Thermoresponsive Hydrogel of Diblock Methylcellulose: Formation of

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Thermoresponsive Hydrogel of Diblock Methylcellulose: Formation of Ribbonlike Supramolecular Nanostructures by Self-Assembly Atsushi Nakagawa,† Frank Steiniger,‡ Walter Richter,‡ Andreas Koschella,§ Thomas Heinze,*,§ and Hiroshi Kamitakahara*,† †

Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan Electron Microscopy Center, Jena University Hospital, Ziegelmühlenweg 1, D-07740 Jena, Germany § Institute for Organic Chemistry and Macromolecular Chemistry, Centre of Excellence for Polysaccharide Research, Friedrich Schiller University of Jena, Humboldtstrasse 10, D-07743 Jena, Germany ‡

S Supporting Information *

ABSTRACT: This article provides detailed insight into the thermoresponsive gelation mechanism of industrially produced methylcellulose (MC), highlighting the importance of diblock structure with a hydrophobic sequence of 2,3,6-tri-O-methylglucopyranosyl units for this physicochemical property. We show herein, for the first time, that well-defined diblock MC self-assembles thermoresponsively into ribbonlike nanostructures in water. A cryogenic transmission electron microscopy (cryo-TEM) technique was used to detect the ribbonlike nanostructures formed by the diblock copolymers consisting of hydrophilic glucosyl or cellobiosyl and hydrophobic 2,3,6-tri-O-methyl-cellulosyl blocks, methyl β-D-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-celluloside 1 (G-236MC, DPn = 10.7, DS = 2.65), and methyl β-D-glucopyranosyl(1→4)-β-D-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-celluloside 2 (GG-236MC, DPn = 28.2, DS = 2.75). Rheological measurements revealed that the gel strength of a dispersion of GG-236MC (2, 2.0 wt %) in water at 70 °C was 3.0 times stronger than that of commercial MC SM-8000, although the molecular weight of GG-236MC (2) having Mw = 8 × 103 g/mol was 50 times smaller than that of SM-8000 having Mw = 4 × 105 g/mol. Cryo-TEM observation suggested that the hydrogel formation of the diblock copolymers could be attributed to the entanglement of ribbonlike nanostructures self-assembled by the diblock copolymers in water. The cryo-TEM micrograph of GG-236MC (2) at 5 °C showed rectangularly shaped nanostructures having a thickness from 11 to 24 nm, although G-236MC (1) at 20 °C showed no distinct self-assembled nanostructures. The ribbonlike nanostructures of GG-236MC (2) having a length ranging from 91 to 864 nm and a thickness from 8.5 to 27.1 nm were detected above 20 °C. Small-angle X-ray scattering measurements suggested that the ribbonlike nanostructures of GG236MC (2) consisted of a bilayer structure with a width of ca. 40 nm. It was likely that GG-236MC (2) molecules were oriented perpendicularly to the long axis of the ribbonlike nanostructure. In addition, wide-angle X-ray scattering measurements revealed that GG-236MC (2) in its hydrogel formed the same crystalline regions as 2,3,6-tri-O-methylcellulose. The influence of the DP of diblock MC with a DS of around 2.7 on the gelation behavior will be discussed.

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INTRODUCTION

temperature. Aqueous solutions of industrially produced MCs with a DS of 1.4−2.0 undergo a sol−gel transition and typically form a physical hydrogel on heating to above approximately 60 °C.12 In contrast, MCs with high DSs are soluble only in cold water.13,14 There have been patent reports on MCs having a low molecular weight (3000−10 000 g/mol) and a high DS (1.8−2.7) as possible soil release agents in detergent compositions.15,16 The gelation is said to be due to the dehydration of hydrated methyl groups of MC in water, and the aggregation through hydrophobic interactions between densely methylated regions causes the formation of cross-linking points and subsequent 3D networks in which water molecules were entrapped, namely,

