Ion-Mediated Gelation of Aqueous Suspensions of Cellulose

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Ion-Mediated Gelation of Aqueous Suspensions of Cellulose Nanocrystals Mokit Chau, Shivanthi E. Sriskandha, Dmitry Pichugin, Héloïse Thérien-Aubin, Dmitro Nykypanchuk, Grégory Chauve, Myriam Methot, Jean Bouchard, Oleg Gang, and Eugenia Kumacheva Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00701 • Publication Date (Web): 23 Jun 2015 Downloaded from http://pubs.acs.org on July 4, 2015

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Ion-Mediated Gelation of Aqueous Suspensions of Cellulose Nanocrystals Mokit Chaua, Shivanthi E. Sriskandhaa, Dmitry Pichugina, Héloïse Thérien-Aubina, Dmitro ,b Nykypanchuk,b Grégory Chauvec, Myriam Méthotc, Jean Bouchardc, Oleg Gangb, Eugenia Kumachevaa,d,e*

a

Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto, Ontario M5S 3H6, Canada

b

Center for Functional Nanomaterials, Brookhaven National Laboratory, Upton, New York 11973, United States c

d

FPInnovations, 570 St. Jean Boulevard, Pointe-Claire, Québec H9R 3J9, Canada

University of Toronto, Department of Chemical Engineering and Applied Chemistry, 200 College Street, Toronto, Ontario M5S 3E5, Canada

e

University of Toronto, The Institute of Biomaterials and Biomedical Engineering, 4 Taddle Creek Road, Toronto, Ontario M5S 3G9, Canada

Keyword: Cellulose nanocrystals, nanofibrillar hydrogels, ions, structure-property relationship

ACS Paragon Plus Environment

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ABSTRACT

Nanofibrillar hydrogels are an important class of biomaterials, with applications as catalytic scaffolds, artificial extracellular matrixes, coatings and drug delivery materials. In the present work, we report the results of a comprehensive study of nanofibrillar hydrogels formed by cellulose nanocrystals (CNCs) in the presence of cations with various charge numbers and radii. We examined sol-gel transitions in aqueous CNC suspensions, and the rheological and structural properties of the CNC hydrogels. At a particular CNC concentration, with an increasing charge and size of a cation, the dynamic shear moduli and the mesh size in the hydrogel increased, which was ascribed to a stronger propensity of CNCs for side-by-side association. The resulting hydrogels had an isotropic nanofibrillar structure. A combination of complementary techniques offered insight into structure-property relationships of CNC hydrogels, important for their potential applications.


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INTRODUCTION Many biological polymers, including proteins and polysaccharides, form hydrogels by the reversible physical association or entanglement of high aspect-ratio structural units called nanofibrils, strands, or filaments.1,2 Nanofibrils are built by the hierarchical assembly of many molecules, and have diameters in the range from tens to hundreds of nanometers and lengths up to micrometers. The assembly of nanofibrils in networks is generally driven by hydrogen bonding, hydrophobic forces, electrostatic forces, or van der Waals interactions. If the concentration of nanofibrils in suspension is sufficiently high and their persistence length is not too large, networks can be formed via entanglement of nanofibrils. The hierarchical nature of nanofibrillar hydrogels strongly impacts their mechanical properties, nonlinear viscoelastic behaviour,3 pore size,4 and thermal stability.5 Nanofibrillar gels formed by biopolymers are generally biocompatible, biodegradable and non-cytotoxic. These properties make them promising candidates for applications in catalytic scaffolding,6 templating polymer composites,7 drug delivery,8 and tissue engineering,9 to name a few. Shape-anisotropic, high-aspect ratio cellulose nanocrystals (CNCs) have recently gained great interest in the materials science and nanoscience fields. These nanoparticles are composed of cellulose molecules packed in a parallel fashion with a helical twist and are held together by hydrogen bonds.10 The diameter and length of CNCs are typically in the range of 10-30 and 50500 nm, respectively, depending on their source. They have a high degree of crystallinity (5488%) and excellent mechanical properties, with an estimated tensile strength of 110-220 GPa. Furthermore, CNCs bear surface hydroxyl groups, which can be used for their chemical functionalization with low-molecular weight molecules or polymers.11 Importantly, CNCs are environmentally friendly, abundant in nature, and inexpensive.11


