Peptide Glycosylation Generates Supramolecular Assemblies from

Mar 1, 2016 - Glycopeptide-based hydrogelators with well-defined molecular structures and varied contents of sugar moieties were prepared via in vitro...
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Peptide Glycosylation Generates Supramolecular Assemblies from Glycopeptides as Biomimetic Scaffolds for Cell Adhesion and Proliferation Jie Liu,† Ziling Sun,‡ Yuqi Yuan,† Xin Tian,‡ Xi Liu,† Guangxin Duan,‡ Yonggang Yang,† Lin Yuan,*,† Hsin-Chieh Lin,*,§ and Xinming Li*,†,⊥ †

College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, China School of Biology and Basic Medical Science, Soochow University, Suzhou, 215123, China § Department of Materials Science and Engineering, National Chiao Tung University, Hsinchu, 300, Taiwan ⊥ State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200433, China ‡

S Supporting Information *

ABSTRACT: Glycopeptide-based hydrogelators with welldefined molecular structures and varied contents of sugar moieties were prepared via in vitro peptide glycosylation reactions. With systematic glucose modification, these glycopeptide hydrogelators exhibited diverse self-assembling behaviors in water and formed supramolecular hydrogels with enhanced thermostability and biostability, in comparison with their peptide analogue. Moreover, because of high water content and similar structural morphology and composition to extracellular matrixes (ECM) in tissues, these self-assembled hydrogels also exhibited great potential to act as new biomimetic scaffolds for mammalian cell growth. Therefore, peptide glycosylation proved to be an effective means for peptide modification and generation of novel supramolecular hydrogelators/hydrogels with improved biophysical properties (e.g., high biostability, increased thermostability, and cell adhesion) which could promise potential applications in regenerative medicine. KEYWORDS: hydrogel, peptide, self-assembly, supramolecular, glycopeptide and self-assembling properties17−19 or nanostructures with high supramolecular orders and improved functionalities.20−24 Although there have been previous reports about glycopeptides from different origins that could self-assemble, the study for the preparation of glycopeptide hydrogelators with precise control of the location and content of carbohydrate moieties on peptide scaffolds was still rare. In this study, we decided to take advantage of site-specific glucose modification reactions in vitro to prepare novel hydrogelators from glycopeptides and investigate the effects of glucose modification on their selfassembling properties and the generation of self-assembled nanomaterials with improved biofunctionalities. In this study, we designed and synthesized five molecules, and each of them consisted of a naphthyl moiety, a tetrapeptide motif (e.g., PhePhe-Asp-Asp), and varied contents of glucose residues (e.g., 2amino-D-glucose) as shown in Scheme 1. The naphthyl moiety and Phe-Phe dipeptide group provided the driving force for supramolecular self-assembling via aromatic−aromatic interactions, and the Asp-Asp segment was subject to site-specific glucose modifications. With systematic glucose modification, we

1. INTRODUCTION Peptide glycosylation is a ubiquitous process in nature for posttranslational modification of cellular proteins by attaching carbohydrates to the side chains of asparagine, serine, and threonine.1 This modification reaction not only introduces enormous structural diversities to encoded proteins, stabilizes their local conformations,2,3 but also generates glycoproteins with enriched functionalities, such as mediating intra- and extracellular trafficking, cell adhesion, cell differentiation, and cell growth via receptor−ligand interactions.4 Because of increased understanding of the biological roles of glycosylation and the advancement of glycochemistry, there has been a growing interest in the synthesis of glycopeptides of different architectures in the applications of vaccine antigens, decoy receptors, and structural mimics of glycoproteins.5−9 Besides, certain peptide-carbohydrate conjugates with extended intermolecular interactions also exhibited additional capabilities for supramolecular self-assemblies and formed nanostructures with spatial arrangement of carbohydrate moieties on their surfaces for various applications,10−16 such as inducing bacterial agglutination, tailoring cell responses, targeting drug delivery. Therefore, peptide glycosylation held great potential to generate supramolecular hydrogelators with novel molecular structures © 2016 American Chemical Society

Received: January 22, 2016 Accepted: March 1, 2016 Published: March 1, 2016 6917

DOI: 10.1021/acsami.6b00850 ACS Appl. Mater. Interfaces 2016, 8, 6917−6924

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Scheme 1. (a) Molecular Structures of Glycopeptide Gelators and (b) Illustration of the Self-Assembling Process of Glycopeptides for the Generation of Supramolecular Hydrogel

obtained glycopeptide hydrogelators with fine-tuned molecular structures and varied contents of glucose moieties, which relied on extended π−π stacking of aromatic naphthalyl and phenyl rings and intermolecular hydrogen bonding between glucoses and amino acids for supramolecular self-assembling. Moreover, because of the high water content, similar structural morphology and composition to extracellular matrixes (ECM) in tissues, these self-assembled hydrogels also exhibited great potential to act as new biomimetic scaffolds for cell adhesion and proliferation.

