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Influence of sonication treatment on supramolecular cellulose microfibril-based hydrogels induced by ionic interaction
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JIEC-2478; No. of Pages 8 Journal of Industrial and Engineering Chemistry xxx (2015) xxx–xxx
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Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
Influence of sonication treatment on supramolecular cellulose microfibril-based hydrogels induced by ionic interaction Nanang Masruchin a, Byung-Dae Park a,*, Valerio Causin b a b
Department of Wood and Paper Sciences, Kyungpook National University, Daegu, 702-701, Republic of Korea Department of Chemical Sciences, University of Padova, via Marzolo 1, 35131, Padova, Italy
A R T I C L E I N F O
Article history: Received 19 December 2014 Received in revised form 12 March 2015 Accepted 29 March 2015 Available online xxx Keywords: Cellulose microfibrils Sonication Ionic cross-linking Hydrogel Drug release
A B S T R A C T
This study investigated effects of sonication treatment on characteristics and drug release behavior of hydrogels prepared by supramolecular cellulose microfibrils (CMFs) isolated by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation which made carboxylate negative charge available on the CMFs’ surface. The hydrogels were fabricated by inducing ionic interactions between negatively charged CMFs and a positive metal ion (Al3+). The sonication time showed no influence on the carboxylate content of CMFs, but it greatly influenced characteristics and drug release behavior of the hydrogels. These results indicate that the sonication time has an impact on hydrogels’ characteristics and drug release behavior. ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Introduction Hydrogels are three dimensional network structure materials that are highly hydrophilic and are able to absorb a large amount of water. Hydrogels are widely used as absorbents, in water purification, in sensors, contact lenses, tissue engineering, and drug delivery systems [1]. Hydrogels can be fabricated from synthetic polymers or natural polymers, where a micro-porous structure is formed by either physical or chemical cross-linking interactions. Cellulose-based hydrogels, generated using a natural polymer, are particularly attractive due to the abundance, renewability, biodegradability, and non-toxicity of cellulose [2]. Although bacterial cellulose (BC)-based hydrogels are commonly utilized in numerous biomedical applications and has been well reviewed for drug delivery systems application [3], such BChydrogels are produced by an expensive and inefficient process [4]. On the other hand, nanocellulose material could be isolated effectively from wood and other lignocellulosic materials on mass production [5–8]. The utilization of nanocellulose material is a bourgeoning field in which systems such as cellulose nanofibrils (CNFs) and cellulose nanowhiskers (CNWs) have been used for
* Corresponding author. Tel.: +82 53 950 5497; fax: +82 53 950 6751. E-mail address: [email protected] (B.-D. Park).
hydrogels. These nanocelluloses are one of the most promising materials for advanced futuristic applications [9–11]. The rationale reason for introducing nanocellulose as drug carrier are the high surface area-to-volume [8] and negative charge of nanocellulose which suggest large amounts of drugs might be bound to the surface of this material with the potential for high payloads and optimal control of dosing [12]. The release of the drug from the hydrogel is linearly correlated to the internal structure of hydrogel and degree of crosslinking [13]. However, studies on pure cellulose hydrogel with regard to drug release are rarely being reported [14]. Recently, cellulose hydrogels have been made by cross-linking cellulose molecules dissolved in various organic solvents. However, the use of these organic solvents limits the applications of cellulose hydrogels because of the undesirable effects of the toxic solvents. Abe and Yano [15] reported the formation of a hydrogel from highly crystalline CNFs by alkaline treatment (9–15 wt% NaOH). The gelation network was formed by entanglement and coalescence of the CNFs; these effects possibly originate from the effect of mercerization on longitudinal shrinkage of the cellulose nanofibers in aqueous alkaline solutions [16]. Syverud et al. [17] evaluated the formation of hydrogels from CNFs isolated by TEMPO-mediated oxidation of cellulose combined with polyethyleneimine and poly-N-isopropylacrylamide-co-allylamineco-methylenebisacrylamide. The aldehyde groups were recognized as suitable reaction sites for cross-linking. Dong et al. [18]
http://dx.doi.org/10.1016/j.jiec.2015.03.034 1226-086X/ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
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successfully synthesized CNFs by TEMPO-oxidation and applied them to the fabrication of a tunable hydrogel with the addition of various cations (mono-, di-, and tri-valent). The anionic carboxylate groups (from oxidation process) and cationic metal salts formed ionic cross-links, resulting in firm hydrogels. Saito et al. [14] investigated the surface carboxylate groups of CNFs that selfaligned into a stiff hydrogel in water by adjusting the pH below the pKa. This hydrogel was reported as having outstanding properties, and aerogels generated from the hydrogel had a large surface area and ultralow density. Compared to CNF-based hydrogels, herein, we propose a quite simple and effective method for producing hydrogels by introducing surface charge on hardwood bleached kraft pulp (HW-BKP) using TEMPO-oxidation followed by different levels of sonication treatment. The suspension was termed cellulose microfibrils (CMFs). The diameters of the CMFs are estimated to be in the range of nano to micron size (7.07 0.99 nm to 10 mm) [19]. The sonication technique is an emerging method for preparing cellulose fibrils with nano size [20]. However, because of the complicated hierarchical structure of the plant cell wall and the interfibrillar hydrogen bonds, the fibers obtained by TEMPO-oxidation are aggregated nanofibers with a wide distribution in width. Therefore, by applying sonication treatments subsequent to TEMPO-oxidation, aggregation of the nanofibrils could be prevented owing to the effect of electrostatic repulsion of the negatively charged cellulose surface [21]. Herein, effect of sonication time on the properties of the oxidized-cellulose suspension is studied in terms of the carboxylate group content, viscosity, transparency, and morphology. Cellulose microfibrils (CMFs) hydrogels were formed by addition of trivalent cations, and the resulting assemblies were characterized in terms of their mechanical properties and chemical and internal structure. Finally, a drug was loaded into the hydrogels to study the behavior of the hydrogels in controlling release drug. The present approach is proposed as a very simple and energy-efficient means of producing cellulose fibril-based hydrogels. Experimental Materials Dried hardwood bleached kraft pulp (HW-BKP) was obtained from Moorim Paper Co., Ltd., and stored in a constant humidity chamber at 25 8C before use. TEMPO, sodium bromide (NaBr), and sodium hypochlorite (NaClO) solutions were purchased and used as received from Sigma-Aldrich. Aluminum nitrate (Al(NO3)3.9H2O) was purchased from Duksan Pure Chemicals Co. Ltd., and was used as received as a cross-linker for surface-charged cellulose. Water (conductivity: 6 mS/cm) from a reverse osmosis system was obtained by purification using a Upure system (ROtech, Daegu, Korea). As a model for drug release evaluation, anhydrous theophylline (MW = 180.16; purity 99%) was purchased from Sigma-Aldrich and used as received.
