Effect of Peptide Sequences on Supramolecular Interactions of

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The Effect of Peptide Sequences on Supramolecular Interactions of Naphthaleneimide/Tripeptide Conjugates Mei-Yu Yeh, Ching-Ting Huang, Tsung-Sheng Lai, Fang-Yi Chen, NienTzu Chu, Dion Tzu-Huan Tseng, Shih-Chieh Hung, and Hsin-Chieh Lin Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01809 • Publication Date (Web): 07 Jul 2016 Downloaded from http://pubs.acs.org on July 15, 2016

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The Effect of Peptide Sequences on Supramolecular Interactions

of

Naphthaleneimide/Tripeptide

Conjugates Mei-Yu Yeh,‡,§ Ching-Ting Huang,† Tsung-Sheng Lai,† Fang-Yi Chen,† Nien-Tzu Chu,† Dion Tzu-Huan Tseng,† Shih-Chieh Hung,‡,# and Hsin-Chieh Lin*,† †

Department of Materials Science and Engineering, National Chiao Tung University, Hsinchu

30010, Taiwan ‡

Integrative Stem Cell Center, China Medical University Hospital, Taichung 40447, Taiwan

§

Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402,

Taiwan #

Graduate Institute of Clinical Medical Science, China Medical University, Taichung 40402,

Taiwan

ACS Paragon Plus Environment

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ABSTRACT: In this study, we reported significant difference in the supramolecular hydrogelation of the newly discovered NI-GFF and NI-FFG on the basis of their phase diagrams. With small difference in peptide chain between NI-GFF and NI-FFG, we observed significant difference in the self-assembly properties; NI-GFF formed a stable gel at neutral pH while NI-FFG did not under the same condition. From spectroscopic and computational studies, the intermolecular π−π interactions and extended hydrogen bonding interactions might reinforce the intermolecular interactions of NI-GFF that may facilitate the formation of the self-assembled nanostructures and the hydrogel. In addition, the AIE-active NI-GFF reveals relatively good biocompatibility compared with that of NI-FFG for two commonly used cell lines, suggesting a promising candidate supramolecular material for biomedical applications. Our results highlight the importance of tripeptide sequences in a self-assembling hydrogel system.

INTRODUCTION Supramolecular hydrogels, a metastable gel state resulting from the self-assembly of small molecules in water, are potential candidate materials in the rapidly expanding field of nanostructured biomaterials.1 Supramolecular hydrogels self-assembled from peptide derivatives have drawn much attention in the recent years because of its similarity to self-assembled proteins in a biological system.2-4 As a result, three major categories of supramolecular peptide hydrogels were established: ionic complimentary peptides,5,6 peptide amphiphiles,7 and π-capped peptide hydrogels.8-12 From a microscopic view, the driving forces behind the formation of a supramolecular hydrogel are weak intermolecular interactions such as hydrogen bonding, π−π stacking and van der Waals forces.13,14 Among these peptide materials, π-capped peptide


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hydrogels have gained increased attention because of tunable composition and simple architecture of the molecular unit. Recently, many terminologies were used to describe this type of molecules, including aromatic-capped peptide hydrogels,15 low-molecular-weight hydrogels,16 aromatic peptide amphilphiles17 and small-molecule hydrogels.18 From the chemical structural features, the molecules are composed of two parts; that is, a hydrophobic part such as aromatic rings,19,20 polyaromatics21,22 or other π-conjugated systems23,24 and a short peptide (usually less than 5 amino acids).25 Therefore, we suggest π-capped peptide hydrogels would be a general terminology for this type of materials. In a recent years, a number of applications based on πcapped peptide hydrogels have been developed, including tissue engineering,8 drug delivery,9 enzyme assay,10 protein separation,11 and wound healing.12 The supramolecular hydrogelation of π-capped dipeptides with the same clogP (P: partition coefficient) have been investigated recently.19,26,27 For example, Saiani and colleagues have studied Fmoc-dipeptides and discovered that Fmoc-FG showed gelation behavior after being heated in water. In contrast, Fmoc-GF formed precipitates instead of a hydrogel under the same condition.26 We recently demonstrated two π-capped dipeptide systems, pentafluorophenyl (PFB)19 and naphthalene diimide (NDI),27 that showed dramatic difference in the hydrogelation properties through inverting the dipeptide sequence (i.e. F (Phe) and G (Gly)). Substantial differences in the self-assembled nanostructures were observed, indicating a promising approach to control hydrogelation properties at the molecular level. Recently, few π-capped tripeptide hydrogelators were developed. Ulijn et al. have developed a novel approach by using enzymes to produce Fmoc-tripeptide hydrogelators that self-assemble into nanofibrous structures.28 Yang and colleagues have studied the anti-degradation properties of the Nap-GFF hydrogel which is potentially useful to be used for long term storage of recombinant proteins at room


