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Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Molecular Mechanisms of Tryptophan−Tyrosine Nanostructures Formation and their Influence on PC-12 Cells Prathyushakrishna Macha,†,‡ Lisa Perreault,§ Yasaman Hamedani,†,‡ Maricris L. Mayes,§ and Milana C. Vasudev*,† †
Department of Bioengineering, ‡Biomedical Engineering and Biotechnology Program, and §Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, Massachusetts 02747, United States
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ABSTRACT: The discovery of self-assembling peptides, which can form well-ordered structures, has opened a realm of opportunity for the design of tailored short peptide-based nanostructures. In this study, a combined experimental and computational approach was utilized to understand the intramolecular and intermolecular interactions contributing to the self-assembly of linear and cyclic tryptophan-tyrosine (WY) dipeptides. The density functional tight binding (DFTB) calculations with empirical dispersive corrections assisted the identification of the lowest energy conformers. Conformer analysis and the prediction of the electronic structure for the monomeric, dimeric, and hexameric forms of the cyclic and linear WY confirmed the contributions of hydrogen bonding, π−π stacking, and CH−π interactions in the stability of the self-assembled nanotubes. The influence of the processing conditions on the morphological and thermal characteristics, as well as the secondary structures of the synthesized nanostructures, were analyzed. Preliminary studies of the influence of the nanotubes on the fate of neuronal cell lines such as, PC-12 cells indicate that the nanotubes promote cellular proliferation, and differentiation in the absence of growth factors. The aspect ratio of the nanotubes played an essential role in cellular interactions where a higher cellular uptake was observed in nanotubes of lower aspect ratios. These results provide insight for future applications of such nanotubes as scaffolds for tissue engineering and nerve regeneration and in drug delivery. KEYWORDS: aromatic peptides, nanotubes, self-assembly, DFTB method, cyclization, secondary structure characterization, PC-12 cells
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INTRODUCTION Self-assembly in nature, specifically in biological molecules such as DNA, peptides, and proteins, is a spontaneous organization of molecules into well-ordered structures.1−3 In the past decade, self-assembling peptides have attracted considerable attention because of properties such as biocompatibility, chemical versatility, and biological recognition abilities. Self-assembly of aromatic peptides is dependent on noncovalent forces including π-stacking, hydrogen bonding, and hydrophobic interactions. Based on variations in the composition of the peptides, a variety of nanostructures such as nanotubes, nanofibers, and nanospheres have been synthesized. One of the most widely studied short selfassembling peptides is diphenylalanine (FF), which has been explored for applications including drug delivery, photoactive materials, antibacterial scaffolds, and sensors.4−8 Although FF is known to self-assemble into highly ordered, entropically favored nanostructures through noncovalent interactions, nanotubes composed of FF are unstable at physiological pH unless chemically modified or vapor deposited.9,10 In contrast, peptides containing other aromatic residues such as tryptophan and tyrosine, which are known to mediate electron transfers in © XXXX American Chemical Society
proteins and can lead to novel cellular interactions, have yet to be studied.11 Tryptophan-based short peptides have been shown to have an inhibitory effect on angiotensin-I converting enzyme (ACE) but have not been studied for supramolecular structural formation and biomaterials applications.12 Aromatic dipeptides contain a conjugated π-electron system that stabilizes the π−π stacking interactions and promotes selfassembly.13 The morphology and properties of the selfassembled nanostructures can be modified based on the knowledge of π−π stacking, hydrogen bonding, and other interactions. The prediction of these interactions in a costeffective way can be achieved by computational studies and simulations. In this study, early stages of the self-assembly of the dipeptides were analyzed using the density functionalbased tight binding (DFTB) method. DFTB is a parametrized version of density functional theory (DFT) that is computationally inexpensive as well as highly accurate for organic systems, leading to efficient calculations. Systematic correlative Received: May 16, 2018 Accepted: September 20, 2018 Published: September 20, 2018 A
