Dynamic and physiologically active scaffolds and their therapeutic use after CNS injury

Abstract

This provides nanostructures that target specific cells to deliver therapeutic cargo, and materials that function as bioactive scaffolds in extracellular space, for use in treatment to prevent permanent paralysis in humans after traumatic injury. [Solution] This application provides a peptide amphiphilic molecule (PA), a supramolecular assembly containing PA, a composition containing PA, and a method of using the same. In a predetermined embodiment, this application provides a supramolecular assembly containing IKVAVPA and growth factor mimetic PA. In a predetermined embodiment, the PA, composition, and supramolecular assembly described in this application are used in a method for treating nerve damage.

Description

[Technical Field]

[0001] Statement regarding financial assistance from the federal government This invention was made possible with government support under contract number FA8650-15-2-5518 from the Air Force Research Laboratory, grant number R01NS104219 from the National Institute on Aging, and grant numbers R21NS107761 and R21NS107761-01A1 from the National Institute of Neurological Disorders and Stroke. The government has certain rights to this invention.

[0002] Statement regarding related applications This application is a national phase application of PCT International Application PCT / US2022 / 017490, filed on 23 February 2022, claiming priority to U.S. Provisional Patent Application No. 63 / 152,498, filed on 23 February 2021, and the entire contents of the aforementioned U.S. Provisional Patent Application are incorporated herein by reference for all purposes.

[0003] This application provides a peptide amphiphilic molecule (PA), a supramolecular assembly containing PA, a composition containing PA, and a method of using the same. In a predetermined embodiment, this application provides a supramolecular assembly containing IKVAVPA and growth factor mimetic PA. In a predetermined embodiment, the PA, composition, and supramolecular assembly described in this application are used in a method for treating nerve damage. Sequence List The computer-readable sequence listing text submitted herein is titled "NWEST_39312_611_SequenceListing_Corrected_ST25", was created on January 4, 2024, has a file size of 6,432 bytes, and is incorporated herein by reference in its entirety. [Background technology]

[0004] Because damaged axons cannot regenerate in the adult central nervous system (CNS), developing therapies to avoid permanent paralysis in humans after traumatic injury remains a major challenge. Nanostructures that target specific cells to deliver therapeutic cargo, and materials that function as bioactive scaffolds in extracellular spaces, are emerging as signaling strategies. However, success is limited because methods for optimizing the design of such nanostructures to obtain high bioactivity from the delivered cargo have not yet been elucidated, and so far, no highly effective therapies exist. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] In a predetermined embodiment, the present application provides a supramolecular assembly. In a predetermined embodiment, the present application provides a supramolecular assembly comprising at least two peptide amphiphilic molecules. In a predetermined embodiment, the at least two peptide amphiphilic molecules comprise at least one IKVAV peptide amphiphilic molecule comprising a hydrophobic segment, a structural peptide segment, a charged peptide segment, and a physiologically active peptide comprising the amino acid sequence IKVAV (SEQ ID NO: 1), and at least one growth factor mimetic peptide amphiphilic molecule. In a predetermined embodiment, the at least one IKVAV peptide amphiphilic molecule comprises a fluorescence anisotropy value of less than 0.3. In a predetermined embodiment, the at least one IKVAV peptide amphiphilic molecule comprises 4s -1 Proton relaxation rate less than ( 1 (Including H-R2). [Means for solving the problem]

[0006] In a given embodiment, the hydrophobic segment is an alkyl chain having 8 to 24 carbon atoms (C 8-24 ) includes. In a given embodiment, the hydrophobic segment is a C16 alkyl chain (C 16) includes. In a given embodiment, the structural peptide segment includes A2G2 (SEQ ID NO: 4). In a given embodiment, the charged peptide segment includes E2, E3, or E4 (SEQ ID NO: 11). In a given embodiment, the physiologically active peptide is linked to the charged peptide segment by a linker. In a given embodiment, the linker is a single glycine (G) residue. In a given embodiment, the IKVAV peptide amphiphilic molecule is C 16 Includes A2G2E4GIKVAV (sequence code 12).

[0007] In a predetermined embodiment, the at least one growth factor mimetic peptide amphiphilic molecule is an alkyl chain having 8 to 24 carbon atoms (C 8-24 The molecule comprises a hydrophobic segment containing ), a structural peptide segment containing V2A2 and A2G2, a charged peptide segment containing E2, E3, or E4, and a growth factor mimetic peptide sequence. In a given embodiment, the growth factor mimetic sequence is a vascular endothelial growth factor (VEGF) mimetic sequence, a fibroblast growth factor 2 (FGF2) 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. In a given embodiment, the growth factor mimetic sequence is an FGF2 mimetic sequence. For example, in a given embodiment, the FGF2 mimetic sequence contains YRSRKYSSWYVALKR (SEQ ID NO: 2). In a given embodiment, the growth factor mimetic peptide is linked to the charged peptide segment by a linker. In a given embodiment, the linker is a single glycine (G) residue.

[0008] In a predetermined embodiment, the at least one growth factor mimetic peptide amphiphilic molecule is C 16 -V2A2E4GYRSRKYSSWYVALKR(Sequence ID 13) or C 16-It contains -A2G2E4GYRSRKYSSWYVALKR (SEQ ID NO: 14). In a given embodiment, the at least one IKVAV peptide amphiphilic molecule is C 16 -It contains -A2G2E4GIKVAV (SEQ ID NO: 12), and the at least one growth factor mimetic peptide amphiphilic molecule is C 16 -It contains -V2A2E4GYRSRKYSSWYVALKR (SEQ ID NO: 13) or C 16 -It contains -A2G2E4GYRSRKYSSWYVALKR (SEQ ID NO: 14). In a given embodiment, the at least one IKVAV peptide amphiphilic molecule is C 16 -It contains -A2G2E4GIKVAV (SEQ ID NO: 12), and the at least one growth factor mimetic peptide amphiphilic molecule is C 16 -It contains -V2A2E4GYRSRKYSSWYVALKR (SEQ ID NO: 13).

[0009] In a given aspect, the present application provides a composition. In a given embodiment, the present application provides a composition containing a supramolecular assembly as described in the present application. In a given embodiment, the present application provides a composition containing at least one IKVAV peptide amphiphilic molecule containing a hydrophobic segment, a structural peptide segment, a charged peptide segment, and a bioactive peptide containing the amino acid sequence IKVAV, and at least one growth factor mimetic peptide amphiphilic molecule. In a given embodiment, the at least one IKVAV peptide amphiphilic molecule and the at least one growth factor mimetic peptide amphiphilic molecule interact to form a supramolecular assembly within the composition.

[0010] In a given embodiment, the at least one IKVAV peptide amphiphilic molecule contains a fluorescence anisotropy value of less than 0.3. In a given embodiment, the at least one IKVAV peptide amphiphilic molecule contains a proton relaxation rate ( -1 H-R2) of less than 4 s 1 In a given embodiment, the hydrophobic segment is an alkyl chain having 8 to 24 carbon atoms (C 8-24) includes, for example, in a given embodiment, the hydrophobic segment is a C16 alkyl chain (C 16 ) includes. In a given embodiment, the structural peptide segment includes A2G2. In a given embodiment, the charged peptide segment includes E2, E3, or E4. In a given embodiment, the physiologically active peptide is linked to the charged peptide segment by a linker. In a given embodiment, the linker is a single glycine (G) residue. In a given embodiment, the IKVAV peptide amphiphilic molecule is C 16 -Includes A2G2E4GIKVAV (sequence number 12).

[0011] In a predetermined embodiment, the at least one growth factor mimetic peptide amphiphilic molecule is an alkyl chain having 8 to 24 carbon atoms (C 8-24 The molecule comprises a hydrophobic segment containing ), a structural peptide segment containing V2A2 (SEQ ID NO: 3) or A2G2 (SEQ ID NO: 4), a charged peptide segment containing E2, E3, or E4 (SEQ ID NO: 11), and a growth factor mimetic peptide sequence. In a given embodiment, the growth factor mimetic sequence is a vascular endothelial growth factor (VEGF) mimetic sequence, a fibroblast growth factor 2 (FGF2) 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. In a given embodiment, the growth factor mimetic sequence is an FGF2 mimetic sequence. In a given embodiment, the FGF2 mimetic sequence contains YRSRKYSSWYVALKR (SEQ ID NO: 2). In a given embodiment, the growth factor mimetic peptide is linked to the charged peptide segment by a linker. In a given embodiment, the linker is a single glycine (G) residue.

[0012] In a predetermined embodiment, the at least one growth factor mimetic peptide amphiphilic molecule is C 16 -V2A2E4GYRSRKYSSWYVALKR(Sequence ID 13) or C 16-A2G2E4GYRSRKYSSWYVALKR (SEQ ID NO: 14) is included. In a given embodiment, the at least one IKVAV peptide amphiphilic molecule is C 16 -A2G2E4GIKVAV (SEQ ID NO: 12) is included, and the at least one growth factor mimetic peptide amphiphilic molecule is C 16 -V2A2E4GYRSRKYSSWYVALKR(Sequence ID 13) or C 16 -A2G2E4GYRSRKYSSWYVALKR (SEQ ID NO: 14) is included. In a given embodiment, the at least one IKVAV peptide amphiphilic molecule is C 16 -A2G2E4GIKVAV (SEQ ID NO: 12) is included, and the at least one growth factor mimetic peptide amphiphilic molecule is C 16 -Includes V2A2E4GYRSRKYSSWYVALKR (sequence number 13).

[0013] The compositions described herein can be used in methods for treating nervous system injuries in a subject. In a given embodiment, the nervous system injury is a central nervous system injury. For example, in a given embodiment, the central nervous system injury is a spinal cord injury.

[0014] In a predetermined embodiment, the present application provides a method for treating a nervous system injury in a subject. In a predetermined embodiment, the present application provides a method for treating a nervous system injury in a subject, comprising providing the subject with the composition described in the present application. In a predetermined embodiment, the nervous system injury is a central nervous system injury. In a predetermined embodiment, the central nervous system injury is a spinal cord injury.

[0015] Other aspects and embodiments of this disclosure may also be conceived by referring to the attached documents and drawings. [Brief explanation of the drawing]