Hydrogels are 3D networks of water-soluble polymers formed in water through either chemical or physical cross-linking1 and are the most attractive soft materials used in many areas such as the biomedical, food, and pharmaceutical industries.2 Many hydrogels undergo a sol−gel transition in response to external stimuli such as the temperature,3,4 pH,5 and coexisting solutes.6 Methylcellulose (MC) is one of the most extensively investigated thermoresponsive polymers7−10 not only because MC is important for many practical applications but also because MC is the simplest cellulose ether derivative for the investigation of structure−property relationships. Industrially produced MCs with a degree of substitution (DS) of ca.1.8 are easy to apply to building materials, cosmetics, thickeners, coating materials, tissue engineering scaffolds, and ceramic materials10,11 because they are soluble in water at room © 2012 American Chemical Society

Received: January 6, 2012 Published: August 1, 2012 12609

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the formation of hydrogels.17 In general, self-assembled fibers and their network, constructed through multiple weak interactions such as van der Waals interactions, hydrogen bonding, and dipole−dipole interactions, are indispensable to physical hydrogel formation.18 The physical gelation process is complex because of the transient and reversible nature of the network formation and stabilization. 2,3 The cryo-TEM technique allows us to know the micro- and nanostructures in water most exactly because for cryo-TEM sample preparation a thin aqueous film of the sample on the TEM grid is rapidly frozen in liquid ethane in order to prevent the formation of ice crystals. In fact, Bodvik et al. have reported that in cryo-TEM micrographs of industrially produced MCs a network of fibrous structures was observed in MC solutions above 45 °C.19 However, it was still unknown how the chemical structure of MCs played a critical role in the formation of fiber structures and subsequent 3D networks because the structure− property relationship of industrially produced MCs with heterogeneous functionalization patterns was complicated. We have recently reported the syntheses and physical properties of diblock copolymers with regioselective functionalization patterns as model compounds for MC to determine a key structure for the thermoreversible gelation of MCs.20,21 As a result, only diblock copolymers having 2,3,6-tri-O-methylglucopyranosyl units, methyl β-D-glucopyranosyl-(1→4)-2,3,6tri-O-methyl-celluloside 1 (G-236MC), and methyl β-Dglucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-2,3,6-tri-Omethyl-celluloside 2 (GG-236MC) aggregated with increasing temperature, accompanied by the formation of a hydrophobic environment. Interestingly, GG-236MC (2) formed a macroscopic hydrogel in water in the temperature range from ambient temperature to ca. 70 °C. The presence of more than ten 2,3,6-tri-O-methyl-glucopyranosyl units was crucially important to thermoreversible hydrogel formation of the diblock copolymers. We also investigated the mechanical properties of a dispersion of a diblock methylcellulose derivative, GG-236MC (2, 2.0 wt %), in water in order to compare the gel strength of a new hydrogel formed by the diblock copolymers with that of industrially produced MC (SM-8000) not only for the elucidation of the structure− property relationships of MC but also for end-use applications. Interestingly, the mechanical strength of a hydrogel of GG236MC (2) was comparable to that of industrially produced MC at a 2.0 wt % concentration at 70 °C, although the molecular weight of GG-236MC (2) was 50 times lower than that of commercial MC. As a consequence, we successfully prepared a new cellulosic supramolecular hydrogelator having a low to moderate molecular weight, resulting from the elucidation of the chemical structure of cross-linking points in commercially produced MC gel by a chemical synthesis approach. In contrast to the polymer hydrogel, there have recently been many reports on supramolecular self-assembled nanofibers and hydrogels consisting of low-molecular-weight compounds such as glycolipids,18,22−24 bolaamphiphiles,25 and peptide amphiphiles.26,27 These amphiphiles are composed of a hydrophobic moiety, generally a single- or double-alkyl tail, linked to a hydrophilic saccharide or oligopeptide headgroup. These compounds are noncovalently self-assembled via well-defined molecular interactions such as hydrophobic interactions and hydrogen bonding, resulting in the formation of nanofibers and hydrogels. Such advanced supramolecular hydrogels, in which small molecules are self-assembled noncovalently, are expected