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Gelation of aqueous CNC suspensions has been achieved in several ways. For example, an increase in CNC concentration in the suspension has led to the formation of lyotropic liquid crystalline gels.12 Alternatively, composite hydrogels have been formed from a mixture of CNCs with a gelling component, e.g., methylcellulose,13 hydroxyethyl cellulose, or hydroxypropyl guar.14 Other methods of the preparation of CNC gels utilized a reduction in electrostatic repulsion between CNCs. More specifically, preparation of CNCs by acid hydrolysis of wood pulp results in CNCs with surface anionic sulphate groups. Electrostatic repulsion between these anionic groups renders CNCs colloidally stable. Desulphation of the CNC surface by heating their suspension in the presence of glycerol led to a decrease in CNC stability and favoured attraction between them, thereby yielding thixotropic CNC hydrogels.15 In this process, however, the replacement of water with glycerol may limit the range of bio-related applications of the CNC hydrogels. An alternative method relies on increasing the ionic strength of CNC suspensions by adding salts. The addition of salts reduces the Debye length of CNCs and suppresses electristatic repulsion between them, thereby leading to dominant attractive CBC interactions, e.g., van der Waals forces and hydrogen bonding. For example, CNC hydrogels have been formed upon the addition of NaCl to an aqueous CNC suspension.16 Gelation using multivalent cations has been studied for 500-2000 nm long carboxylatedecorated cellulose nanofibrils,17 which were significantly longer than CNCs and contained alternating amorphous and crystalline domains. The storage moduli of such hydrogels increased with increasing cation charge number, which enabled the tuning of their mechanical properties. To the best of our knowledge, a systematic study of the properties and structure of CNC hydrogels formed in the presence of cations with different charge numbers and dimensions has not been reported, although CNC gels can offer improved colloidal stability, enhanced


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mechanical properties and propensity for alignment under shear, in comparison with cellulose nanofibrils. In the present work, we report the results of a comprehensive experimental study of CNC hydrogels formed by the addition of cations with varying charge numbers and ionic radii to aqueous CNC suspensions. For these hydrogels, we examined their rheological properties by oscillatory rheometry and structure by scanning electron microscopy, NMR, polarisation optical microscopy, and small-angle X-ray scattering. For a particular CNC concentration, hydrogel stiffness increased with increasing charge number and ionic radius of the added cations. The increase in gel stiffness was accompanied by the increase in mesh size, in contrast to the prediction of conventional poroelastic theory of molecular gels. Both features were attributed to the stronger side-by-side CNC association in the presence of added cations, which led the formation of a stiffer network. As a result, the mechanical properties of the CNC gels could be accurately tuned by varying the type and the concentration of the cations. The established structure-property relationships have important implications for the use of CNC gels as drug delivery vehicles and a scaffolds for tissue engineering.

EXPERIMENTAL Materials An aqueous CNC suspension of 6.43 % w/w was provided by FPInnovations (Quebec, Canada). NaCl, AlCl3 (anhydrous) and SrCl2 (anhydrous) were purchased from Acros Organics. MgCl2 (anhydrous) was supplied by Alfa Aesar. CaCl2 (anhydrous) was purchased from Fisher Scientific. Dialysis membrane (MWCO: 6000 Da) was purchased from Spectrum Laboratories,


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Inc. Dextran analytical standard (Mw=147600 kDa, Mw/Mn=1.47) was purchased from SigmaAldrich. Deuterium oxide (D2O) was supplied by Cambridge Isotope Laboratories, Inc., USA. Deionized water was obtained from a Millipore Milli-Q water purification system. Methods Preparation of CNC suspensions An aqueous suspension with a CNC concentration, CCNC, of 6.43 % w/w was dialyzed against deionized water. The water-to-suspension volume ratio was 100:1, and over the course of dialysis, the water was changed 6 times (the first and the second water changes were 2 and 4 h and subsequent changes were 12 h after the beginning of dialysis, respectively). After dialysis, the CNC suspension was filtered using No. 41 and 42 Whatman filter papers, and reconcentrated by evaporating water under ambient conditions until CCNC=5.53 % w/w was reached. This suspension was diluted with deionized water to prepare CNC suspensions with the required concentrations.

