Letter pubs.acs.org/macroletters
Enzymatic Synthesis of Cellulose Oligomer Hydrogels Composed of Crystalline Nanoribbon Networks under Macromolecular Crowding Conditions Yuuki Hata,† Tomoya Kojima,† Taro Koizumi,‡ Hiromichi Okura,‡ Takamasa Sakai,§,∥ Toshiki Sawada,† and Takeshi Serizawa*,† †
Department of Chemical Science and Engineering, School of Materials and Chemical Technology, and ‡Department of Organic and Polymeric Materials, Graduate School of Science and Engineering, Tokyo Institute of Technology, 2-12-1-H121 Ookayama, Meguro-ku, Tokyo 152-8550, Japan § Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ∥ Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan S Supporting Information *
ABSTRACT: Macromolecular crowding, a solution state with high macromolecular concentrations, was used to promote the crystallization-driven selfassembly of enzymatically synthesized cellulose oligomers. Cellulose oligomers were synthesized via cellodextrin phosphorylase-catalyzed enzymatic reactions in the concentrated solutions of water-soluble polymers, such as dextran, poly(ethylene glycol), and poly(N-vinylpyrrolidone). The reaction mixtures were transformed into cellulose oligomer hydrogels composed of well-grown crystalline nanoribbon networks irrespective of the polymer species. This method was successfully applied in the one-pot preparation of double network hydrogels composed of the nanoribbons and physically cross-linked gelatin molecules through the simple control of reaction temperatures, demonstrating the superior mechanical properties of the composite hydrogels. Our concept that promotes the growth of self-assembled architectures under macromolecular crowding conditions demonstrates a new avenue into developing novel hydrogel materials.
M
Nanocelluloses, such as cellulose nanofiber, cellulose nanocrystal, and bacterial cellulose, obtained from natural resources have recently gained considerable attention as relatively new cellulosic nanomaterials with high crystallinity due to their chemical/thermal stability, lightweight properties, mechanical stiffness, high surface areas, and biocompatibility.17,18 Alternatively, the in vitro synthesis of cellulose oligomers via enzymatic reactions under water-based mild conditions has been investigated to develop freely designed artificial nanocelluloses with unique structural and morphological properties.19−21 For example, the cellulase-catalyzed oligomerization of β-D-cellobiosyl fluoride monomers22−24 and the cellodextrin phosphorylase (CDP)-catalyzed oligomerization of α-D-glucose 1-phosphate (αG1P) monomers from D-glucose primers25,26 have been demonstrated. Particularly in the case of CDPcatalyzed oligomerization, the cellulose oligomer molecules with an average degree of polymerization (DP) of ∼10 formed rectangular sheet-like nanocelluloses with a width of several
acromolecular crowding represents a solution state with high macromolecular concentrations and is observed in biological intracellular environments.1−3 Macromolecular crowding can be in vitro reproduced by concentrated polymer solutions. Water-soluble polymers, such as dextran (Dex),4−6 Ficoll,4,6,7 poly(ethylene glycol) (PEG),4,5,8−13 and poly(Nvinylpyrrolidone) (PVP)7,14 have frequently been used for the production of macromolecular crowding conditions in aqueous solutions. The crowding polymers generate a space inaccessible to colloidal solutes due to the excluded volume effects; therefore, macromolecular crowding affects the interactions of colloidal solutes.11,12,15 In fact, colloidal dispersions of diverse nanoparticles were dramatically stabilized in concentrated PEG solutions due to the depletion repulsion within a certain time period.12 Furthermore, macromolecular crowding decreased the diffusion rate of proteins to inhibit their aggregation.16 Thus, macromolecular crowding has great potential for controlling dynamics of colloidal solutes. However, to the best of our knowledge, macromolecular crowding has hardly been used for the nano-to-micrometer scale control of molecular selfassembly.13 © 2017 American Chemical Society
Received: November 9, 2016 Accepted: February 1, 2017 Published: February 3, 2017 165
DOI: 10.1021/acsmacrolett.6b00848 ACS Macro Lett. 2017, 6, 165−170
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ACS Macro Letters hundred nanometers, a length of less than several micrometers, and a thickness of ∼5 nm, in which the molecules were aligned perpendicular to the base plane to form a lamella crystal with the cellulose II allomorph.25,26 However, the aggregation and subsequent precipitation of the nanocelluloses were not usually avoided during the enzymatic reactions in aqueous buffer solutions, which should suppress the further growth of the nanocelluloses. To broaden the applicability of the enzymatically synthesized nanocelluloses, controlling the crystallization-driven selfassembly of cellulose oligomer molecules during the enzymatic reactions is needed. Therefore, we hypothesized that the aforementioned nanocelluloses produced during enzymatic reactions should grow into greater architectures without aggregation under macromolecular crowding conditions. Herein, we demonstrated the CDP-catalyzed enzymatic synthesis of cellulose oligomers under macromolecular crowding conditions for the crystallization-driven self-assembly into nanoribbon networks (Figure 1). Dex, PEG, and PVP with different
Figure 1. Schematic representation of this study.