on the resin by using HBTU as a coupling reagent. These coupling and deprotection steps were repeated by following standard Fmoc SPPS protocol to elongate the peptide chain. The resin was washed with DMF 3−5 times after each step. At the last step, the resin was washed by DMF, DCM, methanol, and hexane, respectively, which was repeated five times for each solvent. Then the synthetic peptide was cleaved from the resin by using TFA cocktail solution (90% TFA in water), and the resulted product was purified with HPLC by using water−acetonitrile as eluents (from 70:30 to 0:100) to afford hydrogelator 1 at a yield of 68%. 1H NMR (400 MHz, DMSO-d6): δ12.60 (s, 2H), 8.41−8.33 (d, 1H), 8.26−8.16 (m, 2H), 8.08−7.99 (d, 1H), 7.88−7.83 (d, 1H), 7.81−7.71 (m, 2H), 7.60−7.54 (s, 1H), 7.52−7.42 (m, 2H), 7.28−7.08 (m, 12H), 6.45−6.41 (s, 1H), 5.01−4.88 (m, 2H), 4.71−4.38 (m, 7H), 3.77−3.43 (m, 7H), 3.18−2.96 (m, 3H), 2.85−2.61 (m, 5H). MS: calcd M+ = 871.88, obsd (M − H)− = 870.3. Transmission Electron Micrograph (TEM) and Scanning Electron Micrograph (SEM) Characterizations. Each hydrogel sample was placed on a single-crystal silica plate or a carbon-coated Cu grid. The samples for TEM study was stained with uranyl acetate (2.0% (w/v)). The silica plate loaded with gel was dried under vacuum and coated with a thin layer of platinum before SEM measurement. Then TEM and SEM images were recorded on a Tecnai G220 transmission electron microscope and a Hitachi S-4800, respectively. Thioflavin T Binding Assays. The freshly prepared solutions of gelator 1, 2, 3, 4, and 5 were mixed with ThT solutions in PBS buffer to final concentration at 2.3 mM for each gelator and ThT at 100 μM. After 5 min, the samples were subject to ThT fluorescence measurements on a PerkinElmer spectrophotometer with excitation at 440 nm and emission at 480 nm, respectively. The Thioflavin T (ThT) fluorescence microscopy images of each gelator were obtained on an IX71 Olympus microscope, Japan. Circular Dichroism and FTIR Analysis. CD spectra were recorded from 185 to 400 nm by using an Aviv 410 spectrometer under a nitrogen atmosphere. The hydrogels (0.2 mL, 1.0 wt %) were placed evenly on the 1 mm thick quartz cuvette and scanned with a 0.5 nm interval. FTIR spectra of glycopeptide hydrogels were collected on a PerkinElmer spectrophotometer by loading each sample into a KBr cuvette. All the hydrogels were prepared by using deuterium oxide (D2O) as a solvent, and deuterium chloride (DCl) and deuterium generation of NaOH (NaOD) as an acid and a base, respectively. Rheological Measurements. Rheological tests were operated on a Thermo Scientific HAAKE RheoStress 6000 rheometer with a 20 mm parallel plate. A volume of 0.2 mL of each sample was loaded on the parallel plate. Dynamic strain sweep test was run from 0.1 to 10% strain with a frequency at 6.282 rad/s at 25 °C. Dynamic frequency sweep test was tested from 200 rad/s to 0.1 rad/s, and a strain at 0.4% was used to ensure the linearity of dynamic viscoelasticity. Biostability Assays of Hydrogelators. A mass of 1.0 mg of each hydrogelator was dissolved in 5 mL of HEPES buffer at pH = 7.5 as a solution, and then proteinase K was added with concentration at 3.2 units/mL. The mixed solutions were incubated at 37 °C for 24 h, and

2. MATERIALS AND METHODS Fmoc-Phe-OH, Fmoc-Asp(OtBu)−OH and Fmoc-Asp-OtBu were purchased from Shanghai GL Biochem. 2-(Naphthalen-6-yl) acetic acid, 2-amino-D-glucose, proteinase K, and staining dyes for live/dead cell assays were provided by Sigma-Aldrich. All the other compounds were obtained from J&K Chemicals and used without further purification. 1H NMR spectra were obtained on a Unity Inova 400 by using DMSO-d6 as a solvent. CD spectroscopy study was performed on an Aviv 410 spectrometer. Liquid chromatography−mass spectrometry (LC−MS) analysis was conducted on an Agilent 6120 quadrupole LC− MS system with an electrospray ionization (ESI) source. HPLC purification and analysis were carried out on a Waters 600E multisolvent delivery system using a YMC C18 RP column with CH3CN (0.1% of TFA) and water (0.1% of TFA) as eluents. Transmission electron micrograph (TEM) and scanning electron micrograph (SEM) images were recorded on a Tecnai G220 transmission electron microscope and a Hitachi S-4800, respectively. Fluorescence microscopy images were taken on an IX71 Olympus microscope, Japan. Fourier transform infrared spectroscopy (FTIR) characterization was carried out on a PerkinElmer spectrophotometer. Rheological study was taken on a Thermo Scientific HAAKE RheoStress 6000 rheometer. Synthesis of Glycopeptide Hydrogelators. The synthesis processes of hydrogelators were illustrated by using hydrogelator 1 as an example, which was prepared by following standard solid-phase peptide synthesis (SPPS) with the application of 2-chlorotrityl chloride resin (100−200 mesh and 1.3−1.8 mmol/g), Fmoc-Phe-OH, FmocAsp(OtBu)-OH, and Fmoc-Asn(Glc)-OH. First, 1.0 g resin was suspended in dry dichloromethane (DCM) with N2 bubbling for 30 min, and the swelled resin was washed by dry dimethylformamide (DMF) three times. The mixed solution containing the first amino acid and N,N-diisopropylethylamine (DIEA) was added to the reactor containing swelled resin and reacted for 1 h. After removal of solvent and washed by dry DMF three times, the resin was quenched by blocking solution (80:15:5 of DCM/methanol/DIEA) for 10 min, which was repeated twice. Then, the resins were treated with 20% piperidine (in DMF) for 0.5 h to remove the Fmoc-protecting group and was washed thoroughly with DMF three times. The subsequent Fmoc-protected amino acid was added to the reactor and coupled with free amino groups 6918