Preparation of CNC gels Hydrogels of CNCs were prepared by adding a metal chloride, that is, NaCl, MgCl2, AlCl3, CaCl2 or SrCl2 solution to a CNC suspension to reach salt molality in the CNC hydrogel in the range from 1 to 50 mm (we expressed concentrations in molality (m), that is, the number of moles of solute dissolved in 1 kg of solvent). Suspensions with CNC concentration, CCNC, in the range from 0.5 to 4 % w/w were used. Immediately after salt addition, the mixture was shaken. To characterize sol-gel transitions and plot the state diagrams, an inversion test was used.

Scanning electron microscopy


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The structure of CNC gels was imaged using scanning electron microscopy (SEM). Supercritical point drying was used to prepare hydrogel samples. First, a CNC gel was placed in a microporous specimen capsule (30 µm pore size, Canemco-Marivac). Next, water was gradually replaced with methanol by consecutively submerging the capsules into 20, 40, 60, 80 % (v/v) methanol/water mixtures and finally, in pure methanol. Afterwards, the capsule with the CNC gel was placed in an Autosamdri-810 Tousimis critical point drier. The methanol in the sample was exchanged with liquid CO2, which was subsequently brought to a supercritical state and removed by slow venting. The dried gels were sputter-coated with gold and imaged using a Quanta FEI 250 scanning electron microscope (5 kV).

Characterization of rheological properties of CNC gels The rheological properties of CNC gels were studied using an ARES (TA Instruments) rheometer with a parallel plate geometry. The diameter of the plates and the gap between them were 25 mm and 1 mm, respectively. Amplitude strain sweeps were performed to determine the range of linear viscoelastic region (Supporting Information). Following this experiment, dynamic frequency sweeps were performed with oscillatory frequencies between 0.1 to 100 rad/s at a constant strain of 0.5 %.

Polarized optical microscopy Gels and suspensions of CNCs were imaged using polarized optical microscopy (POM). The CNC suspensions at CCNC=4 % w/w were prepared as described above. The CNC suspensions at CCNC=2 % w/w were prepared by diluting the CNC suspension of 2.67 % w/w with deionised water. Gelation was induced by adding a metal chloride solution to a suspension of CNCs. A 0.2


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mm-thick sample of CNC gel was confined between two glass slides to prevent water evaporation and placed between two cross-polarizers (U-AN360 and UP110 U-POT, Olympus, Japan). The sample was imaged using a Canon, EX-F1 digital camera. Determination of mesh size of CNC gels The mesh sizes in CNC hydrogels were determined using pulsed field gradient-NMR (PFG NMR) by measuring the diffusion coefficients of dextran molecules in solution and in CNC gels (Do and D, respectively) 18 Dextran was used as a probe because it does not interact with CNCs.14 The hydrodynamic radius of the dextran molecule was determined by dynamic light scattering (Malvern Zetasizer Nano ZS). A suspension of CNC (0.75 g) in H2O at CCNC of 2.67 or 5.33 % w/w was added into a NMR tube. Then, 0.25 g of a 5 or 50 mm solution of salt in H2O was added and the content of the tube were mixed. After gel formation, 1 g of a 20 mg/mL dextran solution in D2O was introduced in the NMR tube above the gel. The sample was allowed to equilibrate overnight, and the supernatant solution was removed. The NMR spectra were acquired using an Agilent DD2 500MHz instrument equipped with a OneNMR™ direct detect probe with 1H 90°-pulse width of 8.4 µs. Bi-polar pulse pair stimulated echo sequence was used as supplied by Agilent. For the CNC gels, a 2 s saturation pulse was used to suppress water signal. The recycle delay was set to be five-fold the longitudinal relaxation time of the dextran in the gel. The gradient pulse length, δ, was 7 µs and the diffusion delay, ∆, was set to 300 ms. The gradient strength, g, was varied in 15 increments from 1.9 G/cm to a gradient strength that yielded 85-90 % attenuation of the dextran signals around 45 G/cm. All spectra were collected using 32 scans per gradient strength with 8 steady states, acquisition time of 4.5 s, 6 s recycle delay, and 8 kHz spectral window centered on


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the water signal. Attenuations were also measured for dextran in D20 at 10 mg/mL. The signal attenuation was fitted, using Origin, to the following equation