Figure 2. Characterization of the products synthesized via the CDPcatalyzed enzymatic reaction under macromolecular crowding conditions. (a) Photos of the reaction mixtures after the reactions in the presence or absence of the crowding polymers. (b) 1H NMR spectrum and (c) MALDI-TOF-MS of the products prepared with Dex. Numbers in (c) indicate DP of cellulose oligomers. (d) Dynamic viscoelastic property of the hydrogel prepared with Dex.
chemical structure characteristics were used as crowding polymers. Significantly, well-grown and regularly structured nanoribbon networks composed of crystalline cellulose oligomers with the cellulose II allomorph were produced during the reactions, resulting in the formation of novel cellulose oligomer hydrogels. Furthermore, gelatin in a sol state at a temperature of the enzymatic reaction was used as a crowding polymer for cellulose oligomer-based hydrogel formation, followed by the physical cross-linking of gelatin molecules to successfully produce double network (DN) hydrogels. The CDP-catalyzed enzymatic synthesis of cellulose oligomers followed our previously published protocols26 with the exception of the addition of crowding polymers. In brief, αG1P monomers (0.2 M) and D-glucose primers (0.05 M) were incubated with CDP (0.2 U mL−1) in 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid buffer solutions (0.5 M, pH 7.5) in the presence or absence of 10% (w/v) water-soluble polymers, such as Dex, PEG, and PVP, at 60 °C for 3 days (unless otherwise stated). The synthetic details, including other experiments, are summarized in the Supporting Information. The reaction mixtures were changed from transparent into opaque after the reactions (Figure 2a), suggesting the successful synthesis of water-insoluble cellulose oligomers even in the presence of the crowding polymers. The monomer conversions were estimated to be ∼20%, ∼10%, and ∼15% for the reactions in the presence of Dex, PEG, and PVP, respectively, based on the average DP of the products (see below). These conversions
were slightly smaller than that without the crowding polymers (∼35%),26 indicating the possible influences of higher solution viscosities,27 changes in the water activity,28 and changes in the dielectric constant of water solvents29 to the enzymatic reactions. 1 H NMR spectra of the products showed adequate proton signals assignable to cellulose oligomers (Figures 2b and S1) and revealed the average DP of the products to be ∼9 based on the integral ratios for H1α (δ = 5.1), H1β (δ = 4.5), and H1′,1″ (δ = 4.3) protons, irrespective of the crowding polymer species. The DP were slightly smaller than that without the crowding polymers (∼10),26 indicating that the smaller cellulose oligomer molecules were crystallized under macromolecular crowding conditions. This is possibly attributed to the promotion of intermolecular interactions between the produced cellulose oligomers derived from changes in the physicochemical properties of water solvents3,8−10 and an increased collision frequency between the produced cellulose oligomers and the stably dispersed colloidal product12 under macromolecular crowding conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectra (MALDITOF-MS) showed two series of peaks with peak-to-peak mass differences of 162 Da, corresponding to a single glucosyl unit, as well as the mass range from the DP of 6 to at most ∼13 (e.g., the peaks at m/z 1336 and 1352 corresponded to cellooctaose with sodium and potassium ion adducts, respectively; Figures 2c and S2). These observations further supported the 166
DOI: 10.1021/acsmacrolett.6b00848 ACS Macro Lett. 2017, 6, 165−170