[0016] [Figure 1A]Figures 1A-1E show the library of IKVAVPA molecules investigated. (A) Specific chemical structures of the various IKVAVPA molecules used and molecular graphics of supramolecular nanofibers exhibiting IKVAV bioactivity signals. (B) CryoTEM microscope images of the various IKVAVPA molecules in the library and color-coded representations of the RMSF values ​​of the individual IKVAVPA filaments corresponding to these images. (C) Bar graphs of the average RMSF values ​​of the various IKVAVPA molecules (error bars correspond to three independent simulations; *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA and Bonferroni method). (D) SAXS patterns and (E) WAXS profiles of the various IKVAVPA nanofibers (scattering intensities are shifted vertically to avoid overlap, and the Bragg peak corresponding to the β-sheet spacing around 1.35 Å is shown in the gray box). Scale bar: 200 nm. [Figure 1B] Figures 1A-1E show the library of IKVAVPA molecules investigated. (A) Specific chemical structures of the various IKVAVPA molecules used and molecular graphics of supramolecular nanofibers exhibiting IKVAV bioactivity signals. (B) CryoTEM microscope images of the various IKVAVPA molecules in the library and color-coded representations of the RMSF values ​​of the individual IKVAVPA filaments corresponding to these images. (C) Bar graphs of the average RMSF values ​​of the various IKVAVPA molecules (error bars correspond to three independent simulations; *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA and Bonferroni method). (D) SAXS patterns and (E) WAXS profiles of the various IKVAVPA nanofibers (scattering intensities are shifted vertically to avoid overlap, and the Bragg peak corresponding to the β-sheet spacing around 1.35 Å is shown in the gray box). Scale bar: 200 nm. [Figure 1C-1E]Figures 1A-1E show the library of IKVAVPA molecules investigated. (A) Specific chemical structures of the various IKVAVPA molecules used and molecular graphics of supramolecular nanofibers exhibiting IKVAV bioactivity signals. (B) CryoTEM microscope images of the various IKVAVPA molecules in the library and color-coded representations of the RMSF values ​​of the individual IKVAVPA filaments corresponding to these images. (C) Bar graphs of the average RMSF values ​​of the various IKVAVPA molecules (error bars correspond to three independent simulations; *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA and Bonferroni method). (D) SAXS patterns and (E) WAXS profiles of the various IKVAVPA nanofibers (scattering intensities are shifted vertically to avoid overlap, and the Bragg peak corresponding to the β-sheet spacing around 1.35 Å is shown in the gray box). Scale bar: 200 nm. [Figure 2A-2B]Figures 2A-2J show the in vitro effects of supramolecular motion on hNPC signaling. (A) Molecular graphics representation of IKVAVPA nanofibers, showing the chemical structure and position of DPH used as a probe in fluorescence depolarization measurements (top); bar graph of fluorescence anisotropy of IKVAVPA solution (error bars correspond to three independent experiments; no significant difference in ns, ***P<0.0001, one-way ANOVA and Bonferroni method). (B) Chemical structure of the IKVAV peptide sequence, with K residues observed by NMR highlighted (top); bar graph of K relaxation times for various IKVAVPAs investigated (error bars correspond to 3 experiments for each condition; ***P<0.0001 vs. IKVAVPA1, #P<0.05, ###P<0.0001 vs. IKVAVPA2, and +P<0.05, +++P<0.0001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (C) Differentiation conditions used for hNPCs. (D) Representative micrographs of hNPCs treated with IKVAVPA1, PA2, PA4 and PA5; nestin-stem cells (red), ITGB1-receptor (green), and DAPI-nucleus (blue). (E) WB results for ITGB1, p-FAK, FAK, ILK, and TUJ1 in hNPCs treated with laminin and various IKVAVPAs. (F) Representative confocal micrographs of hNPCs treated with IKVAVPA1, PA2, PA4, and PA5; nestin-stem cells (red), SOX2-stem cells (green), TUJ1-neurons (white), and DAPI-nuclei (blue). (G, H) Bar graphs of percentages of SOX2+ / nestin+ stem cells (G) and TUJ1+ neurons (H) treated with various IKVAVPAs (error bars correspond to three independent differentiations; **P<0.01, ***P<0.001 vs. IKVAVPA2 and ##P<0.01, ###P<0.001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (I) Fluorescence anisotropy (left) and K residue relaxation time (right) obtained with IKVAVPA2 nanofibers in the absence (No Ca2+) or presence (Ca2+) of calcium ions (***P<0.001, Student's t-test). (J) WB results of ITGB1, p-FAK, FAK, and ILK in hNPC treated with IKVAVPA2 in the absence (-) or presence (+) of Ca2+.Scale bar: (D) 10mm, (F) 100mm. [Figure 2C]Figures 2A-2J show the in vitro effects of supramolecular motion on hNPC signaling. (A) Molecular graphics representation of IKVAVPA nanofibers, showing the chemical structure and position of DPH used as a probe in fluorescence depolarization measurements (top); bar graph of fluorescence anisotropy of IKVAVPA solution (error bars correspond to three independent experiments; no significant difference in ns, ***P<0.0001, one-way ANOVA and Bonferroni method). (B) Chemical structure of the IKVAV peptide sequence, with K residues observed by NMR highlighted (top); bar graph of K relaxation times for various IKVAVPAs investigated (error bars correspond to 3 experiments for each condition; ***P<0.0001 vs. IKVAVPA1, #P<0.05, ###P<0.0001 vs. IKVAVPA2, and +P<0.05, +++P<0.0001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (C) Differentiation conditions used for hNPCs. (D) Representative micrographs of hNPCs treated with IKVAVPA1, PA2, PA4 and PA5; nestin-stem cells (red), ITGB1-receptor (green), and DAPI-nucleus (blue). (E) WB results for ITGB1, p-FAK, FAK, ILK, and TUJ1 in hNPCs treated with laminin and various IKVAVPAs. (F) Representative confocal micrographs of hNPCs treated with IKVAVPA1, PA2, PA4, and PA5; nestin-stem cells (red), SOX2-stem cells (green), TUJ1-neurons (white), and DAPI-nuclei (blue). (G, H) Bar graphs of percentages of SOX2+ / nestin+ stem cells (G) and TUJ1+ neurons (H) treated with various IKVAVPAs (error bars correspond to three independent differentiations; **P<0.01, ***P<0.001 vs. IKVAVPA2 and ##P<0.01, ###P<0.001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (I) Fluorescence anisotropy (left) and K residue relaxation time (right) obtained with IKVAVPA2 nanofibers in the absence (No Ca2+) or presence (Ca2+) of calcium ions (***P<0.001, Student's t-test). (J) WB results of ITGB1, p-FAK, FAK, and ILK in hNPC treated with IKVAVPA2 in the absence (-) or presence (+) of Ca2+.Scale bar: (D) 10mm, (F) 100mm. [Figure 2D]Figures 2A-2J show the in vitro effects of supramolecular motion on hNPC signaling. (A) Molecular graphics representation of IKVAVPA nanofibers, showing the chemical structure and position of DPH used as a probe in fluorescence depolarization measurements (top); bar graph of fluorescence anisotropy of IKVAVPA solution (error bars correspond to three independent experiments; no significant difference in ns, ***P<0.0001, one-way ANOVA and Bonferroni method). (B) Chemical structure of the IKVAV peptide sequence, with K residues observed by NMR highlighted (top); bar graph of K relaxation times for various IKVAVPAs investigated (error bars correspond to 3 experiments for each condition; ***P<0.0001 vs. IKVAVPA1, #P<0.05, ###P<0.0001 vs. IKVAVPA2, and +P<0.05, +++P<0.0001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (C) Differentiation conditions used for hNPCs. (D) Representative micrographs of hNPCs treated with IKVAVPA1, PA2, PA4 and PA5; nestin-stem cells (red), ITGB1-receptor (green), and DAPI-nucleus (blue). (E) WB results for ITGB1, p-FAK, FAK, ILK, and TUJ1 in hNPCs treated with laminin and various IKVAVPAs. (F) Representative confocal micrographs of hNPCs treated with IKVAVPA1, PA2, PA4, and PA5; nestin-stem cells (red), SOX2-stem cells (green), TUJ1-neurons (white), and DAPI-nuclei (blue). (G, H) Bar graphs of percentages of SOX2+ / nestin+ stem cells (G) and TUJ1+ neurons (H) treated with various IKVAVPAs (error bars correspond to three independent differentiations; **P<0.01, ***P<0.001 vs. IKVAVPA2 and ##P<0.01, ###P<0.001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (I) Fluorescence anisotropy (left) and K residue relaxation time (right) obtained with IKVAVPA2 nanofibers in the absence (No Ca2+) or presence (Ca2+) of calcium ions (***P<0.001, Student's t-test). (J) WB results of ITGB1, p-FAK, FAK, and ILK in hNPC treated with IKVAVPA2 in the absence (-) or presence (+) of Ca2+.Scale bar: (D) 10mm, (F) 100mm. [Figure 2E-2F]Figures 2A-2J show the in vitro effects of supramolecular motion on hNPC signaling. (A) Molecular graphics representation of IKVAVPA nanofibers, showing the chemical structure and position of DPH used as a probe in fluorescence depolarization measurements (top); bar graph of fluorescence anisotropy of IKVAVPA solution (error bars correspond to three independent experiments; no significant difference in ns, ***P<0.0001, one-way ANOVA and Bonferroni method). (B) Chemical structure of the IKVAV peptide sequence, with K residues observed by NMR highlighted (top); bar graph of K relaxation times for various IKVAVPAs investigated (error bars correspond to 3 experiments for each condition; ***P<0.0001 vs. IKVAVPA1, #P<0.05, ###P<0.0001 vs. IKVAVPA2, and +P<0.05, +++P<0.0001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (C) Differentiation conditions used for hNPCs. (D) Representative micrographs of hNPCs treated with IKVAVPA1, PA2, PA4 and PA5; nestin-stem cells (red), ITGB1-receptor (green), and DAPI-nucleus (blue). (E) WB results for ITGB1, p-FAK, FAK, ILK, and TUJ1 in hNPCs treated with laminin and various IKVAVPAs. (F) Representative confocal micrographs of hNPCs treated with IKVAVPA1, PA2, PA4, and PA5; nestin-stem cells (red), SOX2-stem cells (green), TUJ1-neurons (white), and DAPI-nuclei (blue). (G, H) Bar graphs of percentages of SOX2+ / nestin+ stem cells (G) and TUJ1+ neurons (H) treated with various IKVAVPAs (error bars correspond to three independent differentiations; **P<0.01, ***P<0.001 vs. IKVAVPA2 and ##P<0.01, ###P<0.001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (I) Fluorescence anisotropy (left) and K residue relaxation time (right) obtained with IKVAVPA2 nanofibers in the absence (No Ca2+) or presence (Ca2+) of calcium ions (***P<0.001, Student's t-test). (J) WB results of ITGB1, p-FAK, FAK, and ILK in hNPC treated with IKVAVPA2 in the absence (-) or presence (+) of Ca2+.Scale bar: (D) 10mm, (F) 100mm. [Figure 2G-2H]Figures 2A-2J show the in vitro effects of supramolecular motion on hNPC signaling. (A) Molecular graphics representation of IKVAVPA nanofibers, showing the chemical structure and position of DPH used as a probe in fluorescence depolarization measurements (top); bar graph of fluorescence anisotropy of IKVAVPA solution (error bars correspond to three independent experiments; no significant difference in ns, ***P<0.0001, one-way ANOVA and Bonferroni method). (B) Chemical structure of the IKVAV peptide sequence, with K residues observed by NMR highlighted (top); bar graph of K relaxation times for various IKVAVPAs investigated (error bars correspond to 3 experiments for each condition; ***P<0.0001 vs. IKVAVPA1, #P<0.05, ###P<0.0001 vs. IKVAVPA2, and +P<0.05, +++P<0.0001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (C) Differentiation conditions used for hNPCs. (D) Representative micrographs of hNPCs treated with IKVAVPA1, PA2, PA4 and PA5; nestin-stem cells (red), ITGB1-receptor (green), and DAPI-nucleus (blue). (E) WB results for ITGB1, p-FAK, FAK, ILK, and TUJ1 in hNPCs treated with laminin and various IKVAVPAs. (F) Representative confocal micrographs of hNPCs treated with IKVAVPA1, PA2, PA4, and PA5; nestin-stem cells (red), SOX2-stem cells (green), TUJ1-neurons (white), and DAPI-nuclei (blue). (G, H) Bar graphs of percentages of SOX2+ / nestin+ stem cells (G) and TUJ1+ neurons (H) treated with various IKVAVPAs (error bars correspond to three independent differentiations; **P<0.01, ***P<0.001 vs. IKVAVPA2 and ##P<0.01, ###P<0.001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (I) Fluorescence anisotropy (left) and K residue relaxation time (right) obtained with IKVAVPA2 nanofibers in the absence (No Ca2+) or presence (Ca2+) of calcium ions (***P<0.001, Student's t-test). (J) WB results of ITGB1, p-FAK, FAK, and ILK in hNPC treated with IKVAVPA2 in the absence (-) or presence (+) of Ca2+.Scale bar: (D) 10mm, (F) 100mm. [Figure 2I-2J]Figures 2A-2J show the in vitro effects of supramolecular motion on hNPC signaling. (A) Molecular graphics representation of IKVAVPA nanofibers, showing the chemical structure and position of DPH used as a probe in fluorescence depolarization measurements (top); bar graph of fluorescence anisotropy of IKVAVPA solution (error bars correspond to three independent experiments; no significant difference in ns, ***P<0.0001, one-way ANOVA and Bonferroni method). (B) Chemical structure of the IKVAV peptide sequence, with K residues observed by NMR highlighted (top); bar graph of K relaxation times for various IKVAVPAs investigated (error bars correspond to 3 experiments for each condition; ***P<0.0001 vs. IKVAVPA1, #P<0.05, ###P<0.0001 vs. IKVAVPA2, and +P<0.05, +++P<0.0001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (C) Differentiation conditions used for hNPCs. (D) Representative micrographs of hNPCs treated with IKVAVPA1, PA2, PA4 and PA5; nestin-stem cells (red), ITGB1-receptor (green), and DAPI-nucleus (blue). (E) WB results for ITGB1, p-FAK, FAK, ILK, and TUJ1 in hNPCs treated with laminin and various IKVAVPAs. (F) Representative confocal micrographs of hNPCs treated with IKVAVPA1, PA2, PA4, and PA5; nestin-stem cells (red), SOX2-stem cells (green), TUJ1-neurons (white), and DAPI-nuclei (blue). (G, H) Bar graphs of percentages of SOX2+ / nestin+ stem cells (G) and TUJ1+ neurons (H) treated with various IKVAVPAs (error bars correspond to three independent differentiations; **P<0.01, ***P<0.001 vs. IKVAVPA2 and ##P<0.01, ###P<0.001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). (I) Fluorescence anisotropy (left) and K residue relaxation time (right) obtained with IKVAVPA2 nanofibers in the absence (No Ca2+) or presence (Ca2+) of calcium ions (***P<0.001, Student's t-test). (J) WB results of ITGB1, p-FAK, FAK, and ILK in hNPC treated with IKVAVPA2 in the absence (-) or presence (+) of Ca2+.Scale bar: (D) 10mm, (F) 100mm. [Figure 3A]Figures 3A-3L show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, produce differences in corticospinal tract axon growth after SCI. (A) Chemical structures of the two PA molecules used. (B) Molecular graphics of supramolecular nanofibers presenting two bioactive signals (top); cryo-TEM micrographs of coassemblies of IKVAVPA2 and various FGF2PAs (FGF2PA1 and FGF2PA2) (bottom). (C) Storage modulus of each coassembly of IKVAVPA2 (green) and various FGF2PAs (FGF2PA1 (red) and FGF2PA2 (blue)). (D) Fluorescence micrograph of spinal cord (green) injected with IKVAVPA2+FGF2PA1 (red) covalently labeled with Alexa 647. (E) Dot plot of PA scaffold volume as a function of time after transplantation. (F) Schematic diagram showing BDA and PA injection sites (left); fluorescence micrographs of the cerebral cortex (upper right) stained with NeuN-neurons (green), BDA-labeled neurons (red), and DAPI-nuclei (blue), and a transverse spinal cord section (lower right) stained with GFAP-astrocytes (green), BDA-labeled descending axons (red), and DAPI-nuclei (blue). (G) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (H) Representative magnified image of the section in G. (I) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method).(J) WB results (left) and dot plot of normalized values ​​(right) for GAP43 and MBP proteins in the sham group, IKVAVPA2 group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group (**P<0.01, ***P<0.001 vs. sham group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (K) Representative 3D fluorescence micrograph of axonal regrowth labeled with BDA (red), showing myelin basic protein (MBP, green) (left) and laminin (white) (right). (L)J shows the Western blot (WB) results of laminin and fibronectin expression under the conditions described in J (left), and a dot plot of normalized values ​​(right) (***P<0.00 vs. Sham group 1 and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). Data points correspond to 3 animals per group in E, and 4 animals per group in J and L. Scale bars: (D, G) 1500 μm, (F) 25 μm (top) and 200 μm (bottom), (H) 100 μm, and (K) 2 μm. [Figure 3B-3C]Figures 3A-3L show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, produce differences in corticospinal tract axon growth after SCI. (A) Chemical structures of the two PA molecules used. (B) Molecular graphics of supramolecular nanofibers presenting two bioactive signals (top); cryo-TEM micrographs of coassemblies of IKVAVPA2 and various FGF2PAs (FGF2PA1 and FGF2PA2) (bottom). (C) Storage modulus of each coassembly of IKVAVPA2 (green) and various FGF2PAs (FGF2PA1 (red) and FGF2PA2 (blue)). (D) Fluorescence micrograph of spinal cord (green) injected with IKVAVPA2+FGF2PA1 (red) covalently labeled with Alexa 647. (E) Dot plot of PA scaffold volume as a function of time after transplantation. (F) Schematic diagram showing BDA and PA injection sites (left); fluorescence micrographs of the cerebral cortex (upper right) stained with NeuN-neurons (green), BDA-labeled neurons (red), and DAPI-nuclei (blue), and a transverse spinal cord section (lower right) stained with GFAP-astrocytes (green), BDA-labeled descending axons (red), and DAPI-nuclei (blue). (G) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (H) Representative magnified image of the section in G. (I) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method).(J) WB results (left) and dot plot of normalized values ​​(right) for GAP43 and MBP proteins in the sham group, IKVAVPA2 group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group (**P<0.01, ***P<0.001 vs. sham group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (K) Representative 3D fluorescence micrograph of axonal regrowth labeled with BDA (red), showing myelin basic protein (MBP, green) (left) and laminin (white) (right). (L)J shows the Western blot (WB) results of laminin and fibronectin expression under the conditions described in J (left), and a dot plot of normalized values ​​(right) (***P<0.00 vs. Sham group 1 and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). Data points correspond to 3 animals per group in E, and 4 animals per group in J and L. Scale bars: (D, G) 1500 μm, (F) 25 μm (top) and 200 μm (bottom), (H) 100 μm, and (K) 2 μm. [Figure 3D-3F]Figures 3A-3L show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, produce differences in corticospinal tract axon growth after SCI. (A) Chemical structures of the two PA molecules used. (B) Molecular graphics of supramolecular nanofibers presenting two bioactive signals (top); cryo-TEM micrographs of coassemblies of IKVAVPA2 and various FGF2PAs (FGF2PA1 and FGF2PA2) (bottom). (C) Storage modulus of each coassembly of IKVAVPA2 (green) and various FGF2PAs (FGF2PA1 (red) and FGF2PA2 (blue)). (D) Fluorescence micrograph of spinal cord (green) injected with IKVAVPA2+FGF2PA1 (red) covalently labeled with Alexa 647. (E) Dot plot of PA scaffold volume as a function of time after transplantation. (F) Schematic diagram showing BDA and PA injection sites (left); fluorescence micrographs of the cerebral cortex (upper right) stained with NeuN-neurons (green), BDA-labeled neurons (red), and DAPI-nuclei (blue), and a transverse spinal cord section (lower right) stained with GFAP-astrocytes (green), BDA-labeled descending axons (red), and DAPI-nuclei (blue). (G) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (H) Representative magnified image of the section in G. (I) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method).(J) WB results (left) and dot plot of normalized values ​​(right) for GAP43 and MBP proteins in the sham group, IKVAVPA2 group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group (**P<0.01, ***P<0.001 vs. sham group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (K) Representative 3D fluorescence micrograph of axonal regrowth labeled with BDA (red), showing myelin basic protein (MBP, green) (left) and laminin (white) (right). (L)J shows the Western blot (WB) results of laminin and fibronectin expression under the conditions described in J (left), and a dot plot of normalized values ​​(right) (***P<0.00 vs. Sham group 1 and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). Data points correspond to 3 animals per group in E, and 4 animals per group in J and L. Scale bars: (D, G) 1500 μm, (F) 25 μm (top) and 200 μm (bottom), (H) 100 μm, and (K) 2 μm. [Figure 3G]Figures 3A-3L show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, produce differences in corticospinal tract axon growth after SCI. (A) Chemical structures of the two PA molecules used. (B) Molecular graphics of supramolecular nanofibers presenting two bioactive signals (top); cryo-TEM micrographs of coassemblies of IKVAVPA2 and various FGF2PAs (FGF2PA1 and FGF2PA2) (bottom). (C) Storage modulus of each coassembly of IKVAVPA2 (green) and various FGF2PAs (FGF2PA1 (red) and FGF2PA2 (blue)). (D) Fluorescence micrograph of spinal cord (green) injected with IKVAVPA2+FGF2PA1 (red) covalently labeled with Alexa 647. (E) Dot plot of PA scaffold volume as a function of time after transplantation. (F) Schematic diagram showing BDA and PA injection sites (left); fluorescence micrographs of the cerebral cortex (upper right) stained with NeuN-neurons (green), BDA-labeled neurons (red), and DAPI-nuclei (blue), and a transverse spinal cord section (lower right) stained with GFAP-astrocytes (green), BDA-labeled descending axons (red), and DAPI-nuclei (blue). (G) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (H) Representative magnified image of the section in G. (I) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method).(J) WB results (left) and dot plot of normalized values ​​(right) for GAP43 and MBP proteins in the sham group, IKVAVPA2 group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group (**P<0.01, ***P<0.001 vs. sham group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (K) Representative 3D fluorescence micrograph of axonal regrowth labeled with BDA (red), showing myelin basic protein (MBP, green) (left) and laminin (white) (right). (L)J shows the Western blot (WB) results of laminin and fibronectin expression under the conditions described in J (left), and a dot plot of normalized values ​​(right) (***P<0.00 vs. Sham group 1 and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). Data points correspond to 3 animals per group in E, and 4 animals per group in J and L. Scale bars: (D, G) 1500 μm, (F) 25 μm (top) and 200 μm (bottom), (H) 100 μm, and (K) 2 μm. [Figure 3H]Figures 3A-3L show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, produce differences in corticospinal tract axon growth after SCI. (A) Chemical structures of the two PA molecules used. (B) Molecular graphics of supramolecular nanofibers presenting two bioactive signals (top); cryo-TEM micrographs of coassemblies of IKVAVPA2 and various FGF2PAs (FGF2PA1 and FGF2PA2) (bottom). (C) Storage modulus of each coassembly of IKVAVPA2 (green) and various FGF2PAs (FGF2PA1 (red) and FGF2PA2 (blue)). (D) Fluorescence micrograph of spinal cord (green) injected with IKVAVPA2+FGF2PA1 (red) covalently labeled with Alexa 647. (E) Dot plot of PA scaffold volume as a function of time after transplantation. (F) Schematic diagram showing BDA and PA injection sites (left); fluorescence micrographs of the cerebral cortex (upper right) stained with NeuN-neurons (green), BDA-labeled neurons (red), and DAPI-nuclei (blue), and a transverse spinal cord section (lower right) stained with GFAP-astrocytes (green), BDA-labeled descending axons (red), and DAPI-nuclei (blue). (G) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (H) Representative magnified image of the section in G. (I) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method).(J) WB results (left) and dot plot of normalized values ​​(right) for GAP43 and MBP proteins in the sham group, IKVAVPA2 group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group (**P<0.01, ***P<0.001 vs. sham group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (K) Representative 3D fluorescence micrograph of axonal regrowth labeled with BDA (red), showing myelin basic protein (MBP, green) (left) and laminin (white) (right). (L)J shows the Western blot (WB) results of laminin and fibronectin expression under the conditions described in J (left), and a dot plot of normalized values ​​(right) (***P<0.00 vs. Sham group 1 and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). Data points correspond to 3 animals per group in E, and 4 animals per group in J and L. Scale bars: (D, G) 1500 μm, (F) 25 μm (top) and 200 μm (bottom), (H) 100 μm, and (K) 2 μm. [Figure 3I]Figures 3A-3L show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, produce differences in corticospinal tract axon growth after SCI. (A) Chemical structures of the two PA molecules used. (B) Molecular graphics of supramolecular nanofibers presenting two bioactive signals (top); cryo-TEM micrographs of coassemblies of IKVAVPA2 and various FGF2PAs (FGF2PA1 and FGF2PA2) (bottom). (C) Storage modulus of each coassembly of IKVAVPA2 (green) and various FGF2PAs (FGF2PA1 (red) and FGF2PA2 (blue)). (D) Fluorescence micrograph of spinal cord (green) injected with IKVAVPA2+FGF2PA1 (red) covalently labeled with Alexa 647. (E) Dot plot of PA scaffold volume as a function of time after transplantation. (F) Schematic diagram showing BDA and PA injection sites (left); fluorescence micrographs of the cerebral cortex (upper right) stained with NeuN-neurons (green), BDA-labeled neurons (red), and DAPI-nuclei (blue), and a transverse spinal cord section (lower right) stained with GFAP-astrocytes (green), BDA-labeled descending axons (red), and DAPI-nuclei (blue). (G) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (H) Representative magnified image of the section in G. (I) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method).(J) WB results (left) and dot plot of normalized values ​​(right) for GAP43 and MBP proteins in the sham group, IKVAVPA2 group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group (**P<0.01, ***P<0.001 vs. sham group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (K) Representative 3D fluorescence micrograph of axonal regrowth labeled with BDA (red), showing myelin basic protein (MBP, green) (left) and laminin (white) (right). (L)J shows the Western blot (WB) results of laminin and fibronectin expression under the conditions described in J (left), and a dot plot of normalized values ​​(right) (***P<0.00 vs. Sham group 1 and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). Data points correspond to 3 animals per group in E, and 4 animals per group in J and L. Scale bars: (D, G) 1500 μm, (F) 25 μm (top) and 200 μm (bottom), (H) 100 μm, and (K) 2 μm. [Figure 3J]Figures 3A-3L show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, produce differences in corticospinal tract axon growth after SCI. (A) Chemical structures of the two PA molecules used. (B) Molecular graphics of supramolecular nanofibers presenting two bioactive signals (top); cryo-TEM micrographs of coassemblies of IKVAVPA2 and various FGF2PAs (FGF2PA1 and FGF2PA2) (bottom). (C) Storage modulus of each coassembly of IKVAVPA2 (green) and various FGF2PAs (FGF2PA1 (red) and FGF2PA2 (blue)). (D) Fluorescence micrograph of spinal cord (green) injected with IKVAVPA2+FGF2PA1 (red) covalently labeled with Alexa 647. (E) Dot plot of PA scaffold volume as a function of time after transplantation. (F) Schematic diagram showing BDA and PA injection sites (left); fluorescence micrographs of the cerebral cortex (upper right) stained with NeuN-neurons (green), BDA-labeled neurons (red), and DAPI-nuclei (blue), and a transverse spinal cord section (lower right) stained with GFAP-astrocytes (green), BDA-labeled descending axons (red), and DAPI-nuclei (blue). (G) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (H) Representative magnified image of the section in G. (I) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method).(J) WB results (left) and dot plot of normalized values ​​(right) for GAP43 and MBP proteins in the sham group, IKVAVPA2 group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group (**P<0.01, ***P<0.001 vs. sham group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (K) Representative 3D fluorescence micrograph of axonal regrowth labeled with BDA (red), showing myelin basic protein (MBP, green) (left) and laminin (white) (right). (L)J shows the Western blot (WB) results of laminin and fibronectin expression under the conditions described in J (left), and a dot plot of normalized values ​​(right) (***P<0.00 vs. Sham group 1 and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). Data points correspond to 3 animals per group in E, and 4 animals per group in J and L. Scale bars: (D, G) 1500 μm, (F) 25 μm (top) and 200 μm (bottom), (H) 100 μm, and (K) 2 μm. [Figure 3K]Figures 3A-3L show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, produce differences in corticospinal tract axon growth after SCI. (A) Chemical structures of the two PA molecules used. (B) Molecular graphics of supramolecular nanofibers presenting two bioactive signals (top); cryo-TEM micrographs of coassemblies of IKVAVPA2 and various FGF2PAs (FGF2PA1 and FGF2PA2) (bottom). (C) Storage modulus of each coassembly of IKVAVPA2 (green) and various FGF2PAs (FGF2PA1 (red) and FGF2PA2 (blue)). (D) Fluorescence micrograph of spinal cord (green) injected with IKVAVPA2+FGF2PA1 (red) covalently labeled with Alexa 647. (E) Dot plot of PA scaffold volume as a function of time after transplantation. (F) Schematic diagram showing BDA and PA injection sites (left); fluorescence micrographs of the cerebral cortex (upper right) stained with NeuN-neurons (green), BDA-labeled neurons (red), and DAPI-nuclei (blue), and a transverse spinal cord section (lower right) stained with GFAP-astrocytes (green), BDA-labeled descending axons (red), and DAPI-nuclei (blue). (G) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (H) Representative magnified image of the section in G. (I) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method).(J) WB results (left) and dot plot of normalized values ​​(right) for GAP43 and MBP proteins in the sham group, IKVAVPA2 group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group (**P<0.01, ***P<0.001 vs. sham group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (K) Representative 3D fluorescence micrograph of axonal regrowth labeled with BDA (red), showing myelin basic protein (MBP, green) (left) and laminin (white) (right). (L)J shows the Western blot (WB) results of laminin and fibronectin expression under the conditions described in J (left), and a dot plot of normalized values ​​(right) (***P<0.00 vs. Sham group 1 and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). Data points correspond to 3 animals per group in E, and 4 animals per group in J and L. Scale bars: (D, G) 1500 μm, (F) 25 μm (top) and 200 μm (bottom), (H) 100 μm, and (K) 2 μm. [Figure 3L]Figures 3A-3L show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, produce differences in corticospinal tract axon growth after SCI. (A) Chemical structures of the two PA molecules used. (B) Molecular graphics of supramolecular nanofibers presenting two bioactive signals (top); cryo-TEM micrographs of coassemblies of IKVAVPA2 and various FGF2PAs (FGF2PA1 and FGF2PA2) (bottom). (C) Storage modulus of each coassembly of IKVAVPA2 (green) and various FGF2PAs (FGF2PA1 (red) and FGF2PA2 (blue)). (D) Fluorescence micrograph of spinal cord (green) injected with IKVAVPA2+FGF2PA1 (red) covalently labeled with Alexa 647. (E) Dot plot of PA scaffold volume as a function of time after transplantation. (F) Schematic diagram showing BDA and PA injection sites (left); fluorescence micrographs of the cerebral cortex (upper right) stained with NeuN-neurons (green), BDA-labeled neurons (red), and DAPI-nuclei (blue), and a transverse spinal cord section (lower right) stained with GFAP-astrocytes (green), BDA-labeled descending axons (red), and DAPI-nuclei (blue). (G) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (H) Representative magnified image of the section in G. (I) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method).(J) WB results (left) and dot plot of normalized values ​​(right) for GAP43 and MBP proteins in the sham group, IKVAVPA2 group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group (**P<0.01, ***P<0.001 vs. sham group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (K) Representative 3D fluorescence micrograph of axonal regrowth labeled with BDA (red), showing myelin basic protein (MBP, green) (left) and laminin (white) (right). (L)J shows the Western blot (WB) results of laminin and fibronectin expression under the conditions described in J (left), and a dot plot of normalized values ​​(right) (***P<0.00 vs. Sham group 1 and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). Data points correspond to 3 animals per group in E, and 4 animals per group in J and L. Scale bars: (D, G) 1500 μm, (F) 25 μm (top) and 200 μm (bottom), (H) 100 μm, and (K) 2 μm. [Figure 4A-4B]Figures 4A-4E show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, result in differences in angiogenesis. (A) Fluorescence micrographs of transverse spinal cord sections in the undamaged group, IKVAVPA2+FGF2PA1 group, IKVAVPA2+FGF2PA2 group, and sham group; GFAP-astrocytes (green), DiI-labeled vessels (red), and DAPI-nuclei (blue). (B) Dot plots of vascular area fraction, perfusion vessel length, and branching number in transverse sections of each group A (*P<0.05, ***P<0.0001 vs. sham group and ##P<0.001, ###P<0.0001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (C) Fluorescence images of BrdU+ / CD31+ cells in the center of lesions in animals injected with IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2; CD31-vascular tissue (green), BrdU-newly generated cells (red), and DAPI-nuclei (blue). (D) Dot plots of BrdU+ / CD31+ cell counts per mm2 in groups administered with IKVAVPA2 alone, IKVAVPA2+FGF2PA1, IKVAVPA2+FGF2PA2, and physiological saline (sham) (*P<0.05, ***P<0.001 vs. sham group and ###P<0.0001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (E) WB results for CD31 protein (left) and dot plot of normalized values ​​(right) (**P<0.001, ***P<0.0001 vs. Siamese group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). Data points correspond to 6 animals in each group in B and D, and 4 animals in each group in E. Scale bars: (A) 200 μm, (C) 25 μm. [Figure 4C-4E]Figures 4A-4E show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, result in differences in angiogenesis. (A) Fluorescence micrographs of transverse spinal cord sections in the undamaged group, IKVAVPA2+FGF2PA1 group, IKVAVPA2+FGF2PA2 group, and sham group; GFAP-astrocytes (green), DiI-labeled vessels (red), and DAPI-nuclei (blue). (B) Dot plots of vascular area fraction, perfusion vessel length, and branching number in transverse sections of each group A (*P<0.05, ***P<0.0001 vs. sham group and ##P<0.001, ###P<0.0001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (C) Fluorescence images of BrdU+ / CD31+ cells in the center of lesions in animals injected with IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2; CD31-vascular tissue (green), BrdU-newly generated cells (red), and DAPI-nuclei (blue). (D) Dot plots of BrdU+ / CD31+ cell counts per mm2 in groups administered with IKVAVPA2 alone, IKVAVPA2+FGF2PA1, IKVAVPA2+FGF2PA2, and physiological saline (sham) (*P<0.05, ***P<0.001 vs. sham group and ###P<0.0001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (E) WB results for CD31 protein (left) and dot plot of normalized values ​​(right) (**P<0.001, ***P<0.0001 vs. Siamese group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). Data points correspond to 6 animals in each group in B and D, and 4 animals in each group in E. Scale bars: (A) 200 μm, (C) 25 μm. [Figure 5A-5B]Figures 5A-5D show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, produce differences in neuronal survival and functional recovery. (A) Fluorescence micrographs of transverse spinal cord sections corresponding to the undamaged group, IKVAVPA2+FGF2PA1 group, IKVAVPA2+FGF2PA2 group, and sham group; showing NeuN neurons (green), DiI-labeled blood vessels (red), and DAPI nuclei (blue), with dashed lines indicating gray matter (horn). (B) High-magnification images of the anterior horn region of section A; NeuN neurons (green), DiI-labeled blood vessels (red), and DAPI nuclei (blue) (left); ChAT motor neurons (green), DiI-labeled blood vessels (red), and DAPI nuclei (blue) (right). (C) Dot plot showing the number of NeuN+ cells (left) and ChAT+ cells (right) per cross-sectional section (data points correspond to a total of 48 sections, 6 mice per group and 8 sections per mouse; **P<0.01, ***P<0.001 vs. sham group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (D) Experimental timeline of the in vivo experiment (top) and Basso Mouse Scale (BMS) of walking movement (bottom) (error bars correspond to 38 mice per group; **P<0.001, ***P<0.0001 all PA group vs. sham group and ###P<0.0001 vs. IKVAVPA2+FGF2PA2 group and IKVAVPA2 group, two-way ANOVA with repeated measures and Bonferroni method). Scale bars: (A) 200 μm, (B) 25 μm. [Figure 5C-5D]Figures 5A-5D show that two chemically different PA scaffolds, each possessing two identical bioactive sequences, produce differences in neuronal survival and functional recovery. (A) Fluorescence micrographs of transverse spinal cord sections corresponding to the undamaged group, IKVAVPA2+FGF2PA1 group, IKVAVPA2+FGF2PA2 group, and sham group; showing NeuN neurons (green), DiI-labeled blood vessels (red), and DAPI nuclei (blue), with dashed lines indicating gray matter (horn). (B) High-magnification images of the anterior horn region of section A; NeuN neurons (green), DiI-labeled blood vessels (red), and DAPI nuclei (blue) (left); ChAT motor neurons (green), DiI-labeled blood vessels (red), and DAPI nuclei (blue) (right). (C) Dot plot showing the number of NeuN+ cells (left) and ChAT+ cells (right) per cross-sectional section (data points correspond to a total of 48 sections, 6 mice per group and 8 sections per mouse; **P<0.01, ***P<0.001 vs. sham group and ###P<0.001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). (D) Experimental timeline of the in vivo experiment (top) and Basso Mouse Scale (BMS) of walking movement (bottom) (error bars correspond to 38 mice per group; **P<0.001, ***P<0.0001 all PA group vs. sham group and ###P<0.0001 vs. IKVAVPA2+FGF2PA2 group and IKVAVPA2 group, two-way ANOVA with repeated measures and Bonferroni method). Scale bars: (A) 200 μm, (B) 25 μm. [Figure 6A-6C]Figures 6A-6J show data verifying in vitro the difference in cellular signaling between two types of PA scaffolds exhibiting different supramolecular motions. (A) Confocal micrographs of HUVEC treated with IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2; actin-cytoskeleton (red), DAPI-nucleus (blue). (B) Bar graphs of branching numbers in HUVEC treated with laminin+FGF2, IKVAVPA2 alone, IKVAVPA2+FGF2PA1, and IKVAVPA2+FGF2PA2. (C) WB results using condition B (left) and bar graphs of normalized values ​​of active FGFR1 (p-FGFR1) and active ERK1 / 2 (p-ERK1 / 2) relative to total FGFR1 (FGFR1) (right). (D) Confocal micrographs of hNPCs on IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2 coatings; EDU proliferation marker (red), SOX2-neuronal stem cell marker (green), and DAPI-nucleus (blue). (E) Bar graphs of percentages of EDU+ / SOX2+ cells on various coatings. (F) WB results (left) and bar graphs of normalized values ​​of active FGFR1 (p-FGFR1) and b1 integrin (ITGB1) relative to total FGFR1 (FGFR1) (right). (G) 1H-NMR spin-spin relaxation times of aromatic protons in Y and W amino acids in the 6.81 ppm FGF2 mimetic signal (solid line is a simple linear regression best fit). (H) Bar graph of aromatic relaxation times measured in G (error bars correspond to 3 experiments for each condition; *P<0.05, Student's t-test). (I) Fluorescence anisotropy of FGF2PA chemically modified with Cy3 dye (error bars correspond to 3 independent experiments; ***P<0.001, by Student's t-test). (J) Color coding of RMSF values ​​in clusters of FGF2PA (left) and corresponding bar graph (right) (IKVAVPA2 molecules are shown in transparent gray, ions and water molecules are removed for clarity, and the simulation box is shown in blue) (error bars correspond to 5 independent simulations; **P<0.01, by Student's t-test).Error bars correspond to three independent experiments for each condition in B and E, and four independent experiments in C and F; ***P<0.0001 vs. Laminin + FGF2 and ###P<0.0001 vs. IKVAVPA2 + FGF2PA1, one-way ANOVA and Bonferroni method. Scale bars: (A) 200 μm, (D) 100 μm. [Figure 6D-6F]Figures 6A-6J show data verifying in vitro the difference in cellular signaling between two types of PA scaffolds exhibiting different supramolecular motions. (A) Confocal micrographs of HUVEC treated with IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2; actin-cytoskeleton (red), DAPI-nucleus (blue). (B) Bar graphs of branching numbers in HUVEC treated with laminin+FGF2, IKVAVPA2 alone, IKVAVPA2+FGF2PA1, and IKVAVPA2+FGF2PA2. (C) WB results using condition B (left) and bar graphs of normalized values ​​of active FGFR1 (p-FGFR1) and active ERK1 / 2 (p-ERK1 / 2) relative to total FGFR1 (FGFR1) (right). (D) Confocal micrographs of hNPCs on IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2 coatings; EDU proliferation marker (red), SOX2-neuronal stem cell marker (green), and DAPI-nucleus (blue). (E) Bar graphs of percentages of EDU+ / SOX2+ cells on various coatings. (F) WB results (left) and bar graphs of normalized values ​​of active FGFR1 (p-FGFR1) and b1 integrin (ITGB1) relative to total FGFR1 (FGFR1) (right). (G) 1H-NMR spin-spin relaxation times of aromatic protons in Y and W amino acids in the 6.81 ppm FGF2 mimetic signal (solid line is a simple linear regression best fit). (H) Bar graph of aromatic relaxation times measured in G (error bars correspond to 3 experiments for each condition; *P<0.05, Student's t-test). (I) Fluorescence anisotropy of FGF2PA chemically modified with Cy3 dye (error bars correspond to 3 independent experiments; ***P<0.001, by Student's t-test). (J) Color coding of RMSF values ​​in clusters of FGF2PA (left) and corresponding bar graph (right) (IKVAVPA2 molecules are shown in transparent gray, ions and water molecules are removed for clarity, and the simulation box is shown in blue) (error bars correspond to 5 independent simulations; **P<0.01, by Student's t-test).Error bars correspond to three independent experiments for each condition in B and E, and four independent experiments in C and F; ***P<0.0001 vs. Laminin + FGF2 and ###P<0.0001 vs. IKVAVPA2 + FGF2PA1, one-way ANOVA and Bonferroni method. Scale bars: (A) 200 μm, (D) 100 μm. [Figure 6G-6J]Figures 6A-6J show data verifying in vitro the difference in cellular signaling between two types of PA scaffolds exhibiting different supramolecular motions. (A) Confocal micrographs of HUVEC treated with IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2; actin-cytoskeleton (red), DAPI-nucleus (blue). (B) Bar graphs of branching numbers in HUVEC treated with laminin+FGF2, IKVAVPA2 alone, IKVAVPA2+FGF2PA1, and IKVAVPA2+FGF2PA2. (C) WB results using condition B (left) and bar graphs of normalized values ​​of active FGFR1 (p-FGFR1) and active ERK1 / 2 (p-ERK1 / 2) relative to total FGFR1 (FGFR1) (right). (D) Confocal micrographs of hNPCs on IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2 coatings; EDU proliferation marker (red), SOX2-neuronal stem cell marker (green), and DAPI-nucleus (blue). (E) Bar graphs of percentages of EDU+ / SOX2+ cells on various coatings. (F) WB results (left) and bar graphs of normalized values ​​of active FGFR1 (p-FGFR1) and b1 integrin (ITGB1) relative to total FGFR1 (FGFR1) (right). (G) 1H-NMR spin-spin relaxation times of aromatic protons in Y and W amino acids in the 6.81 ppm FGF2 mimetic signal (solid line is a simple linear regression best fit). (H) Bar graph of aromatic relaxation times measured in G (error bars correspond to 3 experiments for each condition; *P<0.05, Student's t-test). (I) Fluorescence anisotropy of FGF2PA chemically modified with Cy3 dye (error bars correspond to 3 independent experiments; ***P<0.001, by Student's t-test). (J) Color coding of RMSF values ​​in clusters of FGF2PA (left) and corresponding bar graph (right) (IKVAVPA2 molecules are shown in transparent gray, ions and water molecules are removed for clarity, and the simulation box is shown in blue) (error bars correspond to 5 independent simulations; **P<0.01, by Student's t-test).Error bars correspond to three independent experiments for each condition in B and E, and four independent experiments in C and F; ***P<0.0001 vs. Laminin + FGF2 and ###P<0.0001 vs. IKVAVPA2 + FGF2PA1, one-way ANOVA and Bonferroni method. Scale bars: (A) 200 μm, (D) 100 μm. [Figure 7] The chemical structures (left) and mass spectra (right) of various IKVAVPA compounds are shown. [Figure 8] The following plots show the root mean squared deviation (RMSD) of various IKVAVPAs over time. [Figure 9] The bar graphs show the normalized values ​​of ITGB1, p-FAK / FAK, ILK, and TUJ1 for hNPC cultured on ornithine coating and those treated with laminin and IKVAVPA libraries (error bars correspond to three independent differentiations: **P<0.01, ***P<0.001 vs. IKVAVPA2 and #P<0.05, ##P<0.01, ###P<0.001 vs. IKVAVPA5, one-way ANOVA and Bonferroni method). [Figure 10A-10B] Figures 10A-10C show the effects of calcium on supramolecular motion and in vitro cell signaling. (A) Anisotropy of IKVAVPA2 and IKVAVPA5 solutions in the absence (No Ca2+) or presence (Ca2+) of calcium (error bars correspond to three independent experiments; **P<0.01, ***P<0.001, Student's t-test). (B) Representative fluorescence micrographs of hNPCs cultured under the conditions described in A. (C) WB results (left) of ITGB1, p-FAK / FAK, ILK, and TUJ1 in hNPC treated with IKVAVPA2 or IKVAVPA5 in the absence (-) or presence (+) of calcium (Ca2+), and corresponding bar graphs (right) showing normalized protein concentrations (calcium alone (+) used as a control; error bars correspond to three independent experiments for each condition; **P<0.01, ***P<0.001, by Student's t-test). Scale bar: 10mm. [Figure 10C]Figures 10A-10C show the effects of calcium on supramolecular motion and in vitro cell signaling. (A) Anisotropy of IKVAVPA2 and IKVAVPA5 solutions in the absence (No Ca2+) or presence (Ca2+) of calcium (error bars correspond to three independent experiments; **P<0.01, ***P<0.001, Student's t-test). (B) Representative fluorescence micrographs of hNPCs cultured under the conditions described in A. (C) WB results (left) of ITGB1, p-FAK / FAK, ILK, and TUJ1 in hNPC treated with IKVAVPA2 or IKVAVPA5 in the absence (-) or presence (+) of calcium (Ca2+), and corresponding bar graphs (right) showing normalized protein concentrations (calcium alone (+) used as a control; error bars correspond to three independent experiments for each condition; **P<0.01, ***P<0.001, by Student's t-test). Scale bar: 10mm. [Figure 11A-11B] Figures 11A-11C show cryo-TEM images and storage moduli of coassemblies of IKVAVPA2 and various FGF2PAs at different molar ratios. (A) Cryo-TEM micrographs (left) and storage moduli (right) of coassemblies of IKVAVPA2 and various FGF2PAs (PA1 or PA2) at molar ratios of 95:5, (B) 90:10, and (C) 80:20 (data points correspond to three replicate experiments for each condition; ns indicates no significant difference, *P<0.05, Student's two-sided t-test). Scale bar: 200nm. [Figure 11C] Figures 11A-11C show cryo-TEM images and storage moduli of coassemblies of IKVAVPA2 and various FGF2PAs at different molar ratios. (A) Cryo-TEM micrographs (left) and storage moduli (right) of coassemblies of IKVAVPA2 and various FGF2PAs (PA1 or PA2) at molar ratios of 95:5, (B) 90:10, and (C) 80:20 (data points correspond to three replicate experiments for each condition; ns indicates no significant difference, *P<0.05, Student's two-sided t-test). Scale bar: 200nm. [Figures 12A-12E]Figures 12A–12F show the characterization of the IKVAVPA2+FGF2PA co-assembly systems. (A) SAXS scattering patterns, (B) WAXS profiles, and (C) FT-IR for IKVAVPA2 (green), IKVAVPA2+FGF2PA1 (red), and IKVAVPA2+FGF2PA2 (blue). (D) High-magnification negative TEM images of IKVAVPA2 (green), IKVAVPA2+FGF2PA1 (red), and IKVAVPA2+FGF2PA2 (blue). (E) Fiber widths for the conditions described in D (error bars correspond to 50 fibers for each condition; **P<0.01 vs. IKVAVPA2 and #P<0.05 vs. IKVAVPA2+FGF2PA1, by one-way ANOVA and Bonferroni method). (F) Optical density (OD) plots at 600 nm for various percentages of IKVAVPA2, FGF2PA1, FGF2PA2 and their coassemblies (left), and photographs of FGF2PA (orange) and its coassemblies with IKVAVPA2 (red) (right) (error bars correspond to 3 replicate experiments for each condition; ***P < 0.0001 vs. its coassembly sample, one-way ANOVA and Bonferroni method). Scale bar: 100 nm. [Figure 12F]Figures 12A–12F show the characterization of the IKVAVPA2+FGF2PA co-assembly systems. (A) SAXS scattering patterns, (B) WAXS profiles, and (C) FT-IR for IKVAVPA2 (green), IKVAVPA2+FGF2PA1 (red), and IKVAVPA2+FGF2PA2 (blue). (D) High-magnification negative TEM images of IKVAVPA2 (green), IKVAVPA2+FGF2PA1 (red), and IKVAVPA2+FGF2PA2 (blue). (E) Fiber widths for the conditions described in D (error bars correspond to 50 fibers for each condition; **P<0.01 vs. IKVAVPA2 and #P<0.05 vs. IKVAVPA2+FGF2PA1, by one-way ANOVA and Bonferroni method). (F) Optical density (OD) plots at 600 nm for various percentages of IKVAVPA2, FGF2PA1, FGF2PA2 and their coassemblies (left), and photographs of FGF2PA (orange) and its coassemblies with IKVAVPA2 (red) (right) (error bars correspond to 3 replicate experiments for each condition; ***P < 0.0001 vs. its coassembly sample, one-way ANOVA and Bonferroni method). Scale bar: 100 nm. [Figures 13A-13D] Figures 13A–13E show the clearing of spinal cord injected with a dual-signal coassembly. (A) Chemical structure and (B) mass spectrum of IKVAVPA2 labeled with Alexa Fluor® 647. (C) CryoTEM image of IKVAVPA2 labeled with Alexa Fluor® 647. (D) Photographs of mouse spinal cord injected with PA before (de-clearing) and after (clearing) tissue clearing. (E) Complete microscopic reconstruction of mouse spinal cord (green) injected with IKVAVPA2 + FGF2PA1 or IKVAVPA2 + FGF2PA2 (both red) after labeling IKVAVPA2 with 1% Alexa Fluor® 647. Scale bars: (C) 100 nm and (E) 1000 μm. [Figure 13E]Figures 13A–13E show the clearing of spinal cord injected with a dual-signal coassembly. (A) Chemical structure and (B) mass spectrum of IKVAVPA2 labeled with Alexa Fluor® 647. (C) CryoTEM image of IKVAVPA2 labeled with Alexa Fluor® 647. (D) Photographs of mouse spinal cord injected with PA before (de-clearing) and after (clearing) tissue clearing. (E) Complete microscopic reconstruction of mouse spinal cord (green) injected with IKVAVPA2 + FGF2PA1 or IKVAVPA2 + FGF2PA2 (both red) after labeling IKVAVPA2 with 1% Alexa Fluor® 647. Scale bars: (C) 100 nm and (E) 1000 μm. [Figure 14A] Figures 14A-14D show the effects of various IKVAVPAs on CST axonal regrowth. (A) Fluorescence micrographs of longitudinal spinal cord sections in the main chain PA group, IKVAVPA1 group, IKVAVPA2 group, and IKVAVPA4 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white longitudinal dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (B) Representative magnified image of section A. (C) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group, +P<0.05, ++P<0.01 vs. IKVAVPA1 and #P<0.05 vs. IKVAVPA4 group, repeated measures of two-way ANOVA and Bonferroni method). (D) WB results of GFAP for the Siamese group, mainchain PA group, IKVAVPA1 group, IKVAVPA2 group, and IKVAVPA4 group (bottom), and corresponding bar graphs (top) showing normalized protein concentrations (data points correspond to 4 animals for each condition; ***P<0.001 vs. Siamese group, #P<0.05 vs. mainchain PA, one-way ANOVA and Bonferroni method). Scale bars: (A) 1500 μm and (B) 100 μm. [Figure 14B]Figures 14A-14D show the effects of various IKVAVPAs on CST axonal regrowth. (A) Fluorescence micrographs of longitudinal spinal cord sections in the main chain PA group, IKVAVPA1 group, IKVAVPA2 group, and IKVAVPA4 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white longitudinal dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (B) Representative magnified image of section A. (C) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group, +P<0.05, ++P<0.01 vs. IKVAVPA1 and #P<0.05 vs. IKVAVPA4 group, repeated measures of two-way ANOVA and Bonferroni method). (D) WB results of GFAP for the Siamese group, mainchain PA group, IKVAVPA1 group, IKVAVPA2 group, and IKVAVPA4 group (bottom), and corresponding bar graphs (top) showing normalized protein concentrations (data points correspond to 4 animals for each condition; ***P<0.001 vs. Siamese group, #P<0.05 vs. mainchain PA, one-way ANOVA and Bonferroni method). Scale bars: (A) 1500 μm and (B) 100 μm. [Figure 14C-14D]Figures 14A-14D show the effects of various IKVAVPAs on CST axonal regrowth. (A) Fluorescence micrographs of longitudinal spinal cord sections in the main chain PA group, IKVAVPA1 group, IKVAVPA2 group, and IKVAVPA4 group; GFAP-astrocytes (green), BDA-labeled axons (red), and DAPI-nuclei (blue); white longitudinal dashed lines indicate the proximal margin (PB), distal margin (DB), and central part of the lesion (LC). (B) Representative magnified image of section A. (C) Schematic diagram of the lesion site and the vertical lines used to count the number of axons crossing at each specified location (top); plot of the number of crossing axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01, ***P<0.001 vs. Siamese group, +P<0.05, ++P<0.01 vs. IKVAVPA1 and #P<0.05 vs. IKVAVPA4 group, repeated measures of two-way ANOVA and Bonferroni method). (D) WB results of GFAP for the Siamese group, mainchain PA group, IKVAVPA1 group, IKVAVPA2 group, and IKVAVPA4 group (bottom), and corresponding bar graphs (top) showing normalized protein concentrations (data points correspond to 4 animals for each condition; ***P<0.001 vs. Siamese group, #P<0.05 vs. mainchain PA, one-way ANOVA and Bonferroni method). Scale bars: (A) 1500 μm and (B) 100 μm. [Figure 15A]Figures 15A-15F show the effects of IKVAVPA2 and various FGF2PA coassemblies on axonal regrowth and glial scar formation. (A) Fluorescence images of longitudinal spinal cord sections of animals injected with physiological saline (sham), IKVAVPA2+FGF2PA1, IKVAVPA2+FGF2PA2, and IKVAVPA2 alone; descending axons (red) and DAPI-nuclei (blue) labeled with BDA; white longitudinal dashed lines indicate the proximal margin (PB) and distal margin (DB). (B) Detailed image of BDA-labeled axons (red) in the center of the lesion under the conditions described in A, with white longitudinal dashed lines indicating the central part of the lesion (LC). (C) Representative images of longitudinal spinal cord sections stained with GFAP-astrocytes (green) and DAPI-nuclei (blue) within the lesion margin under the conditions described in A. (D) WB results using condition A (bottom) and corresponding bar graphs (top) showing normalized protein concentrations of GFAP (data points correspond to 4 animals for each condition; ***P<0.001 vs. Siamese group, one-way ANOVA and Bonferroni method). (E) Representative 3D fluorescence micrographs taken at the center of the lesion, showing BDA-labeled axons (red), GFAP-astrocytes (green), and DAPI-nuclei (blue). (F) 3D micrograph reconstruction of BDA-labeled axonal regrowth in the IKVAVPA2 group, showing coating by myelin basic protein (MBP, green) (top) and laminin (white) (bottom). Scale bars: (A, C) 100 μm, (B) 50 μm, and (E, F) 2 μm. [Figure 15B]Figures 15A-15F show the effects of IKVAVPA2 and various FGF2PA coassemblies on axonal regrowth and glial scar formation. (A) Fluorescence images of longitudinal spinal cord sections of animals injected with physiological saline (sham), IKVAVPA2+FGF2PA1, IKVAVPA2+FGF2PA2, and IKVAVPA2 alone; descending axons (red) and DAPI-nuclei (blue) labeled with BDA; white longitudinal dashed lines indicate the proximal margin (PB) and distal margin (DB). (B) Detailed image of BDA-labeled axons (red) in the center of the lesion under the conditions described in A, with white longitudinal dashed lines indicating the central part of the lesion (LC). (C) Representative images of longitudinal spinal cord sections stained with GFAP-astrocytes (green) and DAPI-nuclei (blue) within the lesion margin under the conditions described in A. (D) WB results using condition A (bottom) and corresponding bar graphs (top) showing normalized protein concentrations of GFAP (data points correspond to 4 animals for each condition; ***P<0.001 vs. Siamese group, one-way ANOVA and Bonferroni method). (E) Representative 3D fluorescence micrographs taken at the center of the lesion, showing BDA-labeled axons (red), GFAP-astrocytes (green), and DAPI-nuclei (blue). (F) 3D micrograph reconstruction of BDA-labeled axonal regrowth in the IKVAVPA2 group, showing coating by myelin basic protein (MBP, green) (top) and laminin (white) (bottom). Scale bars: (A, C) 100 μm, (B) 50 μm, and (E, F) 2 μm. [Figures 15C-15D]Figures 15A-15F show the effects of IKVAVPA2 and various FGF2PA coassemblies on axonal regrowth and glial scar formation. (A) Fluorescence images of longitudinal spinal cord sections of animals injected with physiological saline (sham), IKVAVPA2+FGF2PA1, IKVAVPA2+FGF2PA2, and IKVAVPA2 alone; descending axons (red) and DAPI-nuclei (blue) labeled with BDA; white longitudinal dashed lines indicate the proximal margin (PB) and distal margin (DB). (B) Detailed image of BDA-labeled axons (red) in the center of the lesion under the conditions described in A, with white longitudinal dashed lines indicating the central part of the lesion (LC). (C) Representative images of longitudinal spinal cord sections stained with GFAP-astrocytes (green) and DAPI-nuclei (blue) within the lesion margin under the conditions described in A. (D) WB results using condition A (bottom) and corresponding bar graphs (top) showing normalized protein concentrations of GFAP (data points correspond to 4 animals for each condition; ***P<0.001 vs. Siamese group, one-way ANOVA and Bonferroni method). (E) Representative 3D fluorescence micrographs taken at the center of the lesion, showing BDA-labeled axons (red), GFAP-astrocytes (green), and DAPI-nuclei (blue). (F) 3D micrograph reconstruction of BDA-labeled axonal regrowth in the IKVAVPA2 group, showing coating by myelin basic protein (MBP, green) (top) and laminin (white) (bottom). Scale bars: (A, C) 100 μm, (B) 50 μm, and (E, F) 2 μm. [Figures 15E-15F]Figures 15A-15F show the effects of IKVAVPA2 and various FGF2PA coassemblies on axonal regrowth and glial scar formation. (A) Fluorescence images of longitudinal spinal cord sections of animals injected with physiological saline (sham), IKVAVPA2+FGF2PA1, IKVAVPA2+FGF2PA2, and IKVAVPA2 alone; descending axons (red) and DAPI-nuclei (blue) labeled with BDA; white longitudinal dashed lines indicate the proximal margin (PB) and distal margin (DB). (B) Detailed image of BDA-labeled axons (red) in the center of the lesion under the conditions described in A, with white longitudinal dashed lines indicating the central part of the lesion (LC). (C) Representative images of longitudinal spinal cord sections stained with GFAP-astrocytes (green) and DAPI-nuclei (blue) within the lesion margin under the conditions described in A. (D) WB results using condition A (bottom) and corresponding bar graphs (top) showing normalized protein concentrations of GFAP (data points correspond to 4 animals for each condition; ***P<0.001 vs. Siamese group, one-way ANOVA and Bonferroni method). (E) Representative 3D fluorescence micrographs taken at the center of the lesion, showing BDA-labeled axons (red), GFAP-astrocytes (green), and DAPI-nuclei (blue). (F) 3D micrograph reconstruction of BDA-labeled axonal regrowth in the IKVAVPA2 group, showing coating by myelin basic protein (MBP, green) (top) and laminin (white) (bottom). Scale bars: (A, C) 100 μm, (B) 50 μm, and (E, F) 2 μm. [Figure 16A]Figures 16A-16E show the effects of IKVAVPA2 and various FGF2PA coassemblies on serotonergic neuron regrowth. (A) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, IKVAVPA2+FGF2PA2 group, and IKVAVPA2 group; laminin-ECM (green), 5HT-serotonergic neurons (red), and DAPI-nuclei (blue). (B, C) Representative magnified images of the (B) proximal margin (PB) and (C) distal margin (DB) of the section from A, with white dashed vertical lines indicating PB and DB. (D) Schematic diagram of the lesion site and the vertical lines used to count the number of 5HT axons intersecting at each specified location (top); plot of the number of intersecting axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01 vs. Siamese group and #P<0.05 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method). (E) Representative magnified image of the lesion center (LC) in IKVAVPA2+FGF2PA1. Scale bars: (A) 1500 μm and (B, C) 100 μm (E) 50 μm. [Figure 16B]Figures 16A-16E show the effects of IKVAVPA2 and various FGF2PA coassemblies on serotonergic neuron regrowth. (A) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, IKVAVPA2+FGF2PA2 group, and IKVAVPA2 group; laminin-ECM (green), 5HT-serotonergic neurons (red), and DAPI-nuclei (blue). (B, C) Representative magnified images of the (B) proximal margin (PB) and (C) distal margin (DB) of the section from A, with white dashed vertical lines indicating PB and DB. (D) Schematic diagram of the lesion site and the vertical lines used to count the number of 5HT axons intersecting at each specified location (top); plot of the number of intersecting axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01 vs. Siamese group and #P<0.05 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method). (E) Representative magnified image of the lesion center (LC) in IKVAVPA2+FGF2PA1. Scale bars: (A) 1500 μm and (B, C) 100 μm (E) 50 μm. [Figure 16C]Figures 16A-16E show the effects of IKVAVPA2 and various FGF2PA coassemblies on serotonergic neuron regrowth. (A) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, IKVAVPA2+FGF2PA2 group, and IKVAVPA2 group; laminin-ECM (green), 5HT-serotonergic neurons (red), and DAPI-nuclei (blue). (B, C) Representative magnified images of the (B) proximal margin (PB) and (C) distal margin (DB) of the section from A, with white dashed vertical lines indicating PB and DB. (D) Schematic diagram of the lesion site and the vertical lines used to count the number of 5HT axons intersecting at each specified location (top); plot of the number of intersecting axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01 vs. Siamese group and #P<0.05 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method). (E) Representative magnified image of the lesion center (LC) in IKVAVPA2+FGF2PA1. Scale bars: (A) 1500 μm and (B, C) 100 μm (E) 50 μm. [Figures 16D-16E]Figures 16A-16E show the effects of IKVAVPA2 and various FGF2PA coassemblies on serotonergic neuron regrowth. (A) Fluorescence micrographs of longitudinal spinal cord sections in the sham group, IKVAVPA2+FGF2PA1 group, IKVAVPA2+FGF2PA2 group, and IKVAVPA2 group; laminin-ECM (green), 5HT-serotonergic neurons (red), and DAPI-nuclei (blue). (B, C) Representative magnified images of the (B) proximal margin (PB) and (C) distal margin (DB) of the section from A, with white dashed vertical lines indicating PB and DB. (D) Schematic diagram of the lesion site and the vertical lines used to count the number of 5HT axons intersecting at each specified location (top); plot of the number of intersecting axons (bottom) (error bars correspond to 6 animals in each group; *P<0.05, **P<0.01 vs. Siamese group and #P<0.05 vs. IKVAVPA2 group and IKVAVPA2+FGF2PA2 group, repeated measures of two-way ANOVA and Bonferroni method). (E) Representative magnified image of the lesion center (LC) in IKVAVPA2+FGF2PA1. Scale bars: (A) 1500 μm and (B, C) 100 μm (E) 50 μm. [Figures 17A-17B] Figures 17A-17E show footprint analysis of animals injected with the dual signal co-assembly. (A) Representative photographs of hind limb position during walking 3 months after injury for the Siamese group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group. (B) Bar graph representing the impact force used to form lesions in the spinal cord of animals administered with saline (Siamese), IKVAVPA2+FGF2PA1, IKVAVPA2+FGF2PA2, and IKVAVPA2 (data points represent the 38 animals analyzed; ns indicates no significant difference). (C) Representative footprints of animals injected under various conditions plotted in B. (D, E) Bar graphs showing the stride length in (D) mm and stride width in (E) mm of animals injected under the various conditions described in B (data points correspond to 38 animals for each condition; ***P<0.0001 vs. Siamese group and ###P<0.0001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). [Figures 17C-17E]Figures 17A-17E show footprint analysis of animals injected with the dual signal co-assembly. (A) Representative photographs of hind limb position during walking 3 months after injury for the Siamese group, IKVAVPA2+FGF2PA1 group, and IKVAVPA2+FGF2PA2 group. (B) Bar graph representing the impact force used to form lesions in the spinal cord of animals administered with saline (Siamese), IKVAVPA2+FGF2PA1, IKVAVPA2+FGF2PA2, and IKVAVPA2 (data points represent the 38 animals analyzed; ns indicates no significant difference). (C) Representative footprints of animals injected under various conditions plotted in B. (D, E) Bar graphs showing the stride length in (D) mm and stride width in (E) mm of animals injected under the various conditions described in B (data points correspond to 38 animals for each condition; ***P<0.0001 vs. Siamese group and ###P<0.0001 vs. IKVAVPA2+FGF2PA1 group, one-way ANOVA and Bonferroni method). [Figure 18] Figure 18 shows the 1H-NMR spectrum of the PA coassembly. 1H-NMR spectra of aromatic protons at Y and W amino acids in the FGF2 mimetic sequence in the IKVAV PA2+FGF2 PA1 and IKVAV PA2+FGF2 PA2 systems, recorded at 600 MHz. [Modes for carrying out the invention]