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temperature.29 Although there are examples for π-capped tripeptide hydrogelators, it is still unclear that the self-assembly and hydrogelation properties of π-capped tripeptide with different sequences. In this study, we report a structure–property correlation studied with new 1,8Naphthalimide (NI)/tripeptide conjugates revealed a remarkable influence by the position of benzyl side chain on the gelation and biological properties. Scheme 1. Design and structure of NI-tripeptide supramolecular hydrogelator.

In Scheme 1, we introduced a NI group at the N-terminus of tripeptides of NI-GFF (NI-Gly-LPhe-L-Phe) and NI-FFG (NI-L-Phe-L-Phe-Gly) to elucidate the role of amino-acid side chains in the formation of supramolecular hydrogels as well as photophysical properties in the assemblies. From the structural design, NI chromophore was used because NI molecules possess many advantages such as strong hydrophobicity and photostability.30,31 More importantly, NI chromophores are known to possess aggregation-induced emission (AIE) feature when they aggregate in organic solvent.32,33 Many of AIE luminogens have gained attraction in biological application owing to the strong fluorescent emission in their aggregate state in water. Accordingly, the integration of AIE luminogen in the chemical structure of a supramolecular hydrogelator would be beneficial to understand the role of π−π interactions in the assemblies. Therefore, we synthesized NI-GFF and NI-FFG and investigated the influence of different


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amino acid sequences on the characteristics of self-supporting hydrogels, photophysical properties and their cytotoxicity in vitro.

RESULTS AND DISCUSSION An efficient way to prepare polypeptide molecules is to use solid-phase peptide synthesis (SPPS) which was firstly developed by Merrifield in 1963.34 After his discovery, solid-phase organic reactions have rapidly developed and became a subfield of organic synthesis. Nowadays, this method is the standard synthetic strategy for producing new and functional peptide materials.35,36 Accordingly, we have synthesized NI-GFF and NI-FFG by SPPS using 2chlorotrityl chloride resin with Fmoc-protected amino acids. The synthetic methodology and chemical structures of NI-GFF and NI-FFG are shown in Scheme 2. For NI-GFF, the resin was swelled in anhydrous CH2Cl2 and then Fmoc-L-phenylalanine was loaded onto the resin in anhydrous DMF and DIEA. For deprotection of the Fmoc group, piperidine (20% in DMF) was added. Similarly, diphenylalanine can be generated onto the resin. Fmoc-glycine was further coupled to the free diphenylalanine using HBTU and DIEA as coupling agents. After the deprotection of Fmoc group, 1,8-Naphthalimide-N-acetic acid was coupled to the GFF using HBTU and DIEA as coupling agents. Finally, the peptide derivative was cleaved through treatment with TFA to obtain the target material of NI-GFF. In a similar manner, NI-FFG can be synthesized and their characterization data were collected in the experimental section.


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Scheme 2. The synthetic route and chemical structures of NI-GFF and NI-FFG.

In the research field of supramolecular biomaterials, there is significant interest in exploiting the supramolecular hydrogels because of the intrinsic biocompatibility. However, many reports only provide one gelation pH for each of the hydrogels. We believe this is not enough to understand the intrinsic property of a material, therefore, in this work, we detail studied the phase diagrams of NI-GFF and NI-FFG for perspective understanding the property of supramolecular hydrogelation. Figure 1 shows the phase diagrams of NI-GFF and NI-FFG as a function of pH values and concentrations. Because of different peptide sequence between NI-GFF and NI-FFG, hydrogelation properties were found to depend strongly on the chemical structure of materials. For instance, NI-GFF gel were stable over a wide pH range (pH 4.0-10.0) at 1 wt%, while NIFFG only formed a stable hydrogel in the range of pH 5.0-6.0 at 1 wt%. In addition, the minimum gelation concentrations are 0.5 wt% for NI-GFF and 1.8 wt% for NI-FFG under pH 7.0, suggesting that peptide sequences may play a crucial role to determine hydrogelation properties. With the same clogP values of NI-GFF and NI-FFG, dramatically different gelation


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pH of the hydrogels were observed, which may indicate the different molecular packing and chemical equilibrium between carboxyl and carboxylate for these two amphilphiles in water.37 The successful hydrogelation of NI-GFF at pH 7.0 implies that NI-GFF may be a candidate material in the biological applications.