order to obtain a dilute soluble fraction, the oxidized pulps were filtered and washed with reverse osmosis water several times. Water (200 mL) was added to obtain a 2% (w/w) suspension of the water-insoluble TEMPO-oxidized cellulose fibril suspension. The suspension was subjected to ultrasonic treatment for different times (i.e., 20, 40, and 60 min) using a Sonomasher (power 30%, frequency 20,320 Hz) with a probe diameter of 1 cm. In order to prevent overheating, the sonication process was performed in intervals of 10 min. Carboxyl content measurement The carboxylate group content of the CMFs was measured via conductometric titration according to procedures reported by Saito et al. [22]. In brief, the cellulose suspension with 0.05% solid content was agitated with addition of 80 mL deionized water and 5 mL 0.01 M NaCl to the CMF suspension. The pH of the suspension was adjusted to 2.5–3 by addition of 0.1 M HCl, after which 0.01 M NaOH was added at a rate of 0.1 mL/min up to pH 11. A typical graph for the conductometric titration is shown in Fig. 1. The conductivity decreased until the acid was neutralized by the addition of NaOH. At the end of neutralization (V1), the conductivity remained unchanged to the end of step (V2). Further addition of NaOH increased the conductivity. Conductivity changes were recorded using a conductometer (SevenGo, METTLER TOLEDO, China). As a comparison, CNFs were also isolated from the CMF suspension by centrifugation at 12.300g for 40 min (Labogene 1580 centrifuge, Gyrozen Co., Ltd., Daejeon, Korea) and the carboxylate group content was also measured. The carboxylate group content was determined by means of Eq. (1), presented below: mmol ðV 2 V 1 Þx M NaOH (1) ¼ Carboxyl content g weight of cellulose Viscosity and transparency The viscosity of the sonicated suspensions was measured with a Brookfield Viscometer (model DV-II + Pro). Spindle number 2 with 5 cm diameter was used to achieve accurate viscosity measurements. The measurements were conducted with 250 mL of sample at a temperature 22 8C, using a speed of 60 RPM and torque of 1– 15%. Three replicate measurements were performed for each parameter. The light transmittance of the sonicated suspensions was measured using a UV–VIS spectrophotometer (Optizen 3220UV, Mecasys Co., Ltd., Daejeon, Korea). The concentration of the cellulose suspension was controlled at 0.3% (w/v). Data were collected in the wavelength range of 200 to 1000 nm.
Methods TEMPO-mediated oxidation and sonication Carboxylated cellulose fibrils were obtained by TEMPO/NaBr/ NaClO oxidation in water at pH 10.5 [22]. Cellulose pulps (2 g) were dispersed in 150 mL of water containing diluted TEMPO (0.025 g) and NaBr (0.25 g). NaClO solution (12.5%, 8 mL, 7.85 mmol/g) was added dropwise to the solution to initiate the oxidation. The pH of the mixture was controlled at 10.5 by dropwise addition of 0.5 M NaOH. The reaction was conducted at room temperature (23 8C) for a course of about 75 min until no further change in pH was observed, indicating the end of the reaction. The mixture was neutralized by adding HCl (0.5 M). In
Fig. 1. Typical conductometric titration curve and equation for calculating carboxyl content. Inset shows images of the CMF and CNF suspensions.
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Hydrogelation of CMFs Hydrogelation was performed by adding a tri-valent cation (Al3+) to the suspension of CMFs. A 50 mM solution of Al(NO3)3, 1.6 mmol/g, was added gently to the top of the cellulose microfibril suspension without stirring. Gelation occurred rapidly upon addition of the cation to the suspension in a beaker (25 mL). The hydrogels were kept at room temperature overnight. The hydrogels were then removed from the beakers and rinsed several times with deionized water to ensure that no free cation remained.
measurements were carried out with an X-ray diffractometer (D/Max-2500, Rigaku, Japan). CuKa-1 X-rays with a wavelength of 1.541 A˚ were used for the analysis. Data were acquired by scanning in the 2u range of 108 to 408 in reflection mode, with a step size of 0.028, using a scanning rate of 68/min. The crystallinity of each hydrogel was calculated from the corresponding XRD profile using Eq. (3) [24]: I200 Iam (3) Crystallinityð%Þ ¼ I200
Measurement of properties of hydrogels A rheometer (MARS, HAAKE, Germany) was used to measure the storage and compression strength of the hydrogels. Specimens for rheological testing were cut into circular tubes (4 cm in diameter and 1 cm thick) using a razor blade. A linear viscoelastic regime was obtained at a strain rate of 0.01%. Steady shear tests were carried out using a 35 mm diameter plate and a plate between 0.1 and 100 rad/s. Duplicate measurements were performed at room temperature. In addition, specimens for analysis of the compression strength of the hydrogels were cut to dimensions of 4 cm diameter and 2 cm thickness and compressed using the same rheometer system. The measurements were performed in duplicate at room temperature using a strain rate of 0.1%/min.
where, I200 is the maximum intensity of the crystalline peak between 228 and 238 2u for the cellulose fibrils, and Iam is the minimum intensity between 188 and 198 2u for the cellulose fibrils. Swelling ratio of hydrogels Approximately 0.2 g of an aerogel sample was dipped into 200 mL of water to swell. The water absorption at a given swelling time was determined gravimetrically from the difference in mass before and after swelling. The sample was carefully removed from the water by sieving, and the excess water was blotted with a filter paper. The swelling ratio (SR) was calculated based on Eq. (4): SR ¼
ATR-IR measurement The freeze-dried hydrogels were analyzed using attenuated total reflectance-infrared (ATR-IR) spectroscopy (ALPHA-P model, Bruker Optics, Germany). The absorbance was measured in the wavenumber range from 4000 to 400 cm1. In order to enhance the signal-to-noise ratio, an average of 24 scans was obtained for each sample. FE-SEM analysis Field emission scanning electron microscopy (FE-SEM) (Hitachi S-4800, Japan) analysis was used to examine the internal morphology of the hydrogels. Prior to FE-SEM observation, the hydrogel samples were dipped in liquid nitrogen and then freezedried using a dryer (FDA8508, IlshinBioBase Co., Ltd., Korea) to produce aerogels. The fractured surface aerogel samples were mounted on sample stubs using carbon adhesive tape, and then coated with osmium for 5 s in a Plasma Coater (HPC-1SW, Vacuum Device Inc., Tokyo Japan). Images were acquired at an acceleration voltage of 5 kV. Specific surface and pore size analysis The aerogel samples (0.01–0.02 g) were placed in sample cells and degassed at 323 K for 5 h. The specific surface area and pore size of the aerogels were determined using N2 adsorption and desorption at 77 K in the relative vapor pressure (p/po) range of 0.02–1. The specific surface area was determined using the Brunauer-Emmett-Teller (BET) method. Furthermore, the average pore size of the cellulose fibril-based aerogels was estimated from the nitrogen desorption isotherm using the Barrett-JoynerHalenda (BJH) method. The diameter (d) of the cellulose fibrils of the aerogel were estimated by using Eq. (2), assuming that the fibrils had a cylindrical shape and that the density of the fibrils was rc = 1460 kg m3 [23]: d¼
4
rc BET
(2)
where, rc is the density of cellulose and BET is specific surface area. X-ray diffraction (XRD) analysis of cellulose crystallinity The crystallinity of the cellulose fibril hydrogels was evaluated by X-ray diffraction (XRD) using the aerogel samples. XRD
wt wo wo
(4)
where, W0 and Wt are the weights of the samples before and after swelling in water for a specific time t, respectively. Drug release study Theophylline drug (0.02% w/w wet hydrogel) was loaded into the hydrogel by stirring into the cellulose suspension after sonication treatment. The mixed suspension was subjected to the hydrogelation process. The drug-loaded hydrogels were cut into a rectangular shape with dimensions of 1 cm 1 cm 3 cm. The samples were then immersed in a beaker filled with distilled water. Approximately 3.5 mL of the distilled water from the beaker was collected each hour, and the drug concentration in the water was determined using a UV/vis spectrometer (Optizen 3220UV, Mecasys Co., Ltd., Daejeon, Korea) at a wavelength of 273 nm. Following acquisition of the UV/vis spectrum, the sample was quantitatively added back into the beaker. The drug concentration in the water at different times was calculated using a calibrated standard curve (R2 = 0.999). The cumulative release percentage of the drug was calculated from Eq. (5): Cumulative releaseð%Þ ¼