DOI: 10.1021/acsami.6b00850 ACS Appl. Mater. Interfaces 2016, 8, 6917−6924

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Scheme 2. Synthetic Route for the Preparation of Supramolecular Hydrogelators 1, 2, 3, 4, and 5 via Solid Phase Synthesis

Figure 1. Optical images of hydrogel 1 (0.2 wt %, pH = 5.5), 2 (0.3 wt %, pH = 5.5), 3 (0.3 wt %, pH = 7.0), and 4 (1.0 wt %, pH = 7.4). Transmission electron micrograph (TEM) and scanning electron micrograph (SEM) images of hydrogels 1, 2, 3, and 4, respectively. The scale bar is on behalf of 50 nm. then 100 μL of solution from each sample was taken at each specific time and analyzed by HPLC. Computational Methodology. The ground-state geometries of 1−5 were optimized by using the Hartree−Fock Austin Model 1 (AM1) method; this technique has been demonstrated to reproduce geometric

parameters for many conjugated systems which were consistent with experimental X-ray data. Geometric optimizations of the four-molecule clusters of 1−5 and the packing model of 1 were performed using the DREIDING force field, which was successfully and widely used in many other biological systems. 6919

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ACS Applied Materials & Interfaces Cell Viability Tests. Cell viability was assessed by CCK-8 assay. Mice skin fibroblasts were suspended at a final concentration of 2 × 104 cells/well and cultured in a 96-well flat bottomed microplate at 37 °C. Following 24 h culture, the medium was replaced with complete culture medium supplemented with various concentrations of gelator (20.0 μM, 50.0 μM, 100.0 μM, 200.0 μM, and 500.0 μM). Following the gelator treatment for 24 h, the cells were incubated with cell counting kit-8 solution (CCK-8; Shimadzu Corporation). Subsequently, the absorbance (optical density, OD) at 450 nm was measured using a Thermo Scientific Varioskan Flash spectral scanning multimode reader. Cell viability was expressed as a percentage of the number of control (untreated) cells. Viability in the control group was designated as 100%. All experiments were performed in quintuplicate. Cell Culture on Glycopeptide Hydrogels. Prior to 2D-culture, 50 μL of hydrogelators 1 and 2 solution in PBS buffer (10 mg/mL, pH = 7.4) was added in a 96-well plate for hydrogel formation in 6 h and then covered with 100 μL of Dulbecco’s Modified Eagle’s Medium (DMEM) for buffer exchange, and then the medium in the wells was replaced with fresh medium after 12 h of incubation. Cells were collected from a subconfluent monolayer by trypsin-EDTA treatment, and were resuspended in complete medium containing DMEM, 10% FBS, and 1% Penicillin/Streptomycin solution. Then 100 μL of cell suspension in complete medium containing 1.0 × 104 cells was pipetted into each well. The 96-well plate was placed in incubator at 37 °C under 5% of CO2 atmosphere. Cells were evenly distributed on the gel surfaces by gently pipetting the medium, and the medium was changed every other day. The cultured cells were stained by calcein/ethidium homodimer, imaged by a Olympus IX71 fluorescence microscope and quantified by ImageJ.

