Glucosamine-Based Supramolecular Nanotubes for Human

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Functional Nanostructured Materials (including low-D carbon)

Glucosamine-based Supramolecular Nanotubes for Human Mesenchymal Cell Therapy Satish Talloj, Bill Cheng, Jen-Po Weng, and Hsin-Chieh Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03226 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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ACS Applied Materials & Interfaces

Glucosamine-based Supramolecular Nanotubes for Human Mesenchymal Cell Therapy Satish Kumar Talloj, Bill Cheng, Jen-Po Weng, and Hsin-Chieh Lin* Department of Materials Science and Engineering, National Chiao Tung University, Hsinchu, Taiwan, Republic of China KEYWORDS: Supramolecular chemistry; self-assembly; hydrogels; nanotubes; biocompatibility; wound healing ABSTRACT: Herein, we demonstrate an example of glucosamine-based supramolecular hydrogels that can be used for human mesenchymal cell therapy. We designed and synthesized a series of amino acid derivatives based on a strategy of capping D-glucosamine moiety at C-terminus and fluorinated benzyl group at N-terminus. From a systematic study on chemical structures, we discovered that the glucosamine-based supramolecular hydrogel (PFB-F-Glu) self-assembled with one dimensional nanotubular structures at physiological pH. The self-assembly of newly discovered PFB-F-Glu motif is attributed to synergistic effect of π-π stacking and extensive intermolecular hydrogen bonding network in aqueous medium. Notably, PFB-F-Glu nanotubes is proved to be non-toxic to human mesenchymal stem cells (hMSCs) and showed to enhance hMSCs proliferation while maintaining its pluripotency. Retaining of pluripotency capabilities provides potentially unlimited source of undifferentiated cells for the treatment of future cell therapies. Furthermore, hMSCs cultured on PFB-F-Glu are able to secrete paracrine factors that down-regulate pro-fibrotic gene expression in LPS-treated human skin fibroblasts which demonstrates that PFB-F-Glu nanotubes have the potential to be used for wound healing applications. Overall, this article addresses the importance of chemical design to generate supramolecular biomaterial for stem cell therapy.

INTRODUCTION Supramolecular assembly has received global attention because of their ability to create multifunctional materials through elegant control of molecular interactions and recognition in many fields of science and technology.1-4 It has been proven that the major driving forces behind such type of supramolecular assemblies are weak non-covalent interactions such as π-π, hydrogen bonding interactions, van der Waals forces and electrostatic effects.5,6 The formation of well-defined nano-sized architectures resulting from the molecular self-assembly has opened up demanding applications in chemistry, biology and materials science. Over the past few years, the development of supramolecular hydrogels with low-molecular-weight (LMW) hydrogelators4 has become an area of increasing interest which are made up of self-assembly of aromatic-capped small molecules into one-dimensional networks of nanofibers, nanobelts and nanotubes depending on molecular structures and their self-assembly conditions.7-10 Glucosamine is a part of the structure of polysaccharides chitosan and chitin, widely used for treating arthritic pain and stiffness in osteoarthritis patients,11,12 and serve as a motif in the design of a supramolecular hydrogelators.3,4 It is recently demonstrated that the incorporation of carbohydrate groups to aromatic peptides can enhance bioavailability of peptides and trigger the molecular and morphological properties.13 Peptides with glucosamine moiety at C-terminus can be used as substitute for anti-proliferative drugs to inhibit postoperative scarring formation,14 and antimicrobial activity.15 Similarly, Xu et. al have demonstrated that the attachment of a hydrophobic group to glucosamine (Nap-L-PheGlu) can enhance wound healing and prevents scar formation in a mouse model.16 The position of attachment of the glycosyl unit to the long chain peptides also plays a significant role in peptide– receptor interactions, pharmacological activity and with systematic modification improves cell adhesion17 and proliferation.18 Currently, stem cell researchers provide new advances in understanding and to treat a range of diseases and injuries such as

