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A Bottom-up Synthesis of Vinyl-Cellulose Nanosheets and their Nanocomposite Hydrogels with Enhanced Strength Jianquan Wang, Jiabao Niu, Toshiki Sawada, Ziqiang Shao, and Takeshi Serizawa Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01224 • Publication Date (Web): 17 Oct 2017 Downloaded from http://pubs.acs.org on October 20, 2017
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A Bottom-up Synthesis of Vinyl-Cellulose Nanosheets and their Nanocomposite Hydrogels with Enhanced Strength Jianquan Wang,*,†, ‡ Jiabao Niu,† Toshiki Sawada,‡ Ziqiang Shao,† Takeshi Serizawa*,‡ †
Beijing Engineering Research Center of Cellulose and Its Derivatives, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing, 100081, China
‡
Department of Chemical Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology, Tokyo, 152-8550, Japan
ABSTRACT: Usually extracted nanocellulose from natural resources needs modifying before used as effective nanofillers. In the present study, through an enzymatic polymerization of α-D-glucose 1-phosphate from the primer 2-(glucosyloxy)ethyl methacrylate (GEMA), a novel type of two-dimensional methacrylate-containing cellulose nanosheets (CNS) with a thickness of about 6 nm, named as GEMA-CNS, was directly synthesized under a mild condition by a “bottom-up” method. The structure and morphology of
GEMA-CNS
were
characterized
by
1
H-nuclear
magnetic
resonance
(NMR),
matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF MS), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM) and atomic force microscopy (AFM). Afterwards, the obtained GEMA-CNS was covalently incorporated into poly(ethylene glycol) matrix through thiol-ene Michael addition, fabricating a series of GEMA-CNS based nanocomposite hydrogels. The 1 ACS Paragon Plus Environment
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addition of GEMA-CNS effectively improved the mechanical strength, and altered internal network structures of hydrogels; as well, the swelling/biodegradation behaviors of gels in phosphate buffer saline (pH 7.4) at 37 °C were affected to some degrees. This species of property-tunable hydrogels with GEMA-CNS dosage demonstrates potential applications in tissue engineering. The current presentation opens a new road for direct enzymatic preparation of reactive nanocellulose and its novel applications in nanocomposite materials.
KEYWORDS: cellulose nanosheets; enzymatic synthesis; nanocomposite; hydrogel; enhanced property
INTRODUCTION
Recently, nanocellulose, an emerging class of material stars, has attracted huge attention due to its renewable and eco-friendly nature as well as its high strength and stiffness. Generally, nanocellulose extracted from native cellulose resources includes two types: cellulose nanocrystals (CNC) and cellulose nanofibers (CNF) in regards of their difference in micromorphology and aspect ratios. CNC is produced from pulp cellulose fibers through acid hydrolysis which scissors the hydrogen bonds and removes the amorphous domains to obtain well-defined hard crystalline rods with a diameter of 5-30 nm and length of 100 to 500 nm or even micron scale depending on various cellulose sources.1,2 CNF, with a morphology of soft and long chains (10-100 nm in width and over 1 µm in length), is obtained using mechanical, chemical or a combination of mechanical, chemical or enzymatic processes.2,3 In respect of 2 ACS Paragon Plus Environment
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preparation approach of nanomaterials, the preparation of both CNC and CNF belongs to a “top-down” method. On the contrary, another important class of nanocellulose, bacterial cellulose (BC) is synthesized by bacteria with glucose as a raw material via fermentation, which is a “bottom-up” approach. Usually BC is existed in the form of network structures entangled by cellulosic nanofibers with average diameters of 20-100 nm and micrometer lengths.4 CNC, CNF and BC can be regarded as one-dimensional (1D) nanocellulose.
In addition to the above three types of well-known nanocellulose forms, a novel class of highly crystalline cellulose II type sheet-like nanostructures with a dimension of several nm in thickness, several hundred nm in width, and µm scale in length, which could be nominated as cellulose nanosheets (CNS), was prepared through a “bottom-up” method.5-7 In these reports, the researchers performed in vitro cellodextrin phosphorylase (CDP) mediated polymerization of α-D-glucose 1-phosphate (αG1P) using glucose or cellobiose as a primer, extending enzymatic synthesis of cellulose early pioneered by others, where cellulose products were obtained in the form of mainly type II highly crystalline precipitates.8,9 Hattori et al. investigated the enzymatic synthesis of cellotriose to cellulose II-like substance via endoglucanase I-mediated transglycosylation in an aqueous medium, and proposed that the formation of cellulose II crystals was arisen from self-assembly of higher oligosaccharides.10 This species of sheet-like nanocellulose can be regarded as a novel category of artificial two-dimensional (2D) materials. In recent years, the emergence and rapid growth of 2D nanomaterials such as graphene, metal (hydr)oxides and sulfides, black phosphorous, silicene and so on have promoted a great revolution in new material fields including structural, 3 ACS Paragon Plus Environment
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medical and energy ones.11-14 As a new member of 2D materials family, 2D nanocellulose with the combination of degradable, sustainable and biocompatible natures is prospective to realize some important applications in advanced materials.