DOI: 10.1021/acsabm.8b00121 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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ACS Applied Bio Materials
for imaging the cellular interactions of the nanotubes (stained with phalloidin-rhodamine and nuclear stain, Hoechst). Chemical Characterization. Infrared (FTIR) and Raman spectroscopy were used to study the self-assembly on the various dipeptides. Digilab Excalibur Fourier transform spectrometer 3000 was used for the FTIR measurements. Finely divided samples were combined with crystalline potassium bromide (KBr) to form a pellet. Spectra were obtained at an average of 100 scans (resolution of 2 cm−1), with a range of 400−3600 cm−1 and speed of 2.5 kHz. Raman spectra were collected using WITec’s alpha 300 M confocal Raman system. Averaged spectra from five acquisitions and 4 s integration time were obtained. CD Spectroscopy. Circular dichroism analysis of the cyclic WY, linear WY and heat-treated linear WY nanotubes was performed on a J-810 (Jasco, Inc. Tokyo, Japan) spectropolarimeter averaged over three scans with a range of 190−300 nm, and speed of 20 nm/min. Stock solutions of the nanotubes were diluted in double distilled water to final concentrations of 0.025, 0.8, and 0.125 mg/mL, respectively. Spectral data was processed using the CDSSTR, CONTIN-LL, and SELCON3 methods available in Dichroweb. NMR Spectral Measurements. The lyophilized peptide were dissolved in deuterated DMSO at a concentration of 10 mg/mL and loaded in Bruker AVANCE III HD 400 MHz High-Performance Digital NMR for their respective 1H and 13C NMR spectra. The chemical shifts were analyzed using MestReNova package and structures were drawn in ChemDraw. UV−Vis Measurements. UV−vis absorption of the peptides and nanotubes were measured using an AccuSkan GO spectrophotometer (Thermo-Fisher). Computational Studies. The electronic structures of the monomeric, dimeric, as well as the hexameric structures of the linear and cyclic forms of WY peptide were calculated using the third-order density-functional-based tight-binding method (DFTB3) using parametrization for organic and biological systems (3OB).18,19 This parametrized variation of DFT has excellent accuracy and the computational efficiency is two or three orders faster than conventional DFT methods. The dispersion effects such as hydrogen bonding and π−π stacking, were accurately accounted for by using the Universal Force Field (UFF) dispersion model.20 Initial conformer analysis of both the linear and cyclic forms of WY dipeptides was performed using the Molecular Mechanics Force Field (MMFF) implemented in Spartan 14 to determine all possible energetic and geometrical configurations. The 15 lowest energy conformers of each dipeptide were optimized in the gas phase using the DFTB level of theory. The lowest energy conformer was subsequently used to construct and study the dimeric and hexameric forms of the dipeptides. The computational process was repeated with implicit acetone solvation using the polarizable continuum model (PCM) at the DFTB level of theory.21 Acetone was used as the solvent due to its similarity in dielectric constant to that of HFIP, commonly used in the self-assembly of the nanotubes. Vibrational frequencies were computed to ensure that each stationary point is a local minimum, and reaction energetics were corrected for zero-point energy differences. Cell Culture and Cytotoxicity Studies. Rat pheochromocytoma cells (PC-12) were obtained from the American Type Culture Collection (ATCC) and maintained at 37 °C with 95% humidity and 5% CO2. Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum.22 Cyclic WY nanotubes and heat-treated linear WY nanotubes at concentrations of 1, 0.5, and 0.2 mg/mL were deposited on glass coverslips (5 mm diameter) that were treated with aminosilane (5% in methanol). Cyclic WY and heat-treated WY peptides were deposited on glass coverslips at concentrations of 0.05, 0.02 and 0.01 mg/mL, as an additional control. The coverslips were seeded with PC-12 cells (5 × 103 cells/coverslip) and cultured in 96-well plates for the cell viability assay. Media changes were performed within the first 12−24 h to remove any unattached cells. The cells’ viability and growth were recorded on days 1, 3, and 5 using the methyl tetrazolium (MTT) reagent at a final concentration of 0.5 mg/mL.23 Absorbance
