Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1825—Fibroblast growth factor [FGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1858—Platelet-derived growth factor [PDGF]
  • A61K38/1866—Vascular endothelial growth factor [VEGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/50—Fibroblast growth factor [FGF]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• peptide amphiphiles PAs
• supramolecular assemblies comprising PAs
• compositions comprising PAs
• methods of use thereof provided herein are supramolecular assemblies comprising an IKVAV PA and a growth factor mimetic PA.
• the PAs, compositions, and supramolecular assemblies described herein are used in methods of treating nervous system injury. BACKGROUND The development of therapies to avoid permanent paralysis in humans after traumatic injuries remains a major challenge given the inability of damaged axons to regenerate in the adult central nervous system (CNS).
• CNS central nervous system
• a supramolecular assembly comprising at least two peptide amphiphiles.
• the at least two peptide amphiphiles comprise at least one IKVAV peptide amphiphile comprising a hydrophobic segment, a structural peptide segment, a charged peptide segment, and a bioactive peptide comprising the amino acid sequence IKVAV; and at least one growth factor mimetic peptide amphiphile.
• the at least one IKVAV peptide amphiphile comprises a fluorescence anisotropy value of less than 0.3.
• the at least one IKVAV peptide amphiphile comprises a proton relaxation rate ( 1 H-R 2) of less than 4s -1 .
• the hydrophobic segment comprises an 8-24 carbon alkyl chain (C8-24). In some embodiments, the hydrophobic segment comprises a 16 carbon alkyl chain (C 16 ). In some embodiments, the structural peptide segment comprises A 2 G 2 . In some embodiments, the charged peptide segment comprises E2, E3, or E4. In some embodiments, the bioactive peptide is attached to the charged peptide segment by a linker. In some embodiments, the linker is a single glycine (G) residue. In some embodiments, the IKVAV peptide amphiphile comprises C 16 A 2 G 2 E 4 GIKVAV.
• the at least one growth factor mimetic peptide amphiphile comprises a hydrophobic segment comprising an 8-24 carbon alkyl chain (C 8-24 ), a structural peptide segment comprising V 2 A 2 , A 2 G 2 , a charged peptide segment comprising E 2 , E 3 , or E 4 , and a growth factor mimetic peptide sequence.
• the growth factor mimetic sequence is a vascular endothelial growth factor (VEGF) mimetic sequence, a fibroblast growth factor 2 (FGF-2) mimetic sequence, a Glial cell-derived neurotrophic factor (GDNF) mimetic sequence, a brain-derived neurotrophic factor (BDNF) mimetic sequence, or a Netrin-1 mimetic sequence.
• VEGF vascular endothelial growth factor
• FGF-2 fibroblast growth factor 2
• GDNF Glial cell-derived neurotrophic factor
• BDNF brain-derived neurotrophic factor
• Netrin-1 mimetic sequence vascular endothelial growth factor
• the growth factor mimetic sequence is an FGF-2 mimetic sequence.
• the FGF-2 mimetic sequence comprises YRSRKYSSWYVALKR (SEQ ID NO: 2).
• the growth factor mimetic peptide is attached to the charged peptide segment by a linker.
• the linker is a single glycine (G) residue.
• the at least one growth factor mimetic peptide amphiphile comprises C16V2A2E4GYRSRKYSSWYVALKR or C16A2G2E4GYRSRKYSSWYVALKR.
• the at least one IKVAV peptide amphiphile comprises C 16 A 2 G 2 E 4 GIKVAV and the at least one growth factor mimetic peptide amphiphile comprises C 16 V 2 A 2 E 4 GYRSRKYSSWYVALKR or C 16 A 2 G 2 E 4 GYRSRKYSSWYVALKR.
• the at least one IKVAV peptide amphiphile comprises C16A2G2E4GIKVAV and the at least one growth factor mimetic peptide amphiphile comprises C 16 V 2 A 2 E 4 GYRSRKYSSWYVALKR.
• compositions comprising a supramolecular assembly as described herein.
• composition comprising at least one IKVAV peptide amphiphile comprising a hydrophobic segment, a structural peptide segment, a charged peptide segment, and a bioactive peptide comprising the amino acid sequence IKVAV; and at least one growth factor mimetic peptide amphiphile.
• the at least one IKVAV peptide amphiphile and the at least one growth factor mimetic peptide amphiphile interact to form a supramolecular assembly within the composition.
• the at least one IKVAV peptide amphiphile comprises a fluorescence anisotropy value of less than 0.3.
• the at least one IKVAV peptide amphiphile comprises a proton relaxation rate ( 1 H-R 2) of less than 4s -1 .
• the hydrophobic segment comprises an 8-24 carbon alkyl chain (C8-24).
• the hydrophobic segment comprises a 16 carbon alkyl chain (C 16 ).
• the structural peptide segment comprises A 2 G 2 .
• the charged peptide segment comprises E2, E3, or E4.
• the bioactive peptide is attached to the charged peptide segment by a linker.
• the linker is a single glycine (G) residue.
• the IKVAV peptide amphiphile comprises C 16 A 2 G 2 E 4 GIKVAV.
• the at least one growth factor mimetic peptide amphiphile comprises a hydrophobic segment comprising an 8-24 carbon alkyl chain (C 8-24 ), a structural peptide segment comprising V 2 A 2 or A 2 G 2 , a charged peptide segment comprising E 2 , E 3 , or E 4 , and a growth factor mimetic peptide sequence.
• the growth factor mimetic sequence is a vascular endothelial growth factor (VEGF) mimetic sequence, a fibroblast growth factor 2 (FGF-2) mimetic sequence, a Glial cell-derived neurotrophic factor (GDNF) mimetic sequence, a brain-derived neurotrophic factor (BDNF) mimetic sequence, or a Netrin-1 mimetic sequence.
• VEGF vascular endothelial growth factor
• FGF-2 fibroblast growth factor 2
• GDNF Glial cell-derived neurotrophic factor
• BDNF brain-derived neurotrophic factor
• Netrin-1 mimetic sequence vascular endothelial growth factor
• the growth factor mimetic sequence is an FGF-2 mimetic sequence.
• the FGF-2 mimetic sequence comprises YRSRKYSSWYVALKR (SEQ ID NO: 2).
• the growth factor mimetic peptide is attached to the charged peptide segment by a linker.
• the linker is a single glycine (G) residue.
• the at least one growth factor mimetic peptide amphiphile comprises C 16 V 2 A 2 E 4 GYRSRKYSSWYVALKR or C 16 A 2 G 2 E 4 GYRSRKYSSWYVALKR.
• the at least one IKVAV peptide amphiphile comprises C 16 A 2 G 2 E 4 GIKVAV and the at least one growth factor mimetic peptide amphiphile comprises C 16 V 2 A 2 E 4 GYRSRKYSSWYVALKR or C 16 A 2 G 2 E 4 GYRSRKYSSWYVALKR.
• the at least one IKVAV peptide amphiphile comprises C16A2G2E4GIKVAV and the at least one growth factor mimetic peptide amphiphile comprises C 16 V 2 A 2 E 4 GYRSRKYSSWYVALKR.
• the compositions described herein may be used in a method of treating a nervous system injury in a subject.
• the nervous system injury is a central nervous system injury.
• the central nervous system injury is a spinal cord injury.
• provided herein are methods of treating a nervous system in jury in a subject.
• FIG.1A-1E shows a library of investigated IKVAV PA molecules.
• A Specific chemical structures of IKVAV PA molecules used and molecular graphics representation of a supramolecular nanofiber displaying the IKVAV bioactive signal.
• FIG.2A-2J shows the effect of supramolecular motion on hNPCs signaling in vitro.
• A Molecular graphics representation of an IKVAV PA nanofiber indicating the chemical structure and location of DPH used as a probe in fluorescence depolarization measurements (top); bar graph of fluorescence anisotropy of IKVAV PA solutions (error bars correspond to 3 independent experiments; n.s. no significant, *** P ⁇ 0.0001, one-way ANOVA with Bonferroni).
• D Representative micrographs of hNPCs treated with IKVAV PA1, PA2, PA4 and PA5; NESTIN-stem cells (red), ITGB1-receptor (green), and DAPI-nuclei (blue).
• E WB results of ITGB1, p-FAK, FAK, ILK, and TUJ-1 in hNPCs treated with laminin and the various IKVAV PAs.
• F Representative confocal micrographs of hNPCs treated with IKVAV PA1, PA2, PA4 and PA5; NESTIN-stem cells (red), SOX-2-stem cells (green), TUJ-1-neurons (white), and DAPI-nuclei (blue).
• FIG.3A-3L shows that two chemically different PA scaffolds with two identical bioactive sequences reveal differences in growth of corticospinal axons after SCI.
• A Chemical structures of the two PA molecules used.
• B Molecular graphics representation of a supramolecular nanofiber displaying two bioactive signals (top); cryo-TEM micrographs of IKVAV PA2 co-assembled with FGF2 PAs (FGF2 PA1 and FGF2 PA2) (bottom).
• C Storage modulus of IKVAV PA2 (green) and their respective co-assemblies with FGF2 PAs (FGF2 PA1, red and FGF2 PA2, blue).
• D Fluorescent micrographs of spinal cords (green) injected with IKVAV PA2+FGF2 PA1 (red) covalently labeled with Alexa 647.
• E Dot plot of PA scaffold volume as a function of time after implantation.
• FIG. F Schematic illustration showing the site of BDA and PA injections (left); fluorescent micrographs of the brain cortex (top, right); NeuN- neurons (green), BDA-labelled neurons (red) and DAPI-nuclei (blue) and transverse spinal cord section stained for GFAP-astrocytes (green), BDA-labelled descending axons (red) and DAPI- nuclei (blue) (bottom, right).
• G Fluorescent micrographs of longitudinal spinal cord sections in sham, IKVAV PA2+FGF2 PA1, and IKVAV PA2+FGF2 PA2 groups; GFAP-astrocytes (green), BDA-labelled axons (red) and DAPI-nuclei (blue); vertical white dashed lines indicate the proximal border (PB), the distal border (DB), and the central part of the lesion (LC).
• H Representative magnified images for those in G.
• FIG.4A-4D show that two chemically different PA scaffolds with two identical bioactive sequences reveal differences in angiogenesis.
• A Fluorescent micrographs of transverse spinal cord sections in uninjured, IKVAV PA2+FGF2 PA1, IKVAV PA2+FGF2 PA2 and sham groups; GFAP-astrocytes (green), DiI-labelled blood vessels (red), and DAPI-nuclei (blue).
• A Fluorescent micrographs of transverse spinal cord sections corresponding to uninjured, IKVAV PA2+FGF2 PA1, IKVAV PA2+FGF2 PA2 and sham groups; NeuN-neurons (green), DiI-labelled blood vessels (red) and DAPI-nuclei (blue), dashed lines indicate the grey matter (horn).
