, action potential amplitude, and rise and fall kinetics) as human iPSC-derived MNs grown on A2G2 matrices for this study. In summary, both MEA and patch clamp data highlight the maturity of MNs grown on A2G2 matrices in their ability to perform repetitive firing / higher firing rates, with higher spiking and bursting activity.

[0187] [Table 2]

[0188] Because the A2G2IKVAV PA system can mimic several aspects of ECM dynamism in the CNS, this nanostructure was used as a scaffold to treat spinal cord injury (SCI) in a mouse in vivo model. The ECM is well known to play a key role during and after the development of disease or injury to the CNS. The dynamic temporal and spatial distribution of ECM components represents a powerful therapeutic approach. However, the functional role for the dynamic distribution of ECM in spinal cord injury regeneration remained to be elucidated. To better understand how molecular dynamics affect spinal cord regeneration, the impact of V2A2 and A2G2IKVAV PA was evaluated after spinal cord injury. A robotically controlled impact (71 ± 0.5 KDyn) on the T10 / T11 segment was used to model a severe contusion injury in mice (Figure 4M) (Figure 41a, b). This injury induced immediate lower limb paralysis in mice. 24 hours after injury, V2A2, A2G2 were injected into the injury site, followed by weekly assessment of locomotion using the Basso Mouse Scale (BMS). As controls, saline solution (control) and V2A2E2PA, a non-bioactive scaffold, were used due to its well-known ability to form robust nanoribbons by itself. Immediately after injury, there were no detectable differences between the PA-treated and saline control groups. Two weeks after injury and thereafter, the A2G2 group showed significant and sustained behavioral improvements compared to the control and V2A2. Notably, 12 weeks after injury, the A2G2-treated group showed a mean BMS score of 5.8 ± 1.6, significantly higher than V2A2 (3.8 ± 0.7) (Figure 4N). The control group and the non-bioactive scaffold PA maintained mean scores of 1.9 ± 0.3 and 2.1 ± 0.8, respectively (Figure 4N and Figure 4c). Western blot analysis of injured spinal cords after 12 weeks showed an increase in β-1 integrin (ITGB1) in conditions where IKVAV was presented, especially in A2G2, consistent with the in vitro results shown above ( Figures 4 O,P ).Furthermore, Western blots showed a reduction in the amount of reactive gliosis, characterized by a reduction in the fibrous ECM protein fibronectin, as well as an overexpression of GFAP in V2A2, which was more evident for A2G2. Interestingly, the ECM laminin protein was more highly expressed in the A2G2 condition, suggesting a greater amount of basement membrane in the injured area. Further histological experiments will indicate the cell population responsible for this behavior. Finally, GAP43, a marker associated with neuronal regeneration and growth, was highly expressed in the highly dynamic A2G2PA (Figure 4O,P).

[0189] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations on the scope of the present disclosure, which is defined solely by the appended claims and their equivalents.

[0190] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications (including but not limited to those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure) can be made without departing from the spirit and scope thereof.

[0191] Any patents and publications referenced herein are hereby incorporated by reference in their entirety. [Brief description of the drawings]