were due to the blockage of the ionizable carboxylate groups on the side chain or C-terminal of peptide residues by glucose modification. However, their solubilities can be improved with gentle heating or by changing pH to a slightly basic condition (pH = 9.0). Followed by the adjustment of pH to 7.0 and 7.4, separately, they self-assembled to form stable hydrogels with minimum gelation concentrations at 0.3 or 1.0 wt % (Table 1). These results indicated that site-specific glucose modifications offered a valid approach to prepare novel hydrogelators from glycopeptides with varied molecular structures and selfassembling properties. Transmission electron microscopy (TEM) and scanning electron micrograph (SEM) images revealed the formation of nano- and microstructures in different morphologies by hydrogelators 1−5 (Figure 1). Without glucose modification, hydrogelator 5 formed helical nanofibrous structures with a width around 22 nm (Figure S16A). After glucose modification at different sites, hydrogelators 1−4 self-assembled into welldefined nanofibers with several micrometers in length and 13, 14, 12, and 13 nm in width, respectively (Table 1). In addition, we also identified the formation of hierarchical nanostructures in hydrogel 3, such as the presence of a large amount of thin single fibers and untwisted nanobundles by parallel association of single fibers (Figure 1). These results indicated that the subtle difference on the molecular structures of hydrogelators can result in the formation of nanostructures in high variabilities. SEM images displayed in Figure 1 and Figure S16B further confirmed the formation of high-aspect-ratio nanofibers selfassembled from hydrogelators 1−5, which physically crosslinked each other to assist the formation of stable supramolecular hydrogels. Circular dichroism (CD) studies provided insightful information about the effect of glucose modification on the secondary structures of hydrogelators in supramolecular self-assemblies. The CD spectrum of hydrogel 5 (Figure 2A) showed a broad negative peak at 208 nm and a weak positive band around 193 nm which was a characteristic of β-sheet conformation. The additional bisignated Cotton effects near 233 and 288 nm were attributed to the ordered arrangement of the exposed L-aspartic acid residues and phenyl groups within its helical nanostructures (Figure S16).26 On the other hand, the CD spectra of hydrogels 1−4 revealed the formation of secondary structures which were distinguishable from that of hydrogel 5. For example, hydrogels 1 and 2 exhibited weak positive bands near 226 nm and broad negative bands around 203 nm, which was blue-shifted from the negative peak at 208 nm of hydrogel 5, while hydrogels 3 and 4 were characterized by much broader negative peaks at 208−213 nm and weak troughs at 240 nm, as well as isodichroic points at ∼230 nm. The decreased intensities of negative bands at 203-213 nm and small shifts of CD spectra of hydrogels 1-5 were due to the altered molecular conformations and packing modes of these glycosylated molecules after glycosylation modification, and the subsequent formation of differentially deformed β-sheet structures.3,27 Fluorescent staining with Thioflavin T (ThT), an amyloid-specific dye, further revealed that the nanofibers of 1, 2, 3, 4, and 5 exhibited the common features of amyloid fibrils, based on the dramatic increase of fluorescence emission of ThT, upon binding with fibrils (Figure S17).28 Fourier transform infrared (FTIR) spectroscopy analysis further confirmed the formation of distorted β-sheet structures by hydrogelators 1-4, in comparison with hydrogelator 5. For example, the FTIR spectrum of hydrogel 5 (Figure 2B) showed an amide I peak near 1631 cm−1, indicating the formation of typical β-sheet

3. RESULTS AND DISCUSSION In order to precisely control the molecular arrangement of glucose residues on peptide backbones, we employed a Fmocprotected glycoamino acid (e.g., Fmoc-Asn(Glc)-OH) and solid phase synthesis to prepare gelators 1−5 (Scheme 2 and Figures S1−S14).25 Gelator 5 was used as a reference molecule to compare the effect of glucose modification on the self-assembling properties of other glycopeptides. Gelation testes indicated that all these molecules can behave as efficient hydrogelators to form supramolecular self-assemblies in water at different pHs and concentrations (Figure 1 and Figure S15). Hydrogelators 1 and 2 containing single glucose modification on different sites of their molecules, exhibited good solubilities in water (pH = 7.4) and tended to form transparent hydrogels upon the change of pH to 5.5, with minimal gelation concentrations (MGC) at 0.2 and 0.3 wt %, respectively (Table 1). In comparison, hydrogelator 5, without glucose modification, formed a semitransparent hydrogel at the concentration of 1.0 wt % and pH 4.5 (Figure S15). However, compounds 3 and 4 which contained two or three glucose groups on their molecular structures, showed decreased solubilities in water at neutral pH (pH = 7.4). Their low solubilies Table 1. Summary of the Conditions and Properties of Supramolecular Hydrogels 1−5 sample

1

2

3

4

5

MGC (wt %) pH width of fibers (nm) critical strain (%) G′ (kPa) biostabilitya Tgel−sol (°C)

0.2 5.5 13 ± 1 0.83 4.7 48.5 −b

0.3 5.5 14 ± 2 0.63 16.0 29.4 96

0.3 7.0 12 ± 2 0.17 13.8 52.0 −b

1.0 7.4 13 ± 3 1.46 0.086 53.9 67

1.0 4.5 22 ± 3 0.35 11.5 0 53

a

Compound remained (%) after 24 h. bThe Tgel−sol of hydrogels were more than 100 °C. 6920

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Figure 2. (A) CD spectra, (B) FTIR spectra, (C) frequency sweep profiles of the dynamic storage moduli (G′) and the loss moduli (G″) of hydrogels 1− 5 with concentration at 1.0 wt %, and (D) time-dependent digestion of hydrogelators 1−5 by proteinase K.