W dt W1
(5)
where, Wdt is the weight of released drug at time t and W1 is the total weight of loaded drug in the hydrogel. The drug release test was performed in duplicate for all samples. Results and discussion Carboxylate group content of the oxidized cellulose The oxidation of cellulose with TEMPO produces negatively charged carboxylate groups on the surface of cellulose by primarily converting the C6 primary hydroxyl groups to carboxylate groups with secondary formation of a small amount of aldehyde groups. The carboxyl content of oxidized cellulose treated for various sonication times is presented in Fig. 2. The carboxyl content of the oxidized cellulose fibrils increased from 0.15 mmol/g for HW-BKP to 0.67 mmol/g after the TEMPO-mediated oxidation. Sonication of the oxidized CMFs for different times did not change the carboxyl content of the CMFs. However, when the CNFs were isolated from the CMF suspension, the carboxyl content of the CNFs was
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explained by the fibrillation effect of ultrasound. During sonication of the liquid CMF suspension, the acoustic cavitation generated by high intensity ultrasound leads to the formation, growth, and collapse of bubbles, which consequently provides a sono-chemical effect with an energy of approximately 10–100 kJ/mol [1]. During cavitation, collapse of the bubbles produces intense local heating and high pressure for a very short lifespan [25]. The fibrillation process was facilitated by the presence of surface anionic charge, which leads to internal electrostatic repulsion within the cellulose microfibrils [20]. Typical balloon-like structures (black arrow) were formed after 60 min sonication as illustrated in the inset of Fig. 3. The width of the balloons was more than twice that of the original pulp. This phenomenon is similar to that observed by Uetani and Yano in the formation of nanofibrils by physical treatment using a high speed blender [26]. Fig. 2. Carboxyl content of oxidized cellulose subjected to different sonication time.
determined to be as high as 0.82 mmol/g. This could be due to the fact that the CNFs have a greater surface area than the CMFs, suggesting that the CNFs have a greater number of negative surface charges for cross-linking. Morphology of CMFs The morphology of the CMFs in the suspension was observed with a light microscope as shown in Fig. 3. Notably, sonication treatment reduced the fraction of CMFs in the suspension, suggesting that the fraction of CNFs in the suspension increased. As the sonication time increases, the control pulp fiber is gradually fibrillated into smaller fragments, submicrometer fibrils, and finally nanofibers. As expected, the CMF suspension became more transparent with sonication for longer periods. This could be
Viscosity and transparency of CMF suspension The viscosity of the CMF suspension increased with an increase in the sonication time (Fig. 4a). The initial rise in viscosity can be attributed to the increasing proportion of high surface area CNF that interacted strongly with the aqueous medium [27]. Furthermore, the transparency of the CMF suspension increased from 15% to 45% as the sonication time increased (Fig. 4b). The transparency of the isolated pure CNF suspension was higher (up to 85%) than that of the CMF suspension; thus, the increasing transparency suggests that the sonication treatment leads to the formation of CNFs in the CMFs suspension. A comparison of the appearance of the CMF and CNF suspensions is presented in the inset of Fig. 1. Mechanical properties of cellulose hydrogels The mechanical properties of the cellulose hydrogels were determined by analysis of the storage modulus (G0 ), loss modulus
Fig. 3. Light micrographs of oxidized cellulose suspension treated for different sonication times. Inset illustrates fiber collapse (balloon-like structure) due to the sonication treatment and electrostatic repulsion of the surface charged cellulose.
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Fig. 4. Effect of sonication times on the viscosity (a) and transparency (b) of CMF suspension.
(G00 ) and compression strength using a rheometer. The results of these measurements are presented in Fig. 5. The G0 , G00 values of the microgels did not change significantly in response to changes in the shear rate (Fig. 5a), which suggests that the CMF suspensions were truly cross-linked by the added cation. The high valency aluminum cation may lead to greater electrostatic attraction with multiple adjacent carboxylate groups and produce aggregation within the fibrils [18]. Throughout the applied frequency, the G0 values were always higher than G00 values indicate a stable interaction. As the sonication time increased, the G0 , G00 value increased. The presence of high surface area CNFs promotes entanglement and cross-linking with adjacent cellulose microfibrils. As expected, the compression strength of the hydrogels followed a trend similar to that of the storage modulus (Fig. 5b). In addition, we could not obtain hydrogel from oxidized-cellulose without fibrillation process through sonication treatment. Chemical groups of the cellulose hydrogels The ATR-IR spectra of freeze dried hydrogels treated for different sonication times are shown in Fig. 6. The sonication time did not influence the location and absorption intensity of the hydrogels, which indicated that no chemical changes were induced by increasing the sonication time [28]. Extending the sonication time has been reported to induce a variety of ultrastructural defects only, such as shortening, kinking, and sub-fibrillation during the pulping process [29]. However, oxidation treatment of cellulose pulp led to two new peaks at 1630–1636 and 1743– 1769 cm1, which were respectively assigned to carboxylate and carboxylic groups. Thus, the appearance of these new functional
groups clearly proves the success of the oxidation reaction. The cation-induced cross-linking of the CMFs also resulted in an obvious shift of the –COO– stretching bands to higher wavenumbers, which is attributed to formation of ionic bonds between the cations and the carboxylate groups of the surface-modified cellulose [30]. A second new shoulder peak appeared at around 1743–1769 cm1, attributed to the carboxylic group. This is due to hydrolysis of the metal salt in solution at low pH, where the carboxylic group was formed in the hydrogels prepared by adding Al3+ as reported by Dong et al. [18]. It is proposed that the –COOH groups formed in the hydrogels can influence the swelling behavior of the hydrogels [31,32]. The broad peaks in the region of 3344 to 3349 cm1 and at 1160 cm1 are assigned to OH stretching and OH bending, respectively, and the CH2 stretching frequency appeared as a small band in the range of 2912–2917 cm1. The band at 1038 cm1 is assigned to CO stretching, and the peak at 897 cm1 is attributed to the chemical bond of the b-glycosidic linkage (–C–O–C) of the cellulose chain. Crystallinity of the cellulose hydrogels Since the swelling behavior of the cellulose hydrogel is influenced by the packing density of the cellulose crystal, the cellulose crystallinity of the hydrogels was calculated by using the corresponding freeze-dried aerogels. Fig. 7a shows typical X-ray diffractograms of the microgels. As expected, the diffractograms of all microgels showed a two-peak pattern, and the positions of the peaks were nearly identical. This XRD pattern is typical of cellulose I with 2u reflections at 14.98, 15.78, and 22.68 which are assigned to
Fig. 5. Mechanical properties of the cellulose hydrogels, (a) storage (G0 ) and loss modulus (G0 0 ) and (b) compression strength.