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ACS Macro Letters production of cellulose oligomers via the enzymatic reactions and indicated a certain level of molecular weight distribution. The reaction mixtures were significantly transformed into hydrogels after the reactions in the presence of the crowding polymers; notably, the precipitates of cellulose oligomers were obtained in the absence of the crowding polymers (Figure 2a). When the concentration of Dex was changed between 2 and 20% (w/v), 5% (w/v) was required for hydrogel formation (Figure S3), indicating an essential requirement of macromolecular crowding conditions.5,6 When the concentration of CDP was changed to 0.05 and 0.4 U mL−1 from 0.2 U mL−1, hydrogel formation was not observed at the former concentration (Figure S4). Because the monomer conversions were estimated to ∼1% and ∼35%, respectively, network formation appeared to be insufficient under the former conditions. Dynamic viscoelastic measurements of the disk-shaped hydrogels (diameter: 25 mm; thickness: 2.8 mm) prepared in the presence of Dex showed the storage moduli greater than the loss moduli with slight frequency-dependence in the range of 0.01−1 Hz, suggesting that the system was in a gel state in the frequency range (Figure 2d). The stress−strain curves obtained by the compression test for the cylinder-shaped hydrogel (diameter: 15 mm; thickness: 7.5 mm) prepared in the presence of Dex (Figure S5a) revealed that the hydrogels showed the Young’s modulus of 880 ± 160 Pa, which was comparable to those for hydrogels composed of self-assembling biomolecules such as peptides and proteins.30,31 After the compression test, water solvent was squeezed out from the hydrogel, suggesting a sponge-like porous structure of the hydrogel (Figure S5b,c). More significantly, scanning electron microscopy (SEM) observations for the xerogels prepared after the removal of the crowding polymers by immersion into excess amounts of water revealed that the hydrogels were composed of well-grown nanoribbon networks irrespective of the crowding polymer species (Figures 3a,b and S6). From the SEM images of the nanoribbons prepared in the presence of Dex, the width was estimated to be several hundred nanometers; the length reached more than 10 μm. These nanoribbon morphologies are similar to those of naturally driven bacterial cellulose;32 however, it should be noted that the molecular weight, molecular orientation, and allomorph of cellulose are considerably different (also see below). Atomic force microscopy (AFM) observations for the mechanically broken products prepared in the presence of Dex, PEG, and PVP revealed nanoribbon thicknesses of 5.4 ± 0.4, 5.6 ± 0.4, and 5.8 ± 0.4 nm (Figures 3c and S7), respectively. These values were comparable with that for sheet-like nanocelluloses prepared in the absence of the crowding polymers.25 Wide-angle X-ray diffraction (WAXD) measurements of the products indicated diffraction peaks for d-spacings of 0.72, 0.45, and 0.40 nm, which were assignable to 1 1̅ 0, 1 1 0, and 0 2 0 of the antiparallel cellulose II allomorph, respectively (Figures 3d and S8).25 The attenuated total reflection-Fourier transform infrared absorption spectra of the products showed two sharp peaks at approximately 3441 and 3490 cm−1 for the OH stretching vibration bands derived from intramolecular hydrogen bonds, which also supported the cellulose II allomorph (Figure S9).33 The thickness of the nanoribbons (Figure 3c) was almost consistent with the length of the cellononaose, a cellulose oligomer with an average DP of 9, in the cellulose II allomorph (4.7 nm, see Figure S10). It was therefore concluded
Figure 3. Structural characterization of the nanoribbons synthesized via the CDP-catalyzed enzymatic reaction under macromolecular crowding conditions. (a) SEM images of the xerogels prepared with Dex, PEG, and PVP. (b) SEM image of the xerogel prepared with Dex at a low magnification. (c) AFM image and (d) WAXD profile of the products prepared with Dex.