Figure 3. (A−E) Optimized structures of five-molecule models of 1, 2, 3, 4, and 5 (C, gray; H, white; N, blue; O, red), in which the green dashed lines indicated the well-developed hydrogen bonding networks of amino acids, pink dashed lines showed the intermolecular hydrogen bonding between glucoses and amino acids, and the orange dashed lines signified the extended intermolecular hydrogen bonding between glucoses and amino acids. (F) The packing model of hydrogelator 1 in a nanofibrous structure, which were stabilized by the extended π−π stacking of aromatic naphthalyl and phenyl rings, the extended hydrogen bonding between amino acids and the intermolecular hydrogen bonding between glucoses and amino acids.

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DOI: 10.1021/acsami.6b00850 ACS Appl. Mater. Interfaces 2016, 8, 6917−6924

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Figure 4. Fluorescence images of live/dead assays of (A) NIH/3T3 fibroblast cells and (B) human umbilical vein endothelial cells (HUVEC) cultured on hydrogels 1 and 2 over the course of 5 days. Cell spreading of (C) NIH/3T3 cells and (D) HUVECs on hydrogel 2. Actin filaments were stained with fluorescein-phalloidin and the nuclei were with DAPI. Cell densities of (E) NIH/3T3 cells and (F) HUVECs determined by cell counting with a hemocytometer over the course of a 5-day culture on hydrogels 1 and 2.

structure, which was shifted to 1629 and 1626 cm−1, respectively, for hydrogels 1 and 2.29,30 On the other hand, hydrogels 3 and 4 exhibited two amide I bands near both 1631 and 1675 cm−1, suggesting the coexistence of β-sheet and turn structures.31−33 On the basis of these studies, we can confirm that glucose modifications can alter the molecular conformations of hydrogelators in supramolecular assemblies, but these hydrogelators still preferred to form β structures with extended hydrogen bonding interactions for supramolecular self-assembling. The viscoelastic properties of hydrogels 1−5 were examined by dynamic rheology experiments at the same concentration of 1.0 wt %. Figure 2C showed frequency sweep profiles of the storage moduli (G′) and the loss moduli (G″) of all hydrogels at 25 °C. Their storage moduli (G′) were much higher than their loss moduli (G″) within the investigated oscillating frequency limit (0.1−200 rad/s), indicating the dominantly elastic properties of hydrogels 1−5 (Figure 2C, Figure S18, and Table 1). Among them, hydrogel 2 exhibited the highest storage modulus of 16.0 KPa, and hydrogels 1, 3, and 5 possessed relatively high storage moduli of 4.7, 13.8, and 11.5 kPa, respectively (Table 1). In comparison, hydrogel 4 showed the lowest storage modulus of 86 Pa, suggesting its weak mechanical strength under this condition. Since thermostability and mechanical biostability were two essential requisites of a supramolecular hydrogel for biomedical applications, we evaluated the thermostabilities of all hydrogels by measuring their gel−sol transition temperatures (Tgel−sol). As summarized in Table 1, hydrogel 5 underwent a gel−sol transition above 53 °C. In comparison, hydrogels 2 and 4 showed enhanced thermostabilities with Tgel−sol at 96 and 67 °C, respectively. More importantly, much higher thermostabilities were observed in hydrogels 1 and 3, with Tgel−sol more than 100 °C. These studies indicated that glucose modification held high potentials to enhance the thermostabilities of peptide hydrogels due to the alteration of molecular amphiphilicity for differential

supramolecular interactions.34 Besides, we also identified that glucose modification was also an effective means to increase the biostability of peptide residues by resisting proteolytic digestion.35,36 As shown in Figure 2D, a well resistance to proteinase K digestion was observed with hydrogelators 1−4, as evidenced by that fact that more than 48.5% of 1, 29.4% of 2, 52.0% of 3, and 53.9% of 4 remained after 24 h of incubation. In contrast, hydrogelator 5 without glucose modification was hydrolyzed completely in 4 h after being treated with proteinase K, indicating the low biostability of 5 toward protease digestion. These results confirmed that the increased biostabilities of glycopeptide hydrogelators 1−4 were due to the glucose modifications on peptide backbones, which protected the peptide groups from proteinase recognition. Also, the biostabilities of these glycopeptide hydrogelators showed relatively high dependence on the location and density of glucose residues on the peptide backbone. In order to elucidate the effect of glucose modification on the molecular association of glycopeptides, we generated 3D models of the complexes of hydrogelators 1−5 by using the quantumchemical Austin Model 1 (AM1)37 and DREIDING force field method.38 On the basis of the models shown in Figure 3, hydrogelator 5, without the presence of glucose unit, mainly relied on the extended π−π stacking between aromatic naphthalyl and phenyl rings, and the hydrogen bonding interactions between the oxygen atoms of amide groups of Nap and Phe(2) and the hydrogen atoms of amide groups of Asp(2) and Phe(2) for supramolecular self-assembling (Figure 3E). After glucose modification, additional hydrogen bonding interactions involving glucose, Asp, and Phe groups have been introduced, which intensified the supramolecular interactions of hydrogelators 1, 2, and 3 (Figure 3A−C). However, more complicated hydrogen bonding interactions were observed for hydrogelator 4, which involved the oxygen atoms from the amide group of Phe(1), the glucose residue in the side chain of Asp(2) 6922