hemostats.19 Among them, self-assembled peptide (SAP) hydrogels is one of the novel technique for better wound dressing at a faster rate.20,21 It has been reported that SAPs are capable of recruiting mesenchymal stem cells (MSCs) in skin regeneration.22 Similarly, Galler and co-workers have demonstrated the use of SAP nanofibers as a scaffold for dental stem cells for dental tissue regeneration.23 Likewise, several SAP hydrogels are demonstrated for its use as scaffolds in regenerative medicines.24 Therefore, the above studies signify the ability of SAPs to enhance the properties of MSCs. However, the main drawback for their applications is relatively low stability of the SAP gels.25,26 MSCs are well-known to promote wound healing, but its cell survival rate is extremely low due to apoptosis after being implanted into injury site.27,28 Thus, the development of new supramolecular scaffolding biomaterials with relatively high gel stability and increased efficacy of MSCs were still remains challenging. In this study, we have reported that the hMSCs cultured on supramolecular nanotubes of PFB-F-Glu (Glu: D-glucosamine) show high cell viability, no inhibitory effects on proliferation and pluripotency, and are able to secrete paracrine factors that down-regulate profibrotic gene expression in LPS-treated human skin fibroblasts (Figure 1).

Figure 1. Illustration of the formation of supramolecular nanotubes and the research strategy for stem cell therapy (SA: self-assembly).

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ACS Applied Materials & Interfaces Phenylalanine

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might minimize the available functionality which can participate in self-assembly and hydrogelation.

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Figure 2. Chemical structures of the glucosamine-based amino acid derivatives 1-5 and the analogue of 1 without glucosamine moiety 6. (PFB: pentafluorobenzyl, TFB: trifluorobenzyl, MFB: monofluorobenzyl, B: benzyl groups, F: phenylalanine, G: glycine). Here, we synthesized a series of amino-acid derivatives (1-6) by liquid-phase peptide synthesis (Figure 2 and Scheme S1) and thoroughly characterized by 1H-NMR, 13C-NMR and high resolution mass spectroscopy. After confirming the structures by spectroscopic analysis, we examined the hydrogelation abilities of 1-6 by heating-cooling process. Typically, 5 mg of compound 1 was taken in a 4 mL dram vial and 1 mL of deionized water or PBS buffer solution was added to make a total concentration of 0.5wt%. It was then heated to 80oC until dissolution (approx. 2-5 min) and allowed to cool gradually to room temperature resulted to form a translucent hydrogel and the pH of the hydrogel found to be 7.4, the occurrence of gelation was confirmed by tube inversion method (Figure 3A). In contrast, compounds 2-6 fails to form hydrogels through heating-cooling process with concentration range of 0.5 to 1wt%. Particularly compounds 2 and 3 formed a viscous suspensions while compounds 4-6 resulted in a clear solutions at 0.5wt% (Figure S1B). From our previous study,10 compound 6 (PFB-F) is an efficient building block for hydrogelation at a pH of 5.0, while it only can form a hydrogel at 1wt% through sequential change in pH. In comparison of 1 with 6, the presence of the D-glucosamine at C-terminus of PFB-F revealed that the hydrogelation occurred at 0.5 wt% under physiological pH. This observation prompted us to further investigate the gelation behavior with varied number of fluorine atoms on end-capped benzyl moieties (2-4) of amino acid derivative and also the presence of glycine moiety 5 on pentafluorobenzyl moiety were studied systematically. It was known that the two stacked aromatic interactions take place via non-covalent interactions generally referred to π-stacking or quadrupole interactions in aqueous media.29-31 The stacked interactions of pentafluorophenyl and phenyl moieties of the side chains of phenylalanine may stabilized by quadrupole interactions because of the presence of hydrogen and fluorine atoms in the aromatic rings.10,29-31 Moreover, by minimizing the number of fluorine atoms on benzyl side chain can weaken the π-stacking interactions between fluorinated and non-fluorinated groups.32,33 Nonetheless, we observed some nanofibrous morphologies even in solution or suspension phase for compounds 2-4 (Figure S2). Therefore, we assume that the three basic chemical structural units of pentafluorobenzyl, phenylalanine and D-glucosamine were all needed to trigger the hydrogelation under physiological pH through heating-cooling process, suggesting hydrogelator 1