I = exp −γ 2δ 2 g 2 (∆ − δ / 3)D' I0

(1),

where Io and I are the intensities of the 3.79 ppm dextran signal at zero gradient and at different gradient amplitudes, respectively. The γ, δ, g, and ∆ are the gyromagnetic ratio of the observed ratio (1H), gradient pulse magnitude, gradient pulse length, and diffusion time, respectively. D' is the fitting parameter equal to the diffusion coefficient of a probe. The ratio of D/D0 was related to the hydrodynamic radius of the probe, Rh, the radius of CNC fibrils forming the mesh (Rf), and the radius of the opening between the fibrils, Rp, (half the mesh size) as follows19 2    R + R D π h   = exp −  f  4  R f + Rp   D0  

(2).

A hydrodynamic radius of dextran was determined using dynamic light scattering (Supporting Information). The average mesh sizes of each CNC gel was calculated as 2Rp and was determined from a set of triplicate experiments.

Small-angle X-ray Scattering High-resolution synchrotron-based SAXS measurements were performed at the X9 beamline at the National Synchrotron Light Source, Brookhaven National Laboratory (U.S.A.) For measurements, a CNC gel was placed inside a 1 mm-diameter quartz capillary (Charles Supper, MA) and data were collected using a PILATUS detector. Camera length was calibrated against silver behenate. The data were corrected for sample absorption using a semitransparent beam stop and the background was corrected for water and capillary scattering.


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RESULTS Ionically mediated gelation of CNC suspensions Gelation of the dialyzed CNC suspensions was induced by adding metal chloride solutions of NaCl, CaCl2, MgCl2, SrCl2, or AlCl3. The final salt concentration in the suspension varied from 1 to 50 mm (millimolal), while CCNC, was varied from 0.5 to 4 % w/w. Table 1 (left column) summarizes the notations used in the present work. For example, the Ca50-4 gel was prepared at 50 mm concentration of CaCl2 and at CCNC = 4 % w/w. Table 2 also shows the charge numbers and ionic radii of the added cations.

Table 1. Nomenclature used for the CNC samples and properties of the cations


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Sample

Salt added

Cation charge number

Cation radius* (Å)

Solubility of Salt metal sulphates concentratio in water** n in the gel (g/100g)

(mm)

CNC concentration in geling suspension (% w/w)

CNC0-4

--

--

--

--

0

4.0

CNC0-2

--

--

--

--

0

2.0

Na50-4

NaCl

1+

1.02

28.1

50

Mg50-4

MgCl2

2+

0.72

35.7

50

4.0

Ca50-4

CaCl2

2+

1.00

0.205

50

4.0

Sr50-4

SrCl2

2+

1.18

0.0135

50

4.0

Al50-4

AlCl3

3+

0.54

38.5

50

4.0

Ca5-4

CaCl2

2+

1.00

0.205

Ca50-2

CaCl2

2+

1.00

0.205

4.0

5.0

a

4.0 50

2.0

Ionic radii were taken from ref. 20.

** The solubility of metal sulphates in water are taken from reference 23. In a qualitative study of gelation of CNC suspensions, we focused on sol-gel transitions induced by the addition of metal chlorides containing cations with different ionic radii and charge numbers. Figure 1a-c shows state diagrams characterizing the effect of CCNC and cation concentration on gel formation. The critical concentration of added metal chloride required for gelation reduced with increasing charge number of the added cation. For example, a higher concentration of NaCl was required to trigger gelation, in comparison with MgCl2 or AlCl3. The diagrams re-plotted for the corresponding Debye lengths of CNCs are given in Figure 1a'-c' (the


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calculations of the Debye length are given in Supporting Information). The similarity between the state diagrams shown in Figure 1a'-c' for different added salts implied that screening of electrostatic repulsion between the CNCs played an important role in the formation of CNC network structure. The difference in ionic radii between Mg2+, Ca2+, and Sr2+ cations did not significantly affect sol-gel boundaries in the state diagrams (Supporting Information, Figure S1). The threshold value of CCNC required for the sol-gel transition remained similar for Na+, Mg2+, Al3+, Mg2+, Ca2+, and Sr2+, which suggested that a minimum CCNC of ~1.5 wt % was required to form a network.