Figure 1. The concentration-pH phase diagrams for (a) NI-GFF and (b) NI-FFG. (S: solution, G: gel and P: phase separation) Since the NI-GFF and NI-FFG exhibit different gelation properties, we further examine the influence of the position of glycine in the tripeptide sequence on the properties of self-assembled hydrogel by transmission electron microscopy (TEM). TEM was used to characterize the nanostructure morphology and the self-assembly of NI-GFF and NI-FFG. Figure 2a and 2b


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show the optical (inset of Figure 2a and 2b) and TEM images of NI-GFF and NI-FFG at a concentration of 1 wt% under neutral pH. The appearance of NI-GFF is a transparent gel and its TEM image revealed a uniform fibrous network with diameters of 11.02±2.0 nm. In contrast, NIFFG did not gel water at neutral pH and the TEM image showed the presence of aggregates (see Figure S1 for TEM image of NI-FFG at 1 wt% under pH 6.0). The viscoelastic properties of a hydrogel are important parameters for many biological applications.38 For example, the stiffness of a hydrogel has been proven to direct the differentiation of various cell types.39-41 Therefore, we have determined the rheological properties of NI-GFF gels and the results were shown in Figure S2. NI-GFF has its ability to form stable gels at 1 wt% under neutral condition and the storage moduli (G') was 770 Pa in the frequency range of 0.1–100 rad·s-1, which is necessary to support the mass of a cell.42,43 The G' of NI-GFF are greater than their loss moduli (G") and they are almost independent of frequency, which indicates that NI-GFF behave like elastic solids (see Figure S3 for rheological properties of the NI-FFG gels). Furthermore, the gel-to-sol transition temperature (Tgel-sol) of NI-GFF was investigated by the test tube inverting method. Tgel-sol of NIGFF was 62 oC which is higher than body temperature, thus suggesting good thermal stability. These results suggest that the position of glycine in the tripeptide sequence would dramatically affect the self-assembly, nanostructure morphology and mechanical properties of NI-tripeptides.


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Figure 2. Optical (insets) and negatively stained TEM images of hydrogels for (a) NI-GFF and (b) NI-FFG at a concentration of 1 wt% under pH 7.0 (scale bar: 100 nm). UV-Vis absorption, fluorescence emission, circular dichroic (CD) spectroscopy and FT-IR were used to characterize intermolecular interactions in the assemblies.19,44 Hydrogelators of NIGFF and NI-FFG showed an UV-Vis absorption band around 340 nm at 1 mM in water, which is the characteristic of electronic transition in NI chromophore (Figure S4).30 To explore the π-π intermolecular interactions in the assemblies, the concentration-dependent emission spectra of NI-GFF and NI-FFG were measured in aqueous solutions with the excitation at 340 nm for the NI group (Figure 3a and 3b). We observed the maxima of emission spectra at 396 nm in solution, and these peaks were red-shifted about 15 and 4 nm for NI-GFF and NI-FFG, respectively, when the concentration increased from 500 to 15000 µM. Notably, new emission bands centered around 500 nm (475–575 nm) were only observed in NI-GFF with concentrations of 5.0, 7.5 and 15.0 mM, which indicates relatively stronger π–π interactions in the assembly of NI-GFF. The CD spectra of NI-GFF and NI-FFG were measured at 1 mM in water (Figure S4). We found that only NI-GFF have positive Cotton effects in the range of 300-400 nm, which might be due to NIs in the self-assembled structures. Moreover, NI-GFF revealed a positive band at 195 nm and a negative band at 218 nm in the CD spectrum, which may indicate a well-defined antiparallel β-sheet structure in the assemblies.45 To confirm this, Thioflavin T (ThT) was used because ThT is one of the most used compounds for selectively staining and identifying amyloid fibrils both in vitro and in vivo.46 Through binding ThT molecules to β-amyloid fibrils, a significant enhancement of fluorescence emission at 484 nm can be detected, while unbound ThT is weakly fluorescent at this wavelength. Figure 3c shows the changes in the emission intensities of ThT at different concentrations of NI-GFF and NI-FFG (1–10 mM). The binding