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FE-SEM analysis of hydrogel morphology
Fig. 6. ATR-IR spectra of the cellulose hydrogels prepared using different sonication times.
the 101, 10-1, and 002 planes, respectively [33]. Using these diffractograms, the cellulose crystallinity of the hydrogels was quantified using Eq. (3). The results are presented in Fig. 7b. The sonication time did not influence the crystallinity of the microgels, which indicates that ionic cross-linking and the sonication time do not interfere with the arrangement of the cellulose crystal. Based on studies by Briois et al. [29], at least 3 h of sonication was required to convert the allomorph of cellulose. In our case, cellulose was subjected to a maximum of 1 h sonication in order to obtain the microgels; thus, the gels can be claimed to be energyefficient hydrogels.
The internal morphology of the freeze-dried hydrogels (i.e., aerogels) was characterized using FE-SEM. Fig. 8 shows FE-SEM images of the hydrogels prepared using three different sonication times. Overall, the internal structures of the hydrogels were mesoporous [34]. As discussed in relation to the viscosity and transparency of the CMF suspension, the internal images of the hydrogels also confirm the presence of CNFs (arrowheads in Fig. 8). The structure of the hydrogels became more porous with an increase of the sonication time. These porous structures were created by ionic cross-linking via inter- or intra-interaction of cellulose fibrils. All hydrogels had lamellar structures that were created by trapped frozen water molecules and self-assembly of the cellulose fibrils induced during the freeze-drying process [35]. The use of a short sonication time resulted in the retention of many un-fibrillated pulp fibers (arrows in Fig. 8) in the gels, and a more lamellar structure was observed than with longer sonication times. These observations indicated that the presence of the CNF fraction influenced the internal structure of the hydrogels. Surface area and pore size of the hydrogels The influence of sonication time on the internal structure of the cellulose hydrogels was evaluated by comparison of the specific surface area, fibril diameter, and pore radius of the hydrogels (Fig. 9). Increasing the sonication time led to an increase in the specific surface area of the hydrogels, while the fibril diameter decreased with an increase in the sonication time (Fig. 9a). These results indicate an increase in the CNF fraction, with a surface area of up to 66 m2/g, as the duration of the sonication process was extended [31]. These results are in agreement with
Fig. 7. (a) Typical XRD diffractogram and (b) cellulose crystallinity of cellulose aerogels.
Fig. 8. FE-SEM images of cellulose hydrogels prepared using different sonication times. Arrows indicate cellulose microfibrils and arrowheads show the CNFs.
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Fig. 9. (a) Specific surface area and calculated fibril diameter and (b) pore radius of the hydrogels as a function of sonication time.
the morphological characteristics observed by FE-SEM, which showed that aggregation of small cellulose fibrils was more extensive with the use of 60 min sonication than 20 min sonication. The pore radius of the aerogels was also estimated using the Barrett-Joyner-Halenda (BJH) method (Fig. 9b). The sonication time did not have a significant effect on the pore radius of the hydrogels, except for 40 min sonication. It is believed that the greater the cross-linking of the hydrogels, the smaller the pore radii. However, as reported from FE-SEM analysis in this study (Fig. 8), the porous structure of the aerogels was combined with lamellar structures and fibril aggregates. Thus, the lamellar structure precludes application of the assumption that the fibrils have a uniform cylindrical shape [23]. Swelling behavior of the hydrogels To compare the water absorption behavior of the hydrogels, the swelling ratio of the aerogels prepared from the hydrogels was compared (Fig. 10). All aerogels absorbed water very quickly and reached an equilibrium swelling ratio within 5 min. This phenomenon is comparable to the network swelling of TEMPO-oxidized nanocellulose [36]. The presence of cellulose fibers containing many hydrophilic hydroxyl groups, as well as the presence of carboxylic groups in the hydrogels as indicated by ATR-IR, resulted in high water absorption of the hydrogels from the initial moment of contact with water. After the initial swelling, there was no further swelling of the hydrogels as the time increased. This could be ascribed to the high crystallinity of the CMFs, which prevents swelling of the CMFs. The swelling ratio was lowest for the sample prepared using a sonication time of 60 min. A longer sonication time causes the
Fig. 10. Swelling behavior of hydrogels prepared using different sonication times.
formation of a larger proportion of CNF in the hydrogels, which subsequently resulted in increased cross-linking, leading to the formation of a denser and more compact hydrogel network. This increased the difficulty of water penetration into the hydrogel. As a consequence, the water absorption capability of the hydrogel decreased significantly, leading to a corresponding decrease in the swelling ratio. However, this was not observed with the use of 40 min sonication, which might be attributed to the smaller pore radius of the cellulose fibrils in the hydrogels prepared using 40 min sonication. Drug release of the hydrogels The porous structure, high surface area, and the ability to absorb water enable the use of the cellulose hydrogels as carriers for controlled drug release. The cumulative release of theophylline drug from the hydrogels in water over time is presented in Fig. 11. Release of the drug was quite low (<20%), which could be due to the relatively constant swelling of the hydrogels (equilibrium state) and short exposure time (i.e., 24 h) for the release of the drug via diffusion. The cumulative release of the drug increased sharply within 6 h for all hydrogels, and further increased gradually to the end of the period. The hydrogel prepared using a sonication time of 60 min showed the slowest release behavior. These drug release characteristics are quite consistent with the swelling behavior of the hydrogels presented in Fig. 10. Thus, these results indicate that the hydrogel characteristics, such as morphology, surface area, pore size, hydrogel integrity, and surface charge, had a significant impact on the drug release profile of the hydrogel.
Fig. 11. Drug release behavior of theophylline-loaded hydrogels prepared using different sonication times.
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Conclusions Cellulose microfibrils hydrogels were successfully produced by a simple method in which a positive tri-valent metal cation was added to a CMF suspension that was prepared by the oxidation of cellulose pulp using the TEMPO-oxidation system followed by sonication for different times. The content of carboxylate groups in the CMFs was unaffected by the duration of the sonication treatment. However, the sonication time influenced the proportion of CNFs in the CMF suspension, which in turn influenced the internal structure of the hydrogels. Increasing the sonication time resulted in an increase of the storage modulus, compression strength, and surface area of the microgels, while the calculated fibril diameter decreased. Extending the sonication time did not influence the chemical structure and cellulose crystallinity of the hydrogels. Thus, the internal structure of the hydrogel had a significant impact on the swelling and drug release behavior of the hydrogels, indicating that the internal structure could be controlled based on the sonication time to manipulate the release of drugs from the hydrogels. Acknowledgments This research was supported by the Korea–Italy Bilateral Joint Research Program through the National Research Foundation (NRF) of Korea and funded by the Ministry of Education, Science, and Technology (grant no. 2013K1A3A1A25037202). Valerio Causin gratefully acknowledges the support of the Italian Ministry of Foreign Affairs, Direzione Generale per la Promozione del Sistema Paese. References [1] C. Chang, L. Zhang, Carbohydr. Polym. 84 (2011) 40–53. [2] L. Alexandrescu, K. Syverud, A. Gatti, G. Chinga-Carrasco, Cellulose 20 (2013) 1765–1775. [3] M.M. Abeer, M.C.I.M. Amin, C. Martin, J. Pharm. Pharmacol. 66 (2014) 1047–1061. [4] A.M. Bochek, Fibre Chem. 40 (2008) 192–197.
