Functional Self-Assembling Peptide Nanofiber Hydrogels Designed

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Functional self-assembling peptide nanofiber hydrogels designed for nerve regeneration Yuqiao Sun, Wen Li, Xiaoli Wu, Na Zhang, Yongnu Zhang, Songying Ouyang, Xiyong Song, Xinyu Fang, Ramakrishna Seeram, Wei Xue, Liumin He, and Wutian Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11473 • Publication Date (Web): 31 Dec 2015 Downloaded from http://pubs.acs.org on January 1, 2016

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ACS Applied Materials & Interfaces

Functional Self-Assembling Peptide Nanofiber Hydrogels Designed for Nerve Degeneration Yuqiao Sun1a, Wen Li2a, Xiaoli Wu2, Na Zhang3, Yongnu Zhang1, Songying Ouyang6, Xiyong Song6, Xinyu Fang2, Ramakrishna Seeram3,4, Wei Xue1, Liumin He1,2*, Wutian Wu2,3,5*

1

Department of Biomedical Engineering, College of Life Science and Technology, Jinan University, Guangzhou, China

2

School of Biomedical Science, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, PR China 3

Guangdong-Hongkong-Macau Institute of CNS Regeneration (GHMICR), Jinan University, Guangzhou, China

4

Center for Nanofibers and Nanotechnology, Department of Mechanical Engineering, Faculty of Engineering, National University of Singapore, Singapore 5

State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, PR China. 6

National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.

a

These authors contributed equally to this work.

*

To whom correspondence should be addressed.

HE: Tel: 8620-8524338, E-mail: [email protected] WU: Tel:852-28199187, Fax:852-28170857, Email:[email protected]

ACS Paragon Plus Environment


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Abstract Self-assembling peptide (SAP) RADA16-I (Ac-(RADA)4-CONH2) has been suffering from a main drawback associated with low pH, which damages cells and host tissues upon direct exposure. In this study, we presented a strategy to prepare nanofiber hydrogels from two designer SAPs at neutral pH. RADA16-I was appended with functional motifs containing cell adhesion peptide RGD and neurite outgrowth peptide IKVAV. The two SAPs were specially designed to have opposite net charges at neutral pH, the combination of which created a nanofiber hydrogel (-IKVAV/-RGD) characterized by significantly higher G′ than G″ in a viscoelasticity examination. Circular

dichroism,

Fourier

transform

infrared

spectroscopy,

and

Raman

measurements were performed to investigate the secondary structure of the designer SAPs, indicating that both the hydrophobic/hydrophilic properties and electrostatic interactions of the functional motifs play an important role in the self-assembling behavior of the designer SAPs. The neural progenitor cells (NPCs)/stem cells (NSCs) fully embedded in the 3D-IKVAV/-RGD nanofiber hydrogel survived, whereas those embedded within the RADA 16-I hydrogel hardly survived. Moreover, the -IKVAV/-RGD nanofiber hydrogel supported NPC/NSC neuron and astrocyte differentiation in a 3D environment without adding extra growth factors. Studies of three nerve injury models, including sciatic nerve defect, intracerebral hemorrhage, and spinal cord transection, indicated that the designer -IKVAV/-RGD nanofiber hydrogel provided a more permissive environment for nerve regeneration than the RADA 16-I hydrogel. Therefore, we reported a new mechanism that might be beneficial for the synthesis of SAPs for in vitro 3D cell culture and nerve regeneration.

Keywords: self-assembling peptides, nanofiber, hydrogel, 3D cell culture, nerve regeneration


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Introduction Advances in molecular science have significantly contributed to the rapidly emerging fields of bionanotechnology and to the development of novel biomaterials1-4. In this context, nano-scaled biomaterials can be fabricated at the molecular scale from bottom-up self-assembly, which processes one molecule at a time through synthesis and one unit at a time through self-assembly5-7. Self-assembling peptides (SAPs) are skillfully designed peptides that can undergo ordered self-assembly into stable secondary structures (α-helix, β-sheet, or random coil), further forming various aggregation states such as fibrils, fibril networks, membranes, and gels8-10. Such characteristics make SAPs particularly attractive in the fields of medicine, biotechnology, and nanotechnology. Zhang’s group pioneered the investigation of SAPs discovered in yeast7,

11

, which were characterized by their structurally

alternating ionic hydrophilic and hydrophobic amino acids, creating periodic repeats of distinct polar and nonpolar surfaces7, 10-12. RADA16-I (Ac-(RADA)4-CONH2) is a widely investigated representative SAP, which comprised repeated segments of positively charged arginine (R), hydrophobic alanine (A), and negatively charged aspartic acids (D)6. This SAP belonged to the family of ionic self-complementary peptides. Special amphiphilic features arise at pH 7, where the net charge on the molecule is almost zero. With high propensity to form highly stable β-sheet structures, RADA16-I could self-assemble into nanofibers with ~10 nm of fiber diameter and eventually form a 3D hydrogel consisting of >99.5% water by increasing pH to 7 or by exposure to salt solutions11, 13. Such hydrogel was documented to support neuronal cell attachment, differentiation, neurite outgrowth, and functional synapse formation between the attached neurons14. Short bioactive peptide motifs that acted as analogues of the extracellular matrix (ECM) proteins were incorporated into nanofibers through conjugation with RADA16-I. Thus, the 3D macroscopic hydrogel could present functional motifs to cell membrane receptors at a high density, generating a permissive niche potential for tissue regeneration15-18. Recently, synergistic effects were achieved by incorporating two or more essential



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signals in one 3D nanofiber hydrogel in an intimate fashion19. The development of such strategy was important to advanced medicine. However, RADA16-I type SAPs suffered from a great drawback associated with low pH, which could damage cells in in situ 3D cell cultures and even host tissues upon direct injection. The peptide solutions should be neutralized before cell seeding or transplantation in vivo20-21, in which case a solid hydrogel has already formed. Most of the SAP nanofiber hydrogels were therefore applied in 2D cell culture system14,

22

. Considerable efforts have been devoted toward the modification of

RADA 16-I by attaching residues derived from ECM molecules and growth factors for in situ 3D cell culture; however, the cells within the matrices migrated from or penetrated the top-surface of the assembled scaffolds17. A large amount of media was immediately needed to equilibrate the gel to physiological pH if cells were directly embedded in the SAP aqueous solution20-21. Here, we reported a strategy to prepare nanofiber hydrogels from two oppositely charged SAPs that carried different biological signals at neutral pH. Two functional SAPs, namely, RADA16-RGD (Ac-(RADA)4-DGDRGDS) and RADA16-IKVAV (Ac-(RADA)4-RIKVAV) were specifically designed by directly conjugating short bioactive

peptide

motifs

to

the

parent

molecule,

RADA16-I.