• B High- magnification images of the ventral horn area for slices in A (left); NeuN-neurons (green), DiI- labelled blood vessels (red), and DAPI-nuclei (blue); ChAT-motor neurons (green), DiI-labelled blood vessels (red), and DAPI-nuclei (blue) (right).
• FIG.6A-6J show data validating cell signaling differences in vitro between two PA scaffolds exhibiting different supramolecular motion.
• A Confocal micrographs of HUVECs treated with IKVAV PA2+FGF2 PA1 and IKVAV PA2+FGF2 PA2; ACTIN-cytoskeleton (red), DAPI-nuclei (blue).
• B Bar graph of the number of branches in HUVECs treated with laminin+FGF-2, IKVAV PA2 alone, IKVAV PA2+FGF2 PA1, and IKVAV PA2+FGF2 PA2.
• C WB results (left) and bar graphs of the normalized values for active FGFR1 (p-FGFR1) vs total FGFR1 (FGFR1) and active ERK1/2 (p-ERK1/2) using the conditions in B (right).
• G 1 H-NMR spin-spin relaxation time of the aromatic protons in Y and W amino acids in the FGF2 mimetic signal at 6.81 ppm (solid lines are single linear best fits).
• H Bar graph of the aromatic relaxation times measured in G (error bars correspond to 3 runs per condition; * P ⁇ 0.05 student’s t-test).
• I Fluorescence anisotropy of FGF2 PAs chemically modified with Cy3 dye (error bars correspond to 3 independent experiments; *** P ⁇ 0.001 student’s t-test).
• FIG.7 shows the chemical structure (left) and mass spectra (right) of IKVAV PAs.
• FIG.8 shows plots representing the root mean square deviation (RMSD) vs. time of IKVAV PAs.
• FIG.9 shows bar graphs of the normalized values for ITGB1, p-FAK/FAK, ILK, and TUJ-1 in hNPCs cultured on ornithine coatings and treated with laminin and the library of IKVAV PAs (error bars correspond to 3 independent differentiations; ** P ⁇ 0.01, *** P ⁇ 0.001 vs. IKVAV PA2 and # P ⁇ 0.05, ## P ⁇ 0.01, ### P ⁇ 0.001 vs. IKVAV PA5, one-way ANOVA with Bonferroni).
• FIG.10A-10C shows the effect of calcium on supramolecular motion and in vitro cell signaling.
• A Anisotropy of IKVAV PA2 and IKVAV PA5 solutions in the absence (No Ca 2+ ) or presence (Ca 2+ ) of calcium (error bars correspond to 3 independent experiments; ** P ⁇ 0.01, *** P ⁇ 0.001, Student’s T-test).
• B Representative fluorescent micrographs of hNPCs cultured on the conditions mentioned in A.
• FIG.11A-11C show cryo-TEM images and storage modulus of IKVAV PA2 co- assembled with FGF2 PAs at different molar ratios.
• FIG. 12A-12F show the characterization of co-assembled IKVAV PA2+FGF2 PAs systems.
• FIG. 13A-13E show cleared spinal cords injected with dual signal co-assemblies.
• A Chemical structures and (B) mass spectra of the Alexa Fluor® 647-labeled IKVAV PA2.
• FIG. 14A-14D show the effect of IKVAV PAs on CST axon regrowth.
• A Fluorescent micrographs of longitudinal spinal cord sections in Backbone PA, IKVAV PA1, IKVAV PA2, and IKVAV PA4 groups; GFAP-astrocytes (green), BDA-labelled axons (red) and DAPI-nuclei (blue); vertical white dashed lines indicate the proximal border (PB), the distal border (DB), and the central part of the lesion (LC).
• B Representative magnified images for those in A.
• C Schematic lesion site and vertical lines used to count the number of axons crossing at each location indicated (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals per group; * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 vs sham, + P ⁇ 0.05, ++ P ⁇ 0.01 vs. IKVAV PA1 and # P ⁇ 0.05 vs. IKVAV PA4 groups, repeated measures of two-way ANOVA with Bonferroni).
• FIG. 15A-15F show the effect of IKVAV PA2 co-assembled with FGF2 PAs on axonal regrowth and glial scar formation.
• A Fluorescence images of longitudinal spinal cord sections of animals injected with saline solution (sham), IKVAV PA2+FGF2 PA1, IKVAV PA2+FGF2 PA2, and IKVAV PA2 alone; BDA-labelled descending axons (red) and DAPI-nuclei (blue); vertical white dashed lines indicate the proximal border (PB) and the distal border (DB).
• B Detailed images of BDA-labeled axons (red) in the center of the lesion for the conditions mentioned in A; vertical white dashed lines indicate the central part of the lesion (LC).
• C Representative images of longitudinal spinal cord sections stained for GFAP-astrocytes (green) and DAPI-nuclei (blue) within the lesion border in conditions mentioned in A.
• D WB results (bottom) and corresponding bar graph representing the normalized protein levels for GFAP using the conditions in A (top) (data points correspond to 4 animals per condition; *** P ⁇ 0.001 vs. sham, one-way ANOVA with Bonferroni).
• E Representative 3D fluorescence micrographs taken in the center of the lesion of BDA-labeled axon (red), GFAP-astrocytes (green), and DAPI-nuclei (blue).
• FIG. 16A-16E show the effect of IKVAV PA2 co-assembled with FGF2 PAs on serotoninergic neuronal regrowth.
• A Fluorescent micrographs of longitudinal spinal cord sections in sham, IKVAV PA2+FGF2 PA1, IKVAV PA2+FGF2 PA2 and IKVAV PA2 groups; Laminin-ECM (green), 5HT-serotoninergic neurons (red) and DAPI-nuclei (blue).
• B, C Representative magnified images of the (B) proximal border (PB) and (C) distal border (DB) for those in A; vertical white dashed lines indicate the PB and DB.
• FIG.17A-17E shows footprint analysis of animals injected with dual signal co-assemblies.
• A Representative photographs of mouse hindlimb positioning when walking after 3 months post- injury in Sham, IKVAV PA2+FGF2 PA1, IKVAV PA2+FGF2 PA2 groups.
• B Bar graph representing the impact force used to create the lesion in the spinal cords of animals treated with saline solution (Sham), IKVAV PA2+FGF2 PA1, IKVAV PA2+FGF2 PA2, IKVAV PA2 (data points show 38 animals analyzed; n.s. indicates not significant).
• C Representative footprints of animals injected with the various conditions plotted in B.
• peptide amphiphile is a reference to one or more peptide amphiphiles and equivalents thereof known to those skilled in the art, and so forth.
• the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc.
• the term “consisting of” and linguistic variations thereof denotes the presence of recited feature(s), element(s), method step(s), etc.
• amino acid refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomers, unless otherwise indicated, if their structures allow such stereoisomeric forms.
• Natural 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).
• Unnatural amino acids include, but are not limited to, azetidinecarboxylic 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-aminoisbutyric acid, 2-aminopimelic acid, tertiary-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 (“
• amino acid analog refers to a natural or unnatural amino acid where one or more of the C-terminal carboxy group, the N-terminal amino group and side-chain bioactive group has been chemically blocked, reversibly or irreversibly, or otherwise modified to another bioactive group.
• aspartic acid-(beta-methyl ester) is an amino acid analog of aspartic acid
• N-ethylglycine is an amino acid analog of glycine
• 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.
• peptide refers an oligomer to short polymer of amino acids linked together by peptide bonds.
• peptides are of about 50 amino acids or less in length.
• a peptide may comprise natural amino acids, non-natural amino acids, amino acid analogs, and/or modified amino acids.
• a peptide may be a subsequence of naturally occurring protein or a non-natural (artificial) sequence.
• artificial refers to compositions and systems that are designed or prepared by man, and are not naturally occurring.
• an artificial peptide, peptoid, or nucleic acid is one comprising a non-natural sequence (e.g., a peptide without 100% identity with a naturally-occurring protein or a fragment thereof).
• a “conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge.
• 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).
• Naturally occurring residues may be divided into classes based on common side chain properties, for example: polar positive (or basic) (histidine (H), lysine (K), and arginine (R)); polar negative (or acidic) (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); non-polar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine.
• a “semi-conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class.
• a conservative or semi-conservative amino acid substitution may also encompass non-naturally occurring amino acid residues that have similar chemical properties to the natural residue. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties.
• Embodiments herein may, in some embodiments, be limited to natural amino acids, non-natural amino acids, and/or amino acid analogs.
• Non-conservative substitutions may involve the exchange of a member of one class for a member from another class.
• sequence identity refers to the degree of which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits.
• sequence similarity refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) differ only by conservative and/or semi- conservative amino acid substitutions.
• the “percent sequence identity” 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 specified window, etc.), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity.
• a window of comparison e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.
• peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C.
• any gaps in aligned sequences are treated as mismatches at that position.
• Any polypeptides described herein as having a particular percent sequence identity or similarity (e.g., at least 70%) with a reference sequence ID number may also be expressed as having a maximum number of substitutions (or terminal deletions) with respect to that reference sequence.
• a sequence having at least Y% sequence identity (e.g., 90%) with SEQ ID NO:Z may have up to X substitutions (e.g., 10) relative to SEQ ID NO:Z, and may therefore also be expressed as “having X (e.g., 10) or fewer substitutions relative to SEQ ID NO:Z.”
• the term “nanofiber” refers to an elongated or threadlike filament (e.g., having a significantly greater length dimension that width or diameter) with a diameter typically less than 100 nanometers.
• the term “supramolecular” refers to the non-covalent interactions between molecules (e.g., polymers, macromolecules, etc.) and the multicomponent assemblies, complexes, systems, and/or fibers that form as a result.
• molecules e.g., polymers, macromolecules, etc.
• self-assemble and “self-assembly” refer to formation of a discrete, non-random, aggregate structure from component parts; said assembly occurring spontaneously through random movements of the components (e.g. molecules) due only to the inherent chemical or structural properties and attractive forces of those components.
• peptide amphiphile refers to a molecule that, at a minimum, includes a hydrophobic segment, a structural peptide segment and/or charged peptide segment (often both).
• a peptide amphiphile includes a bioactive peptide (e.g. an IKVAV peptide, a growth factor mimetic peptide).
• a peptide amphiphile includes a linker (e.g. G).
• the peptide amphiphile may express a net charge at physiological pH, either a net positive or negative net charge, or may be zwitterionic (i.e., carrying both positive and negative charges).
• Certain peptide amphiphiles consist of or comprise: (1) a hydrophobic, non-peptide segment (e.g., comprising an acyl group of six or more carbons), (2) a structural peptide segment; (3) a charged peptide segment, and (4) a bioactive peptide segment (e.g. IKVAV peptide, growth factor mimetic peptide).