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of ThT with NI-GFF at 10 mM induces a substantial fluorescence enhancement of ca. 200 folds as compared to that without adding any hydrogelators. In contrast, the emission intensities of ThT slightly increase with concentrations of NI-FFG in water, revealing that the self-assembly of NI-GFF may have well-developed secondary conformation compared with that of NI-FFG (Figure 3c). To further study hydrogen bonding interactions, FT-IR spectra were measured in trifluoroethanol (TFE) and in water (Figure S5).47 FT-IR spectrum of NI-GFF in water showed the typical amide I bands at 1665 cm-1 and 1635 cm-1 which were shifted to lower wavenumbers compared with those in TFE, thus indicating the formation of extended hydrogen-bonding networks may be through the amide groups in the assemblies.


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Figure 3. Normalized concentration-dependent fluorescence spectra for (a) NI-GFF and for (b) NI-FFG at pH 7.0. (c) Emission intensities of ThT (20 µM) at various concentrations of hydrogelators of NI-GFF (blue squares) and NI-FFG (black squares). (d) Optimized molecular packing structure of NI-GFF. (e) Visualization of the weak interactions for NI-GFF dimer (left) and for NI-FFG dimer (right) in real space. The gradient isosurface method was used and the scale runs from -0.02 (min) to 0.02 (max). C: gray, H: white, N: blue, O: red.


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According to the spectroscopic results, we then optimized a packing model for molecules in the fibrils (Figure 3d). It may invoke a packing of consecutive hydrogelators with aromatic π–π interactions occurring between end-capped NI groups and benzyl side chains and β-sheet-like structure from extended hydrogen bonding between amide and carboxyl groups of the tripeptides. To get more insight into the self-assembly of NI-GFF and NI-FFG, we further investigated intermolecular interactions by using the newly discovered gradient isosurface method.48-50 The intermolecular interactions of dimers of NI-GFF and NI-FFG, such as van der Waals interactions, hydrogen bonds, and other weak repellent interactions can be visualized in real space within this methodology.48-50 As displayed in Figure 3e, the intermolecular interactions of the NI-GFF dimer revealed a continuous wave function overlap with electronic attraction effect, van der Waals effect and relatively weak repellent effect. Notably, the intermolecular attractions between NI and phenyl ring of phenylalanine in NI-GFF dimer were also essential. In contrast, the dimer of NI-FFG exhibits a relatively significant repulsion effect between two molecules and aromatic-aromatic interactions are relatively weak (Figure 3e). These results are consistent with the UV-Vis absorption, fluorescence emission, CD and FT-IR results. There are few reports that aggregation of NI chromophores in organic solvent could exhibit AIE properties.32,33 Since the NI/peptide conjugates are partially water soluble, it is possible to test AIE characteristics for self-assembly of NI-GFF and NI-FFG in aqueous solution. To explore whether hydrogelators NI-GFF and NI-FFG have AIE characteristics in the assemblies, the fluorescence spectra of NI-GFF and NI-FFG were measured in a series of DMSO/water mixtures with different volume fractions of water (fw) at 500 µM. Note that NI-GFF or NI-FFG was completely dissolved in DMSO. In Figure 4, NI-GFF is almost non-luminescent in DMSO


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while with gradual addition of water into DMSO at fw ≤ 60 vol%, the emission intensity remains silent and is almost unchanged in the profile. When fw ≥ 70 vol%, the emission is exponentially intensified with fw. At fw = 99 vol%, an around 120-fold enhancement of emission has been observed as compared to that in DMSO. Similar enhancement was observed in the emission spectra of NI-FFG and these results reveal that NI-GFF and NI-FFG are AIE-active in the assemblies in water.

Figure 4. (a,c) Fluorescence emission spectra (λex: 340 nm) of (a) NI-GFF and (c) NI-FFG at 500 µM at pH 7.0 in DMSO solutions containing various water fractions (vol %). (b,d) Relative fluorescence intensity (I/I0) plotted with respect to the water fraction in DMSO for (b) NI-GFF and (d) NI-FFG; I0: fluorescence intensity of NI-GFF or NI-FFG in pure DMSO.