IKVAV

(Ile-Lys-Val-Ala-Val) is a laminin sequence known to interact with mammalian neurons and promote neurite outgrowth, whereas RGD (Arg-Gly-Asp) is a well-known cell adhesion ligand derived from fibronectin. Oligopeptides containing RGD or IKVAV epitopes were found to promote the adhesion, differentiation, or neurite outgrowth of neural progenitor cells (NPCs)/stem cells (NSCs)23-24, embryonic neurons22,

25-26

, and mesenchymal stem cells (MSCs)27-28 to various extents when

immobilized onto various substrates. These two designer SAPs were opposite charged in aqueous solution at physiological pH, and a 3D nanofiber hydrogel was formed when these SAPs were combined together. The two bioactive motifs were thereby incorporated in one 3D hydrogel scaffold, yielding a synergistic promotion effect on nerve cell response and nerve regeneration. Constant neutral pH was kept during the self-assembly process so that bioactive molecules and cells could be fully embedded


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in the 3D environment. Moreover, the hydrogel of the designer SAPs can be directly delivered to living tissues through simple injection without causing acid corrosion and damaging host tissues. To test our hypothesis, the survival and differentiation of NPCs/NSCs within the 3D bioactive nanofiber hydrogel were investigated. Its application in promoting nerve recovery after injury was also evaluated in three different nerve injury models, including sciatic nerve defect, intracerebral hemorrhage, and spinal cord transection.

Results In the current study, RADA16-I was appended with IKVAV and RGD, which were derived from laminin and fibronectin, respectively (Table 1). RADA 16-IKVAV and RADA 16-RGD showed zeta potentials of 19.7 and -18.6, respectively, at pH 7. The opposite charges were in good agreement with our original design. As shown in Fig. 1, the combination of the two neutral solutions led to the formation of solid hydrogel immediately. The cell culture medium that diffused into the hydrogel did not show detectable change of red color, indicating that the formed hydrogel had neutral pH. A rotational rheometer was employed to investigate the viscoelasticity of the two designer peptide solutions (Fig. 1E). Both RADA16-IKVAV and RADA16-RGD exhibited G′>G″, indicating that the designer SAPs had certain elastic properties despite good fluidity. However, -IKVAV/-RGD showed a significantly higher G′ than G″ after combining RADA16-IKVAV and RADA16-RGD solutions, indicating that a characteristic solid hydrogel was formed. The rheological measurement was in good agreement with the actual observation. The stable hydrogel was further confirmed by the minimal sensibility to frequency by G′ and G″. Meanwhile, both of RADA 16-IKVAV and RADA 16-RGD solutions exhibited a shear-thinning behavior as indicated in Fig S1. In our design, RADA 16-IKVAV and RADA 16-RGD will be simultaneously injected into the lesion and form hydrogel in situ. The rheological property demonstrates the feasibility of in vivo implantation through simple needle injection. AFM was employed to observe the structure of the designer SAPs. As shown in



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Fig. 2, nanofibers were present in each designer SAP solution. The entanglement and non-covalent interactions among the existing nanofibers might be responsible for the elastic character of RADA16-IKVAV and RADA16-RGD solutions. However, the two designer SAPs showed quite different morphologies. Aqueous RADA16-IKVAV presented uniform nanofibers with diameters of 22.5±2.6 nm on the length scale of hundreds of nanometers; whereas RADA16-RGD self-assembled into long nanofibers with two diameter distributions, which were 18.6±3.2 nm and 46.2±4.9 nm. RADA16-I SAPs were documented to have high propensities to self-assemble into fibril units with high stability even at a low pH of 2.29 Therefore, we presumed that the fibers were practically formed in the solutions of the two designer SAPs at pH 7. RADA16-IKVAV diameter coincided with the number of amino acids in the peptides; whereas RADA16-RGD fell out of its theoretical diameter21, which suggested the lateral association of multiple subunits30. A nanofiber network was observed in -IKVAV-RGD hydrogel. High fiber entanglement and even twisting (arrows in Fig. 2C) were found to form thick fibrils, which were then bridged by short thin fibers. The fiber diameters ranged from 15 nm to 50 nm. Such phenomenon suggested the interactions between RADA 16-IKVAV and RADA 16-RGD nanofibers upon combination, which was expected to influence the association of RADA 16-RGD nanofibers. The morphologies of SAPs were observed by TEM using uranyl acetate as a negative stain. Ribbon-like nanofibers were observed in both solutions of RADA 16-IKVAV and RADA 16-RGD, which were similar to the RADA 16-I morphology investigated by Cormier et al.30. As documented previously, the rod-like RADA 16-I molecule and those after modification assemble into a stable β-sheet double-tape structure6, which then associated into ribbons31. Thus, our study herein documented the ribbon-like morphology of functionalized RDAD 16-I that could twist, creating narrow lines as indicated by the arrow (Fig. 2D and 2E). Fiber aggregation was observed in the -IKVAV/-RGD hydrogel after the combination of RADA 16-IKVAV and RADA 16-RGD. Interestingly, an association of parallel fibers that created an evenly layered ribbon was detected, as indicated by the arrow (Fig. 2F).


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Analysis of surface structures using AFM measurements and TEM via negative staining suffer from limitations because the drying procedure also induces flattening of the fiber structures on the surface29. Therefore, cryogenic-TEM that could provide structural details under hydrated conditions was employed to achieve a reliable examination of fiber structure. Our cryogenic-TEM characterization showed that the SAP structures agreed with the observation by AFM (Figs. 2D-F). Thin fibers of different lengths were detected in RADA16-IKVAV, whereas thicker fibers were observed

in

RADA16-RGD.

The

combination

of

RADA16-IKVAV

and

RADA16-RGD resulted in a fibrous network, which was composed of single fibers and thick ones formed by bundles of fibers. The sample concentration for cryogenic-TEM characterization was 10 mg/mL for cell culture in vitro and in vivo transplantation; thus, the results presented a reliable structure of the SAP nanofibers for actual applications. Therefore, in addition to the hydrogel presented by Stupp et al. 19

, we successfully presented a method for constructing 3D nanofibrous hydrogel from

two oppositely charged SAPs carrying different biological signals at neutral pH. Circular dichroism (CD) spectroscopy, which could provide basic information to understand the secondary structure of peptides, was utilized to investigate the assembling behavior of the designer SAPs.