Figure 1. Effect of CNC and salt concentration on gelation of CNC suspension at 25 °C . (a-c) State diagrams for a) NaCl, b) MgCl, and c) AlCl solutions of various concentrations were added to CNC suspensions of various concentrations. (a'-c') State diagrams as in (a-c), respectively, plotted for the corresponding Debye lengths of CNCs. The sol and gel states are indicated as filled triangles and filled squares, respectively. The solid lines represent the boundaries between the sol and gel states.


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Qualitatively similar sol-gel transitions were observed at 25 and 37 °C (the physiological temperature), as shown in Supporting Information, Figure S2, suggesting that CNC gels are stable at physiological temperature and may be suitable for cell culture and in vivo applications, if their cytotoxicity is appropriate.

Rheological properties of CNC gels The rheological properties were studied for the CNC gels formed by adding 50 mm of a particular metal chloride to a suspension at CCNC=4 % w/w. Prior to the experiments, amplitude strain sweeps were performed on the gels to determine the region of the linear viscoelastic response (Supporting Information). Dynamic frequency sweeps were performed at 0.5 % strain. Figure 2 shows the effect of the cation charge number and ionic radii on the storage modulus (G’) and loss modulus (G′′) of the hydrogels. Over the entire range of oscillatory frequencies from 0.1 to 100 rad/s and for all the samples examined, the value of G′ was greater than G′′, which signified gel-like properties of the sample. Furthermore, for all the gels the values of loss factor (tan δ = G′′/ G′) were significantly smaller than unity, which suggested that elastic behaviour dominated.21 Table 2 summarizes the rheological characteristics of the gels, that is, the values of G′, G′′, |G*| (the magnitude of the complex modulus), and tan δ for the CNC gels, all at the oscillatory frequency of 1 rad/s. The complex shear modulus (G* = G′ + iG′′) characterized the rigidity of a gel subjected to deformation below the yield stress.21


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Figure 2. Dynamic frequency sweeps for a) Na50-4 (circles), Mg50-4 (squares), and Al50-4 (triangles); and b) Mg50-4 (circles), Ca50-4 (squares), and Sr50-4 (triangles). The variations in the storage moduli, G′, and loss moduli, G′′, are shown with close and open symbols, respectively. The dynamic frequency sweeps were performed at 0.5 % strain.

Table 2. Rheological properties and dimensions of CNC gelsa Sample

G'

G''

(kPa)

(kPa)

tan δ

|G*| (kPa)

Average mesh sizeb (nm)

Na50-4

1.5

0.1

0.08

1.5

71 ± 6

Mg50-4

7.7

1.0

0.13

7.8

80 ± 2

Ca50-4

10.0

1.4

0.14

10.1

85 ± 1

Sr50-4

11.8

1.7

0.14

12.0

92 ± 7

Al50-4

13.9

2.2

0.16

14.1

83 ± 1

Ca5-4

1.6

0.4

0.12

3.0

79± 0.4

Ca50-2

0.6

0.1

0.09

0.6

156 ± 5

a

Rheological experiments were performed at 0.5 % strain at a frequency of 1 rad/s. The uncertainty in the mesh size represents a standard deviation, based a set of triplicate experiments. b


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The results presented in Figure 2a indicate that both elastic and viscous contributions to gel rigidity increased with an increasing cation charge number in the order Na+ < Mg2+ < Al3+. This trend was consistent with an earlier study of gels formed by cellulose nanofibers functionalized with carboxylate surface groups.17 The increase in gel strength of CNCs with an increasing cation charge number was caused by the reduction in Debye length and stronger screening of electrostatic repulsion between the CNCs, thereby making van der Waals and hydrogen bonding interactions dominant forces favouring CNC association, in agreement with the DerjaguinLandau-Verwey-Overbeek (DLVO) theory.22 Figure 2b shows the variation in G′ and G′′ for the hydrogels formed in the presence of divalent cations with various ionic radii. The values of G' and G′′ for the oscillatory frequency of 1 rad/s are also shown in Table 2. The increase in ionic radii of the cations led to an increase in G′ and G′′ values over the entire frequency range. The change in rigidity (Mg50-4 < Ca50-4 < Sr50-4) correlated with the change in solubility of the metal sulphates in water: the solubility of MgSO4, CaSO4, and SrSO4 are 35.7, 0.205, and 0.0135 g per 100 g of water, respectively23. This trend suggested that the divalent cations, whose metal sulphates have low solubility in water, could bridge two adjacent sulphate half-ester groups of CNCs. Stronger association between sulphate groups and metal ions of varying ionic radii could be rationalized by using the hard-soft acid-base acid-base (HSAB) theory and resulted in the formation of more rigid gels. A similar bridging (cross-linking) effect by cations of larger ionic radii has been demonstrated for acidic groups decorating cellulose nanofibrils.17 Gels formed in the presence of Ca2+ cations were used to study the effect of salt and CNC concentrations on the rheological properties of the system (Figure 3). Upon the increase of Ca2+ cation concentration from 5 to 50 mm (corresponding to the Debye length of CNCs from 2.35 to