• lipophilic moiety or “hydrophobic moiety” refers to the moiety (e.g., an acyl, ether, sulfonamide, or phosphodiester moiety) disposed on one terminus (e.g., C-terminus, N-terminus) of the peptide amphiphile, and may be herein and elsewhere referred to as the lipophilic or hydrophobic segment or component.
• the hydrophobic segment should be of a sufficient length to provide amphiphilic behavior and aggregate (or nanosphere or nanofiber) formation in water or another polar solvent system.
• a linear acyl chain is the lipophilic group (saturated or unsaturated carbons), palmitic acid.
• other lipophilic groups may be used in place of the acyl chain such as steroids, phospholipids and fluorocarbons.
• structural peptide or “structural peptide segment” refer to a portion of a peptide amphiphile, typically disposed between the hydrophobic segment and the charged peptide segment.
• the structural peptide is generally composed of three to ten amino acid residues with non-polar, uncharged side chains (e.g., His (H), Val (V), Ile (I), Leu (L), Ala (A), Phe (F)) selected for their propensity to form hydrogen bonds or other stabilizing interactions (e.g., hydrophobic interactions, van der Waals' interactions, etc.) with structural peptide segments of adjacent structural peptide segments.
• the structural peptide segment has a propensity to form ⁇ -helix and/or ⁇ -sheet secondary structures.
• assemblies of peptide amphiphiles having structural peptide segments display linear or 2D structure when examined by microscopy and/or ⁇ -helix and/or ⁇ -sheet character when examined by circular dichroism (CD).
• the structural peptide segment has a low propensity to for ⁇ -helix and/or ⁇ -sheet secondary structures.
• the structural peptide segment has a total propensity for forming ⁇ -sheet conformations of 4 or less.
• assemblies of peptide amphiphiles having structural peptide segments with a total propensity for forming ⁇ -sheet conformations of 4 or less display a less ordered character (e.g.
• nanofibers of peptide amphiphiles having structural peptide segments with a total propensity for forming ⁇ -sheet conformations of 4 or less display a propensity to form random coil structures.
• beta ( ⁇ )-sheet-forming peptide segment refers to a structural peptide segment that has a propensity to display ⁇ -sheet-like character (e.g., when analyzed by CD).
• amino acids in a beta ( ⁇ )-sheet-forming peptide segment are selected for their propensity to form a beta-sheet secondary structure.
• suitable amino acid residues selected from the twenty 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 their propensity to form beta sheets).
• suitable amino acid residues selected from the twenty 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 their propensity to form beta sheets).
• non-naturally occurring amino acids of similar beta-sheet forming propensity may also be used.
• Peptide segments capable of interacting to form beta sheets and/or with a propensity to form beta sheets are
• 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 residue that have a net positive or negative charge under physiologic conditions.
• a charged peptide segment may be acidic (e.g., negatively charged), basic (e.g., positively charged), or zwitterionic (e.g., having both acidic and basic residues).
• carboxy-rich peptide segment refers to a peptide sequence of a peptide amphiphile that comprises one or more amino acid residues that have side chains displaying carboxylic acid side chains (e.g., Glu (E), Asp (D), or non-natural amino acids).
• a carboxy-rich peptide segment may optionally contain one or more additional (e.g., non-acidic) amino acid residues.
• Non- natural amino acid residues, or peptidomimetics with acidic side chains could be used, as will be evident to one ordinarily skilled in the art.
• amino acids there may be from about 2 to about 7 amino acids, and or about 3 or 4 amino acids in this segment.
• amino-rich peptide segment refers to a peptide sequence of a peptide amphiphile that comprises one or more amino acid residues that have side chains displaying positively-charged acid side chains (e.g., Arg (R), Lys (K), His (H), or non-natural amino acids, or peptidomimetics).
• a basic peptide segment may optionally contain one or more additional (e.g., non-basic) amino acid residues.
• bioactive peptide refers to amino acid sequences that mediate the action of sequences, molecules, or supramolecular complexes associated therewith.
• Peptide amphiphiles and structures (e.g., nanofibers) bearing bioactive peptides (e.g., an IKVAV peptide) exhibit the functionality of the bioactive peptide.
• a “bioactive peptide” comprising the bioactive amino acid sequence IKVAV (SEQ ID NO: 1) is referred to herein as an “IKVAV peptide amphiphile” or an “IKVAV PA”.
• a “bioactive peptide” is a peptide comprising a growth factor mimetic peptide sequence.
• a bioactive peptide comprising a growth factor mimetic peptide sequence is referred to herein as a “growth factor mimetic peptide amphiphile” or a “growth factor mimetic PA”.
• biocompatible refers to materials and agents that are not toxic to cells or organisms.
• a substance is considered to be “biocompatible” if its addition to cells in vitro results in less than or equal to approximately 10% cell death, usually less than 5%, more usually less than 1%.
• biodegradable as used to describe the polymers, hydrogels, and/or wound dressings herein refers to compositions degraded or otherwise “broken down” under exposure to physiological conditions.
• a biodegradable substance is a broken down by cellular machinery, enzymatic degradation, chemical processes, hydrolysis, etc.
• a wound dressing or coating comprises hydrolyzable ester linkages that provide the biodegradability.
• physiological conditions relates 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 tissues.
• chemical conditions e.g., pH, ionic strength
• biochemical e.g., enzyme concentrations
• the physiological pH ranges from about 7.0 to 7.4.
• the terms “treat,” “treatment,” and “treating” refer to reducing the amount or severity of a particular condition, disease state (e.g., CNS injury), or symptoms thereof, in a subject presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete treatment (e.g., total elimination of the condition, disease, or symptoms thereof).
• Treatment encompasses any administration or application of a therapeutic or technique for a disease (e.g., in a mammal, including a human), and includes inhibiting the disease, arresting its development, relieving the disease, causing regression, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process.
• the terms “prevent,” “prevention,” and preventing” refer to reducing the likelihood of a particular condition or disease state (e.g., CNS injury) from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete or absolute prevention.
• preventing CNS injury refers to reducing the likelihood of CNS injury occurring in a subject not presently experiencing or diagnosed with a CNS injury.
• a composition or method need only reduce the likelihood of CNS injury, not completely block any possibility thereof.
• prevention encompasses any administration or application of a therapeutic or technique to reduce the likelihood of a disease developing (e.g., in a mammal, including a human). Such a likelihood may be assessed for a population or for an individual.
• co-administration and “co-administering” refer to the administration of at least two agent(s) or therapies to a subject (e.g., a supramolecular assembly described herein and one or more therapeutic agents).
• the co- administration of two or more agents or therapies is concurrent.
• a first agent/therapy is administered prior to a second agent/therapy.
• the formulations and/or routes of administration of the various agents or therapies used may vary.
• the appropriate dosage for co-administration can be readily determined by one skilled in the art.
• the respective agents or therapies are administered at lower dosages than appropriate for their administration alone.
• co-administration is especially desirable in embodiments where the co- administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.
• a potentially harmful agent e.g., toxic
• co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.
• PAs peptide amphiphiles
• compositions comprising PAs
• supramolecular assemblies comprising PAs
• methods of use thereof are provided herein.
• the peptide amphiphile molecules and compositions of the embodiments described herein are synthesized using preparatory techniques well-known to those skilled in the art, preferably, by standard solid-phase peptide synthesis, with the addition of a fatty acid in place of a standard amino acid at the N-terminus (or C-terminus) of the peptide, in order to create the lipophilic segment (although in some embodiments, alignment of nanofibers is performed via techniques not previously disclosed or used in the art (e.g., extrusion through a mesh screen).
• Synthesis typically starts from the C-terminus, to which amino acids are sequentially added using either a Rink amide resin (resulting in an --NH2 group at the C-terminus of the peptide after cleavage from the resin), or a Wang resin (resulting in an --OH group at the C-terminus).
• peptide amphiphiles having a C-terminal moiety that may be selected from the group consisting of --H, --OH, --COOH, --CONH2, and --NH2.
• peptide amphiphiles comprise a hydrophobic segment (i.e. a hydrophobic tail) linked to a peptide.
• the peptide comprises a structural peptide segment.
• the structural peptide segment is a hydrogen-bond- forming segment, or beta-sheet-forming segment.
• the structural peptide segment has the propensity to form random coil structures (e.g. a total propensity for forming ⁇ - sheet conformations of 4 or less).
• the structural peptide segment has a low propensity to form ordered secondary structures and therefore possesses a relatively high level of internal motion.
• the peptide comprises a charged segment (e.g., acidic segment, basic segment, zwitterionic segment, etc.).
• the peptide further comprises linker or spacer segments for adding solubility, flexibility, distance between segments, etc.
• peptide amphiphiles comprise a spacer segment (e.g., peptide and/or non- peptide spacer) at the opposite terminus of the peptide from the hydrophobic segment.
• the spacer segment comprises peptide and/or non-peptide elements.
• the spacer segment comprises one or more bioactive groups (e.g., alkene, alkyne, azide, thiol, etc.).
• various segments may be connected by linker segments (e.g., peptide (e.g., GG) or non-peptide (e.g., alkyl, OEG, PEG, etc.) linkers).
• 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 that is linked to the N- or C-terminal amino acid through an acyl bond.
• PA molecules self-assemble (e.g., into cylindrical micelles (a.k.a., nanofibers)) to bury the lipophilic segment in their core and display the bioactive peptide on the surface.
• the structural peptide undergoes intermolecular hydrogen bonding to form beta sheets that orient parallel to the long axis of the micelle.
• the structural peptide displays weak intermolecular hydrogen bonding, resulting in a less rigid beta-sheet conformation within the nanofibers.
• a hydrophobic (e.g., hydrocarbon and/or alkyl/alkenyl/alkynyl tail, or 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 ranges there between) is covalently coupled to peptide segment (e.g., a peptide a structural peptide segment and a charged peptide segment
• a plurality of such PAs will self-assemble in water (or aqueous solution) into a nanostructure (e.g., nanofiber).
• the relative lengths of the peptide segment and hydrophobic segment result in differing PA molecular shape and nanostructural architecture.
• a broader peptide segment and narrower hydrophobic segment results in a generally conical molecular shape that has an effect on the assembly of PAs (See, e.g., J. N. Israelachvili Intermolecular and surface forces; 2nd ed.; Academic: London San Diego, 1992; herein incorporated by reference in its entirety).
• Other molecular shapes have similar effects on assembly and nanostructural architecture.
• the pH of the solution may be changed (raised or lowered) or multivalent ions, such as calcium, or charged polymers or other macromolecules may be added to the solution.
• the hydrophobic segment is a non-peptide segment (e.g., alkyl/alkenyl/alkynyl group).
• the hydrophobic segment comprises an alkyl chain (e.g., saturated) of 4-25 carbons (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25), fluorinated segments, fluorinated alkyl tails, heterocyclic rings, aromatic segments, pi-conjugated segments, cycloalkyls, oligothiophenes etc.