Both RADA16-IKVAV

and

RADA16-RGD exhibited characteristic β-sheet CD spectra with a strong positive band around 195 nm and a strong negative band at 215 nm (Fig. 3A). Thus, the CD spectra demonstrated at the molecular scale the key structural component of our designer SAPs used to drive assembly into fibers. According to CDNN (CD Spectroscopy Deconvolution Software) analysis, α helix, β-sheet, and random coil structures coexisted in the RADA16-IKVAV solution (Table S1). However, a distinctly different spectrum resulted from the combination of RADA16-IKVAV and RADA16-RGD. Only one strong negative band was observed around 219 nm, which was slightly red shifted compared with the spectra before combination. Yokoi et al. attributed this behavior to peptide aggregation 12. Moreover, the strong positive band around 195 nm disappeared in the CD spectrum of -IKVAV/-RGD hydrogel, indicative of the appearance of more mixed secondary structures in the



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-IKVAV/-RGD hydrogel. Considering that the fiber diameters showed a large distribution range in the -IKVAV/-RGD hydrogel, we expected that the interactions between RADA16-IKVAV and RADA16-RGD fibers such as ionic bridges and hydrogen bonding might initiate β-turns and random coil conformations in -IKVAV/-RGD. The content of -IKVAV/-RGD random coils increased compared with those in RADA16-IKVAV and RADA16-RGD, according to the CDNN analysis (Table S1). The experimental CD intensity of -IKVAV/-RGD hydrogel was compared with the mathematical average of the two individual peptides as shown in Figure S2. Although both of them exhibited characteristic β-sheet CD spectra, the intensities were quite different. The actual -IKVAV/-RGD hydrogel showed a lower intensity as compared to the mathematical average of the two individual peptides. The difference indicated that the interactions between RADA 16-IKVAV and RADA 16-RGD diminished β-sheet structure. Therefore, the disappearance of the band at 195 nm might have resulted from the significant spectral overlap of random coil negative band, which is characteristically located around 200 nm, and a positive band of β-sheet around 195 nm. Consequently, no clear brands were unambiguously presented in the CD spectrum of -IKVAV/-RGD. The secondary structures of the designer SAP were further investigated by FTIR spectroscopy. As shown in Fig. 3B, the amide I region between the wave numbers 1700 and 1600 cm-1, which was the most informative region regarding the secondary structure of proteins. A strong absorption peak around 1615 cm-1 in RADA16-IKVAV spectrum was assigned to intermolecular β-sheets, whereas the peak around 1635 cm-1 was ascribed to intramolecular β-sheets32. The region between 1647 cm-1 to 1650 cm-1 was associated with the presence of α helix or random coil structures. In addition, another main peak located around 1672 cm-1 was observed, suggesting the presence of β-turns. A shoulder was observed around 1684 cm-1, which was assigned to β-sheets or β-turns, or both. In RADA16-RGD spectrum, the peak assigned to intermolecular β-sheets shifted to 1613 cm-1. A weak peak around 1640 cm-1 was assigned to α helix, where the peak at 1649 cm-1 might correspond to α helices and random coils. Similar to RADA16-IKVAV, RADA16-RGD also showed a main peak around 1672 cm-1 that


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was assigned to β-turns with a shoulder around 1684 cm-1, which was associated with β-sheets, β-turns, or both. However, the -IKVAV/-RGD FTIR spectrum presented only a main peak at 1672 cm-1 with shoulders around 1684 cm-1 and 1663 cm-1. The new shoulder around 1663 cm-1 was assigned to β-turns 33. The main peak associated with intermolecular β-sheets present in RADA16-IKVAV and RADA16-RGD spectra disappeared after the combination; instead, a broad peak between 1620 and 1658 cm-1 was

observed.

Therefore, the interactions between

RADA16-IKVAV

and

RADA16-RGD nanofibers might influence the interactions among the existing nanofibers before combination, causing the increase of random coils. This was in good agreement with the CD results. Raman spectroscopy is a powerful tool that can be used for the detailed investigation of protein conformations and is capable of revealing changes in the conformations and microenvironments of amino acid residues34. Thus, in addition to FTIR and CD, we investigated the designer SAP structures through Raman spectroscopy in the range of 900 cm-1 to 2000 cm-1. In this range, various peaks assigned to different vibration modes were located, such as the stretching of C-C, C-N, and C=O35; the bending modes of N-H36; and those associated with CH2 and CH3 deformation37. The location of the amide I band of proteins depended mainly on the strength of hydrogen bonding interactions (C=O···H) involving amide groups, and the strength of dipole-dipole interactions between carboxyl groups38. Therefore, information on the secondary structure of proteins could be obtained from the analysis of these bands from α-helices, β-sheets, and unstructured β-strands39-41. The Raman spectra (top) and second derivative spectra (bottom) of aqueous RADA 16-IKVAV, RADA 16-RGD, and -IKVAV/-RGD hydrogel are presented in Fig. 3C. The asymmetrical peak of RADA 16-IKVAV amide I band in the 1300 cm-1 to 1500 cm-1 regions generated two clear peaks in the second derivative spectrum, which were located at around 1624 and 1665 cm-1. The band at around 1624 cm-1 was a result of NH2 bending36; whereas the band at 1665 cm-1 was associated to β-sheets38, 42, in which the hydrogen bond is formed between the C=O and NH groups from adjacent chains arranged either in parallel or anti-parallel modes43. The β-sheet confirmation of



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RADA 16-IKVAV was further proven by the appearance of amide III band at round 1236 cm-1. RADA 16-RGD showed only a single peak located at 1645 cm-1, which was positioned at the edge between α-helices and β-turns44. According to Wen’s study, loose β-sheets and disordered structures occurred at about 1640 cm-1 43. Considering the β-sheets in RADA 16-RGD as indicated by the aforementioned CD and FTIR analyses, we expected loose β-sheet and β-turn structures in RADA 16-RGD. After combining the aqueous RADA 16-KVAV and RADA 16-RGD, only a single peak with higher intensity than RADA 16-RGD in 1645 cm-1 was observed. The band ascribed to the fine β-sheets at 1665 cm-1 disappeared in the Raman spectrum of -IKVAV/-RGD hydrogel. Therefore, the combination of RADA 16-IKVAV and RADA 16-RGD resulted in more mixed secondary structures with α-helices, β-turns, and disordered structures co-existing in the -IKVAV/-RGD hydrogel. In addition to various modes of the backbone structures in SAPs, CH2 and CH3 deformation modes peaking at approximately 1442 cm-1 in the Raman spectrum could provide new means to investigate the self-assembling behavior of SAP. CH2/CH3 deformation modes were documented to be extremely sensitive to self-assembly, showing higher intensity in non-assembled structures, such as α-helices or β-strands, than in β-sheet fibers42. Taraballi et al. attributed such preference to non-assembled structures to the hydrophobic nature, which kept the molecules into α-helices and disordered β-strand conformations without relevant reciprocal interactions42. In our study, RADA 16-IKVAV had more nonpolar amino acids than RADA 16-RGD (e.g., Val, Ala, and Ile), resulting in stronger hydrophobic forces as shown by more intense bands at 1442 cm-1. However, in Taraballi’s study, RADA16 I-ALK aggregated into globular structures rather than self-assembling into nanofibers; whereas RADA 16-IKVAV formed fine nanofibers, as indicated by AFM and TEM. We presumed that the like-charge repulsions between the motifs would confront the hydrophobic attraction. RADA 16-IKVAV self-assembled into β-sheets as a result of the counterbalanced forces45. The motifs of RADA 16-RGD mainly included polar amino acids with high hydrophilicity, which resulted in high and varying surface area exposed to water when combined with repulsion caused by the net like charge from