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Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
Influence of sonication treatment on supramolecular cellulose microfibril-based hydrogels induced by ionic interaction Nanang Masruchin a, Byung-Dae Park a,*, Valerio Causin b a b
Department of Wood and Paper Sciences, Kyungpook National University, Daegu, 702-701, Republic of Korea Department of Chemical Sciences, University of Padova, via Marzolo 1, 35131, Padova, Italy
A R T I C L E I N F O
Article history: Received 19 December 2014 Received in revised form 12 March 2015 Accepted 29 March 2015 Available online xxx Keywords: Cellulose microfibrils Sonication Ionic cross-linking Hydrogel Drug release
A B S T R A C T
This study investigated effects of sonication treatment on characteristics and drug release behavior of hydrogels prepared by supramolecular cellulose microfibrils (CMFs) isolated by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation which made carboxylate negative charge available on the CMFs’ surface. The hydrogels were fabricated by inducing ionic interactions between negatively charged CMFs and a positive metal ion (Al3+). The sonication time showed no influence on the carboxylate content of CMFs, but it greatly influenced characteristics and drug release behavior of the hydrogels. These results indicate that the sonication time has an impact on hydrogels’ characteristics and drug release behavior. ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Introduction Hydrogels are three dimensional network structure materials that are highly hydrophilic and are able to absorb a large amount of water. Hydrogels are widely used as absorbents, in water purification, in sensors, contact lenses, tissue engineering, and drug delivery systems [1]. Hydrogels can be fabricated from synthetic polymers or natural polymers, where a micro-porous structure is formed by either physical or chemical cross-linking interactions. Cellulose-based hydrogels, generated using a natural polymer, are particularly attractive due to the abundance, renewability, biodegradability, and non-toxicity of cellulose [2]. Although bacterial cellulose (BC)-based hydrogels are commonly utilized in numerous biomedical applications and has been well reviewed for drug delivery systems application [3], such BChydrogels are produced by an expensive and inefficient process [4]. On the other hand, nanocellulose material could be isolated effectively from wood and other lignocellulosic materials on mass production [5–8]. The utilization of nanocellulose material is a bourgeoning field in which systems such as cellulose nanofibrils (CNFs) and cellulose nanowhiskers (CNWs) have been used for
* Corresponding author. Tel.: +82 53 950 5497; fax: +82 53 950 6751. E-mail address: [email protected] (B.-D. Park).
hydrogels. These nanocelluloses are one of the most promising materials for advanced futuristic applications [9–11]. The rationale reason for introducing nanocellulose as drug carrier are the high surface area-to-volume [8] and negative charge of nanocellulose which suggest large amounts of drugs might be bound to the surface of this material with the potential for high payloads and optimal control of dosing [12]. The release of the drug from the hydrogel is linearly correlated to the internal structure of hydrogel and degree of crosslinking [13]. However, studies on pure cellulose hydrogel with regard to drug release are rarely being reported [14]. Recently, cellulose hydrogels have been made by cross-linking cellulose molecules dissolved in various organic solvents. However, the use of these organic solvents limits the applications of cellulose hydrogels because of the undesirable effects of the toxic solvents. Abe and Yano [15] reported the formation of a hydrogel from highly crystalline CNFs by alkaline treatment (9–15 wt% NaOH). The gelation network was formed by entanglement and coalescence of the CNFs; these effects possibly originate from the effect of mercerization on longitudinal shrinkage of the cellulose nanofibers in aqueous alkaline solutions [16]. Syverud et al. [17] evaluated the formation of hydrogels from CNFs isolated by TEMPO-mediated oxidation of cellulose combined with polyethyleneimine and poly-N-isopropylacrylamide-co-allylamineco-methylenebisacrylamide. The aldehyde groups were recognized as suitable reaction sites for cross-linking. Dong et al. [18]
http://dx.doi.org/10.1016/j.jiec.2015.03.034 1226-086X/ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
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successfully synthesized CNFs by TEMPO-oxidation and applied them to the fabrication of a tunable hydrogel with the addition of various cations (mono-, di-, and tri-valent). The anionic carboxylate groups (from oxidation process) and cationic metal salts formed ionic cross-links, resulting in firm hydrogels. Saito et al. [14] investigated the surface carboxylate groups of CNFs that selfaligned into a stiff hydrogel in water by adjusting the pH below the pKa. This hydrogel was reported as having outstanding properties, and aerogels generated from the hydrogel had a large surface area and ultralow density. Compared to CNF-based hydrogels, herein, we propose a quite simple and effective method for producing hydrogels by introducing surface charge on hardwood bleached kraft pulp (HW-BKP) using TEMPO-oxidation followed by different levels of sonication treatment. The suspension was termed cellulose microfibrils (CMFs). The diameters of the CMFs are estimated to be in the range of nano to micron size (7.07 0.99 nm to 10 mm) [19]. The sonication technique is an emerging method for preparing cellulose fibrils with nano size [20]. However, because of the complicated hierarchical structure of the plant cell wall and the interfibrillar hydrogen bonds, the fibers obtained by TEMPO-oxidation are aggregated nanofibers with a wide distribution in width. Therefore, by applying sonication treatments subsequent to TEMPO-oxidation, aggregation of the nanofibrils could be prevented owing to the effect of electrostatic repulsion of the negatively charged cellulose surface [21]. Herein, effect of sonication time on the properties of the oxidized-cellulose suspension is studied in terms of the carboxylate group content, viscosity, transparency, and morphology. Cellulose microfibrils (CMFs) hydrogels were formed by addition of trivalent cations, and the resulting assemblies were characterized in terms of their mechanical properties and chemical and internal structure. Finally, a drug was loaded into the hydrogels to study the behavior of the hydrogels in controlling release drug. The present approach is proposed as a very simple and energy-efficient means of producing cellulose fibril-based hydrogels. Experimental Materials Dried hardwood bleached kraft pulp (HW-BKP) was obtained from Moorim Paper Co., Ltd., and stored in a constant humidity chamber at 25 8C before use. TEMPO, sodium bromide (NaBr), and sodium hypochlorite (NaClO) solutions were purchased and used as received from Sigma-Aldrich. Aluminum nitrate (Al(NO3)3.9H2O) was purchased from Duksan Pure Chemicals Co. Ltd., and was used as received as a cross-linker for surface-charged cellulose. Water (conductivity: 6 mS/cm) from a reverse osmosis system was obtained by purification using a Upure system (ROtech, Daegu, Korea). As a model for drug release evaluation, anhydrous theophylline (MW = 180.16; purity 99%) was purchased from Sigma-Aldrich and used as received.
