Viscoelastic properties of physical cellulosic bionogels of cholinium lysinate M. Mar Villar-Chavero ⁎, Juan C. Domínguez, M. Virginia Alonso, Victoria Rigual, Mercedes Oliet, Francisco Rodriguez Chemical Engineering and Materials Department, Complutense University of Madrid, Av. Complutense S/N, 28040 Madrid, Spain Abstract Novel ionogels with different cellulose contents, namely, 0.5, 1, 1.5 and 2 wt%, were formulated with cholinium lysinate (ChLys), and the rheological properties were evaluated at 3 and 7 days postgelation. Because of the biobased compounds contained in these ionogels, in this work, they are denoted as bionogels. These materials have great potential to yield functional biomaterials for use in the medical/pharmacological sector. Some knowledge of how cellulose is dissolved in ChLys was necessary to formulate the bionogels. The dissolution time was studied for each bionogel, with the dissolution times being 3, 4, 4.5, and 6.5 h for 0.5, 1, 1.5, and 2% cellulose, respectively. The bionogel with a 2% cellulose load had the highest rheological properties, i.e. elastic modulus (G′), loss modulus (G″) and complex viscosity (η*), on the studied postgelation days: G′ (3 days): 0.7–50 kPa, G′ (7 days): 1–100 kPa, G″ (3 days): 0.1–10 kPa, and G″ (7 days): 0.2–20 kPa, η* (3 days): 0.2–200 kPa s and η* (7 days): 0.4–300 kPa s. The postgelation time is an important parameter in the formulation of bionogels, since at 3 days postgelation, the networks continued to be constituted. Regarding classification, these bionogels were weak physical gels. 1. Introduction In recent years, third-generation ionic liquids have acquired great interest due to their high biodegradability and low toxicity [1]. Within this generation are cholinium amino acid ionic liquids (ChAAILs), which are composed of a cholinium cation (N,N,N-trimethylethanolammonium) and an amino acid anion [2,3]. Choline is an essential micronutrient that participates in different biological functions; therefore, it is a biocompatible and nontoxic biocompound [4]. Amino acids are biomolecules obtained from natural and renewable raw materials and are one of the most abundant classes of organic compounds in nature [2,5]. Therefore, ChAAILs are considered biodegradable, biocompatible, nontoxic, and more cost-effective relative to conventional ionic liquids [6,7]. The application of ChAAILs is focused on the industrial (e.g., green catalysis, CO2 capture), biomass processing (for biomass pretreatment), and pharmaceutical/medical chemistry (e.g., biosensors, drug delivery) sectors [2,3]. For applications in pharmaceutical/medical chemistry, the study and development of new materials has acquired much relevance in recent years [1]. Ionogels are gel materials comprised of two components, an ionic liquid as the dispersed phase and a continuous solid phase consisting of organic (e.g., biopolymers), inorganic (e.g., metals, ceramics), or hybrid organic-inorganic compounds [8,9]. The nature of the interactions between the ionic liquid and the solid phase can be physical or chemical. Physical interactions are noncovalent, such as hydrogen bonds, π-π interactions or van der Waals forces; meanwhile, chemical interactions are covalent bonds and, therefore, form irreversible 3D networks [9]. Depending on these types of interactions, ionogels exhibit different thermal and viscoelastic properties [10]. Cellulose, a polysaccharide consisting of glucose units linked through β-(1–4) glycosidic linkages, is a perfect candidate for the solid phase of ionogels. The solubility of cellulose in conventional ionic liquids and, therefore, in the formation of cellulosic ionogels is well-known and has been previously reported [11–14]. This solubilization lies in the ability of the conventional ionic liquid to form hydrogen bonds with cellulose, i.e., through physical interactions [15]. The same ability has been found by Scarpellini, Ortolani, Nucara, Baldassarre, Missori, Fastampa and Caminiti [16] for ChAAILs, and they proposed an interaction mechanism between cellulose and cholinium glycinate (ChGly) based on hydrogen bonds between NH2౼ and COO– of glycine and\\OH of cellulose [16]. For this reason, novel biobased ChAAILs-cellulose ionogels can be developed, and are