• the hydrophobic segment comprises an acyl/ether chain (e.g., saturated) of 2-30 carbons (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30).
• the hydrophobic segment comprises an 8-24 carbon alkyl chain (C 8-24 ). In some embodiments, the hydrophobic segment comprises a 16 carbon alkyl chain (C 16 ).
• PAs comprise one or more peptide segments. Peptide segment may comprise natural amino acids, modified amino acids, unnatural amino acids, amino acid analogs, peptidomimetics, or combinations thereof. In some embodiments, peptide segment comprise at least 50% sequence identity or similarity (e.g., conservative or semi-conservative) to one or more of the peptide sequences described herein. In some embodiments, peptide amphiphiles comprise a charged peptide segment. The charged segment may be acidic, basic, or zwitterionic.
• peptide amphiphiles comprise an acidic peptide segment.
• 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 sequence.
• the acidic peptide segment comprises up to 7 residues in length and comprises at least 50% acidic residues.
• an acidic peptide segment comprises (Xa) 1-7 , wherein each Xa is independently D or E.
• an acidic peptide segment comprises E 2-4 .
• an acidic peptide segment comprises E2.
• an acidic peptide segment comprises E3.
• an acidic peptide segment comprises E4.
• peptide amphiphiles comprise a basic peptide segment.
• 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 sequence.
• the basic peptide segment comprises up to 7 residues in length and comprises at least 50% basic residues.
• an acidic peptide segment comprises (Xb) 1-7 , wherein each Xb is independently R, H, and/or K.
• peptide amphiphiles comprises a structural peptide segment.
• the structural peptide segment is a beta-sheet-forming segment. In some embodiments, the structural peptide segment displays weak hydrogen bonding and lacks secondary structure. In some embodiments, the structural peptide segment displays weak hydrogen bonding and has the propensity to form random coil structures rather than rigid beta- sheet conformations. 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., V 2 A 2 , V 3 A 3 , A 2 V 2 , A 3 V 3 , or other combinations of V and A residues, etc.).
• V 2 A 2 , V 3 A 3 , A 2 V 2 , A 3 V 3 or other combinations of V and A residues, etc.
• the structural peptide segment comprises 4 or more consecutive A and/or V residues, or conservative or semi-conservative substitutions thereto.
• the structural peptide segment comprises V 2 A 2.
• the structural peptide segment comprises an alanine and glycine-rich peptide segment (e.g. A2G2, A3G3, or other combinations of A and G residues, etc.).
• the structural peptide segment comprises A 2 G 2 .
• the structural peptide segment comprises a glycine-rich peptide segment.
• the structural peptide segment comprises G3 or G4.
• the structural peptide segment has a total propensity for forming ⁇ -sheet conformations 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.
• the total propensity for forming ⁇ -sheet conformations may be calculated as the sum of the propensity for forming ⁇ -sheet conformations of each amino acid in the structural peptide segment.
• the propensity of each amino acid for forming ⁇ -sheet conformations and methods for calculating the same are described in, for example, Fujiwara, K., Toda, H. & Ikeguchi, M.
• the structural peptide segment may comprise any suitable number and combination of amino acids to achieve a total propensity for forming ⁇ -sheet conformations of 4 or less.
• Table 1 Amino acid Propensities for ⁇ -sheet conformations
• a structural peptide segment having a total propensity for forming ⁇ -sheet conformations of 4 or less indicates that the amino acids within the structural peptide segment have weaker interactions with neighboring molecules.
• the structural peptide segment may display weak hydrogen-bonding abilities. Accordingly, such structural peptide segments and the peptide amphiphiles comprising the same may create more dynamic supramolecular assemblies.
• an A2G2 structural peptide segment may display random coil structures rather than rigid beta-sheet conformations.
• a bioactive peptide amphiphile (e.g. an IKVAV peptide amphiphile) comprises a relatively low fluorescent anisotropy value.
• Anisotropy is calculated using the following equation: Where ⁇ ⁇ represents the parallel intensity to the excitation plane, ⁇ is the perpendicular intensity to the excitation plane, g is grating factor (G-factor) that represents the intensity ratio of the sensitivity of the detection system for vertically and horizontally polarized light.
• the bioactive peptide amphiphile (e.g. IKVAV peptide amphiphile) comprises a fluorescence anisotropy value of less than 0.3 (e.g.
• the bioactive peptide amphiphile comprises a relatively low proton relaxation rate ( 1 H-R 2 ).
• Lower proton relaxation rates are indicative of higher motion, thus facilitating the formation of dynamic supramolecular assemblies with high degrees of internal motion.
• the relaxation rate for the methylene protons attached to the e carbon of the K residue in the IKVAV sequence may be measured by transverse-relaxation nuclear magnetic resonance (T2-NMR) spectroscopy.
• the IKVAV peptide amphiphile comprises a proton relaxation rate ( 1 H-R 2 ) of less than 4s -1 . In some embodiments, the IKVAV peptide amphiphile comprises a proton relaxation rate ( 1 H-R2) of less than 3s -1 .
• peptide amphiphiles comprise a non-peptide spacer or linker segment. In some embodiments, the non-peptide spacer or linker segment is located at the opposite terminus of the peptide from the hydrophobic segment. In some embodiments, the spacer or linker segment provides the attachment site for a bioactive group.
• the spacer or linker segment provides a reactive group (e.g., alkene, alkyne, azide, thiol, maleimide etc.) for functionalization of the PA.
• a spacer or linker further comprises additional bioactive groups, substituents, branches, etc.
• the linker segment is a single glycine (G) residue.
• G glycine
• the characteristics (e.g., shape, rigidity, hydrophilicity, etc.) of a PA supramolecular structure depend upon the identity of the components of a peptide amphiphile (e.g., lipophilic segment, acidic segment, structural peptide segment, bioactive segment, etc.).
• a peptide amphiphile e.g., lipophilic segment, acidic segment, structural peptide segment, bioactive segment, etc.
• nanofibers, nanospheres, intermediate shapes, and other supramolecular structures are achieved by adjusting the identity of the PA component parts.
• characteristics of supramolecular nanostructures of PAs are altered by post-assembly manipulation (e.g., heating/cooling, stretching, etc.).
• a peptide amphiphile comprises: (a) a hydrophobic tail comprising an alkyl chain of 8-24 carbons; (b) a structural peptide segment (e.g., comprising A2G2 or G4); and (c) a charged segment (e.g., comprising E2-E4)
• any PAs within the scope described herein, comprising the components described herein, or within the skill of one in the field, may find use herein.
• peptide amphiphiles comprise a bioactive moiety (e.g., IKVAV peptide).
• a bioactive moiety is the most C-terminal or N-terminal segment of the PA.
• the bioactive moiety is attached to the end of the charged segment. In some embodiments, the bioactive moiety is exposed on the surface of an assembled PA structure (e.g., nanofiber).
• a bioactive moiety is typically a peptide, but is not limited thereto.
• the bioactive moiety is a peptide identified in the extracellular matrix (ECM).
• ECM extracellular matrix
• the bioactive moiety may be a peptide sequence found in collagens, elastins, fibronectins, or laminins. In some embodiments, the bioactive moiety is a peptide sequence found in laminins.
• the bioactive moiety may 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.
• the bioactive moiety is a peptide sequence found in laminin-1.
• the bioactive moiety is the peptide sequence IKVAV (SEQ ID NO: 1).
• the bioactive moiety is a recombinant peptide.
• a bioactive moiety is a peptide sequence that binds a peptide or polypeptide of interests, for example, an ECM protein.
• a peptide amphiphile comprises (e.g., from C-terminus to N- terminus or from N-terminus to C-terminus): bioactive peptide (e.g., IKVAV peptide) - charged segment (e.g., comprising E 2-4 , etc.) – structural peptide segment (e.g., comprising A 2 G 2 , G 4 ) - hydrophobic tail (e.g., comprising an alkyl chain of 8-24 carbons).
• bioactive peptide e.g., IKVAV peptide
• structural peptide segment e.g., comprising A 2 G 2 , G 4
• hydrophobic tail e.g., comprising an alkyl chain of 8-24 carbons
• a peptide amphiphile comprises (e.g., from C-terminus to N- terminus or from N-terminus to C-terminus): bioactive peptide (e.g., IKVAV peptide) – flexible linker (e.g. comprising G, etc.) - charged segment (e.g., comprising E 2-4 , etc.) – structural peptide segment (e.g., comprising A2G2, G4) - hydrophobic tail (e.g., comprising an alkyl chain of 8-24 carbons).
• bioactive peptide e.g., IKVAV peptide
• flexible linker e.g. comprising G, etc.
• - charged segment e.g., comprising E 2-4 , etc.
• structural peptide segment e.g., comprising A2G2, G4
• hydrophobic tail e.g., comprising an alkyl chain of 8-24 carbons
• a bioactive PA comprising IKVAV as the bioactive peptide, also referred to herein as an “IKVAV peptide amphiphile”.
• the IKVAV peptide amphiphile comprises (e.g., from C-terminus to N-terminus or from N-terminus to C-terminus): IKVAV- charged segment (e.g., comprising E 2-4 ) – structural peptide segment (e.g., comprising A 2 G 2 , G 4 ) - hydrophobic tail (e.g., comprising an alkyl chain of 8-24 carbons).
• the peptide amphiphile further comprises a linker.
• the peptide amphiphile comprises a single glycine residue linking the bioactive peptide (e.g. IKVAV) to the charged peptide segment.
• the IKVAV peptide amphiphile comprises C16A2G2E4GIKVAV.
• the IKVAV peptide amphiphile comprises C16G4E4GIKVAV.
• the bioactive moiety is a growth factor mimetic peptide.
• the growth factor mimetic peptide comprises a growth factor mimetic sequence.
• the growth factor mimetic sequence is a vascular endothelial growth factor (VEGF) mimetic sequence, a fibroblast growth factor 2 (FGF-2) mimetic sequence, a Glial cell-derived neurotrophic factor (GDNF) mimetic sequence, a brain-derived neurotrophic factor (BDNF) mimetic sequence, or a Netrin-1 mimetic sequence.
• VEGF vascular endothelial growth factor
• FGF-2 fibroblast growth factor 2
• GDNF Glial cell-derived neurotrophic factor
• BDNF brain-derived neurotrophic factor
• Netrin-1 mimetic sequence vascular endothelial growth factor
• the growth factor mimetic sequence is an FGF-2 mimetic sequence.
• the FGF-2 mimetic sequence comprises YRSRKYSSWYVALKR (SEQ ID NO: 2).
• a bioactive PA comprising a growth factor mimetic sequence as the bioactive peptide.
• growth factor mimetic peptide amphiphile comprising (e.g., from C-terminus to N-terminus or from N-terminus to C-terminus): growth factor mimetic peptide sequence - charged segment (e.g., comprising E 2-4 , etc.) – structural peptide segment (e.g., comprising A2G2, V2A2.) - hydrophobic tail (e.g., comprising an alkyl chain of 8-24 carbons).