Figure 3. A) Optical image of hydrogel for compound 1 in DIwater. B) Negatively stained TEM image of hydrogel 1 (scale bar: 50 nm) inset: magnified images of the nanotubes. C) AFM image of hydrogel 1 (scale bar: 500 nm) insets: magnified image and cross sectional area for height distribution. D) SEM image for hydrogelator 1 (scale bar: 5 µm). Transmission electron microscope (TEM) was used to visualize the morphologies for all the compounds (1-6). Interestingly, the self-assembly of 1 revealed the formation of long and thin flexible cross-linked nanotube-like networks (Figure 3B). Here, we noticed that nanotube-like structures appeared as two light shells of parallel lines separated by a dark centre (Figure 3B, insets), which suggested to be a hollow tubular structure filled with uranyl acetate, in negative staining.34,35 These nanotube-like structures exhibited a uniform external diameter of approximately 10.9 ± 1.1 nm in width and that extended to several micrometres in length. Notably, the compounds 2-5 exhibited fibrous morphology (Figure S2) and compound 6 did not show any nanostructures by healing-cooling method at 0.5 wt%. Since compound 1 exhibited unique nanotube-like structures as revealed by TEM, we further investigated the nano-sized structures of compound 1 by atomic force microscopy (AFM) and scanning electron microscope (SEM). The AFM image in figure 3C showed the formation of self-assembled one-dimensional nanostructures with a mean height of 10.4 ± 0.7 nm, which was well resembled with the width observed by TEM.36,37 In consideration with the height and width based on AFM and TEM, it was evident that the formation of a hollow nanotubular structures occured in the hydrogel of 1. Moreover, we also captured the co-existence of helical and twisted ribbon-like structures (Insets, Figure 3C), albeit rare, suggested to be a final step of matured nanotube formation arising from the closure of twisted/helical ribbons structure.38 This was a direct evidence of structural transition from ribbon-like structures, twisting of ribbons into helical ribbons and finally to nanotubes. Further characterization of hydrogel 1 by scanning electron microscope (SEM) showed that these self-assembled nanostructures were entangled into dense three-dimensional nanostructured networks (Figure 3D). These nanostructures


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formed a consistent, porous three-dimensional network throughout the hydrogel matrix, which also proved that compound 1 forms long-range, instead of local, nanostructures.39 In addition compound 1 was an ambidextrous gelator which could form both hydrogel and an organogel. The organogels formed by this material in ethanol or in acetonitrile exhibited only nanofibrils (Figure S3), indicating glycosylated peptide molecule assembled into nanotubular structure only in aqueous media. The effect of ethanol and water as co-solvent mixtures on hydrogelation and molecular self-assembly were also investigated simultaneously. Interestingly, structural transition from nanotubes to coiled fibres and finally to nanofibers were observed when solvent switched from water to ethanol at different ratios from 8:2 to 1:9 (Figure 4 and Figure S4). The aim of this co-solvent study was to determine the significance of aqueous system in self-assembly. In a single solvent system containing water it showed tubular assembly while in ethanol, nanofibrils were observed. Once the hydrogelator 1 mixed with water and ethanol at various ratios with fixed concentration of 0.5 wt%, we noticed the formation of coiled or twisted structures. Thus we concluded that, the role of aqueous solvent in hydrogelation was significant in the self-assembling of the tubular network.

indicated the disruption of inter/intra-molecular hydrogen bonding.40 FTIR spectrum of compound 1 also showed the presence of intermolecular hydrogen bonding.41 In solution phase, the carbonyl stretching vibration for amides displays at 1646 cm-1, upon self-assembly in aqueous solvent the stretching absorption band for carbonyl amide shifted towards lower wavenumber at 1638 cm-1 (Figure 5C), also the stretching vibration of the glycosyl ring appeared at 1013 cm-1 in solution and shifted to 1003 cm-1, respectively, after gelation (Figure S6B). This significant shifting of peaks towards lower wavenumber in gel phase suggested the existence of intermolecular hydrogen bonding.42 Wide-angle X-ray scattering (WAXS) experiment was performed to understand the molecular packing of compound 1. The freeze dried hydrogel (xerogels) provided information regarding the molecular arrangement after self-assembly (Figure S9). The intense peak at around 2θ =19.9° with d=4.44Å may be attributed to the spacing of hydrogen bonding networks and a broad signal at 2θ=26.5° with d=3.50Å corresponding to aromatic π-π interactions.43 Thioflavin T (ThT) test was used to investigate the β-sheet type of structure. ThT was a benzothiazine dye specifically used for detection of amyloid fiber formation both in vivo and in vitro.44 ThT showed a significant florescence enhancement when aggregated to β-sheet amyloid fibrils at 484 nm, whereas non aggregated ThT showed a relatively low fluorescence emission at this wavelength.45,46 From Figure 5D, the emission intensities of thioflvain T (20 µM) for compound 1 increased linearly with increasing concentrations (500-5000 µM).