Most recently, our group enriched CDP-mediated αG1P oligomerization using several β-D-glucosyl primers with various substituent groups, obtaining various nanostructures such as sheets, rods or ribbons depending on the nature of substitute moieties on the primers.15-17 Besides the easy operation and mild reaction condition in aqueous system, the great virtue of this enzymatic preparation for nanocellulose is the simultaneous achievement of synthesis and functionalization, only by choosing a suitable β-D-glucosyl primer. Undoubtably, this is much convenient relative to traditional post-modification of CNC and CNF, which are usually accomplished via esterification or etherification of hydroxyl or carboxyl groups on their surfaces.18
Nanocellulose has been widely studied for applications in polymer nanocomposites including plastics, rubbers, fibers, resins, liquid crystals, gels, and so on.3,4,19-22 Most of them were mechanically improved via interfacial chemical bonding, so that the functionalization of nanocellulose is very important to meet actual demands. As a novel form of nanocellulose, enzymatically synthesized oligocellulose nanoassembly carrying reactive groups is promising for reinforcement of other materials, and this is still a research gap so far. Only one report with regard to enzymatically synthesized non-functional nanocellulose based composite hydrogels has been published, where the CDP-mediated αG1P polymerization was in situ undertaken in concentrated gelatin solutions, generating double-network physical hydrogels 4 ACS Paragon Plus Environment
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with enhanced mechanics.23 Considering the structural novelty and operational facility of enzymatically synthesized nanocellulose, in this study, we aim to prepare a novel vinyl-nanocellulose through CDP-mediated enzymatic reaction, followed by the fabrication of composite hydrogels strengthened by this special nanofiller via covalent bonding, as illustrated in Scheme 1. To this end, methacrylate-carrying cellulose nanosheets (GEMA-CNS) is synthesized for the first time via CDP-catalyzed αG1P oligomerization from the primer 2-(glucosyloxy)ethyl methacrylate (GEMA), and subsequently the obtained vinyl-nanosheets are utilized as reactive fillers to enhance poly(ethylene glycol) (PEG) hydrogels
constructed
by
a
star
tetra-thiol
PEG
(PTE-200SH)
and
a
linear
PEG-dimethacrylate (PEG-DMA) through thiol-ene Michael addition in physiological phosphate buffered saline (pH 7.4, PBS7.4) environment.
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Scheme 1. Enzymatic synthesis of GEMA-CNS and fabrication of its composite hydrogels.
EXPERIMENTAL SECTION
Materials. 50 wt% GEMA aqueous solution was purchased from Nippon Fine Chemical Co. Ltd, Japan. αG1P disodium salt n-hydrate and Dulbecco’s phosphate buffered saline (D-PBS) were supplied from Wako Pure Chemical Industries, Japan. EM stainer was bought from Nissin EM Corporation, Japan. Sunbright® PTE-200SH was obtained from NOF Corporation, Japan. L-Glutathione reduced (GSH) trypsin from porcine pancreas (BioReagent grade) was purchased from Sigma-Aldrich. Polyethylene glycol 1000 dimethacrylate (PEG1000-DMA) was a gift from Shin-Nakamura Chemical Co. Ltd, Japan. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Bio West. Cell counting kit (CCK) was purchased from Dojindo. Penicillin-streptomycin solution was purchased from Gibco. All other reagents were purchased from Nacalai Tesque. CDP was expressed and purified according to the procedure described in our previous literature.15 Milli-Q ultrapure water (>18.2 MΩ·cm) was used throughout all of the experiments.
Enzymatic synthesis of GEMA-CNS. GEMA-CNS was prepared according to our published method.15,16 Briefly, the monomer αG1P (200 mM) was enzymatically polymerized in the presence of GEMA (150 mM) as a primer, under the catalysis of CDP (0.2 U/mL) in 500 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.5) for 3 days at 60 °C. In this study, the scale of 50 mL was performed. The water-insoluble products were washed by ultrapure water through centrifugation/redispersion cycles more than 5 times. 6 ACS Paragon Plus Environment
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Finally 22 mL of GEMA-CNS dispersion (22.3 mg/mL) was obtained, with the monomer conversion of about 25 %, which was stored at 4 °C.