proposed to be denoted as bionogels in the present work. The study of viscoelastic properties is of great importance for gel-like materials due to the fact that the mechanical properties of these materials are determined, and it is possible to evaluate the mechanism and factors involved in the gelation process [17]. Moreover, bionogels formulated with ChAAILs have not been developed or rheologically characterized to date. The aim of this research is to formulate novel cellulosic bionogels containing cholinium lysinate (ChLys), a ChAAILs, and to study the influence of the cellulose load and the evolution over time on thei rheological properties. In this way, nontoxic, biodegradable, and biocompatible bionogels can be developed for the production of functional biomaterials with potential uses in the medical/pharmaceutical sector. 2. Experimental section 2.1. Materials The materials used for the formulation of the bionogels were ChLys with a purity of 90%, acquired from Iolitec GmbH, and Avicel® PH-101 microcrystalline cellulose with a degree of polymerization of 230, obtained from Sigma-Aldrich. Both compounds were dried in a vacuum oven for 12 h at 40 °C before formulation of the bionogels. 2.2. Dissolution of cellulose in ChLys Microcrystalline cellulose was dissolved in ChLys under magnetic stirring (400 rpm) at 100 °C under an N2 atmosphere. The percentages of cellulose added to obtain each bionogel were 0.5, 1, 1.5, and 2 wt%. The cellulose was added in one dosage, excepting the concentrations of 1.5 and 2%, for which the cellulose was introduced in two equivalent doses to avoid aggregates and improve the dissolution process. Aliquots were taken every 30 min and visualized under a Carl Zeiss Axio Scope A1 microscope equipped with a Zeiss AxioCam ICc1 to determine the dissolution time for each cellulose concentration. 2.3. Formulation of bionogels The formulation of the bionogels is schematized in Fig. 1. This formulation was conducted under the same conditions used for the dissolution of cellulose (100 °C, 400 rpm and inert atmosphere). Fully dissolved cellulose solutions were poured into a steel plate covered with PET film at room temperature and kept until to gelation. After 3 and 7 days, samples of 25 mm in diameter were cut to evaluate the rheological properties of the bionogels Fig. 1. Schematic of the process followed to obtain the cellulosic bionogels 2.4. Rheological characterization Rheological characterization was conducted with an ARES rheometer (TA Instruments) under isothermal conditions, using serrated parallel plates of 25 mm in diameter and a gap of 2 mm. The linear viscoelastic region of the bionogels was determined through a strain sweep test. These tests were conducted in the strain range of 0.007 to 0.3% at 25 °C and 50 Hz (the maximum frequency used in the dynamic frequency tests). Previously, the stability of the bionogels was checked by time sweep tests (data not shown). The rheological spectra were generated from dynamic frequency sweep tests conducted from 0.01 to 50 Hz with a fixed strain of 0.01% (within the linear viscoelastic region) at 25 °C. These spectra were obtained at 3 and 7 days postgelation (from the day the bionogel was developed) to evaluate the influence of time on the viscoelastic properties of the bionogels. 2.5. Attenuated total reflectance Fourier Transform infrared (FTIR/ATR) analyses Attenuated total reflectance Fourier Transform infrared (FTIR/ATR) spectra of the cellulose, ChLys, and the bionogels were recorded to test the type of interaction between cellulose and ChLys. A Jasco 4700 spectrometer with a Golden Gate TM diamond accessory Speac 10542 was employed. FTIR/ATR spectra were measured between 4000 and 800 cm−1 , using 64 scans and a resolution of 2 cm−1 . 