Figure 4. TEM images of 1 in water: ethanol co-solvent system with the ratios from 8:2 to 1:9 at 0.5 wt% for compound 1 (Scale bar = 50 nm); Gels: A) 8:2 B) 7:3 C) 6:4 D) 5:5 (1:1) and in solutions: E) 4:6 F) 3:7. To get a deeper insight on molecular interactions in aqueous media for compound 1 in solution and hydrogel state, we employed fluorescence spectroscopy, FTIR, variable temperature NMR techniques. The emission maximum (λmax) for 1 in solution phase (250 µM) appeared at 304 nm and the λmax in its gel phase exhibited a remarkable bathochromic shift to 365 nm (Figure 5A). This red shift in the emission spectra can be attributed to molecular aggregation through π-π stacking of aromatic rings in the gel state.10 Although compounds 2-4 could not form gel at 0.5wt%, we collected the emission spectra of suspension/solution of 2-4 at 0.5wt%. It was observed that for compounds 3 and 4 since one or no fluorine atoms attached to benzyl group, no red shift in the emission spectra was observed, however for compound 2, a partial aggregation of aromatic π-stacking was observed (Figure S5), due to which it resulted as a viscous suspension at 0.5wt%. To gain further insight of the self-assembly in 1, we performed temperature-dependent 1H-NMR in DMSO-d6. It was noticed that the amide -NH signals at 8.4 and 7.9 ppm were shifted to 8.1 and 7.5 ppm, respectively, after raising the temperature from 298 K to 338 K (Figure 5B). Similarly, the hydroxyl signals (-OH) of glucosamine residue at 6.5, 4.9, and 4.7 ppm were also shifted to up field region. These observations

Figure 5. A) Normalized emission spectra of 1 in solution (blue) and gel state (red), B) Variable temperature 1H-NMR spectra of 1 from 25oC to 65oC in DMSO-d6 solvent. (Red arrow: amide (– NH) signals, blue arrow: shifting of Hydroxyl (–OH) signals), C) FTIR spectra of 1 in solution and in gel phase, D) Emission intensities of Thioflavin T (20 µM, λex = 440 nm) at various concentrations for compound 1. E) Bright field image and F) Polarized optical microscopic image of hydrogel 1 at 0.5 wt% stained with Congo Red solution (scale bar = 100 nm).


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However, for compound 6, very slight increase in emission intensities in water was observed (Figure S8). This result emphasizes the importance of glucosamine for the formation of well-developed secondary structure. We also employed Congo red staining to further confirm the existence of β-sheet structure in the assemblies of the gel 1. In Figure 5F, an apple green birefringence was observed under polarized optical microscope upon staining with congo red solution (Figure 5E represents bright field), which suggested the characteristic of β-sheet structure.47

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the gel-to-sol transition temperature (Tgel-sol) of hydrogel 1 was 59oC which is higher than human body temperature (37oC) thus is beneficial for use in tissue engineering applications.49 We further studied the bio-stability of the hydrogel 1 with proteinase K, a highly active enzyme that could hydrolyse the peptidic bonds.50 After 24 hours of incubation at 37oC with proteinase K gel 1 showed 85% of the compound remained (Figure S10B). This study demonstrated that gel 1 had high resistance towards protease digestion, which is beneficial for its use as biomaterial.3,4,17,51

Figure 7. (A) Cell numbers and (B) viability of 3A6-RFP cultured onto different surfaces were examined over 7 days (n=3). *, P < 0.05; **, P < 0.01; PFB-F-Glu (1) vs PFB-F (6), n.s., not significant.