analysis of theoretical and experimental results from linear and cyclic tryptophan-tyrosine (WY) dipeptides and their subsequent assembly lead to the identification of the lowest energy conformers for the formation of the nanotubes. In this study, nanotubes composed of linear and cyclic WY dipeptides were synthesized. Additionally, the influence of heat treatment on the stability and aspect ratio of the synthesized linear dipeptide nanotubes was studied. PC-12 cells isolated from the rat adrenal pheochromocytoma are commonly used as models for neural differentiation. In this study, PC-12 cells were used to evaluate the cytotoxicity of synthesized nanotubes and to understand their influence on cellular proliferation and differentiation.14 PC-12 cells respond to external environmental cues such as chemical composition, topography, and the elasticity of the surface. Additionally, growth factors and nanostructures have been shown to have a pronounced effect on cellular fate due to increased interactions, which can promote adhesion, alignment, spreading, and changes in morphology and gene expression.15,16 The exact mechanism behind the topographical effect observed when cells are cultured on a nanotubular surface is not clear; however, there are several hypotheses. One promising hypothesis is that morphological changes alter the function and protein behavior, which influences environmental biochemistry and cytoskeletal reorganization, resulting in highly polarized cells. Earlier studies indicated that substrates with structures larger than the filopodia resulted in the development of a higher number of filopodia; however, outgrowth of the neurites was limited. In contrast, microscale structures resulted in longer neurites, and fewer cells with filopodia.17 To stimulate neurite outgrowth, previous studies have examined the combination of topographical factors with significant amounts of neuronal growth factor (NGF). The current study investigates the influence of topography and chemical composition of the nanotubes on the behavior of PC12 cells in the absence of NGF.
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EXPERIMENTAL METHODS
Synthesis of Nanotubes. Linear tryptophan-tyrosine (H-TrpTyr-OH), and cyclic tryptophan-tyrosine (Cyclo (-Trp-Tyr)) peptides were purchased from Bachem (Switzerland). To form nanotubes, we dissolved the linear dipeptide at 100 mg/mL concentration in an acidified solution of 1,1,1, 3,3,3-hexafluoro-2propanol (HFIP 99%, Sigma) containing 20 μL of 0.1 M hydrochloric acid (HCl). This solution was further diluted in water, followed by an overnight incubation at 4 °C. The nanotubes were lyophilized and subjected to heat treatment both in a vacuum and microwave oven, at their glass−liquid transition temperatures (167 °C), which lead to their cyclization. Similarly, cyclic WY dipeptide was dissolved at 100 mg/mL concentration in 100 μL of HFIP and diluted to 5 mL in water (final conc. 2 mg/mL). Upon dilution in water, nanotubes formed instantaneously. Morphological Characterization. Scanning electron microscope JSM-5610 (JEOL Inc., USA) with accelerating voltage of 0.5 to 30 kV was used in the morphological characterization of the synthesized nanotubes. The diluted nanotubes were deposited on clean aminosilane treated silicon wafers and air-dried at room temperature for SEM imaging. Samples were sputter coated with 4 nm gold before imaging. High-resolution transmission electron micrographs of the nanotubes were obtained by depositing the solution of nanotubes on lacey carbon grids with carbon backing and imaged at 200 kV using the FEI Talos F200X in STEM mode. An inverted fluorescence microscope, DMI8 (Leica Microsystems, Germany) and a confocal microscope, Zeiss 710 (Carl Zeiss, Thornwood, NY, USA) were used B
DOI: 10.1021/acsabm.8b00121 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 1. DFTB optimized structures of linear WY peptide: (a) monomer, (b) dimer, (c) hexamer. SEM and HR-STEM micrographs of (d) linear WY nanotubes and (e) heat-treated linear WY nanotubes.