[0192] [Figure 1]Figure 1A-L. Characterization of supramolecular nanofibers formed by peptide amphiphiles (PAs). (A) Schematic molecular structure of IKVAV PA, which contains (B) V2A2, (C) A2G2, and (D) VEVA2. (E-G) Cryo-TEM micrographs of (E) V2A2, (F) A2G2, and (G) VEVA2 in KCl and NaCl aqueous solutions ([PA] = 0.01 wt%, [KCl] = 3 mM, [NaCl] = 150 mM). (H-J) SEM micrographs of (H) V2A2, (I) A2G2, and (J) VEVA2. (K) FT-IR spectra (1500-1800 cm-1) of film samples of V2A2 (red), A2G2 (blue), and VEVA2 (orange). (L) WAXS profiles of V2A2 (red), A2G2 (blue), and VEVA2 (orange) in KCl and NaCl aqueous solutions ([PA] = 5.3 mM, [KCl] = 3 mM, [NaCl] = 150 mM). [Diagram 2]Figure 2A-I. Dynamics within supramolecular IKVAV PA induce distinct effects on human motor neuron (MN) signals. (A) Fluorescence depolarization profile (λex=336 nm, λem=450 nm) at 25 °C of DPH (2.8 µM) embedded in IKVAV PA containing V2A2 (red), A2G2 (blue), and VEVA2 (orange) in KCl and NaCl aqueous solutions ([PA]=100 µM, [KCl]=3 mM, [NaCl]=150 mM). (B) Self-assembled structure and coarse-grained view of a single IKVAV PA fiber containing V2A2 (red), A2G2 (blue), and VEVA2 (orange) PA after 10 µs. The micrograph contains a partial periodic image through the fiber-forming axis within the simulation box (in blue). Water and ions have been omitted for clarity. (C) Kinetic analysis of IKVAV PA containing V2A2 (red), A2G2 (blue), and VEVA2 (orange) structures formed in B. (D) Fluorescence emission spectrum (λex=336 nm) at 25 °C of DPH (2.8 μM) embedded in IKVAV PA containing V2A2 (red), A2G2 (blue), and VEVA2 (orange) in aqueous KCl and NaCl solutions ([PA]=100 μM, [KCl]=3 mM, [NaCl]=150 mM). (E) Water content analysis of IKVAV PA containing V2A2 (red), A2G2 (blue), and VEVA2 (orange). (F) Representative three-dimensional SIM micrographs (left) and shaded reconstructions (right) of MN neurites (TUJ-1, green) containing b-1 integrin (pink, ITGB1) cultured on A2G2 and laminin. (G) Intensity analysis of ITGB1 in MNs cultured on A2G2 (blue) and laminin (black) matrices at 72 h. (H) and (I) Western blot analysis of ITGB1 and downstream kinases (ILK, pFAK, FAK) in MNs cultured on the supramolecular IKVAV matrix containing V2A2 (red), A2G2 (blue), and VEVA2 (orange) at 72 h. Scale bar: 10 mm. Data are the average of at least four independent differentiations.All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001). [Diagram 3]Figure 3A-O. Effect of intermolecular cohesion of A2G2-containing IKVAV PA on human motor neuron (MN) maturation. (A) Schematic of human MN differentiation and its culture on supramolecular matrix. (B) Western blot and (C) normalized protein expression levels (after 60 days) related to integrin activation (ITGB1), signaling pathways (ILK) and motor neuron maturation (ChAT) in MNs cultured on supramolecular IKVAV matrix containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black). All values ​​were normalized to actin. (D) Schematic of integrins and signaling pathways mediating cell behavior upon dynamic presentation of A2G2-containing IKVAV. All values ​​were normalized to actin or total FAK expression. (E) Volcano plots depicting proteomic changes of MNs cultured on A2G2 vs laminin. Mean log2 (fold change) vs P-value (-log10) are shown. Proteins up- and downregulated by 2-fold change and FDR<0.05 are labeled by green and red dots, respectively. (F) Subset of most significant GO terms enriched in up- and downregulated groups of proteins identified in MNs cultured in A2G2- vs. laminin-coated. (G) Cell viability assessed by LDH concentration released into cell medium (day 60). (H) Quantification of ChAT+ cells in MNs cultured on IKVAV PA containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) matrix (after 60 days). (I,J) Confocal microscopy images of human MNs cultured on (I) A2G2 and (J) laminin matrix. (K) Analysis of total neurite processes in human MNs cultured on IKVAV PA containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) matrix (after 60 days). (L) Sholl analysis of dendritic branching of MNs cultured in vitro for 60 days on V2A2 (red), A2G2 (blue), VEVA2 (orange), and laminin (gray).(M) Representative representative confocal micrographs (after 60 days) of human MNs cultured on IKVAV PA and laminin (white) matrices containing V2A2 (red), A2G2 (blue), VEVA2 (orange). Cells were stained with motor neuron marker ChAT (green), microtubule-associated protein-2 (MAP-2, red) and nuclei (DAPI). (N) DAPI channel micrograph of the image shown in M. (O) Histogram analysis of cell distribution on the different matrices referred to in M ​​and N. Scale bars: (M, N) 100 μm. Data are the average of at least three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001). [Figure 4]Figure 4A-P. Impact of molecular dynamics within supramolecular assemblies on functional maturation and regeneration. (A,B) Representative confocal micrographs (day 50) of synaptic vesicles (postsynaptic PS95 and presynaptic SYN-1) distributed along human MN neurites cultured on (A) A2G2 and (B) laminin matrices. (C) Intensity analysis of PSD95 and SYN-1 on MNs cultured on A2G2 and laminin coatings. (D) Western blots and (E) normalized protein levels of presynaptic and postsynaptic markers (SYN-1 and PS95, respectively) in human MNs cultured on A2G2 (blue) and laminin (black). All values ​​were normalized to actin. (F) Brightfield images of human MNs cultured for 40 days on MEA plates coated with A2G2 supramolecular peptide amphiphiles. Plots representing (G) the difference in spike counts and (H) the number of bursts per electrode and (I) synchrony index in MN cultures in various IKVAV PA, laminin or glial coatings. (J) Two-photon microscopy images (days 48-49) of human MNs cultured on A2G2 and laminin matrix and filled with Texas Red dextran through