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the charged faces. Hence, assembly and aggregation would be hindered, resulting in β-turn conformation of the motifs45-46. The otherwise stable β-sheet structures of the RADA16-I backbone would be subsequently disturbed, resulting in loose β-sheets and disordered structures42. Such analysis was consistent with the morphologies of nanofibers observed in the AFM and TEM images. Thin nanofibers were detected in aqueous RADA 16-IKVAV, whereas thick nanofibers were found in aqueous RADA 16-RGD. Opposite charges were both involved in the -RGD motifs (Arg vs. Asp); thus, nanofiber bundling and aggregation occurred because of the electrostatic attraction of the random coils attached to the edge of the core RADA 16 backbone. When combining RADA 16-IKVAV and RADA 16-RGD together, the motifs of opposite charges would attract with relevant reciprocal interactions, which eliminates net charges. Charge screening between the polar head motifs reduced the electrostatic repulsions that pre-existed in RADA 16-IKVAV and RADA 16-RGD, forming the stable supramolecular aggregation along their stretched backbone47. Such aggregation occurred

via

hydrogen

bonding

and

electrostatic

interactions

from

the

ionic-complementary residues. Therefore, the resultant hydrogel showed better stability and mechanical properties than aqueous RADA 16-IKVAV and RADA 16-RGD, the viscosity of which was aroused by windings of nanofiber in the solutions. Charge screening by addition of ions could decrease the surface tension and cause more inter-chain interactions as a result of the expansion of peptide backbone47. Therefore, we assumed that inter-molecular interactions between RADA 16-IKVAV and RADA 16-RGD occurred, although both of them assembled into nanofibers before combination. The formation of β-sheets required at least two β-extended peptides to be correctly oriented in close proximity to form the cross-strand hydrogen bonds. Therefore, the probability to form fine β-sheets between RADA 16-IKVAV and RADA 16-RGD was rather low, which was consistent with our CD, FTIR, and Raman spectroscopy analysis of the secondary structures. Moreover, strong hydrophobic features of RADA16-IKVAV after charge screening induced the formation of intra-molecular conformations, which prevented the self-assembly of peptides in fine β-sheets and formed loose β-sheets instead; even though β-sheet



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inter-molecular interactions might probably be stable once they were formed42. Combining the analysis above, we proposed a molecular model to interpret the self-assembling behavior of the designer SAPs as indicated in Fig. 4. The two functional motifs were conjugated to the C-terminus of the self-assembling peptide RADA16 (Fig. 4A). Several amino acids were specially inserted between RADA 16 and the functional motifs to obtain opposite charges. The repeats of RADA aligned as antiparallel β-sheets (represented by arrows) with non-covalent interactions as core. On one hand, the functional motifs attached to the edge of bilayered RADA 16-I formed clusters (Fig. 4B). On the other hand, the functional motifs of -IKVAV explored an extended conformation out from RADA 16-I core as a result of the counterbalanced forces of hydrophobic attractions and like charge repulsions, which did not seriously influence the β-sheets of RADA 16-I. However, the strongly hydrophilic -RGD motifs extended out from RADA 16-I core toward the opposite directions as a result of like charge repulsions, forming random coils. Such predominant repulsions would split the bilayered β-sheet structure of RADA 16-I, resulting in loose β-sheets and disordered structures. Nanofiber bundling and aggregation would occur because of the electrostatic attraction of the random coils in the -RGD motifs (Arg vs. Asp), resulting in thick fibers. When RADA 16-IKVAV and RADA 16-RGD were combined, the oppositely charged -IKVAV and -RGD motifs came in contact with one other. This contact formed salt-bridged pairs on the edge of the bilayered β-sheet structure of RADA 16-I and/or in the overlay mode. Either form could influence the pre-existing secondary structures in aqueous RADA 16-IKVAV and RADA 16-RGD. In the current study, we presented a 3D nanofiber hydrogel from two designer SAP aqueous solutions at physiological pH. This hydrogel was expected to benefit 3D cell culture and could be delivered to living tissues through direct injection. To verify our hypothesis, cell viability within the designer SAP hydrogel was examined first. NPC/NSC spheres of E14.5 embryos of GFP-transgenic rats were directly embedded within the SAP nanofiber hydrogels. As shown in Fig. 5, the sphere did not change its morphology during the seven days of cultivation within the RADA16-I hydrogel, and


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no processes were found around the sphere. On the contrary, the sphere cultured within -IKVAV/-RGD hydrogel outgrew numerous long processes from the sphere in the 3D environment. A single cell dissociated from the sphere was also found to outgrow many long processes (Fig. 5B). To further evaluate cell viability, single NPCs/NSCs were embedded within the two SAP nanofiber hydrogels, and Live/Dead Cell Viability Assay Kit was utilized. Dead cells marked by red fluorescence demonstrated strong cytotoxicity of RADA16-I. Moreover, round cell morphologies with no processes indicated that rapid cell death occurred. In contrast, numerous green cells with long processes were observed within the -IKVAV/-RGD hydrogel. Therefore, the results confirmed our hypothesis that the designer SAP nanofiber hydrogel overcame low pH involved in the application of RADA16-I and could support 3D cell growth. This finding is extremely important for biological research and regenerative medicine because nearly all native cells are embedded in a 3D microenvironment in the body. Few NPCs/NSCs survived when directly embedded within RADA16-I aqueous solution before gelation. Thus, the differentiation within -IKVAV/-RGD nanofiber hydrogel was investigated. NPCs/NSCs from GFP-transgenic SD rats were stained with two well-described markers, namely, β-tubulin for neurons and GFAP for astrocytes, seven days after seeding. As shown in Fig. 6, neurons with long processes and branches were observed within the 3D -IKVAV/-RGD nanofiber hydrogel. Astrocytes with a multipolar glial morphology and long processes were observed. Notably, no extra growth factors and adhesion proteins were added in the designer SAP hydrogel or in the culture medium in the cell viability and differentiation study. This was in sharp contrast to conventional 2D cell cultures, in which case a variety of soluble growth factors were needed to maintain cell viability. Quantitative investigation was performed by calculating the percentage of the total cell population as defined by GFP positive cells. The percentages of neurons and astrocytes were 34.7±1.4% and 10.3±2.1%, respectively, when NPCs/NSCs were seeded on laminin-coated coverslip, which is generally used to culture cells. The β-tubulin+ and GFAP+ percentages were 48.6±7% and 5%, respectively, when embedded in 3D