order to obtain a dilute soluble fraction, the oxidized pulps were filtered and washed with reverse osmosis water several times. Water (200 mL) was added to obtain a 2% (w/w) suspension of the water-insoluble TEMPO-oxidized cellulose fibril suspension. The suspension was subjected to ultrasonic treatment for different times (i.e., 20, 40, and 60 min) using a Sonomasher (power 30%, frequency 20,320 Hz) with a probe diameter of 1 cm. In order to prevent overheating, the sonication process was performed in intervals of 10 min. Carboxyl content measurement The carboxylate group content of the CMFs was measured via conductometric titration according to procedures reported by Saito et al. [22]. In brief, the cellulose suspension with 0.05% solid content was agitated with addition of 80 mL deionized water and 5 mL 0.01 M NaCl to the CMF suspension. The pH of the suspension was adjusted to 2.5–3 by addition of 0.1 M HCl, after which 0.01 M NaOH was added at a rate of 0.1 mL/min up to pH 11. A typical graph for the conductometric titration is shown in Fig. 1. The conductivity decreased until the acid was neutralized by the addition of NaOH. At the end of neutralization (V1), the conductivity remained unchanged to the end of step (V2). Further addition of NaOH increased the conductivity. Conductivity changes were recorded using a conductometer (SevenGo, METTLER TOLEDO, China). As a comparison, CNFs were also isolated from the CMF suspension by centrifugation at 12.300g for 40 min (Labogene 1580 centrifuge, Gyrozen Co., Ltd., Daejeon, Korea) and the carboxylate group content was also measured. The carboxylate group content was determined by means of Eq. (1), presented below: mmol ðV 2 V 1 Þx M NaOH (1) ¼ Carboxyl content g weight of cellulose Viscosity and transparency The viscosity of the sonicated suspensions was measured with a Brookfield Viscometer (model DV-II + Pro). Spindle number 2 with 5 cm diameter was used to achieve accurate viscosity measurements. The measurements were conducted with 250 mL of sample at a temperature 22 8C, using a speed of 60 RPM and torque of 1– 15%. Three replicate measurements were performed for each parameter. The light transmittance of the sonicated suspensions was measured using a UV–VIS spectrophotometer (Optizen 3220UV, Mecasys Co., Ltd., Daejeon, Korea). The concentration of the cellulose suspension was controlled at 0.3% (w/v). Data were collected in the wavelength range of 200 to 1000 nm.
Methods TEMPO-mediated oxidation and sonication Carboxylated cellulose fibrils were obtained by TEMPO/NaBr/ NaClO oxidation in water at pH 10.5 [22]. Cellulose pulps (2 g) were dispersed in 150 mL of water containing diluted TEMPO (0.025 g) and NaBr (0.25 g). NaClO solution (12.5%, 8 mL, 7.85 mmol/g) was added dropwise to the solution to initiate the oxidation. The pH of the mixture was controlled at 10.5 by dropwise addition of 0.5 M NaOH. The reaction was conducted at room temperature (23 8C) for a course of about 75 min until no further change in pH was observed, indicating the end of the reaction. The mixture was neutralized by adding HCl (0.5 M). In
Fig. 1. Typical conductometric titration curve and equation for calculating carboxyl content. Inset shows images of the CMF and CNF suspensions.
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Hydrogelation of CMFs Hydrogelation was performed by adding a tri-valent cation (Al3+) to the suspension of CMFs. A 50 mM solution of Al(NO3)3, 1.6 mmol/g, was added gently to the top of the cellulose microfibril suspension without stirring. Gelation occurred rapidly upon addition of the cation to the suspension in a beaker (25 mL). The hydrogels were kept at room temperature overnight. The hydrogels were then removed from the beakers and rinsed several times with deionized water to ensure that no free cation remained.
measurements were carried out with an X-ray diffractometer (D/Max-2500, Rigaku, Japan). CuKa-1 X-rays with a wavelength of 1.541 A˚ were used for the analysis. Data were acquired by scanning in the 2u range of 108 to 408 in reflection mode, with a step size of 0.028, using a scanning rate of 68/min. The crystallinity of each hydrogel was calculated from the corresponding XRD profile using Eq. (3) [24]: I200 Iam (3) Crystallinityð%Þ ¼ I200
Measurement of properties of hydrogels A rheometer (MARS, HAAKE, Germany) was used to measure the storage and compression strength of the hydrogels. Specimens for rheological testing were cut into circular tubes (4 cm in diameter and 1 cm thick) using a razor blade. A linear viscoelastic regime was obtained at a strain rate of 0.01%. Steady shear tests were carried out using a 35 mm diameter plate and a plate between 0.1 and 100 rad/s. Duplicate measurements were performed at room temperature. In addition, specimens for analysis of the compression strength of the hydrogels were cut to dimensions of 4 cm diameter and 2 cm thickness and compressed using the same rheometer system. The measurements were performed in duplicate at room temperature using a strain rate of 0.1%/min.
where, I200 is the maximum intensity of the crystalline peak between 228 and 238 2u for the cellulose fibrils, and Iam is the minimum intensity between 188 and 198 2u for the cellulose fibrils. Swelling ratio of hydrogels Approximately 0.2 g of an aerogel sample was dipped into 200 mL of water to swell. The water absorption at a given swelling time was determined gravimetrically from the difference in mass before and after swelling. The sample was carefully removed from the water by sieving, and the excess water was blotted with a filter paper. The swelling ratio (SR) was calculated based on Eq. (4): SR ¼
ATR-IR measurement The freeze-dried hydrogels were analyzed using attenuated total reflectance-infrared (ATR-IR) spectroscopy (ALPHA-P model, Bruker Optics, Germany). The absorbance was measured in the wavenumber range from 4000 to 400 cm1. In order to enhance the signal-to-noise ratio, an average of 24 scans was obtained for each sample. FE-SEM analysis Field emission scanning electron microscopy (FE-SEM) (Hitachi S-4800, Japan) analysis was used to examine the internal morphology of the hydrogels. Prior to FE-SEM observation, the hydrogel samples were dipped in liquid nitrogen and then freezedried using a dryer (FDA8508, IlshinBioBase Co., Ltd., Korea) to produce aerogels. The fractured surface aerogel samples were mounted on sample stubs using carbon adhesive tape, and then coated with osmium for 5 s in a Plasma Coater (HPC-1SW, Vacuum Device Inc., Tokyo Japan). Images were acquired at an acceleration voltage of 5 kV. Specific surface and pore size analysis The aerogel samples (0.01–0.02 g) were placed in sample cells and degassed at 323 K for 5 h. The specific surface area and pore size of the aerogels were determined using N2 adsorption and desorption at 77 K in the relative vapor pressure (p/po) range of 0.02–1. The specific surface area was determined using the Brunauer-Emmett-Teller (BET) method. Furthermore, the average pore size of the cellulose fibril-based aerogels was estimated from the nitrogen desorption isotherm using the Barrett-JoynerHalenda (BJH) method. The diameter (d) of the cellulose fibrils of the aerogel were estimated by using Eq. (2), assuming that the fibrils had a cylindrical shape and that the density of the fibrils was rc = 1460 kg m3 [23]: d¼
4
rc BET
(2)
where, rc is the density of cellulose and BET is specific surface area. X-ray diffraction (XRD) analysis of cellulose crystallinity The crystallinity of the cellulose fibril hydrogels was evaluated by X-ray diffraction (XRD) using the aerogel samples. XRD
wt wo wo
(4)
where, W0 and Wt are the weights of the samples before and after swelling in water for a specific time t, respectively. Drug release study Theophylline drug (0.02% w/w wet hydrogel) was loaded into the hydrogel by stirring into the cellulose suspension after sonication treatment. The mixed suspension was subjected to the hydrogelation process. The drug-loaded hydrogels were cut into a rectangular shape with dimensions of 1 cm 1 cm 3 cm. The samples were then immersed in a beaker filled with distilled water. Approximately 3.5 mL of the distilled water from the beaker was collected each hour, and the drug concentration in the water was determined using a UV/vis spectrometer (Optizen 3220UV, Mecasys Co., Ltd., Daejeon, Korea) at a wavelength of 273 nm. Following acquisition of the UV/vis spectrum, the sample was quantitatively added back into the beaker. The drug concentration in the water at different times was calculated using a calibrated standard curve (R2 = 0.999). The cumulative release percentage of the drug was calculated from Eq. (5): Cumulative releaseð%Þ ¼