Fabrication of hydrogels via thiol-ene Michael addition. 80 mg of PTE-200SH and 47.3 µL of 10 wt% PEG1000-DMA in PBS7.4 were mixed with an appropriate amount of 10× PBS7.4, H2O and/or GEMA-CNS dispersion. The total volume of reactant mixture was 900 µL, in which GEMA-CNS contents were adjusted from 0 to 2.0 wt%, and the reactant medium was PBS7.4. The mixtures were ultrasonicated for 20 min so as to uniformly disperse and debubble before transformed into home-made molds (diameter = 8 mm, height = 5 mm). Through changing the amount of GEMA-CNS, five species named as CNS0, CNS0.5, CNS1.0, CNS1.5 and CNS2.0, were fabricated, respectively, where the number following CNS meant the mass percentage content of GEMA-CNS in the gel. As a comparison, pure PTE-200SH gel was prepared without use of PEG1000-DMA and GEMA-CNS, and it was nominated as PTE-200SH. The detailed recipes for various hydrogels are listed in Table 1.
Table 1. The detailed recipes for different hydrogels Sample
PTE-200SH CNS0 CNS0.5 CNS1.0 CNS1.5 CNS2.0 a
PTE-200SH (mg)
10 wt% PEG1000-DMA
GEMA-CNS
10×PBS7.4
H2O
dispersion a (µL)
(µL)
(µL)
80 80 80 80 80 80
(µL) 0 47.3 47.3 47.3 47.3 47.3
0 0 202 404 606 808 (–116) b
81 81 81 81 81 81
739 692 490 288 86 /
The concentration is 22.3 mg/mL; b 116 µL of supernatant was removed after centrifugation.
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Cytotoxicity measurement of hydrogels. In a 96-well cell culture PP plate, reaction solutions (50 µL/well) were fed to fabricate hydrogel samples of CNS0, CNS1.0 and CNS2.0, and 9 samples were prepared for every formulation under the same condition as described above. Before the cell culture, the obtained gels were washed using ultrapure water for 3 days with every 12 h exchange of fresh water, followed by sterilization with 70 % ethanol for 24 h and subsequent exchange of DMEM culture medium. Human cervical cancer cells (HeLa cell line) were cultured in DMEM containing 10 % FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL). The cells were seeded on the hydrogels and incubated at 37 °C with 5 % CO2 for 3 days. Non-adhered cells were removed, and the wells were washed using D-PBS. The number of living cells was quantified using a CCK with 60 min incubation.
Swelling/degradation of hydrogels. For the experiments to check the presence of disulfide bonds in gels, about 100 mg of pre-weighed hydrogels were soaked in 2 mL of PBS7.4 solution containing 10 mM GSH for 1 week at 37 °C. The undissolved gels were weighed, and their mass values were divided by respective original ones before immersion to get swelling ratios.
For the swelling/degradation studies under physiological conditions, the pre-weighed hydrogels were swollen in PBS7.4 solution at 37 °C. At certain time intervals, the hydrogels were taken out, and wiped with wet filter papers to remove surface water, followed by mass determination. Mass ratios of swollen hydrogels to initial ones were defined as swelling ratios.
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In above measurements, three hydrogel samples of the same recipe were used, and the average value was taken.