that the cellulose oligomers in the nanoribbons were aligned perpendicular to the base plane, similarly to the conventional rectangular sheet-like nanocelluloses, as schematically illustrated in Figure 1.25 The enzymatic reactions in the absence of the crowding polymers produced crystalline cellulose oligomers with rectangular sheet-like morphologies as precipitates,25,26 whereas those in the presence of crowding polymers produced the hydrogels composed of well-grown nanoribbon networks. These observations suggested that macromolecular crowding increased dispersion stability of the colloidal products due to the depletion repulsion12 and the decrease in diffusion rates,16 thus, leading to the promotion of crystal growth. In addition, the steric repulsion derived from crowding polymers adsorbed onto the colloidal products34 might contribute to the stabilization. Our previous study demonstrated that when oligo(ethylene glycol) (OEG)-bearing β-D-glucose was used as a primer for the CDP-catalyzed enzymatic reactions, hydrogels composed of well-grown nanoribbon networks of OEG-bearing cellulose oligomers were produced.35 The gelation was attributed to the dispersion stabilization of colloidal products due to the OEG chains, followed by the successful growth into nanoribbon networks. Therefore, this study is regarded as an 167
DOI: 10.1021/acsmacrolett.6b00848 ACS Macro Lett. 2017, 6, 165−170
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ACS Macro Letters
to ∼25%, which was smaller than that without the crowding polymers,26 indicating a slight influence of gelatin to the enzymatic reaction. Furthermore, the cellulose oligomers similarly grew into nanoribbon networks with a thickness of 5.8 ± 0.5 nm (Figures 4f and S12), further confirming our proposed mechanism for the production of well-grown cellulose oligomer architectures via the enzymatic synthesis under macromolecular crowding conditions. The cylinder-shaped hydrogels (diameter: 15 mm; thickness: 7.5 mm) composed of cellulose/gelatin, cellulose alone, and gelatin alone were prepared to evaluate their mechanical properties (Figure 5a). The stress−strain curves were obtained
alternative system to promote the crystallization-driven selfassembly of enzymatically synthesized cellulose oligomers for hydrogel formation. Despite unique properties of hydrogels, their inferior mechanical properties are often an obstacle to various applications. Recently, DN gels that consist of two different networks were developed to construct hydrogels with excellent mechanical performance.36 To construct DN gels via the enzymatic synthesis of cellulose oligomers under macromolecular crowding conditions, we focused on gelatin, which forms physically cross-linked hydrogels below certain temperatures.37 We hypothesized that the cellulose oligomer hydrogels were produced in a sol-state gelatin solution at temperatures of the enzymatic reaction and that the subsequent reduction in temperature led to the gelation of gelatin components, followed by the one-pot preparation of DN hydrogels. The reaction mixture after the enzymatic reaction at 60 °C in the presence of 8% (w/v) gelatin was successfully transformed into hydrogels (Figure 4a), followed by reduction in temperature to 25 °C
Figure 5. Compression tests of the cellulose/gelatin composite hydrogels. (a) Photos of the hydrogels prepared with gelatin applied to compression tests. (b) Stress−strain curves and (c) Young’s moduli of the hydrogels. Young’s moduli were presented as the average of five individual trials, and the error bars represent the standard deviation of those trials.
using a compression test (Figure 5b). The Young’s modulus for the cellulose/gelatin composite hydrogel was estimated to 10.1 ± 1.1 kPa, which was approximately 6-fold greater than those of cellulose alone (1.8 ± 0.7 kPa) and gelatin alone (1.7 ± 1.0 kPa) (Figure 5c). In other words, the Young’s modulus for the composite hydrogel was approximately three times the sum of the component moduli. These observations strongly indicated the successful formation of DN hydrogels composed of cellulose nanoribbon and gelatin networks. It is noted that the cellulose/gelatin composite hydrogel held water solvent even after compression, similarly to gelatin alone, while water solvent was squeezed out from the hydrogel composed of cellulose alone (Figure S13). To the best of our knowledge, such physically cross-linked DN hydrogels with excellent mechanical properties have hardly been reported.38,39 Notably, the apparent concentration of cellulose oligomers in the hydrogel was estimated to ∼0.9% (w/v), which was substantially smaller than that of gelatin (8% (w/v)). This observation indicated that the cellulose oligomers with lower concentrations efficiently formed the network structures in the hydrogels. It was therefore suggested that the rational design and control of the enzymatic reaction using unique crowding polymers had great potential for systematically producing useful
Figure 4. Characterization of the products synthesized via the CDPcatalyzed enzymatic reaction in gelatin solutions. (a) Photo of the reaction mixtures after the reactions in the presence of gelatin. Photos of the hydrogels composed of (b) cellulose/gelatin and (c) cellulose alone. (d) 1H NMR spectrum and (e) WAXD profile of the products prepared with gelatin. (f) SEM image of the xerogel prepared with gelatin.