• growth factor mimetic peptide amphiphile comprising (e.g., from C-terminus to N-terminus or from N-terminus to C-terminus): growth factor mimetic peptide sequence - charged segment (e.g., comprising E 2-4 , etc.) – structural peptide segment (e.g., comprising A2G2, V2A2.) - hydrophobic tail (e.g., comprising an alkyl chain of 8-24 carbons).
• the peptide amphiphile further comprises In some embodiments, the peptide amphiphile further comprises a linker.
• the peptide amphiphile comprises a single glycine residue linking the growth factor mimetic peptide sequence to the charged peptide segment.
• a growth factor mimetic peptide amphiphile comprising (e.g., from C-terminus to N-terminus or from N-terminus to C-terminus): a growth factor mimetic peptide sequence – flexible linker (e.g.
• a PA further comprises an attachment segment or residue (e.g., G) for attachment of the hydrophobic tail to the peptide potion of the PA.
• compositions comprising at least two peptide amphiphiles as described herein. In some embodiments, provided herein are compositions comprising at least one IKVAV peptide amphiphile and at least one growth factor mimetic peptide amphiphile. In some embodiments, provided herein is a composition comprising at least one IKVAV peptide amphiphile comprising a hydrophobic segment, a structural peptide segment, a charged peptide segment, and a bioactive peptide comprising the amino acid sequence IKVAV and at least one growth factor mimetic peptide amphiphile.
• the IKVAV peptide amphiphile comprises a hydrophobic segment comprising an 8-24 carbon alkyl chain (C8-24), a structural peptide segment comprising AAGG, a charged peptide segment comprising E 4 , a linker (e.g. G), and the IKVAV peptide sequence.
• the growth factor mimetic peptide amphiphile comprises a hydrophobic segment comprising an 8-24 carbon alkyl chain (C8-24), a structural peptide segment comprising V2A2 or A 2 G 2 , a charged peptide segment comprising E 2 , E 3 , or E 4 , and a growth factor mimetic peptide sequence.
• the at least one IKVAV peptide amphiphile and the at least one growth factor mimetic peptide amphiphile interact to form a supramolecular assembly within the composition.
• supramolecular assemblies comprising at least two peptide amphiphiles described herein.
• a supramolecular assembly is a nanofiber.
• a supramolecular assembly comprising at least two bioactive peptide amphiphiles as described herein.
• provided herein is a supramolecular assembly comprising an IKVAV peptide amphiphile and a growth factor mimetic peptide amphiphile.
• a supramolecular assembly comprising an IKVAV peptide amphiphile comprising a hydrophobic segment, a structural peptide segment, a charged peptide segment, and a bioactive peptide comprising the amino acid sequence IKVAV and a growth factor mimetic peptide amphiphile.
• the IKVAV peptide amphiphile comprises a hydrophobic segment comprising an 8-24 carbon alkyl chain (C8-24), a structural peptide segment comprising AAGG, a charged peptide segment comprising E 4 , a linker (e.g. G), and the IKVAV peptide sequence.
• the growth factor mimetic peptide amphiphile comprises a hydrophobic segment comprising an 8-24 carbon alkyl chain (C8-24), a structural peptide segment comprising V2A2 or A 2 G 2 , a charged peptide segment comprising E 2 , E 3 , or E 4 , and a growth factor mimetic peptide sequence.
• supramolecular assemblies e.g. nanostructures, such as nanofibers
• a bioactive moiety e.g., IKVAV peptide amphiphiles
• the compositions or supramolecular assemblies e.g.
• nanostructures, such as nanofibers) described herein comprise a molar ratio of IKVAV PAs to growth factor mimetic PAs of about 90:10.
• the molar ratio of IKVAV PA:growth factor mimetic PA is about 99:1, about 98:2, about 97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about 91:9: about 90:10, about 89:11, about 88:12, abut 87:13, about 86:14, or about 85:15.
• the ratio of IKVAV PA to growth factor mimetic determines the mechanical characteristics (e.g., liquid or gel) of the nanofiber material and under what conditions the material will adopt various characteristics (e.g., gelling upon exposure to physiologic conditions, liquifying upon exposure to physiologic conditions, etc.).
• the compositions and supramolecular assemblies described herein additionally describe one or more filler PAs.
• the term “filler PA” or “diluent PA” are used interchangeably herein to refer to a PA comprising a hydrophobic segment, a structural peptide segment, and a charged peptide segment as described herein, but lacking a bioactive moiety (e.g.
• a filler PA is a non-bioactive PA molecule having highly charged glutamic acid residues on the terminal end of the molecule (e.g., surface-displayed end). These negatively charged PAs allow for the gelation to take place between nanofibers via ionic crosslinks.
• a filler PA is a non-bioactive PA molecule having highly charged lysine residues on the terminal end of the molecule (e.g., surface-displayed end). These positively charged PAs allow for the gelation to take place under basic conditions.
• the filler PAs provide the ability to incorporate other bio-active PAs molecules into the nanofiber matrix while still ensuring the ability of the nanofibers solution to gel.
• the solutions are annealed for increased viscosity and stronger gel mechanics.
• These filler PAs have sequences are described in, for example, U.S. Pat. No.8,772,228 (e.g., C 16- VVVAAAEEE), which is herein incorporated by reference in its entirety.
• the PA nanofiber described herein exhibit a small cross-sectional diameter (e.g., ⁇ 25 nm, ⁇ 20 nm, ⁇ 15nm, about 10 nm, etc.).
• the small cross-section of the nanofibers ( ⁇ 10 nm diameter) allows the fibers to permeate the brain parenchyma.
• the PAs, compositions, and supramolecular assemblies described herein find use in treating or preventing a nervous system injury in a subject.
• the PAs, compositions, and supramolecular assemblies (e.g. nanofibers) described herein may be used for methods of treatment of nervous system injury in a subject.
• the PAs, compositions, and supramolecular assemblies described herein may be used in methods for treatment of prevention of injury to the central nervous system (CNS), including the brain and the spinal cord, or the peripheral nervous system (PNS), including the nerves and ganglia outside of the brain and spinal cord.
• the PAs, compositions, and supramolecular assemblies described herein may be used for treatment or prevention of injury to the CNS or PNS in a subject.
• the injury is a spinal cord injury.
• the spinal cord injury may be cervical, lumbar, thoracic, sacral, or any combination thereof.
• the injury may be a traumatic injury.
• a traumatic injury refers to an injury caused by trauma, for example trauma such as that caused by an automobile accident, a fall, violence, sports injury, surgical injury, and the like.
• trauma such as that caused by an automobile accident, a fall, violence, sports injury, surgical injury, and the like.
• the PAs, compositions, and supramolecular assemblies described herein may be used for the treatment of traumatic spinal cord injury.
• the PAs, compositions, and supramolecular assemblies described herein may be used for the treatment of traumatic brain injury (TBI).
• TBI traumatic brain injury
• the injury may be a non-traumatic injury.
• the injury may be a non-traumatic injury to the CNS (e.g., the brain and/or the spinal cord) or the PNS caused by, for example, cancer, multiple sclerosis, inflammation, arthritis, spinal stenosis, tumors, blood loss, and the like.
• the composition comprising PAs and/or supramolecular assemblies (e.g. nanofibers) as described herein is provided to a subject suspected of having a traumatic spinal cord injury.
• the composition may be provided to the 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.
• the composition may be provided to the subject to treat the injury.
• treating the injury may prevent worsening of one or more symptoms associated with the injury.
• treating the injury may reduce the severity of and/or eliminate one or more symptoms associated with the injury.
• the composition is used to promote vascularization, nerve regeneration, functional recovery, and/or to limit the damage after spinal cord injury.
• the composition may be provided to a subject at any suitable point following injury (e.g. traumatic spinal cord injury) to treat the injury.
• the composition may be provided to the subject within 24 hours of the 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 from injury.
• the composition may be provided to the subject after a duration longer than 24 hours has passed following injury or diagnosis of injury.
• the composition may be administered in any suitable amount, depending on factors including the age of the subject, weight of the subject, severity of the injury, and the like.
• the composition may be administered in combination with other suitable treatments for injury or preventative measures to prevent the severity of the injury from worsening.
• compositions described herein are formulated for delivery to a subject.
• Suitable routes of administrating the composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.
• the PA compositions are administered parenterally.
• parenteral administration is by intrathecal administration, intracerebroventricular administration, 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.
• the motions of molecules within scaffold fibrils were intensified, which resulted in notable differences in vascular growth, axonal regeneration, myelination, survival of motor neurons, reduced gliosis, and functional recovery. Accordingly, the signaling of cells by ensembles of molecules can be optimized by tuning their internal motions.
• Pharmacological signaling of cells usually proceeds through strong binding of small organic molecules to proteins that activate or inhibit particular responses.
• An emerging signaling strategy is to use nanostructures that target specific cells to deliver a therapeutic cargo, or materials functioning as bioactive scaffolds in the extracellular space.
• Described herein is a supramolecular scaffold of nanoscale fibrils that integrates two different orthogonal biological signals, the laminin signal IKVAV which promotes differentiation of neural stem cells into neurons and to extend axons, and the fibroblast growth factor-2 (FGF-2) mimetic peptide YRSRKYSSWYVALKR (SEQ ID NO: 2), which activates the receptor FGFR1 to promote cell proliferation and survival.
• FGF-2 fibroblast growth factor-2 mimetic peptide YRSRKYSSWYVALKR
• the two signals were placed at the termini of two different peptides with alkyl tails, known as peptide amphiphiles (PAs), that copolymerize noncovalently in aqueous media to form supramolecular fibrils.
• PAs peptide amphiphiles
• Different domains that alter the physical properties of a potential scaffold therapy to restore functional recovery in vivo after hind limb paralysis in a murine model of severe spinal cord injury (SCI) were investigated herein.
• SCI therapies that avoid permanent paralysis in humans after traumatic injuries remains a major challenge given the inability of damaged axons to regenerate in the adult central nervous system (CNS).
• WAXS Wide-angle X-ray analysis
• FD fluorescence depolarization
• PA2 and PA5 had the lowest anisotropy values (0.21 and 0.18, respectively) indicating they formed the most dynamic supramolecular assemblies, PA4 had intermediate dynamics (0.30), and the remaining PAs had less intense supramolecular motion (0.40 to 0.37) (Fig.2A).
• Molecular dynamics in the IKVAV epitope were also measured using transverse-relaxation nuclear magnetic resonance (T2-NMR) spectroscopy. These experiments obtained the relaxation rate for the methylene protons attached to the e carbon (H ⁇ ) of the K residue in the IKVAV sequence (observed at 2.69 to-2.99 parts per million).