3. Results and discussion 3.1. Dissolution time of cellulose in ChLys Some of the images acquired to determine the dissolution times of each concentration of cellulose in ChLys are shown in Fig. 2. Cellulose microparticles could be observed at short times of dissolution (1.5 and 3 h) for all cellulose concentrations. For all samples, the amount of cellulose microparticles observed decreased as the time increased until complete dissolution was reached (Fig. 2). This behavior and the capability of ChLys to dissolve cellulose are also found in the literature [18]. The dissolution times for the studied concentrations are shown in Fig. 3. The complete dissolution times became longer as the cellulose load was increased. This effect was because the viscosity was greater when the amount of dissolved cellulose was increased, and therefore, the dissolution required more time to complete [19] Fig. 2. Microscope images of the dissolution of cellulose in ChLys at different concentrations and times. Certain particles have been marked with red circles to denote their presence in the microscope images Fig. 3. Dissolution times of cellulose in ChLys at different cellulose concentrations. 3.2. Linear viscoelastic region of the bionogels (LVR) The linear viscoelastic regions (LVRs) associated with each bionogel were determined through strain sweep tests. The results obtained at 3 and 7 days postgelation are shown in Fig. 4. Fig. 4. Strain sweep tests for all bionogels at 50 Hz and 25 °C at a) 3 days and b) 7 days postgelation. In the LVR, the elastic modulus (G′) is almost constant throughout variation of the strain. In this region, the internal structure network of the material does not change with applied strain. The limits of the LVRs (γc) for the bionogels with cellulose concentrations of 1, 1.5, and 2% at 3 days postgelation were 0.034, 0.027, and 0.024%, respectively. Thus, the bionogels with 1.5 and 2% cellulose exhibited similar γc values, which were lower than that of the 1% cellulose bionogel. For the bionogel with 0.5% cellulose, the γc value was not observed within the studied range. This means that the bionogel with 0.5% cellulose presented the most deformability (with deformability understood as the ability to accept high strains without modifying the internal structure), followed by the bionogel with concentration of 1% cellulose, and, finally, the bionogels with 1.5 and 2% cellulose. At 7 days postgelation, the γc values of the bionogels with 1, 1.5, and 2% cellulose were similar: 0.022, 0.018, and 0.019%, respectively. The exception was the bionogel with 0.5% cellulose, whose γc was not reached within the strain range used in the test, which was similar to the case observed at 3 days postgelation; therefore, this bionogel presented an LVR for all strains used in the test. Consequently, the bionogel with 0.5% cellulose did not present a clear yielding behavior within the studied range [20], since its structure had a weak network, and therefore, it exhibited the most capacity for deformation in comparison with the other samples, which were more solid-like. These different results obtained at 3 and 7 days postgelation indicated that for 3 days, the rheological properties of the bionogels were evolving, i.e., the gel networks were not fully constituted. In general, the obtained γc values were lower than those found for natural biopolymer gels and conventional ionogels containing natural biopolymers, which present γc values of ~1% and 1.1–1.8%, respectively [21,22]. Thus, in the present work, the formulated bionogels had less capacity for deformation than do these types of gels. 3.3. Rheological spectra The rheological spectra showed that the elastic modulus (G′) was higher than the loss modulus (G″), and G′ was almost constant in all cases (Fig. 5). Therefore, the formulated bionogels at 3 and 7 days postgelation exhibited dominant elastic behavior. For this reason, the studied region of frequencies in the bionogels spectra belonged to the plateau region [23], i.e., the internal structure of the bionogels is gel type because there were interactions between cellulose and Chlys Fig. 5. Rheological spectra of the formulated bionogels at 3 days (a and b) and 7 days postgelation (c and d). At 3 days postgelation, there was a strong influence of the cellulose content. The bionogel with 2% cellulose (Fig. 5a) exhibited the highest values of G′, e.g., at 4 Hz, the G′ values were 1.5, 2.8, 7.7, and 30 kPa Table 1 Values of parameters of the model G′ ~ ωa , the ratios of G′/G″ at 4 Hz and 45 Hz, for the cellulosic bionogels at 3 and 7 days of post-gelation. Cellulose concentration (%) a G0 (Pa) R2 G′/G″ (4 Hz) G′/G″ (45 Hz) 3 days 2 0.129 ± 0.003 25491 ± 1 0.961 6.5 3.2 1.5 0.060 ± 0.003 7450 ± 