to be highly advantageous with respect to traditional hydrogels formed by the entanglement of water-soluble polymers because the properties of a supramolecular hydrogel such as the ability to respond to thermal stimuli and pH sensitivity can be easily tuned by adjusting the 1D chemical structures of a small molecule.28 These supramolecular architectures are predominantly developed by the well-defined design of the chemical structure. The syntheses of numerous examples of amphiphiles having different building blocks are therefore required to understand how the 1D structure influences supramolecular morphologies such as fibers, tapes, nanotubes, vesicles, and spherical micelles. Whereas the supramolecular nanofibers and hydrogels of synthetic amphiphiles having a hydrophobic group such as a long alkyl chain have already been reported and extensively studied, to the best of our knowledge, there are no reports of supramolecularly self-assembled nanostructures formed by amphiphilic diblock copolymers consisting of only sugar chains in both hydrophilic and hydrophobic blocks. Selfassembled fibrous structures and their network should be formed in aqueous solution because a diblock MC derivative, GG-236MC (2), formed a hydrogel. It is of crucial importance to investigate not only how GG-236MC (2) forms a hydrogel but also what makes the hydrogel of GG-236MC (2) relatively tough. We report here for the first time hierarchical ribbonlike nanostructures formed by β-(1→4)-linked saccharide derivatives with diblock structure, as detected by a cryo-TEM technique in combination with small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) measurements.

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EXPERIMENTAL SECTION

Materials. Syntheses of methyl β-D-glucopyranosyl-(1→4)-2,3,6tri-O-methyl-celluloside 1 (G-236MC) and methyl β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-celluloside 2 (GG-236MC) were described in our previous paper.20 The chemical structures of compounds 1 and 2 are shown in Figure 1. Compounds 1 and 2 have almost the same DS, although the DPn of compound 2 is approximately three times larger than that of compound 1.

Figure 1. Chemical structures of diblock copolymers consisting of hydrophilic glucosyl or cellobiosyl and hydrophobic 2,3,6-tri-Omethyl-glucopyranosyl blocks. Rheological Measurements. Rheological measurements were carried out using a Haake RS-6000 rheometer (Germany) with a cone−plate sensor system (cone plate C60/1) at 25 and 70 °C. Cryo-Transmission Electron Microscopy (Cryo-TEM). For cryo-TEM of GG-236MC (2) at 5 °C (the representative example for sample preparation is described), the aqueous dispersion was stored for some months in the refrigerator at 9 °C. One hour before measurement, the dispersion was taken out of the refrigerator. During this time, the temperature of the sample increased to around 15−18 °C. Then the sample was precooled in a temperature-controlled water 12610

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bath at 3 °C for 20 min. After the sample was precooled, a volume of 5 μL of the dispersion was placed onto a grid covered by a perforated carbon support foil (Quantifoil Micro Tools Jena). The grid was blotted with a self-made autocontrolled blotting system equipped with a climatic chamber (5 °C) and then plunged rapidly into liquid ethane. The frozen sample was transferred with the Gatan-626 single tilt cryotransfer system to a Philips-CM120 cryo-electron microscope. Small-Angle X-ray Scatting (SAXS) Measurements. SAXS experiments were carried out on a Bruker Nanostar with Cu Kα radiation (λ = 0.15405 nm) and a position-sensitive HiStar detector. The samples were prepared in a sealed quartz capillary with a diameter of 2 mm at 5, 30, 50, and 70 °C. SAXS data were fitted with a cylinder model using NANOFIT software. Wide-Angle X-ray Scattering (WAXS) Measurements. WAXS experiments were carried out at approximately 15 °C on a Rigaku Ultima IV diffractometer. Nickel-filtered Cu Kα radiation was used at 40 kV and 40 mA.

Figure 3. Dynamic storage modulus G′ and dynamic loss modulus G″ of a dispersion of GG-236MC (2, 2.0 wt %) in water as a function of angular frequency ω at 25 °C {G′ (▲) and G″ (△)} and 70 °C {G′ (●) and G″ (○)}.