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To further study the cytotoxicity of the hydrogelators for biomedical applications, MTT [3(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay was used to evaluate the in vitro cytotoxicity of NI-GFF and NI-FFG. CTX-TNA2 and MCF-7 cells were used to examine the cell viability ratios of the hydrogelators compared to those without the hydrogelators in the medium as control experiment. It has been demonstrated that the preliminary biocompatibility of π-capped peptide hydrogelators could be evaluated with the concentrations range of 10-500 µM.19 Figure 5a and 5b show the cell viabilities of NI-GFF and NI-FFG for CTX-TNA2 cell line. After incubating CTX-TNA2 cells with hydrogelators at 37 °C for 48 h, the cell viability ratio exceed ~90% for NI-GFF and ~82% for NI-FFG at 500 µM. Similar conditions were used to culture MCF-7 cells with the hydrogelators and the results were shown in Figure 5c and 5d. Notably, after being incubated MCF-7 cells with hydrogelators at 500 µM for 48 h, the cell viabilities were ~92% and ~55% for NI-GFF and NI-FFG, respectively. The cytotoxicity tests of the NI-GFF and NI-FFG gels at 2wt% for CTX-TNA2 and MCF-7 cells are also studied (Figure S6). From the phase diagram in Figure 1, it is clear that the NI-GFF is easier to form self-assembled aggregates compared with that of NI-FFG at neutral condition. If that is the case, it may result in less cytotoxic for NI-GFF because less hydrogelators can diffuse into the cells and that may potentially reduce the cytotoxicity of the cells. Furthermore, we examined the biostability of hydrogelators through the incubation of NI-GFF or NI-FFG with proteinase K, a powerful protease that hydrolyzes a wide range of peptidic molecules.51 In Figure S7, we observed that a better resistance to enzymatic digestion for NI-GFF compared with that of NIFFG, thus indicating the better self-assembled properties of NI-GFF at physiological condition would result in better biostability. These results reveal that NI-GFF is relatively biocompatible


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compared with that of NI-FFG, thus implying the NI-GFF may be a candidate material in the applications of tissue engineering and drug delivery.

Figure 5. Viability ratio measured by MTT assay on (a, b) CTX-TNA2 and (c, d) MCF-7 cells in the presence of 10, 50, 100, 200 and 500 µM of (a, c) NI-GFF and (b, d) NI-FFG for 48 h. Blue for 10 µM, white for 50 µM, light gray for 100 µM, gray for 200 µM and dark gray for 500 µM.

CONCLUSION In this study, we demonstrate the first example of NI/tripeptide conjugates that form supramolecular hydrogels. The detail study of the phase diagrams of NI-GFF and NI-FFG revealed that significant difference in the self-assembly. NI-GFF forms a stable gel at pH 7.0 while NI-FFG did not under the same condition. From spectroscopic and computational studies, the intermolecular π−π interactions and extended hydrogen bonding interactions might reinforce


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the intermolecular interactions of NI-GFF that may facilitate the formation of the self-assembled nanostructures and the hydrogel. In addition, NI-GFF has AIE property and excellent biocompatibility for CTX-TNA2 and MCF-7 cell lines, thus making it a potential candidate material for biomedical applications.

EXPERIMENTAL SECTION Synthesis of Naphthalimide-Gly-Phe-Phe (NI-GFF): The NI-GFF was prepared through SPPS using 2-chlorotrityl chloride resin, Fmoc-L-phenylalanine, Fmoc-glycine, and 1,8Naphthalimide-N-acetic Acid. The resin (2.4 g) was swelled in anhydrous CH2Cl2 for 30 min and then Fmoc-L-phenylalanine (1.55 g, 4.00 mmol; 1.55 g, 4.00 mmol) was loaded onto the resin in anhydrous N,N-dimethylformamide and N,N-diisopropylethylamine (DIEA; 1.65 mL, 10.00 mmol) for 1 h. For deprotection of the Fmoc group, piperidine (20% in DMF) was added and the sample left for 20 min; this procedure was repeated twice (each time for 2 min). Fmoc-glycine (1.20 g, 4.00 mmol) was coupled to the free amino group using O-(benzotriazol-1-yl)-N,N,N´,N´tetramethyluraniumhexafluorophosphate

(HBTU)