(Figure 4b). The hydrogel was stable even after the removal of the gelatin molecules by immersion into excess amounts of water at 60 °C, suggesting formation of networks composed of cellulose oligomers (Figure 4c). The average DP of the synthesized cellulose oligomers was estimated to ∼9, and the cellulose oligomers formed the cellulose II allomorph (Figures 4d,e and S11). These observations are the same as those obtained with the aforementioned crowding polymers. The monomer conversion in the presence of gelatin was estimated 168
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(5) White, D. A.; Buell, A. K.; Knowles, T. P. J.; Welland, M. E.; Dobson, C. M. J. Am. Chem. Soc. 2010, 132, 5170−5175. (6) Lee, C. F.; Bird, S.; Shaw, M.; Jean, L.; Vaux, D. J. J. Biol. Chem. 2012, 287, 38006−38019. (7) Wang, Y.; Sarkar, M.; Smith, A. E.; Krois, A. S.; Pielak, G. J. J. Am. Chem. Soc. 2012, 134, 16614−16618. (8) Nakano, S.; Karimata, H.; Ohmichi, T.; Kawakami, J.; Sugimoto, N. J. Am. Chem. Soc. 2004, 126, 14330−14331. (9) Yu, H.; Gu, X.; Nakano, S.; Miyoshi, D.; Sugimoto, N. J. Am. Chem. Soc. 2012, 134, 20060−20069. (10) Eggers, D. K.; Valentine, J. S. J. Mol. Biol. 2001, 314, 911−922. (11) Khripin, C. Y.; Arnold-Medabalimi, N.; Zheng, M. ACS Nano 2011, 5, 8258−8266. (12) Zhang, X.; Servos, M. R.; Liu, J. J. Am. Chem. Soc. 2012, 134, 9910−9913. (13) Saeidi, N.; Karmelek, K. P.; Paten, J. A.; Zareian, R.; DiMasi, E.; Ruberti, J. W. Biomaterials 2012, 33, 7366−7374. (14) Charlton, L. M.; Barnes, C. O.; Li, C.; Orans, J.; Young, G. B.; Pielak, G. J. J. Am. Chem. Soc. 2008, 130, 6826−6830. (15) Zhou, H.-X.; Rivas, G.; Minton, A. P. Annu. Rev. Biophys. 2008, 37, 375−397. (16) Breydo, L.; Reddy, K. D.; Piai, A.; Felli, I. C.; Pierattelli, R.; Uversky, V. N. Biochim. Biophys. Acta, Proteins Proteomics 2014, 1844, 346−357. (17) Klemm, D.; Kramer, F.; Moritz, S.; Lindström, T.; Ankerfors, M.; Gray, D.; Dorris, A. Angew. Chem., Int. Ed. 2011, 50, 5438−5466. (18) Moon, R. J.; Martini, A.; Nairn, J.; Simonsen, J.; Youngblood, J. Chem. Soc. Rev. 2011, 40, 3941−3994. (19) Kobayashi, S.; Sakamoto, J.; Kimura, S. Prog. Polym. Sci. 2001, 26, 1525−1560. (20) Kadokawa, J. Chem. Rev. 2011, 111, 4308−4345. (21) Shoda, S.; Uyama, H.; Kadokawa, J.; Kimura, S.; Kobayashi, S. Chem. Rev. 2016, 116, 2307−2413. (22) Kobayashi, S.; Kashiwa, K.; Kawasaki, T.; Shoda, S. J. Am. Chem. Soc. 1991, 113, 3079−3084. (23) Lee, J. H.; Brown, R. M.; Kuga, S.; Shoda, S. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 7425−7429. (24) Tanaka, H.; Koizumi, S.; Hashimoto, T.; Kurosaki, K.; Kobayashi, S. Macromolecules 2007, 40, 6304−6315. (25) Hiraishi, M.; Igarashi, K.; Kimura, S.; Wada, M.; Kitaoka, M.; Samejima, M. Carbohydr. Res. 2009, 344, 2468−2473. (26) Serizawa, T.; Kato, M.; Okura, H.; Sawada, T.; Wada, M. Polym. J. 2016, 48, 539−544. (27) Homchaudhuri, L.; Sarma, N.; Swaminathan, R. Biopolymers 2006, 83, 477−486. (28) Sasaki, Y.; Miyoshi, D.; Sugimoto, N. Biotechnol. J. 2006, 1, 440−446. (29) Nakano, S.; Kitagawa, Y.; Yamashita, H.; Miyoshi, D.; Sugimoto, N. ChemBioChem 2015, 16, 1803−1810. (30) Aulisa, L.; Dong, H.; Hartgerink, J. D. Biomacromolecules 2009, 10, 2694−2698. (31) Raub, C. B.; Putnam, A. J.; Tromberg, B. J.; George, S. C. Acta Biomater. 