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indicating the formation of a well-developed gel network.29 Between 25 and 70 °C, G′ increased by an order of magnitude. The G′ value of a dispersion of GG-236MC (2, 2.0 wt %) in water at 70 °C was approximately 7600 Pa, whereas the G′ value of a 2.0 wt % aqueous solution of industrially produced MC (SM-8000, weight-average molecular weight Mw = 4 × 105 g/mol) at 70 °C was approximately 2500 Pa.30,31 As a consequence, the rheological measurement revealed that a dispersion of diblock methylcellulose having Mw = 8.1 × 103 g/ mol, GG-236MC (2, 2.0 wt %), in water formed a thermoresponsive hydrogel above ambient temperature (25 °C) and the gel strength of GG-236MC (2) was 3.0 times stronger than that of commercial MC SM-8000 having Mw = 4 × 105 g/mol. The interesting results concerning the rheological measurement of the GG-236MC (2) hydrogel prompted us to investigate the detailed structure of the diblock copolymers in water on the micro- and nanoscales using a cryo-TEM technique. Cryo-TEM Observation. A cryo-TEM technique was employed to investigate the temperature-dependent nanostructures formed in a 0.2 wt % aqueous solution of G-236MC (1, DPn = 10.7 and DS = 2.65) and a dispersion of GG-236MC (2, 0.2 wt %, DPn = 28.2, and DS = 2.75). The cryo-TEM micrographs of the G-236MC (1) aqueous solution frozen from 5 to 20 °C showed no distinct structures such as fibrous structures (Figure 4a,b). At 50 °C, many ribbonlike supramolecular nanostructures were clearly seen. From the periodic change in the width of the elongated fibrous structures with minimum dimensions at crossover points, a twisted ribbon morphology can be concluded (as indicated by arrows in Figures 4c−f). A tilt series of micrographs in avi format is also available in the Supporting Information. In the micrographs at 70 °C, a solution structure consists of ribbonlike supramolecular nanostructures together with disklike and multilamellar nanostructures that were composed of stacked layers 3 to 4 nm thick (Figure 4l). In contrast, the cryo-TEM micrograph of GG-236MC (2) at 5 °C showed rectanglar nanostructures having a thickness of 11 to 24 nm (tilt series of micrographs in avi format in the Supporting Information), although the cryo-TEM micrographs of G-236MC (1) at 20 °C showed no distinct self-assembled nanostructures. Ribbonlike supramolecular nanostructures were clearly observed in the solution held at 20 °C. In addition, GG236MC (2) formed twisted ribbon structures at 20 °C (indicated by the arrows in Figure 5f,h,i,,l). With increasing temperature to 50 and 70 °C, the number of such ribbonlike supramolecular nanostructures increased, and the homogeneous solution structure consisting of predominantly the

RESULTS AND DISCUSSION We have recently investigated the temperature-dependent behavior and thermoreversible gelation of diblock MC derivatives as model compounds for industrial MC to determine a key structure in the thermoreversible gelation of an aqueous solution of MC.20,21 As a result, DSC, fluorescence, and DLS measurements revealed that only diblock copolymers having a sequence of 2,3,6-tri-O-methyl-glucopyranosyl units formed hydrophobic environments in water and aggregated with increasing temperature. In particular, the diblock copolymer consisting of hydrophilic cellobiosyl blocks and a hydrophobic 2,3,6-tri-O-methyl-cellulosyl block, GG-236MC (2, DPn = 28.2, DS = 2.75), exhibited thermoreversible gelation. We have also reported that the gelation temperature of diblock copolymers depended on the molecular weight when the DS values were almost the same.21 Figure 2 shows photographs of dispersions of GG-236MC (2, 1.0 and 2.0 wt %, DPn = 28.2, and DS = 2.75) in water at 25

Figure 2. Photographs of dispersions of GG-236MC (2, 1.0 and 2.0 wt %, DPn = 28.2, and DS = 2.75) in water at 25 and 70 °C.