Figure 2. DFTB optimized structures of cyclic WY peptide: (a) monomer, (b) dimer (c) hexamer; and (d) SEM and HR-STEM micrographs of bundled cyclic WY nanotubes. measurements were carried out at 570 nm using a Multiskan FC microplate reader (Thermo Scientific). PC-12 cells on nanotubes were fixed at 72 h using 2.5% glutaraldehyde (primary fixative) and 1% osmium tetroxide (secondary fixative) in water. Following fixture, samples were washed with phosphate-buffered saline (PBS) and further dried using hexamethyldisilazane (HMDS) as a chemical alternative to critical point drying.24
while both the cyclic WY peptide and heat-treated linear WY peptide were tubular and shorter in comparison to the linear WY nanotubes. Cyclic WY and heat-treated linear WY nanotubes had similar aspect ratios (average: 27), while the linear WY nanotubes had an average aspect ratio of 56, as indicated by the analysis of the electron microscopy images using ImageJ software. This result suggests that heat induces structural changes in the linear WY nanostructures and cyclization is occurring as indicated by the appearance of tubular structures similar to the cyclic WY nanotubes (Figures 1e and 2d). These changes in the heat-treated WY nanotubes could be attributed to stronger bonds and improved stacking interactions between the cyclic aromatic groups. Phase changes
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RESULTS AND DISCUSSION SEM and TEM imaging of the self-assembled linear WY nanotubes, heat-treated linear WY nanotubes, and cyclic WY nanotubes (Figures 1d, e and 2d) indicate that the linear WY peptide formed ribbon-like structures with high aspect ratios C
DOI: 10.1021/acsabm.8b00121 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 3. (a). 1H spectra of 0.5, 1, and 2 mg/mL of cyclic WY peptide nanotubes in DMSO-d6. (b) 1H spectra of 1, 2, and 5 mg/mL heat-treated linear WY peptide nanotubes.
Figure 4. (a) FTIR spectra of linear WY, cyclic WY, and heat-treated linear WY nanotubes. (b) Calculated IR spectra of linear WY monomer, dimer, and hexamer. (c) Calculated IR spectra of cyclic WY monomer, dimer, and hexamer.
in the dipeptide samples were measured as a function of temperature using DSC and TGA. DSC analysis of the linear WY dipeptide indicates the onset of phase change around 140 °C. Release of the excess free energy from amorphous molecules due to loss of randomness is indicated by the exothermic peak at 155.5 °C and heat flow out of the sample. The mid glass−liquid transition at 165.95 °C of the linear WY monomer was observed both in the TGA and DSC measurements and this temperature was utilized for the heattreatment of the linear WY nanotubes. Major weight loss was observed at 355.7 °C, indicative of the decomposition of the
dipeptides (Figure S1). Chromatograms of the cyclic and heattreated linear WY nanotubes obtained using HPLC coupled with a hybrid quadrupole time-of-flight mass spectrometry show prominent molecular weight (MW) peaks at 350 g/mol with a retention time of 0.376 min and at 700 g/mol. The drop of 18 g/mol in the heat-treated linear WY nanotubes is attributed to the loss of a water molecule and the formation of an additional amide bond and the peak at 700 g/mol indicates the formation of dimer units (Figures S2 and S3). Proton and 13C NMR analysis of the peptides, demonstrate similarities in corresponding protons and carbons of WY cyclic peptide and D
DOI: 10.1021/acsabm.8b00121 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 5. (a) Raman spectra of linear, cyclic dipeptides, and nanotubes. (b) Gas phase computational Raman spectra of WY cyclic monomer, dimer, and hexamer. (c) Gas phase computational Raman spectra of WY linear monomer, dimer, and hexamer.