W dt W1
(5)
where, Wdt is the weight of released drug at time t and W1 is the total weight of loaded drug in the hydrogel. The drug release test was performed in duplicate for all samples. Results and discussion Carboxylate group content of the oxidized cellulose The oxidation of cellulose with TEMPO produces negatively charged carboxylate groups on the surface of cellulose by primarily converting the C6 primary hydroxyl groups to carboxylate groups with secondary formation of a small amount of aldehyde groups. The carboxyl content of oxidized cellulose treated for various sonication times is presented in Fig. 2. The carboxyl content of the oxidized cellulose fibrils increased from 0.15 mmol/g for HW-BKP to 0.67 mmol/g after the TEMPO-mediated oxidation. Sonication of the oxidized CMFs for different times did not change the carboxyl content of the CMFs. However, when the CNFs were isolated from the CMF suspension, the carboxyl content of the CNFs was
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explained by the fibrillation effect of ultrasound. During sonication of the liquid CMF suspension, the acoustic cavitation generated by high intensity ultrasound leads to the formation, growth, and collapse of bubbles, which consequently provides a sono-chemical effect with an energy of approximately 10–100 kJ/mol [1]. During cavitation, collapse of the bubbles produces intense local heating and high pressure for a very short lifespan [25]. The fibrillation process was facilitated by the presence of surface anionic charge, which leads to internal electrostatic repulsion within the cellulose microfibrils [20]. Typical balloon-like structures (black arrow) were formed after 60 min sonication as illustrated in the inset of Fig. 3. The width of the balloons was more than twice that of the original pulp. This phenomenon is similar to that observed by Uetani and Yano in the formation of nanofibrils by physical treatment using a high speed blender [26]. Fig. 2. Carboxyl content of oxidized cellulose subjected to different sonication time.
determined to be as high as 0.82 mmol/g. This could be due to the fact that the CNFs have a greater surface area than the CMFs, suggesting that the CNFs have a greater number of negative surface charges for cross-linking. Morphology of CMFs The morphology of the CMFs in the suspension was observed with a light microscope as shown in Fig. 3. Notably, sonication treatment reduced the fraction of CMFs in the suspension, suggesting that the fraction of CNFs in the suspension increased. As the sonication time increases, the control pulp fiber is gradually fibrillated into smaller fragments, submicrometer fibrils, and finally nanofibers. As expected, the CMF suspension became more transparent with sonication for longer periods. This could be
Viscosity and transparency of CMF suspension The viscosity of the CMF suspension increased with an increase in the sonication time (Fig. 4a). The initial rise in viscosity can be attributed to the increasing proportion of high surface area CNF that interacted strongly with the aqueous medium [27]. Furthermore, the transparency of the CMF suspension increased from 15% to 45% as the sonication time increased (Fig. 4b). The transparency of the isolated pure CNF suspension was higher (up to 85%) than that of the CMF suspension; thus, the increasing transparency suggests that the sonication treatment leads to the formation of CNFs in the CMFs suspension. A comparison of the appearance of the CMF and CNF suspensions is presented in the inset of Fig. 1. Mechanical properties of cellulose hydrogels The mechanical properties of the cellulose hydrogels were determined by analysis of the storage modulus (G0 ), loss modulus
Fig. 3. Light micrographs of oxidized cellulose suspension treated for different sonication times. Inset illustrates fiber collapse (balloon-like structure) due to the sonication treatment and electrostatic repulsion of the surface charged cellulose.
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Fig. 4. Effect of sonication times on the viscosity (a) and transparency (b) of CMF suspension.
(G00 ) and compression strength using a rheometer. The results of these measurements are presented in Fig. 5. The G0 , G00 values of the microgels did not change significantly in response to changes in the shear rate (Fig. 5a), which suggests that the CMF suspensions were truly cross-linked by the added cation. The high valency aluminum cation may lead to greater electrostatic attraction with multiple adjacent carboxylate groups and produce aggregation within the fibrils [18]. Throughout the applied frequency, the G0 values were always higher than G00 values indicate a stable interaction. As the sonication time increased, the G0 , G00 value increased. The presence of high surface area CNFs promotes entanglement and cross-linking with adjacent cellulose microfibrils. As expected, the compression strength of the hydrogels followed a trend similar to that of the storage modulus (Fig. 5b). In addition, we could not obtain hydrogel from oxidized-cellulose without fibrillation process through sonication treatment. Chemical groups of the cellulose hydrogels The ATR-IR spectra of freeze dried hydrogels treated for different sonication times are shown in Fig. 6. The sonication time did not influence the location and absorption intensity of the hydrogels, which indicated that no chemical changes were induced by increasing the sonication time [28]. Extending the sonication time has been reported to induce a variety of ultrastructural defects only, such as shortening, kinking, and sub-fibrillation during the pulping process [29]. However, oxidation treatment of cellulose pulp led to two new peaks at 1630–1636 and 1743– 1769 cm1, which were respectively assigned to carboxylate and carboxylic groups. Thus, the appearance of these new functional
groups clearly proves the success of the oxidation reaction. The cation-induced cross-linking of the CMFs also resulted in an obvious shift of the –COO– stretching bands to higher wavenumbers, which is attributed to formation of ionic bonds between the cations and the carboxylate groups of the surface-modified cellulose [30]. A second new shoulder peak appeared at around 1743–1769 cm1, attributed to the carboxylic group. This is due to hydrolysis of the metal salt in solution at low pH, where the carboxylic group was formed in the hydrogels prepared by adding Al3+ as reported by Dong et al. [18]. It is proposed that the –COOH groups formed in the hydrogels can influence the swelling behavior of the hydrogels [31,32]. The broad peaks in the region of 3344 to 3349 cm1 and at 1160 cm1 are assigned to OH stretching and OH bending, respectively, and the CH2 stretching frequency appeared as a small band in the range of 2912–2917 cm1. The band at 1038 cm1 is assigned to CO stretching, and the peak at 897 cm1 is attributed to the chemical bond of the b-glycosidic linkage (–C–O–C) of the cellulose chain. Crystallinity of the cellulose hydrogels Since the swelling behavior of the cellulose hydrogel is influenced by the packing density of the cellulose crystal, the cellulose crystallinity of the hydrogels was calculated by using the corresponding freeze-dried aerogels. Fig. 7a shows typical X-ray diffractograms of the microgels. As expected, the diffractograms of all microgels showed a two-peak pattern, and the positions of the peaks were nearly identical. This XRD pattern is typical of cellulose I with 2u reflections at 14.98, 15.78, and 22.68 which are assigned to
Fig. 5. Mechanical properties of the cellulose hydrogels, (a) storage (G0 ) and loss modulus (G0 0 ) and (b) compression strength.