the patch electrode. (K) Percentage of neurons grown on A2G2 and laminin matrix that can repeatedly fire action potentials. (L) Representative examples of action potentials from MNs grown on A2G2 and laminin matrix. MNs grown on A2G2 were significantly larger and had faster rise and fall velocities. (M) Schematic of a dorsoventral contusion injury in a mouse spinal cord. (N) Basso Mouse Scale (BMS) scores for animals treated with saline solution (control), V2A2 (red) and A2G2 (blue). (O) Western blot and (P) normalized protein levels of beta-1 integrin receptor (ITGB1), various ECM proteins (fibronectin and laminin), astroglial GFAP and neuronal GAP43 markers in spinal cords treated with saline solution (control), V2A2 (red) and A2G2 (blue). All values ​​were normalized to GAPDH. In vivo data are the average of at least eight animals.All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001). [Diagram 5] Figure 5A-F. MARTINI bead type and charge for (a) V2A2, (b) A2G2, (c) VEVA2 and (d) VE(-)VA2. (e) Charge amount of PA. (f) Raw radial distribution function for each scaffold bead of V2A2 (insert), showing in blue the area integrated to give the water contacts for the first solvation sphere. [Figure 6] Figure 6A-F. Liquid chromatography-mass spectrometry of IKVAV PA. (ac) Analytical high-performance liquid chromatography (analytical HPLC) traces of purified (a) C16V2A2E4GIKVAV, (b) C16A2G2E4GIKVAV (c) C16VEVA2E4GIKVAV. (d-f) Electrospray ionization mass spectrometry (ESI-MS) of (d) C16-V2A2E4-GIKVAV, (e) C16A2G2E4GIKVAV, and (f) C16VEVA2E4GIKVAV. [Figure 7] Figure 7A-I. Cryo-TEM and SEM images of IKVAV PA. (a-c) Schematic molecular structures, (a, d, g) C16V2A2E4GIKVAV, (b, e, h) C16A2G2E4GIKVAV, (c, f, i) C16VEVA2E4GIKVAV, (d-f) cryo-TEM and (g-i) SEM micrographs. [Figure 8] Figure 8A-D. TEM images of DPH-embedded IKVAV PA. (a) Schematic molecular structure of 1,6-diphenyl-1,3,5-hexatriene (DPH). (b-d) TEM micrographs of DPH-embedded PA (b) C16-V2A2E4GIKVAV, (c) C16A2G2E4GIKVAV, and (d) C16VEVA2E4GIKVAV stained with uranyl acetate ([PA] = 100 μM, [DPH] = 2.8 μM in aqueous KCl and NaCl solution ([KCl] = 3 mM, [NaCl] = 150 mM). [Figure 9]Figure 9A-F. TEM images of TMA-DPH-embedded IKVAV PA. (a) Schematic molecular structure of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH). (b-d) TEM micrographs of TMA-DPH-embedded IKVAV PA (b) C16V2A2E4GIKVAV (V2A2), (c) C16A2G2E4GIKVAV (A2G2), and (d) C16VEVA2GIKVAV (VEVA2) stained with uranyl acetate ([PA] = 100 μM, [TMA-DPH] = 2.8 μM in KCl and NaCl aqueous solutions ([KCl] = 3 mM, [NaCl] = 150 mM). (e) Fluorescence depolarization profiles (λex=336 nm, λem= 450 nm) of TMA-DPH (2.8 μM) embedded in V2A2, A2G2 and VEVA2 in KCl and NaCl aqueous solutions at 25 °C ([PA]=100 μM, [KCl]=3 mM, [NaCl]=150 mM). (f) Fluorescence emission spectra (λex=336 nm) of TMA-DPH (2.8 μM) embedded in V2A2, A2G2 and VEVA2 in KCl and NaCl aqueous solutions at 25 °C ([PA]=100 μM, [KCl]=3 mM, [NaCl]=150 mM). [Figure 10] Figure 10. DPH location to hydrocarbon tails within lipid bilayers. [Figure 11] Figure 11A-F. Simulation results for IKVAV PA nanofibers. (a-c) Self-assembled structures and coarse-grained representations of single fibers of (a) V2A2, (b) A2G2, and (c) VEVA2 IKVAV PA after 10 μs. Fibers are shown transparent with all but the C16 tail to show the patch core. Micrographs include partial periodic images through the fiber forming axis. Simulation boxes are shown in blue. Water and ions have been omitted for clarity. (d-f) Graphs representing the root mean square deviation (RMSD) versus time for (d) V2A2, (e) A2G2, and (f) VEVA2. RMSD plots are the average of five independent simulations. [Figure 12]Figure 12A-E. Simulation results of E-charged VE(-)VA2 PA placed in the β-sheet region. (a, b) Self-assembled structure of VE(-)VA2 fiber after 10 μs. (a) β-sheet region is represented in orange, IKVAV in green, and the rest of the fiber in black. (b) Fiber is represented all transparent except for C16 tail to show patch core. Micrograph includes partial periodic image through fiber forming axis. Simulation box is shown in blue. Water and ions are omitted for clarity. (c) Water contact graph of VE(-)VA2 compared to V2A2, A2G2, and VEVA2. (d) Root mean square deviation (RMSD) vs. time graph of dynamism analysis of VE(-)VA2 compared to V2A2, A2G2, and VEVA2 in (a-b). Analysis in c-d is the result of five independent simulations. [Figure 13] Figure 13A-F. Liquid chromatography-mass spectrometry of scrambled VKIVA PA. (a-c) Analytical high-performance liquid chromatography (analytical HPLC) traces of purified (a) C16-V2A2E4-GVKIVA, (b) C16A2G2E4GVKIVA, (c) C16VEVA2E4GVKIVA. (d-f) Electrospray ionization mass spectrometry (ESI-MS) of (d) C16-V2A2E4-GIKVAV, (e) C16A2G2E4GVKIVA, and (f) C16VEVA2E4GVKIVA. [Figure 14]Figure 14A-H. Simulation results of scrambled VKIVA-PA. (a-f) Self-assembled structures and coarse-grained representations of single fibers of (A,b) V2A2 (red), (c,d) A2G2 (blue), and (e,f) VEVA2 (orange) VKIVA after 10 μs. Color code is displayed in the PA array. Micrographs include partial periodic images through the fiber forming axis. Simulation boxes are displayed in blue. Water and ions are omitted for clarity. (b,d,f) Fibers are displayed all transparent except for the C16 tail to show their patchiness. The A2G2 equilibrated structure differs from the typical fiber structure. (g) Water contact graphs of IKVAV and VKIVA-PA. (h) C16 cluster analysis of scrambled VKIVA and IKVAV PA. Quantification of the number of C16 clusters has been used as a qualitative measure of disorder. [Figure 15] Figure 15A-F. Cryo-TEM micrographs of scrambled VKIVA-PA. (a-c) Schematic molecular structures and (d-f) Cryo-TEM micrographs of the scrambled IKVAV sequence (a, d) C16-V2A2E4GVKIVA, (b, e) C16A2G2E4GVKIVA, and (c, f) C16VEVA2E4GVKIVA. Scale bar: 500 nm. [Figure 16] Figure 16A-F. Liquid chromatography-mass spectrometry of skeletal PA. (a-c) Analytical high-performance liquid chromatography (analytical HPLC) traces of purified (a) C16V2A2E4, (b) C16A2G2E4, and (c) C16VEVA2E4. (d-f) Electrospray ionization mass spectrometry (ESI-MS) of (d) C16V2A2E4, (e) C16A2G2E4, and (f) C16VEVA2E4. [Figure 17] Figure 17A-F. Cryo-TEM micrographs of scaffold-PA. (a-c) Schematic molecular structures and (d-f) Cryo-TEM micrographs of scaffold sequences (a, d) C16V2A2E4, (b, e) C16A2G2E4, and (c, f) C16VEVA2E4. [Figure 18]FIG. 18A-K. Analysis of other laminin mimetic sequences. (a, c, e, g, i) Cryo-TEM micrographs and (b, d, f, h, j) self-assembled structure micrographs of C16A2G2E4G fibers with bioactive epitopes (a, b) LGTIPG, (c, d) LRGDN, (e, f) PDGSR, (g, h) RGD, (i, j) YIGSR sequences after 10 μs. The simulated micrographs contain partial periodic images through the fiber forming axis. The simulation boxes are shown in blue. Water and ions are omitted for clarity. (k) Water contact analysis for LGTIPG, LRGDN, PDGSR, RGD, YIGSR sequences. IKVAV was used as a reference. [Figure 19] Figure 19A-I. Short-term analysis (72 h) of hiPSC-derived MNs on IKVAV PA matrices. (a) Schematic of the experimental paradigm. (b) Spectral illumination micrographs of MNs labeled with TUJ-1 (red) on A2G2 IKVAV PA covalently linked to alexa-488 dye (green) after 72 h. (c) Quantification of cell numbers / mm2 under various conditions at 72 h. (d, e) Representative confocal micrographs of MNs cultured on IKVAV PA supramolecular fibers and laminin (white) coatings containing V2A2 (red), A2G2 (blue), VEVA2 (orange). (d, e) Cells were consistently stained with neuronal (TUJ-1 and MAP-2, red) and motor neuron (ISL1 / 2 and ChAT, green) markers. Nuclear DNA was stained with DAPI, blue. (f-h) Quantification of (f) ISL1 / 2+ and (g) ChAT+ (h) TUJ-1+ MN percentages cultured on IKVAV PA supramolecular fibers containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) coatings at 72 h. (i) Cell viability was assessed by LDH assay at 72 h. Scale bar: 50 μm. Data are the average of at least four independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001), ns: not significant. [Figure 20]FIG. 20A-I. Short-term analysis of FOXA2 populations on IKVAV PA matrices. (a, b) Representative confocal micrographs of MNs cultured on (a) A2G2 IKVAV PA and (b) laminin matrices at 72 hours. Cells were stained with the floor plate cell marker FOXA-2 (green), neuronal marker TUJ-1 (red) and nuclear marker DAPI (blue). (c) Western blot and (d) normalized levels of protein expression associated with the floor plate marker FOXA-2 and proliferation marker PH3 in MNs cultured on IKVAV PA supramolecular fibers containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) at 72 hours. (e, f) Representative confocal micrographs of MNs cultured on (e) A2G2 IKVAV PA and (f) laminin matrices at 72 hours. Cells were stained with floor plate marker FOXA-2 (green), neuronal marker TUJ-1 (white), proliferation marker KI67 (red) and nuclear marker DAPI (blue). (g-i) Quantification of (g) FOXA2+, (h) KI67+ and (i) FOXA2+ / KI67+ MN percentages cultured on IKVAV PA supramolecular fiber and laminin (black) matrix containing V2A2 (red), A2G2 (blue), VEVA2 (orange) at 72 h. All values ​​were normalized to actin. Scale bar: 25 μm. Data are the average of at least three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001). [Figure 21]Figure 21A-G. ITGB1 expression in human MNs cultured on IKVAV PA. (a-d) Representative three-dimensional confocal micrographs of ITGB1 receptor expression in neurites of MNs (labeled with TUJ-1 in green) cultured on IKVAV PA supramolecular fibers containing V2A2, A2G2, VEVA2 and laminin coatings at 72 h. (f, g) Three-dimensional shadow reconstructions of confocal imaging conditions referenced in c and d. (e) Intensity analysis of ITGB1 in MNs cultured on V2A2 (red) and VEVA2 (orange) matrices at 72 h. Scale bar: 2 μm. [Figure 22] Figure 22A-F. Effect of αITGB1 blocking antibodies on IKVAV PA attachment and viability of MNs. (a) Schematic of hiPSC-derived MN differentiation and subsequent culture on supramolecular matrix in the presence of αβ-1 or β-4 ITG antibodies for 72 h. (b) Cell viability of MNs treated with ITGB1 assessed by LDH levels released in the cell medium at 72 h. (c, d) Representative confocal micrographs of MNs treated with (c) β-1 integrin or (d) β-4 integrin blocking antibodies cultured on IKVAV PA supramolecular fibers containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) matrix at 72 h. Cells were stained with neuronal marker TUJ-1 (red), motor neuron marker ISL1 / 2 (green) and nuclei were stained with DAPI (blue). (e, f) Quantification of cell number / mm2 for conditions referred to in c and d, respectively. Scale bar: 50 μm. Data are the average of three independent differentiations. All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001), ns: not significant. [Figure 23]Figure 23A-F. Effect of mechanical properties on laminin-associated signaling. (a) Schematic diagram of hiPSC-derived MN differentiation and its culture on different supramolecular matrix thicknesses. (b) Elastic modulus (gray and white graphs) and storage modulus (dark grey graphs) of thin coating and thick gel of supramolecular IKVAV matrix containing V2A2, A2G2 and VEVA2. (c,e) Western blot analysis and (d,f) normalized expression levels of ITGB1 receptor activation and signaling pathways (ILK, p-FAK, FAK) in human MNs cultured on (c,d) thick coating or (e,f) thin coating of supramolecular IKVAV matrix containing V2A2, A2G2, VEVA2 at 72 h. Laminin coating was used as a control. Data are the average of at least three independent experiments or differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001). [Figure 24] Figure 24A-I. Profilometry of IKVAV PA and laminin coating. (a-h) Representative images of IKVAV PA containing (a,b) V2A2, (c,d) A2G2, (e,f) VEVA2 and (g,h) laminin coating after 3 days (top images) and 60 days (bottom images) in vitro. (i) Thickness analysis of IKVAV PA and laminin coating referenced in a-h. Data are the average of four independent experiments. All values ​​are presented as mean ± SD; ANOVA (***P<0.001). [Diagram 25] Figure 25A-L. FM characterization of thin and thick IKVAV PA gels. (A-c) AFM topography images of thin coatings of IKVAV PA containing V2A2, (b) A2G2, and (c) VEVA2; (d-f) elastic modulus distributions of thin coatings from a-c; (g-i) optical microscopy images of thick gels of IKVAV PA indentation experiments containing V2A2, A2G2, and VEVA2; (j-l) elastic modulus distributions of thick gels for the conditions referenced in g-i. [Figure 26]Figure 26A-F. Rheological measurements of IKVAV PA gels. (a-c) Strain sweeps showing storage modulus G' and loss modulus G'' in the shear strain range of 0-100 for supramolecular IKVAV PA containing (a) V2A2, (b) A2G2, and (c) VEVA2. (d-f) Frequency sweeps showing storage modulus G' for angular frequency range of 0-100 rad / s for the conditions referenced in a-c. Data are the average of n=3 gels. All values ​​are presented as mean ± SD. [Figure 27] Figure 27A-C. Time course of human MNs cultured on IKVAV PA matrix. Representative confocal micrographs of MNs cultured on IKVAV PA supramolecular fiber and laminin (black) matrix containing V2A2, A2G2, VEVA2 at (a) 30 days, (b) 45 days, and (c) 60 days in vitro. Cells were stained with the neuronal marker MAP-2 (red), the motor neuron marker ChAT (green), and nuclei were stained with DAPI (blue). Scale bar: 100 μm. [Figure 28] FIG. 28A-F. Long-term culture of human MNs on IKVAV PA matrix. (a, c, e) Representative brightfield images and (b, d, f) confocal micrographs of MNs cultured on IKVAV PA supramolecular fiber and laminin matrix containing V2A2, A2G2, VEVA2 at (a, b) 30 days, (c, d) 45 days, and (e, f) 60 days in vitro. Cells were stained with the neuronal marker MAP-2 (red), the motor neuron marker ChAT (green), and nuclei were stained with DAPI (blue). Scale bar: 25 μm. Data are the average of six independent differentiations. [Figure 29]FIG. 29A-D. Characterization of human MNs on IKVAV PA at different time points. (a, c) Western blot analysis and (b, d) normalized protein levels of (a, b) ITGB1 receptor activation, motor neuron markers ChAT and ISL1 / 2 and (ILK-1), and (c, d) neuronal markers MAP-2 and TUJ-1 in human MNs cultured on supramolecular IKVAV matrix containing V2A2 (red), A2G2 (blue), VEVA2 (orange). Data are the average of at least three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001), ns: not significant. [Diagram 30] Figure 30A-C. Differential ITGB1 expression in human MNs cultured on A2G2 IKVAV PA and laminin. (a, b) Representative three-dimensional SIM micrographs of MN neurites (TUJ-1, green) and ITGB1 receptor expression (pink) cultured on (a) A2G2 and (b) laminin matrices at 60 days. (c) Intensity analysis of ITGB1 in MNs cultured on A2G2 (blue) and laminin matrices at 60 days in vitro. Scale bar: 10 μm. [Diagram 31]Figure 31A-C. Assessment of MN maturation by gene ontology (GO) analysis of differentially expressed proteins in MNs cultured on A2G2 IKVAV PA and laminin-coated surfaces. Tandem mass spectrometry revealed differentially expressed proteins between MNs plated on laminin-coated surfaces, which are associated with GO terms directly related to human MN maturation (Ho et al., 2016). (a) From the differentially expressed proteins in the A2G2 condition, we identified 21 of 58 (36%) GO terms related to human MN maturation. MN maturation-related terms were also identified from the list of upregulated (b) and downregulated (c) proteins in cultures plated on A2G2 IKVAV PA versus laminin-coated surfaces. Bar graphs represent p-values ​​of MN maturation-related GO terms identified in the protein dataset. Pie charts represent the percentage of GO terms obtained in this study compared to previously described GO terms related to MN maturation ( Ho et al., 2016 ). [Diagram 32] Figure 32A-C. Morphological analysis of human motor neurons (MNs) on IKVAV PA matrices. (a, b) Representative confocal micrographs of single human MNs cultured on IKVAV PA supramolecular fiber and laminin matrices containing V2A2, A2G2, VEVA2 at 60 days in vitro. Cells were stained with (a) the neuronal marker MAP-2 (red). (b, c) Analysis of (b) soma size and (c) number of primary neurites of MN cultures at 60 days. Scale bar: 25 μm. Data are the average of at least three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001). [Diagram 33]Figure 33A-D. IKVAV PA coating after 60 days in vitro. (a, b) Representative structured illumination microscopy (SIM) micrographs of human MNs cultured on IKVAV PA containing V2A2, A2G2, and VEVA2. MNs were stained with TUJ-1 (red) and IKVAV PA was covalently linked with alexa-488 dye (green). (c, d) Scanning electron micrographs showing neurites of MNs cultured on IKVAV PA containing A2G2. Scale bars: (c) 5 μm, (d) 2 μm. [Diagram 34] Figure 34A-C. Synthesis and liquid chromatography-mass spectrometry of IKVAV peptide for modification of glass coverslips. (a) Schematic molecular structure of IKVAV peptide. (b) Analytical high-performance liquid chromatography (analytical HPLC) trace of purified IKVAV peptide. (c) Electrospray ionization mass spectrometry (ESI-MS) of IKVAV peptide. [Diagram 35] Figure 35A-E. Effect of immobilized IKVAV on MN attachment. (a) Schematic of immobilized IKVAV peptide on coverslip surface. (b) Bright field images. (c, d) Representative confocal micrographs of MNs