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-IKVAV/-RGD hydrogel. Therefore, the designer SAP hydrogel promoted neuron growth compared with astrocytes. Such preference was similar to Stupp’s study48, which was linked to the amplification of bioactive epitope presentation by the nanofibers to cells. To evaluate its application in promoting nerve regeneration, the designer SAP hydrogel transplantation was examined in both peripheral nervous system (PNS) and central nervous system (CNS). Three different injury models were exploited, including sciatic nerve defect, spinal cord transection, and hematencephalon. As shown in Fig. 7, robust axons were observed to regenerate into the designer SAP graft in the sciatic nerve defect. Moreover, numerous Schwann cells migrated into the graft and were found to be in close association with regenerated axons (Fig. 7D). However, the axons and Schwann cells were found to grow around the porous graft or along the walls of the cavities in the RADA 16-I graft (Fig. 7A), just as water flows around an obstruction. Seldom did axons regenerate into the hydrogel bulk. In the spinal cord injury model, SAP solutions were immediately injected into the lesion after 2 mm of spinal cord was removed. After two months, numerous axons were found regenerated in -IKVAV/-RGD graft. The regenerated axons, however, grew along the wall surfaces of the cavities formed in the RADA 16-I graft. The long axis of the cavities was seldom parallel to the spinal cord and most of the cavities were closed, thus, the regenerated axons seemed to wander in the cavities. Similar to sciatic nerve defect, axons were also found to grow around the whole transplanted RADA 16-I graft rather than crossing the SAPs. In the ICH model, regenerated nerve fibers were observed in the -IKVAV/-RGD graft, whereas no fibers were regenerated in the RADA 16-I graft. Such phenomenon agreed with our previous study49. Moreover, similar to the structure in the sciatic nerve defect model, cavities were formed in the RADA 16-I graft of the ICH model. One significant difference between CNS and PNS is that the CNS lacks Schwann cells, which provide nutrient support, growth-promoting molecules, and growth factors for neurons and axons. Therefore, we investigated if Schwann cells could be


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recruited into the SAP grafts after transplantation into the spinal cord lesions. More Schwann cells were observed within the -IKVAV/-RGD graft than within the RADA 16-I graft two weeks after transplantation (Fig. 8). Moreover, Schwann cells were distributed on the cavity walls of the RADA 16-I graft rather than within the material bulk as shown in -IKVAV/-RGD graft. Close association between Schwann cells and regenerated axons were found in both -IKVAV/-RGD and RADA 16-I grafts (Fig. 8A and 8C). The immigrating Schwann cells probably myelinated or ensheathed the axons regenerated in the grafts. To verify this hypothesis, immunofluorescence cytochemistry staining of myelin basic protein (MBP) and nerve fibers was performed. As shown in the Figure, MBP-positive expression was detected to be closely associated with axons, especially in the -IKVAV/-RGD graft (Fig. 8B). Therefore, the evaluations in all the injury models indicated that the designer -IKVAV/-RGD SAPs could provide a more permissive environment for nerve regeneration and promote myelination than RADA 16-I. Discussion Until now, biomaterial science still faces the challenge of developing a well-controlled culture system for 3D cell growth in vitro and a permissive environment for tissue regeneration. RADA16-I is a well-documented synthetic peptide that can self-assemble into nanofibers and mimic natural extracellular matrix depending on pH and ionic strength6, 11. More SAPs have been widely exploited by modifying

RADA16-I

with

short

peptide

motifs4,

17,

21-22

.

The

hydrophobic/hydrophilic profiles and charge distributions of the motifs play an important role in secondary structures and subsequently influence the self-assembling behavior of the functional SAPs42. However, low pH remains to be the main drawback of the RADA 16-I SAPs, despite great efforts to overcome it. In the current study, we synthesized two designer SAPs by appending RADA16-I with motifs from laminin (IKVAV) and fibronectin (RGD). Special amino acids were inserted between RADA 16 and the functional motifs, achieving the positively and negatively charged SAPs at physiological pH, respectively. Strong hydrophobic attractions between



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-IKVAV motifs resulted from the hydrophobic amino acids in the -IKVAV sequence. The β-sheet structure of the core RADA 16-I was not seriously influenced as a result of the counterbalanced forces of hydrophobic attractions and like charge repulsions. However, the like charge repulsions of -RGD motif dominated because of its high hydrophilicity, which consequently disturbed the stable β-sheet structure of the RADA16-I backbone. The loose secondary structure would induce lateral association of multiple subunits, resulting in thick fibers and even fiber bundles. Salt-bridged pairs were formed on the edge of the bilayered β-sheet structures of RADA 16 upon the combination of aqueous RADA 16-IKVAV and RADA 16-RGD. The intermolecular interactions between RADA 16-IKVAV and RADA 16-RGD disturbed the previous secondary structures because of strong electrostatic interactions between the -IKVAV and -RGD motifs. This behavior eliminated the fine β-sheets of RADA16-IKVAV, although both motifs already assembled into nanofibers. These interactions primarily contributed to the mechanical properties of the hydrogel from our two designer SAPs, as indicated by the significant increase of G′ after the combination of RADA 16-IKVAV and RADA 16-RGD. The stable hydrogel was further confirmed by the minimal sensibility of G′ and G″ to frequency where the interactions/junctions between fibers were relatively permanent50. Such assembling mechanism was different from RADA16-I, which self-assembled into neutral fibers as a result of ionic intramolecular self-complementation and formed hydrogels through the entanglement of fibers51. Therefore, a stable and bioactive hydrogel from a nanofiber network was formed from two oppositely charged SAPs carrying different biological signals at neutral pH. Such designer SAPs showed great advantages over RADA 16-I SAP and those modified by various motifs. -IKVAV/-RGD hydrogel showed better cell survival and viability than RADA 16-I hydrogel when directly embedded into SAP solutions before gelation. Cell viability could be improved by immediately adjusting RADA 16-I solution to neutral pH. The operation, however, was inconvenient for 3D cell biological studies of large quantity in vitro. Moreover, spontaneous in vivo gelation of RADA 16-I would take some time, which would probably damage cell viability in the