• IKVAV PA2 and PA5 induced substantially higher concentrations of active ITGB1 and the downstream effectors integrin-linked kinase (ILK) and phospho-focal adhesion kinase (p-FAK) relative to the rest of the IKVAV PAs, the IKVAV peptide, and laminin or ornithine coatings as controls (Fig.2, D and E, and FIG.9).
• ILK integrin-linked kinase
• p-FAK phospho-focal adhesion kinase
• hNPCs upregulated the neuronal form of ⁇ -TUBULIN (TUJ-1 + ) when treated with IKVAV PAs, this induction (which reflects neuronal differentiation commitment) was higher for IKVAV PA2 and PA5 (20.5 ⁇ 1 % and 20.7 ⁇ 1.2 %, respectively), the two most dynamic supramolecular fibrils (Fig.2, F to H).
• IKVAV PA4 which showed an intermediate neuronal differentiation commitment (PA4: 14 ⁇ 1.2 %), had a lower percentage of induction of TUJ-1 + neuronal cells (PA1: 8.2 ⁇ 0.7 %, PA3: 7.5 ⁇ 0.6 %, PA6: 7.9 ⁇ 1.3 %, PA7: 7.4 ⁇ 0.6 %, and PA8: 7.5 ⁇ 0.5 %).
• SUnSET technique puromycin-based protein synthesis analysis
• IKVAV PA2 was both miscible and could form hydrogels with similar mechanical properties when mixed with either FGF2 PA1 or FGF2 PA2, particularly at a molar ratio of 90:10 (Fig.3, A to C, Fig.11A-11C, Table 3). Furthermore, both FGF2 PAs alone formed highly aggregated short fibers that further contributed to immiscibility with other IKVAV PAs such as PA1, PA4 or PA5. Table 3. List of PA co-assemblies used. The miscible and gel-forming binary systems with similar mechanical properties, IKVAV PA2 with either FGF2 PA1 or FGF2 PA2, were taken forward to in vivo experiments (Fig.3A and fig. 12).
• the fluorescent materials were injected into the spinal cord 24 h post-injury and their volume was measured at 1, 2, 4, 6, and 12 weeks by fully reconstructing spinal cords using spinning disk confocal microscopy (see Fig.3D).
• the soft materials biodegraded gradually within a period of 1 to 12 weeks after implantation, and no differences in biodegradation rate among the three experimental materials were observed (see Fig.3E and fig. 13).
• Bilateral injections of biotinylated dextran amine (BDA) were administered 10 weeks after the injury into the sensorimotor cortex in order to trace the corticospinal tracts (CST), which mediate voluntary motor function (Fig.3F).
• mice injected with IKVAV PA2 alone or co-assembled with FGF2 PA2 which shares the same A2G2 non-bioactive domain as IKVAV PA2
• a modest, but increased axon regrowth was observed compared to the sham condition.
• injections of IKVAV PA2 co-assembled with FGF2 PA1 led to robust corticospinal axon regrowth across the lesion site, even surpassing its distal border (Fig.3, G and H, and Fig.15).
• MBP myelin basic protein
• a functional vessel network was investigated by transcardially injecting a glucose solution containing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye that incorporates into endothelial cell membranes (Fig.4A).
• DiI 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
• Fig.4A a lipophilic carbocyanine dye that incorporates into endothelial cell membranes
• Transverse spinal cord sections of the most bioactive co- assembly group showed NeuN + neurons near the newly generated vessels in the dorsal region similar to the uninjured control group (Fig.5A). Furthermore, neurons (NeuN + cells) that were also ChAT + (motor neurons) were only found in the ventral horn when PAs were utilized, showing a significantly higher number in the most bioactive system relative to other groups (Fig. 5, B and C). The lack of any double BrdU + /NeuN + neurons within the lesion in any of the groups suggested the absence of local neurogenesis. It was evaluated whether the observed axonal regeneration, angiogenesis, and local neuronal cell survival led to behavioral improvement in injured animals.
• BMS Basso Mouse Score
• CG-MD simulations supported the T2-NMR and FD results above by yielding higher values of RMSF for FGF2 PA molecules in the most bioactive co-assembly.
• the simulations also revealed that FGF2 PA molecules form clusters in both co-assemblies (slightly larger in the most bioactive system) with a distribution of mobilities (RMSF values) (Fig.6J).
• RMSF values mobilities
• the decreases in bioactivity in one of the systems could be attributed to differences in the extent of co-assembly between the two PA molecules bearing signals.
• 1D 1 H-NMR, diffusion ordered spectroscopy (DOSY), and T2-NMR of methylene units in alkyl tails indicate the occurrence of co-assembly in both systems (Table 4). Table 4.
• SAXS profiles showed a slope in Guinier region of – 1.2 for IKVAV PA2 and –1.6 for IKVAV PA2+FGF2 PA1 and IKVAV PA2+FGF2 PA2, indicating different degree of transition between cylindrical fibers (-1.0) to ribbons (-2.0).
• PA co-assemblies IKVAV PA2+FGF2 PA1 and IKVAV PA2+FGF2 PA2 showed consistent ⁇ ⁇ -sheet spacing on WAXS, corroborated by the FT-IR spectra, showing similar ⁇ ⁇ - sheet signature in amide I region to IKVAV PA2.
• High-magnification transmission electron microscopy (TEM) with negative staining of the co-assemblies showed an average fiber width of 13.7 ⁇ 0.3 and 15.6 ⁇ 0.2 nm for IKVAV PA2+FGF2 PA1 and IKVAV PA2+FGF2 PA2 respectively.
• the amount FGF2 PAs incorporated into the IKVAV PA2 was analyzed based on light scattering intensity (optical density) at 600 nm (O.D.600 nm).
• FGF2 PAs PA1 and PA2
• IKVAV PA2 enhances its solubility, leading to their assembly.
• the O.D. showed similar values to IKVAV PA2 alone (0 mol % of FGF2 PA) and significantly lower than FGF2 PAs free in solution.
• mice injected with backbone PA regrown axons within the lesion were hardly observed, whereas some regrowth of axons for IKVAV PA1 was observed.
• IKVAV PA4 which was found to have intermediate supramolecular motion and bioactivity in vitro (between IKVAV PA1 and IKVAV PA2), also showed an intermediate level of axon regrowth in vivo.
• hNPCs on co-assembled systems hNPCs were seeded on top of the PA coatings and were found to attach and survive in a similar way for all PA conditions tested over one week.
• IKVAV PA2+FGF2 PA2 and laminin resulted in an increased percentage of neuronal progenitor cells PAX6 + indicative of more differentiated cells.
• P- FGFR-1 was highly expressed in hNPCs seeded on IKVAV PA2+FGF2 PA1 or treated with native FGF-2, while active ITGB1 exhibited significantly higher levels in cells cultured on all PA conditions containing IKVAV PA2.
• IKVAV PA2+FGF2 PA1 triggered higher expression of the neural stem cell marker SOX-2, neuronal marker ⁇ -Tubulin-III (TUJ-1), and the postmitotic marker PH3 in a similar way to cells seeded on laminin coatings treated with native FGF-2.
• a highly agile and physically plastic supramolecular scaffold could be more effective at signaling receptors in cell membranes undergoing rapid shape fluctuations.
• An alternative explanation for the cause of the recovery could be broadly more favorable interactions of the molecularly dynamic scaffolds with the protein milieu of the ECM.
• the scaffolds described herein provide great opportunities in the structural design of dynamics to optimize the bioactivity of therapeutic supramolecular polymers.
• IKVAV PAs IKVAV PAs (IKVAV PA1: C 16 VVAAEEEEGIKVAV and IKVAV PA2: C 16 AAGGEEEEGIKVAV, IKVAV PA3: C 16 VAGGEEEEGIKVAV, IKVAV PA4: C16AVGGEEEEGIKVAV, IKVAV PA5: C16AAAAEEEEGIKVAV, IKVAV PA6: C 16 GGGGEEEEGIKVAV, IKVAV PA7: C 16 VAAAEEEEGIKVAV, IKVAV PA8: C 16 AVAAEEEEGIKVAV ), FGF2 PAs (FGF2 PA1: C16VVAAEEEEGYRSRKYSSWYVALKR and FGF2 PA2: C16AAGGEEEEGYRSRKYSSWYVALKR), their scrambled versions scr-IKVAV PAs
• FGF2 PAs and their scrambled versions were purified under acidic conditions (0.1% TFA v/v in the water and CH3CN) using a Phenomenex Kinetex C8 column, (C8 stationary phase, 5 ⁇ m, 100 ⁇ pore size, 150 x 30 mm).
• LC-MS liquid chromatography-mass spectrometry
• Electrospray ionization mass (ESI-mass) spectrometry was performed in positive scan mode on an Agilent model 6510 Quadrupole Time-of-Flight LC-MS.
• ESI-mass Electrospray ionization mass
• PAs with a covalently linked dye an Alexa Fluor®-647 labeled-IKVAV PA2 and Cy3-labeled-FGF2 PAs, were synthesized with an added cysteine or azidolysine on the C- terminus of the sequences above respectively.
• the purified IKVAV PAs were dissolved with tris(2-carboxyethyl) phosphine (TCEP) hydrochloride (5 equiv.
• TCEP tris(2-carboxyethyl) phosphine
• PA solutions were annealed at 80 °C for 30 min and then slowly cooled at 1 °C per minute to reach a final temperature of 27 °C using a thermocycler (Eppendorf PCR Thermocycler) for even and controlled heating and cooling of all samples.
• thermocycler Eppendorf PCR Thermocycler
• To prepare a PA coated substrate 24-, 12-, or 6-well polystyrene cell culture plate or 12 mm and 18 mm glass coverslips (German Glass, Chemglass Life Science) were coated with poly-D-lysine (0.01 mg/mL, Sigma-Aldrich) for 3 h at 37 °C.
• PA powder was reconstituted in sterile Isotonic Saline Sodium Chloride, 0.9 % (w/v) (Ricca Chemical) at a concentration of 1 mg/100 ⁇ l.
• the resulting PA solution was then adjusted to a pH of 7.4 using 1 ⁇ L additions of sterile 1 M NaOH, followed by co-assembly of IKVAV PA2 with 10 mol % FGF2 PA1 or FGF2 PA2 (see table 3). After mixing, the solutions were sonicated and annealed.
• TEM Transmission electron microscopy
• TEM imaging was carried out on a JEOL 1230 microscope with a LaB6 filament at 100 kV accelerating voltage, equipped with a Gatan 831 CCD camera. A cold finger was introduced for sample stabilization during imaging.
• the specimen was blotted in an environment with 100 % humidity at room temperature (blot force: 3, blot total: 1-2, wait time: 0.5-1 s, blot time: 3 s, drain time: 0-1 s), and plunged into a liquid ethane reservoir cooled by liquid nitrogen.