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0.78 nm, respectively) at CCNC=4 % w/w, the magnitude of the complex modulus, |G*|, increased from 1.6 to 8.2 kPa (Table 2). Similarly, increasing CCNC from 2 to 4 % w/w at Ca2+ cation concentration of 50 mm increased the value of |G*| from 0.59 to 8.2 kPa, respectively. Thus a 10fold increase in divalent salt concentration resulted in a 6-fold increase in gel rigidity, while a two-fold increase in CCNC resulted in a 17-fold increase in |G*|. Thus we conclude that (i) the mechanical properties of the CNC hydrogels were more sensitive to changes in CCNC than in the concentration of added cations and (ii) the addition of cations can be used to fine-tune the mechanical properties of CNC hydrogels.

Figure 3. Dynamic frequency sweeps for Ca50-2 (circles), Ca5-4 (squares), and Ca50-4 (triangles). The storage moduli, G′, and loss moduli, G′′, are shown in close and open symbols, respectively. The experiments were performed at 0.5 % strain.

The values of G′ and G" shown in Figures 2 and 3 were dependent on the oscillatory frequency. At low frequencies (from 0.1 to 1 rad/s), the value of G′ increased with frequency, while G" decreased. At higher oscillatory frequencies (from 1 to 100 rad/s), both G′ and G" increased with frequency. These trends were rationalized as follows. At low frequency, the dissipation of energy occurred due to the motion of large sections of the CNC network,24 while at


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high frequencies, the motion of small segments of the network (associated with a higher rigidity of the CNC network) was experimentally accessed. The hysteresis of the rheological properties of the gels was examined by repeating a dynamic frequency sweep immediately after completion of the first one. For Mg50-4, Al50-4, Ca50-4, Sr50-4, and Ca50-2 gels (Supporting Information, Figure S4), similar values of G′ and G" were obtained at high frequencies in both experiments, which implied that the gels either did not change, or rapidly recovered their structure after shear deformation. Significant hysteresis was observed for Na50-4 and Ca5-4: over the entire frequency range, the value of G′ strongly increased in the second experiment (Supporting Information S4 and S5), which was ascribed to the shear-induced reorganization of CNC fibrils in the gel.

Characterization of hydrogel structure Figure 4 shows scanning electron microscopy (SEM) images of the CNC hydrogels. The samples were prepared by the supercritical point drying method extensively used for imaging of hydrogel structures.17,25,26 All the hydrogels prepared in the presence of different cations exhibited a similar nanofibrillar network structure, with a random orientation of nanofibrils on the length scale of several micrometres. Thus the structure of CNC hydrogels was further characterized by their mesh size by measuring the diffusion coefficients of the free dextran molecular probe in solution and in the CNC hydrogels in pulsed field gradient-NMR experiments. Table 2 shows the mesh sizes of the CNC hydrogels formed in the presence of different cations. At 50 mm salt concentration and CCNC=4 % w/w, the mesh size increased in gels formed by adding cations with higher charge numbers and larger ion sizes. We attribute the increase in


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mesh size to the same two factors that affected gel rigidity. First, reduction in the Debye length of the CNCs (which for Na50-4, Ca50-4, and Al50-4 was 1.34, 0.78, and 0.55 nm, respectively) led to the screening of electrostatic repulsion between the CNCs. Second, increase in the cation radius enhanced CNC attraction via increasing metal-ligand affinity between the metal acid and the sulphate groups. Both effects favored association of CNCs and led to the formation of denser and/or thicker fibrils, which for a particular CCNC formed a gel with a larger mesh.