[0017] definition Any methods and materials similar to or equivalent to those described herein may be used to implement or test the embodiments described herein, but this application describes certain preferred methods, compositions, apparatus and materials. However, before describing the materials and methods of this application, it should be noted that the specific molecules, compositions, techniques or protocols described herein can be modified according to routine experimentation and optimization, and therefore the present invention is not limited to these specific molecules, etc. Likewise, the terms used herein are intended to describe only specific aspects or embodiments and do not limit the scope of the embodiments described herein.

[0018] Unless otherwise defined, all scientific and technical terms used in this application have the same meaning as those widely understood by those ordinary skill in the art to which this invention pertains. However, in the event of any conflict, this specification, including its definitions, shall prevail. Accordingly, the following definitions shall apply to the embodiments described herein.

[0019] Unless otherwise evident from the context, the singular indefinite and definite articles used in this application and claims include plural references. Therefore, for example, "peptide amphiphilic molecule" means one or more peptide amphiphilic molecules and their equivalents known to those skilled in the art, and the same applies to other terms.

[0020] As used in this application, the term "contains" and its inflections mean the presence of one or more specified features, elements, process steps, etc., without excluding the presence of one or more other features, elements, process steps, etc. Conversely, the term "composed of" and its inflections mean the presence of one or more specified features, elements, process steps, etc., excluding one or more unspecified features, elements, process steps, etc., with the exception of impurities that are normally present. The term "essentially composed of" means one or more specified features, elements, process steps, etc., and one or more other features, elements, process steps, etc., that do not substantially affect the fundamental properties of the composition, system, or method. Many embodiments described in this application are described using the open term "contains". Such embodiments also encompass multiple closed "composed of" and / or "essentially composed of" embodiments, which may also be paraphrased using the term "contains" in the claims or specification.

[0021] The term "amino acid" refers to natural amino acids, unnatural amino acids, and amino acid analogs, and unless otherwise specified, they all exist as D- and L-forms where possible based on their structure.

[0022] Examples of 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).