• the vitrified samples were stored in liquid nitrogen and then transferred to a Gatan 626 Cryo-TEM holder.
• Cryo-TEM images were obtained using a JEOL1230 electron microscope operating with a LaB6 filament at an accelerating voltage of 100 kV, equipped with a Gatan 831 CCD camera.
• the resulting dehydrated samples were mounted on stubs using carbon adhesive tape (Electron Microscopy Sciences) and in some cases, carbon glue (Electron Microscopy Sciences).
• Samples were coated with approximately 10 nm of osmium (Filgen, OPC-60A) to make the sample surface conductive for imaging. All images were taken with an accelerating voltage of 2 kV with a Hitachi SU8030 SEM instrument.
• SAXS Small-angle X-ray scattering
• MAXS medium-angle X-ray scattering
• WAXS wide- angle X-ray scattering
• Samples were oscillated at a rate of 10 ⁇ L/sec in the capillary with a syringe pump during sample measurement to prevent damage due to beam overexposure.
• the scattering intensity was recorded in the interval 0.002390 ⁇ q ⁇ 4.4578 ⁇ -1 .
• the acquired 2D scattering data were then reduced to 1D intensity vs. wavevector plots via azimuthal integration around the beam center in GSAS-II software.
• HT High Tension
• FT-IR Fourier Transformed Infrared
• FT-IR spectroscopy FT-IR spectra were recorded on a Bruker Tensor 37 FT-IR spectrometer. Samples were prepared in deuterated water (D2O) and placed between two CaF2 windows with a spacing of 50 ⁇ m. Final spectra are the result of 25 scans with 1 cm -1 resolution and atmospheric CO 2 and H 2 O were background subtracted.
• DPH was exited at 336 nm and emission was recorded at 450 nm on an ISS model PC1 spectrofluorometer with a 300 W xenon arc lamp with power of 18 A.
• Excitation slit and emission slit widths were set as 1 mm (8 nm bandwidth).
• Anisotropy was calculated using the following equation: Where represents the parallel intensity to the excitation plane, ⁇ is the perpendicular intensity to the excitation plane, g is grating factor (G-factor) that represents the intensity ratio of the sensitivity of the detection system for vertically and horizontally polarized light. G-factors were determined individually in each measurement. Results were averaged based on 34 iterations from two measurements.
• Cy3 was exited at 535 nm and emission was recorded at 575 nm.
• Rheology PA materials were prepared using the method described above for in vitro studies. An MCR302 Rheometer (Anton Paar) was used for all rheological studies. The instrument stage was set to 37°C to simulate in vitro and in vivo conditions. The PA solution (150 ⁇ L) was placed on the sample stage and 30 ⁇ L of 25 mM CaCl2 solution was pipetted onto the underside of a 25 mm cone plate positioned above the material. The plate was slowly lowered to the measuring position and a humidity collar was used to enclose the sample plunger and prevent sample evaporation during each 45 min experimental run.
• Optical density (O.D.) PA materials were prepared using the method described above for in vitro studies. The PA solutions were further diluted with a 1x saline solution to a total volume of 300 ⁇ L.100 ⁇ L of these suspensions was pipetted into triplicate wells of a 96 well plate and their optical density was recorded at 600 nm using a Cytation3 cell imaging multi-mode reader (BioTek).
• Borosilicate glass coverslips (12 mm in diameter; Fisher Scientific) were modified with synthetic IKVAV peptide.
• Borosilicate glass coverslips were cleaned with 2 % (v/v) micro-90 detergent (Sigma-Aldrich) for 30 min at 60 °C, rinsed six times with distilled water, rinsed with ethanol and then dried. Coverslips were plasma-etched (Harrick Plasma PDC-001-HP) with O 2 for 30 sec, then immediately incubated in a 2 % (v/v) solution of (3-aminopropyl) triethoxysilane (Sigma–Aldrich) in ethanol for 15 min.
• IKVAV peptide was then prepared at 50 nmol/mL in a 1.25 mg/mL solution of 1-ethyl-3-(dimethylaminopropyl) carbodiimide (Acros Organics) with 2 % DMF (Sigma–Aldrich). Coverslips were incubated with this solution for 3.5 h at 40 °C. After incubation, coverslips were washed with 100 % acetic anhydride (Fisher Chemical), 2 M hydrochloric acid (Fisher Chemical), and 0.2 M sodium bicarbonate in succession.
• NMR experiments NMR spectra were acquired at 600 MHz on a Tecmag NMR spectrometer using a Doty diffusion probe with a sweep width of 6 kHz and 16k data points or at 600 MHz on a Brucker Neo system with QCI-F cryoprobe.
• NMR spectra for IKVAV PAs were recorded at 25 °C using TFA-d, H2O/D2O in 9/1 ratio (D2O contains 0.05 wt.% 3-(trimethylsilyl) propionic-2,2,3,3-d 4 acid, sodium salt) as solvents. Chemical shifts are reported in part per million (ppm). Structural assignment was performed using 1 H, 1 H-gCOSY, 1 H, 13 C-gHSCQAD, TOCSY and NOESY. Multiplicities are quoted as singlet (s), doublet (d), multiplets (m), doublet of doublets (dd), doublet of doublet of doublets (ddd), triplet (t), quartet (q).
• the 90 ⁇ pulse width was 15 ⁇ s and typical spectra required 32 scans. Additional scans (512) were required for accurate estimation of the aromatic signal intensity since the epitope containing aromatic protons was present at only 10 mol %.
• the diffusion coefficients were measured by pulse-field gradient NMR using the longitudinal eddy-current delay with bipolar pulse pairs pulse sequence with a maximum gradient strength of 53.5 G/cm and 16 values for the gradient strength.
• the peak intensity I was measured and fit to the Stejskal–Tanner equation: where I 0 is the intensity in the absence of the gradient pulse, D is the diffusion coefficient, ⁇ is the proton gyromagnetic ratio, g is the gradient strength, ⁇ is the length of the pulse field gradient pulse (2 ms) and ⁇ is the diffusion delay (0.1 s).
• the radius of gyration R g was calculated from the Stokes–Einstein equation as: where k B is the Boltzmann constant, T is the temperature, and ⁇ is the viscosity.
• the spin-spin relaxation rates were measured using the Carr–Purcell–Gill–Meiboom pulse sequence with a delay time of 0.2 ms in a variable loop.
• Diffusion-ordered spectroscopy (DOSY) PA materials were mixed (90 mol % of IKVAV PA2 + 10 mol % FGF2 PAs) and sonicated as described above and then lyophilized. Samples were then dissolved in D 2 O water and solubilized in 1 equiv. NaOD at a concentration of 6 mM in standard 5 mm NMR tube with 0.25 mM sodium trimethylsilylpropanesulfonate (DSS) as a chemical shift reference and an intensity standard. After sonication for 20 min, the samples were annealed at 80 °C for 30 min, followed by slow cooling at room temperature.
• DOSY Diffusion-ordered spectroscopy
• Diffusion coefficients were measured using pulse-field gradient NMR using the stimulated echo pules sequence with a 2 ms gradient pulse and a 100 ms diffusion delay time using a maximum gradient strength of 53 G/cm. 1. Simulation procedures The PAs were created in Avogadro and transformed to MARTINI force field coarse-grained (CG) representation using a modified version of martinize.py to include the palmitoyl tail and using coiled coil secondary structure for the peptide. The last two E residues (furthest from aliphatic tail) as well as the K and R residues in the epitopes were charged, while the first two E residues were treated as neutral as this was found to be ideal for fiber formation in previous simulations.
• CG coarse-grained
• the final charge is -1 for the IKVAV PAs and +3 for FGF2 PAs. Simulations were made in two steps in a cubic box 21.5 ⁇ 21.5 ⁇ 21.5 nm 3 solvated with CG water and with enough ions to neutralize the systems. Firstly, 300 IKVAV PAs were randomly disposed on the box with a minimum intermolecular space of 3 ⁇ . This gives a concentration of 50 mM (7.3-7.8 wt % for IKVAV PAs). This is within the range of concentrations commonly used to accelerate self-assembly simulations, which can be up to 10 times higher than the experimental system used. These systems were equilibrated for 10 ⁇ s (FIG.8).
• the fibers formed were centered in the box and 33 FGF2 PAs (10 mol %) were added randomly around the fibers with a minimum allowance of 3 ⁇ . These systems were then equilibrated for 10 ⁇ s in five independent simulations per system. All visualizations were rendered using visual molecular dynamics (VMD). Coarse-grained molecular dynamic (CG-MD) simulations were performed in GROMACS 5.0.4, which was also used for the analysis of the simulations. A cut-off of 1.1 nm was used for intermolecular interactions using reaction field with a relative dielectric constant of 15 for electrostatics and potential-shift for Lennard–Jones interactions. All systems were minimized to 5000 steps or until the forces between atoms converged below 2000 pN.
• VMD visual molecular dynamics
• CG-MD Coarse-grained molecular dynamic
• RMSF root mean square fluctuations
• HUVEC treatments and coatings Treatments were prepared by dissolving the co-assembled PAs in media without serum. The total concentration of FGF2 PAs per treatment was 0.5 ⁇ M. FGF-2 native protein (Peprotech) was resuspended and used at 0.25 nM.
• FGF-2 native protein Peprotech
• PA coatings PAs were painted on the coverslips (German Glass, Chemglass Life Science) or tissue culture plates as described in the Co-assembled PA preparation section.
• hNPCs Human neural progenitor cell
• NPCs Neural progenitor cells
• N2B27 medium 50 % DMEM: F12, 50 % Neurobasal, supplemented with NEAA, Glutamax, N2 and B27; Gibco
• SB431542, DNSK International and LDN-193189, Tocris dual SMAD inhibitors
• NPCs were dissociated with neural rosette selection reagent (STEMCELL Technologies) to obtain NPCs, which are expanded in N2B27 (Gibco) medium supplemented with bFGF (Millipore).
• NPCs were dissociated with Accutase (Innovative Cell technology), and cultured in the distinct platforms with DMEM: F12+N2+B27 medium with hyclone penicillin-streptomycin (GE Healthcare) and ascorbic acid (0.2 ⁇ g/mL; Sigma-Aldrich).
• hNPCs Human neural progenitor cell cultures treated with IKVAV PAs or seeded on co- assembled PA coatings
• IKVAV PA treatments For IKVAV PA treatments, hNPCs were cultured on ornithine coatings (German Glass, Chemglass Life Science) in 6 well and 24 well plates at a density of 500,000 cells/well and 80,000 cells/well respectively.
• IKVAV PA2 and IKVAV PA5 were also mixed with 5 mM CaCl2 at a ratio PA:CaCl26:1 to treat hNPCs.
• hNPCs were cultured on the different IKVAV PAs or laminin coatings in 6 well and 24 well plates at densities mentioned above.