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case of cell transplantation. Our results also demonstrated that the -IKVAV/-RGD hydrogel supported NPCs/NSCs differentiation towards neural and glial phenotypes in the 3D environment, even without extra growth factors and adhesion proteins except for those in the cell media. In the 3D environment, functional motifs present on the nanofiber surfaces surrounded the cells in all dimensions, thus providing a favorable environment similar to native tissues. Large cavities were formed in RADA 16-I grafts in all three nerve injury models, including PNS and CNS. This phenomenon might be related to exothermal properties during gelation, which was actually an acid-base neutralization reaction. -IKVAV/-RGD hydrogel was formed according to electrostatic interactions between opposite charges, thus, the process was mild and did not damage the host tissue. Regenerated nerves grew along the walls of the cavities or around the whole grafts rather than into the grafts, as observed in -IKVAV/-RGD grafts. Therefore, our designer SAPs provided a more permissive environment for nerve regeneration. Moreover, our study indicated that -IKVAV/-RGD grafts were favorable for Schwann cell recruitment, which probably myelinated or ensheathed the axons regenerated in the grafts. Therefore, in addition to the nanofibrous hydrogel presented by Stupp19, we successfully presented a method to construct 3D nanofibrous hydrogel from two SAPs carrying different biological signals at neutral pH. The designer SAP nanofibrous hydrogel scaffolds may have extensive applications in regenerative medicine. Conclusion In this investigation, we reported two oppositely charged SAPs carrying IKVAV and RGD epitopes, which underwent self-assembly into a stable nanofiber hydrogel at neutral pH. The designer SAP hydrogel overcame low pH associated with the use of RADA 16-I SAP. NPCs/NSCs showed good survival when fully embedded in the 3D environment of the nanofiber hydrogel from -IKVAV/-RGD. Moreover, NPCs/NSCs within the designer SAP hydrogel differentiated into neurons and astrocytes without adding extra soluble growth factors. In vivo studies indicated that the designer SAPs hydrogel provided a more permissive environment for Schwann cell recruitment and nerve regeneration than RADA 16-I hydrogel. These results indicated that our



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designer SAPs hydrogel provided a promising environment for true 3D cell culture as well as axon regrowth across the lesion site after nerve injury. Thus, the designer SAPs hydrogel might become a potential material for nerve regeneration. Moreover, our study described a flexible and simple approach in designing SAP nanofiber hydrogels for nanotechnology applications. This approach facilitates the incorporation of various biochemically and medically desired features, so that tailor-made functional materials can be produced. Materials and methods Materials RADA16-I with a concentration of 1% (v/w, pH 3-4) was purchased from BD Biosciences (Cambridge, MA). The two functionalized peptides RADA16-RGD and RADA16-IKVAV were custom-synthesized by American Peptide Company, Inc. with the purity above 95%. The peptide sequences are listed in Table 1. The peptide powders were dissolved in distilled water with concentration of 1% (v/w), filter-sterilized with an Acrodisc Syringe Filter (0.2 µm HT Tuffrun membrane, Pall Corp., Ann Arbor, MI) for subsequent use. Both of the peptide solutions were carefully titrated to neutral liquor using 1 M Tris solution. The hydrogel formed by RADA16-RGD and RADA16-IKVAV was defined as -IKVAV/-RGD in this article. Characterization of SAPs Rheological experiments were carried out in upper cone geometries (diameter 20 mm) on the rheometer plate using a Malvern KNX2100 instrument. The samples (1%) were scooped onto the rheometer plate so that no air gap was found within the cone. Frequency sweep experiments were performed as a function of angular frequency (0.1–10 rad s-1) at a fixed strain of 0.01% at 25 °C, and the storage modulus (G′) and the loss modulus (G′′) were plotted against angular frequency (ω). The Shear-thinning experiment was performed at 25℃, increasing the shear rate from a minimum of 0.1s-1 to maximum of 100 s-1, the shear viscosity (Pa. s) was plotted against shear rate(s-1). AFM imaging was performed on a Multimode-V (Bruker) operating in ScanAsyst mode in air. Then, 2.5 µL designer SAP solutions (50 µg/mL in Milli-Q water) were


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adsorbed on a freshly cleaved mica surface and air dried for at least 12 h before imaging. Height images were acquired using a silicon cantilever (BudgetSensors, Innovative Solutions Bulgaria Ltd.) with a nominal force constant of 5 N/m and resonant frequency of 150 kHz. The morphology of the self-assembled amphiphilic peptides were observed on a FEI Tecnai Spirit (120 kV) transmission electron microscope (TEM). Before the observation, the solution of the peptides (10 mg/mL) were applied to a copper grid and stained with a 2% (w/v) uranyl acetate solution. Cryogenic-TEM was performed on a FEI Tecnai 20 microscope operating at 200 kV. Peptide solutions (10 mg/mL) were applied to a copper TEM grid with a holey carbon support film. The sample specimen was blotted under a controlled environment and vitrified using a Vitrobot Mark IV (FEI) device. Waiting time was 8 s, blotting time 4 s, and blot force was 2. CD spectra were collected using a Chriscan (Applied Photophysics, Ltd.) spectrophotometer. Far-UV CD spectra were recorded from 260 nm to 190 nm with the cell holder temperature controlled at 25 °C. SAP solutions with concentration of 100 µg/mL in Milli-Q water were tested. Spectra obtained after buffer subtraction was corrected for protein concentration and smoothed using the Savitsky-Golay function. FTIR measurements were performed on a Vertex 70 (BRUCKER OPTIC) instrument. Measurements were performed with gelators in air and with D2O in the gel

state

at

room

temperature.

The

concentrations

of

RADA16-IKVAV,

RADA16-RGD, and their combination are 2% and the samples were prepared at least 12 h before measurement. The spectra were collected within the wavelength range of 1700 cm-1 to 1600 cm-1 at 1 cm-1 resolution and smoothed using the Savitsky-Golay function after buffer subtraction. Raman spectra were collected on solutions of RADA16-IKVAV, RADA16-RGD, and their combination at room temperature. Measurements were carried out using the 633 nm line of a He-Ne laser as the light source, whereas the scattered light signal in backscattering geometry was collected by a Raman spectrometer (Renishaw inVia). The signal was detected using a CCD with a spectral resolution of 1 cm-1, by averaging 10 spectra with acquisition time of 180 s each. This region was fitted with



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Origin 8.5. A Gaussian interpolation function was employed and deconvolution of the amide I region (1500 cm-1 to 1800 cm-1) for each spectra was performed. In vitro cell study The

spinal

cord

of

embryonic

day

14.5

(E14.5)