an additional 2, 5 diketopiperazine ring, contributes to the stronger interactions, when compared to the linear monomer which has a single amide bond and a free amine group. (Figures 1a and 2a).29 Fourier transformed infrared (FTIR) spectra were analyzed to elucidate the structural and chemical differences between the various monomers and nanotubes (Figure 4). Specific peaks assigned to the C−H, CC, CO, N−H, O−H groups were analyzed because they are H-bond donors. Hbond acceptors and aromatic carbons, which are crucial in noncovalent interactions such as van der Waals interactions and hydrogen bonds, lead to self-assembly. The high-intensity peak found in the range of 1600−1700 cm−1 was attributed to Amide I (CO, carbonyl group) stretching, shedding light on the dipeptides backbone conformation. Deconvolution of the secondary structure region of the linear and heat−treated linear WY nanotubes indicate a major contribution from the βsheet conformation and the absence of α-helical conformation. In contrast, the cyclic WY peptide was shown to have an αhelical structure in combination with β-strands and β-turns (Figure S6). The NH stretching mode was observed as a single peak in the cyclic monomers and cyclized tubes due to the presence of a secondary amine group. However, in the linear monomers and nanotubes, this stretching mode appears as two peaks, owing to the existence of a primary amine group. Between different samples, slight variations in the FTIR peak values were observed. This difference could be a result of changes in the bonds force constant resulting from atoms participating in interactions such as H-bonding.30,31 Groundstate IR peaks of the cyclic and linear WY peptides were calculated and compared with the experimental findings. Notable peaks observed in the experimental FTIR spectra of the cyclic and linear WY peptides, were consistent with the computational results (Figure 4 and Table S1). The distinct difference between the monomeric, dimeric, and hexameric spectra were observed in their IR intensity. As the number of monomers involved in the arrangement increased, the peak intensities increased correspondingly, i.e., the dimer peaks were
heat-treated linear WY peptides. The free amine peak at 4.35 ppm, which is present in linear WY peptide spectra but not in the heat-treated WY linear peptide indicates the loss of free amine due to heat-induced cyclization. Further, the presence of strong carboxylic amide proton signals in the cyclic WY and heat-treated WY linear peptides suggest the cyclic structure of the peptides (Figure 4).25,26 Hydrogen bonding is an important factor to the intermolecular interactions leading to the ordered morphology and structural rigidity observed in the cyclic and heat-treated linear WY nanotubes. The contribution of the hydrogen bonding interaction in the supramolecular structural formation was studied using the concentrationdependent NMR as shown in Figure 3.27,28 Proton NMR spectra of cyclic WY and heat-treated WY nanotubes were measured at 25 °C with varying concentrations in DMSO-d6 (Figure 3a, b). An overall upfield shift of 0.05 ppm was observed in the indole N−H, phenolic hydroxyl protons of both WY cyclic nanotubes (10.83 → 10.78 ppm) and WY heat-treated nanotubes (11.00 → 10.95 ppm). Similarly, overall shifts of 0.04 and 0.03 ppm were observed in case of the amide protons of 2,5-diketopiperazine rings of both cyclic WY (7.75 → 7.71 ppm, 7.56 → 7.52 ppm) and heat-treated WY nanotubes (7.69 → 7.66 ppm,7.35 → 7.32 ppm) respectively. These shifts as a function of concentration, suggest a strong intermolecular hydrogen bonding contributed by the amide and hydroxyl protons in the process of selfassembly. DFTB calculations suggest that the most stable conformer of the linear WY dipeptide is characterized by intermolecular hydrogen bonding of the carboxylic hydroxyl group with the carbonyl of the amide. A nearly parallel orientation between the aromatic side chains with ∼4 Å distance between the two aromatic rings was observed, which confirmed that π−π stacking and this stacking interaction contributes to the stabilization of this molecule. On the other hand, the diketopiperazine in cyclic WY is characterized by a slightly puckered boat structure with the aromatic side chains in pseudoaxial and pseudoequatorial positions. The presence of E
DOI: 10.1021/acsabm.8b00121 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 6. (a) HOMO−LUMO plots of linear WY dipeptide. (b) Simulated absorption spectra for linear WY dipeptide. (c) HOMO−LUMO plots of cyclic WY dipeptide. (d) Simulated absorption spectra for cyclic WY dipeptide.