• co-assembled experiments hNPCs were cultured on the different co-assembled PA coatings in 6 well and 24 well plates at a density of 400,000 cells/well and 50,000 cells/well respectively.
• hNPCs were fed 4 times a week with DMEM: F12+N2+B27 medium with hyclone penicillin-streptomycin and ascorbic acid.
• the age of animals used in the study was in the range of 10-16 weeks.8 independent in vivo experiments were carried out (injury +PA injection) and each of the 8 experiments had animals of the exact same age which were injected with at least the 4 main treatment groups: Sham, IKVAV PA2 alone, IKVAV PA2+FGF2 PA1, and IKVAV PA2+FGF2 PA2. Animals were anesthetized using 2.5 % isoflurane gas with oxygen. A laminectomy was performed to expose the spinal cord at the T10-T11 spinal level. A severe contusive injury was performed using the Infinite Horizon Spinal Cord Impactor system (IH-0400 Precision Systems and Instrumentation) with 85 kdyn of impact force and a dwell time of 60 s.
• IH-0400 Precision Systems and Instrumentation Infinite Horizon Spinal Cord Impactor system with 85 kdyn of impact force and a dwell time of 60 s.
• mice After the lesion, the skin was sutured using 9 mm wound clips (BD Biosciences) and the animals were recovered on a heating pad to maintain body temperature. Bladders were manually expressed daily during the entire 12 weeks of the experiment. Animal inclusion and exclusion criteria All mice were evaluated in an open-field environment 24 h after the lesion and animals exhibiting any hindlimb movements (score higher than 0 in the BMS score) were discarded from the study. Mice that passed this inclusion criterion were randomized into experimental groups for PA injection and were thereafter evaluated blind to their experimental condition.
• PAs dissolved in sterile NaCl 0.9% solution at 1 wt % were injected 24 h after SCI using a glass capillary micropipette (Sutter Instruments, Novato, CA) (outer diameter, 100 ⁇ m) coated with Sigmacote (Sigma-Aldrich) to reduce surface tension as described elsewhere (19).
• the capillaries were loaded onto a Hamilton syringe using a female Luer adaptor (World Precision Instruments) controlled by a Micro4 microsyringe pump controller (World Precision Instruments). Under isoflurane anesthesia, autoclips were removed and the injury site was exposed. At 24 h post injury, the laminectomy in the spinal column was still intact and the bruise created by the lesion was visible.
• a stereotaxic Kopf apparatus was used to position the micropipette just dorsal to the lesion.
• the micropipette was lowered to a depth of 750 ⁇ m measured from the dorsal surface of the cord, and 4-6 ⁇ L of the diluted amphiphile solution was injected at 1 ⁇ L/min.
• the micropipette was withdrawn at intervals of 250 ⁇ m to leave a trail (ventral to dorsal) of PA within the cord.
• the pipette was left in place for 2-3 additional minute to allow material gelation, after which it was withdrawn, and the wound was closed with 9 mm wound clips.
• Hindlimb locomotor evaluation The motor function was evaluated with the locomotor open-field rating scale on the Basso Mouse Scale (BMS). A team of two experienced examiners evaluated each animal for 5 to 10 minutes and assigned a defined score for each hindlimb. For the footprint analysis, the hindlimbs of the mice were dipped in colored dyes. A narrow runway (80 cm length and 4 cm width) was lined with white paper as the animal walked across. The stride length was defined as the distance from the start to the end of a step with the back paw. The stride width was defined as the distance from the left outermost toe to the right outermost toe. All measurements were taken on each side for three consecutive steps and were averaged.
• BMS Basso Mouse Scale
• Anterograde BDA corticospinal tracing 14 days before perfusion, 6 animals were used per condition to trace the corticospinal tract. The animals were anesthetized using 2.5 % isoflurane gas in oxygen. The head of each mouse was stabilized using the stereotaxic frame (Stoelting Co.). An 8 mm incision was made through the skin along the midline of the skull using a surgical scalpel blade. Injections of 0.25 ⁇ L of 10 % biotinylated dextran amines (BDA, molecular weight 10,000, Thermo Fisher Scientific) were placed into each hemisphere spanning the motor cortex.
• BDA biotinylated dextran amines
• BrdU incorporation was analyzed by immunohistochemistry (rat-anti-BrdU, 1:1000; Abcam) at 12 weeks after PA injection. DiI labelling After deep anesthesia and shortly before perfusion, 20 ⁇ L/mL of DiI (Sigma-Aldrich) in PBS solution with 5 w/v % of glucose was transcardially injected into 6 animals per condition, as previously described (21).
• mice were transcardially perfused using an isotonic solution containing 4 % of paraformaldehyde (PFA, in 0.4 M phosphate buffer, pH 7.4) Animal sacrifice and tissue processing All animals were sacrificed using an overdose of carbon dioxide and transcardially perfused with phosphate buffered saline (PBS) followed by 4 % paraformaldehyde (PFA, Sigma- Aldrich) in isotonic solution (0.4 M phosphate buffer, pH 7.4). The spinal cords were fixed for 4- 6 h in 4 % PFA and overnight in PBS containing 30 % of sucrose (Sigma-Aldrich).
• PFA paraformaldehyde
• the spinal cords were then frozen in PBS containing 30 % sucrose (Sigma-Aldrich) and 15 % gelatin (Sigma-Aldrich) and sectioned on a Leica CM1850 cryostat at 40 ⁇ m thick. 5.
• Biological assays for in vitro and in vivo Immunofluorescence For immunofluorescence, fixed primary cultures or free-floating tissue sections (40 ⁇ m Thick) were incubated in blocking buffer containing 0.02 % Triton-X (Sigma-Aldrich), 1 % NHS (Gibco) and PBS 1X (Gibco). Samples were incubated with primary antibodies over night at 4 °C.
• Alexa-488, Alexa-555 and/or Alexa-647 (1:500, Thermofisher) were used as secondary antibodies.
• DAPI (1:500, Thermofisher) was used to stain nuclei.
• the samples were mounted on coverslips with Immu-Mount (Fisher Scientific) solution for imaging.
• Western blot (WB) RIPA buffer (Thermofisher) with a cocktail of Halt Protease and Phosphatase Inhibitors (Thermofisher) was used to extract protein from in vitro and in vivo samples.
• spinal cord tissue was then sonicated using a horn sonicator (Branson) to break up the tissue.
• a PierceTM BCA Protein Assay Kit (Thermofisher) was used to determine protein content for all samples used. Protein extracts obtained from cell cultures or tissue were separated using an SDS– polyacrylamide gel and electro-transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked for 30 minutes to 1 hour using a 5 % milk solution (Bio-Rad) and incubated over night with primary antibodies. Corresponding secondary HRP-conjugated antibodies (1:1000, ThermoFisher) were used for 1 h at room temperature. Radiance Bioluminescent ECL substrate (Azure Biosystems) was used to detect protein signal. The membranes were imaged using the Azure Biosystems imager and densitometry analysis were performed using ImageJ software.
• Antibodies for immunofluorescence and western blot The following primary antibodies were used for in vitro and/or in vivo studies: rabbit anti-GFAP (1:1000, Dako, Z0334), rabbit anti-laminin B1 (1:1000, Sigma-Aldrich), rabbit anti- Actin (1:2000, Sigma-Aldrich, A2066), mouse anti-GAPDH (1:2000, Cell Signaling, 97166), goat anti-5HT (1:1000, Abcam, mab66047), rabbit anti-CD31 (1:100, BD Pharmigen, 550274 ) mouse anti-Neurofilament (NF, 1:2000, Millipore, MAB1592 ), rabbit anti-GAP43 (1:2000, Cell Signaling, 8945), goat anti-Sox-2 (1:1000, Abcam, ab110145), Streptavidin Alexa FluorTM 555 Conjugate (1:500, Thermofisher, S32355), rabbit anti-PH3 (1:1000, Cell Signaling, 9701), rat anti-B
• Blood vessel analysis To assess in vitro tubulogenesis and in vivo angiogenesis, an ImageJ (Fiji) script was established to automatically calculate 1) area fraction of blood vessels, 2) blood vessels length, and 3) number of branches. Images were processed, binarized and analyzed. Newly generated blood vessels in vivo were identified by quantifying the amount of CD31/BrdU double-positive cells within the region of interest. Functional blood vessels were identified using DiI staining in 8 sections within the lesion per mouse.6 animals per group treatment were used in this analysis. The quantified cross sections were chosen as the first serial cross sections within the lesion that had DiI staining.
• Neurite tracking Axons labeled using BDA (Thermofisher, N7167) or stained with 5HT (Abcam) were quantified using Imaris® software version 9.3 as previously described (21). Lines were drawn across longitudinal spinal cord sections from the proximal border (PB) to the distal border (DB) of the SCI lesion at consistent distances and the number of axons intercepting the lines drawn was counted by researchers that were blinded to the experimental conditions. Multiple sections through the middle of the cord, where BDA or 5HT staining was denser, were counted per mouse and expressed as total intercepts per location, per animal.6 animals per group treatment were used in these analyses.
• Degradation studies and tissue clearing Degradation studies of the PAs injected into the lesioned spinal cord were performed by covalently labeling the IKVAV sequence with Alexa-647 (click) fluorescent dye.
• spinal cords were perfused, extracted, and cleared using benzyl alcohol-benzyl benzoate (BABB, Sigma-Aldrich). After clearing, spinal cord tissue became fluorescent at 488 nm.
• Full reconstructions were performed using spinning disk confocal microscopy and analyzed using Imaris software. Three spinal cords per group treatment and time point were used in these studies.
• Imaging A Nikon A1R confocal laser-scanning microscope with GaAsP detectors, Nikon W1 Dual Cam Spinning Disk Confocal and Nikon A1RMP+ Multiphoton were used to visualize and image fluorescent cells, sections or full cleared spinal cord samples.
• Nikon Ti2 Widefield was used to acquire larger sections of spinal cords.
• Image quantification and analysis For in vitro cell quantification, image files were imported into NIH Image J (1.51) software and the “analyze particles” and “cell counter” functions were used to measure the total number of cells in a determined area. Serial tissue sections were stained with NeuN and ChAT using free-floating immunostaining were quantified using Nikon Elements software.
• the rostral and caudal borders of the lesions were chosen as the first cross ⁇ sections that had NeuN staining in all four gray matter horns.8 sections within the lesion were chosen per animal and expressed as number of neurons per section.
• Automated multichannel image acquisition, image stitching, and z-stack reconstruction (36-40 mm thick) were carried out on a Nikon GasP R1 confocal microscope to image the entire selected cross ⁇ section for NeuN and Chat markers for all conditions. Fluorescence intensity of tissue sections stained for GFAP was analyzed using NIH Fiji Software. Scanned images with constant exposure settings in the various microscopes mentioned above were used for this analysis. Single channel immunofluorescence images were used to analyze the number of fluorescent positive pixels along the area selected in each image.

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