Figure 6. 3A6-RFP cells were seeded onto normal tissue culture plate (TCP), D-Glucosamine (Glu), PFB-F (6) and PFB-F-Glu (1) coated surfaces for 24 hours. Cell morphology of each sample was examined the next day under 10X (scale bar; 200 nm) and 40X (scale bar; 50 nm) magnification. To explore the gel 1 as a potential candidate biomaterial for use in regenerative medicine, gel stiffness and stability were studied. The rheological property of a hydrogel is one of the characteristic features to judge a gel material for biomedical applications.48 The hydrogel 1 exhibited relatively high storage modulus (G′=8.7×103 Pa) compared to its loss modulus (G′′=2.6×103 Pa), indicating gel 1 was elastic material (Figure S10A). Notably, the G′ of 1 is sufficient to support the mass of a cell (G′>100 Pa).10 Importantly,

To further investigate the potential application of hydrogel PFBF-Glu (1) in regenerative medicine, we exposed 3A6-RFP, which is a hMSC cell line, that has been engineered to express intracellular red fluorescent proteins (RFP) constitutively,52 to different surface coated materials for 24 hours and examined the cell morphology in each group the following day (Figure 6). 3A6RFP After 24 hours of exposing the materials to the cells, 3A6RFP displayed elongated structures when exposed to either simple glucosamine hydrochloride solution (Glu) or PFB-F (6). In comparison, those 3A6-RFP seeded onto PFB-F-Glu at 0.5wt% had similar morphology as those seeded onto tissue culture plate (TCP). Thus, the data indicated that either the Glu or PFB-F component in PFB-F-Glu did not affect the morphology of 3A6RFP. To further characterize the biocompatibility of PFB-F-Glu, cell number and viability of 3A6-RFP were investigated over 7 days of cell culture. (Figure 7A). Compared to the 3A6-RFP on TCP, those that were seeded onto Glu-coated or PFB-F-coated surfaces had lower cell counts over the 7 days period. In contrast, the cell count for 3A6-RFP cultured on PFB-F-Glu, were similar to those cultured on TCP. Moreover, there was a significant difference in count between PFB-F-Glu and PFB-F group, indicating PFB-F alone could inhibit cell proliferation. To ensure the lower cell count seen in the Glu and PFB-F groups were not due to cell toxicity, therefore, cell viability in each group was analysed by haemocytometer. Briefly, 3A6 cells were exposed to compounds PFB-F-Glu and PFB-F at their gelation concentrations and simple glucosamine hydrochloride solution at 0.5wt% concentration to examine the cell viability in each case after being incubated for 7 days of cell culture. Compared to the TCP group on each day, there was no significant difference in the cell viability of other groups (Figure 7B). Thus, the data indicates PFB-F could inhibit hMSC proliferation, whereas PFB-F-Glu had no effect on cell proliferation. Moreover, it was likely that there was a relationship between the low proliferation rate with the morphology changes seen in either Glu or PFB-F group. Previous studies have demonstrated that there is a correlation between cell morphology and expression level of pluripotent markers in hMSCs when they undergoes differentiation.53 Thus, the changes seen in 3A6-RFP cell morphology when being seeded onto PFB-F


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but not when seeded onto PFB-F-Glu, were investigated for the three pluripotency markers (NANOG, OCT4, and SOX2) expression levels (Figure 8A). Compared to the 3A6-RFP seeded onto TCP, there was a significant reduction in all three pluripotent markers in those that were exposed to either Glu or PFB-F over the 7 days of culture. Conversely, the pluripotency of 3A6-RFP that exposed to PFB-F-Glu over the 7 days period did not showed any significant changes, except for SOX2 which started to decreases from day 5 onwards. Since the reduction in the expression level of pluripotent markers indicated the cells have the capability to differentiate into another cell type, so we then asked the question whether cells seeded onto PFB-F or PFB-FGlu could differentiate into another cell type (Figure 8B). Four early specific lineage markers were examined, including SOX9 (chondrogenic),54 FAPB4 (adipogenic),55 Nestin (neural),56 and ALP (oestrogenic).57 Since D-glucosamine is known to be a strong inducer of chondrogenic differentiation of hMSCs,58,59 the exposure of 3A6-RFP resulted in significant expression of SOX9 genes. In contrast either PFB-F or PFB-F-Glu did not resulted in significant expression of SOX9 or any other specific lineage markers. It was likely that PFB-F alone could not direct differentiation of hMSCs into a specific lineage in the absence of a well-defined differentiation media.