[0023] Non-natural amino acids include, but are not limited to, azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, naphthylalanine ("naph"), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine ("tBuG"), 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, homoproline ("hPro" or "homoP"), hydroxylysine, allohydroxylysine, 3-hydroxyproline ("3Hyp"), and 4-hydroxy Examples include proline ("4Hyp"), isodesmosine, alloisoleucine, N-methylalanine ("MeAla" or "Nime"), N-alkylglycines including N-methylglycine ("NAG"), N-methylisoleucine, N-alkylpentylglycines including N-methylpentylglycine ("NAPG"), N-methylvaline, naphthylalanine, norvaline ("Norval"), norleucine ("Norleu"), octylglycine ("OctG"), ornithine ("Orn"), pentylglycine ("pG" or "PGly"), pipecolic acid, thiothioproline ("ThioP" or "tPro"), homolysine ("hLys"), and homoarginine ("hArg").

[0024] The term "amino acid analog" refers to natural or non-natural amino acids in which one or more of the C-terminal carboxyl group, N-terminal amino group, and side-chain bioactive groups are reversibly or irreversibly chemically blocked, or otherwise modified to another bioactive group. For example, aspartic acid (β-methyl ester) is an amino acid analog of aspartic acid, N-ethylglycine is an amino acid analog of glycine, and 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.

[0025] As used in this application, the term "peptide" refers to an oligomer or short-chain polymer of amino acids linked together by peptide bonds. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), peptides are approximately 50 amino acids or less in length. Peptides may include natural amino acids, unnatural amino acids, amino acid analogs, and / or modified amino acids. Peptides may be subsequences of naturally occurring proteins or unnatural (artificial) sequences.

[0026] As used in this application, the term "artificial" means compositions and systems that are artificially designed or produced and do not exist in nature. For example, an artificial peptide, peptoid, or nucleic acid is a peptide, peptoid, or nucleic acid that contains a non-natural sequence (e.g., a peptide that does not have 100% identity with a naturally occurring protein or a fragment thereof).

[0027] As used in this application, a “conservative” amino acid substitution means substituting an amino acid in a peptide or polypeptide with another amino acid that has similar chemical properties such as size and charge. For the purposes of this disclosure, each of the following eight groups includes amino acids that are mutually conservative substitutions. 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).

[0028] Naturally occurring residues can be classified based on their common side-chain properties, for example, as follows: polar positive charge (or basic) (histidine (H), lysine (K), and arginine (R)); polar charge (or acidic) (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); nonpolar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); nonpolar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used in this application, "semi-conservative" amino acid substitution means substituting an amino acid in a peptide or polypeptide with another amino acid within the same classification.

[0029] In certain embodiments, unless otherwise specified, conserved or semi-conserved amino acid substitutions may also include non-natural amino acid residues having chemical properties similar to those of natural residues. These non-natural residues are generally incorporated by chemical peptide synthesis rather than by synthesis in biological systems. Examples of these include, but are not limited to, peptidomimetics and other inversions or reversals of amino acid moieties. The embodiments described herein may, in certain embodiments, be limited to natural amino acids, non-natural amino acids, and / or amino acid analogs.

[0030] Non-conservative substitutions can include exchanging members of one classification for members of another classification.

[0031] As used in this application, the term "sequence matching degree" means the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same continuous composition of monomer subunits. The term "sequence similarity degree" means the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) differ only in conserved and / or semi-conservative amino acid substitutions. The "percentage of sequence matching degree" (or "percentage of sequence similarity degree") is calculated by (1) optimally aligning the two sequences and comparing them within a comparison frame (e.g., length of the longer sequence, length of the shorter sequence, specified frame, etc.), (2) determining the number of positions containing matching (or similar) monomers (e.g., the same amino acid exists in both sequences, or similar amino acids exist in both sequences), and calling this the number of match positions, (3) dividing the number of match positions by the total number of positions within the comparison frame (e.g., length of the longer sequence, length of the shorter sequence, specified frame), and (4) multiplying the result by 100 to obtain the percentage of sequence matching degree or the percentage of sequence similarity degree. For example, if both peptides A and B are 20 amino acids long and all amino acids except one are identical, then peptides A and B have a 95% sequence agreement. If the amino acids at the non-identical positions have the same biophysical characteristics (for example, both are acidic), then peptides A and B will have a 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to some of the amino acids in peptide C, then peptides C and D have a 70% sequence agreement, but peptide D has a 93.3% sequence agreement with respect to the optimal comparison frame of peptide C. For the purpose of calculating the "percentage of sequence agreement" (or "percentage of sequence similarity") in this application, any gaps in the aligned sequences are considered to be mismatches at those positions.

[0032] A whole polypeptide described in this application as having a specific percentage of sequence agreement or similarity (e.g., at least 70%) with respect to a reference sequence number can also be described as having an upper limit of substitutions (or terminal deletions) with respect to the reference sequence. For example, a sequence having at least Y% (e.g., 90%) of sequence agreement with respect to sequence number Z (e.g., 100 amino acids) can have up to X (e.g., 10) substitutions with respect to sequence number Z, and therefore can be described as having "X (e.g., 10) or fewer substitutions with respect to sequence number Z".

[0033] As used in this application, the term "nanofiber" generally refers to elongated or thread-like filaments with a diameter of less than 100 nanometers (for example, whose length is significantly greater than its width or diameter).

[0034] As used in this application, the term "supramolecular" (e.g., "supramolecular complex," "supramolecular interaction," "supramolecular fiber," "supramolecular polymer," etc.) means non-covalent interactions between molecules (e.g., polymers, macromolecules, etc.) and the resulting multi-component assemblies, complexes, systems, and / or fibers.

[0035] As used in this application, the terms “self-assembling” and “self-assembly” mean that separate, non-random aggregated structures are formed from component parts, and such assembly occurs spontaneously through the random movement of these components, which is solely due to the chemical or structural properties and attractive forces inherent to the components (e.g., molecules).

[0036] As used in this application, the term “peptide amphiphilic molecule” means a molecule comprising at least a hydrophobic segment, a structural peptide segment, and / or a charged peptide segment (often both). In a given embodiment, the peptide amphiphilic molecule comprises a physiologically active peptide (e.g., IKVAV peptide, growth factor mimetic peptide). In a given embodiment, the peptide amphiphilic molecule comprises a linker (e.g., G). The peptide amphiphilic molecule can exhibit a net charge of either a net positive charge or a net negative charge at physiological pH, or can be amphoteric (i.e., possess both positive and negative charges). A given peptide amphiphilic molecule comprises or includes (1) a hydrophobic non-peptide segment (e.g., containing an acyl group with 6 or more carbon atoms), (2) a structural peptide segment, (3) a charged peptide segment, and (4) a physiologically active peptide segment (e.g., IKVAV peptide, growth factor mimetic peptide).

[0037] As used in this application and the appended claims, the terms “lipophilic portion” or “hydrophobic portion” refer to a portion (e.g., acyl, ether, sulfonamide, or phosphodiester portion) located at one end (e.g., C-terminus, N-terminus) of a peptide amphiphilic molecule, and may also be referred to as a lipophilic or hydrophobic segment or component in this application and other literature. The hydrophobic segment should be of sufficient length to provide amphiphilic behavior and aggregate (or nanosphere or nanofiber) formation in water or another polar solvent system. Therefore, with respect to the embodiments described in this application, the hydrophobic component is of formula:C n-1 H 2n-1 It is preferable to include one linear acyl chain of C(O)- (wherein n=2 to 25). In a given embodiment, the linear acyl chain is a lipophilic group (saturated or unsaturated carbon) or palmitic acid. On the other hand, other lipophilic groups such as steroids, phospholipids, and fluorocarbons may be used instead of the acyl chain.

[0038] The terms "structural peptide" and "structural peptide segment" are used synonymously in this application and refer to a portion of an amphiphilic peptide molecule that is generally located between a hydrophobic segment and a charged peptide segment. A structural peptide generally consists of 3 to 10 amino acid residues containing a nonpolar, uncharged side chain (e.g., His(H), Val(V), Ile(I), Leu(L), Ala(A), Phe(F)) selected for its tendency to form hydrogen bonds or other stabilizing interactions (e.g., hydrophobic interactions, van der Waals interactions, etc.) with adjacent structural peptide segments. In certain embodiments, the structural peptide segment tends to form an α-helix and / or β-sheet secondary structure. In certain embodiments, an aggregate of amphiphilic peptide molecules having a structural peptide segment exhibits a linear or 2D structure when examined by microscopy and / or exhibits α-helix and / or β-sheet characteristics when examined by circular dichroism (CD) measurement.

[0039] In a given embodiment, the structural peptide segment has a low tendency to form α-helices and / or β-sheet secondary structures. For example, in a given embodiment, the structural peptide segment has a total tendency of 4 or less to form β-sheet conformations. In a given embodiment, an assembly of peptide amphiphilic molecules having a structural peptide segment with a total tendency of 4 or less to form β-sheet conformations exhibits low-order characteristics (e.g., low-order secondary structures such as low-rigidity β-sheet conformations). Such PAs are considered advantageous because they have a relatively high degree of internal motion and can form dynamic supramolecular assemblies. In a given embodiment, nanofibers of peptide amphiphilic molecules having a structural peptide segment with a total tendency of 4 or less to form β-sheet conformations (e.g., A2G2 (SEQ ID NO: 4), GGGG (SEQ ID NO: 7)) tend to form random coil structures.

[0040] As used in this application, the term “beta(β) sheet-forming peptide segment” refers to a structural peptide segment that tends to exhibit β-sheet-like characteristics (for example, when analyzed by CD). In a given embodiment, the amino acids in the beta(β) sheet-forming peptide segment are selected based on their tendency to form a β-sheet secondary structure. Examples of suitable amino acid residues selected from 20 naturally occurring amino acids include (in order of their tendency to form β-sheets) Met(M), Val(V), Ile(I), Cys(C), Tyr(Y), Phe(F), Gln(Q), Leu(L), Thr(T), Ala(A), and Gly(G). Alternatively, non-natural amino acids with similar β-sheet-forming tendencies may be used. Peptide segments capable of interacting to form β-sheets and / or peptide segments that tend to form β-sheets are known (see, for example, Mayo et al. Protein Science (1996), 5:1301-1315, the entire disclosure of which is incorporated herein by reference).

[0041] As used in this application, the term "charged peptide segment" refers to a part of an amphiphilic peptide molecule that has a high content (e.g., >50%, >75%) of charged amino acid residues or amino acid residues that have a net positive or negative charge under physiological conditions. A charged peptide segment can be acidic (e.g., positively charged), basic (e.g., positively charged), or amphoteric (e.g., having both acidic and basic residues).

[0042] As used in this application, the terms “carboxylate-rich peptide segment,” “acidic peptide segment,” and “loaded electropeptide segment” refer to the peptide sequence of a peptide amphiphilic molecule containing one or more amino acid residues having a side chain that presents a carboxylic acid side chain (e.g., Glu(E), Asp(D), or a non-natural amino acid). The carboxylate-rich peptide segment may optionally contain one or more other (e.g., non-acidic) amino acid residues. As will be obvious to those with ordinary skill in the art, non-natural amino acid residues having an acidic side chain, or peptidomimetics, may also be used. The segment may contain about 2 to about 7 amino acids and / or about 3 or 4 amino acids.

[0043] As used in this application, the terms “amino-rich peptide segment,” “basic peptide segment,” and “positively charged peptide segment” refer to the peptide sequence of a peptide amphiphilic molecule containing one or more amino acid residues having a side chain that presents a positively charged acidic side chain (e.g., Arg(R), Lys(K), His(H), or a non-natural amino acid, or peptidomimetics). The basic peptide segment may optionally contain one or more other (e.g., non-basic) amino acid residues. As is obvious to those with ordinary skill in the art, non-natural amino acid residues having a basic side chain may also be used. This segment may contain about 2 to about 7 amino acids and / or about 3 or 4 amino acids.

[0044] As used in this application, the term "bioactive peptide" refers to an amino acid sequence that mediates the action of a sequence, molecule, or supramolecular complex associated with it. A peptide amphiphilic molecule and structure (e.g., nanofiber) containing a bioactive peptide (e.g., IKVAV peptide) demonstrates the function of the bioactive peptide. In a given embodiment, a "bioactive peptide" containing the bioactive amino acid sequence IKVAV (SEQ ID NO: 1) is referred to in this application as an "IKVAV peptide amphiphilic molecule" or "IKVAVPA". In a given embodiment, the "bioactive peptide" is a peptide containing a growth factor mimetic peptide sequence. A bioactive peptide containing a growth factor mimetic peptide sequence is referred to in this application as a "growth factor mimetic peptide amphiphilic molecule" or "growth factor mimetic PA".

[0045] As used in this application, the term "biocompatible" means materials and substances that are non-toxic to cells or living organisms. In a given embodiment, a substance is considered "biocompatible" if, when added to cells in vitro, the cell death rate is approximately 10% or less, usually less than 5%, and more generally less than 1%.

[0046] As used in this application for polymers, hydrogels, and / or wound dressings, “biodegradable” means a composition that decomposes or “disintegrates” in other ways when exposed to physiological conditions. In a given embodiment, a biodegradable substance disintegrates by cellular mechanisms, enzymatic degradation, chemical processes, hydrolysis, etc. In a given embodiment, the wound dressing or coating contains hydrolyzable ester bonds that provide biodegradability.

[0047] As used in this application, the term "physiological conditions" refers to the range of chemical (e.g., pH, ionic strength) and biochemical (e.g., enzyme concentration) conditions that may be encountered in the intracellular or extracellular fluid of tissues. In most tissues, the physiological pH is approximately 7.0 to 7.4.

[0048] As used in this application, the terms “to treat,” “to cure,” and “for therapeutic purposes” mean reducing the amount or severity of a particular pathological condition or disease state (e.g., CNS injury) in an object that is currently suffering from or has developed said condition. This term does not necessarily mean complete cure (e.g., complete elimination of the pathological condition, disease, or its symptoms). “Treatment” encompasses any administration or application of a therapeutic agent or technology for a disease (e.g., in mammals, including humans), and includes suppression of disease, prevention of its onset, mitigation of disease, induction of regression, restoration or repair of loss, impairment or reduction of function, or stimulation of inefficient processes.

[0049] As used in this application, the terms “prevent,” “prevention,” and “for preventive purposes” mean reducing the probability of a particular pathological condition or disease state (e.g., CNS injury) occurring in subjects who do not currently have or have developed the said condition. This term does not necessarily mean complete or absolute prevention. For example, “preventing CNS injury” means reducing the probability of CNS injury occurring in subjects who do not currently have or have not been diagnosed with CNS injury. To “prevent CNS injury,” the composition or method is only required to reduce the probability of CNS injury, and does not need to completely eliminate the possibility. “Prevention” includes any administration or application of therapeutic agents or techniques to reduce the probability of occurrence (e.g., in mammals, including humans). Such probabilities can be evaluated for a population or for individuals.

[0050] As used in this application, the terms “combined administration” and “administered in combination” mean administering at least two drugs or therapies (e.g., the supramolecular aggregate described in this application and one or more therapeutic agents) to a target. In a given embodiment, the combined administration of two or more drugs or therapies is simultaneous. In other embodiments, the first drug / therapy is administered before the second drug / therapy. As will be obvious to those skilled in the art, the formulations and / or routes of administration of the various drugs or therapies used can be diverse. The appropriate dosage for combined administration can be easily determined by those skilled in the art. In a given embodiment, when drugs or therapies are administered in combination, they are administered at a lower dose than the dose appropriate for administering each drug or therapy alone. Therefore, combined administration is particularly desirable in embodiments where the combined administration of drugs or therapies reduces the required dose of a potentially harmful (e.g., toxic) drug, and / or where the combined administration of two or more drugs increases the target's sensitivity to one of the beneficial effects of the drugs as a result of the combined administration of other drugs.

[0051] Detailed explanation This application provides a peptide amphiphilic molecule (PA), a composition containing PA, a supramolecular assembly containing PA, and a method for using the same.

[0052] In a predetermined embodiment, the present application provides a peptide amphiphilic molecule. In a predetermined embodiment, the peptide amphiphilic molecule and composition of the embodiment described herein are synthesized using manufacturing techniques well known to those skilled in the art (although in the predetermined embodiment, the alignment of nanofibers is carried out by a technique not previously disclosed or used in the art (e.g., mesh screen extrusion)), and are preferably synthesized by a standard solid-phase peptide synthesis method, except that a fatty acid is added to the N-terminus (or C-terminus) of the peptide instead of a standard amino acid to form a lipophilic segment. Synthesis generally begins from the C-terminus, and amino acids are sequentially added using Rink amide resin (which yields an -NH2 group at the C-terminus of the peptide after cleavage from the resin) or Wang resin (which yields an -OH group at the C-terminus). Accordingly, the predetermined embodiment described herein encompasses a peptide amphiphilic molecule having a C-terminal portion that can be selected from the group consisting of -H, -OH, -COOH, -CONH2, and -NH2.

[0053] In a given embodiment, the peptide amphiphilic molecule includes a hydrophobic segment (i.e., a hydrophobic tail) linked to the peptide. In a given embodiment, the peptide includes a structural peptide segment. In a given embodiment, the structural peptide segment is a hydrogen bond forming segment or a beta sheet forming segment. In a given embodiment, the structural peptide segment has a tendency to form a random coil structure (for example, the total tendency to form a β-sheet conformation is 4 or less). In a given embodiment, the structural peptide segment has a relatively high level of internal motion because it has a low tendency to form an ordered secondary structure.

[0054] In a given embodiment, the peptide includes a charged segment (e.g., an acidic segment, a basic segment, an amphoteric segment, etc.). In a given embodiment, the peptide further includes a linker or spacer segment to increase solubility, flexibility, distance between segments, etc. In a given embodiment, the amphiphilic peptide molecule includes a spacer segment (e.g., a peptide and / or non-peptide spacer) at the peptide end opposite to the hydrophobic segment. In a given embodiment, the spacer segment includes a peptide and / or non-peptide element. In a given embodiment, the spacer segment includes one or more physiologically active groups (e.g., an alkene, an alkyne, an azide, a thiol, etc.). In a given embodiment, various segments can be linked by a linker segment (e.g., a peptide (e.g., GG) or a non-peptide (e.g., alkyl, OEG, PEG, etc.) linker).

[0055] The lipophilic or hydrophobic segment is generally composed of a fatty acid or other acid that is added to the N- or C-terminus of the peptide after the final amino acid coupling and linked to the N- or C-terminal amino acid via an acyl bond. In aqueous solution, PA molecules self-assemble (for example, as cylindrical micelles (also known as nanofibers)), embedding the lipophilic segment in their center and presenting the bioactive peptide on their surface. In a given embodiment, the structural peptide generates intermolecular hydrogen bonds, forming a beta-sheet oriented parallel to the long axis of the micelle. In a given embodiment, the structural peptide has weak intermolecular hydrogen bonds, resulting in a low-rigidity beta-sheet conformation inside the nanofiber.

[0056] In a given embodiment, a hydrophobic (e.g., hydrocarbon and / or alkyl / alkenyl / alkynyl tail, or steroid such as cholesterol) segment of sufficient length (e.g., 2-carbon, 3-carbon, 4-carbon, 5-carbon, 6-carbon, 7-carbon, 8-carbon, 9-carbon, 10-carbon, 11-carbon, 12-carbon, 13-carbon, 14-carbon, 15-carbon, 16-carbon, 17-carbon, 18-carbon, 19-carbon, 20-carbon, 21-carbon, 22-carbon, 23-carbon, 24-carbon, 25-carbon, 26-carbon, 27-carbon, 28-carbon, 29-carbon, 30-carbon or longer, or any range in between) segment covalently bonds with a peptide segment (e.g., peptide, structural peptide segment, and charged peptide segment) to form a peptide amphiphilic molecule. In a given embodiment, multiple such PAs self-assemble in water (or aqueous solution) to form a nanostructure (e.g., nanofiber). In various embodiments, different PA molecular shapes and nanostructure architectures can be obtained as a result of the relative lengths of the peptide segment and the hydrophobic segment. For example, when the peptide segment is wide and the hydrophobic segment is narrow, a conical molecular shape is generally obtained, which affects the PA aggregate (see, for example, JNIsraelachvili Intermolecular and surface forces; 2nd ed.; Academic: London San Diego, 1992, the entire disclosure thereof is incorporated herein by reference). Other molecular shapes have similar effects on the aggregate and nanostructure architecture.

[0057] In certain embodiments, the pH of the solution may be changed (increased or decreased) to induce self-assembly of the peptide amphiphilic molecule aqueous solution, or polyvalent ions such as calcium, charged polymers, or other macromolecules may be added to the solution.

[0058] In a given embodiment, the hydrophobic segment is a non-peptide segment (e.g., an alkyl / alkenyl / alkynyl group). In a given embodiment, the hydrophobic segment includes (e.g., saturated) alkyl chains having 4 to 25 carbon atoms (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25), fluorinated segments, fluorinated alkyl tails, heterocycles, aromatic segments, π-conjugated segments, cycloalkyls, oligothiophenes, etc. In a given embodiment, the hydrophobic segment comprises (e.g., saturated) acyl / ether chains having 2 to 30 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). In a given embodiment, the hydrophobic segment comprises alkyl chains having 8 to 24 carbon atoms (C 8-24 ) includes. In a given embodiment, the hydrophobic segment is a C16 alkyl chain (C 16 ) includes.

[0059] In a given embodiment, PA comprises one or more peptide segments. The peptide segments may include natural amino acids, modified amino acids, unnatural amino acids, amino acid analogs, peptidomimetics, or combinations thereof. In a given embodiment, the peptide segments have at least 50% (e.g., conserved or semi-conserved) sequence matching or similarity to one or more peptide sequences described in this application.

[0060] In a given embodiment, the peptide amphiphilic molecule includes a charged peptide segment. The charged segment may be acidic, basic, or amphoteric.

[0061] In a given embodiment, the peptide amphiphilic molecule includes an acidic peptide segment. For example, in a given embodiment, the acidic peptide includes one or more (e.g., one, two, three, four, five, six, seven or more) acidic residues (D and / or E) in its sequence. In a given embodiment, the acidic peptide segment includes up to 7 residues in length and contains at least 50% acidic residues. In a given embodiment, the acidic peptide segment is (Xa) 1-7 The formula includes, where each Xa is independently D or E. In a given embodiment, the acidic peptide segment is E 2-4 This includes, for example, in a given embodiment, the acidic peptide segment includes E2. In a given embodiment, the acidic peptide segment includes E3. In another embodiment, the acidic peptide segment includes E4.

[0062] In a given embodiment, the peptide amphiphilic molecule includes a basic peptide segment. For example, in a given embodiment, the acidic peptide includes one or more (e.g., one, two, three, four, five, six, seven or more) basic residues (R, H, and / or K) in its sequence. In a given embodiment, the basic peptide segment includes up to 7 residues in length and contains at least 50% basic residues. In a given embodiment, the acidic peptide segment is (Xb) 1-7 The formula includes, where each Xb is independently R, H, and / or K.

[0063] In a given embodiment, the peptide amphiphilic molecule includes a structural peptide segment. In a given embodiment, the structural peptide segment is a beta-sheet forming segment. In a given embodiment, the structural peptide segment has weak hydrogen bonding and no secondary structure. In a given embodiment, the structural peptide segment has weak hydrogen bonding and tends to form a random coil structure rather than a rigid beta-sheet conformation. In a given embodiment, the structural peptide segment has a high content of one or more H, I, L, F, V, G, and A residues. In a given embodiment, the structural peptide segment includes an alanine and valine-rich peptide segment (e.g., V2A2 (SEQ ID NO: 3), V3A3 (SEQ ID NO: 19), A2V2 (SEQ ID NO: 31), A3V3 (SEQ ID NO: 16), or other combinations of V and A residues). In a given embodiment, the structural peptide segment includes four or more consecutive A and / or V residues, or their conserved or semi-conservative substitutions. In a given embodiment, the structural peptide segment includes V2A2 (SEQ ID NO: 3). In a given embodiment, the structural peptide segment comprises an alanine and glycine-rich peptide segment (e.g., A2G2 (SEQ ID NO: 4), A3G3 (SEQ ID NO: 17), or other combinations of A and G residues). In a given embodiment, the structural peptide segment comprises A2G2 (SEQ ID NO: 4). In a given embodiment, the structural peptide segment comprises a glycine-rich peptide segment. For example, in a given embodiment, the structural peptide segment comprises G3 or G4 (SEQ ID NO: 7).

[0064] In a predetermined embodiment, the structural peptide segment has a total tendency to form a β-sheet conformation of 4 or less (for example, less than 4, less than 3.9, less than 3.8, less than 3.7, less than 3.6, less than 3.5, less than 3.4, less than 3.3, less than 3.2, less than 3.1, less than 3.0, less than 2.9, less than 2.8, less than 2.7, less than 2.6, less than 2.5, less than 2.4, less than 2.3, less than 2.2, less than 2.1, less than 2.0, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, or less than 1).

[0065] The overall tendency to form a β-sheet conformation can be calculated as the sum of the β-sheet conformation tendencies of each amino acid in the structural peptide segment. The β-sheet conformation tendencies of each amino acid and the method of calculation are described, for example, in Fujiwara, K., Toda, H. & Ikeguchi, M. Dependence of α-helical and β-sheet amino acid propensities on the overall protein fold type. BMC Struct Biol 12, 18 (2012), and the entire disclosure is incorporated herein by reference. Typical values ​​are shown in Table 1 below. To calculate the overall tendency to form a β-sheet conformation of a structural peptide segment, the values ​​shown in the "Total Residues" column for each amino acid in Table 1 are summed up. For example, in the case of the A2G2 structural peptide segment, the overall tendency to form a β-sheet conformation is 0.75 + 0.75 + 0.67 + 0.67 = 2.84. The structural peptide segment may contain any appropriate number and combination of amino acids such that the total tendency to form a β-sheet conformation is 4 or less.

[0066] [Table 1]

[0067] In a given embodiment, if the total tendency of a structural peptide segment to form a β-sheet conformation is 4 or less, then the amino acids within the structural peptide segment are judged to have weak interactions with neighboring molecules. For example, the structural peptide segment may exhibit weak hydrogen bonding ability. Therefore, such a structural peptide segment and the amphiphilic peptide molecule containing such a segment can form a more dynamic supramolecular assembly. For example, the A2G2 structural peptide segment may exhibit a random coil structure rather than a rigid β-sheet conformation.