GFP-transgenic/non-GFP-transgenic Sprague-Dawley rats were dissected in cooled HBSS and dissociated mechanically. The cells were collected by centrifugation and resuspended in DMEM/F12 with B27 supplement (2%, Gibco), N2 (1%, Gibco), epidermal growth factor (EGF, 20 ng/mL; Gibco), basic fibroblast growth factor (bFGF, 20 ng/mL; Sigma), penicillin (100 U/mL), and streptomycin (100 µg/mL). In this study, the primary NPCs/NSCs were used after isolation. In situ 3D cell culture investigation After isolation, NPCs/NSCs were resuspended at a concentration of 500,000 cells/µL in 1% solutions of RADA16-I, RADA16-RGD, and RADA16-IKVAV. RADA16-RGD and RADA16-IKVAV solutions were quickly mixed together for gelation in a 24-well plate. Then, 600 mL DMEM/F12 with 1% B27 supplement (Gibco), 1% fetal calf serum, and 1% penicillin-streptomycin was carefully added in the culture medium. The cell density cultured on a PLL-coated coverslip was 100,000 cells/well in a 24-well plate. The medium was changed every three days and after being cultured for one week, the cells in the peptide hydrogels were stained with LIVE/DEAD Viability/Cytotoxicity Assay Kit (Invitrogen) according to protocol. In brief, the cell-encapsulated peptide hydrogels were rinsed three times with 0.1 M phosphate buffer saline (pH 7.4) for 10 min each time. Then, the hydrogels were incubated in 0.3 mL of 0.1 M phosphate buffer containing 2 mM of calcein-AM and 4 mM of ethidium homodimer (EthD-III) (Viability/Cytotoxity Assay Kit for Live & Dead Animal Cells, Biotium, USA) for one hour at 37 °C. Then, the hydrogels were rinsed three times (10 min each time) and fixed with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min. Immunocytochemistry (ICC) was performed to investigate the differentiation of NPCs/NSCs within the functionalized SAP hydrogel. Briefly, after one week of culture, the samples were rinsed three times with PBS solution and fixed in 4%


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formaldehyde in 0.1 M PBS for 30 min at room temperature. The samples were blocked with 20% norm donkey serum and 0.1% Triton X-100 in PBS for 30 min at room temperature. Then, the samples were incubated in mouse primary antibody against β-tubulin type III (1:1000; Sigma) overnight at 4 °C, and treated with fluorescent Alexa 568 donkey anti-mouse secondary antibody (1:1000; Invitrogen) for two hours at room temperature. Finally, the sections were mounted using mounting medium containing 4′,6-diamidino-2-phenylindole to counterstain the nuclei. The images were taken using a confocal microscope (Zeiss LSM 700 META). In vivo study In the sciatic nerve defect model, a 5 mm gap was made at the middle of the right sciatic nerve trunk using an operating microscope. Both the proximal and distal nerve stumps were inserted into a biodegradable polymer, poly-L-(lactic acid) electrospun conduit and fixed with 11-0 suture. RADA 16-I and RADA 16-IKVAV/RADA 16-RGD were then separately injected into the gap within the conduit. Antibiotics (Amoxycillin) and analgesics (Buprenorphine, Meloxicam, and Acetaminophen) were used for post-operative care. The rats were allowed standard access to food and water ad lib throughout the study, following the guidelines from the University of Hong Kong for animal care and use. Mice experimental ICH was performed according to our previous study49. In brief, ICH was induced by a slow injection of 0.2 U collagenase IV (Sigma-Aldrich, St. Louis, USA) in 1.0 µL saline into the left striatum for over 10 min. At 3.5 h after collagenase injection, aspiration was achieved by gentle suction through a 1 mL syringe attached to a 23-G needle. The needle was placed at the same stereotactic coordinates as the collagenase injection. Four aspirations were performed over 15 min, followed by injection of 10 µL 1% RADA16-I or 1% RADA16-IKVAV/RADA 16-RGD solution into the lesion via a 25-G needle. Finally, the burr hole was sealed with bone wax and the wound was sutured. In the spinal cord transection model, a 2 mm-long block of spinal cord at the 8th thoracic segment was cut and removed using a combination of scissors and aspiration with a 23-G blunt needle. Complete ventral and lateral transection was ensured with



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visual verification and care was taken to preserve the dura mater. After sufficient hemostasis, RADA16-RGD and RADA16-IKVAV were quickly and equivalently blended and injected within the lesion cavity to bridge the gap. After the operations, the dura and the opened musculature were sequentially sutured and the skin was closed using wound clips. All animals were allowed to recover from anesthesia in a small animal incubator set at 30 °C. After full recovery, the rats were returned to their cages and given water and food ad libitum. Buprenorphine were given (0.05 mg/kg SC, twice daily) for analgesia when the animal showed signs of discomfort (such as chewing, anorexia, and other signs of pain). Then, 2 mL of Enrofloxacin (25 mg/ml oral preparation) was added to 500 mL water and administered for 7 days to prevent urinary tract infection. As a control, RADA 16-I solution was transplanted following the above mentioned operation. Histology and Immunohistochemistry The animals were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) eight weeks after surgery. Spinal cord segments containing the lesion/peptide site were dissected, post-fixed overnight at 4 °C, transferred to 30% sucrose for 48 h at 4 °C, and then embedded in optimum cutting temperature compound. Serial longitudinal cryosections (18 µm) were cut with a cryotome and mounted onto glass slides. After drying at 37 °C for 2 h and washing with PBS for 10 min, the samples were stained with ICC following the procedure described in the in vitro study section. The slides which centered at the lesion were presented. Acknowledgments The authors thank for funding supports from the National Program on Key Basic Research Project (973 Program, 2014CB542205), Hong Kong Health and Medical Research Fund (02132826), Foundation for Distinguished Young Talents in Higher Education of Guangdong (Yq2013023), Pearl River Nova Program of Guangzhou (1317000404), China Postdoctoral Science Foundation (2013M540684), the Leading


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Talents of Guangdong Province (87014002), National Natural Science Foundation of China (31570875, 81590761 and 31200559). The authors wish to acknowledge the support from the Hong Kong Scholars Program (XJ2012024)

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51. Chen, P., Self-Assembly of Ionic-Complementary Peptides: a Physicochemical Viewpoint. Colloid Surface A 2005, 261 (1-3), 3-24.

Code RADA16-I RADA16-RGD RADA16-IKVAV

Sequences Ac-(RADA)4-CONH2 Ac-(RADA)4-DGDRGDS Ac-(RADA)4-RIKVAV

Net charge, pH 7.4 ζ, pH7.4 Neutral 0

MW, Da 1713

—

-18.6

2416

+

19.7

2451

Table 1 Physicochemical properties for self-assembling peptides used in this study.