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FE-SEM analysis of hydrogel morphology
Fig. 6. ATR-IR spectra of the cellulose hydrogels prepared using different sonication times.
the 101, 10-1, and 002 planes, respectively [33]. Using these diffractograms, the cellulose crystallinity of the hydrogels was quantified using Eq. (3). The results are presented in Fig. 7b. The sonication time did not influence the crystallinity of the microgels, which indicates that ionic cross-linking and the sonication time do not interfere with the arrangement of the cellulose crystal. Based on studies by Briois et al. [29], at least 3 h of sonication was required to convert the allomorph of cellulose. In our case, cellulose was subjected to a maximum of 1 h sonication in order to obtain the microgels; thus, the gels can be claimed to be energyefficient hydrogels.
The internal morphology of the freeze-dried hydrogels (i.e., aerogels) was characterized using FE-SEM. Fig. 8 shows FE-SEM images of the hydrogels prepared using three different sonication times. Overall, the internal structures of the hydrogels were mesoporous [34]. As discussed in relation to the viscosity and transparency of the CMF suspension, the internal images of the hydrogels also confirm the presence of CNFs (arrowheads in Fig. 8). The structure of the hydrogels became more porous with an increase of the sonication time. These porous structures were created by ionic cross-linking via inter- or intra-interaction of cellulose fibrils. All hydrogels had lamellar structures that were created by trapped frozen water molecules and self-assembly of the cellulose fibrils induced during the freeze-drying process [35]. The use of a short sonication time resulted in the retention of many un-fibrillated pulp fibers (arrows in Fig. 8) in the gels, and a more lamellar structure was observed than with longer sonication times. These observations indicated that the presence of the CNF fraction influenced the internal structure of the hydrogels. Surface area and pore size of the hydrogels The influence of sonication time on the internal structure of the cellulose hydrogels was evaluated by comparison of the specific surface area, fibril diameter, and pore radius of the hydrogels (Fig. 9). Increasing the sonication time led to an increase in the specific surface area of the hydrogels, while the fibril diameter decreased with an increase in the sonication time (Fig. 9a). These results indicate an increase in the CNF fraction, with a surface area of up to 66 m2/g, as the duration of the sonication process was extended [31]. These results are in agreement with
Fig. 7. (a) Typical XRD diffractogram and (b) cellulose crystallinity of cellulose aerogels.
Fig. 8. FE-SEM images of cellulose hydrogels prepared using different sonication times. Arrows indicate cellulose microfibrils and arrowheads show the CNFs.
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Fig. 9. (a) Specific surface area and calculated fibril diameter and (b) pore radius of the hydrogels as a function of sonication time.
the morphological characteristics observed by FE-SEM, which showed that aggregation of small cellulose fibrils was more extensive with the use of 60 min sonication than 20 min sonication. The pore radius of the aerogels was also estimated using the Barrett-Joyner-Halenda (BJH) method (Fig. 9b). The sonication time did not have a significant effect on the pore radius of the hydrogels, except for 40 min sonication. It is believed that the greater the cross-linking of the hydrogels, the smaller the pore radii. However, as reported from FE-SEM analysis in this study (Fig. 8), the porous structure of the aerogels was combined with lamellar structures and fibril aggregates. Thus, the lamellar structure precludes application of the assumption that the fibrils have a uniform cylindrical shape [23]. Swelling behavior of the hydrogels To compare the water absorption behavior of the hydrogels, the swelling ratio of the aerogels prepared from the hydrogels was compared (Fig. 10). All aerogels absorbed water very quickly and reached an equilibrium swelling ratio within 5 min. This phenomenon is comparable to the network swelling of TEMPO-oxidized nanocellulose [36]. The presence of cellulose fibers containing many hydrophilic hydroxyl groups, as well as the presence of carboxylic groups in the hydrogels as indicated by ATR-IR, resulted in high water absorption of the hydrogels from the initial moment of contact with water. After the initial swelling, there was no further swelling of the hydrogels as the time increased. This could be ascribed to the high crystallinity of the CMFs, which prevents swelling of the CMFs. The swelling ratio was lowest for the sample prepared using a sonication time of 60 min. A longer sonication time causes the
Fig. 10. Swelling behavior of hydrogels prepared using different sonication times.
formation of a larger proportion of CNF in the hydrogels, which subsequently resulted in increased cross-linking, leading to the formation of a denser and more compact hydrogel network. This increased the difficulty of water penetration into the hydrogel. As a consequence, the water absorption capability of the hydrogel decreased significantly, leading to a corresponding decrease in the swelling ratio. However, this was not observed with the use of 40 min sonication, which might be attributed to the smaller pore radius of the cellulose fibrils in the hydrogels prepared using 40 min sonication. Drug release of the hydrogels The porous structure, high surface area, and the ability to absorb water enable the use of the cellulose hydrogels as carriers for controlled drug release. The cumulative release of theophylline drug from the hydrogels in water over time is presented in Fig. 11. Release of the drug was quite low (<20%), which could be due to the relatively constant swelling of the hydrogels (equilibrium state) and short exposure time (i.e., 24 h) for the release of the drug via diffusion. The cumulative release of the drug increased sharply within 6 h for all hydrogels, and further increased gradually to the end of the period. The hydrogel prepared using a sonication time of 60 min showed the slowest release behavior. These drug release characteristics are quite consistent with the swelling behavior of the hydrogels presented in Fig. 10. Thus, these results indicate that the hydrogel characteristics, such as morphology, surface area, pore size, hydrogel integrity, and surface charge, had a significant impact on the drug release profile of the hydrogel.
Fig. 11. Drug release behavior of theophylline-loaded hydrogels prepared using different sonication times.
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Conclusions Cellulose microfibrils hydrogels were successfully produced by a simple method in which a positive tri-valent metal cation was added to a CMF suspension that was prepared by the oxidation of cellulose pulp using the TEMPO-oxidation system followed by sonication for different times. The content of carboxylate groups in the CMFs was unaffected by the duration of the sonication treatment. However, the sonication time influenced the proportion of CNFs in the CMF suspension, which in turn influenced the internal structure of the hydrogels. Increasing the sonication time resulted in an increase of the storage modulus, compression strength, and surface area of the microgels, while the calculated fibril diameter decreased. Extending the sonication time did not influence the chemical structure and cellulose crystallinity of the hydrogels. Thus, the internal structure of the hydrogel had a significant impact on the swelling and drug release behavior of the hydrogels, indicating that the internal structure could be controlled based on the sonication time to manipulate the release of drugs from the hydrogels. Acknowledgments This research was supported by the Korea–Italy Bilateral Joint Research Program through the National Research Foundation (NRF) of Korea and funded by the Ministry of Education, Science, and Technology (grant no. 2013K1A3A1A25037202). Valerio Causin gratefully acknowledges the support of the Italian Ministry of Foreign Affairs, Direzione Generale per la Promozione del Sistema Paese. References [1] C. Chang, L. Zhang, Carbohydr. Polym. 84 (2011) 40–53. [2] L. Alexandrescu, K. Syverud, A. Gatti, G. Chinga-Carrasco, Cellulose 20 (2013) 1765–1775. [3] M.M. Abeer, M.C.I.M. Amin, C. Martin, J. Pharm. Pharmacol. 66 (2014) 1047–1061. [4] A.M. Bochek, Fibre Chem. 40 (2008) 192–197.
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