Figure 7. Cell viability assay using MTT reagent of PC-12 cells grown on a substrate coated with (a) cyclic WY nanotubes and (b) heat-treated linear WY nanotubes at different time points (1, 3, and 5 days) at varying concentrations in comparison with control.
cm−1 in Raman signatures, was observed to be negligent. The O−H stretch in the cyclic nanotubes, and the heat-treated linear nanotubes was observed at 3415 cm−1 while in the linear monomer it was a broader peak ∼3400 cm−1 (Figure 5a).32−35 A similar analysis of the calculated Raman spectra indicate the peaks were in agreement with the experimental Raman spectrum for both cyclic and linear WY dipeptides. In the lower frequency region of the cyclic and linear WY dipeptide gas-phase spectra, there are prominent peaks attributed to the amide region of the dipeptides (Figure 5b, c). The amide peaks in the monomer, dimer, and hexamer structures, as discussed previously, have a higher intensity due to increases in amide bonds within the system (Figure 5b, c). The secondary structure prediction from FTIR and Raman spectroscopy were in strong agreement with the CD spectra (Figure S8a), and energy level calculations using DFTB (Figure 6). The analysis of the CD spectra of both the heattreated linear and linear WY nanotubes, using Dichroweb revealed the predominant secondary structure contributors to be β-strands and β-turns. The positive peaks at 220 and 200 nm, respectively, both in linear and heat-treated linear WY
twice the intensity of the monomer peaks and the hexamer peaks were about six times the intensity of the monomer peaks. The band around 1670−1680 cm−1 seen in the computational spectra of the linear WY system was attributed to β-turns and β-strands, corresponding well with experimental data. Raman scattering peaks of the peptides and nanotubes show characteristic peaks near 840 cm−1 which are tyrosine Fermi doublet peaks resulting from benzene ring breathing and are intensified due to π-electron interactions (Figure 5 and Table S1). Notable variations in peak positions of the peptide and the nanotubes include the Amide III peak in the peptides at 1257 cm−1, which has right-shifted in the nanotubes, suggesting its role in the self-assembly of the nanotubes. Differences in the N−C stretch, skeletal stretches, and N−H bending peaks were observed between the peptides and nanotubes. In the peptides as well as in the nanotubes, peaks in the Amide I region at 1626 cm−1 were characterized by carbonyl groups mixed with frequencies like C−N stretching and N−H deformation. An additional weak peak ∼1680 cm−1 in the nanotubes suggests the existence of β-sheets. The characteristic N−H stretch peak, usually found ∼3200−3300 F
DOI: 10.1021/acsabm.8b00121 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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Figure 8. Confocal and SEM imaging of PC-12 cells on nanotube surfaces indicate elongated cells and neurite outgrowths. (a, b) Elongated cells on cyclic WY nanotubes substrate with some engulfed nanotubes (green arrows) and neurite outgrowths (yellow arrows). (c) Rounded morphology in cells exposed to cyclic WY peptide (Control). (d) Rounded morphology in cells on untreated coverslips (Control) (e, f) Elongated cells on thecyclic WY nanotubes substrate with the indicated presence of growth cones (orange arrows) and neurite out-growth (yellow arrows). (g) Rounded morphology in cells exposed to cyclic WY peptide (Control) . (h) Rounded morphology in cells on untreated coverslips (Control). (i, j) Elongated cells on heat-treated linear WY nanotubes substrate with some engulfed nanotubes (green arrows) and neurite outgrowths (yellow arrows). (k) Rounded morphology in cells exposed to heat-treated linear WY peptide (Control). (l) Rounded morphology in cells on untreated coverslips (Control). (m, n) SEM of heat-treated linear WY nanotubes with the indicated presence of growth cones (orange arrows) and neurite out-growth (yellow arrows). (o) Rounded morphology in cells exposed to heat-treated linear WY peptide (Control). (p) Rounded morphology in cells on untreated coverslips (Control).