[0068] In a given embodiment, the bioactive peptide amphiphilic molecule (e.g., the IKVAV peptide amphiphilic molecule) exhibits relatively low fluorescence anisotropy. The anisotropy is calculated using the following formula.

[0069]

number

[0070] In a predetermined embodiment, the bioactive peptide amphiphilic molecule (e.g., IKVAV peptide amphiphilic molecule) has a fluorescence anisotropy value of less than 0.3 (e.g., less than 0.3, less than 0.29, less than 0.25, less than 0.24, less than 0.23, less than 0.22, less than 0.21, or less than 0.2).

[0071] In a predetermined embodiment, the bioactive peptide amphiphilic molecule (e.g., IKVAV peptide amphiphilic molecule) has a proton relaxation rate ( 1 The H-R2 is relatively low. A lower proton relaxation rate means higher mobility, making it easier to form dynamic supramolecular assemblies with high internal mobility. For example, the relaxation rate of the methylene proton bound to the e-carbon of the K residue in the IKVAV sequence can be measured by transverse relaxation nuclear magnetic resonance (T2-NMR) spectroscopy. In a given embodiment, the amphiphilic molecule of the IKVAV peptide has a proton relaxation rate ( 1 H-R2) 4s -1It is less than. In a given embodiment, the IKVAV peptide amphiphilic molecule has a proton relaxation rate ( 1 H-R2) 3s -1 It is less than.

[0072] In a given embodiment, the peptide amphiphilic molecule includes a non-peptide spacer or linker segment. In a given embodiment, the non-peptide spacer or linker segment is located at the peptide terminus opposite the hydrophobic segment. In a given embodiment, the spacer or linker segment provides a linking site for a bioactive group. In a given embodiment, the spacer or linker segment provides a reactive group (e.g., alkenes, alkynes, azides, thiols, maleimides, etc.) for functionalization of the PA. In a given embodiment, the spacer or linker is a substantially linear chain of CH2, O, (CH2)2O, O(CH2)2, NH, and C=O groups (e.g., CH2(O(CH2)2)2NH, CH2(O(CH2)2)2NHCO(CH2)2CCH, etc.). In a given embodiment, the spacer or linker further includes other bioactive groups, substituents, branching, etc. In a given embodiment, the linker segment is a single glycine (G) residue.

[0073] The peptide amphiphilic molecules suitable for use in the materials described in this application, methods for producing PA and related materials, amino acid sequences for use in PA, and materials used together with PA are protected by the following patents, namely, U.S. Patent Nos. 9,044,514, 9,040,626, 9,011,914, 8,772,228, 8,748,569, 8,580,923, 8,546,338, 8,512,693, 8,450,271, 8,236,800, 8,138,140, ​​and 8,124,583, U.S. This is described in Japanese Patent No. 8,114,835, U.S. Patent No. 8,114,834, U.S. Patent No. 8,080,262, U.S. Patent No. 8,076,295, U.S. Patent No. 8,063,014, U.S. Patent No. 7,851,445, U.S. Patent No. 7,838,491, U.S. Patent No. 7,745,708, U.S. Patent No. 7,683,025, U.S. Patent No. 7,554,021, U.S. Patent No. 7,544,661, U.S. Patent No. 7,534,761, U.S. Patent No. 7,491,690, U.S. Patent No. 7,452,679, U.S. Patent No. 7,371,719, and U.S. Patent No. 7,030,167, and the entirety of the disclosures therein is incorporated herein by reference.

[0074] The properties of the PA supramolecular structure (e.g., shape, rigidity, hydrophilicity, etc.) vary depending on the type of components of the peptide amphiphilic molecule (e.g., lipophilic segment, acidic segment, structural peptide segment, bioactive segment, etc.). For example, by adjusting the type of PA component moiety, nanofibers, nanospheres, intermediate shapes, and other supramolecular structures can be achieved. In certain embodiments, the properties of the PA supramolecular nanostructure are altered by post-assembly operations (e.g., heating / cooling, stretching, etc.).

[0075] In a given embodiment, the peptide amphiphilic molecule comprises (a) a hydrophobic tail containing an alkyl chain having 8 to 24 carbon atoms, (b) a structural peptide segment (including, for example, A2G2 (SEQ ID NO: 4) or G4 (SEQ ID NO: 7)), and (c) a charged segment (including, for example, E2-E4). In a given embodiment, any PA within the scope described herein that contains the components described herein, or any PA within the scope conceivable to those skilled in the art, may be used herein.

[0076] In a given embodiment, the peptide amphiphilic molecule includes a bioactive moiety (e.g., IKVAV peptide). In a particular embodiment, the bioactive moiety is the C-terminus or N-terminus of the PA. In a given embodiment, the bioactive moiety is ligated to the end of the charged segment. In a given embodiment, the bioactive moiety is exposed on the surface of the assembled PA structure (e.g., nanofiber). The bioactive moiety is generally a peptide, but is not limited to peptides.

[0077] In a given embodiment, the bioactive moiety is a peptide identified in the extracellular matrix (ECM). For example, the bioactive moiety may be a peptide sequence present in collagen, elastin, fibronectin, or laminin. In a given embodiment, the bioactive moiety is a peptide sequence present in laminin. For example, the bioactive moiety may be present in laminin-1, laminin-2, laminin-3, laminin-4, laminin-5, laminin-6, laminin-7, laminin-8, laminin-9, laminin-10, laminin-11, laminin-12, laminin-13, laminin-14, or laminin-15. In a given embodiment, the bioactive moiety is a peptide sequence present in laminin-1. In a particular embodiment, the bioactive moiety is the peptide sequence IKVAV (SEQ ID NO: 1). In a given embodiment, the bioactive moiety is a recombinant peptide. In a given embodiment, the physiologically active portion is a peptide sequence that binds to the peptide or polypeptide of interest (e.g., an ECM protein).

[0078] In a given embodiment, the peptide amphiphilic molecule is composed of a bioactive peptide (e.g., IKVAV peptide) and (e.g., E) (e.g., E 2-4 It comprises a charged segment (including, for example, A2G2 (SEQ ID NO: 4), G4 (SEQ ID NO: 7)), a structural peptide segment (including, for example, A2G2 (SEQ ID NO: 4), G4 (SEQ ID NO: 7)), and a hydrophobic tail (including, for example, an alkyl chain having 8 to 24 carbon atoms).

[0079] In a given embodiment, the peptide amphiphilic molecule comprises a bioactive peptide (e.g., IKVAV peptide) (e.g., from the C-terminus to the N-terminus or from the N-terminus to the C-terminus), a flexible linker (e.g., including G), and (e.g., E 2-4 It comprises a charged segment (including, for example, A2G2 (SEQ ID NO: 4), G4 (SEQ ID NO: 7)), a structural peptide segment (including, for example, A2G2 (SEQ ID NO: 4), G4 (SEQ ID NO: 7)), and a hydrophobic tail (including, for example, an alkyl chain having 8 to 24 carbon atoms).

[0080] In a predetermined embodiment, the present application provides a physiologically active PA (also referred to in the present application as "IKVAV peptide amphiphilic molecule") containing IKVAV as the physiologically active peptide. In a predetermined embodiment, the IKVAV peptide amphiphilic molecule comprises IKVAV (for example, from the C-terminus toward the N-terminus or from the N-terminus toward the C-terminus) and (for example, E 2-4 It comprises a charged segment (including A2G2, G4, etc.), a structural peptide segment (including A2G2, G4, etc.), and a hydrophobic tail (including an alkyl chain having 8 to 24 carbon atoms, etc.). In a given embodiment, the peptide amphiphilic molecule further comprises a linker. For example, in a given embodiment, the peptide amphiphilic molecule comprises a single glycine residue linking the physiologically active peptide (e.g., IKVAV) to the charged peptide segment. In a given embodiment, the IKVAV peptide amphiphilic molecule comprises C 16 -Contains A2G2E4GIKVAV (SEQ ID NO: 12). In a given embodiment, the IKVAV peptide amphiphilic molecule is C 16 -Includes G4E4GIKVAV (sequence number 18).

[0081] In a given embodiment, the physiologically active portion is a growth factor mimetic peptide. In a given embodiment, the growth factor mimetic peptide includes a growth factor mimetic sequence. In a given embodiment, the growth factor mimetic sequence is a vascular endothelial growth factor (VEGF) mimetic sequence, a fibroblast growth factor 2 (FGF2) 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. In a given embodiment, the growth factor mimetic sequence is an FGF2 mimetic sequence. In a given embodiment, the FGF2 mimetic sequence includes YRSRKYSSWYVALKR (SEQ ID NO: 2).

[0082] In a predetermined embodiment, the present application provides a physiologically active PA containing a growth factor mimetic sequence as the physiologically active peptide. Such a peptide amphiphilic molecule is referred to in the present application as a "growth factor mimetic peptide amphiphilic molecule".

[0083] In a predetermined embodiment, the present application provides a growth factor mimetic peptide sequence (for example, from the C-terminus to the N-terminus or from the N-terminus to the C-terminus) and (for example, E 2-4 The present invention provides a growth factor mimetic peptide amphiphilic molecule comprising a charged segment (including, for example, A2G2 (SEQ ID NO: 4), V2A2 (SEQ ID NO: 3)), a structural peptide segment (including, for example, A2G2 (SEQ ID NO: 4), V2A2 (SEQ ID NO: 3)), and a hydrophobic tail (including, for example, an alkyl chain having 8 to 24 carbon atoms). In a given embodiment, the peptide amphiphilic molecule further comprises a linker. For example, in a given embodiment, the peptide amphiphilic molecule comprises a single glycine residue linking the growth factor mimetic peptide sequence to the charged peptide segment.

[0084] In a predetermined embodiment, the present application provides a growth factor mimetic peptide sequence (for example, from the C-terminus to the N-terminus or from the N-terminus to the C-terminus), a flexible linker (for example, containing G, etc.), and (for example, E 2-4 The present invention provides a growth factor mimetic peptide amphiphilic molecule comprising a charged segment (including, for example, A2G2 (SEQ ID NO: 4), V2A2 (SEQ ID NO: 3)), a structural peptide segment (including, for example, A2G2 (SEQ ID NO: 4), V2A2 (SEQ ID NO: 3)), and a hydrophobic tail (including, for example, an alkyl chain having 8 to 24 carbon atoms). In a given embodiment, the growth factor mimetic peptide amphiphilic molecule is C 16 -V2A2E4GYRSRKYSSWYVALKR(Sequence ID 13) or C 16 -Contains A2G2E4GYRSRKYSSWYVALKR (sequence number 14).

[0085] In a given embodiment, the PA further includes a binding segment or residue (e.g., G) for linking the hydrophobic tail to the peptide portion of the PA.

[0086] In a given embodiment, the present application provides a composition comprising at least two of the peptide amphiphilic molecules described herein. In a given embodiment, the present application provides a composition comprising at least one IKVAV peptide amphiphilic molecule and at least one growth factor mimetic peptide amphiphilic molecule. In a given embodiment, the present application provides a composition comprising at least one IKVAV peptide amphiphilic molecule comprising a hydrophobic segment, a structural peptide segment, a charged peptide segment, and a physiologically active peptide comprising the amino acid sequence IKVAV, and at least one growth factor mimetic peptide amphiphilic molecule. In a given embodiment, the IKVAV peptide amphiphilic molecule comprises an alkyl chain having 8 to 24 carbon atoms (C 8-24 The molecule comprises a hydrophobic segment containing ), a structural peptide segment containing AAGG (SEQ ID NO: 4), a charged peptide segment containing E4 (SEQ ID NO: 11), a linker (e.g., G), and the IKVAV (SEQ ID NO: 1) peptide sequence. In a given embodiment, the growth factor mimetic peptide amphiphilic molecule comprises an alkyl chain (C) having 8 to 24 carbon atoms.8-24 The composition comprises a hydrophobic segment containing ), a structural peptide segment containing V2A2 (SEQ ID NO: 3) or A2G2 (SEQ ID NO: 4), a charged peptide segment containing E2, E3, or E4 (SEQ ID NO: 11), and a growth factor mimetic peptide sequence. In a given embodiment, the at least one amphiphilic IKVAV peptide molecule and the at least one amphiphilic growth factor mimetic peptide molecule interact to form a supramolecular assembly within the composition.

[0087] In a predetermined embodiment, the present application provides a supramolecular assembly comprising at least two of the peptide amphiphilic molecules described herein. In a predetermined embodiment, the supramolecular assembly is a nanofiber. In a predetermined embodiment, the present application provides a supramolecular assembly comprising at least two of the bioactive peptide amphiphilic molecules described herein. In a predetermined embodiment, the present application provides a supramolecular assembly comprising an IKVAV peptide amphiphilic molecule and a growth factor mimetic peptide amphiphilic molecule. In a predetermined embodiment, the present application provides an IKVAV peptide amphiphilic molecule 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 amphiphilic molecule. In a predetermined embodiment, the IKVAV peptide amphiphilic molecule comprises an alkyl chain having 8 to 24 carbon atoms (C 8-24 The molecule comprises a hydrophobic segment containing ), a structural peptide segment containing AAGG (SEQ ID NO: 4), a charged peptide segment containing E4 (SEQ ID NO: 11), a linker (e.g., G), and an IKVAV peptide sequence (SEQ ID NO: 1). In a given embodiment, the growth factor mimetic peptide amphiphilic molecule comprises an alkyl chain (C) having 8 to 24 carbon atoms. 8-24 It contains a hydrophobic segment containing ), a structural peptide segment containing V2A2 (SEQ ID NO: 3) or A2G2 (SEQ ID NO: 4), a charged peptide segment containing E2, E3, or E4 (SEQ ID NO: 11), and a growth factor mimetic peptide sequence.

[0088] In a given embodiment, a supramolecular assembly (e.g., a nanostructure such as a nanofiber) is assembled from a first type of PA containing a physiologically active moiety (e.g., an IKVAV peptide amphiphilic molecule) and a growth factor mimetic PA. In a given embodiment, the composition or supramolecular assembly (e.g., a nanostructure such as a nanofiber) described herein has a molar ratio of IKVAVPA to growth factor mimetic PA of about 90:10. In a given embodiment, the molar ratio of IKVAVPA: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, about 87:13, about 86:14, or about 85:15. In a given embodiment, the ratio of IKVAVPA to the growth factor mimetic determines the mechanical properties of the nanofiber material (e.g., liquid or gel) and the conditions under which the material exhibits various properties (e.g., gelling when exposed to physiological conditions, liquefying when exposed to physiological conditions, etc.).

[0089] In a given embodiment, the composition and supramolecular assembly described herein further comprises one or more filler PAs. The terms “filler PA” or “diluent PA” are used synonymously herein and mean PAs that include hydrophobic segments, structural peptide segments, and charged peptide segments as described herein, but do not contain physiologically active moieties (e.g., PAs that do not contain the IKVAV peptide sequence, PAs that do not contain the growth factor mimetic peptide sequence, etc.). In a given embodiment, the filler PA is a non-physiologically active PA molecule having a highly charged glutamic acid residue at the end of the molecule (e.g., the end presented on the surface). These charged PAs can induce gelation between nanofibers by ion crosslinking. In a given embodiment, the filler PA is a non-physiologically active PA molecule having a highly charged lysine residue at the end of the molecule (e.g., the end presented on the surface). These positively charged PAs can induce gelation under basic conditions. The filler PAs enable the incorporation of other physiologically active PA molecules into the nanofiber matrix while maintaining the gelling ability of the nanofiber solution. In a given embodiment, the solution is annealed to increase its viscosity and enhance its gel dynamics. These fillers PA are, for example, arranged as described in U.S. Patent No. 8,772,228, the entirety of which is incorporated herein by reference (e.g., C 16 It has -VVVAAAEEE (sequence number 19)).

[0090] In a given embodiment, the PA nanofiber described in this application has a small cross-sectional diameter (e.g., <25 nm, <20 nm, <15 nm, about 10 nm, etc.). In this given embodiment, because the cross-sectional diameter of the nanofiber is small (about 10 nm in diameter), the nanofiber can penetrate the brain parenchyma.

[0091] In a given embodiment, the PA, compositions, and supramolecular assemblies described herein are used for the treatment or prevention of nervous system injury in a subject. In a given embodiment, the PA, compositions, and supramolecular assemblies (e.g., nanofibers) described herein can be used in a method for treating nervous system injury in a subject. For example, the PA, compositions, and supramolecular assemblies described herein can be used in a method for treating or preventing injury to the central nervous system (CNS), including the brain and spinal cord, or to the peripheral nervous system (PNS), including nerves and ganglia outside the brain and spinal cord. In a given embodiment, the PA, compositions, and supramolecular assemblies described herein can be used for the treatment or prevention of CNS or PNS injury in a subject. In a given embodiment, the injury is a spinal cord injury. The spinal cord injury may be of the cervical, lumbar, thoracic, sacral, or any combination thereof.

[0092] The injury may be a traumatic injury. A traumatic injury means an injury resulting from trauma (for example, an injury caused by a car accident, fall, violence, sports injury, surgical injury, etc.). For example, the PA, composition, and supramolecular assemblies described in this application can be used in the treatment of traumatic spinal cord injury. As another example, the PA, composition, and supramolecular assemblies described in this application can be used in the treatment of traumatic brain injury (TBI). Alternatively, the injury may be a non-traumatic injury. For example, the injury may be a non-traumatic injury of the CNS (e.g., brain and / or spinal cord) or PNS resulting from, for example, cancer, multiple sclerosis, inflammation, arthritis, spinal stenosis, tumor, blood loss, etc.

[0093] In a given embodiment, a composition comprising PA and / or supramolecular aggregates (e.g., nanofibers) as described herein is provided to a subject suspected of having traumatic spinal cord injury. For example, the composition can be provided to a subject exhibiting one or more symptoms, including loss of sensation and / or motor control in one or more body areas (e.g., hands, arms, legs, feet, etc.), hypotension, inability to regulate blood pressure, inability to regulate body temperature, inability to sweat below the injury site, chronic pain, and / or spinal edema. The composition can be provided to the subject to treat the injury. In a given embodiment, treatment of the injury can prevent the worsening of one or more symptoms associated with the injury. In a given embodiment, treatment of the injury can reduce the severity of the injury and / or eliminate one or more symptoms associated with the injury. In a given embodiment, the composition is used to promote angiogenesis, nerve regeneration, functional recovery, and / or limit post-spinal cord injury disability.

[0094] The composition can be provided to a subject at any appropriate time after the injury (e.g., traumatic spinal cord injury) to treat the injury. For example, the composition can be provided to the subject within 24 hours of the injury (e.g., within 24 hours, 12 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour). In a predetermined embodiment, the composition can be provided to the subject more than 24 hours after the injury or diagnosis of the injury.

[0095] The composition can be administered in any appropriate amount depending on factors including the age of the subject, the subject's weight, and the severity of the injury. The composition can be administered in combination with other treatments appropriate for the injury or with preventive measures to prevent the worsening of the injury.

[0096] In a given embodiment, the composition described herein is formulated to be delivered to a subject. Suitable routes of administration of the composition described herein include, but are not limited to, topical, subcutaneous, transdermal, intradermal, intrafocal, intraarticular, intraperitoneal, intrabladderal, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, and intracerebroventricular administration. In a given embodiment, the PA composition is administered parenterally. In a given embodiment, parenteral administration is carried out by intrathecal, intracerebroventricular, or intraparenchymal administration. The PA composition described herein may be administered as a monoactive agent or in combination with other pharmaceuticals, such as other drugs used to treat neurological injuries in a subject. [Examples]

[0097] [Example 1] Supramolecular motion in a bioactive scaffold promotes recovery from spinal cord injury.

[0098] Summary: This embodiment describes supramolecular polymers of peptide amphiphilic molecules containing two different signals. These supramolecular polymers were tested in a mouse model of severe spinal cord injury. The first signal activates the transmembrane receptor β1 integrin, and the second signal activates the basic fibroblast growth factor 2 receptor. By mutating the peptide sequence of the amphiphilic monomer in the non-bioactive domain, the movement of molecules within the scaffold fibril was activated, resulting in significant changes in vascular growth, axonal regeneration, myelinization, motor neuron survival, suppression of gliosis, and functional recovery. Therefore, by regulating the internal movement of molecules, it is possible to optimize cellular signaling through the assembly of these molecules.

[0099] Introduction: Pharmacological signaling within cells typically proceeds through strong binding of proteins and small organic molecules that activate or inhibit specific responses. Nanostructures targeting specific cells to deliver therapeutic cargo, and materials that function as bioactive scaffolds in extracellular space, are emerging as signaling strategies. Molecular design of receptor signaling materials and the relationship between such signals and molecular movement within artificial scaffolds remain underdeveloped areas in this field. This example describes a nanoscale fibril supramolecular scaffold integrating two distinct orthogonal biological signals: the laminin signal IKVAV, which promotes differentiation from neural stem cells into neurons and axonal extension, and the fibroblast growth factor 2 (FGF2) mimetic peptide YRSRKYSSWYVALKR (SEQ ID NO: 2), which activates the receptor FGFR1 and promotes cell proliferation and survival. These two signals were positioned at the terminals of two different peptides (referred to as peptide amphiphilic molecules (PAs)) having alkyl tails that non-covalently copolymerize in an aqueous medium to form supramolecular fibrils. In this study, we investigated various domains that modify the physical properties of potential scaffold therapy to restore functional recovery in vivo after hindlimb paralysis in a mouse model of severe spinal cord injury (SCI). Since damaged axons cannot regenerate in the adult central nervous system (CNS), developing SCI therapies to avoid permanent paralysis in humans after traumatic injury remains a significant challenge. In this study, we discovered that slight mutations in the tetrapeptide sequences of these domains, while maintaining the same density of both biological signals, dramatically altered the cellular biological response in vitro and functional recovery from SCI in mice in vivo.

[0100] result: To investigate nanofiber-type supramolecular polymers with various physical properties that each present two identical signals, we synthesized libraries of various IKVAVPAs (IKVAVPA1-PA8) with tetrapeptide domains controlling physical behavior using various sequences of amino acids V, A, and G (see Figure 1A, Figure 7, and Table 2 for a list of the PAs used and their peptide sequences). These amino acids were selected considering that the tendency of molecules within the fibril to form highly cohesive β-sheets as a result of their hydrogen bond density was a key factor. These interactions suppress the mobility of PA molecules within the fibril. For example, V2A2 (PA1) has a high tendency to form a β-sheet structure due to its valine content, while A2G2 (PA2) is a potentially low-order segment and does not form a secondary structure (see Figure 1A). Other sequences were selected as potential candidates for intermediate levels of mobility. All IKVAVPAs utilized the E4G sequence, which provides high water solubility between this segment and the bioactive signal.

[0101] [Table 2]

[0102] Cryo-transmission electron microscopy (cryo-TEM) revealed that all IKVAVPA forms nanofibers after supramolecular polymerization in water (Figure 1B). Furthermore, synchrotron X-ray solution small-angle scattering (SAXS) confirmed filament formation, with the exception of PA5 (slope = -0.2), which suggests a mixture of filaments and spherical micelles, the slopes of the Guigné regions ranged from -1 to -1.7 (Figure 1D). Coarse-grained molecular dynamics (CG-MD) simulations using the MARTINI force field (13) were used to compare the physical behavior of various assemblies in the library (Figure 8). These simulations suggested that the molecules within each IKVAVPA fiber exhibited different degrees of internal dynamics (Figure 1B). The numerical values ​​of a parameter defined as root-mean-square fluctuation (RMSF), a measure of the average displacement of PA molecules during the last 5 ms of the simulation, were determined by simulation, suggesting differences in the ability of molecules to change position internally over sufficient distances (on the order of nanometers) (Figure 1C). These simulations indicate that molecules in PA2 fibers actually have high internal mobility, similar to PA5, which contains only G residues. Wide-angle X-ray analysis (WAXS) also revealed that internal order (β-sheet Bragg peaks with d-lattice spacing of 4.65 Å) exists in all IKVAVPA except those with low RMSF values ​​(PA2 and PA5) (Figure 1E).

[0103] To investigate the differences in dynamics among various IKVAVPAs, the degree of fluorescence depolarization (FD) was measured by encapsulating 1,6-diphenyl-1,3,5-hexatriene (DPH) within PA nanofibers and measuring the microviscosity of the internal hydrophobic core. PA2 and PA5 had the lowest anisotropy values ​​(0.21 and 0.18, respectively) and were judged to have formed the most dynamic supramolecular assemblies, PA4 had intermediate dynamics (0.30), and the other PAs had low supramolecular mobility (0.40-0.37) (Figure 2A). Molecular dynamics in the IKVAV epitopes were also measured using transverse relaxation nuclear magnetic resonance (T2-NMR) spectroscopy. In these experiments, the relaxation rate of the methylene proton bound to the e-carbon of the K residue in the IKVAV sequence (H) was observed (observed at parts per million from 2.69 to 2.99).ε The relaxation rate was measured. IKVAVPA1 showed the best relaxation rate (low motility), while IKVAVPA2 and PA5 showed the lowest relaxation rates among the IKVAVPA libraries (each 1 H-R2 = 2.7 ± 0.1 and 2.6 ± 0.003 s -1 ), which was consistent with higher kinetic levels (Figure 2B and Table 3). Consistent with the FD results, IKVAVPA4 exhibits supramolecular motion at an intermediate level between PA1 and PA2 (or PA5). Taken together, the simulations and FD, WAXS, and T2-NMR measurements are consistent with three levels of supramolecular motion in the library of molecules investigated.

[0104] [Table 3]

[0105] Supramolecular motion and in vitro physiological activity In vitro experiments were conducted to investigate whether the IKVAV signaling pathway is equally physiologically active in libraries of IKVAVPA. To demonstrate the physiological activity of IKVAVPA, neural progenitor cells (hNPCs) derived from human embryonic stem cells were treated with various IKVAVPA fibers or recombinant protein laminin in solution (Figure 2C). PA filaments closely associate with cells, and when their surfaces present signals, they can activate receptors.