Figure 4. Scanning electron microscopy images of hydrogels, (a) Na50-4, (b) Mg50-4, (c) Al504, (d) Ca50-4, and (e) Sr50-4. The scale bars are 1 µm. The variation in CCNC at constant salt content (e.g., at 50 mm CaCl2) also influenced the gel mesh size. A decrease in CCNC from 4 to 2 % w/w led to an increase in mesh size from 85 to 156 nm (Table 2). In this case, a larger mesh size was the result of a smaller number of associating CNCs and a lower number of contact points per gel volume.


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At CCNC=4 % w/w, with a 10-fold reduction in the concentration of Ca2+ cations (an increase in Debye length from 0.78 to 2.35 nm), the mesh size decreased from 85 to 79 nm for Ca50-4 and Ca5-4, respectively. This effect occurred due to a weaker association between the CNCs at a lower cation concentration. Figure 5 shows polarized optical microscopy (POM) images of the CNC suspension (sample CNC0-4) and CNC gels at CCNC=4 % w/w. The POM image of the CNC0-4 sample exhibited a streaked texture, referred to as “pre-cholesteric order” (Figure 6a).12,27 Upon the introduction of 50 mm of salt (Figure 5b-f), the streaked texture of all the gels was replaced with a marble-like texture. At low concentration of CaCl2 at 5 mm, the streaked structure was partly preserved, although the density of the streaks increased (Figure 6a).

Figure 5. Polarization optical microscopy images of a) the CNC suspension (sample CNC0-4) and CNC gels of b) Na50-4, c)Mg50-4, d) Al50-4, e) Ca50-4, and f) Sr50-4. The scale bars are 100 µm.

The variation in CCNC also affected the gel texture examined by POM. At CCNC=2 % w/w, the CNC suspension phase-separated into anisotropic and isotropic phases,28 and the POM image of the gel featured small chiral nematic domains (tactoids) dispersed in an isotropic continuous


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phase (Figure 6b). In the presence of 50 mm of CaCl2, both Ca50-4 and Ca50-2 featured a similar marble-like texture (Figures 5e and 6c, respectively).

Figure 6. Polarization optical microscopy images of a) Ca5-4 gel, b) CNC0-2 suspension and c) Ca50-2 gel. The scale bars are 100 µm.

Characterization of the gel structure by small angle X-ray scattering Small angle X-ray scattering (SAXS) profile of the CNC0-4 sample revealed well-defined interference peaks with the correlation length of ∼40 nm (Figure 7). We used the rod model to describe the scattering profile of this system.29 The fit of the experimental profile to the theoretical one yielded inter-rod distance of ~40 nm and the average CNC diameter of ∼6 nm. (the small value of the CNC diameter was ascribed to their whisker-like geometry). Thus longrange repulsive interactions between the CNCs led to well-defined distances between the nanofibrils. In contrast, in the scattering vector range from 0.005 to 0.12 Å-1, corresponding to length scales from approximately 5 to 125 nm, no features could be attributed to a well-defined correlation length in the CNC gels prepared with addition of cations. Such behaviour can be explained either by a highly irregular distance between the CNCs associating in fibrils and/or the dense packing of CNCs in fibrils. In the latter case, the scattering contrast diminished due to the negligible gap between the CNCs, which resulted in the featureless SAXS profiles.


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Figure 7. SAXS intensity profiles for CNC0-4 suspension and CNC gels. The fitting for CNC0-4 is shown with the solid line. The SAXS intensity profiles arbitrarily were shifted for easier visualization.

Since the average mesh size in the CNC gels was below 120 nm (Table 1), we conclude that the gels obtained in the presence of cations did not exhibit an ordered structure with the characteristic length scale up to 125 nm (the upper limit of SAXS measurements). Thus the birefringence of these gels had a different origin. The CNC gels formed without the addition of cations (Figure 5a and Figure 6b) exhibited a pre-cholesteric order with the length scale exceeding 125 nm, in addition to the periodic distance between CNCs in the fibrils. Gels formed in the presence of cations showed a "marble" POM structure, which originated most probably, only from the CNC association side-by-side.