Figure 8. The effect of coated materials on 3A6-RFP pluripotency was examined by analysing (A) Pluripotent markers expressions (NANOG, OCT4 and SOX2) over 7 days of cell culture. (B) Expression levels of different early lineage differentiation markers at day 7 of culturing SOX9, FAPB4, Nestin and ALP. (C) Expression level of anti-fibrotic genes in the different cell samples were examined (n=3). Cultured media from each of 3A6-RFP samples were transferred to WS1 cell cultures that were pre-exposed to 200 ng/mL LPS for 24 hours. (D) The expression levels of profibrotic genes in WS1 were examined after exposing to different LPS treated 3A6-RFP conditioned media (n=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

Since PFB-F-Glu was demonstrated to have great biocompatibility with 3A6-RFP, in which both the cell pluripotency and cell viability were not affected, it was decided to investigate its potential application in hMSC therapy. MSCs have been showed to promote wound healing by down regulating skin fibrosis,60,61 the expressions of anti-fibrotic genes in 3A6-RFP were examined after the cells were cultured onto PFB-F-Glu for 24 hours followed by another 24 hours exposure to LPS (Figure 8C). The expressions of both TGFβ3 and COX2 are known to be up-regulated in MSC after exposure to LPS which mimics the inflamed microenvironment in a tissue injury site.62-64 Compared to the TCP control, the gene expression of either TGFβ3 or COX2 was significantly lower in the 3A6-RFP cultured on Glu or PFBF, suggesting either material could affect hMSC activity in addition to the down regulation in pluripotent marker expressions. In contrast, the 3A6-RFP cultured on PFB-F-Glu had similar expression levels of TGFβ3 and COX2 as the TCP control, indicating PFB-F-Glu do not have inhibitory effect on the expression of anti-fibrotic genes in MSCs. The two anti-fibrotic genes, TGFβ3 and COX2, would later get translated into proteins, and that the TGFβ3 and the downstream product of COX2, PGE2 would be secreted into the culture media.65-67 To examine whether these secreted compounds had any therapeutic activity, an in vitro anti-fibrotic assay was set up using human skin fibroblast cell line, WS1 (Figure 8D).68 WS1 cells were first pre-treated with 200 ng/mL of LPS for 24 hours, in which the media was then removed and treated with 3A6-conditioned media from each sample in Figure 8C. Compared to the untreated WS1, the LPS-treated WS1 had strong expression of two fibrotic genes,69 COLA1 and CTGF. After the LPS pre-treated WS1 were treated with the conditioned media from LPS-treated 3A6-RFP cultured on PFB-F-Glu. Although down-regulation in two fibrotic genes was also seen in the WS1 treated with the conditioned media from either LPStreated 3A6-RFP cultured on Glu or PFB-F, the level of down regulation, however it is not as good significant as the conditioned media taken from LPS-treated 3A6 cultured on PFB-F-Glu.

CONCLUSIONS In summary, we demonstrated an example of the formation of Dglucosamine-based supramolecular nanotubular networks in a hydrogel. These nanotubes could be utilized for various applications in peptide based biomaterials. The formation of one dimensional nanotubular networks was confirmed by TEM and supported by AFM and SEM analytical techniques. Temperaturedependent 1H-NMR, fluorescence spectroscopy, FTIR, Thioflavin T (ThT) test and congo red staining techniques provided the useful information on intermolecular interactions and molecular packing in the assemblies, which suggested the formation of nanotubes was attributed to synergistic effect of π-π stacking and intermolecular hydrogen bonding interactions. Interestingly, the PFB-F-Glu gel showed great biocompatibility with hMSCs in which the cell pluripotency and viability were not affected by this material after cell culturing. Using a D-glucosamine-based supramolecular hydrogel, hMSCs have been showed to secrete paracrine factors that down-regulate pro-fibrotic gene expression in LPS-treated human skin fibroblasts. Overall, this work signifies the importance of chemical design in the formation of unique nano-sized structures and could be a future candidate biomaterial for use in tissue engineering and in stem cell therapy.