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ACS Applied Materials & Interfaces and the C-terminal of peptide (e.g., end-capped glucose) with the hydrogen atoms from the glucoses on Asp(2), Asp(1), and endcapped glucose. And the glucose unit on the C-terminal of hydrogelator 4 (Figure 3D) may interfere the intermolecular interactions between the amide groups of glycopeptide and thus reduced its supramolecular assembling ability. The high water content and similar structural morphology and composition of these glycopeptide hydrogels to extracellular matrixes (ECM) in tissues,39,40 also stimulated us to explore the potentials of these hydrogels to work as biomimetic extracellular matrixes for cell culture. After confirming the excellent biocompatibilities of these hydrogelators (Figure S19), and the high stabilities of hydrogels 1 and 2 in cell culture media (Figure S20), we seeded NIH 3T3 and HUVEC cells on hydrogels 1 and 2, respectively, and examined their cell adhesion and proliferation behaviors over the course of a 5-day culture. The live−dead assays shown in Figure 4A,B revealed that two types of cells can attach to and spread well on both hydrogels after 1 day incubation, as exemplified by the appearance of a typical spindle or polyhedral-like morphology. Cell attachment and spreading of both types of cells on the surface of glycopeptide hydrogel at day 1 was further assessed by cytoskeletal F-actin staining by using fluorescein-phalloidin. As observed in Figure 4C,D, both types of cells showed the formation of highly elongated stress actin filaments (green) surrounding their nuclei (blue) inside cells, which can promote the extensive spreading of cells on hydrogel. Because of favorable interactions with hydrogels 1 and 2, these cells tended to form cell clusters on hydrogel 1 and 2 at day 3, and the cell densities increased gradually with time. At day 5, we observed the formation of confluent monolayers on hydrogels 1 and 2 from both types of cells. Cell density quantifications further confirmed the steady increase of cell densities of 3T3 (>7-fold) and HUVEC (>4.5-fold) on both hydrogels (Figure 4E,F). In comparison, both 3T3 HUVEC cultured on hydrogel 5 showed slower rates of proliferation and lower cell densities than those on hydrogel 1 and 2 over the course of a 5-day culture (Figure S21), which indicated that glucose clusters on the surface of nanofibers in hydrogels 1 and 2 can provide a biomimetic coating to regulated cell adhesion and growth via multivalent sugar−lectin and sugar−sugar interactions.41,42

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported financially by the National Natural Science Foundation of China (Grants 21374070 and 21344002) to L. Yuan and X. Li, the Natural Science Foundation of Jiangsu Province (Grant BK20151218) to X. Li, State Key Laboratory of Molecular Engineering of Polymers (Fudan University) to X. Li, the National Science Council of the Republic of China (Grant NSC 102-2113-M-009-006-MY2), the “Aim for the Top University” program of the National Chiao Tung University and Ministry of Education, Taiwan, R.O.C. to H.-C. Lin, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We thank the National Center for High-Performance Computing of Taiwan for computer time and facilities.

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REFERENCES

(1) Ritchie, G. E.; Moffatt, B. E.; Sim, R. B.; Morgan, B. P.; Dwek, R. A.; Rudd, P. M. Glycosylation and the Complement System. Chem. Rev. 2002, 102, 305−319. (2) Dwek, R. A. Glycobiology: Toward Understanding the Function of Sugars. Chem. Rev. 1996, 96, 683−720. (3) Bosques, C. J.; Tschampel, S. M.; Woods, R. J.; Imperiali, B. Effects of Glycosylation on Peptide Conformation: A Synergistic Experimental and Computational Study. J. Am. Chem. Soc. 2004, 126, 8421−8425. (4) Moremen, K. W.; Tiemeyer, M.; Nairn, A. V. Vertebrate Protein Glycosylation: Diversity, Synthesis and Function. Nat. Rev. Mol. Cell Biol. 2012, 13, 448−462. (5) Sears, P.; Wong, C. H. Carbohydrate Mimetics: A New Strategy for Tackling the Problem of Carbohydrate-Mediated Biological Recognition. Angew. Chem., Int. Ed. 1999, 38, 2300−2324. (6) Seitz, O. Glycopeptide Synthesis and the Effects of Glycosylation on Protein Structure and Activity. ChemBioChem 2000, 1, 214−246. (7) Seeberger, P. H.; Haase, W. C. Solid-Phase Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries. Chem. Rev. 2000, 100, 4349−4394. (8) Grogan, M. J.; Pratt, M. R.; Marcaurelle, L. A.; Bertozzi, C. R. Homogeneous Glycopeptides and Glycoproteins for Biological Investigation. Annu. Rev. Biochem. 2002, 71, 593−634. (9) Liu, Y. F.; Sun, Z. Y.; Chen, P. G.; Huang, Z. H.; Gao, Y.; Shi, L.; Zhao, Y. F.; Chen, Y. X.; Li, Y. M. Glycopeptide Nanoconjugates Based on Multilayer Self-Assembly as an Antitumor Vaccine. Bioconjugate Chem. 2015, 26, 1439−1442. (10) Lim, Y.-B.; Park, S.; Lee, E.; Ryu, J.-H.; Yoon, Y.-R.; Kim, T.-H.; Lee, M. Tunable Bacterial Agglutination and Motility Inhibition by SelfAssembled Glyco-Nanoribbons. Chem. - Asian J. 2007, 2, 1363−1369. (11) Ikeda, M.; Ueno, S.; Matsumoto, S.; Shimizu, Y.; Komatsu, H.; Kusumoto, K.-i.; Hamachi, I. Three-Dimensional Encapsulation of Live Cells by Using a Hybrid Matrix of Nanoparticles in a Supramolecular Hydrogel. Chem. - Eur. J. 2008, 14, 10808−10815. (12) Liao, S. W.; Yu, T.-B.; Guan, Z. De Novo Design of SaccharidePeptide Hydrogels as Synthetic Scaffolds for Tailored Cell Responses. J. Am. Chem. Soc. 2009, 131, 17638−17646.