DISCUSSION Ionically mediated gelation of CNC suspensions yielded isotropic nanofibrillar gels. The rheological properties and structure (mesh size) of the gels depended on the type of cation introduced to the CNC suspension. To emphasize this effect, we plotted the variations in |G*| and mesh size as a function of the Debye length of the CNCs for gels formed by addition of different


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salts at constant CCNC and salt concentration (Figure 8). The change in |G*| and mesh size followed the same trend: with increasing charge number and ionic radii of the cation added, the value of |G*| and mesh size increased. Since with increasing cation charge number, repulsionbirefringence of these gels had a different origin. The CNC gels formed without the addition of cations (Figure 5a and Figure 6b) exhibited a pre-cholesteric order with the length scale exceeding 125 nm, in addition to the periodic distance between CNCs in the fibrils. Gels formed in the presence of added salts showed a "marble" POM structure, which originated most probably, only from side-by-side CNC association. between the CNCs reduced, association of the CNCs resulted in stronger and/or thicker fibrils. Therefore, the value of |G*| increased as Al50-4>Mg50-4>Na50-4, and for a given CCNC gels with a larger mesh size formed. We stress that for hydrogels Mg50-4, Ca50-4, and Sr50-4 at the same Debye length, the values of |G*| and mesh size increased with increasing ionic radii, which implied that an effect other than electrostatic screening (bridging) could be in play.

Figure 8. Variations in a) the complex modulus, |G*|, and b) the mesh size, both plotted as a function of Debye length for various gel samples containing 50 mm metal chloride and 4 % w/w CNCs. The values of |G*| are obtained from dynamic frequency sweeps at an oscillatory frequency of 1 rad/s with 0.5 % strain.


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Increasing rigidity with increasing mesh size was an unexpected result and was contrary to the conventional poroelastic theory,30,31 which rationalizes that gel rigidity increases with reducing mesh size. In the case of ionicallly mediated CNC gelation, due to the hierarchical structure of nanofibrillar hydrogels, they became stronger with increasing mesh size.

CONCLUSIONS In summary, we studied the properties of nanofibrillar CNC gels formed by adding cations with different charge numbers and ionic radii to aqueous CNC suspensions. Addition of ions with a higher charge number CNC suspensions reduced the Debye length of the CNCs, thereby favouring their association in a network structure with thicker mesh walls and/or higher stiffness. Bridging the sulphate groups of the CNCs by the metal cations with larger ionic radii also contributed in CNC gelation. As an ionic radius of the cation increased at the same Debye length of the CNCs, the CNCs gels became stiffer and the mesh size increased. Importantly, increase in mesh size paralleled with increase in gel rigidity, a unique feature for these hierarchically assembled hydrogels. The ability to vary the mechanical properties of CNC gels without significant change in mesh size is the advantage of ion-mediated gelation, in comparison with changing CNC concentration in suspensions. The structure-property relationships established in the present work are useful for fine-tuning the structure and mechanical properties of CNC hydrogels, which can be used for tissue engineering, pharmaceutical and industrial sol-gel applications.

ASSOCIATED CONTENT


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Transmission electron microscopy image of CNCs, state diagram of CNC dispersions, strain amplitude sweeps, hysteresis in shear of CNC gels, scanning electron microscopy image for Ca50-2 and Ca5-4, hydrodynamic radius of dextran probe, 1H NMR spectra and diffusion coefficients of dextran probe in solution and in CNC gels. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected].

Author Contributions MC, SS, DP and HT-A contributed in the preparation, characterization of hydrogel properties, data analysis and interpretation of the results; DN and OG conducted SAXS experiments and interpreted their results; GC, MM and JB prepared and characterized CNC properties. EK provided guidance in the experiments and in the interpretation of the results. The manuscript was written by all the authors. ACKNOWLEDGMENTS MC and SES acknowledge support of NSERC CREATE IDEM program. The authors thank Battista Calvieri for his assistance in the preparation of hydrogel samples for SEM imaging. Ilya Gourevich is acknowledged for his assistance with SEM imaging. We thank Professor Jan Lagerwall for his insight in the interpretation of the results of POM imaging. SAXS experiments were carried out at the Center for Functional Nanomaterials, Brookhaven National Laboratory,


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supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886. ABBREVIATIONS CNC, cellulose nanocrystal, SEM, scanning electron microscopy, PFG-NMR, pulse-field gradient nuclear magnetic resonance.

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For table of contents use only


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Ion-Mediated Gelation of Aqueous Suspensions of Cellulose

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