[0106] The activation of the transmembrane receptor β1 integrin (ITGB1) expressed in the presence of various IKVAVPAs and laminin was evaluated using the activity-specific antibody HUTS4. Activation of the receptor's intracellular signaling pathway was also investigated. Fluorescence confocal microscopy and Western blot (WB) analysis revealed that IKVAVPA2 and PA5 induced substantially higher concentrations of active ITGB1 and downstream effectors, integrin-binding kinase (ILK) and phosphorylated focal adhesion kinase (p-FAK), compared to other IKVAVPAs, IKVAV peptides, and laminin or ornithine coatings as controls (Figures 2D and E, and Figure 9). PA4 showed intermediate levels of activation, corresponding to intermediate supramolecular motion compared to other PAs in the library. PAs exhibiting the VVIAK scramble sequence showed the lowest level of cellular activation of ITGB1. Furthermore, pretreatment with the ITGB1 antibody inhibited the binding of hNPC to all IKVAVPAs, suggesting that the IKVAV-ITGB1 interaction mediates this process.

[0107] When hNPCs were processed with various IKVAVPAs, neuronal β-tubulin was upregulated (TUJ1 + However, this induction rate (reflecting involvement in neuronal differentiation) was particularly high for the two most dynamic supramolecular fibrils, IKVAVPA2 and PA5 (20.5±1% and 20.7±1.2%, respectively) (Figure 2F~H). IKVAVPA4 showed intermediate involvement in neuronal differentiation (PA4: 14±1.2%), while other IKVAVPAs showed TUJ1 + The induction rate of nerve cells was low (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%). Using a puromycin-based protein synthesis analysis method (SUnSET), it was verified that similar protein translation levels were obtained under all conditions, confirming that the observed differences were not related to metabolic activity.

[0108] In vitro experiments were conducted by treating hNPC with a mixture of 5 mM CaCl2, which electrostatically crosslinks the IKVAVPA (PA2 and PA5), which exhibits the highest biological activity, with the loaded PA fibers, and then applying the mixture. 2+ FD and T2-NMR experiments confirmed that the addition of Ca suppresses supramolecular motion (Figure 2I). 2+ By adding ions to the medium, supramolecular motion was reduced, which also decreased the activation of ITGB1 and its downstream intracellular pathways (ILK, p-FAK / FAK) (Figure 2J and Figures 10a-10c). These results indicate that a mutation has been introduced into the tetrapeptide amino acid sequence in the non-bioactive domain of IKVAVPA, resulting in a strong positive correlation between dynamics and in vitro physiological activity.

[0109] SCI model: Axonal regrowth and glial scar formation Next, we tested the ability of dual-signal fibrils to enhance functional recovery in vivo after SCI. Since IKVAVPA1, PA3, PA4, PA6, PA7, and PA8 showed only low levels of in vitro bioactivity, these PAs were not used in combination with FGF2PA. Nanofibers presenting both signals simultaneously were also tested, so the dual system needed to be miscible and, upon injection into the injury site and contact with physiological fluid, form a hydrogel with similar mechanical properties. Only IKVAVPA2 was miscible and formed a hydrogel with similar mechanical properties, particularly when mixed with FGF2PA1 or FGF2PA2 in a molar ratio of 90:10 (Figures 3A-C, 11A-11C, Table 4). Furthermore, both FGF2PAs, alone, formed highly aggregated short fibers, further contributing to miscibility with other IKVAVPAs such as PA1, PA4, or PA5.

[0110] [Table 4]

[0111] A binary system of IKVAVPA2 and FGF2PA1 or FGF2PA2, which forms a miscible gel with similar mechanical properties, was developed for in vivo experiments (Figures 3A and 12). In a mouse model established for SCI (20), a saline solution of a 90:10 molar ratio IKVAVPA2 and FGF2PA1 or FGF2PA2 core assembly was injected into the spinal cord of mice 24 hours after severe contusion. IKVAVPA2, the single signaling system with the highest biological activity, was used as a control in all in vivo experiments. The total PA solution gelled in situ upon delivery to the spinal cord and localized to the injury site. To track and quantify the biodegradation of the biologically active scaffold as a function of time, PA molecules were fluorescently labeled with Alexa 647 dye. Fluorescent material was injected into the spinal cord 24 hours after injury, and its volume was measured at 1, 2, 4, 6, and 12 weeks after transplantation by completely reconstructing the spinal cord using spinning disk confocal microscopy (see Figure 3D). The soft material gradually biodegraded within 1 to 12 weeks after transplantation, and no difference in biodegradation rate was observed among the three experimental materials (see Figures 3E and 13).

[0112] To track the corticospinal tract (CST), which mediates voluntary motor function, biotinylated dextranamine (BDA) was administered bilaterally 10 weeks after sensorimotor cortical injury (Figure 3F). Anterior labeled CST axonal regrowth was evaluated 12 weeks after injury in both the all-PA group and the sham (saline-only infusion) group. This method required quantifying the number of labeled axons that regrow beyond the proximal lesion margin. IKVAVPA1 and PA fibers without physiologically active signals on their surface ("main chain PA") were injected as controls.

[0113] In mice injected with physiological saline, little axonal regrowth was observed within the lesion. However, with IKVAVPA1, some axonal regrowth was observed, and the fibers showed low motility (Figures 3G and 14). On the other hand, in mice injected with IKVAVPA2 alone or as a co-assembly with FGF2PA2 (which has the same A2G2 non-bioactive domain as IKVAVPA2), a slight increase in axonal regrowth was observed compared to the sham condition. In contrast, when a co-assembly of FGF2PA1 (which contains the V2A2 non-bioactive domain instead of A2G2) and IKVAVPA2 was injected, strong corticospinal tract axonal regrowth occurred throughout the lesion site, even extending beyond its distal edge (Figures 3G and H, and Figure 15). In this group, total axonal regrowth within the lesion was twice that of the group using the IKVAVPA2 and FGF2PA2 co-assembly, and 50 times that of the sham group (Figure 3I). Serotonin axons (5HT) are also thought to play a role in walking function, and similarly regrow within the central part of the lesion, showing a similar trend to CST (Figure 16).

[0114] While we do not intend to be overly theoretical, the observation of CST and 5HT axonal regrowth is thought to be partly due to the absence of prominent astrocyte scarring, which is a severe impairment of axonal regeneration. In the sham group and the main chain PA group, this impairment was observed as a dense population of reactive astrocytes expressing high levels of GFAP at the lesion margin, but the density of glial scarring was low in the overall physiologically active PA group (Figure 3H, Figure 16). Consistent with these results, WB analysis revealed that only the core assembly with the highest physiological activity (IKVAVPA2+FGF2PA1) showed higher concentrations of growth-related protein 43 (GAP-43) in the growth cone of regenerating axons (Figure 3J).

[0115] Next, we investigated whether the PA scaffold could induce remyelination of corticospinal tract axons three months after injury. In the IKVAVPA2+FGF2PA1 configuration, high levels of myelin basic protein (MBP) were detected within the lesion, particularly wrapped around the regrowing axons (Figures 3J and K). Furthermore, under these conditions, many growing axons within the lesion were associated with high levels of laminin and low levels of fibronectin, suggesting a reduction in the fibrotic core (Figures 3K and L, Figure 15). These histological and biochemical findings suggest that physical differences between two supramolecular coassemblies carrying two types of bioactive signals can significantly enhance post-injury nerve regeneration outcomes.

[0116] SCI model: Angiogenesis, cell survival, and functional recovery The influence of both dual signaling coassemblies on angiogenesis at the site of injury is crucial for complete anatomical and functional regeneration, and this influence was investigated next. Compared to uninjured tissue sections, transverse spinal cord sections of Siamese mice showed a significant degree of tissue degeneration extending more than 2.0 mm axially from the center of the lesion. In these cases, a significant decrease in vascular area fraction, vascular length, and branching was observed compared to uninjured controls (Figures 4A and B). The presence of a functional vascular network was investigated by transcardiac injection of a glucose solution containing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye incorporated into the endothelial cell membrane (Figure 4A). In the group administered the PA scaffold, abdominal tissue structure was highly preserved, and the functional vascular network was maintained. When the co-assembly with the highest biological activity was administered, vascular area fraction, vascular length, and branching increased, particularly in the dorsal region (Figures 4A and B). There were no significant differences in these parameters between the IKVAVPA2 monotherapy group and the low-biological-activity co-assembly group (IKVAVPA2 + FGF2PA2), suggesting that the mimetic FGF2 angiogenesis signaling pathway was not functioning optimally with IKVAVPA2 + FGF2PA2.

[0117] To determine the origin of blood vessels within the lesion, 5'-bromo-2'-deoxyuridine (BrdU), a thymidine analog, was injected intraperitoneally during the first week after injury. Twelve weeks after injury, newly formed blood vessels were observed within the lesion in the core assembly group with the highest physiological activity. This was due to BrdU compared to samples from all other groups. + / CD31 + A significant increase in cell count was confirmed by WB analysis (Figure 4C and D). The IKVAVPA2+FGF2PA2 co-assembly and IKVAVPA2 alone resulted in a very slight but significant increase in angiogenesis compared to the sham group.

[0118] The effects of both dual-signal coassemblies on neuronal survival, spinal circuit maintenance, and local function were investigated. Natural FGF2 appears to be associated with increased neuronal survival after SCI. Transverse spinal sections of the coassembly group with the highest physiological activity showed increased neuronal survival near newly formed blood vessels in the dorsal region, similar to the undamaged control group. + The neuron is shown (Figure 5A). Furthermore, when PA is used, ChAT is also activated simultaneously. + (motor neurons) are also neurons (NeuN) + Only BrdU cells were detected in the anterior horn, and the system with the highest physiological activity had a significantly larger number of BrdU cells compared to the other groups (Figures 5B and C). In all groups, double BrdU cells were present in the lesion. + / NeuN + No neurons were observed, suggesting the absence of localized neurogenesis.

[0119] We evaluated whether observed axonal regeneration, angiogenesis, and local neuronal survival led to behavioral improvements in injured animals. For this purpose, we assessed walking motor recovery in all groups over 12 weeks post-injury using the Basso Mouse Score (BMS) open field walking motor score and footprint analysis (Figures 5D and 17). From one week after injury onward, all PA groups showed significantly sustained behavioral improvements compared to the sham group. Interestingly, three weeks after injury, mice administered with the core assembly with the highest bioactivity showed significant functional recovery (5.9±0.5) compared to mice injected with IKVAVPA2+FGF2PA2 and IKVAVPA2 alone (4.4±0.5 and 4.3±0.5, respectively) (Figure 5D). Quantitative analysis of footprints revealed that mice administered with the core assembly with the highest bioactivity had significantly larger stride length and width compared to the other groups (Figure 17). In summary, these data suggest that neuronal survival and functional recovery observed in dual-signal systems are remarkably related to differences in the chemical composition of each non-bioactive tetrapeptide.

[0120] In vitro results in human endothelial cells and neural progenitor cells Based on the above results, the in vitro physiological activity of FGF2 signaling in both coassemblies was evaluated using human umbilical vein endothelial cells (HUVECs). Natural FGF2 enhances endothelial cell proliferation and reticular formation. When HUVECs were cultured on the coassembly or FGF2 protein with the highest physiological activity, extensive branching and formation of vascular-like capillary networks were observed within 48 hours (Figures 6A and B). Western blotting analysis was also performed to verify whether the in vitro physiological activity observed in the FGF2PA1 and IKVAVPA2 coassemblies was related to the intracellular FGF2 signaling pathway. HUVECs treated with the coassembly or natural FGF2 with the highest physiological activity were found to have high concentrations of p-FGFR1 and the downstream protein p-ERK1 / 2, which activate endothelial cell proliferation and migration (Figure 6C). Systems containing scrambled FGF2 mimetic sequences showed no physiological activity whatsoever.

[0121] To demonstrate the simultaneous bioactivity of IKVAV and FGF2 signaling in both core assemblies, double-positive EdU was used. + / SOX2 + The effects of these molecules on hNPC proliferation were evaluated in vitro by quantifying the induction of ITGB1 and pFGFR-1 (Figures 6D-F). These experiments suggest that FGF2 signaling is nearly non-functional in the less physiologically active core assembly, while IKVAV signaling appears to be functionally maintained in both core assemblies. These results are consistent with findings from SCI experiments.

[0122] Physical experiments and computer simulations on supramolecular motion We investigated the potential physical reasons for the decrease in in vitro and in vivo physiological activity when the tetrapeptide following the alkyl tail in FGF2PA was mutated from V2A2 to A2G2. The differences in intermolecular dynamics between these two coassemblies were examined by T2-NMR spectroscopy and FD (Figure 6G-I). The relaxation rates of aromatic protons at the Y and W amino acids, which are present only in the FGF2 mimetic signal, were measured. These rates were slower in the coassembly with the highest physiological activity, suggesting more active supramolecular motion in the signaling peptide. 1 H-R2 = 80.9 / 18.9s of the core assembly with lower physiological activity -1 49.3■11s -1 (Figures 6G and H). FD experiments were performed on the two types of coassemblies using FGF2PA molecules covalently labeled with Cy3 dye (cryo-TEM images showed that the dye did not disrupt the supramolecular assemblies). Lower anisotropy was detected in the coassembly with the highest biological activity, indicating high mobility of FGF2 signaling molecules within the nanofibers (Figure 6I).

[0123] CG-MD simulations showed that the co-assembly with the highest biological activity had a higher RMSF value for the FGF2PA molecule, supporting the T2-NMR and FD results mentioned above. The simulations also revealed that the FGF2PA molecule forms clusters in both co-assemblies (slightly larger in the system with the highest biological activity) based on the distribution of mobility (RMSF value) (Figure 6J). The decrease in biological activity in one of the systems is thought to be due to the difference in the degree of co-assembly between the two PA molecules that carry the signal. However, the 1D of the methylene unit in the alkyl tail... 1 Based on 1H-NMR, diffusion-aligned spectroscopy (DOSY), and T2-NMR, it was determined that co-assembly occurred in both systems (Table 5).

[0124] [Table 5]

[0125] In systems containing only IKVAVPA, the tetrapeptide V2A2 (present in FGF2PA1) exhibited the lowest mobility, so the result of higher mobility obtained for the FGF2PA1 molecule was contrary to intuitive expectations. The lower mobility of the FGF2PA2 molecule in the coassembly with IKVAVPA2 was thought to be due to the strong interactions between hydrogen bonds and side chain contacts between the identical tetrapeptides present in both molecules. On the other hand, the two molecules in the highly physiologically active IKVAVPA2+FGF2PA1 coassembly contain two different tetrapeptides, so it is thought that strong interactions did not occur between the two molecules, which did not lead to higher supramolecular mobility.

[0126] The strong interaction and low mobility of IKVAVPA2 and FGF2PA2 are demonstrated by the fact that the CD spectrum remains almost unchanged when FGF2PA2 is added to IKVAVPA2. On the other hand, when the less interacting FGF2PA1 is added to IKVAVPA2, the CD spectrum changes, suggesting disruption of the secondary structure. Therefore, the high mobility detected by NMR in the FGF2PA1 molecule is thought to indicate a high degree of freedom in the translational motion of clusters within the fibril or the vertical motion of signal transduction clusters entering and leaving the fibril.

[0127] Additional results: Core Assembly Characterization When IKVAVPA2 was co-assembled with various FGF2PAs (IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2), both systems formed long, ribbon-like structures with a molar ratio of 90:10 (Figure 3B). The morphological changes after co-assembly were confirmed by SAXS and WAXS profiles and FT-IR spectra. According to the SAXS profile, the slope of the Guinier region was -1.2 for IKVAVPA2, and -1.6 for IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2, indicating that the degree of transition from cylindrical fibers (-1.0) to ribbons (-2.0) differed. Furthermore, the PA co-assemblies (IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2) showed consistent β-sheet spacing in WAXS, which was supported by FT-IR spectra, showing similar β-sheet discrimination characteristics in the amide I region as IKVAVPA2. High-magnification transmission electron microscopy (TEM) of the core assemblies with negative staining revealed that IKVAVPA2+FGF2PA1 and IKVAVPA2+FGF2PA2 had average fiber widths of 13.7±0.3 nm and 15.6±0.2 nm, respectively. The fiber widths of both systems were significantly larger than those of the main portion alone (IKVAVPA2 = 10.6±0.1 nm), which was attributed to the addition of FGF2PA to the system. The amount of FGF2PA incorporated into IKVAVPA2 was analyzed based on the light scattering intensity (optical density) at 600 nm (OD600 nm). Various mol% concentrations of FGF2PA (PA1 and PA2) showed higher values ​​than when the same percentage was co-assembled with IKVAVPA2. Therefore, while FGF2PA has low solubility on its own, its solubility increases with IKVAVPA2, leading to their aggregation. In the case of coassemblies of low percentage FGF2PA (5-10 mol%) and IKVAVPA2, the OD (Oxygen Demand) was comparable to that of IKVAVPA2 alone (FGF2PA 0 mol%) and significantly lower than that of free FGF2PA in solution.

[0128] PA control in the SCI model: IKVAVPA1 fibers, IKVAVPA4 fibers, and PA fibers (main chain PA) lacking physiologically active signals on their surface were injected as controls. In mice injected with main chain PA, almost no axonal regrowth was observed within the lesion, but some axonal regrowth was observed in IKVAVPA1. Interestingly, IKVAVPA4 was found in vitro to have intermediate supramolecular motion and physiological activity (between IKVAVPA1 and IKVAVPA2), and it also showed intermediate levels of axonal regrowth in vivo.

[0129] hNPC on the Core Assembly System When hNPCs were seeded on PA coatings, they adhered and survived similarly under all PA conditions tested for one week. The number of seeded hNPCs was the same under all conditions, but when hNPCs were cultured on IKVAVPA2+FGF2PA1 or with natural FGF2, the growth of EdU was different. + and SOX2 + The percentage of cells increased, and this percentage significantly decreased when cells were cultured on IKVAVPA2+FGF2PA2, IKVAVPA2 alone, or commercial laminin (Figure 6E). After one week, IKVAVPA2+FGF2PA1 increased the number of SOX2s to a level comparable to that of the natural FGF2 protein. + The cell pool was maintained. Conversely, cells seeded on IKVAVPA2+FGF2PA2, IKVAVPA2, and laminin were PAX6, which are neuronal progenitor cells. +The percentage of increased, indicating that cell differentiation was progressing. Next, the expression of ITGB1 and FGFR-1 was tracked by WB to test whether the physiological activity observed in IKVAVPA2+FGF2PA1 was actually related to ITGB1 and FGFR-1, respectively. p-FGFR1 was highly expressed in hNPCs seeded on IKVAVPA2+FGF2PA1 or hNPCs treated with natural FGF2, but active ITGB1 was significantly higher in cells cultured under all PA conditions including IKVAVPA2. Differences in phenotypic profiles were also observed between cells seeded on the IKVAVPA2 and FGF2PA1 core assembly and cells seeded on the IKVAVPA2 and FGF2PA2 core assembly. IKVAVPA2+FGF2PA1 induced higher expression of the neural stem cell marker SOX2, the neuronal marker β-tubulin III (TUJ1), and the post-mitotic marker PH3, similar to cells seeded on laminin coatings treated with natural FGF2.

[0130] Core assembly system 1 1H-NMR, DOSY, and relaxation NMR 1 ¹H-NMR spectra revealed that in both coassemblies, the peak originating from the methylene proton was broader and lower in intensity compared to IKVAVPA2 alone, and these results were confirmed by DOSY. Proton relaxation rate data was also obtained using T2-NMR, and significantly higher values ​​were detected in the coassembly compared to IKVAVPA2 (IKVAVPA2+FGF2PA1: 1 H-R2 = 59.6 ± 1.9 s -1 IKVAVPA2+FGF2PA2: 1 H-R2 = 52.7 ± 1.7 s -1 , and IKVAVPA2: 1 H-R2 = 6.4 ± 0.1 s -1 These results indicate that a co-assembly of both signals occurs in both systems.

[0131] Consideration This example describes a bioactive scaffold in which active supramolecular motion was demonstrated by physical and computer analyses, and therefore, is expected to improve functional recovery from SCI in a mouse model. In a one-dimensional scaffold of non-covalently polymerized bioactive molecules, it was anticipated that the multivalent effect would help cluster receptors for effective signal transduction. It was also anticipated that the internal structure of the supramolecular scaffold would restrict free motion and preferentially direct signals to receptors perpendicular to its fibril axis. However, a surprising finding in this endeavor was that the intensity of molecular motion within the bioactive fibrils, measured on the bench, correlated with improved axon regrowth, neuron survival, vascular regeneration, and functional recovery from SCI. Computer simulations and experimental data suggest that bioactivity is enhanced by vertical translation on a nanometer scale within or from the aggregate to the receptor site. While not intended to be theoretical, highly agile and physically plastic supramolecular scaffolds are thought to be effective in signaling to receptors present in the cell membrane under rapid shape fluctuations. The cause of recovery can also be broadly explained from the perspective that the interaction between the ECM protein environment and the molecular dynamics scaffold is more favorable. In short, the scaffold described in this application provides an excellent opportunity for dynamic structural design to optimize the physiological activity of therapeutic supramolecular polymers.

[0132] Materials and methods Characterization of peptide amphiphilic molecular nanostructures: Synthesis and Purification PA Synthesis and Preparation: Using a CEM model Liberty Blue microwave peptide synthesizer with linkamide MBHA resin as the support, various IKVAVPA (IKVAVPA1:C) were synthesized by the standard fluorenyl methoxycarbonyl (Fmoc) solid-phase peptide synthesis method. 16 -VVAAEEEEGIKVAV(sequence code 20), IKVAVPA2:C 16 -AAGGEEEEGIKVAV(sequence code 12), IKVAVPA3:C 16 -AVGGEEEEGIKVAV(sequence code 21), IKVAVPA4:C 16-VAGGEEEEGIKVAV (SEQ ID NO: 22), IKVAVPA5:C 16 -AAAAEEEEGIKVAV (SEQ ID NO: 25), IKVAVPA6:C 16 -GGGGEEEEGIKVAV (SEQ ID NO: 18), IKVAVPA7:C 16 -VAAAEEEEGIKVAV (SEQ ID NO: 23), IKVAVPA8:C 16 -AVAAEEEEGIKVAV (SEQ ID NO: 24), various FGF2PA (FGF2PA1:C 16 -VVAAEEEEGYRSRKYSSWYVALKR (SEQ ID NO: 13) and FGF2PA2:C 16 -AAGGEEEEGYRSRKYSSWYVALKR (SEQ ID NO: 14), and their scrambled sequences as various scr-IKVAVPA (VVIAKPA1:C 16 -VVAAEEEEGVVIAK (SEQ ID NO: 27) and VVIAKPA2:C 16 -AAGGEEEEGVVIAK (SEQ ID NO: 28)) and various scr-FGF2PA (scr-FGF2PA1:C 16 -VVAAEEEEGWRSKKYSLYYVASRR (SEQ ID NO: 29) and scr-FGF2PA2:C 16 -AAGGEEEEGWRSKKYSLYYVASRR (SEQ ID NO: 30)), and the main chain PA(C 16 -VVAAEE (SEQ ID NO: 19)) were synthesized (for a list of material names and corresponding peptide sequences, see Table 2; C 16 is a palmitoyl group). An automatic coupling reaction was carried out using 4 equivalents of Fmoc-protected amino acids, 4 equivalents of N,N'-diisopropylcarbodiimide (DIC), and 8 equivalents of ethyl cyano(hydroxyimino)acetate (Oxyma pure).

[0133] The Fmoc group was removed with a 20% 4-methylpiperidine DMF solution. The peptide was cleaved from the resin using a standard solution of 95% TFA, 2.5% water, and 2.5% triisopropylsilane (TIS), and precipitated with cold ether. Next, various IKVAVPAs, their scramble sequences, and main-chain PAs were purified under basic conditions by reverse-phase high-performance liquid chromatography (HPLC) using a Shimadzu Prominence modular HPLC system, two LC-20AP delivery units, an SPD-M20A diode array detector, and an FRC-10A fraction collector, with a Phenomenex Gemini NX-C18 column (C18 stationary phase, 5 μm, pore size 110 Å, 150 × 30 mm) and an H2O / CH3CN gradient containing 0.1% (v / v) NH4OH as the eluent at a flow rate of 25.0 mL / min. Various FGF2PAs and their scrambled sequences were purified under acidic conditions (aqueous solution containing 0.1% (v / v) TFA / CH3CN solution) using a Phenomenex Kinetex C8 column (C8 stationary phase, 5 μm, pore size 100 Å, 150 × 30 mm). The purity of various lyophilized PAs was analyzed by liquid chromatography-mass spectrometry (LC-MS) using an Agilent Model 1200 Infinity Series binary LC gradient system with either a Phenomenex Jupiter 4μm Proteo 90Å column (C12 stationary phase, 4μm, pore size 90Å, 1×150mm) or a Phenomenex Gemini C18 column (C18 stationary phase, 5μm, pore size 110Å, 150×1mm), using an H2O / CH3CN gradient containing 0.1% (v / v) formic acid or NH4OH as the eluent at a flow rate of 50μL / min. Electrospray ionization mass (ESI-mass) analysis was performed in positive scan mode on an Agilent Model 6510 quadrupole time-of-flight LC-MS.

[0134] For various PAs covalently bonded with dyes, cysteine ​​or azidridine was added to the C-terminus of the above sequence, respectively, and IKVAVPA2 labeled with Alexa Fluor®-647 and various FGF2PAs labeled with Cy3 were synthesized. The purified IKVAVPAs were dissolved in a pH 8 Tris buffer solution of tris(2-carboxyethyl)phosphine (TCEP) hydrochloride (5 equivalents relative to PA) and reacted with Alexa Fluor®-647 (Thermo Fisher), which was functionalized with maleimide. The purified FGF2PAs were dissolved in N,N-dimethylformamide (DMF) and reacted with Cy3-DBCO (Click Chemistry Tools), which was functionalized with dibenzocyclooctin. The various final PA products labeled with dyes were purified by HPLC, lyophilized, and stored until use.