and 70 °C. Although a dispersion of GG-236MC (2, 1.0 wt %) in water forms a fluid gel even at 70 °C, a dispersion in water (2.0 wt %) forms stable hydrogels at 25 and 70 °C. Thus, we investigated the gel strength of a 2.0 wt % hydrogel of GG236MC (2) at 25 and 70 °C. Mechanical Property of a Thermoresponsive Hydrogel Formed by Diblock Methylcellulose. Figure 3 shows the dynamic storage modulus G′ and dynamic loss modulus G″ as a function of the angular frequency ω for a dispersion of GG236MC (2.0 wt %, 2, DPn = 28.2, and DS = 2.75) in water at 25 and 70 °C. The G′ values at 25 °C (783 Pa at 0.9 rad s−1) and 70 °C (7416 Pa at 0.9 rad s−1) were around 15 and 10 times higher than the G″ values at comparable temperatures, and both moduli were almost independent of frequency ω, 12611

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Figure 4. Cryo-TEM micrographs of G-236MC (1) at (a) 5, (b) 20, (c−f) 50, and (g−k) 70 °C. (l) Enlarged view of a multilamellar structure shown in k.

graphs of GG-236MC (2) at 5, 20, 50, and 70 °C. Figure 6 shows the size distribution histograms, temperature-dependent average length and thickness, and schematic representation of the ribbonlike nanostructure. GG-236MC (2) formed nanostructures at 5 °C, although the solution was apparently clear. At 5 °C, ribbonlike supra-

supramolecular nanostructures was visible. There were few multilamellar nanostructures in the case of a dispersion of GG236MC (2) in water (not shown). Size Distributions of Self-Assembled Nanostructures. The length and thickness of ca. 600 ribbonlike supramolecular nanostructures were measured directly from cryo-TEM micro12612

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Figure 5. Cryo-TEM micrographs of GG-236MC (2) at (a−c) 5, (d−f) 20, (g−i) 50, and (j−l) 70 °C.

and the thickness ranged from 9.0 to 27.1 nm. The average lengths of supramolecular structures at 5, 20, 50, and 70 °C were 150, 310, 228, and 144 nm, respectively. Meanwhile, the average thicknesses at 5, 20, 50, and 70 °C were 16.4, 13.6, 13.4, and 13.7 nm, respectively. The self-assembled nanostructures of GG-236MC (2, DPn = 28.2 and DS = 2.75) became

molecular nanostructures of GG-236MC (2) had a length ranging from 53 to 479 nm and a thickness ranging from 11.2 to 24.5 nm. At 20 °C, the length ranged from 91 to 864 nm and the thickness ranged from 8.5 to 27.1 nm. At 50 °C, the length ranged from 61.3 to 672 nm and the thickness ranged from 7.2 to 27.0 nm. At 70 °C, the length ranged from 51.6 to 559 nm 12613