Characterization and measurements. GEMA-CNS dispersion was lyophilized for 36 h to obtain powder samples for Fourier transform infrared spectroscopy (FTIR) absorption, 1H nuclear magnetic resonance (1H-NMR) and X-ray diffraction (XRD) measurements. FTIR spectra were obtained on a JASCO FT/IR-4100 spectrometer. 1H-NMR spectra were recorded on a Bruker DPX-300 spectrometer after dissolution of GEMA-CNS powder in 4 wt% NaOD/D2O. XRD was measured on a Rigaku MiniFlex 300/600+ with Cu Kα radiation (λ = 0.154 nm). The matrix-assisted laser desorption/ionization time-of-flight mass spectrum (MALDI-TOF MS) was recorded on a Shimadzu AXIMA-performance mass spectrometer equipped with a nitrogen laser (λ =337 nm) and pulsed ion extraction, which was operated at an accelerating potential of 20 kV with a linear-positive ion mode. For MALDI-TOF MS measurement, 5 µL of the mixture containing 2, 5-dihydroxybenzoic acid (10 mg/mL, 5 µL), trifluoroacetic acid/acetonitrile solution (0.2 vol%, 15 µL), and diluted GEMA-CNS dispersion (500 ppm, 5 µL) was mounted onto a sample target plate. Zeta potential and particle size distribution of GEMA-CNS dispersion were measured with Malvern zetasizer Nano ZS90 based on the principle of phase analysis light scattering, and they were analyzed through Herry and Stockes-Einstein equations, respectively. Transmission electron microscopy (TEM) was performed on a Hitachi H-7650 Zero A microscope operated at 100 kV, for which the sample was prepared as follows: 15 µL of 100 ppm GEMA-CNS diluted dispersion was coated on a Cu-grid and left for 1 h; then excessive liquid was absorbed by 9 ACS Paragon Plus Environment
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filter paper, followed by staining with a Nissin EM stainer for 0.5 h, which was dried in a desiccator for at least 12 h. As for atomic force microscopy (AFM), 10 µL of diluted GEMA-CNS dispersion (100 ppm) was dropped on a piece of mica plate, which was dried in a desiccator overnight; then the sample was observed using a Shimadzu SPM-9600 with a non-contact mode.
Compression performance of hydrogels was measured on a Shimadzu Autograph AGS-X 100 N, with a compression rate of 2 mm/min and the maximum strain of 80 %. Cryo-scanning electron microscopy (Cryo-SEM) was performed on a Helios Nanolab 600i. Before SEM observation, the hydrogel sample was rapidly immersed in liquid nitrogen at -210 °C for a while, and was transferred to a cryostat chamber, where the frozen sample was fractured to reveal cross-sectional surface, and then being sublimated for 15 min at -90 °C, followed by platinum-coating for 60 s at 10 mA.
RESULTS AND DISCUSSION
Preparation and characterization of GEMA-CNS. In this work, GEMA was selected as a substrate primer for αG1P propagation to provide vinyl groups on the produced nanocellulose surface, so as to further explore the utilization for reactive fillers. As shown in Figure 1(a), the transparent precursor solution of αG1P (200 mM), GEMA (150 mM) and CDP (0.2 U/mL) in pH 7.5 HEPES (500 mM) buffer solution became turbid through 3 days of incubation at 60 °C, implying the generation of water-insoluble resultants. Through multiple washing with pure water, viscous dispersion with the concentration of 22.3 mg/mL was 10 ACS Paragon Plus Environment
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obtained, in which no sedimentation was found even after one year of storage at 4 °C. Zeta potential and size analysis measurement towards the diluted dispersion (100 ppm) shows that the average hydrodynamic size of particles is 287 nm (PDI 0.09) with Zeta-potential of -13.4 ± 0.8 mV, as shown in Figure S1. Such a lower PDI indicates the near-monodisperse size of particles inside the dispersion. Here, the dispersion of artificial nanocellulose with about 500 mg of dried mass was obtained in the scale of 50 mL volume through one-step method, and the implementation of this scale was based on the preliminary exploration of 1 and 10 mL, so it can be inferred that greater scale could be easily expanded due to the mild condition and eco-friendly reaction medium. Compared with the functionalization of traditional nanocellulose such as CNC and CNF, this method is much facile, avoiding the usage of strong acid or oxidant for their production and subsequent post-modification.
In this study, the decision of every reactant concentration in recipe was on the basis of our previous studies,7,15 and the only difference is that the primer GEMA was fed at three times dosage of those in the previous systems. It is known that GEMA has two types of stereoisomers, i.e., α- and β-types, and their ratio was calculated to be around 2:1 according to the respective peak areas for β-H at 4.92 ppm and α-H at 4.47 ppm (Figure 1(b)). So GEMA was dosed at the concentration of 150 mM instead of 50 mM in consideration of the specificity of CDP to β-type substrate.15-17,24 In Figure 1(b), the comparison of GEMA and GEMA-CNS 1H-NMR spectra demonstrates the successful incorporation of GEMA into resultants based on the emergence of methacryloxyethyl characteristic signals in
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GEMA-CNS. Meanwhile, the unavailability of α-type GEMA is also confirmed by 1H-NMR spectrum of GEMA-CNS, where the β-H on GEMA is not found in products.
Further, according to the peak areas at both 5.4 and 4.1-4.3 ppm, which correspond to a proton on vinyl group (CH2=C