2010, 6, 4657−4665. (32) Fink, H.-P.; Purz, H. J.; Bohn, A.; Kunze, J. Macromol. Symp. 1997, 120, 207−217. (33) Nelson, M. L.; O’Connor, R. T. J. Appl. Polym. Sci. 1964, 8, 1311−1324. (34) Tadros, T. F. Colloid Stability: The Role of Surface Forces, Part I; Tadros, T. F., Ed.; Wiley-VCH: Weinheim, Germany, 2006; Vol. 1, pp 1−22. (35) Nohara, T.; Sawada, T.; Tanaka, H.; Serizawa, T. Langmuir 2016, 32, 12520. (36) Gong, J. P. Soft Matter 2010, 6, 2583−2590. (37) Gómez-Guillén, M. C.; Giménez, B.; López-Caballero, M. E.; Montero, M. P. Food Hydrocolloids 2011, 25, 1813−1827. (38) Li, C.; Rowland, M. J.; Shao, Y.; Cao, T.; Chen, C.; Jia, H.; Zhou, X.; Yang, Z.; Scherman, O. A.; Liu, D. Adv. Mater. 2015, 27, 3298−3304.
and versatile hydrogel materials in a one-pot preparation manner. In conclusion, the CDP-catalyzed enzymatic synthesis of cellulose oligomers was demonstrated under macromolecular crowding conditions prepared using concentrated water-soluble polymers. The reaction mixtures in sol states were successfully transformed into hydrogels composed of well-grown nanoribbon networks of crystalline cellulose oligomers after the enzymatic reactions irrespective of the crowding polymer species. The reaction system was significantly applied to the production of physically cross-linked DN hydrogels composed of cellulose nanoribbon and gelatin networks via the sol−gel transition of gelatin solutions at controlled temperatures. Enzymatic syntheses under aqueous mild conditions can be regarded as environmentally friendly, low-energy, and sustainable bioprocesses to produce functional oligomers/polymers and their assembled structures in a single step. Since other primers can be applied to the CDP-catalyzed enzymatic reactions,35,40,41 the nanoribbons will be readily functionalized. Our findings open a new avenue into developing unique biobased softmaterials via the precise control of precipitationdriven enzymatic reactions under macromolecular crowding conditions. Further investigations into the enzymatic synthesis of functional soft materials with such unique structures and their potential applications are now in progress.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00848. Experimental details and Figures S1−13 (PDF).
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Takeshi Serizawa: 0000-0002-4867-8625 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors wish to thank the Division of Materials Analysis Ookayama (Tokyo Tech) for the SEM observations and WAXD measurements. This study was partially supported by the Funding Program for Next Generation World-Leading Researchers (NEXT Program), the Grants-in-Aids for Scientific Research (26288056, 26620174, and 16K14075) from the Japan Society for the Promotion of Science, and the collaborative research with JX Nippon Oil and Energy Corporation.
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