1 0.864 6.2 2.6 1 0.091 ± 0.004 2649 ± 1 0.863 4.0 1.8 0.5 0.157 ± 0.004 1336 ± 1 0.950 2.6 1.7 7 days 2 0.167 ± 0.005 49805 ± 1 0.938 5.6 3.1 1.5 0.171 ± 0.005 21526 ± 1 0.934 4.1 2.5 1 0.144 ± 0.008 15536 ± 1 0.802 5.5 3.4 0.5 0.142 ± 0.003 2190 ± 1 0.966 2.9 1.2 for the bionogels with 0.5, 1, 1.5, and 2% cellulose, respectively. In this respect, the bionogel with 2% cellulose presented the most solid-like behavior. Regarding G″, the trend of the results was the same (Fig. 5b). For example, at 4 Hz frequency, the values of G″ were 0.6, 0.7, 1.2 and 4.6 kPa for the bionogels with 0.5, 1, 1.5 and 2% cellulose, respectively; therefore, the bionogel with 2% cellulose had the most viscous behavior. For all samples, the values of G′ were greater than those of G″. Note that this influence was greater when the cellulose load was lower. At 7 days postgelation, the values of G′ for the samples containing 0.5, 1, 1.5, and 2% cellulose at 4 Hz were 2, 18, 26, and 61 kPa, respectively. The G′ values presented the same behavior as that exhibited at 3 days postgelation (Fig. 5c), i.e., as the cellulose load was increased, the G′ increased, with exception of the sample with 1% cellulose, whose behavior was similar to that of the bionogel with 1.5% cellulose. In the case of G″, the same tendency detected for G′ was observed (Fig. 5d), in that the bionogel with 2% cellulose exhibited higher values of G″, e.g., at 4 Hz, the values were 0.8, 3.2, 6.3, and 11 kPa for the bionogels with 0.5, 1, 1.5, and 2% cellulose, respectively. As observed at 3 days postgelation, the values of G′ were greater than those of G″. The postgelation time had an influence on the studied rheological properties. Thus, at 7 days postgelation, all moduli increased compared to the values at 3 days postgelation. For instance, for the bionogel with a 2% cellulose load, at 3 days, the values of G′ (4 Hz) and G″ (4 Hz) were 51 and 58% lower, respectively, than those at 7 days. This could have been due to the interactions between ChLys and cellulose having increased because of the longer interaction time, i.e., at 3 days, the networks were not fully constituted (as mentioned in Section 3.2). The formulated bionogels showed rheological properties and spectra similar to those obtained by different authors for other gels, such as hydrogels, for use in the medical/pharmaceutical sector. Slavutsky and Bertuzzi [24], Lee, Kim and Jeong [25], and Quah, Smith, Preston, Laughlin and Bhatia [26] developed hydrogels containing pectin and brea gum, hyaluronic acid, and alginate, respectively, obtaining rheological spectra with shapes similar to those obtained in this work. In addition, the moduli acquired by Quah, Smith, Preston, Laughlin and Bhatia [26], who reported G′ values between 0.2 and 10 kPa and G″ values from 0.02 to 1 kPa, were consistent with those obtained in this research (G′: 0.7–100 kPa, G″: 0.1–20 kPa). Thus, these novel bionogels can potentially be used in this sector to obtain functional biomaterials. 3.4. Gel strength and classification of bionogels A power-law model (Eq. (1)) was applied to the obtained elastic moduli (G′) to model its response to the frequency, according to the polymer dynamics theory [27] as follows: G’ = G0 ωa where G0 (Pa·sa ) is the gel strength, the measurement of the elastic energy stored in a unit volume of the network, which is the intercept with the log G′-axis (Eq. (2)) [28], and a is the slope of the log G′-log frequency curve, which is particular to each material. The parameters of this modeling are shown in Table 1. G0 = limω→0 G’(ω) The interpretation of the parameter a from the point of view of the internal structure of the material is associated with the type of interactions inside the material. Thus, if a is zero, the material is formed by covalent interactions, a situation known as a “true gel”. Contrarily, if a is N0, physical interactions are responsible for maintaining the gel-like structure [17,28]. On the studied days postgelation, the values of parameter a were higher than 0; therefore, the formulated bionogels were physical gels. On the other hand, the gel strength, G0, varied as the cellulose load and postgelation time changed, as shown in Table 1. Thus, as the cellulose load in the bionogel was