[0135] PA Co-assembly Fabrication In in vitro experiments, after lyophilization, the PA powder was reconstituted in a solution of 150 mM NaCl and 3 mM KCl, and the pH was adjusted to 7.4 by adding 1 μL of 1 M NaOH to ensure cell compatibility and material consistency. Various physiologically active PAs were mixed in various mol%s (see Table 3) and sonicated at 10% intensity for 10 seconds in three separate sessions using a horn-type sonicator. After annealing the PA solution at 80°C for 30 minutes, all samples were slowly cooled at a rate of 1°C per minute using a thermocycler (Eppendorf PCR Thermocycler) to reach a final temperature of 27°C. To prepare PA-coated supports, poly-D-lysine (0.01 mg / mL, Sigma-Aldrich) was coated onto 24-well, 12-well, or 6-well polystyrene cell culture plates or 12 mm and 18 mm glass coverslips (German Glass, Chemglass Life Science) at 37°C for 3 hours. Next, the plates were rinsed three times with MilliQ water and dried for 4 hours. Various PAs were applied to coverslips or tissue culture plates by pipetting a thin, even coating of material (8-30 μL of annealed PAs (1 wt%)) across the entire surface. The PA coatings were incubated at room temperature for 3 hours. The plates were gently rinsed with culture medium before subsequent use. In experiments with dye-labeled PAs, various FGF2PAs labeled with Cy3 were co-assembled with their corresponding unlabeled PAs at 1 mol% (see Table 4).

[0136] In in vivo experiments, after lyophilization, the PA powder was reconstituted with 0.9% (w / v) sterile isotonic sodium chloride saline (Ricca Chemical) at a concentration of 1 mg / 100 μl. Next, 1 μL of sterile 1 M NaOH was added to the resulting PA solution to adjust the pH to 7.4, and then a co-assembly of IKVAVPA2 and 10 mol% FGF2PA1 or FGF2PA2 was added (see Table 3). After mixing, the solution was sonicated and annealed. In experiments with dye-labeled PA, Alexa-647-labeled IKVAVPA2 was co-assembled with their corresponding unlabeled PAs at a concentration of 1 mol% (see Table 4).

[0137] Transmission electron microscopy (TEM) To prepare negative-stained TEM specimens, 7 μL of PA sample solution (prepared to 1 wt% PA by diluting 150 mM NaCl and 3 mM KCl 10-fold with MilliQ water) was dropped onto the glossy side of a glow-discharged grid (300 mesh copper with carbon film, Electron Microscopy Sciences). The grid was then rinsed three times with MilliQ water to remove excess salt. Next, the grid was stained with filtered 2 wt% uranyl acetate aqueous solution and air-dried. TEM imaging was performed using a JEOL1230 microscope equipped with a Gatan 831 CCD camera and LaB6 filament at an acceleration voltage of 100 kV. A cold finger was used to stabilize the sample during imaging.

[0138] Cryo-transmission electron microscopy (cryo-TEM) Cryo-TEM plunge-frozen samples were prepared using the FEI model Vitrobot Mark IV (FEI). 7 μL of sample solution ([PA] = 0.05~0.1 wt% aqueous solution) was dropped onto a plasma-cleaned TEM grid (300-mesh copper with a lace-like carbon film, Electron Microscopy Sciences) and fixed with tweezers attached to the Vitrobot. The samples were blotted at room temperature and 100% humidity (blot intensity: 3, total blot count: 1~2, waiting time: 0.5~1 second, blotting time: 3 seconds, drain time: 0~1 second), and then immersed in a liquid ethane reservoir cooled with liquid nitrogen. After storing the glassy samples in liquid nitrogen, they were transferred to a Gatan 626 cryo-TEM holder. Cryo-TEM images were acquired using a JEOL1230 electron microscope equipped with a Gatan 831 CCD camera and operated at an acceleration voltage of 100 kV using a LaB6 filament.

[0139] Scanning electron microscopy (SEM) PA coatings, either with or without cells attached, were fixed for 20 minutes with a mixture of paraformaldehyde (2.0%, Electron Microscopy Sciences) and glutaraldehyde (2.5%, Electron Microscopy Sciences) dissolved in phosphate-buffered saline (1×, Gibco). The fixative was removed, and the water was replaced with ethanol by incubating the sample in a gradient of ethanol solution with gradually increasing concentrations of 200 proof ethanol (Decon Laboratories, Inc.) (30 → 100%). Excess water was removed using critical point drying (Tousimis Samdri-795). A 20-minute purge cycle was used to ensure sufficient water exchange. The resulting dehydrated samples were mounted on stubs using carbon adhesive tape (Electron Microscopy Sciences), and in some cases, carbon adhesive (Electron Microscopy Sciences). The samples were coated with approximately 10 nm of osmium (Filgen, OPC-60A) to make the sample surface conductive for imaging. All images were captured using a Hitachi SU8030 SEM instrument at an acceleration voltage of 2kV.

[0140] X-ray scattering method X-ray small-angle scattering (SAXS), medium-angle scattering (MAXS), and wide-angle scattering (WAXS) experiments were conducted at beamline 5-ID-D of the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) Synchrotron Research Center at the Advanced Photon Source facility located at Argonne National Laboratory, USA. 100-150 μL of sample solution (aqueous solutions of NaCl and KCl ([NaCl]=150 mM and [KCl]=3 mM) with [PA]=1-3 w / v% (approximately 6-10 mM)) was introduced into a capillary flow cell of a fixed diameter, and X-rays were irradiated for irradiation times of 0.5 seconds, 2 seconds, 3 seconds, or 10 seconds. During sample measurement, the sample was vibrated within the capillary at a rate of 10 μL / sec using a syringe pump to prevent damage from excessive beam irradiation. Data was acquired at an X-ray energy of 17 keV (l=0.83 Å) using a triple-area detector system. 0.002390 <q<4.4578Å -1 Scattering intensity was recorded in the specified interval. The wave vector q was defined as q = (4π / λ)sin(θ / 2), where θ is the scattering angle. The acquired 2D scattering data was then converted into a 1D intensity plot against the wave vector by integral of the azimuthal angle around the beam center using GSAS-II software. A background scattering pattern was obtained from a sample containing 150 mM NaCl and 3 mM KCl. This background data was then subtracted from the experimental data. All data was analyzed using the Irena software package running on IgorPro software.

[0141] Circular dichroism (CD) spectroscopy Each PA sample was diluted to a concentration of 0.01–0.04 wt% with H2O (salt-free sample) or a buffer solution containing 150 mM NaCl and 3 mM KCl (high salt concentration). CD spectra were recorded using a JASCO Model J-815 spectropolarimeter with a 0.5 mm path length quartz cell. Sensitivity was set to standard mode, and continuous scan mode was used at a scan speed of 100 nm per minute. The high-voltage (HT) voltage of each sample was recorded to prevent measurement saturation. The cumulative values ​​of three measurements were used, and the buffer sample was subtracted as background to obtain the final spectrum. The final spectrum was normalized to the final concentration of each sample using the molar-average molecular weight.

[0142] Fourier transform infrared (FT-IR) spectroscopy FT-IR spectra were recorded using a Bruker Tensor 37FT-IR spectrometer. Samples were prepared with heavy water (D2O) and placed between two CaF2 windows separated by 50 μm. The final spectrum was obtained at 1 cm². -1 The results were obtained from 25 scans at a given resolution, with atmospheric CO2 and H2O subtracted as background.

[0143] Fluorescence polarization depolarization method 100 μL of an aqueous solution of annealed PA ([PA]=6 mM, [KCl]=3 mM, [NaCl]=150 mM) was added to a THF solution of 1,6-diphenyl-1,3,5-hexatriene (DPH; 1.4 mM) (2 μL), and the mixture was incubated at 25°C for 30 minutes. Then, the mixture was diluted with an aqueous solution of KCl and NaCl (1900 μL, [KCl]=3 mM, [NaCl]=150 mM), incubated at 25°C for 10-30 minutes to obtain a solution of PA (300 μM) embedded with DPH (1.4 μM). Next, to obtain IKVAVPA2 (A2G2) or IKVAVPA5 (G4) in the presence of CaCl2, 5 mM CaCl2 was added to the aqueous solution of PA embedded with DPH so that the molar ratio of PA:CaCl2 was 6:1. An ISS model PC1 spectrofluorometer equipped with a 300W xenon arc lamp with an 18A power supply was used to excite DPH at 336 nm, and emission was recorded at 450 nm. The excitation slit width and emission slit width were set to 1 mm (bandwidth 8 nm). Anisotropy was calculated using the following formula.

[0144]

number

[0145] Cy3-functionalized FGF2PA sample for fluorescence testing Various FGF2PAs functionalized with Cy3 DBCO dyes were added in 1 mol% to an aqueous PA solution of an IKVAV / FGF2 mixture in a molar ratio of 90:10 ([PA] total A solution ([KCl]=6mM, [NaCl]=3mM, [NaCl]=150mM) was prepared. 100 μL of this solution was diluted with an aqueous solution of KCl and NaCl (1900 μL, [KCl]=3mM, [NaCl]=150mM), and the system was incubated at 25°C for 10 minutes to equilibrate. The final concentrations of PA and dye were 300 μM and 0.3 μM, respectively. Cy3 was excited at 535 nm, and luminescence was recorded at 575 nm.

[0146] Rheology PA material was prepared using the method described above for in vitro testing. An MCR302 rheometer (Anton Paar) was used for all rheological tests. The instrument stage was set to 37°C to simulate in vitro and in vivo conditions. 150 μL of PA solution was dropped onto the sample stage, and 30 μL of 25 mM CaCl2 solution was pipetteed onto the underside of a 25 mm cone plate placed above the material. The plate was slowly lowered to the measurement position, and a humidifying collar was used to surround the sample plunger to prevent evaporation of the sample during each 45-minute experiment. After each experiment, the sample was equilibrated for 30 minutes at a constant angular frequency of 10 [rad / s] and 0.1% strain. After reaching the plateau, the storage modulus and loss modulus (G' and G") were recorded.

[0147] Optical density (OD) PA materials were prepared using the method described above for the in vitro test. The PA solution was further diluted with 1× physiological saline to a total volume of 300 μL. 100 μL of these suspensions were pipetted into three wells of a 96-well plate, and their optical density was recorded at 600 nm using a Cytation3 cell imaging multimode reader (BioTek).

[0148] Immobilization of IKVAV peptide onto glass surface Borosilicate glass coverslips (12 mm in diameter; Fisher Scientific) were modified with synthetic IKVAV peptide. The borosilicate glass coverslips were washed with 2% (v / v) Micro 90 detergent (Sigma-Aldrich) at 60°C for 30 minutes, rinsed six times with distilled water, rinsed with ethanol, and then dried. The coverslips were plasma-etched with O2 for 30 seconds (Harrick Plasma PDC-001-HP), and immediately incubated in a 2% (v / v) ethanol solution of (3-aminopropyl)triethoxysilane (Sigma-Aldrich) for 15 minutes. The coverslips were then rinsed twice with ethanol and twice with water, and then oven-dried. Next, the IKVAV peptide was prepared to a concentration of 50 nmol / mL in a 1.25 mg / mL solution of 1-ethyl-3-(dimethylaminopropyl)carbodiimide (Acros Organics) containing 2% DMF (Sigma-Aldrich). The coverslips were incubated in the presence of this solution at 40°C for 3.5 hours. After incubation, the coverslips were sequentially washed with 100% anhydride acetic acid (Fisher Chemical), 2M hydrochloric acid (Fisher Chemical), and 0.2M sodium bicarbonate. After rinsing with excess water, the samples were sonicated in 4M urea for 10 minutes, then in 1M NaCl for 10 minutes, rinsed with excess water, and dried at 100°C for 1 hour.

[0149] NMR experiment NMR spectra were acquired at 600 MHz using a Tecmag NMR spectrometer with a Doty diffusion probe having a sweep width of 6 kHz and 16 k data points, or at 600 MHz using a Brucker Neo system equipped with a QCI-F cryoprobe.

[0150] NMR spectra of various IKVAVPAs were recorded at 25°C using a solvent prepared by adding TFA-d to H2O / D2O in a 9 / 1 ratio (D2O containing 0.05 wt% of 3-(trimethylsilyl)propion-2,2,3,3-d4 sodium salt). Chemical shifts are reported in parts per million (ppm). 1 H,1 H-Gcosy, 1 H, 13 Structure assignment was carried out using C-gHSCQAD, TOCSY and NOESY. The multiplicities are represented as singlet (s), doublet (d), multiplet (m), doublet of doublets (dd), doublet of doublets of doublets (ddd), triplet (t), quartet (q). The 90° pulse width was 15 μs and a typical spectrum required 32 scans. Since the epitope containing aromatic protons was only present at 10 mol%, more scans (512 scans) were required for an accurate estimation of the aromatic signal intensity.

[0151] The diffusion coefficient was measured by pulsed magnetic field gradient NMR using a bipolar pulse pair sequence with the longitudinal eddy current delay and varying the gradient strength value in 16 steps with a maximum gradient strength of 53.5 G / cm. The peak intensity I was measured and fitted to the Stejskal-Tanner equation:

[0152]

Equation

[0153] The rotational radius R was calculated from the Stokes-Einstein equation as follows g as follows.

[0154]

Equation

[0155]

number

[0156] Peak Attribution IKVAVPA1: 1 H-NMR(600MHz,TFA-d1):δ0.83(t,3H,J=6.6Hz,Pal-CH3),0.88(t,3H,J=7.2Hz,Ile-C (δ) H3), 0.92-1.01 (m, 24H, 4xVal-C (γ) H3),1.25(m,24H,several Pal-CH2),1.49(m,9H,3xAla-C (β) H3), 1.67(m, 8H, 4xGlu-C (β) H2), 2.1(m, 8H, 4xGlu-C (γ) H2), 2.24(m, 3H, Pal-C (α) H2), 2.59(m,3H,3xVal-C (β) H), 2.81(t, 2H, J=7.2Hz, Lys-C (ε) -H2),3.88(m,Lys-C (α) H2, Ile-C (α) H2), 3.97(m, 4H, 4xGlu-C (α) H2), 4.01(m, 3H, 3xVal-C (α) H), 4.38-4.5 (m, 6H, Gly-C (α) H2,3xAla-C (α) H, Val-C (α) H), 4.62-4.75 (m, Ala-C (α) H2). IKVAVPA2: 1H-NMR(600MHz,TFA-d1):δ0.80(t,3H,J=6.5Hz,Pal-CH3),0.86(t,3H,J=7.4Hz,Ile-C (δ) H3),0.92-0.96(m,12H,2xVal-C (γ) H3),1.01(dd,J=10.9Hz,6.7Hz,Ile-C (γ) H3),1.21-1.33(m,24H,several Pal-CH2),1.45(d,J=7.1Hz,Ile-C (β) H3),1.49(m,9H,3xAla-C (β) H3),1.66(m,2H,Lys-C (β) H2),1.72(m,2H,Lys-C (γ) H2),1.85(m,8H,4xGlu-C (β) Η2),2.1(m,8H,4xGlu-C (γ) Η2),2.25(m,2H,Pal-C (α) Η2),2.52(t,2H,J=7.1Hz,Lys-C (ε) -H2),4.1-4.3(m,2H,Lys-C (α) H,Ile-C (α) H),4.4-4.5(m,2H,2xGlu-C (α) H),4.60-4.67(m,2H,2xGlu-C (α) H),4.71-4.86(m,12H,3xGly-C (α) H2,3xAla-C (α) H,3xVal-C (α) H). IKVAVPA3: 1 H-NMR(600MHz,TFA-d1):δ0.93(t,3H,J=6.8Hz,Pal-CH3),0.99(t,3H,J=7.1Hz,Ile-C (δ) H3),1.04-1.16(m,18H,3xVal-C (γ) H3,3H,Ile-C (β) H3),1.25-1.32(m,24H,several Pal-CH2),1.51(m,6H,2xAla-C (β) H3)1.59-1.63(m,8H,4xGlu-C (β)Η2),1.80(dq,J=12.9,1H,Val-C (β) H),2.25(m,2H,Pal-C (α) Η2,4H,2xGlu-C (β) Η2),2.39(m,4H,2xGlu-C (β) Η2),2.66(t,2H,J=7.1Hz,Lys-C (ε) -H2),3.86-4.01(m,5H,Ile-C (α) H,2xGlu-C (α) H,Lys-C (α) H,Val-C (α) H),4.55-4.62(m,2H,2xGlu-C (α) H),4.77-4.82(m,6H,3xGly-C (α) H2),4.86-4.92(m,3H,2xAla-C (α) H,Val-C (α) H),4.98-5.01(m,1H,Val-C (α) H). IKVAVPA4: 1 H-NMR(600MHz,TFA-d1):δ0.80(t,3H,J=6.9Hz,Pal-CH3),0.86(t,3H,J=7.2Hz,Ile-C (δ) H3),0.91-1.06(m,18H,3xVal-C (γ) H3,3H,Ile-C (β) H3),1.21-1.28(m 24H,several Pal-CH2),1.45-1.51(m,8H,4xGlu-C (β) Η2),1.56(m,6H,2xAla-C (β) H3)1.71(dq,J=12.9,1H,Val-C (β) H),1.83(m,2H,Pal-C (α) Η2,4H,2xGlu-C (β) Η2),2.13(m,4H,2xGlu-C (γ) Η2),2.7(t,2H,J=7.3Hz,Lys-C (ε) -H2),4.14-4.22(m,5H,Ile-C (α) H,2xGlu-C (α) H,Lys-C (α) H,Val-C(α) H),4.24-4.29(m,2H,2xGlu-C (α) H),4.43-4.52(m,6H,3xGly-C (α) H2),4.62-4.68(m,3H,2xAla-C (α) H,Val-C (α) H),4.73-4.86(m,1H,Val-C (α) H). IKVAVPA5: 1 H-NMR(600MHz,TFA-d1):δ0.78(t,3H,J=6.8Hz,Pal-CH3),0.84(t,3H,J=7.3Hz,Ile-C (δ) H3),0.90-0.94(m,12H,2xVal-C (γ) H3,3H,Ile-C (β) H3),1.19-1.29(m 24H,several Pal-CH2),1.44-1.49(m,11H,4xGlu-C (β) Η2,Ala-C (β) H3),1.88(m,2H,Pal-C (α) Η2),2.11(m,8H,4xGlu-C (γ) Η2),2.54(t,2H,J=6.9Hz,Lys-C (ε) -H2),4.13-4.29(m,5H,Ile-C (α) H,2xGlu-C (α) H,Lys-C (α) H,Val-C (α) H),4.41-4.47(m,2H,2xGlu-C (α) H),4.62-4.67(m,8H,4xGly-C (α) H2),4.72-4.79(m,2H,Ala-C (α) H,Gly-C (α) H2),4.73-4.86(m,1H,Val-C (α) H). IKVAVPA6: 1 H-NMR(600MHz,TFA-d1):δ0.82(t,3H,J=6.9Hz,Pal-CH3),0.88(t,3H,J=7.2Hz,Ile-C (δ) H3),0.93-1.06(m,18H,3xVal-C (γ)H3,3H,Ile-C (β) H3),1.23-1.21(m,24H,several Pal-CH2),1.46-1.51(m,20H,4xGlu-C (β) Η2,4xAla-C (β) H3),1.79-1.93(m,2H,Pal-C (α) Η2,8H,4xGlu-C (γ) Η2),2.09-2.20(m,8H,4xGlu-C (γ) Η2),2.62(t,2H,J=6.9Hz,Lys-C (ε) -H2),4.17(d,1H,J=17Hz,Ile-C (α) H2),4.21(d,1H,J=17Hz,Ile-C (β) H2),4.45-4,49(m,5H,4xGlu-C (α) H2,Val-C (α) H),4.55(d,J=7.33Hz,1H,Val-C (α) H),4.61-4.71(m,4H,Ala-C (α) H),4.79-4.82(m,2H,Val-C (α) H,Lys-C (α) H),4.87(m,1H,Val-C (α) H). IKVAVPA7: 1 H-NMR(600MHz,TFA-d1):δ0.84(t,3H,J=7.1Hz,Pal-CH3),0.89(t,3H,J=7.1Hz,Ile-C (δ) H3),0.89-1.03(m,18H,3xVal-C (γ) H3,3H,Ile-C (β) H3),1.17-1.24(m,24H,several Pal-CH2),1.38-1.54(m,20H,4xGlu-C (β) Η2,4xAla-C (β) H3),1.84-1.91(m,2H,Pal-C (α) Η2,8H,4xGlu-C (γ) Η2),2.01-2.11(m,8H,4xGlu-C (γ) Η2),2.72(t,2H,J=6.9Hz,Lys-C (ε)-H2),4.14(d,1H,J=16.7Hz,Ile-C (α) H2),4.21(d,1H,J=16.7Hz,Ile-C (β) H2),4.29-4.35(m,5H,4xGlu-C (α) H2,Val-C (α) H),4.51(d,J=7.12Hz,1H,Val-C (α) H),4.67-4.73(m,4H,Ala-C (α) H),4.72-4.81(m,2H,Val-C (α) H,Lys-C (α) H),4.88(m,1H,Val-C (α) H). IKVAVPA8: 1 H-NMR(600MHz,TFA-d1):δ0.85(t,3H,J=7.2Hz,Pal-CH3),0.86(t,3H,J=7.1Hz,Ile-C (δ) H3),0.91-1.06(m,12H,2xVal-C (γ) H3,3H,Ile-C (β) H3),1.11-1.20(m,24H,several Pal-CH2),1.29-1.44(m,20H,4xGlu-C (β) Η2,4xAla-C (β) H3),1.81-1.92(m,2H,Pal-C (α) Η2,8H,4xGlu-C (γ) Η2),2.02-2.14(m,8H,4xGlu-C (γ) Η2),2.65(t,2H,J=7.1Hz,Lys-C (ε) -H2),4.09(d,1H,J=17.1Hz,Ile-C (α) H2),4.13(d,1H,J=17.1Hz,Ile-C (β) H2),4.21-4.30(m,4H,4xGlu-C (α) H2),4.54-4.62(m,5H,Ala-C (α) H),4.69-4.84(m,2H,Val-C (α) H,Lys-C (α) H),4.86-4.91(m,1H,Val-C (α) H).

[0157] Diffusion-aligned spectroscopy (DOSY) PA materials were mixed (90 mol% IKVAVPA2 + 10 mol% various FGF2PA), sonicated as described above, and then freeze-dried. Next, using 0.25 mM sodium trimethylsilylpropanesulfonate (DSS) as the chemical shift standard and intensity standard in a standard 5 mm NMR tube, the sample was dissolved in D2O water and solubilized in 1 equivalent of 6 mM NaOD. After sonication for 20 minutes, the sample was annealed at 80°C for 30 minutes and then slowly cooled to room temperature. The diffusion coefficient was measured by pulsed magnetic field gradient NMR using a stimulated echo pulse series with a gradient pulse width of 2 ms and a diffusion delay time of 100 ms, and a maximum gradient intensity of 53 G / cm.

[0158] 1. Simulation Procedure Various PAs were constructed in Avogadro and converted to MARTINI coarse-grained (CG) force field representation using a modified version of martinize.py for palmitoyl tail addition and the coiled-coil secondary structure of the peptides. The last two E residues (farthest from the aliphatic tail) and the K and R residues within the epitope are charged, while the first two E residues were considered neutral as previous simulations had shown they are ideal for fiber formation. The final charge was assumed to be -1 for IKVAVPA and +3 for FGF2PA. CG water and 21.5×21.5×21.5 nm solvated with enough ions to neutralize the system. 3The simulation was performed in two stages using a cubic box. First, 300 IKVAVPA molecules were randomly placed in the box with a minimum intermolecular gap of 3 Å. This yielded a concentration of 50 mM (7.3-7.8 wt%) for IKVAVPA. This is within a widely used concentration range to accelerate self-assembly simulations and can be increased up to 10 times higher than the experimental system used. These systems were brought to equilibrium for 10 μs (Figure 8). The formed fibers were placed in the center of the box, and 33 FGF2PA molecules (10 mol%) were randomly added around the fibers with a minimum gap of 3 Å. Next, these systems were brought to equilibrium for 10 μs in five independent simulations for each system.

[0159] Full visualization was performed using Visual Molecular Dynamics (VMD). Coarse-grained molecular dynamics (CG-MD) simulations were performed using GROMACS 5.0.4, which was also used for the simulation analysis. A relative permittivity of 15 for electrostatic action and a 1.1 nm cutoff were used for intermolecular interactions using a reaction field based on the potential shift of the Lennard-Jones interaction. The entire system was minimized to 5000 steps or until the interatomic forces converged to less than 2000 pN. Self-assembly simulations were performed using a 25 fs time step with an NPT ensemble using a velocity rescaling algorithm for temperature (303 K, τT=1 ps) and the Berensen method for pressure (1 bar, τP=3 ps). The simulation was performed at 100,000,000 steps, corresponding to an effective time of 10 μs.

[0160] Clustering was measured by setting the cutoff distance of PA within the same cluster to 0.6 nm (Figure 6J). Dynamics were measured using the root-mean-square fluctuations (RMSF) during the last 5 μs of five simulations, using systems that equilibrated after 5 μs when expressed in terms of root-mean-square fluctuations (RMSF). Therefore, some FGF2PA did not bind to the fibers before the last 5 μs, resulting in a shielding effect on the results, and such systems were excluded from this analysis. For visualization, RMSF was converted to a β coefficient, and all results were normalized to obtain comparable color codes.

[0161] 2. In vitro cell culture Human umbilical vein (HUVEC) culture Human umbilical vein cells (HUVECs) (donor pools: LONZA, Allendale, New Jersey) were grown in T-75 cell culture flasks using complete medium supplemented with 1% penicillin-streptomycin (Endo GRO-VEGF complete medium kit, Millipore) until they reached 70-80% confluence in each experiment (P2-P4). The medium was changed every 3 days.

[0162] HUVEC treatment and coating The treatment solution was prepared by dissolving the PA co-assembly in serum-free medium. The total concentration of FGF2PA per treatment solution was 0.5 μM. FGF2 natural protein (Peprotech) was resuspended and used at 0.25 nM. In Western blotting experiments, HUVEC cells were treated after in vitro culture in 12-well plates at a density of approximately 150,000 cells / well for 2 days. After adding the treatment solution in vitro for 2 hours to 2 days, the cell lysate was collected or the cells were fixed. For PA coating, PA was applied to coverslips (German Glass, Chemglass Life Science) or tissue culture plates as described in the "PA Co-assembly Preparation" section. Three independent experiments were performed for each condition, with three slides for each condition.