Materials and Methods: Pentafluorophenyl acetic acid purchased from Oakwood chemicals, 2,4,6 trifluorophenylaceticacid from Matrix scientific, 4-fluorophenylaceticacid, N-hydroxysuccinimide, Diisopropylcarbodiimide, 4-Dimethylaminopyridine (DMAP) from Alfa-aesar, Phenylaceticacid and D-Glucosamine hydrochloride from Acros-Organics, L-Phenylalanine from Carbosynth limited (UK), Thioflavin T (ThT) from Sigma aldtich, Proteinase K from dongshengbio Cat# N9016 . Anhydrous grade solvents were purchased from Sigma Aldrich and used as received. Instruments Used: All 1H-NMR and 13C-NMR spectra were characterized using a 300 MHz Varian Unity Inova spectrophotometer with deuterated dimethylsulfoxide (DMSO-d6) at room temperature. Mass spectra were recorded using a Q-TOF Micro MS spectrometer. FT-IR spectra were measured using a Perkin Elmer spectrum 100 series spectrometer, and were collected at a resolution of 4 cm-1 using a detector by averaging scans (20 scans). UV-Vis absorption and circular dichroic spectra (CD) were measured with a spectropolarimeter (JASCO J-815, cell length 1 mm, 25 °C) from 190 to 450 nm and the concentration of peptides for UV and CD measured at gelation concentration. C18 Reverse phase HPLC (waters 1525) with UV detector (2489) were used for purification. Fluorescence emission spectra were obtained using a Hitachi F4500 fluorescence spectrometer at an excitation wavelength of λ = 260 nm with the concentrations of 250, 500, 1000, 5000, 7500, 15000 µM, and also at their gelation concentration. Transmission Electron Microscopy (TEM): The images were obtained using a Hitachi HT7700 transmission electron microscope at an accelerating voltage of 100 kV. Hydrogels were applied directly onto 200 mesh carbon-coated copper grids. An excess amount of the hydrogel was carefully removed by capillary action (filter paper), and the grids were then immediately stained with uranyl acetate for 30 seconds. Excess stain was removed by capillary action, and the grids were allowed to air dry for 48 hours. Atomic Force Microscope (AFM): 2 µL of the 0.5wt% hydrogel was applied onto 200 mesh carboncoated copper grids and rinsed with DI-water and allowed to air dried for 48 hours. The copper grid was sticked onto freshly cleaved silicon wafer and the images were obtained by tapping mode TISCO atomic force scanning probe microscope. Scanning electron microscopy (SEM): Samples were visualized with a JEOL JSM-6700F scanning electron microscope at an accelerating voltage of 15 kV and a working distance of 6.3 mm. Hydrogels were applied onto silicon wafer and freeze died for 48 h to result a white fluffy powder. A thin layer of platinum was coated onto the samples by plasma sputtering before SEM analysis. Wide-Angle X-Ray Scattering (WAXS) analysis: The freeze dried hydrogel (xerogel) at a concentration of 0.5wt% were analyzed by D8 ADVANCE Bruker AXS GmbH. The X-ray measurement source is Cu K-α radiation with an average wavelength of 1.5406 Å. Each pattern was scattered over a scattering angle (2θ) range from 5o to 30o.