4. CONCLUSIONS In summary, we introduced here a series of new supramolecular hydrogelators from glycopeptides which were prepared from site-specific glucose modification reactions in vitro on peptide residues. With the presence of glucose ligands, these hydrogelators and hydrogels exhibited improved biophysical and biofunctional properties, in comparison with their peptide analogue, such as increased thermostabilities and improved biostabilities. Besides, because of their excellent biocompatibilities and high stabilities in cell culture media, hydrogels 1 and 2 also exhibited great potentials to work as new biomimetic scaffolds to promote cell adhesion and growth. Therefore, we expected that peptide glycosylation could offer an alternative way for peptide modification and generation of novel supramolecular hydrogelators and hydrogels with improved biostability, thermostability, and cell adhesion properties for biomedical applications.

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Synthetic route for the preparation of Fmoc-Asn(Glc)OH, TEM and SEM images of hydrogel 5, cytotoxicity, and gel stabilities in culture media (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b00850. 6923

DOI: 10.1021/acsami.6b00850 ACS Appl. Mater. Interfaces 2016, 8, 6917−6924

Research Article

ACS Applied Materials & Interfaces (13) Wang, W.; Wang, H.; Ren, C.; Wang, J.; Tan, M.; Shen, J.; Yang, Z.; Wang, P. G.; Wang, L. A Saccharide-Based Supramolecular Hydrogel for Cell Culture. Carbohydr. Res. 2011, 346, 1013−1017. (14) Huang, J.; Bonduelle, C.; Thevenot, J.; Lecommandoux, S.; Heise, A. Biologically Active Polymersomes from Amphiphilic Glycopeptides. J. Am. Chem. Soc. 2012, 134, 119−122. (15) Du, X.; Zhou, J.; Guvench, O.; Sangiorgi, F. O.; Li, X.; Zhou, N.; Xu, B. Supramolecular Assemblies of a Conjugate of Nucleobase, Amino Acids, and Saccharide Act as Agonists for Proliferation of Embryonic Stem Cells and Development of Zygotes. Bioconjugate Chem. 2014, 25, 1031−1035. (16) Restuccia, A.; Tian, Y. F.; Collier, J. H.; Hudalla, G. A. SelfAssembled Glycopeptide Nanofibers as Modulators of Galectin-1 Bioactivity. Cell. Mol. Bioeng. 2015, 8, 471−487. (17) Roytman, R.; Adler-Abramovich, L.; Kumar, K. S. A.; Kuan, T.-C.; Lin, C.-C.; Gazit, E.; Brik, A. Exploring the Self-Assembly of Glycopeptides Using a Diphenylalanine Scaffold. Org. Biomol. Chem. 2011, 9, 5755−5761. (18) Birchall, L. S.; Roy, S.; Jayawarna, V.; Hughes, M.; Irvine, E.; Okorogheye, G. T.; Saudi, N.; De Santis, E.; Tuttle, T.; Edwards, A. A.; Ulijn, R. V. Exploiting CH-Pi Interactions in Supramolecular Hydrogels of Aromatic Carbohydrate Amphiphiles. Chem. Sci. 2011, 2, 1349−1355. (19) Ogawa, Y.; Yoshiyama, C.; Kitaoka, T. Helical Assembly of Azobenzene-Conjugated Carbohydrate Hydrogelators with Specific Affinity for Lectins. Langmuir 2012, 28, 4404−4412. (20) Pati, D.; Shaikh, A. Y.; Das, S.; Nareddy, P. K.; Swamy, M. J.; Hotha, S.; Sen Gupta, S. Controlled Synthesis of O-Glycopolypeptide Polymers and Their Molecular Recognition by Lectins. Biomacromolecules 2012, 13, 1287−1295. (21) Krannig, K.-S.; Schlaad, H. pH-Responsive Bioactive Glycopolypeptides with Enhanced Helicity and Solubility in Aqueous Solution. J. Am. Chem. Soc. 2012, 134, 18542−18545. (22) Kramer, J. R.; Deming, T. J. Glycopolypeptides with a RedoxTriggered Helix-to-Coil Transition. J. Am. Chem. Soc. 2012, 134, 4112− 4115. (23) Quadir, M. A.; Martin, M.; Hammond, P. T. Clickable Synthetic Polypeptides-Routes to New Highly Adaptive Biomaterials. Chem. Mater. 2014, 26, 461−476. (24) Mildner, R.; Menzel, H. Hydrophobic Spacers Enhance the Helicity and Lectin Binding of Synthetic, pH-Responsive Glycopolypeptides. Biomacromolecules 2014, 15, 4528−4533. (25) Zhang, Y.; Muthana, S. M.; Farnsworth, D.; Ludek, O.; Adams, K.; Barchi, J. J., Jr.; Gildersleeve, J. C. Enhanced Epimerization of Glycosylated Amino Acids During Solid-Phase Peptide Synthesis. J. Am. Chem. Soc. 2012, 134, 6316−6325. (26) Zhang, Y.; Yang, Z. M.; Yuan, F.; Gu, H. W.; Gao, P.; Xu, B. Molecular Recognition Remolds the Self-Assembly of Hydrogelators and Increases the Elasticity of the Hydrogel by 10(6)-fold. J. Am. Chem. Soc. 2004, 126, 15028−15029. (27) Andreotti, A. H.; Kahne, D. Effects of Glycosylation on Peptide Backbone Conformation. J. Am. Chem. Soc. 1993, 115 (8), 3352−3353. (28) Biancalana, M.; Koide, S. Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim. Biophys. Acta, Proteins Proteomics 2010, 1804, 1405−1412. (29) Xu, X.-D.; Liang, L.; Cheng, H.; Wang, X.-H.; Jiang, F.-G.; Zhuo, R.-X.; Zhang, X.-Z. Construction of Therapeutic Glycopeptide Hydrogel as a New Substitute for Antiproliferative Drugs to Inhibit Postoperative Scarring Formation. J. Mater. Chem. 2012, 22, 18164− 18171. (30) Hughes, M.; Debnath, S.; Knapp, C. W.; Ulijn, R. V. Antimicrobial Properties of Enzymatically Triggered Self-Assembling Aromatic Peptide Amphiphiles. Biomater. Sci. 2013, 1, 1138−1142. (31) Reches, M.; Gazit, E. Self-Assembly of Peptide Nanotubes and Amyloid-Like Structures by Charged-Termini-Capped Diphenylalanine Peptide Analogues. Isr. J. Chem. 2005, 45, 363−371. (32) Pashuck, E. T.; Stupp, S. I. Direct Observation of Morphological Tranformation from Twisted Ribbons into Helical Ribbons. J. Am. Chem. Soc. 2010, 132, 8819−8821.