(1.52

g,

4.00

mmol)

and

N,N-

diisopropylethylamine (DIEA) (1.65 mL, 10.00 mmol) as coupling agents for 30 min. Again, the sample was treated with piperidine (20% in DMF) for 20 min; this procedure was repeated twice (each time for 2 min). Finally, 1,8-Naphthalimide-N-acetic acid (1.02 g, 4.00 mmol) was coupled to the free amino group using HBTU (1.52 g, 4.00 mmol) and DIEA (1.65 mL, 10.00 mmol) as coupling agents. After the reaction mixture had been stirred overnight, the peptide derivative was cleaved through treatment with CF3CO2H overnight. The resulting solution was dried by air and then DI water was added to precipitate the target product. The solid was dried under vacuum to remove residual solvent (0.157 g). 1H NMR (300 MHz, DMSO-d6) δ (ppm):


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2.65-2.80 (m, 1H), 2.90-3.15 (m, 3H), 3.55-3.70 (m, 1H), 3.70-3.85 (m, 1H), 4.40-4.50 (m, 1H), 4.55-4.65 (m ,1H), 4.74 (s, 2H), 7.15-7.35 (m, 10H), 7.92 (dd, J = 7.6, 7.6, 2H), 8.07 (d, J = 8.4, 1H), 8.4 (d, J = 7.8, 1H) 8.45-8.60 (m, 5H);

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C NMR (300 MHz, DMSO-d6) δ (ppm) = 37.6,

38.6, 42.7, 43.2, 54.5, 122.9, 127.2, 127.3, 128.2, 128.4, 128.9, 129.1, 130.0, 130.1, 131.8, 132.3, 135.5, 138.7, 164.3, 167.9, 169.1, 171.9 ; MS [ESI-] : m/z(%) calcd : 606.21, obsvd : 605.30 [MH]-. Synthesis of Naphthalimide-Phe-Phe-Gly (NI-FFG): 2-Chlorotrityl chloride resin (2.40 g) was swelled in anhydrous CH2Cl2 for 30 min and then Fmoc-glycine (1.20 g, 4.00 mmol) was loaded onto the resin in anhydrous DMF and DIEA (1.65 mL, 10.00 mmol) over a period of 1 h. For deprotection of the Fmoc group, piperidine (20% in DMF) was added and the sample left for 20 min; this procedure was repeated twice (each time for 2 min). Fmoc-L-phenylalanine (1.55 g, 4.00 mmol; 1.55 g, 4.00 mmol) was coupled to the free amino group, using HBTU (1.52 g, 4.00 mmol) and DIEA (1.65 mL, 10.00 mmol) as coupling agents, over a period of 1 h. Again, the sample was treated with piperidine (20% in DMF) for 20 min; this procedure was repeated twice (each time for 2 min). Finally, 1,8-Naphthalimide-N-acetic acid (1.02 g, 4.00 mmol) was coupled to the free amino group using HBTU (1.52 g, 4.00 mmol) and DIEA (1.65 mL, 10.00 mmol). After the reaction mixture had been stirred overnight, the peptide derivative was cleaved through treatment with CF3CO2H overnight. The resulting solution was concentrated through exposure to the air and then DI water was added to precipitate the target product. The solid was dried under vacuum to remove any residual solvent, providing a white solid (0.139 g). 1H NMR (300 MHz, DMSO-d6) δ (ppm): 2.75-2.95 (m, 2H), 2.95-3.15 (m, 2H), 3.70-3.90 (m, 2H), 4.50-4.65 (m, 2H), 4.70 (s, 2H), 7.15-7.40 (m, 10H), 7.92 (dd, J = 7.5 7.5, 2H), 8.15-8.30 (m, 2H), 8.53 (d, J = 7.2, 4H) ; 13C NMR (300 MHz, DMSO-d6) δ (ppm) = 38.4, 38.6, 43.1, 54.8, 122.8, 127.1, 127.2,