increased, the strength became greater, to such an extent that this parameter for the bionogel with 2% cellulose was 1808 and 2174% higher than that of the 0.5% cellulose bionogel at 3 and 7 days postgelation, respectively. These results suggested that as the cellulose load was increased, the interactions between cellulose and ChLys became higher. These interactions were physical due to the hydrogen bond formed between the amines and carboxylate of the ChLys with the hydroxyl group of the cellulose chain. Electrostatic interactions between the nitrogen of choline and the terminal oxygen of lysine could also occur, both with cellulose and with itself, which is in concordance play a fundamental role in the gelation of bionogels due to their hygroscopic nature, helping in the formation of physical crosslinking points [13]. The proposed plausible mechanism of cellulosic bionogels formation is shown in Fig. 6. The influence of the postgelation time on G0 was the same as that on the elastic and loss moduli. Fig. 6. Plausible mechanism of cellulosic bionogels formation with ChLys: (a) dissolution of cellulose and (b) interactions in the bionogel. To ensure that the interaction forces in the bionogels were physical, FTIR/ATR tests were performed. The spectra of the cellulose and ChLys exhibited the characteristic bands of these compounds (Fig. 7). The cellulose spectrum showed bands between 3400 and 3200 cm−1 corresponding to O-H stretching vibrations. The band at 1433 cm−1 is associated with the intermolecular hydrogen attraction at C6 of the aromatic ring group. The bands at 1160 and 1110 cm−1 are due to C-O-C stretching and ring asymmetry stretching, respectively [29,30]. On the other hand, ChLys spectrum exhibited a broad band between 3450 and 3100 cm−1 associated with O-H and N-H stretchings. The band at 1565 cm−1 corresponds to asymmetric stretching of C-O. The 1475 cm−1 band is associated with C-H stretching vibration of CH3. In addition, the band at 1389 cm−1 is observed due to the CO2 symmetric stretching. At 1091 cm−1 the band corresponding to the CHn rocking/twisting is detected. Finally, the band at 955 cm−1 is related to C-N stretching [31]. The interaction between the cellulose and ChLys was physical because the spectra of the formulated bionogels showed overlapped bands corresponding to the cellulose and ChLys bands. In addition, there were no new bands indicating the formation of chemical bonds. Note that as the cellulose load was increased the characteristic bands of cellulose in the bionogels spectra were intensified. Fig. 7. FTIR/ATR spectra of the cellulose, ChLys and the formulated bionogels To verify the physical interactions in the bionogels, they were washed with water (a more polar solvent than ChLys). In Fig. 8a, it can be observed that immediately after putting the bionogels in water, the ChLys free of interactions with cellulose was removed from the bionogel, and therefore, as the cellulose load increased, the opacity of the bionogel increased. Water replaced ChLys in its position through the creation of new hydrogen bonds, forming hydrogels. This effect can be observed in Fig. 8b. The same behavior was found for cellulosic bionogels formulated with conventional ionic liquids [18,22]. Fig. 8. Images of the bionogels submerged in water (a) and the formed hydrogels (b) Regarding the classification of these novel bionogels, the relation G′/ G″ was evaluated. Table 1 contains the G′/G″ ratios at 4 Hz as an example of this relation at a certain frequency for assessment. Additionally, the G′/G″ ratio at 45 Hz has also been included, with the objective of evaluating the behavior at high frequencies of the spectrum. According to the G′/G″ ratios at 4 Hz, the bionogels can be classified as weak physical gels because these ratios ranged between 2.6 and 6.5 (N10 corresponds to strong gels, and b10 is related to weak gels [32,33]). This type of gel is similar to that found by others authors examining different biopolymer-based ionogels, containing compounds such as agarose or guar gum [34,35]. Although the concept of a weak gel is applied for all formulated bionogels and for both studied postgelation times, as the percentage of cellulose varied, there were different levels of “weakness”. Thus, the