Figure 1 Preparation of hydrogel from two designer SAPs at pH 7. (A) RADA 16-IKVAV solution (10 mg/ml), labeled as P1 on the bottle. (B) RADA 16-RGD solution (10 mg/ml), labeled as P2 on the bottle. (C) Hydrogel was formed when combining P1 and P2 together. (D) Cell culture medium was added into (C), and the medium diffused into the hydrogel did not change its red color. (E) Rheological measurements of different SAPs solution (10 mg/ml) over an angular frequency range of 0.1-10s-1. Figure 2. Structures of designer SAPs. (A-C) AFM phase images of different designer SAPs. (D-F) TEM images of different designer SAPs by negative staining at room temperature. (G-I) Cryo-TEM images of different designer SAPs. The dimensions of AFM images were 2 × 2 µm2. Scale bars of cryo-TEM images were 100 nm. Figure 3 Secondary structure investigation. (A) CD spectra of different SAPs. (B) FTIR spectra of different SAPs. (C) CD spectra of different SAPs. Figure 4 A proposed molecular model for dynamic reassembly of self-assembling


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RADA16-IKVAV, RADA16-RGD as well as combination of the two designer SAPs. a) Amino acid sequence and molecular model of RADA16-I, RADA16-IKVAV and RADA16-RGD. b) Bilayer structures of RADA16-IKVAVA and RADA16-RGD. Color schemes for self-assembling ‘‘core’’ RADA 16-I were: blue (basic), white (hydrophobic), red (acidic), whereas in the motifs: green (polar), dark gray (hydrophobic) and light gray (hydrophilic residues). c) Nanofibers were formed in aqueous RADA16-IKVAV and RADA16-RGD as well as in –IKVAV/-RGD hydrogel. , and green (polar)The was composed by alternating basic residues (blue) with acid residues (red) and hydrophobic ones (white). Polar neutral residues (green) are present in added functional motifs. Figure 5 Cell viability within different SAP hydrogel. NPC/NSC spheres were from GFP-transgenic SD rats while single cells were from SD rats. Single cells and spheres were cultured for 7 day. (A and B) Maximum intensity projection of Z stack images of cell spheres within RADA 16-I hydrogel and RADA 16-IKVAV/-RGD hydrogel. (C and D) Cell viability was measured by live/dead staining of NPCs/NSCs within RADA 16-I hydrogel and RADA 16-IKVAV/-RGD hydrogel. Live cells were stained fluorescent green, and dead cells appeared red. Scale bars in (A) and (B) were 50 µm. Figure 6 NPCs/NSCs from GFP-transgenic SD rats differentiation within RADA 16-IKVAV/RADA 16-RGD hydrogel (A) and on laminin-coated coverslips (B) after cultivation for 7 days. Scale bar in (B): 50 µm Figure 7 Axon regeneration 8 weeks after implanting different SAPs in different nerve defect model. (A, D) Sciatic nerve defect model. (B, E) Spinal cord transection. (C, F) The hematencephalon model. * indicated of RADA16 I grafts while the dashed line was the boundary between the grafts and the host tissue. The slides were located at the middle positions of the lesion. Scale bars were 100 µm. Figure 8 Myelination of regenerated axons within different SAPs 8 weeks after implantation in spinal cord. Green axons were myelinated in many cases and were also closely associated with Schwann cells. (A and B) Axons myelination within -IKVAV/-RGD. (C and D) Axons myelination within -IKVAV/-RGD. * defined the grafted SAPs in the spinal cord which indicated that the axons grew into the -IKVAV/-RGD hydrogel while on the walls of the cavity in the RADA 16-I hydrogel. The slides were located at the middle positions of the lesion. Scale bars were 100 µm.



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Table of Contents (TOC)


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Table of Contents (TOC) 265x96mm (150 x 150 DPI)



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Figure 1 Preparation of hydrogel from two designer SAPs at pH 7. (A) RADA 16-IKVAV solution (10 mg/ml), labeled as P1 on the bottle. (B) RADA 16-RGD solution (10 mg/ml), labeled as P2 on the bottle. (C) Hydrogel was formed when combining P1 and P2 together. (D) Cell culture medium was added into (C), and the medium diffused into the hydrogel did not change its red color. (E) Rheological measurements of different SAPs solution (10 mg/ml) over an angular frequency range of 0.1-10s-1. 194x57mm (150 x 150 DPI)


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Figure 2. Structures of designer SAPs. (A-C) AFM phase images of different designer SAPs. (D-F) TEM images of different designer SAPs by negative staining at room temperature. (G-I) Cryo-TEM images of different designer SAPs. The dimensions of AFM images were 2 × 2 µm2. Scale bars of cryo-TEM images were 100 nm. 317x317mm (72 x 72 DPI)



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Figure 3 Secondary structure investigation. (A) CD spectra of different SAPs. (B) FTIR spectra of different SAPs. (C) CD spectra of different SAPs. 180x135mm (150 x 150 DPI)


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Figure 4 A proposed molecular model for dynamic reassembly of self-assembling RADA16-IKVAV, RADA16RGD as well as combination of the two designer SAPs. a) Amino acid sequence and molecular model of RADA16-I, RADA16-IKVAV and RADA16-RGD. b) Bilayer structures of RADA16-IKVAVA and RADA16-RGD. Color schemes for self-assembling ‘‘core’’ RADA 16-I were: blue (basic), white (hydrophobic), red (acidic), whereas in the motifs: green (polar), dark gray (hydrophobic) and light gray (hydrophilic residues). c) Nanofibers were formed in aqueous RADA16-IKVAV and RADA16-RGD as well as in –IKVAV/-RGD hydrogel. 164x96mm (150 x 150 DPI)



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Figure 5 Cell viability within different SAP hydrogel. NPC/NSC spheres were from GFP-transgenic SD rats while single cells were from SD rats. Single cells and spheres were cultured for 7 day. (A and B) Maximum intensity projection of Z stack images of cell spheres within RADA 16-I hydrogel and RADA 16-IKVAV/-RGD hydrogel. (C and D) Cell viability was measured by live/dead staining of NPCs/NSCs within RADA 16-I hydrogel and RADA 16-IKVAV/-RGD hydrogel. Live cells were stained fluorescent green, and dead cells appeared red. Scale bars in (A) and (B) were 50 µm. 120x99mm (150 x 150 DPI)


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Figure 6 NPCs/NSCs from GFP-transgenic SD rats differentiation within RADA 16-IKVAV/RADA 16-RGD hydrogel (A) and on laminin-coated coverslips (B) after cultivation for 7 days. Scale bar in (B): 50 µm 129x130mm (150 x 150 DPI)



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Figure 7 Axon regeneration 8 weeks after implanting different SAPs in different nerve defect model. (A, D) Sciatic nerve defect model. (B, E) Spinal cord transection. (C, F) The hematencephalon model. * indicated of RADA16 I grafts while the dashed line was the boundary between the grafts and the host tissue. The slides were located at the middle positions of the lesion. Scale bars were 100 µm. 196x114mm (150 x 150 DPI)


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Figure 8 Myelination of regenerated axons within different SAPs 8 weeks after implantation in spinal cord. Green axons were myelinated in many cases and were also closely associated with Schwann cells. (A and B) Axons myelination within -IKVAV/-RGD. (C and D) Axons myelination within -IKVAV/-RGD. * defined the grafted SAPs in the spinal cord which indicated that the axons grew into the -IKVAV/-RGD hydrogel while on the walls of the cavity in the RADA 16-I hydrogel. The slides were located at the middle positions of the lesion. Scale bars were 100 µm.\r\n 133x99mm (150 x 150 DPI)


Functional Self-Assembling Peptide Nanofiber Hydrogels Designed

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