cultured on APTES and IKVAV peptide coating. Cells were stained with neuronal markers (c) ISL1 / 2 (green) and TUJ-1 (red) and (d) ChAT (green) and MAP-2 (red). Nuclei were stained with DAPI (blue). (e) Quantification of cell number per mm2 cultured on APTES and immobilized peptide (IKVAV). IKVAV PA containing V2A2, A2G2, VEVA2 and laminin matrix were used as controls. Scale bar: 100 μm. Data are the average of three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001). [Diagram 36]FIG. 36A-C. Human MN attachment on scrambled VKIVA PA. (a) Representative bright field and (b) confocal images of MNs cultured on scrambled VKIVA PA arrays containing V2A2, A2G2, and VEVA2. Cells were stained with ChAT, MAP-2, and DAPI in b. (c) Quantification of cell numbers per mm2 culture for conditions referenced in a, b. IKVAV PA containing V2A2, A2G2, VEVA2 and laminin matrix were used as controls. Scale bar: 100 μm. Data are the average of three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01, and ***P<0.001). [Figure 37]Figure 37A-O. Effects of IKVAV PA on other human iPSC-derived motor neurons (11a cell line). (a, b) Representative micrographs of MN-11a cultured for 72 hours on V2A2, A2G2, VEVA2 IKVAV PA supramolecular fibers and laminin matrix. Cells were stained with neuronal and motor neuron markers, TUJ-1 (red) and ISL1 / 2 (green) in (a); MAP-2 (red) and ChAT (green), respectively. Nuclei were stained with DAPI (blue). Quantification of cell number per mm2 at 72 hours (c) cultured on IKVAV PA supramolecular fibers containing V2A2, A2G2, VEVA2 and laminin coating. (c-f) Quantification of (c) TUJ-1+, (d) ISL1 / 2+ (f) ChAT+ MN percentages. (g) Representative confocal micrographs of human MN11a cultured on IKVAV PA containing V2A2, A2G2, VEVA2 and laminin matrix at 60 days. Cells were stained with MN markers ChAT (green), microtubule associated protein-2 (MAP-2, red) and nuclei (DAPI). (h) DAPI channel micrograph of the image shown in g. (i) Histogram analysis of cell distribution on the different matrices referenced in g and h. (j) Quantification of cell number per mm2 at 60 days and (k) quantification of ChAT+MN percentage cultured on IKVAV PA supramolecular fibers containing V2A2 (red), A2G2 (blue), VEVA2 (orange) and laminin (black) coating at 60 days. (l-m) Scanning electron microscope (SEM) micrographs of MN cultured on IKVAV PA containing (l) A2G2 and (m) laminin. (n) Western blots and (o) normalized protein levels of ITGB1 receptor activation, neuronal marker MAP-2 and motor neuron marker ChAT. All values ​​were normalized to actin. Data are the average of three independent differentiations: Scale bars (a,b) 50 μm, (g,h) 100 μm, (l,m) 5 μm. Data are the average of three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001). [Figure 38]Figure 38A-L. Effects of IKVAV PA on human iPSC-derived cortical neurons (CxNs). (a) Schematic of iPSC-derived CxN differentiation and its culture on supramolecular matrix. (b-d) Representative micrographs of CxNs cultured on V2A2 (red), A2G2 (blue), VEVA2 (orange) IKVAV PA supramolecular fibers and laminin (white) matrix at (b) 72 h and (c, d) 60 days. Cells were stained with neuronal markers TUJ-1 (red), MAP-2 (green) and NEUN (red), as well as cortical marker SATB-2 (green). Nuclei were stained with DAPI (blue). (e) Quantification of cell number per mm2 at 72 h. (f) Cell viability assessed by release of LDH in cell medium at 60 days. (g-j) Quantification of the percentage of (g) TUJ-1+, (h) MAP2+, (i) NEUN+ and (j) SATB-2+ neurons cultured on IKVAV PA supramolecular fibers containing V2A2, A2G2, VEVA2 and laminin (black) coatings at 60 days. (k) Western blot and (l) normalized protein levels of ITGB1 receptor activation, neuronal markers MAP-2, TUJ-1 and NEUN, and presynaptic marker SYP. All values ​​were normalized to actin. Data are the average of three independent differentiations. Scale bars: (b) 100 μm, (c, d) 50 μm. Data are the average of three independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001). [Figure 39]FIG. 39A-G. Expression of functional markers in human motor neurons (MNs) cultured on V2A2 and VEVA2 IKVAV PA. (a, b) Representative confocal micrographs of synaptic vesicles (SYN-1, presynaptic, and PS95, postsynaptic) distributed along the neurites of MNs cultured on IKVAV and (b) laminin matrices containing A2G2 after 40 days in vitro. (c, d) Mean intensity analysis of (c) PSD95 and (d) SYN-1 in MNs cultured on V2A2 and VEVA2. (f) Western blot analysis and (g) normalized protein levels of synaptic markers PSD95, SYN-1, and SYP, as well as neuronal marker DCX, in human MNs cultured on supramolecular IKVAV matrices containing V2A2, A2G2, VEVA2 and laminin coatings. Scale bar: data are the average of at least three independent differentiations. All values ​​are presented as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001). [Diagram 40] FIG. 40A-F. Electrophysiological studies of human MNs cultured on IKVAV PA. (a) Schematic of multi-electrode array plate (MEA plate) for recording neuronal cultures. (b, c) Schematic of neurons (green) plated on (b) IKVAV PA and (c) laminin-coated MEA plate. (d) Bright field image of MNs cultured on IKVAV PA containing V2A2, A2G2, VEVA2 and laminin-coated MEA plate. (e) Weighted average firing rate and (f) number of burst electrodes in the same conditions as referenced in d. Scale bar: 100 μm. Data are the average of two independent differentiations. All values ​​are shown as mean ± SD; ANOVA (*P<0.05, **P<0.01 and ***P<0.001). [Diagram 41] Figure 41A-C. Spinal cord injury study in mice. (a) Schematic of spinal cord injury at T10 / 11 in mouse model. (b) Impact force graphs of animals treated with V2A2, A2G2, V2A2E2 and saline solution (control). (c) Basso Mouse scale (BMS) for the conditions described in (b).