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128.2, 128.9, 129.0, 130.1, 130.2, 131.8, 132.3, 135.4, 138.6, 164.1, 167.4, 171.5, 172.0 ; MS [ESI+] : m/z(%) calcd : 606.21 ; obsvd : 629.20 [M+Na]+. Preparation of Peptide Hydrogels: Supramolecular hydrogel was prepared by suspending the NI-GFF in purified water, followed by increasing pH to a value greater than 8 with the addition of 1 N NaOH. Because the deprotonation of the carboxylic acid end group that can increase the water solubility, a clear solution was obtained. Subsequently, 1 N HCl was added into this solution until the pH value reached 7.0 and an stable hydrogel was formed at pH 7.0 (1 wt%). This gel was self-supporting because it was able to invert the container without any gel collapsing. The gel of NI-FFG can be prepared similarly. Transmission Electron Microscopy: Images were obtained using a Hitachi HT7700 transmission electron microscope operated at an accelerating voltage of 100 kV. Hydrogels were applied directly onto 200-mesh carbon-coated copper grids. The excess of the hydrogel was carefully removed through capillary action (filter paper) and then the grids were immediately stained with uranyl acetate for 30 s. Excess stain was removed through capillary action and then the grids were left to air-dry. Inverted Tube Method. Gelation tests were performed by weighing a compound (2.0 mg) in a screw-capped 2-mL vial (diameter: 10 mm), adding a solvent (0.20 mL), sealing the vial tightly, heating it until the compound had dissolved, and then cooling the vial to room temperature. Gelation was considered to have occurred when a solid-like material was obtained that did not exhibit gravitational flow (inverted test tube method) during a period of 5 min. Rheological Tests. Rheological tests were conducted using a TA rheometer (DHR-1) and a 40-mm parallel plate. The hydrogel sample (400 µL, 1 wt %) was placed on the parallel plate for


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the angular frequency sweep test (test range: 0.1–100 rad s–1; strain, 0.8%; 15 points per decade; sweep mode, “log”; temperature, 25 °C). Cell Viability. The biocompatibility of the NI-GFF and NI-FFG were measured through MTT cell viability tests. CTX-TNA2 and MCF-7 were seeded in 24-well plates at a density of 50,000 cells per well, in the presence of Dulbecco’s modified Eagle’s medium (DMEM, 0.5 mL) containing 10% FBS and 1% penicillin/streptomycin, and incubated for 24 h. Test compounds of various concentrations (10, 50, 100, 200, 500 µM) were added during plating of the cells. After 24 and 48 h, the medium was replaced with fresh medium supplemented with MTT (4 mg mL–1, 0.5 mL per well). After another 4 h, the medium containing MTT was removed and DMSO (0.5 mL per well) was added to dissolve the formazan crystals. Each 24-well was transferred to a 96well plate. The optical density of the resulting solution was measured at 595 nm, using an absorbance microplate reader (Infinite F50, TECAN). Cells that had not been subjected to treatment with the test compounds were used as the control. The cell viability percentage was calculated using the expression ODsample/ODcontrol. Computational Details. The geometries of the model compounds were optimized by DFT/631G* method.52 The visualization of weak interactions of the NI-GFF and NI-FFG dimers were conducted using Multiwfn 2.6 software53 in real space. Multiwfn 2.6 deals with reduced density gradient (RDG) in real space as equation:

RDG (r ) =

1 1 2 3

∇ρ (r )

×

2 × (3 × π )

ρ (r )

4 3

where ρ(r) is electron density and can be defined as: 2 2

ρ (r ) = ∑η i ϕi (r ) = ∑ηi ∑ C j ,i χ j (r ) i

i

j


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where ϕ is orbital wave function mentioned above, ηi is occupation number of orbital i, χ is basis function. C is coefficient matrix, the element of ith row jth column corresponds to the expansion coefficient of orbital j respect to basis function i. The graphic displays of π–π interactions were then drawn using VMD 1.9. This visualization method was successfully and widely used in many other works.48-50

ASSOCIATED CONTENT Supporting Information TEM images, rheological properties, UV-Vis absorption, CD, FT-IR, cell viability as well as biostability of gels, 1H NMR and 13C NMR spectra were collected in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work has been financially supported by the Ministry of Science and Technology of the Republic of China, Taiwan (grant MOST 104-2119-M-009-006-); the “Aim for the Top University” program of the National Chiao Tung University and Ministry of Education, Taiwan,


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R.O.C.; the Novel Bioengineering and Technological Approaches to Solve Two Major Health Problems in Taiwan sponsored by the Taiwan Ministry of Science and Technology Academic Excellence Program under Grant Number: MOST 105-2633-B-009-003; the National Center for High-Performance Computing of Taiwan for computer time and facilities.

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TOC


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Effect of Peptide Sequences on Supramolecular Interactions of

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