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DPn = 10.7 and DS = 2.65) having fewer than ten 2,3,6-tri-Omethyl-glucopyranosyl units exhibited no gelation as recently reported. This was likely because G-236MC (1) formed disklike and multilamellar nanostructures rather than ribbonlike nanostructures. SAXS Measurements of GG-236MC (2). A dispersion of GG-236MC (2, DPn = 28.2 and DS = 2.75) was investigated by SAXS measurements to obtain more precise information on the structure and dimensions of the nanostructures. Figure 7 shows the diffractograms obtained for a dispersion of GG-236MC (2, 0.2 wt %) in water at 5, 30, 50, and 70 °C. According to the analysis by NANOFIT software, the scattering curves were well fitted to the cylinder model with a short height, that is, the disklike model. The radii of the cylinders were 19.5, 16.8, 18.4, and 17.5 nm at 5, 30, 50, and 70 °C, respectively, as shown in Figure 7b. The radii were in good agreement with the molecular length (ca. 14 nm) of GG-236MC (2) calculated from DPn = 28.2. Furthermore, at increasing temperature from 5 to 30 °C, the height of the cylinder increased from 2.1 to 3.4 nm and remained constant (6.3 nm) above 50 °C (Figure 7b). The results from NANOFIT analysis suggested the following temperature-dependent self-assembled ribbonlike nanostructures. The SAXS experiments suggested that GG-236MC molecules (2) were oriented perpendicular to the long axis of ribbonlike nanostructure with bilayer structure having a width of ca. 40 nm. The cylinder model having a height of 2−6 nm was stacked horizontally with respect to the thickness direction of ribbonlike nanostructures, though the average thickness of ribbonlike nanostructures from cryo-TEM micrographs was ca. 14 nm. At 5 °C, the ribbonlike nanostructures consisted of the periodic diffraction structure having a height of 2 nm in the thickness direction as a result of the loose packing between molecules. At 30 °C, the height of the periodic structure increased to 3.4 nm as dehydration proceeded. Above 50 °C, the periodic height remained constant (6.3 nm) likely because of the dehydration between GG-236MC (2) molecules. WAXS Measurement of GG-236MC (2). The crystalline morphology of the 2.0 wt % gel of GG-236MC (2, DPn = 28.2 and DS = 2.75) was investigated at approximately 15 °C by WAXS measurement. The diffraction patterns of (a) 2,3,6-triO-methylcellulose (powder sample, DPn = 201, Mw/Mn = 2.71), (b) a 2.0 wt % gel of GG-236MC (2), and (c) the dried sample prepared from a 2.0 wt % gel of GG-236MC (2) are shown in Figure 8. The diffractogram of (a) 2,3,6-tri-O-methylcellulose showed diffraction peaks at 2θ = 7.6 and 19.9. The diffraction peaks of (b) the gel and (c) the dried sample of GG236MC (2) coincided well with those of (a) 2,3,6-tri-O-methylcellulose. The crystal lattice of GG-236MC (2) would be same as that of 2,3,6-tri-O-methyl-cellulose because GG-236MC (2) had a segment consisting of approximately twenty-seven 2,3,6tri-O-methyl-glucopyranosyl units. Zugenmaier et al. have concluded from X-ray and electron diffraction data that the crystal structure of 2,3,6-tri-O-methylcellulose was regarded as orthorhombic with four chains running through the unit cell and axis values of a = 0.46 nm, b = 4.32 nm, and c = 1.04 nm (fiber axis).32 Furthermore, two pairs of parallel chains of 2,3,6tri-O-methyl-cellulose were packed in an antiparallel fashion because only chains running antiparallel have been detected in single crystals grown from solutions. There have been some reports on the WAXS measurement of commercial MC in the gel state.8,33 Kato et al. have reported that the peak position of the X-ray diffraction pattern of the MC gel coincided well with those of 2,3,6-tri-O-methylcellulose, indicating that the cross-

Figure 6. Size distribution histograms of a dispersion of GG-236MC (2, 0.2 wt %) in water obtained by measuring the length and thickness on cryo-TEM micrographs at (a) 5, (b) 20, (c) 50, and (d) 70 °C. (e) Average length and thickness vs temperature: (●) length and (○) thickness. (f) Schematic representation of the ribbonlike nanostructure.

larger with increasing temperature from 5 to 20 °C, indicating the growth of ribbonlike nanostructures along the axis. With increasing temperature from 20 to 70 °C, the length of the ribbonlike nanostructures decreased in the direction of the long axis. It was found that diblock MCs with different DP values showed different temperature-dependent behaviors, even if the DS values of diblock MCs were almost the same. G-236MC (1, 12614

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Figure 7. (a) Scattering intensity as a function of the scattering vector for a dispersion of GG-236MC (2, 0.2 wt %) in water at increasing temperature: (●) 5, (△) 30, (■) 50, and (◊) 70 °C. (b) Radius of the nanostructure obtained by NANOFIT software using the cylinder model as a function of temperature for a dispersion of GG-236MC (2, 0.2 wt %).