Claims

1. Hydrophobic tail, A structural peptide segment having an overall propensity for beta-sheet structure formation of 4 or less; a charged peptide segment, and Bioactive peptides A peptide amphiphile comprising:

2. The hydrophobic tail is an alkyl chain having 8 to 24 carbon atoms (C ８－２４ 2. The peptide amphiphile of claim 1, comprising:

3. The hydrophobic tail is an alkyl chain having 16 carbon atoms (C １６ 3. The peptide amphiphile of claim 2, comprising:

4. A peptide amphiphile according to any one of claims 1 to 3, wherein said structural peptide segments have an overall propensity for forming β-sheet structure of 3.5 or less.

5. 5. The peptide amphiphile of claim 4, wherein the structural peptide segments have an overall propensity for beta-sheet structure formation of 3 or less.

6. The structural peptide segment is A ２ G ２ The peptide amphiphile according to any one of claims 1 to 5, comprising:

7. The peptide amphiphile of any one of claims 1 to 6, wherein the charged peptide segment comprises an acidic, basic, or zwitterionic peptide segment.

8. The charged peptide segment is ２－４ The peptide amphiphile according to any one of claims 1 to 7, comprising:

9. The peptide amphiphile of any one of claims 1 to 8, wherein the biologically active peptide comprises IKVAV.

10. The peptide amphiphile of any one of claims 1 to 9, wherein the bioactive peptide is attached to the charged peptide segment by a linker.