nanotubes, correspond to the n−π* and π−π* transitions, confirming the presence of β-sheets. In contrast, the cyclic WY dipeptides have α-helices, along with β-sheets and turns. The maximum peak at 190 nm and the minimum peak at 213 nm correspond to the π → π* transitions of α-helices (Figure S8a).36 The HOMO and LUMO from the DFTB calculation corresponds to π and π* (Figure 6a, c). The HOMO of both linear and cyclic monomers is delocalized around the tryptophan ring. The LUMO of the cyclic form indicates that it is delocalized in the tryptophan ring, whereas the LUMO of the linear form shows that it is delocalized on both the aromatic side chains. The first peak in the simulated absorption spectra is mainly due to HOMO (π) to LUMO (π*) transition (Figure 6b, d). Additionally, for linear WY, the dominant transition around 231 nm is mostly due to π → π* transition of the tyrosine portion of the molecule. The strong band ∼191 nm is attributed to π → π* transition of the peptide bond. The weak peak at 213 is due to n → π* transition of the peptide bond while the peak at 270 nm is primarily due to intermolecular charge transfer. On the other hand, for the WY cyclic monomer, all the major peaks are mainly due to π → π* transitions. The weak peak at around 235 nm can be attributed to intermolecular charge transfer.
The average MTT absorption of the PC-12 cells cultured on the cyclic and heat-treated linear WY nanotubes substrate were compared to a negative control with no nanotubes (Figure 7). The MTT absorption increased with the number of days in culture, indicating an overall increase in cell proliferation and activity. The cellular proliferation assessed on the varying nanotube concentrations suggest that lower concentrations, such as 0.2 mg/mL were marginally lower in cytotoxicity in comparison to the higher concentrations of 0.5 and 1 mg/mL, respectively. Both the cyclic and heat-treated linear WY nanotubes demonstrated good cell viability and proliferation, in comparison with the control (untreated glass coverslip). All concentrations of the heat-treated linear and cyclic WY nanotubes showed comparable cell viability with the control on day 1. Interestingly, lower concentrations of heat-treated WY nanotubes (0.2 mg/mL) indicated higher cell proliferation (17−22% higher) compared to the control sample grown on untreated coverslips on days 3 and 5 as shown in Figure 7. But the cyclic WY nanotubes demonstrated a lower cell viability than the untreated substrates (control) on days 3 and 5. An additional control of the PC-12 cells cultured in the presence of cyclic and heat-treated linear WY peptides were found to be nontoxic but peptide concentrations used was an order of G
DOI: 10.1021/acsabm.8b00121 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
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higher in the heat-treated WY and cyclic nanotubes with lower aspect ratios, than in the linear WY nanotubes. The morphology of the cells and the nuclear distortion may consequently modify gene expression, affecting the fate and behavior of the cells. The active mechanism for neurite formation in PC-12 cells involves the formation of directionally specific growth cones that are sensitive and motile at the growing axon tips. These growth cones assist in path-finding, or the establishment and rewiring of neuronal circuitry following damage, and could influence neural regeneration.38−41 Though nanomaterial scaffolds for neural regeneration face many shortcomings such as biocompatibility, biodegradability, and the presence of toxic monomers and cross-linking agents, self-assembling short peptide-based nanostructures may address some of the issues associated with scaffolds. However, further studies are required to establish the potential of aromatic peptide-based scaffolds as a functional material for neural regeneration.
magnitude lower than the nanotubes due to solubility issues (Figure S9a). Comparison of toxicity of heat-treated FF nanotubes with an equivalent concentration of heat-treated WY nanotubes is shown in Figure S9b. The P value was calculated using the regression analysis between average values measured in the mentioned groups compared with the control. A 90% confidence interval was used for the study, and at P < 0.1, the samples did not have any cytotoxic effects on the cells. For the various concentrations of cyclic and heat-treated linear WY nanotubes at the four different time points tested, the P values were