At 5 °C, G-236MC (1) dissolved well and was hydrated, whereas GG-236MC (2) was dispersed in water and formed self-assembled nanostructures. As the temperature increases, the surrounding water molecules near the diblock copolymer are detached, resulting in the diblock copolymers being dehydrated to form the crystalline regions consisting of the sequences of 2,3,6-tri-O-methyl-glucopyranosyl units. The WAXS measurement suggested the presence of these crystalline regions in the gel state. The endothermic peak of the dehydration phenomena has been detected in the DSC curve at around 30 °C.21 The DSC studies on the endothermic dehydration phenomenon of commercial MC have been reported by many researchers.9,17,29,34−36 However, there has been no consensus on the dehydration at the endothermic peak temperature. In contrast, DSC measurements of GG-236MC (2) in combination with cryo-TEM, SAXS, and WAXS measurements strongly suggested that the dehydration occurred at 30 °C because of the hydrophobic interaction between the sequences of 2,3,6-tri-O-methyl-glucopyranosyl units. At around 30 °C, the formation and growth of crystalline regions accompanied by the expulsion of water molecules and the entanglement of ribbonlike nanostructures accompanied by the entrapment of water occurred almost simultaneously. GG236MC (2, DPn = 28.2 and DS = 2.75) formed a hydrogel at 2.0 wt % concentration at 25 °C within 1 h. This fact indicated the formation of self-assembled nanostructures below endothermic temperature at approximately 30 °C at a heating rate of 3.5 °C/min in the DSC curve. Note that this gelation behavior at temperature lower than the dehydration temperature detected by DSC is similar to that of MC reported by Joshi et al.37 Thus, we concluded that the difference between cryoTEM and DSC attributed to their thermal history. The diblock copolymers self-assemble in water, resulting in the formation of ribbonlike supramolecular nanostructures by hydrophobic interaction between the sequence of 2,3,6-tri-O-methylglucopyranosyl units. The formation of hydrophobic environments of self-assembled nanostructures has been analyzed by a fluorescent probe method.21 Further increases in temperature cause the growth of nanostructures.

Figure 8. Wide angle X-ray diffractograms of (a) 2,3,6-tri-Omethylcellulose, (b) GG-236MC (2), a 2.0 wt % gel sample, and (c) GG-236MC (2), a dried sample.

linking loci of MC gels consists of crystallites of 2,3,6-tri-Omethyl-glucopyranose sequences.8 However, the gel structure and gelation mechanism of MC are still uncertain because commercial MC has a heterogeneous structure. In summary, the WAXS measurement of GG-236MC (2) having a welldefined structure strongly suggested that the gelation of GG236MC (2) was attributed to the close packing between the sequences of 2,3,6-tri-O-methyl-glucopyranose units. This intermolecular interaction was of fundamental significance not only for the gelation of commercial MC8 but also for that of present real diblock MC (2, DPn = 28.2 and DS = 2.75). Possible Mechanism for the Self-Assembly and Gelation of G-236MC (1) and GG-236MC (2). On the basis of the results from cryo-TEM, SAXS, and WAXS, the selfassembling mechanism of diblock copolymers G-236MC (1, DPn = 10.7 and DS = 2.65) and GG-236MC (2, DPn = 28.2 and DS = 2.75) is illustrated in Figure 9. 12615

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Figure 9. Schematic representation of the possible gelation mechanism of G-236MC (1) and GG-236MC (2).

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CONCLUSIONS The mechanism of hydrogel formation of real diblock MC having more than ten 2,3,6-tri-O-methyl-glucopyranosyl residues such as GG-236MC (2, DPn = 28.2 and DS = 2.75) was investigated, and the importance of ribbonlike nanostructures consisting of GG-236MC for the supramolecular hydrogel formation was proven by means of some analytical methods tested. The cryo-TEM technique unraveled for the first time that the ribbonlike nanostructures were fabricated by selfassembly of the cellulosic diblock copolymer consisting of only saccharide chains in both hydrophilic and hydrophobic blocks. The GG-236MC is a new class of middle-molecular-weight hydrogelator. These hydrogels had a relatively tough mechanical strength compared to that of a polymer hydrogel of MC. The cryo-TEM technique also allowed us to observe the detailed morphological nanostructures formed in an aqueous solution of G-236MC (1, DPn = 10.7 and DS = 2.65) and a dispersion of GG-236MC (2, DPn = 28.2 and DS = 2.75) in water. Cryo-TEM observations clearly revealed that ribbonlike supramolecular nanostructures and multilamellar nanostructures were formed in water from diblock MC derivatives depending on the temperature. We found from those results that a supramolecular hydrogel forms only when many ribbonlike supramolecular nanostructures exist. At lower temperature such as 5 °C, aqueous solution does not gelate because of the absence of ribbonlike nanostructures. Furthermore, when the number of multilamellar and disklike nanostructures increased instead of increasing the number of ribbonlike nanostructures, a hydrogel also did not form in the case of G-236MC (1, DPn = 10.7 and DS = 2.65). We also demonstrated that fibrous structures such as the ribbonlike nanostructures of GG-236MC (2, DPn = 28.2, and DS = 2.75) indeed formed via the hydrophobic interaction between 2,3,6tri-O-methyl-glucopyranosyl blocks by means of WAXS, which was also the underlying interaction and structure for the formation of a hydrogel, the same as for industrially produced MC. These key findings are attributable to the synthesis and characterization of well-defined diblock methylcelluloses with