11. The peptide amphiphile of claim 10 , wherein the linker is a single glycine (G) residue.

12. The peptide amphiphile is ８‐２４ -A ２ G ２ E ４ A peptide amphiphile according to any one of claims 1 to 11, comprising (G) IKVAV.

13. A nanofiber comprising a peptide amphiphile according to any one of claims 1 to 12.

14. further comprising one or more filler peptide amphiphiles; 14. The nanofiber of claim 13, wherein the filler peptide amphiphile does not include a bioactive moiety.

15. 15. The nanofiber of claim 14, wherein the filler peptide amphiphile comprises a hydrophobic non-peptide tail, a structural peptide segment, and a charged peptide segment.

16. A pharmaceutical composition comprising a peptide amphiphile according to any one of claims 1 to 12 or a nanofiber according to any one of claims 13 to 15.

17. 17. The pharmaceutical composition of claim 16 for use in treating nervous system injury in a subject.

18. 18. The pharmaceutical composition of claim 17, wherein the nervous system injury is a central nervous system injury.

19. The pharmaceutical composition of claim 17, wherein the nervous system injury is a spinal cord injury.

20. The pharmaceutical composition of any one of claims 17 to 19, wherein the composition is formulated for parenteral administration.

21. 1. A pharmaceutical composition for use in treating nervous system injury in a subject, comprising: comprising self-assembled peptide amphiphile nanofibers; at least a portion of the peptide amphiphile comprises a hydrophobic tail, a structural peptide segment, a charged peptide segment, and an IKVAV bioactive peptide; A pharmaceutical composition wherein said structural peptide segments have an overall propensity for beta-sheet structure formation of 4 or less.

22. The hydrophobic tail is an alkyl chain having 8 to 24 carbon atoms (C ８－２４ 22. The pharmaceutical composition of claim 21 , comprising:

23. The hydrophobic tail is an alkyl chain having 16 carbon atoms (C １６ 23. The pharmaceutical composition of claim 22, comprising

24. The pharmaceutical composition according to any one of claims 21 to 23, wherein said structural peptide segments have an overall propensity for forming β-sheet structure of 3.5 or less.

25. 25. The pharmaceutical composition of any one of claims 21 to 24, wherein said structural peptide segments have an overall propensity for beta-sheet structure formation of 3 or less.

26. The pharmaceutical composition of any one of claims 21 to 25, wherein the charged peptide segment comprises an acidic, basic, or zwitterionic peptide segment.

27. The charged peptide segment is ２－４ The pharmaceutical composition according to any one of claims 21 to 26, comprising:

28. The pharmaceutical composition of any one of claims 21 to 27, wherein the structural peptide segment comprises AAGG.

29. The peptide amphiphile is ８‐２４ -A ２ G ２ E ４ The pharmaceutical composition of claim 28, comprising (G) IKVAV.

30. The pharmaceutical composition according to any one of claims 21 to 29, wherein the nervous system injury is a central nervous system injury.

31. 31. The pharmaceutical composition of claim 30, wherein the nervous system injury is a spinal cord injury.

32. The pharmaceutical composition according to any one of claims 21 to 31, wherein the pharmaceutical composition is formulated for parenteral administration.

33. comprising self-assembled peptide amphiphile nanofibers; at least a portion of said peptide amphiphiles comprises a hydrophobic tail, a structural peptide segment, a charged peptide segment, and an IKVAV bioactive peptide, said structural peptide segment having an overall propensity for beta-sheet structure formation of 4 or less; There is a scaffold.

34. The hydrophobic tail is an alkyl chain having 8 to 24 carbon atoms (C ８－２４ 34. The scaffold of claim 33, comprising:

35. The hydrophobic tail is an alkyl chain having 16 carbon atoms (C １６ 34. The scaffold of claim 33, comprising:

36. 36. The scaffold of any one of claims 33 to 35, wherein said structural peptide segments have an overall propensity for beta-sheet structure formation of 3.5 or less.

37. 37. The scaffold of any one of claims 33 to 36, wherein said structural peptide segments have an overall propensity for beta-sheet structure formation of 3 or less.

38. 38. The scaffold of any one of claims 33 to 37, wherein the charged peptide segments comprise acidic, basic, or zwitterionic peptide segments.

39. The charged peptide segment is ２－４ The scaffold of any one of claims 33 to 38, comprising:

40. 40. The scaffold of any one of claims 33 to 39, wherein the structural peptide segment comprises AAGG.

41. The peptide amphiphile is ８‐２４ -A ２ G ２ E ４ 41. The scaffold of claim 40, comprising: (G) an IKVAV.

42. A cell cultured on the scaffold according to any one of claims 33 to 41.

43. 43. The cell of claim 42, wherein the cell is a neuron.

44. The cell of claim 42, wherein the cell is a hiPSC-derived motor neuron.

45. A method of culturing cells, comprising contacting the cells with the scaffold of any one of claims 33 to 41.

46. 46. ​​The method of claim 45, wherein the cell is a neuron.

47. 47. The method of claim 46, wherein the cell is a hiPSC-derived motor neuron.

48. A scaffold according to any one of claims 33 to 41; Cells cultured on the scaffold; A system that includes:

49. 49. The system of claim 48, wherein the cell is a neuron.

50. The system of claim 49, wherein the cell is a hiPSC-derived motor neuron.

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