[0163] Human neural progenitor cell (hNPC) culture Neural progenitor cells (NPCs) were differentiated from human embryonic stem cells HUES64 (Harvard University) (see Figure 2C). In summary, mTESR medium (STEMCELL Technologies) was dispensed into Matrigel (Thermo Fisher) coated plates, and stem cells were grown to 70% confluence. The medium was then replaced with N2B27 medium (50% DMEM:F12, 50% Neurobasal; Gibco) containing dual SMAD inhibitors (SB431542, DNSK International and LDN-193189, Tocris) and cultured for 12 days to induce the generation of neural progenitor cells. Neuralization was enhanced by adding laminin to the medium from day 5 to day 12. Next, nerve rosettes were isolated using a nerve rosette selection reagent (STEMCELL Technologies) to obtain NPCs, which were then grown in N2B27 (Gibco) medium supplemented with bFGF (Millipore). In preparation for experiments on various PA platforms, the NPCs were detached using Accutase (Innovative Cell Technology) and cultured on individual platforms dispensed with DMEM:F12+N2+B27 medium supplemented with hyclone penicillin-streptomycin (GE Healthcare) and ascorbic acid (0.2 μg / mL; Sigma-Aldrich).

[0164] Treatment of human neural progenitor cell (hNPC) cultures with various IKVAVPAs or seeding onto PA core assembly coatings In the IKVAVPA treatment, hNPCs were cultured at a density of 500,000 cells / well and 80,000 cells / well, respectively, on ornithine-coated (German Glass, Chemglass Life Science) 6-well plates and 24-well plates. hNPCs were treated with 60 mM IKVAVPA ([PA]=6 mM, [KCl]=3 mM, [NaCl]=150 mM) or recombinant laminin protein (10 mg / mL) in solution. IKVAVPA2 and IKVAVPA5 were mixed with 5 mM CaCl2 so that the PA:CaCl2 ratio was 6:1, and hNPCs were treated. In the 2D experiment, hNPCs were cultured at the above density on various IKVAVPA or laminin coatings of 6-well plates and 24-well plates. In the core assembly experiment, hNPCs were cultured at a density of 400,000 cells / well and 50,000 cells / well, respectively, on various core assembly PA coatings of 6-well plates and 24-well plates.

[0165] The hNPCs were replenished with DMEM:F12 + N2 + B27 medium supplemented with hyclone penicillin-streptomycin and ascorbic acid four times a week. After 24 hours, 72 hours, 1 week or 2 weeks in vitro, cell lysates were collected or fixed for WB or ICC, respectively. At least three independent experiments were performed in triplicate for each condition investigated.

[0166] Surface sensing of translation (SUnSET) hNPCs cultured in the presence of various IKVAVPA treatment solutions were pulsed with puromycin (20 μM, Sigma-Aldrich) for 10 minutes at 37°C. Two hours prior to the puromycin pulse, the cells were pretreated with cycloheximide (100 mg / mL, Calbiochem, Millipore) as a translation inhibitor control. 30 μg of protein extract was obtained and loaded onto Mini-PROTEAN TGX stain-free gel (4-20% gradient, BIO-RAD). Total protein signal was detected using ChemiDoc™ XRS+ (Bio-Rad), and newly synthesized proteins were detected by Western blotting using an anti-puromycin antibody (mouse anti-puromycin 1:5000, Millipore).

[0167] Cell viability assay HUVEC and hNPC were treated or cultured in vitro for 2 days to 2 weeks in various PA co-assembly systems. The culture medium was removed, and the cells were rinsed once with 1×HBSS (Gibco). Cell viability was evaluated using the Calcein AM / Ethidium Homodimer 1 viability assay (Invitrogen). The HBSS solution of Calcein AM / Ethidium Homodimer 1 was added to each well at room temperature (RT) for 20 minutes. The solution was removed, the samples were rinsed three times with HBSS (Gibco), and the coverslips were mounted in preparation for imaging.

[0168] EdU analysis In the hNPC proliferation assay, 2 μM of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU, Thermo Fisher) was incorporated into the culture medium for 24 hours. At the specified time, the cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 minutes. After fixation, the samples were washed twice with PBS and then stained at room temperature in the dark for 30 minutes with the Click-iT® EdU cell proliferation kit containing 11 mM CuSO4 (taken from 50 mM stock solution) and 1 μg / mL Alexa Fluor-555 azide (taken from 0.5 mg / mL Thermo Fisher product). Next, the staining cocktail was removed and the cells were washed with PBS. Then, hNPC was counterstained at room temperature for 20 minutes with an anti-rabbit SOX2 antibody and a PBS solution of 5 μg / mL 4',6-diamidino-2-phenylindole (DAPI, Thermo Fisher) according to the immunofluorescence protocol described below. Next, the cells were washed with PBS and mounted using an Immu-Mount (Fisher Scientific) in preparation for imaging.

[0169] 4. In vivo trials: All animal care and treatment were carried out in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals. All treatments were approved by the Northwestern University Institutional Animal Care and Use Committee.

[0170] Surgical procedure All experiments were conducted at the Simpson Querrey Institute at Northwestern University. 182 CD1 mice (Charles River) were included in this study (Siamese group: N=38, IKVAVPA2 monotherapy group: N=38, IKVAVPA2 + FGF2PA1 group: N=38, IKVAVPA2 + FGF2PA2 group: N=38, IKVAVPA1 group: N=12, main chain PA group: N=12, IKVAVPA4 group: N=6). The animals used in the study were 10-16 weeks old. Eight independent in vivo experiments were conducted (injury + PA injection), and in each of the eight experiments, animals of exactly the same age were injected with at least four major treatment groups: Siamese group, IKVAVPA2 monotherapy group, IKVAVPA2 + FGF2PA1 group, and IKVAVPA2 + FGF2PA2 group. The animals were anesthetized using 2.5% isoflurane gas and oxygen. Laminectomy was performed to expose the spinal cord at the T10-T11 level. A severe contusion was created using the Infinite Horizon Spinal Cord Impactor system (IH-0400, Precision Systems and Instrumentation) with an impact force of 85 kDyn and a duration of 60 seconds. After injury, the skin was sutured using 9 mm wound clips (BD Biosciences), and the animals were allowed to recover on a heating pad to maintain body temperature. Manual abdominal pressure urination was performed daily throughout the entire 12-week experimental period.

[0171] Animal Eligibility and Exclusion Criteria All mice were evaluated in an open-field environment 24 hours after injury, and any animals showing any hindlimb movement (BMS score higher than 0) were excluded from the study. Mice that met this eligibility criterion were randomly assigned to the PA injection group, and then evaluated in a blind state, concealing the experimental conditions.

[0172] Injection procedure Twenty-four hours after SCI, various PAs were injected using a glass capillary micropipette (Sutter Instruments, Novato, CA) (outer diameter 100 μm) coated with Sigmacote (Sigma-Aldrich) to reduce surface tension, as described in other literature (19) (100 μm). The capillary was attached to a Hamilton syringe using a female Luer adapter (World Precision Instruments) controlled by a Micro4 microsyringe pump controller (World Precision Instruments). Under isoflurane anesthesia, the autoclip was removed to expose the injury site. Twenty-four hours after injury, the laminectomy of the spine remained intact, and the wound formed by the injury was visible. Using a Kopf stereotactic system, the micropipette was positioned immediately dorsal to the lesion. The micropipette was lowered to a depth of 750 μm from the dorsal surface of the spinal cord, and 4-6 μL of diluted amphiphilic molecular solution was injected at a rate of 1 μL / min. The micropipette was withdrawn at 250 μm intervals to leave a trajectory of PA within the spinal cord (from ventral to dorsal). At the end of the injection, the pipette was held for an additional 2-3 minutes to allow the material to gel before being withdrawn, and the incision was closed with a 9 mm wound clip.

[0173] Hindlimb gait movement evaluation Motor function was assessed using the Basso Mouse Scale (BMS) open-field gait score scale. Two experienced examiners evaluated each animal for 5–10 minutes, assigning a defined score to each hind limb. For footprint analysis, the hind limbs of the mice were dipped in dye. A narrow runway (80 cm long and 4 cm wide) was covered with white paper, and the animals were allowed to walk across it. Stride length was defined as the distance from the start to the end of each step made by the hind limbs. Stride width was defined as the distance from the outermost toe of the left foot to the outermost toe of the right foot. All measurements were taken over three consecutive steps on each side and averaged.

[0174] Anterior BDA corticospinal tract tracing Fourteen days prior to perfusion, the corticospinal tract was traced using six mice for each condition. Animals were anesthetized with isoflurane gas at a concentration of 2.5% in oxygen. The heads of each mouse were fixed using a stereotactic device (Stoelting Co.). An 8 mm incision was made in the scalp along the midline of the skull using a surgical scalpel. 0.25 μL of 10% biotinylated dextranamine (BDA, molecular weight = 10,000, Thermo Fisher Scientific) was injected into each hemisphere, which is the region of the motor cortex. The following coordinates were used: Rostral-caudal from the bregma: ±0.0 mm, 0.5 mm, 1.0 mm; Lateral: ±1.0 mm, 1.5 mm; Depth: 0.7 mm. Sections were treated with a secondary antibody (Thermo Fisher) labeled with a conjugate of streptavidin and 555 to detect BDA.

[0175] BrdU injection PA was injected into the spinal cord 24 hours later, and 5-bromo-2'-deoxyuridine (BrdU, Sigma-Aldrich) was injected intraperitoneally (5 mg per 10 g of body weight) for 7 days (1 pulse per day) using 6 rats for each condition. BrdU uptake was analyzed 12 weeks after PA injection by immunohistochemistry (rat anti-BrdU, 1:1000, Abcam).

[0176] DiI label Immediately after deep anesthesia and before perfusion, as previously described (21), 20 μL / mL of DiI (Sigma-Aldrich) was dissolved in a PBS solution containing 5 w / v% glucose and injected transcardially into 6 animals under each condition. Immediately thereafter, the animals were perfused transcardially with an isotonic solution containing 4% paraformaldehyde (PFA dissolved in 0.4 M phosphate buffer at pH 7.4).

[0177] Animal sacrificial deaths and tissue disposal All animals were euthanized by transcardiac perfusion with carbon dioxide overdose, followed by isotonic solution containing 4% paraformaldehyde (PFA, Sigma-Aldrich) (0.4 M phosphate buffer at pH 7.4). The spinal cord was fixed with 4% PFA for 4-6 hours and then fixed overnight with PBS containing 30% sucrose (Sigma-Aldrich). Next, the spinal cord was frozen in PBS containing 30% sucrose (Sigma-Aldrich) and 15% gelatin (Sigma-Aldrich) and sectioned to a thickness of 40 μm using a Leica CM1850 cryostat.

[0178] 5. Bioassays for in vitro and in vivo use Immunofluorescence For immunofluorescence, fixed primary cultures or free-floating tissue sections (40 μm thick) were incubated in a blocking buffer containing 0.02% Triton-X (Sigma-Aldrich), 1% NHS (Gibco), and 1× PBS (Gibco). The samples were incubated overnight at 4°C with the primary antibody. Alexa-488, Alexa-555, and / or Alexa-647 (1:500, Thermofisher) were used as secondary antibodies. Nuclei were stained using DAPI (1:500, Thermofisher). Finally, the samples were mounted in coverslips with Immu-Mount (Fisher Scientific) solution in preparation for imaging.

[0179] Western blot (WB) Proteins were extracted from in vitro and in vivo samples using RIPA buffer (Thermofisher) supplemented with the Halt protease / phosphatase inhibitor cocktail (Thermofisher). Subsequently, in the in vivo assay, spinal cord tissue was sonicated using a Branson horn-type sonicator to disrupt the tissue. The protein content of all samples used was determined using the Pierce® BCA protein assay kit (Thermofisher). Protein extracts obtained from cell cultures or tissues were separated using an SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad) by electrophoresis. The membrane was blocked with 5% skim milk solution (Bio-Rad) for 30 minutes to 1 hour and incubated overnight with the primary antibody. The corresponding HRP-labeled secondary antibody (1:1000, ThermoFisher) was used at room temperature for 1 hour. Protein signals were detected using Radiance bioluminescent ECL substrate (Azure Biosystems). We used Azure Biosystems imager to image the membrane and performed densitometry analysis using ImageJ software.

[0180] Antibodies for immunofluorescence and Western blotting The following primary antibodies were used in 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-Sox2 (1:1000, Abcam, ab110145), streptavidin Alexa Fluor(registered trademark) 555 conjugate (1:500, Thermofisher, S32355), rabbit anti-PH3 (1:1000, Cell Signaling, 9701), rat anti-BrdU (1:1000, Abcam, ab6326), rabbit anti-MBP (1:500, Abcam, ab40390), mouse anti-NeuN (1:1000, Millipore, MAB377), goat anti-ChAT (1:1000, Millipore, AB144P), rabbit anti-FGFR1 (1:500, Cell Signaling, 3472), p-FGFR1 (1:500, Cell (Signalling, 3471), mouse anti-ITGB1 (1:250, Millipore, clone HUTS-4, MAB2079Z), rabbit anti-p-ERK1 / 2 (1:1000, Abcam, ab196883), TUJ1 (1:2000, Biolegend, 802001 and 801213), mouse anti-Pax6 (1:500, Millipore, AD2.38), rabbit anti-Ki67 (1:500, Abcam, ab15580), rabbit anti-nestin (1:1000 Millipore, ABD69), rabbit anti-ILK (1:500, Cell Signaling, 3862), rabbit anti-p-FAK (1:1000, Cell Signaling, 3281S), rabbit anti-FAK (1:1000, Cell Signaling, 3285S).

[0181] Blood vessel analysis To evaluate in vitro luminal structure formation and in vivo angiogenesis, an ImageJ (Fiji) script was configured to automatically calculate 1) vascular area fraction, 2) vascular length, and 3) branching number. Images were processed, binarized, and analyzed. Newly formed vessels in vivo were identified by quantifying the amount of CD31 / BrdU double-positive cells within the region of interest. Functional vessels were identified using DiI staining in 8 sections within the lesion per mouse. Six mice were used for each treatment group in this analysis. The quantified sections were selected as the first continuous section within the DiI-stained lesion.

[0182] Tracking nerve processes Axons labeled with BDA (Thermofisher, N7167) or stained with 5HT (Abcam) were quantified using (21) Imaris® software version 9.3 as previously described. Lines were drawn at regular intervals across longitudinal spinal cord sections from the proximal margin (PB) to the distal margin (DB) of the SCI lesion, and researchers, unaware of the experimental conditions, counted the number of axons intersecting the drawn lines. Multiple sections passing through the center of the spinal cord with dense BDA or 5HT staining were counted for each individual, and the total number of crossovers per location per individual was expressed. Six individuals were used for each treatment group in these analyses.

[0183] Decomposition test and tissue clearing The degradation of PA injected into injured spinal cords was performed by covalently labeling the IKVAV sequence with the Alexa-647 (click) fluorescent dye. Spinal cords were perfused, extracted, and cleared using benzyl alcohol-benzyl benzoate (BABB, Sigma-Aldrich) at various time points after PA injection (2, 4, 6, and 12 weeks). After clearing, the spinal cord tissue fluoresced at 488 nm. Complete reconstruction was performed using spinning disk confocal microscopy and analyzed using Imaris software. Three spinal cords were used for each dosing group and each time point in these studies.

[0184] Imaging Fluorescent cells, sections, or fully cleared spinal cord samples were visualized and imaged using a Nikon A1R confocal laser scanning microscope equipped with a GaAsP detector, a Nikon W1 dual-cam spinning disk confocal microscope, and a Nikon A1RMP+ multiphoton confocal laser microscope. Larger cross-sections of the spinal cord were obtained using a Nikon Ti2 wide-field microscope.

[0185] Image quantification and analysis For in vitro cell quantification, image files were imported into NIH Image J(1.51) software, and the total number of cells within a specified region was measured using the "Particle Analysis" and "Cell Counter" functions. Serial tissue sections were stained with NeuN and ChAT using free-floating immunohistochemistry and quantified using Nikon Elements software. The rostral and caudal margins of the lesion were selected as the first section stained with NeuN at all four corners of the gray matter. Eight sections were selected from the lesion per animal, and the number of neurons per section was expressed. Automated multi-channel image acquisition, image stitching, and z-stack reconstruction (36-40 mm thickness) were performed using a Nikon GasP R1 confocal microscope, and the entire selected section for NeuN and ChAT markers was imaged under all conditions.

[0186] The fluorescence intensity of GFAP-stained tissue sections was analyzed using NIH Fiji software. Images scanned with the various microscopes mentioned above under constant exposure settings were used for this analysis. Single-channel immunofluorescence images were used, and the number of fluorescently positive pixels was analyzed along selected regions in each image. The "Plot Profile" function of ImageJ software was used to obtain the average pixel value relative to the position along the drawn line. Five cross-sections passing through the center of the spinal cord were counted per mouse to plot GFAP against distance. Six mice were used for image quantification for each treatment group.

[0187] statistical analysis Data were analyzed using GraphPad Prism software (version 9.12). For each statistical analysis, QQ plots or frequency distribution plots (histograms) were used for rapid identification of the normal distribution. The Shapiro Wilk test and the D'Agostino-Pearson omnibus normality test were also used to test whether the sample data fit a Gaussian distribution. Comparisons between experimental group pairs were performed using unpaired Student's t-tests. Comparisons of three or more groups were performed using one-way ANOVA and independent pairwise post-hoc analysis using the Bonferroni method. Mouse BMS behavior scores were compared between the PA-administered group and the saline-administered group or PA group using repeated measures of two-way ANOVA and the Bonferroni method. Statistical tests and parameters, including definitions and the number of experiments, are reported in the corresponding figure descriptions. In vitro data were represented using bar graphs. Error bars correspond to 30 images per experiment and 3 independent experiments for each condition. In vivo data are represented using dot plots, with each data point representing a value from one in vivo animal or one tissue section (in the overall staining experiment, ICC was independently repeated using tissues from six different animals to obtain comparable results. Eight images, 36–40 mm thick, were taken from each animal in each group). Unless otherwise specified, all data are expressed as mean ± mean standard error (SEM).

Claims

1. A supramolecular assembly comprising at least two types of peptide amphiphilic molecules, wherein the at least two types of peptide amphiphilic molecules are a. At least one IKVAV peptide amphiphilic molecule comprising a hydrophobic segment, a structural peptide segment, a charged peptide segment, and a physiologically active peptide containing the amino acid sequence IKVAV (SEQ ID NO: 1); b. The supramolecular assembly comprising at least one growth factor mimetic peptide amphiphilic molecule.

2. The supramolecular assembly according to claim 1, wherein the at least one IKVAV peptide amphiphilic molecule contains a fluorescence anisotropy value of less than 0.

3.

3. The aforementioned at least one IKVAV peptide amphiphilic molecule, 4s －１ Proton relaxation rate less than ( １ H-R ２ A supramolecular assembly according to claim 1 or 2, comprising )

4. The hydrophobic segment is an alkyl chain having 8 to 24 carbon atoms (C ８－２４ A supramolecular assembly according to any one of claims 1 to 3, comprising )

5. The hydrophobic segment is an alkyl chain having 16 carbon atoms (C １６ The supramolecular assembly according to claim 4, comprising )

6. The structural peptide segment is A ２ G ２ A supramolecular assembly according to any one of claims 1 to 5, including the above.

7. The charged peptide segment is E ２ , E ３ , or E ４ The supramolecular assembly according to any one of claims 1 to 6, which comprises

8. The supramolecular assembly according to any one of claims 1 to 7, wherein the physiologically active peptide is linked to the charged peptide segment by a linker.

9. The supramolecular assembly according to claim 8, wherein the linker is a single glycine (G) residue.

10. The above-mentioned at least one IKVAV peptide amphiphilic molecule is C １６ A ２ G ２ E ４ A supramolecular assembly according to any one of claims 1 to 9, comprising GIKVAV.

11. The above-mentioned at least one growth factor mimetic peptide amphiphilic molecule comprises an alkyl chain having 8 to 24 carbon atoms (C ８－２４ A hydrophobic segment containing ) and V ２ A ２ Or A ２ G ２ A structural peptide segment containing E ２ , E ３ , or E ４ A supramolecular assembly according to any one of claims 1 to 10, comprising a charged peptide segment containing and a growth factor mimetic peptide sequence.

12. The supramolecular assembly according to claim 11, wherein the growth factor mimetic sequence is a vascular endothelial growth factor (VEGF) mimetic sequence, a fibroblast growth factor 2 (FGF2) 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.

13. The supramolecular assembly according to claim 12, wherein the growth factor mimetic sequence is an FGF2 mimetic sequence.

14. The supramolecular assembly according to claim 13, wherein the FGF2 mimetic sequence includes YRSRKYSSWYVALKR (SEQ ID NO: 2).

15. The supramolecular assembly according to any one of claims 12 to 14, wherein the growth factor mimetic sequence is linked to the charged peptide segment by a linker.

16. The supramolecular assembly according to claim 15, wherein the linker is a single glycine (G) residue.

17. The above-mentioned at least one growth factor mimetic peptide amphiphilic molecule is C １６ V ２ A ２ E ４ GYRSRKYSSWYVALKR or C １６ A ２ G ２ E ４ A supramolecular assembly according to any one of claims 1 to 16, comprising GYRSRKYSSWYVALKR.

18. The above-mentioned at least one IKVAV peptide amphiphilic molecule is C １６ A ２ G ２ E ４ The GIKVAV is included, and the at least one growth factor mimetic peptide amphiphilic molecule is C １６ V ２ A ２ E ４ GYRSRKYSSWYVALKR or C １６ A ２ G ２ E ４ A supramolecular assembly according to any one of claims 1 to 17, comprising GYRSRKYSSWYVALKR.

19. The above-mentioned at least one IKVAV peptide amphiphilic molecule is C １６ A ２ G ２ E ４ The GIKVAV is included, and the at least one growth factor mimetic peptide amphiphilic molecule is C １６ V ２ A ２ E ４ The supramolecular assembly according to claim 18, comprising GYRSRKYSSWYVALKR.

20. A composition comprising a supramolecular assembly according to any one of claims 1 to 19.

21. a. At least one IKVAV peptide amphiphilic molecule comprising a hydrophobic segment, a structural peptide segment, a charged peptide segment, and a physiologically active peptide containing the amino acid sequence IKVAV (SEQ ID NO: 1); b. A composition comprising at least one growth factor mimetic peptide amphiphilic molecule, The composition wherein at least one IKVAV peptide amphiphilic molecule and at least one growth factor mimetic peptide amphiphilic molecule interact to form a supramolecular assembly within the composition.

22. The composition according to claim 21, wherein the at least one IKVAV peptide amphiphilic molecule contains a fluorescence anisotropy value of less than 0.

3.

23. The aforementioned at least one IKVAV peptide amphiphilic molecule, 4s －１ Proton relaxation rate less than ( １ H-R ２ The composition according to claim 21 or 22, comprising )

24. The hydrophobic segment is an alkyl chain having 8 to 24 carbon atoms (C ８－２４ The composition according to any one of claims 21 to 23, comprising )

25. The hydrophobic segment is an alkyl chain having 16 carbon atoms (C １６ The composition according to claim 24, comprising ).

26. The structural peptide segment is A ２ G ２ A composition according to any one of claims 21 to 25, comprising:

27. The charged peptide segment is E ２ , E ３ , or E ４ A composition according to any one of claims 21 to 26, comprising:

28. The composition according to any one of claims 21 to 27, wherein the physiologically active peptide is linked to the charged peptide segment by a linker.

29. The composition according to claim 28, wherein the linker is a single glycine (G) residue.

30. The above-mentioned at least one IKVAV peptide amphiphilic molecule is C １６ A ２ G ２ E ４ A composition according to any one of claims 21 to 29, comprising GIKVAV.

31. The above-mentioned at least one growth factor mimetic peptide amphiphilic molecule comprises an alkyl chain having 8 to 24 carbon atoms (C ８－２４ The composition according to any one of claims 21 to 30, comprising a hydrophobic segment containing ), a structural peptide segment containing VVAA or AAGG, a charged peptide segment containing EE, EEE, or EEEE, and a growth factor mimetic peptide sequence.

32. The composition according to claim 31, wherein the growth factor mimetic sequence is a vascular endothelial growth factor (VEGF) mimetic sequence, a fibroblast growth factor 2 (FGF2) 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.

33. The composition according to claim 32, wherein the growth factor mimetic sequence is an FGF2 mimetic sequence.

34. The composition according to claim 33, wherein the FGF2 mimetic sequence comprises YRSRKYSSWYVALKR (SEQ ID NO: 2).

35. The composition according to any one of claims 31 to 34, wherein the growth factor mimetic sequence is linked to the charged peptide segment by a linker.

36. The supramolecular assembly according to claim 35, wherein the linker is a single glycine (G) residue.

37. The above-mentioned at least one growth factor mimetic peptide amphiphilic molecule is C １６ V ２ A ２ E ４ GYRSRKYSSWYVALKR or C １６ A ２ G ２ E ４ A composition according to any one of claims 21 to 36, comprising GYRSRKYSSWYVALKR.

38. The above-mentioned at least one IKVAV peptide amphiphilic molecule is C １６ A ２ G ２ E ４ The GIKVAV is included, and the at least one growth factor mimetic peptide amphiphilic molecule is C １６ V ２ A ２ E ４ GYRSRKYSSWYVALKR or C １６ A ２ G ２ E ４ A composition according to any one of claims 21 to 37, comprising GYRSRKYSSWYVALKR.

39. The at least one IKVAV peptide amphiphile is C １６ A ２ G ２ E ４ GIKVAV, and the at least one growth factor mimetic peptide amphiphile is C １６ V ２ A ２ E ４ GYRSRYSSWYVALKR, the composition according to claim 38.

40. A composition according to any one of claims 21 to 39, for use in a method for treating nerve damage in a subject.

41. The composition according to claim 40, wherein the nerve damage is central nervous system damage.

42. The composition according to claim 41, wherein the central nervous system injury is a spinal cord injury.

43. A method for treating nerve damage in a subject, comprising providing the subject with a composition according to any one of claims 21 to 39.

44. The method according to claim 43, wherein the nerve damage is central nervous system damage.

45. The method according to claim 44, wherein the central nervous system injury is a spinal cord injury.

Images