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Rheological tests: Rheological tests were conducted on Anton Paar rheometer. 25 mm parallel plate was used during the experiment. 200 µL of hydrogel sample was placed on the parallel plate. Angular frequency sweep test: Test range (0.1 to 100 rads-1 frequency, strain = 0.8 %), 13 points per decade. Sweep mode is “log” and temperature maintaining at 25oC. Inverted Tube Method: For glucosamine related compounds 1 mg of compounds in a 2 mL vial (diameter 10 mm) with a screw cap and the addition of 0.20 mL water. The vial was heated until the compound dissolved, at which time the vial was cooled to room temperature. Gelation is considered to have occurred when the material is obtained that does not exhibit gravitational flow (confirmed by inverted test tube method for 5 min). Thioflavin T (ThT) test: Different concentrations of samples (500-5000 µM) were prepared in water. Then freshly prepared ThT (20 µM) were added and aged for 24 hours. Then measured using Fluorescence emission spectra using a Hitachi F-4500 fluorescence spectrometer at an excitation wavelength of λ = 440 nm, with slits 5 nm. The ThT emission intensities were detected at 484 nm. Congo Red Staining: To a freshly prepared saturated sodium chloride ethanol solution (80%) was added Congo Red stain and the resulting solution was vortexed and filtered. Congo Red solution was pipetted onto a glass microscope slide, and then the 0.5 wt% hydrogel was placed underneath the surface of the congo red solution and stained for approximately 1-2 min. After the excess Congo Red solution was removed by blotting, images of the sample placed between crossed polarizers were obtained with ZEISS microscope. Proteinase K tests: 1.5 mg of each compound was dissolved under heating in 3 mL PBS buffer at pH=7.3. Subsequently, proteinase K were added in the concentration of 5 units/mL and incubated at 37oC for 24 h. 100 µL of sample were taken out each time (2h, 4h, 6h, 8h, 12h, and 24h) and analyzed by reverse phase C18 HPLC column (UV detector at λ = 220 and 254 nm wavelength (retention time: 15-16 minutes). Cells: Human mesenchymal stem cell line (3A6-RFP) was a gift from Dr. Shih-Chieh Hung (China Medical University, Taiwan), and was cultured in Dulbecco’s modified Eagle’s medium (DMEM) low glucose containing 10% fetal calf serum. Human WS1 skin fibroblast (CRL-1502, ATCC) were cultured in Minimum Essential Medium, (Cat# 30-2033, ATCC) with the addition of fetal bovine serum to a final concentration of 10%. Quantitative PCR (qPCR) Analysis: Total RNA was isolated from cells using TRI Reagent (1 mL/~1 × 107 cells). Subsequently, 1 µg of RNA was transcribed into cDNA using random primer mixer (ProtoScript M-MuLV First Strand cDNA synthesis kit, New England BioLabs) and amplified during 40 cycles by PCR utilizing specific primers (Table S1). For quantitative real-time PCR (qPCR), 1 µL cDNA was mixed with 0.5 µL forward and reverse primers (10 µM each) and 10 µL


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Power SYBR Green PCR Master Mix (Thermo Scientific Fisher) followed by adding RNAase free water to make up to 20 µL per sample. The β-actin (ACTB) expression was used as an endogenous control and to normalize the PCR results. Preparation of 3A6-RFP conditioned media: A 24-well plate was pre-coated with glucosamine (Cat# G4875, Sigma-Aldrich), PFB-F or PFB-F-glucosamine at the designated wells, in which the plate was kept at 4oC until further use. 1×105 cells/mL of 3A6-RFP were seeded to each well, and the whole plate was incubated at 37oC for one overnight. Subsequently, the culture media in each well were replaced with serum-free media containing 200 ng/mL of lipopolysaccharide (LPS, Cat# L4391, Sigma Aldrich), and the whole plate was kept in 37oC for another overnight. The media in each well collected and stored at -80oC, and the cells were harvested for qPCR analysis. Anti-fibrotic assay: A 24-well plate was seeded with 1×105 cells/mL of human WS1 skin fibroblast in each well, and was incubated at 37oC for one overnight. Next day, the culture media in each well were replaced with serum-free media containing 200 ng/mL LPS (Cat# L4391, Sigma Aldrich) and the whole plate was incubated at 37oC for another overnight. The LPS effects were then neutralized by replacing the serum-free media with the 3A6-RFP conditioned media. After one night of incubation at 37oC, the media were discarded and cells were harvested for qPCR analysis.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Synthetic route, experimental protocols, optical images of hydrogels, TEM images, WAXS, PL Spectra, FTIR spectra, ThT data, rheological results and Proteinase K tests.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT The study was supported by the Ministry of Science and Technology of the Republic of China, Taiwan (grant MOST 106-2113-M009-010-; MOST 106-2622-E-009-014-CC3; MOST 106-3114-B039-002-).

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Glucosamine-Based Supramolecular Nanotubes for Human

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