bionogel with 2% cellulose presented as a well structured gel, with G′/G″ (4 Hz) ≈ 7 at 3 days and G′/G″ (4 Hz) ≈ 6 at 7 days, whereas the bionogel with 0.5% cellulose had G′/G″ values of approximately 2.5 and 2 times less than that of the 2% cellulose bionogel at 3 and 7 days postgelation, respectively. Therefore, the bionogel with 0.5% cellulose was the weakest bionogel at the studied postgelation times. These results are corroborated by the values of the G′/G″ ratio obtained near the end of the test (45 Hz) because the vicinity of the G′ and G″ of this bionogel was higher than that of other bionogels at this frequency. This means that this bionogel is close to the leathery region at high frequencies [23]. Cholinium-based chemical cellulosic ionogels have been found in the literature, with moduli approximately 1000 and 200 Pa for elastic and loss moduli, respectively [4,36]. These values are lower than those obtained in this work for physical cellulosic bionogels. Thus, ChLysbased bionogels showed greater rheological properties than do chemical cholinium-based ionogels Table 2 Parameters of the power-law model of the complex viscosity of the bionogels Cellulose concentration (%) q (Pa·sp ) p R2 3 days 2 8058 ± 1 0.170 ± 0.005 0.997 1.5 3516 ± 1 0.177 ± 0.006 0.997 1 2507 ± 1 0.138 ± 0.008 0.994 0.5 369 ± 1 0.160 ± 0.004 0.998 7 days 2 4137 ± 1 0.131 ± 0.003 0.999 1.5 1202 ± 1 0.060 ± 0.003 0.999 1 440 ± 1 0.105 ± 0.005 0.997 0.5 226 ± 1 0.173 ± 0.005 0.997 3.5. Complex viscosities of the bionogels The results of the complex viscosities (η*) for the range of studied frequencies are shown in Fig. 9. Fig. 9. Complex viscosity (η*) of the cellulosic bionogels at 3 (a) and 7 (b) days postgelation. All cellulosic bionogels studied on different days postgelation presented a pseudoplastic-like behavior because the viscosity decreased when the frequency was increased [22]. The complex viscosities were fitted to a power-law model, as follows: η*= q ωp−1 where q (Pa·sp ) provides the resistance to deformation at frequencies near zero, and p is a measure of the behavior of the material relative to the frequency. The parameters of the model of complex viscosities using the power law (Eq. (3)) are shown in Table 2. The results of the parameter p confirmed this pseudoplastic behavior (p b 1) for all formulated bionogels. The complex viscosity of the materials exhibited a strong dependence on the cellulose load. The parameter q values, which are a measure of the consistency of the material, increased as the cellulose load in the bionogel became greater. For instance, the bionogels with 1.5, 1 and 0.5% cellulose showed q values of 3, 2, and 21-fold at 3 days and of 3, 9 and 18-fold at 7 days postgelation, respectively, those of the bionogel with 2% cellulose. These data are consistent with the η* values obtained in a previous work for cellulosic physical ionogels (q: 462–2500 Pa·sp , η*: 0.01–300 kPa) [22]. The obtained results confirm that a higher cellulose load provides more consistency because more hydrogen bonds are formed. The effects of the postgelation time on the studied parameters were the same as those found in the studied spectra. 4. Conclusions Novel cellulosic bionogels were developed successfully using ChLys along with cellulose loads of 0.5, 1, 1.5, and 2 wt%. The obtained rheological properties of these bionogels revealed a strong dependence on the cellulose load, with the bionogel containing 2% cellulose being the most consistent and strongest and exhibiting the most solid-like behavior among the formulated bionogels. The interactions between ChLys and cellulose were physical and became more effective as the cellulose load in the bionogel was increased. The formulated bionogels were classified as weak physical gels due to the relation of their moduli. Due to the biocompatible and biodegradable characteristics of their compounds, these bionogels have great prospect for use in the medical/ pharmacological sector. Acknowledgments The authors are grateful for the financial support of the “Ministerio de Ciencia, Innovación y Universidades” under the funded project CTQ2017-88623-R and Victoria Rigual thanks the contract BES-2014- 067788. 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