(33) Segman-Magidovich, S.; Lee, M.-r.; Vaiser, V.; Struth, B.; Gellman, S. H.; Rapaport, H. Sheet-Like Assemblies of Charged Amphiphilic Alpha/Beta-Peptides at the Air-Water Interface. Chem. Eur. J. 2011, 17, 14857−14866. (34) Asensio, J. L.; Arda, A.; Canada, F. J.; Jimenez-Barbero, J. Carbohydrate-Aromatic Interactions. Acc. Chem. Res. 2013, 46, 946− 954. (35) Sola, R. J.; Griebenow, K. Effects of Glycosylation on the Stability of Protein Pharmaceuticals. J. Pharm. Sci. 2009, 98, 1223−1245. (36) Culyba, E. K.; Price, J. L.; Hanson, S. R.; Dhar, A.; Wong, C.-H.; Gruebele, M.; Powers, E. T.; Kelly, J. W. Protein Native-State Stabilization by Placing Aromatic Side Chains in N-Glycosylated Reverse Turns. Science 2011, 331, 571−575. (37) Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P. The Development and Use of Quantum-Mechanical Molecular-Models.76. AM1 - A New General-Purpose Quantum-Mechanical MolecularModel. J. Am. Chem. Soc. 1985, 107, 3902−3909. (38) Mayo, S. L.; Olafson, B. D.; Goddard, W. A. Dreding - A Generic Force-Field for Molecular Simulations. J. Phys. Chem. 1990, 94, 8897− 8909. (39) Lee, K. Y.; Mooney, D. J. Hydrogels for Tissue Engineering. Chem. Rev. 2001, 101, 1869−1879. (40) Slaughter, B. V.; Khurshid, S. S.; Fisher, O. Z.; Khademhosseini, A.; Peppas, N. A. Hydrogels in Regenerative Medicine. Adv. Mater. 2009, 21, 3307−3329. (41) Kaltner, H.; Stierstorfer, B. Animal lectins as cell adhesion molecules. Cells Tissues Organs 1998, 161 (1−4), 162−179. (42) Hardy, J. G.; Pfaff, A.; Leal-Egana, A.; Mueller, A. H. E.; Scheibel, T. R. Glycopolymer Functionalization of Engineered Spider Silk Protein-based Materials for Improved Cell Adhesion. Macromol. Biosci. 2014, 14, 936−942.

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DOI: 10.1021/acsami.6b00850 ACS Appl. Mater. Interfaces 2016, 8, 6917−6924