One of the important factors in the formation of ribbonlike nanostructures and subsequent hydrogel is the molecular weight of a hydrophobic 2,3,6-tri-O-methyl-glucopyranosyl block. From our earlier publications, more than ten 2,3,6-triO-methyl-glucopyranosyl units are necessary to form a hydrogel.21 The diblock copolymer having a shorter hydrophobic 2,3,6-tri-O-methyl-glucopyranosyl block such as G236MC (1, DPn = 10.7 and DS = 2.65) would form disk or spherical micelles rather than a ribbonlike structure. For example, the diblock co-oligomer consisting of a fully methylated cello-tetrasaccharide and unmodified cello-oligosaccharide forms self-assembled ellipsoidal particles.38 In this study, cryo-TEM micrographs of G-236MC (1, DPn = 10.7 and DS = 2.65) having fewer than ten 2,3,6-tri-O-methylglucopyranosyl units proved that the molecular weight of a hydrophobic block influenced the morphology of the selfassembled structure (disk micelle, multilamellar structure, and ribbonlike structure). Consequently, in the case of G-236MC (1), the number of ribbonlike supramolecular nanostructures decreased at 70 °C because of the production of disklike and multilamellar nanostructures. As a result, a hydrogel did not form. In contrast, GG-236MC (2, DPn = 28.2, and DS = 2.75) having more than ten 2,3,6-tri-O-methyl-glucopyranosyl units formed ribbonlike supramolecular nanostructures, the number of which increased with increasing temperature, resulting in the formation of a hydrogel by the entanglement of fibrous structures and the entrapment of water molecules in their network. In the case of cryo-TEM of commercial MC (Supporting Information), sharply defined micrographs were not obtained, compared to the diblock copolymers. In addition, as far as we know, there have been no reports on the detailed structure of the fibrous structure of commercial MC. The welldefined structure of diblock copolymers 1 and 2 would be a reason that their clear micrographs compared with that of commercial MC with a complicated structure could be obtained. 12616

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regioselective functionalization patterns to elucidate the chemical structure of cross-linking points in industrially produced MC hydrogels.20,21 In the future, a novel synthesis method for diblock MC analogues with DS 2.23 based on click chemistry in our recent paper39 will permit us to synthesize diblock MC analogues with DS ≈ 1.8 and to understand the gelation mechanism of diblock MC with almost the same DS (∼1.8) as that of commercial MC.

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ASSOCIATED CONTENT

S Supporting Information *

A tilt series of cryo-TEM micrographs. A cryo-TEM micrograph of commercial MC. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(T.H.) Tel: +49 3641 948 270. Fax: +49 3641 948 272. Email: [email protected]. (H.K.) Tel: +81-75-7536255. Fax: +81-75-753-6300. E-mail: [email protected]. ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Professors F. Nakatsubo and T. Takano for conducting their research. EKO Instruments Co., Ltd. Osaka, Japan is gratefully acknowledged for rheological measurements. We thank Bruker AXS K.K., Yokohama, Japan for SAXS measurements. This investigation was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan (nos. 21580205 and 24380092) and by the Japan-Germany bilateral research program from the Japanese Society for the Promotion of Sciences (JSPS) and the German Science Foundation (DFG, grant no. 446 JAP 113/341/0-1).

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